Lustri, WR and Oliveira Barud, HG present the same contributions in this book chapter manuscript.

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### **Crystalline Nanocellulose — Preparation, Modification, and Properties**

Mikaela Börjesson and Gunnar Westman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61899

### **Abstract**

Cellulose is a linear biopolymer found naturally in plant cells such as wood and cotton. It is the worlds most abundant polymer in nature and possesses properties such as good biocompatibility, low cost, low density, high strength, and good mechanical properties. By mechanical or chemical treatment, the cellulose fibers can be converted into cellulose nanofibers (CNFs) or cellulose nanocrystals (CNCs) that possess outstanding properties compared with the original cellulosic fiber but also when compared with other materials normally used as reinforcements in composite materials such as Kevlar or steel wires. This review will describe the nanocellulose materials preparation techniques and cellu‐ lose sources, chemical modification both on the crystalline surface and during hydrolysis and its many properties and its use in biocomposite materials. Nanocellulose in its differ‐ ent forms shows an increasing interest in application areas such as packaging, paper and paperboard, food industry, medical and hygiene products, paints, cosmetics, and optical sensors

**Keywords:** Nanocellulose, cellulose nanocrystals, hydrolysis, chemical modification, bio‐ composites

### **1. Introduction**

Cellulose (*Latin:* rich in small cells) is a biopolymer found naturally in, for example, plant cells such as wood and cotton. It is the most abundant polymer in nature and is the main constituent in the cell wall of trees and plants. Cotton have the highest cellulose content of the plants with about 90% cellulose, compared to wood that has about 40–50% cellulose content or bast fibers, such as flax, hemp, or ramie, which have about 70–80% cellulose content [1–2]. Besides wood and plants, cellulose can also be found in various bacterial species, algae, and tunicates, a sea animal that consists of proteins and carbohydrates.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The cellulose polymer is a linear homo-polysaccharide consisting of D-anhydroglucopyranose units (AGU) linked together by β-1,4-glycosidic bonds. Every other AGU is turned 180° with respect to its neighbor and two AGU next to each other form a cellobios unit, the smallest repeating unit in the polymer (Figure 1). The degree of polymerization (DP) is a measure of how many AGUs there is in the polymer and since no polymer is homogenous in length, the molecular weight distribution will have an important influence on the fibers properties. The DP of a cellulose polymer can be as high as 10,000 in wood cellulose and even higher in, for example, native cotton plant fibers. After degradation reactions and purification processes, the DP is reduced to about 300–1700 in wood cellulose [3–4].

The cellulose molecule contains three different kinds of AGU: a reducing end group that contains a free hemiacetal or aldehyde at the C1 position, a non-reducing end group with a free hydroxyl group at the C4 position, and internal glucose rings joined at the C1 and the C4 positions. The internal glucose units are predominant due to the long chain lengths. Each internal AGU has three hydroxyl groups. The hydroxyl group at the C6 position is a primary alcohol, while the hydroxyl groups at the C2 and C3 positions are secondary alcohols. These hydroxyl groups are all possible sites for chemical modification of cellulose where the hydroxyl group at the C6 position is the most reactive [5].

**Figure 1.** The molecular structure of a cellulose polymer where the cellobios is the smallest repeating unit in the poly‐ mer. The reducing end group can be either a free hemiacetal or an aldehyde.

Due to the linear and quite regular structure of cellulose and the many hydroxyl groups in the molecule, cellulose polymers can form ordered crystalline structures held together with hydrogen bonds. These crystalline regions give important mechanical properties to the cellulose fibers. The hydroxyl groups in the cellulose polymer can form hydrogen bonds between different cellulose polymers (intermolecular hydrogen bonds) or within the polymer itself (intramolecular hydrogen bonds). The intramolecular bonds give stiffness to the polymer chain, while the intermolecular bonds allow the linear polymers to form sheet structures. The high crystallinity and the many hydrogen bonds in the cellulose fibers make cellulose insoluble in water and in most conventional organic solvents [6].

The structure of a cellulose fiber can be divided into three different levels: the molecular level, the supramolecular level, and the morphological level. The molecular level was described in the beginning of this section (Figure 1). The supramolecular level is the polymer chains ordered in crystalline and non-crystalline regions due to hydrogen bonds and the morphological level consists of the cellulose fiber and its cell walls (Figure 2).

The cellulose polymer is a linear homo-polysaccharide consisting of D-anhydroglucopyranose units (AGU) linked together by β-1,4-glycosidic bonds. Every other AGU is turned 180° with respect to its neighbor and two AGU next to each other form a cellobios unit, the smallest repeating unit in the polymer (Figure 1). The degree of polymerization (DP) is a measure of how many AGUs there is in the polymer and since no polymer is homogenous in length, the molecular weight distribution will have an important influence on the fibers properties. The DP of a cellulose polymer can be as high as 10,000 in wood cellulose and even higher in, for example, native cotton plant fibers. After degradation reactions and purification processes, the

The cellulose molecule contains three different kinds of AGU: a reducing end group that contains a free hemiacetal or aldehyde at the C1 position, a non-reducing end group with a free hydroxyl group at the C4 position, and internal glucose rings joined at the C1 and the C4 positions. The internal glucose units are predominant due to the long chain lengths. Each internal AGU has three hydroxyl groups. The hydroxyl group at the C6 position is a primary alcohol, while the hydroxyl groups at the C2 and C3 positions are secondary alcohols. These hydroxyl groups are all possible sites for chemical modification of cellulose where the hydroxyl

**Figure 1.** The molecular structure of a cellulose polymer where the cellobios is the smallest repeating unit in the poly‐

Due to the linear and quite regular structure of cellulose and the many hydroxyl groups in the molecule, cellulose polymers can form ordered crystalline structures held together with hydrogen bonds. These crystalline regions give important mechanical properties to the cellulose fibers. The hydroxyl groups in the cellulose polymer can form hydrogen bonds between different cellulose polymers (intermolecular hydrogen bonds) or within the polymer itself (intramolecular hydrogen bonds). The intramolecular bonds give stiffness to the polymer chain, while the intermolecular bonds allow the linear polymers to form sheet structures. The high crystallinity and the many hydrogen bonds in the cellulose fibers make cellulose insoluble

The structure of a cellulose fiber can be divided into three different levels: the molecular level, the supramolecular level, and the morphological level. The molecular level was described in the beginning of this section (Figure 1). The supramolecular level is the polymer chains ordered in crystalline and non-crystalline regions due to hydrogen bonds and the morphological level

DP is reduced to about 300–1700 in wood cellulose [3–4].

160 Cellulose - Fundamental Aspects and Current Trends

group at the C6 position is the most reactive [5].

mer. The reducing end group can be either a free hemiacetal or an aldehyde.

in water and in most conventional organic solvents [6].

consists of the cellulose fiber and its cell walls (Figure 2).

**Figure 2.** The different levels of the cellulose structure: (i) the molecular structure of a cellulose polymer, (ii) the poly‐ mers ordered into microfibrils with crystalline and non-crystalline regions, (iii) several microfibrils assembled together to form a macrofibril, and (iv) the different layers in the cell wall. Numbers (ii) and (v) show cellulose microfibrils (CMFs) and cellulose nanocrystals (CNCs), respectively. *Illustration: Jari Sundqvist.*

Figure 2 shows the different levels of the cellulose structure where the cellulose polymers (i) are aggregated to form microfibrils (ii), which are long bundles of cellulose molecules stabi‐ lized by hydrogen bonds. Several microfibrils are assembled together to form a macrofibril (iii), which are oriented in different layers in the cell wall (iv) and the different layers differ in fibril direction, densities, and textures. There are four distinct layers in the cell wall named P (primary), S1, S2, and S3 (secondary) and each layer is mainly composed of a combination of the three polymers: cellulose, hemicellulose, and lignin. The S2 layer in the cell wall of higher plants, for example wood, has the highest quantity of cellulose. Between the cells there is a middle lamella (ML) consisting of pectin, a polysaccharide that binds the cells together providing the fiber stability. Numbers (ii) and (v) in Figure 2 represent nanocellulose, where (ii) shows cellulose microfibrils (CMFs) and (v) shows cellulose nanocrystals (CNCs).

The natural fiber strength and stiffness in cellulose fibers comes from the formation of the microfibrils (ii). Microfibrils have a wide range from 2 to 30 nm depending on cellulose source and a length that can be several micrometers. The fibrils are assembled into long threadlike bundles of cellulose molecules stabilized by hydrogen bonds [7–8].

Cellulose has been known for about 150 years and is a renewable and biodegradable polymer and has for a long time been used as energy source, building material, and clothing. By chemical modification on the cellulose polymers, cellulose derivatives such as cellulose ethers and cellulose ester can be prepared, which have opened up for many novel material and applications for cellulose such as coatings, films, membranes, new building materials, drilling techniques, pharmaceuticals, and food products. Also the regeneration process of cellulose has contributed to novel techniques such as spinning of fibers and the viscose process.

Nanotechnology, in the recent years, has gotten huge interest in many industries and nano‐ technology has opened up for many new possibilities, such as in the forest industry and cellulose-based products. Nanotechnology is defined as the understanding and control of matter with at least one dimension measuring from 1 to 100 nm. By mechanical treatments or chemical modifications on cellulose pulp, nanometer-sized cellulose such as cellulose nanofi‐ brils (CNFs) and cellulose nanocrystals (CNCs) can be produced. Nanocellulose has shown extraordinary properties compared with the bulk material but also with other materials such as Kevlar, carbon fibers, or stainless steel [9–10].

Nanocellulose is not yet a fully commercial product but the first factory for production and disposal of CNC was opened in 2012 by CelluForce in Canada that produces about one tonne of CNC per day. There are also some pilot plants for nanocellulose production located all over the world and one of the first pilot plants for nanocellulose was opened by Innventia in Sweden in 2011 and the first pilot plant in the United States opened in 2012 in Madison and is the country's leading producer of nanocellulose materials where they produce both CNCs and TEMPO-based cellulose nanofibrils. The aim of the pilot plant is to aid the commercialization of nanocellulose materials by providing researchers and early adopters in the area with working quantities of nanocellulose.

The most recent news about the production of CNC was announced by MoRe Research on May 2015. A new pilot plant for production of CNC will be started in Örnsköldsvik in north of Sweden on 2016 and will be the first of its kind in Europe [11]. The operations of the pilot plant are based on the Israeli start-up company Melodeas technology. Melodea have devel‐ oped a unique process that allows utilizing the sludge of the pulp and paper industry as a source for CNC production.

By expanding the knowledge in the area of nanocellulose and finding ways on how to control its properties during processing, new avenues in product development can be opened. Biobased products with outstanding properties can be produced and used as a replacement for fossil fuel-based products. There are some reviews about nanocellulose, most of them about nanocellulose as a reinforcement material in biocomposites or the use of bacterial nanocellulose (BNC) in medical or surgical products [4, 9, 12–15]. This review will deal with the current knowledge of isolation of nanocellulose, mainly derived from wood fibers, and chemical modification of cellulose nanocrystals, as well as the role of nanocellulose in biocomposites.

### **2. Nanocellulose**

Nanocellulose, or variously termed nanocrystals, whiskers, rods, nanofibrils, or nanofibers, is when the cellulose fiber or crystal has at least one dimension within the nanometer size range. In 2011, TAPPI released a roadmap for the development of international standards of nano‐ cellulose [16] where they stated the abbreviations for different nanocelluloses as: cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and cellulose microfibrils (CMFs), which will be the denomination used throughout this review. For cellulose nanocrystals, the noncrystalline regions are hydrolyzed and the remaining crystals will be in nanometer size range in all dimensions (Figure 3). For cellulose nanofibrils CNFs, sometimes also termed cellulose microfibrils (CMFs), some of the interfibrillated hydrogen bonds will break and form fibers with micrometer size in length (non-crystalline regions still present) and nanometer size in width. Bacterial nanocellulose (BNCs), on the other hand, are synthesized by special bacteria and grown as microfibrils in a culture medium. BNC microfibrils can also be hydrolyzed into bacteria nanocrystals by an acid hydrolysis similar to CNC.

Nanotechnology, in the recent years, has gotten huge interest in many industries and nano‐ technology has opened up for many new possibilities, such as in the forest industry and cellulose-based products. Nanotechnology is defined as the understanding and control of matter with at least one dimension measuring from 1 to 100 nm. By mechanical treatments or chemical modifications on cellulose pulp, nanometer-sized cellulose such as cellulose nanofi‐ brils (CNFs) and cellulose nanocrystals (CNCs) can be produced. Nanocellulose has shown extraordinary properties compared with the bulk material but also with other materials such

Nanocellulose is not yet a fully commercial product but the first factory for production and disposal of CNC was opened in 2012 by CelluForce in Canada that produces about one tonne of CNC per day. There are also some pilot plants for nanocellulose production located all over the world and one of the first pilot plants for nanocellulose was opened by Innventia in Sweden in 2011 and the first pilot plant in the United States opened in 2012 in Madison and is the country's leading producer of nanocellulose materials where they produce both CNCs and TEMPO-based cellulose nanofibrils. The aim of the pilot plant is to aid the commercialization of nanocellulose materials by providing researchers and early adopters in the area with

The most recent news about the production of CNC was announced by MoRe Research on May 2015. A new pilot plant for production of CNC will be started in Örnsköldsvik in north of Sweden on 2016 and will be the first of its kind in Europe [11]. The operations of the pilot plant are based on the Israeli start-up company Melodeas technology. Melodea have devel‐ oped a unique process that allows utilizing the sludge of the pulp and paper industry as a

By expanding the knowledge in the area of nanocellulose and finding ways on how to control its properties during processing, new avenues in product development can be opened. Biobased products with outstanding properties can be produced and used as a replacement for fossil fuel-based products. There are some reviews about nanocellulose, most of them about nanocellulose as a reinforcement material in biocomposites or the use of bacterial nanocellulose (BNC) in medical or surgical products [4, 9, 12–15]. This review will deal with the current knowledge of isolation of nanocellulose, mainly derived from wood fibers, and chemical modification of cellulose nanocrystals, as well as the role of nanocellulose in biocomposites.

Nanocellulose, or variously termed nanocrystals, whiskers, rods, nanofibrils, or nanofibers, is when the cellulose fiber or crystal has at least one dimension within the nanometer size range. In 2011, TAPPI released a roadmap for the development of international standards of nano‐ cellulose [16] where they stated the abbreviations for different nanocelluloses as: cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and cellulose microfibrils (CMFs), which will be the denomination used throughout this review. For cellulose nanocrystals, the noncrystalline regions are hydrolyzed and the remaining crystals will be in nanometer size range

as Kevlar, carbon fibers, or stainless steel [9–10].

162 Cellulose - Fundamental Aspects and Current Trends

working quantities of nanocellulose.

source for CNC production.

**2. Nanocellulose**

**Figure 3.** Part of a cellulose fiber where the crystalline and non-crystalline regions are shown. Acid hydrolysis removes the non-crystalline regions and only crystalline parts will remain (CNC). Mechanical treatment of the fibers will re‐ main both the non-crystalline and the crystalline regions but some of the interfibrillar bonds will break, creating fibrils in nanometer size in width and micrometer size in length (CNF).

Nanocellulose has generated a high interest, especially as a filler in biocomposites. Some beneficial attributes of nanocellulose are its sustainability, abundance, mechanical properties such as large surface to volume ratio, high tensile strength and stiffness, high flexibility, and good electrical and thermal properties [17]. Cellulose and nanocellulose have been classified as safe, both to handle and to consume [18–22]. Cellulose and some of its derivatives are approved by the European Food Safety Authority (E-number: E460-E466 and E468-E469) [23] and the U.S. Food and Drug Administration (FDA) for use as additives in food products.

There are, however, some disagreements if nanocellulose should be seen as non-toxic just because its origin, cellulose, is. A recent study by Yanamala et al. found inflammatory responses in the lungs of mice that have been exposed to CNC derived from wood [24]. Since nanomaterials are a quite new area, more studies on health and safety aspects and other analysis methods might be necessary to develop.

Some application areas for nanocellulose are listed below [12–13]:

**Paper, paperboard, and packaging:** One of the applications for nanocellulose in the paper and paperboard industry is to enhance the fiber-fiber bond strength and have a reinforcement effect on paper materials [25].

**Composite materials:** Nanocellulose has many beneficial and unique properties and is commonly used as filler or reinforcement in biocomposites (see Section 7 in this review).

**Food industry:** Nanocellulose can form emulsions and dispersions and is suitable for use in food products as thickeners or stabilizers [22].

**Medical and hygiene products:** Nanocellulose has good absorption properties and can be used in, for example, tissues, non-woven products, or diapers.

**Other applications:** Films, painting, cosmetics, automotive, etc.

There are different types of nanocellulose depending on, for example, the cellulose source and treatment. Different cellulose sources give different characteristics and also different aspect ratios (L/d, where L is the length and d is the diameter). The aspect ratio is a measure of length and width of the cellulose crystals or fibers. Almost particular crystals have a low aspect ratio (L/d = 1), while microfibrils can have a very high aspect ratio due to its small diameter (nm) and long fibrils (nm-µm). The higher the aspect ratio, the higher is the reinforcement capacity when incorporated in composite materials. Also, the more surface area of the fillers, the more contact will the filler have with the polymer matrices [17, 26]. Typical aspect ratio for CNC ranges from 1 to 100 [15, 27] and for CNF 15–100 [27].

### **3. Cellulose Nanocrystals (CNC)**

Cellulose nanocrystals (CNCs) generally have a width of about 2–30 nm and could be several hundreds of nanometers in length and are formed during acid hydrolysis of cellulose fibers where a selective degradation of the more accessible, disordered parts takes place. Since the non-crystalline regions (see Figure 3) act as structural defects in the microfibril, it is responsible for the transverse cleavage of the microfibrils into short monocrystals under acidic hydrolysis [28]. In the early stage of the hydrolysis the acid diffuses into the non-crystalline parts of the cellulose fiber and hydrolyzes the glycosidic bonds. After these, more easily accessible glycosidic bonds in the polymer are hydrolyzed and finally hydrolysis occurs at the reducing end group and at the surface of the nanocrystals. The harder it is for the acid to hydrolyze the glycosidic bonds the slower is the reaction [29–30]. The hydrolysis of the reducing end groups and the surface of the nanocrystals will make the nanocrystals charged depending on what acid is used. By using a 64 wt% sulphuric acid solution, 0.5–2% sulfate groups will be attached to the surface of the nanocrystal [31]. Due to the charged sulfate groups, CNC will form stable colloidal dispersion when diluted in water to specific concentrations [32–35].

Cellulose nanocrystals have been hydrolyzed from many different cellulose sources such as hardwood pulp [30], softwood pulp [36–39], microcrystalline cellulose (MCC) [40–41], sisal [42], cotton [39, 43–45], wheat straw [7, 46], rice straw [43, 47], bacterial cellulose [39, 48], algae [40, 49], banana fibers [17], sugar beet [50–53], and tunicin [39, 45, 53–60]. Other biopolymers that have been reported to form nanocrystals during acid hydrolysis are chitin [61–62], potato pulp [63–66], yellow pea [67], and waxy maize [68–69]. The different types of cellulose sources give some different structures of the nanocrystals and the aspect ratio will differ for the different sources. The dimensions and aspect ratio for some different cellulose sources are shown in Table 1.


**Table 1.** Geometrical characteristics of nanocrystals from different cellulose sources.

### **3.1. Acidic treatment on cellulose**

**Food industry:** Nanocellulose can form emulsions and dispersions and is suitable for use in

**Medical and hygiene products:** Nanocellulose has good absorption properties and can be used

There are different types of nanocellulose depending on, for example, the cellulose source and treatment. Different cellulose sources give different characteristics and also different aspect ratios (L/d, where L is the length and d is the diameter). The aspect ratio is a measure of length and width of the cellulose crystals or fibers. Almost particular crystals have a low aspect ratio (L/d = 1), while microfibrils can have a very high aspect ratio due to its small diameter (nm) and long fibrils (nm-µm). The higher the aspect ratio, the higher is the reinforcement capacity when incorporated in composite materials. Also, the more surface area of the fillers, the more contact will the filler have with the polymer matrices [17, 26]. Typical aspect ratio for CNC

Cellulose nanocrystals (CNCs) generally have a width of about 2–30 nm and could be several hundreds of nanometers in length and are formed during acid hydrolysis of cellulose fibers where a selective degradation of the more accessible, disordered parts takes place. Since the non-crystalline regions (see Figure 3) act as structural defects in the microfibril, it is responsible for the transverse cleavage of the microfibrils into short monocrystals under acidic hydrolysis [28]. In the early stage of the hydrolysis the acid diffuses into the non-crystalline parts of the cellulose fiber and hydrolyzes the glycosidic bonds. After these, more easily accessible glycosidic bonds in the polymer are hydrolyzed and finally hydrolysis occurs at the reducing end group and at the surface of the nanocrystals. The harder it is for the acid to hydrolyze the glycosidic bonds the slower is the reaction [29–30]. The hydrolysis of the reducing end groups and the surface of the nanocrystals will make the nanocrystals charged depending on what acid is used. By using a 64 wt% sulphuric acid solution, 0.5–2% sulfate groups will be attached to the surface of the nanocrystal [31]. Due to the charged sulfate groups, CNC will form stable

Cellulose nanocrystals have been hydrolyzed from many different cellulose sources such as hardwood pulp [30], softwood pulp [36–39], microcrystalline cellulose (MCC) [40–41], sisal [42], cotton [39, 43–45], wheat straw [7, 46], rice straw [43, 47], bacterial cellulose [39, 48], algae [40, 49], banana fibers [17], sugar beet [50–53], and tunicin [39, 45, 53–60]. Other biopolymers that have been reported to form nanocrystals during acid hydrolysis are chitin [61–62], potato pulp [63–66], yellow pea [67], and waxy maize [68–69]. The different types of cellulose sources give some different structures of the nanocrystals and the aspect ratio will differ for the different sources. The dimensions and aspect ratio for some different cellulose sources are

colloidal dispersion when diluted in water to specific concentrations [32–35].

food products as thickeners or stabilizers [22].

164 Cellulose - Fundamental Aspects and Current Trends

in, for example, tissues, non-woven products, or diapers.

ranges from 1 to 100 [15, 27] and for CNF 15–100 [27].

**3. Cellulose Nanocrystals (CNC)**

shown in Table 1.

**Other applications:** Films, painting, cosmetics, automotive, etc.

When cellulose pulp is treated with an acid, the pulp will start to degrade. The degradation will start with the most accessible parts of the fiber, followed by the reducing end groups and the crystal surfaces. Acid concentration, reaction time, and reaction temperature are some of the most important parameters for controlling the acid hydrolysis of wood pulp. A reaction time that is too long will hydrolyze the cellulose crystals completely and a reaction time that is too short will give a high degree of polymerization (DP) due to large undispersed fibers [30]. The reaction temperature and time correlate to each other and a higher reaction temperature, shorten the reaction time. Not only time and temperature affect the properties of nanocellulose, but also the acid concentration and acid to pulp ratio [29]. After hydrolyzation and purification through dialysis, small crystalline rod-like particles will be yielded in an aqueous suspension.

The nanocrystalline cellulose that is formed through the acidic treatment is of colloidal dimensions and forms an aqueous suspension when stabilized. The critical concentration of the colloidal suspension, which is the lowest concentration where the whiskers self-organize, depends on particle size, acidic treatment, preparation conditions, aspect ratio, and ionic strength [29–30, 33, 70–71]. Revol et al. [36] showed that microcrystals from bleached Kraft wood pulp spontaneously ordered in a crystalline phase above the critical concentration. This observation was also observed for cellulose whiskers from cotton and the critical concentration ranging from 2 wt% to 10 wt% depending on the preparation conditions.

The self-ordered nanocrystals form a chiral nematic ordering as seen in Figure 4. This helical, self-ordered structure has catalytic and photonic crystal benefits and researchers have tried to introduce chirality into porous inorganic solids by using CNC as a template to improve properties in other materials for applications such as optical filters or sensors [72]. From many aqueous CNC solutions, a bluish color can be observed that is due to the helical chiral nematic ordering and the length of the pitch gap (p). CNCs are able to absorb visible light and de‐ pending on the length of the pitch gap, different wavelengths are absorbed and the reflected light emits different colors. Therefore, different colored CNC film can be seen if the films are sufficiently thin.

**Figure 4.** At certain concentrations, the nanocrystals self-organize into a chiral nematic ordering where the length of the pitch gap (p) in the helical structure can absorb different wavelengths and emit wavelengths with different colors. *Illustration: Jari Sundqvist.*

Sulfuric acid (H2SO4) is the most common acid for nanocellulose preparation through chemical hydrolysis but it is possible to use other acids as well. Hydrochloric acid (HCl), hydrobromic acid (HBr), and phosphoric acid (H3PO4) have been used for CNC preparation but compared to sulfuric acids, hydrochloric acid, and hydrobromic acid, will not have any surface charges and a stable colloidal dispersion is, therefore, harder to form (Figure 5). Phosphoric acid will give charged phosphate groups on the nanocrystal surface (Figure 5c). Regarding the process industry, sulfuric acid is a more suitable choice of acid compared to hydrochloric acid. In 2008, sulfuric acid was the most produced chemical in the U.S., which was almost 10-fold more compared to hydrochloric acid, making sulfuric acid economically beneficial due to its larger quantities [73].

**Figure 5.** The surface polymer chain of CNC can be modified with different functional groups to give different surface characteristics. The surface modification of CNC depends on the isolation process and further treatment of the nano‐ crystals. The figure shows some examples of different functional groups attached to the surface cellulose polymers where (a) shows the surface polymer chain after H2SO4 hydrolysis, (b) after HCl or HBr hydrolysis, (c) after H3PO4 hydrolysis, (d) after H2SO4 hydrolysis followed by a surface cationization, and (e) after a HCl/HBr hydrolysis followed by a TEMPO-oxidation.

### *3.1.1. Isolation of CNC using sulfuric acid*

**Figure 4.** At certain concentrations, the nanocrystals self-organize into a chiral nematic ordering where the length of the pitch gap (p) in the helical structure can absorb different wavelengths and emit wavelengths with different colors.

Sulfuric acid (H2SO4) is the most common acid for nanocellulose preparation through chemical hydrolysis but it is possible to use other acids as well. Hydrochloric acid (HCl), hydrobromic acid (HBr), and phosphoric acid (H3PO4) have been used for CNC preparation but compared to sulfuric acids, hydrochloric acid, and hydrobromic acid, will not have any surface charges and a stable colloidal dispersion is, therefore, harder to form (Figure 5). Phosphoric acid will give charged phosphate groups on the nanocrystal surface (Figure 5c). Regarding the process industry, sulfuric acid is a more suitable choice of acid compared to hydrochloric acid. In 2008, sulfuric acid was the most produced chemical in the U.S., which was almost 10-fold more compared to hydrochloric acid, making sulfuric acid economically beneficial due to its larger

**Figure 5.** The surface polymer chain of CNC can be modified with different functional groups to give different surface characteristics. The surface modification of CNC depends on the isolation process and further treatment of the nano‐ crystals. The figure shows some examples of different functional groups attached to the surface cellulose polymers where (a) shows the surface polymer chain after H2SO4 hydrolysis, (b) after HCl or HBr hydrolysis, (c) after H3PO4 hydrolysis, (d) after H2SO4 hydrolysis followed by a surface cationization, and (e) after a HCl/HBr hydrolysis followed

*Illustration: Jari Sundqvist.*

166 Cellulose - Fundamental Aspects and Current Trends

quantities [73].

by a TEMPO-oxidation.

Sulfuric acid hydrolysis of cellulose pulp is a heterogeneous process where the acid diffuses into the pulp fiber and cleaves the glycosidic bonds in the cellulose polymer. Depending on reaction times, the hydrolysis could also occur on the crystalline regions and some of the hydroxyl groups on the crystalline surface will convert into sulfate groups (e.g., conversion of cellulose-OH to cellulose-OSO3 − H+ , Figure 5a). Other side reactions are also possible during acid hydrolysis such as dehydration and oxidation [36, 74]. In the cellulose pulp sample, hemicelluloses or pectin might be present and these polysaccharides will also undergo hydrolysis but at faster rates due to their higher reactivity. As the acid hydrolysis proceeds, the DPs are expected to decrease since the non-crystalline regions in the microfibril will be removed. Due to the loss of non-crystalline regions the crystallinity will increase and also the insolubility against water because the crystalline parts are less accessible (see Figure 6) [74].

The sulfuric acid hydrolyze reaction has been optimized by several researchers and one general way to produce CNC from sulfuric acid is by using a 64 wt% sulfuric acid solution at 45 °C for 45–60 min with constant stirring, followed by quenching the suspension with 10-fold deionized water, concentrate the CNC through centrifugation and dialysis against deionized water until constant neutral pH is achieved. To achieve separate crystals, the suspension has to be sonicated repeatedly [29, 33, 75].

The sulfuric acid hydrolysis reaction conditions on softwood pulp has been evaluated [30, 74]. When using a 64 wt% sulfuric acid solution, an acid to pulp ratio of 8.75 ml/g and treated the pulp at 45 °C at two different reaction times, 25 and 45 min, the longer reaction time showed a less polydisperse length distribution and a higher sulfur content than the shorter reaction time did. When the acid to pulp ratio is increased to 17.5 ml/g and the pulp is treated for 45 min at 45 °C, a smaller length and polydispersity was observed but the effect was not that large compared with the much higher acid to pulp ratio. Also, different sulfuric acid concentrations on softwood Kraft pulp and its effect on DP, crystallinity, crystal size, and yield was observed [74]. Three different sulfuric acid concentrations (16 wt%, 40 wt%, and 64 wt%) with the acid to pulp ratio 8.75 ml/g and three different reaction temperatures (45 °C, 65 °C, and 85 °C) were evaluated at a reaction time of 25 min. Both higher acid concentration and higher reaction temperature resulted in a lower DP, individually. The yield should be lower for CNC due to the loss of the non-crystalline regions and for the pulp samples hydrolyzed with a 64 wt% sulfuric acid, the yield drops at all three temperatures that were tested. Figure 6 shows a diagram where the crystal size, DP, and amount of crystallinity are marked in relation to each other. The highest crystallinity was obtained for the highest concentration of sulfuric acid, which also gives the smallest crystals and lowest DP.

Dong et al. studied the preparation of CNC from cotton fibers where the reaction time and temperature were in focus [29]. A 64 wt%, sulfuric acid was used and the acid to pulp ratio was 8.75 ml/g. The temperatures tested were 26 °C, 45 °C, and 65 °C, and the reaction times were 15 min up to 18 h. For low temperatures (26 °C), the reaction time needed to be very long (18 h) to produce CNC that could form an ordered suspension. At 65 °C, the reaction was hard to control and already after 15 min a color change was noted, indicating side reactions such as dehydration. After 1 h at 65 °C, the whole suspension had turned from white to black. The

**Figure 6.** Crystallinity size in relation to the DP. During acid hydrolysis, the DP will decrease and the cellulose crystal‐ linity will increase, due to loss of non-crystalline regions and, as a result, smaller crystal size will be obtained.

most optimal and easy handled reaction parameters for a stable colloidal CNC suspension was performed at 45 °C for 1 h.

Dong and co-workers also studied the relationship between reaction time and crystallinity size of the cellulose particles for hydrolysis with a 64 wt% sulphuric acid at 45 °C for 10–240 min [29]. The reaction was fast from the beginning but slows down in the later stages. The initial rapid decrease was due to the acid that diffused into the non-crystalline regions and hydro‐ lyzed the most accessible glycosidic bonds. The harder it was to hydrolyze the glycosidic bonds the slower was the reaction. After 1 h at 45 °C, the crystallinity size became relatively stable.

### *3.1.2. Isolation of CNC using alternative acids*

There are other acids besides sulfuric acid that can be used for the hydrolysis of cellulose fibers into CNC, for example, hydrochloric acid, which when compared to sulphuric acid will not give any charged groups on the cellulose crystal surface (Figure 5b). The lack of charged groups could be suitable for the study of enzymatic degradation, which is hard to study when surface groups can hinder the substrate recognition by the enzymes [37].

The procedure for a hydrochloric acid hydrolysis was described by Araki and co-workers. The hydrolysis was prepared by treating Kraft pulp with 30 ml/g of a 4 N hydrochloric acid at 80 °C for 225 min. The suspension was then centrifuged repeatedly until the sample reached pH 4 and the CNC became non-sedimenting and the supernatant became turbid. The supernatant was collected, purified, and neutralized by dialysis against deionized water and finally sonicated to disperse the suspension [37–38].

Hydrobromic acid and phosphoric acid are two other acids that have been used in the preparation of CNC. Lee and co-workers studied HBr-CNC for their use in PVA composites [76] and the HBr-CNC was prepared by adding 40 ml/g 1.5–2.5 M hydrobromic acid to MCC. The hydrolysis was quenched after 4 h at 100 °C. The HBr-CNC showed a decrease in DP of the higher acid concentration used and a higher thermal stability both for the CNC and the composite films.

Zhang and co-workers [77] studied the effect of four different acids for CNC hydrolysis on bamboo cellulose where 25 ml/g acid with concentration of 6.5 M was added to the cellulose samples and the hydrolysis occurred for 2 h at 60 °C. The different acids were a sulphuric acid solution, hydrochloric acid solution, phosphoric acid solution, and a mixture of acetic acid and nitric acid solution (ratio 10:1). Sulfuric acid gave the highest crystallinity index followed by phosphoric acid. Hydrochloric acid and the mixed acetic and nitric acid solution gave the lowest crystallinity index due to the higher tendency to promote the breakage of the hydrogen bonds in crystalline regions of cellulose. Both hydrochloric acid and the mixed acetic acid and nitric acid solution have better capability to swell cellulose, thereby facilitating the breakage of intra- and intermolecular hydrogen bonds in the crystalline regions.

Since no surface groups will be attached on the cellulose crystals when prepared from hydrochloric acid or hydrobromic acid, the colloidal dispersion is not as stable for HCl/HBr-CNC as for CNC prepared from sulfuric acid. A comparison between sulfuric acid and hydrochloric acid have been made on waxy maize starch and on softwood Kraft pulp, where it was found that CNC prepared from hydrochloric acid showed a higher risk for agglomerate in aqueous medium. The charged groups on CNC prepared from sulfuric acid limits their ability to flocculate [37, 78]. The CNC prepared from hydrochloric acid showed a thixotropic behavior easier than for CNC from sulfuric acid. This was due to particle aggregation, which is formed in static conditions but is destroyed by shear flow. By increasing the distance between the particles, the ability to aggregate reduces. Colloidal suspensions are often stabilized by electrostatic repulsion or by steric hindrance such as grafted polymers.

Araki et al. described a method to attach sulfate groups to the cellulose crystals prepared from a hydrochloric acid hydrolysis through an esterification reaction with sulfuric acid (postsulfonation) [38, 79]. The esterification reaction was carried out with a 65 wt% sulfuric acid solution added to CNC prepared from hydrochloric acid and the mixture was reacted in a shaken water bath for 2 h at 60 °C. After reaction, the sample was diluted with a large amount of cold water and washed by repeated centrifugation and decantation cycles, followed by dialysis and sonication. Through this method it was possible to control the surface charge on the cellulose nanocrystals.

Stable aqueous systems of CNC prepared from hydrochloric acid hydrolysis have been achieved by grafting polyethylene glycol (PEG) onto the nanocrystals. The grafting was performed by using an oxidative carboxylation-amidation procedure known as TEMPO [80– 81]. The grafting of nanocrystals acts as a steric hindrance in the CNC suspension and inhibits particle aggregation and enhances a stable colloidal CNC solution. An example of charged surface groups from TEMPO-oxidations is shown in Figure 5e.

### **4. Cellulose Nanofibril (CNF)**

most optimal and easy handled reaction parameters for a stable colloidal CNC suspension was

**Figure 6.** Crystallinity size in relation to the DP. During acid hydrolysis, the DP will decrease and the cellulose crystal‐ linity will increase, due to loss of non-crystalline regions and, as a result, smaller crystal size will be obtained.

Dong and co-workers also studied the relationship between reaction time and crystallinity size of the cellulose particles for hydrolysis with a 64 wt% sulphuric acid at 45 °C for 10–240 min [29]. The reaction was fast from the beginning but slows down in the later stages. The initial rapid decrease was due to the acid that diffused into the non-crystalline regions and hydro‐ lyzed the most accessible glycosidic bonds. The harder it was to hydrolyze the glycosidic bonds the slower was the reaction. After 1 h at 45 °C, the crystallinity size became relatively stable.

There are other acids besides sulfuric acid that can be used for the hydrolysis of cellulose fibers into CNC, for example, hydrochloric acid, which when compared to sulphuric acid will not give any charged groups on the cellulose crystal surface (Figure 5b). The lack of charged groups could be suitable for the study of enzymatic degradation, which is hard to study when surface

The procedure for a hydrochloric acid hydrolysis was described by Araki and co-workers. The hydrolysis was prepared by treating Kraft pulp with 30 ml/g of a 4 N hydrochloric acid at 80 °C for 225 min. The suspension was then centrifuged repeatedly until the sample reached pH 4 and the CNC became non-sedimenting and the supernatant became turbid. The supernatant was collected, purified, and neutralized by dialysis against deionized water and finally

Hydrobromic acid and phosphoric acid are two other acids that have been used in the preparation of CNC. Lee and co-workers studied HBr-CNC for their use in PVA composites [76] and the HBr-CNC was prepared by adding 40 ml/g 1.5–2.5 M hydrobromic acid to MCC. The hydrolysis was quenched after 4 h at 100 °C. The HBr-CNC showed a decrease in DP of

performed at 45 °C for 1 h.

168 Cellulose - Fundamental Aspects and Current Trends

*3.1.2. Isolation of CNC using alternative acids*

sonicated to disperse the suspension [37–38].

groups can hinder the substrate recognition by the enzymes [37].

Cellulose nanofibrils (CNFs), sometimes termed cellulose microfibrils (CMFs), are unlike CNC produced by mechanical treatment that preserves the non-crystalline parts in the microfibril as well as the length of the fibrils. Microfibrils are long, threadlike bundles of cellulose molecules that are stabilized by hydrogen bonds mainly between the many hydroxyl groups in the cellulose polymer. By using mechanical treatment, forces will peel the fibers and the interfibrillar bonds between the cellulose molecules will break and give nanofibrils with a diameter in nanodimensions and fiber length ranging from nanometer to micrometer. A common mechanical treatment for CNF is refining the pulp, followed by homogenization, which will individualize the nanofibrils and give a stable dispersion when diluted in water. Another technique to produce CNF is through regeneration and electrospinning of a cellulose polymer melt [4].

CNF can be produced from many different types of cellulose sources. A huge area of interest for CNF is in applications such as composite materials where the high aspect ratio of CNF and long flexible microfibrils is suitable for use as reinforcement in a polymer matrix. Bhatnagar and Sain studied the possibility to use cellulose nanofibers from plant cells due to its high abundance and low costs [82]. CNF were prepared from flax fibers, rutabaga, Kraft pulp, and hemp fibers using chemo-mechanical treatments before they were used as a filler in a polyvinyl alcohol (PVA) matrix. The use of plant fibers as reinforcements in composites did improved the mechanical properties in the composite material compared with the pure PVA [82].

### **4.1. Mechanical treatment of cellulose fibers**

The mechanical treatment of pulp fibers consists of refining the pulp, followed by a highpressure homogenization process to obtain individualized cellulose nanofibrils. In order to purify the cellulose fibers and decrease the amount of lignin, hemicellulose, and pectin, chemical treatments might be necessary before the mechanical treatment. There are mainly two different treatments to extract the fibers before homogenization of the nanofibril solution: using a refiner or by cryochrusching.

In the refining process, a dilute suspension of cellulose (1–2 wt%) is treated with forces in a refiner or blender equipped with bars, against which the fibers are subjected to repeated cyclic stresses which changes the morphology and size of the fibers [4, 52, 64]. The refining process is carried out before the individualization of the cellulose fibers because refining causes external fibrillation by peeling the external cell wall layers (P and S1 layers, see Figure 2) and exposing the S2 layer, which is the layer with the highest quantity of cellulose. Refining also cause internal fibrillation that unlooses the fiber wall, which is suitable in the following homogenization process [4].

Another treatment to extract cellulose fibers is cryochrusching. A liquid nitrogen-frozen pulp is mechanically crushed and the ice crystals, which are formed within the pulp cell wall, leads to release the cellular wall fragments [50]. These smaller fragments are later diluted in water before their homogenization to a CNF suspension in a homogenizer.

The individualization of the nanofibrils takes place in a homogenizer. In the homogenization process, refined and diluted suspensions of cellulose fibers are pumped at high pressure and fed through a spring high-pressure loaded valve assembly. The valve is opened and closed very rapidly, which lead to large pressure drops with shearing and impact forces affecting on the fibers. The number of passes through the valve will vary depending on the starting material. The combination of high pressure and forces on the cellulose fibers gives a high degree of microfibrillation, resulting in a CNF solution [4, 50, 64].

To ease the mechanical treatment of microfibers and to reduce the energy consumption, chemical treatment on the cellulose fibers can be used as a pre-treatment before the refining takes place. The pre-treatment can be enzymatic [83] or by introduction of charged groups to the fiber surface, e.g., through a carboxylation or by TEMPO-oxidation [84]. The pre-treatment will affect the surface of the fibers, which will make it easier to peel the fiber in the refining process and by that reduce the energy consumption..

### **5. Bacterial Nanocellulose (BNC)**

as well as the length of the fibrils. Microfibrils are long, threadlike bundles of cellulose molecules that are stabilized by hydrogen bonds mainly between the many hydroxyl groups in the cellulose polymer. By using mechanical treatment, forces will peel the fibers and the interfibrillar bonds between the cellulose molecules will break and give nanofibrils with a diameter in nanodimensions and fiber length ranging from nanometer to micrometer. A common mechanical treatment for CNF is refining the pulp, followed by homogenization, which will individualize the nanofibrils and give a stable dispersion when diluted in water. Another technique to produce CNF is through regeneration and electrospinning of a cellulose

CNF can be produced from many different types of cellulose sources. A huge area of interest for CNF is in applications such as composite materials where the high aspect ratio of CNF and long flexible microfibrils is suitable for use as reinforcement in a polymer matrix. Bhatnagar and Sain studied the possibility to use cellulose nanofibers from plant cells due to its high abundance and low costs [82]. CNF were prepared from flax fibers, rutabaga, Kraft pulp, and hemp fibers using chemo-mechanical treatments before they were used as a filler in a polyvinyl alcohol (PVA) matrix. The use of plant fibers as reinforcements in composites did improved the mechanical properties in the composite material compared with the pure PVA [82].

The mechanical treatment of pulp fibers consists of refining the pulp, followed by a highpressure homogenization process to obtain individualized cellulose nanofibrils. In order to purify the cellulose fibers and decrease the amount of lignin, hemicellulose, and pectin, chemical treatments might be necessary before the mechanical treatment. There are mainly two different treatments to extract the fibers before homogenization of the nanofibril solution:

In the refining process, a dilute suspension of cellulose (1–2 wt%) is treated with forces in a refiner or blender equipped with bars, against which the fibers are subjected to repeated cyclic stresses which changes the morphology and size of the fibers [4, 52, 64]. The refining process is carried out before the individualization of the cellulose fibers because refining causes external fibrillation by peeling the external cell wall layers (P and S1 layers, see Figure 2) and exposing the S2 layer, which is the layer with the highest quantity of cellulose. Refining also cause internal fibrillation that unlooses the fiber wall, which is suitable in the following

Another treatment to extract cellulose fibers is cryochrusching. A liquid nitrogen-frozen pulp is mechanically crushed and the ice crystals, which are formed within the pulp cell wall, leads to release the cellular wall fragments [50]. These smaller fragments are later diluted in water

The individualization of the nanofibrils takes place in a homogenizer. In the homogenization process, refined and diluted suspensions of cellulose fibers are pumped at high pressure and fed through a spring high-pressure loaded valve assembly. The valve is opened and closed very rapidly, which lead to large pressure drops with shearing and impact forces affecting on

before their homogenization to a CNF suspension in a homogenizer.

polymer melt [4].

170 Cellulose - Fundamental Aspects and Current Trends

**4.1. Mechanical treatment of cellulose fibers**

using a refiner or by cryochrusching.

homogenization process [4].

The last kind of nanocellulose that will be discussed is the bacterial nanocellulose (BNC), which is synthesized from special bacteria that build up nanofibers with nanometer size in diameter and up to micrometer size in length. There are several reviews and research reports found in the literature describing BNC and its structure and properties [4, 12–13]. Unlike other cellulose sources, bacterial cellulose is grown to nanofibers by special bacteria, such as *Acetobacter* species, cultivated in a culture medium. The bacteria produce BNC by synthesizing cellulose and building up bundles of microfibrils [85].

Acetobacter is a microorganism present everywhere in nature where sugar fermentation occurs and it is involved in the conversion of ethanol to acetic acid. Acetobacter has for many years been used in the fermentation industry for mass production of acetic acid and is important in several other industries where acetic acid is of importance.

BNC have unique properties like an extremely fine and pure fiber network structure, high degree of polymerization (up to 8,000), good mechanical properties such as high mechanical strength, biocompatibility, and water holding capability [4, 13]. The main application area for bacterial nanocellulose is in medical health and surgical applications such as bandages for wound healing or skin burns or as a substitute for medical materials such as blood vessels. Some other application areas where BNC can be found include the food industry and the paper and packaging industry [86–88]. The use of BNC in composites is also of interest, which have been studied by Grunert and Winter [89], where BNC fibrils first where hydrolyzed to bacterial nanocrystals and then used as a reinforcement in cellulose acetate butyrate (CAB) films.

The procedure that Tokoh et al. used to produce bacterial cellulose from Acetobacter species was to cultivate the bacteria in a culture medium for three days followed by liberation of the bacteria by rinsing the synthesized sheet with distilled water. The cells were gathered by centrifugation and re-suspended in the control medium [85]. The bacterial cellulose is micro‐ meter long threadlike bundles of nanofibrils, which can be hydrolyzed to nanocrystals by e.g., sulphuric acid hydrolysis, as described in section 3.1.1.

Some disadvantages with BNC are the low availability of the bacterial cellulose, the inefficient process in synthesizing bacteria cellulose and the high costs, which makes it hard to make BNC commercially attractive. The traditional process in synthesizing BCN cannot produce the high quantities that would be requested for a commercialization of BNC and further process development needs to be performed for a large scale production of bacteria cellulose [88].

### **6. Chemical modification on nanocellulose**

During sulfuric acid hydrolysis of cellulose, starch or chitin, sulfate groups will cover the surface of the nanocrystals. If using hydrochloric acid instead, the sulfate groups can be attached to the nanocrystal surfaces afterwards by an esterification reaction with sulfuric acid [37–38, 79]. In this way, the amount of charged groups on the cellulose crystal surface can be controlled. By using chemical modification on nanocrystals or nanofibers, the properties of the nanocrystal or nanofiber could be changed and controlled in specific ways.

Figure 5 shows some different surface treatments on CNC where a TEMPO-oxidation or cationization of the CNC surface can give charged side groups which will give electrostatic repulsion between the crystals and prevent aggregation, especially on CNC prepared from hydrochloric acid or hydrobromic acid.

Most of the literature that describes chemical modification of nanoparticles had the aim of improving the interfacial compatibility between the nanocrystals and various polymer matrices. A better compatibility could enhance the mechanical properties of the composite materials and addition of just a few percent of nanocellulose to a polymer matrix could improve the mechanical properties of the significant composite material [27, 35, 62, 90]. Most of the literature about chemical modification on nanocellulose describes a surface modification of the crystals or fibrils. For surface modification of CNC, there are mainly two different proce‐ dures, surface compatibilization or co-polymerization. In surface compatibilization the main idea is to attach a small molecular agent to the cellulose polymer, where the molecular agent contains at least one reactive moiety. For co-polymerization the molecular agent needs at least two functional groups, one that can react with the hydroxyl groups on the cellulose polymer and one that can covalently bond to a polymer matrix. The co-polymerization can be performed in different ways such as grafting, radical reactions, or by the use of organometallics [27].

Chemical modification on chitin nanowhiskers has been performed to change the surface properties of the nanocrystals to make it possible to have in organic solvents [62]. The reagents used to modify the chitin nanowhiskers were alkenyl succinic anhydride, phenyl isocyanate, and isopropenyl-α,α'-dimethylbenzyl isocyanate. The chemically modified chitin whiskers showed stable suspensions in toluene and were used in composite films casted from a toluene solution and all of the results showed that the chemically modified whiskers improved the adhesion between the filler and the matrix. The results also showed that the chemical treatment decreased the mechanical performance of the composites. The loss of performance could be due to the destruction of the 3-dimensional network of the chitin whiskers that occurs in the unmodified composites [61–62]. Nanocrystals from waxy maize starch have also been chem‐ ically modified with alkenyl succinic anhydride and phenyl isocyanate [90] to study the crystal structure of the nanoparticles after chemical treatment. X-ray diffraction analysis confirmed that the crystalline nanostructure was preserved and contact angle measurements concluded that the surface chemical modification enhancing the non-polar nature of waxy maize starch nanocrystals. A non-polar nanocrystal allows the use of non-polar polymers as a matrix for composite materials.

2,3-epoxypropyltrimethylammonium chloride (EPTMAC) have been used for surface catio‐ nization on nanocrystals from cotton cellulose under aqueous alkaline conditions [91]. The surface cationization resulted in an electrostatically stabilized CNC suspension due to the cationic trimethylammonium groups on the crystalline surface (Figure 5d). The morphology and dimensions of the nanocrystals were not affected by the alkaline conditions but the sulfate groups on the nanocrystals surface were hydrolyzed. A decrease in total charge density was observed due to the loss of surface sulfate groups that resulted in an increased tendency for the modified CNC to form thixotropic gels.

Cationic CNC as a reinforcement in fiber networks such as paper or paperboard has been patented [25]. Additions of 1–5 wt% of cationic CNC are expected to increase the strength in the fiber-fiber joints and in that way improve the mechanical performance of the fiber material. Applications for these types of materials can be found in the packaging industry.

### **6.1. Chemical modification of nanocellulose during acid hydrolysis**

commercially attractive. The traditional process in synthesizing BCN cannot produce the high quantities that would be requested for a commercialization of BNC and further process development needs to be performed for a large scale production of bacteria cellulose [88].

During sulfuric acid hydrolysis of cellulose, starch or chitin, sulfate groups will cover the surface of the nanocrystals. If using hydrochloric acid instead, the sulfate groups can be attached to the nanocrystal surfaces afterwards by an esterification reaction with sulfuric acid [37–38, 79]. In this way, the amount of charged groups on the cellulose crystal surface can be controlled. By using chemical modification on nanocrystals or nanofibers, the properties of the

Figure 5 shows some different surface treatments on CNC where a TEMPO-oxidation or cationization of the CNC surface can give charged side groups which will give electrostatic repulsion between the crystals and prevent aggregation, especially on CNC prepared from

Most of the literature that describes chemical modification of nanoparticles had the aim of improving the interfacial compatibility between the nanocrystals and various polymer matrices. A better compatibility could enhance the mechanical properties of the composite materials and addition of just a few percent of nanocellulose to a polymer matrix could improve the mechanical properties of the significant composite material [27, 35, 62, 90]. Most of the literature about chemical modification on nanocellulose describes a surface modification of the crystals or fibrils. For surface modification of CNC, there are mainly two different proce‐ dures, surface compatibilization or co-polymerization. In surface compatibilization the main idea is to attach a small molecular agent to the cellulose polymer, where the molecular agent contains at least one reactive moiety. For co-polymerization the molecular agent needs at least two functional groups, one that can react with the hydroxyl groups on the cellulose polymer and one that can covalently bond to a polymer matrix. The co-polymerization can be performed in different ways such as grafting, radical reactions, or by the use of organometallics [27].

Chemical modification on chitin nanowhiskers has been performed to change the surface properties of the nanocrystals to make it possible to have in organic solvents [62]. The reagents used to modify the chitin nanowhiskers were alkenyl succinic anhydride, phenyl isocyanate, and isopropenyl-α,α'-dimethylbenzyl isocyanate. The chemically modified chitin whiskers showed stable suspensions in toluene and were used in composite films casted from a toluene solution and all of the results showed that the chemically modified whiskers improved the adhesion between the filler and the matrix. The results also showed that the chemical treatment decreased the mechanical performance of the composites. The loss of performance could be due to the destruction of the 3-dimensional network of the chitin whiskers that occurs in the unmodified composites [61–62]. Nanocrystals from waxy maize starch have also been chem‐ ically modified with alkenyl succinic anhydride and phenyl isocyanate [90] to study the crystal structure of the nanoparticles after chemical treatment. X-ray diffraction analysis confirmed

nanocrystal or nanofiber could be changed and controlled in specific ways.

**6. Chemical modification on nanocellulose**

hydrochloric acid or hydrobromic acid.

172 Cellulose - Fundamental Aspects and Current Trends

There are some literatures available for chemical modification on nanocellulose where the modification takes part on the produced nanocellulose. Most of these modifications are to improve the interface between cellulose and a polymer matrix in biocomposites. There are not that many studies done on chemical modification during acid hydrolysis but some available studies are among other the acidic hydrolysis with sulfuric acid where sulfate groups are attached to the CNC surface. In recent years, studies on the hydrolysis reaction of CNC with phosphoric acid has been performed with the aim of creating phosphoric groups on the surface. During the phosphoric acid hydrolysis, reagents, such as acetic anhydride, have been added to a phosphoric acid hydrolysis to obtain acetylated CNC in a single-step procedure [92].

It would be interesting to study if more traditional organic synthesis on hydroxyl groups in acidic environments could be used for chemical modifications during acid hydrolysis from pulp to CNC. There are at least (in theory) two possible ways to achieve chemically modified nanocrystals during hydrolysis: (1) to find alternative reagents to sulfuric acid that at the same time hydrolyzes the cellulose fiber and modify the nanocrystals in some way, or (2) add a reagent to the sulfuric acid (or other acid that hydrolyze cellulose fibers) that could react with the nanocrystals and is functional in acidic and aqueous conditions.

Attempts of using heteropoly acids (HPA) to catalyze organic reactions in water, was made on cellulose where HPAs were used to hydrolyze cellulose polymers into glucose units [93– 94]. HPA is a promising green solid acid catalyst that can replace environmentally harmful liquid acid catalysts. HPA ionic liquids have also been used to catalyze the conversion of sucrose and starch into glucose.

Chemical synthesis that works under acidic conditions and in the presence of water is desirable for CNC modification during hydrolysis. Fischer esterification could be an option where a carboxylic acid reacts with the hydroxyl groups on the nanocellulose. In a Fischer esterification, a carboxylic acid is refluxed with an alcohol in the presence of an acid catalyst. In the case of CNC, a carboxylic acid could react with the hydroxyl groups on cellulose in presence of a sulfuric acid as catalyst.

In the production of acrylamide describe in a patent by McNae [95], one intermediate to acrylamide is acrylamide sulfate, containing 60 wt% sulfuric acid. By increasing the amount of sulphuric acid to a 66 wt% solution and still have the unsaturated acrylate groups left in the solution, unsaturated ester groups could be attached to the cellulose nanocrystal during acid hydrolysis. The authors research group has performed an acid hydrolysis with acrylamide sulfate on microcrystalline cellulose (MCC, Avicel PH-101) with a procedure similar to the one described earlier [29, 33]: the hydrolysis occurred for 2 h at 45 °C with an acid to pulp ratio of 8 g/ml. During the acid hydrolysis, the MCC solution shifted from white to black, which, according to Dong, Revol, and Gray, concluded that side reactions such as dehydration take place when the hydrolysis turns black [29]. After the hydrolysis, the solution was quenched in deionized water before centrifugation, decantation, and dialysis against deionized water.

Figure 7a shows an atomic force microscope (AFM) picture of the acrylamide sulfate hydro‐ lyzed nanocrystals were all the crystals are within the nanometer-sized area in all dimensions. The AFM picture in Figure 7a is similar to CNC produced in the same way but with 64 wt% sulfuric acid (Figure 7b-c). This indicated that the acrylamide sulfate solution could hydrolyze the MCC to nanoparticles, but the process need further optimization since the solution changed color, meaning the hydrolysis went too far. In the AFM picture (Figure 7a), small particles can be seen that further indicates that the hydrolysis went too far. Fourier-Transform Infrared (FTIR) analysis of the acrylamide sulfate hydrolyzed CNC showed new absorption bands at 1,718 cm−1 and at 811 cm−1 corresponding to unsaturated ester groups and alkene groups, respectively, which correlates well to the expected surface group attachment. More studies on the material needs to be performed for further evaluation.

**Figure 7.** AFM picture of CNC from (a) acrylamide sulfate hydrolyzed MCC and a schematic picture of CNC with an acrylate functional group, (b) AFM picture of CNC that has been fully dried, and (c) dried to a high viscous gel. All the crystal dimensions are within nanometer size.

### **6.2. Drying of nanocellulose**

carboxylic acid reacts with the hydroxyl groups on the nanocellulose. In a Fischer esterification, a carboxylic acid is refluxed with an alcohol in the presence of an acid catalyst. In the case of CNC, a carboxylic acid could react with the hydroxyl groups on cellulose in presence of a

In the production of acrylamide describe in a patent by McNae [95], one intermediate to acrylamide is acrylamide sulfate, containing 60 wt% sulfuric acid. By increasing the amount of sulphuric acid to a 66 wt% solution and still have the unsaturated acrylate groups left in the solution, unsaturated ester groups could be attached to the cellulose nanocrystal during acid hydrolysis. The authors research group has performed an acid hydrolysis with acrylamide sulfate on microcrystalline cellulose (MCC, Avicel PH-101) with a procedure similar to the one described earlier [29, 33]: the hydrolysis occurred for 2 h at 45 °C with an acid to pulp ratio of 8 g/ml. During the acid hydrolysis, the MCC solution shifted from white to black, which, according to Dong, Revol, and Gray, concluded that side reactions such as dehydration take place when the hydrolysis turns black [29]. After the hydrolysis, the solution was quenched in deionized water before centrifugation, decantation, and dialysis against deionized water.

Figure 7a shows an atomic force microscope (AFM) picture of the acrylamide sulfate hydro‐ lyzed nanocrystals were all the crystals are within the nanometer-sized area in all dimensions. The AFM picture in Figure 7a is similar to CNC produced in the same way but with 64 wt% sulfuric acid (Figure 7b-c). This indicated that the acrylamide sulfate solution could hydrolyze the MCC to nanoparticles, but the process need further optimization since the solution changed color, meaning the hydrolysis went too far. In the AFM picture (Figure 7a), small particles can be seen that further indicates that the hydrolysis went too far. Fourier-Transform Infrared (FTIR) analysis of the acrylamide sulfate hydrolyzed CNC showed new absorption bands at 1,718 cm−1 and at 811 cm−1 corresponding to unsaturated ester groups and alkene groups, respectively, which correlates well to the expected surface group attachment. More studies on

**Figure 7.** AFM picture of CNC from (a) acrylamide sulfate hydrolyzed MCC and a schematic picture of CNC with an acrylate functional group, (b) AFM picture of CNC that has been fully dried, and (c) dried to a high viscous gel. All the

the material needs to be performed for further evaluation.

crystal dimensions are within nanometer size.

sulfuric acid as catalyst.

174 Cellulose - Fundamental Aspects and Current Trends

When preparing nanocellulose from acid hydrolysis, the nanocrystals are in a water suspen‐ sion. By removing some of the water, a critical concentration will be reached. Above the critical concentration, the CNC will be in a stable colloidal dispersion. The critical concentration for CNC is normally between 2 and 10 wt% [36].

Fully dried CNC prepared, for example, through freeze-drying, will give thin lamellar white flakes that can be re-dispersed in water. Figures 7b and 7c show two different AFM pictures of CNC where different amounts of water have been removed, one fully dried (Figure 7b) and the other one dried to a high viscous gel with a CNC content of 8.9% (Figure 7c). The two samples, which were taken from the same CNC batch, were then re-dispersed in deionized water to the same CNC concentration of 4.15%. The CNC have been prepared from MCC (Avicel PH-101) hydrolyzed in a 64 wt% sulphuric acid at 45 °C for 2 h. Both pictures show crystals in the nanometer size area.

The AFM pictures in Figure 7b–c show no significant differences on the dimensions of the nanocrystals. Both of the two samples show thixotropic behavior, where the fully dried sample showed more tendency to thixotropy compared to the high viscous gel sample. Figure 8 shows the fully dried sample re-dispersed in water to a CNC concentration of 4.15%. In static mode it, behaves as a gel and immediately after agitation, it behaves more like a liquid.

**Figure 8.** Thixotropic behavior of the fully dried CNC sample re-dispersed in water to a CNC concentration of 4.15%. (a) In static mode and (b) immediately after agitation.

### *6.2.1. CNC films*

CNC films were prepared from the two different CNC samples shown in Figures 7b–c, one sample that had been freeze-dried and one that was dried to a high viscous gel. The two samples were re-dispersed in water to a CNC concentration of ca 1% and sonicated to destroy eventual aggregates. The water was thereafter allowed to evaporate in ambient conditions until two solid air dried CNC films was obtained from the two different samples (Figure 9).

**Figure 9.** Scanning electron microscope (SEM) pictures of CNC films. (a) CNC from the high viscous gel suspension and (b) CNC from the freeze-dried sample.

The two films shown in Figure 9 show very different appearances, where the film from the high viscous gel sample is transparent and the film from the freeze-dried sample shows no transparency at all. When a never dried CNC suspension evaporates, the CNC concentration will gradually increase and once the critical concentration for a stable colloidal dispersion is exceeded, a self-ordered phase will form. This ordered form will remain as the CNC concen‐ tration increase further until all water has evaporated. A solid semitransparent CNC film will be left where the crystals are tightly packed in the same ordered crystalline form. For suffi‐ ciently thin films, colors can be seen in the film.

For CNC that has been fully dried or dried far above its critical concentration, particle aggregation occurs, leading to loss of properties or functionality [96]. To overcome this problem, surfactants or surface modification can be used to prevent aggregation during drying [80]. Beck, Bouchard, and Berry studied the difference between freeze-dried CNC films and CNC films from an aqueous CNC solution. The CNC film from the aqueous solution could be re-dispersed again but not the freeze-dried CNC film. Beck and co-workers found out that by adding a sodium cation (Na+ ) to the CNC solution before drying, the freeze-dried Na+ -CNC was completely re-dispersed in water to give colloidal CNC suspensions [96].

By studying CNC films with FTIR analysis, Dong and Gray explained the phenomena as intermolecular hydrogen bonding from the cellulose backbone was much stronger in the CNC film without sodium added (the S-H bonding from the sulfate groups in the nanocrystals) than in the CNC film with sodium added (S-Na) [70]. The freeze-dried CNC-film shown in Figure 9b did not have any cations added and this might be the reason why the freeze-dried CNC did not behave like the film from the high viscous gel sample in Figure 9a.

The importance of knowing how to re-disperse fully dried CNC is necessary for many applications of CNC. It is convenient, when handling CNC, to fully dry it to minimize its shipment size, weight, cost, and the inhibition of bacterial and fungal growth. Many applica‐ tions also require a dried CNC that can be re-dispersed in water or in organic solvents for chemical modification or for nanocomposite manufacture [96].

### **7. Biobased composites**

**Figure 9.** Scanning electron microscope (SEM) pictures of CNC films. (a) CNC from the high viscous gel suspension

The two films shown in Figure 9 show very different appearances, where the film from the high viscous gel sample is transparent and the film from the freeze-dried sample shows no transparency at all. When a never dried CNC suspension evaporates, the CNC concentration will gradually increase and once the critical concentration for a stable colloidal dispersion is exceeded, a self-ordered phase will form. This ordered form will remain as the CNC concen‐ tration increase further until all water has evaporated. A solid semitransparent CNC film will be left where the crystals are tightly packed in the same ordered crystalline form. For suffi‐

For CNC that has been fully dried or dried far above its critical concentration, particle aggregation occurs, leading to loss of properties or functionality [96]. To overcome this problem, surfactants or surface modification can be used to prevent aggregation during drying [80]. Beck, Bouchard, and Berry studied the difference between freeze-dried CNC films and CNC films from an aqueous CNC solution. The CNC film from the aqueous solution could be re-dispersed again but not the freeze-dried CNC film. Beck and co-workers found out that by

By studying CNC films with FTIR analysis, Dong and Gray explained the phenomena as intermolecular hydrogen bonding from the cellulose backbone was much stronger in the CNC film without sodium added (the S-H bonding from the sulfate groups in the nanocrystals) than in the CNC film with sodium added (S-Na) [70]. The freeze-dried CNC-film shown in Figure 9b did not have any cations added and this might be the reason why the freeze-dried CNC did

The importance of knowing how to re-disperse fully dried CNC is necessary for many applications of CNC. It is convenient, when handling CNC, to fully dry it to minimize its shipment size, weight, cost, and the inhibition of bacterial and fungal growth. Many applica‐ tions also require a dried CNC that can be re-dispersed in water or in organic solvents for

was completely re-dispersed in water to give colloidal CNC suspensions [96].

not behave like the film from the high viscous gel sample in Figure 9a.

chemical modification or for nanocomposite manufacture [96].

) to the CNC solution before drying, the freeze-dried Na+


and (b) CNC from the freeze-dried sample.

176 Cellulose - Fundamental Aspects and Current Trends

adding a sodium cation (Na+

ciently thin films, colors can be seen in the film.

There are a lot of reviews available regarding nanomaterials as reinforcements in composite materials [4, 9, 12–15, 27, 35, 97–100] and the huge interest of nanomaterials in composite products is due to the many advantages of the biobased nanomaterial. Some of the beneficial properties with nanomaterials from bioresources are listed below [4, 15]:


Composite materials are widely used in many applications today and consists of a polymeric matrix and a filler material as reinforcement. The fillers used are often synthetic such as glass fiber, carbon, or aramid. The addition of a filler to a polymeric material enhances the mechan‐ ical and thermal properties of the composite material, compared to the polymeric material itself, because of their high performance and great versatility. Today, there is a strong focus on environmental issues and the synthetic fillers used in composite materials as they cause problems at the end-of-life disposal due to their partial combustibility and the high demand on techniques for recycling of the materials [82]. By replacing the synthetic fillers with natural ones, such as cellulose fibers, starch, or chitin there will be many positive environmental benefits and advantages including lower costs, lower density, good thermal properties, and biodegradability [99].

By using nanomaterials as fillers in composite materials improved stiffness, strength, tough‐ ness, barrier properties, and flame retardancy can be achieved compared to the pure polymer material. The addition of only a few percent (1–5 wt%) of nanomaterial is enough for these improvements due to the large surface area of the nanoparticles [98].

Compared to carbon nanotubes, which is the strongest nanofiber produced today, cellulose nanofibers from wood only have 25% of the strength but the costs for cellulose nanofibers are much lower than the costs for carbon nanotubes, which makes the wood-based nanomaterial more attractive for certain applications [4]. The reinforcing ability of the cellulose whiskers comes from its high surface area and good mechanical properties. Table 2 shows the tensile strength and elastic modulus (E-module) of some different reinforcement materials [9].


**Table 2.** Tensile strength and E-module for different reinforcement materials used in composite materials.

The difference between hardwood fibers with diameters in micrometer size and nanometer size (CNF) have been evaluated when used as reinforcement in a polyurethane matrix [101]. Two different filler concentrations were used for both the cellulose fibers (CF, 8.5 and 18.7 wt %) and the CNF (7.5 and 16.5 wt%). Both mechanical and thermal behavior were evaluated for the composite materials and the results shows that both the tensile strength and E-modulus were improved with increased CF and CNF content, compared to a pure polyurethane sample. The CNF composite showed a larger increase in mechanical properties than CF, and are therefore more effective as reinforcement than the micrometer-sized cellulose fibers. The improvement in mechanical properties for CNF compared to CF is due to the smaller fiber dimensions and higher aspect ratio, which gives a better incorporation of the nanofibrils in the polymer matrix.

The cellulosic three-dimensional network of intermolecular hydrogen bonds between the cellulose molecules gives strong interactions between the fillers (fibers or fibrils) or between the fillers and the polymeric matrix, resulting in better composite properties compared to the pure polymer matrix alone. Another important effect of fibers or CNF is the high flexibility of the cellulose polymer that gives a tangling effect with the polymer matrix that contributes to improved mechanical and thermal properties [101–103].

The first reported composites from cellulose whiskers was in 1995 where nanowhiskers from tunica was mixed with a co-polymer of 35 wt% styrene and 65 wt% butyl acrylate [56–57]. Nanowhiskers prepared from tunicate are frequently reported in literature for their use as filler material in biocomposites [54, 56–57, 104].

Azizi Samir et al. compared composite materials where 6 wt% CNF and CNC from sugar beet pulp were used as fillers in a latex composite material [51]. Both CNF and CNC enhanced mechanical and thermal behavior compared to the unfilled polymer matrix. The reinforcing effect for nanocellulose fillers occurs most probably from the cellulose hydrogen bonding network within the polymer matrix. Between the two different nanocellulose fillers, the CNF showed the largest improvements in mechanical and thermal behavior due to its morphology. The more flexible and hairy nanofibrils showed a tangling effect compared to the more particular cellulose crystals. Improved thermal behavior for CNF composites from potato pulp [64] and eucalyptus pulp [105] has also been reported, with similar results as described above. Table 3 shows some different cellulose sources that have been used as reinforcement fillers in polymer matrices, either as CNF or as CNC. There is a wide variety of the polymer matrixes used, both synthetic polymers and natural polymers, such as starch, polylactic acid (PLA), and cellulose acetate butyrate (CAB).

**Material Tensile strength (GPa) E-module (GPa)**

**Table 2.** Tensile strength and E-module for different reinforcement materials used in composite materials.

The difference between hardwood fibers with diameters in micrometer size and nanometer size (CNF) have been evaluated when used as reinforcement in a polyurethane matrix [101]. Two different filler concentrations were used for both the cellulose fibers (CF, 8.5 and 18.7 wt %) and the CNF (7.5 and 16.5 wt%). Both mechanical and thermal behavior were evaluated for the composite materials and the results shows that both the tensile strength and E-modulus were improved with increased CF and CNF content, compared to a pure polyurethane sample. The CNF composite showed a larger increase in mechanical properties than CF, and are therefore more effective as reinforcement than the micrometer-sized cellulose fibers. The improvement in mechanical properties for CNF compared to CF is due to the smaller fiber dimensions and higher aspect ratio, which gives a better incorporation of the nanofibrils in the

The cellulosic three-dimensional network of intermolecular hydrogen bonds between the cellulose molecules gives strong interactions between the fillers (fibers or fibrils) or between the fillers and the polymeric matrix, resulting in better composite properties compared to the pure polymer matrix alone. Another important effect of fibers or CNF is the high flexibility of the cellulose polymer that gives a tangling effect with the polymer matrix that contributes to

The first reported composites from cellulose whiskers was in 1995 where nanowhiskers from tunica was mixed with a co-polymer of 35 wt% styrene and 65 wt% butyl acrylate [56–57]. Nanowhiskers prepared from tunicate are frequently reported in literature for their use as filler

Azizi Samir et al. compared composite materials where 6 wt% CNF and CNC from sugar beet pulp were used as fillers in a latex composite material [51]. Both CNF and CNC enhanced mechanical and thermal behavior compared to the unfilled polymer matrix. The reinforcing effect for nanocellulose fillers occurs most probably from the cellulose hydrogen bonding network within the polymer matrix. Between the two different nanocellulose fillers, the CNF showed the largest improvements in mechanical and thermal behavior due to its morphology. The more flexible and hairy nanofibrils showed a tangling effect compared to the more particular cellulose crystals. Improved thermal behavior for CNF composites from potato pulp [64] and eucalyptus pulp [105] has also been reported, with similar results as described above.

improved mechanical and thermal properties [101–103].

material in biocomposites [54, 56–57, 104].

CNC 7.5–7.7 110–220 Kevlar 3.5 124–130 Steel wire 4.1 210 Carbon fiber 1.5–5.5 150–500 Carbon nanotubes 11–63 270–950

178 Cellulose - Fundamental Aspects and Current Trends

polymer matrix.


**Table 3.** Different cellulose sources as reinforcement fillers in polymer matrices.

A problem with using cellulose fibers as reinforcement in polymer composites is the hydro‐ philic properties of the cellulose fibers when mixed with hydrophobic polymer matrices. To overcome this problem, the cellulose fibers can be modified in different ways, either by coating the cellulose nanoparticles with surfactants or by chemically modifying the cellulose surface with hydrophobic groups. The use of surfactants is the easiest method but a very high amount of surfactants is needed to coat the surface of the fillers, which causes problems in composite applications. Chemical modification of nanocellulose was described earlier (Section 6) and most of the chemical modifications on CNC is to improve the incorporation between CNC and the more hydrophobic polymer matrix.

Researchers have shown that it is possible to use tunicin whiskers in organic solvents without using any modification on the whiskers [60]. The tunicin whiskers were dispersed in dime‐ thylformamide (DMF) without any other additives and the stability of the suspension was found to be as good as in water. The stability is due to the high value of the dielectric constant of DMF and the wettability of the tunicin whiskers. These results open up new ways of using CNC as reinforcements in hydrophobic polymers as matrix.

There is a huge interest in nanocomposites from biobased resources in many different industries today due to the many benefits with nanocellulose compared to other conventional filler materials. Some industries interested in nanocomposites from cellulose-based materials are the automotive, aerospace, medical and health, packaging and forest industries.

### **8. Summary and outlook**

This review has described the properties of nanocellulose and its meaningfulness in the society as a material with extraordinary properties that could be used as reinforcement in paper or composite materials. The addition of few weight percent nanocellulose to a polymer matrix could improve the mechanical properties of the material, compared to pure polymer matrix alone. The use of nanocellulose in the society has also been successful in application areas such as packaging, paper and paperboard, food industry, medical and hygiene products, paints, cosmetics, etc.

The different preparation methods for nanocellulose have been optimized for many years, and good techniques that also work on a larger scale have been developed. For bacterial cellulose, there is an interest to develop a synthesis that could be suitable for larger scale production but the process today is too expensive and takes a long time. The commercialization of nanocel‐ lulose is not yet fully developed, but there are manufacturers as well as pilot plants distributed in different places in the world providing nanocellulose for researchers and early adapters to aid in the commercialization of the products.

Different preparation techniques were briefly described and the techniques used depend on what type of nanocellulose is requested and what cellulose source is used. Cellulose nanofibers (CNFs) are prepared from a mechanical treatment while cellulose nanocrystals (CNCs) are hydrolyzed by acids. Bacterial nanocellulose (BNC), on the other hand, is synthesized to very pure and fine microfibrils from bacteria in a cultivated medium. The main focus in the review was on the acid hydrolysis of cellulose pulp to CNC.

The production of CNC hydrolysis could be made from different acids but it is most common to use a sulfuric acid solution due to a better colloidal suspension and high crystallinity. Sulfuric acid will convert some of the hydroxyl groups on the nanocrystal surface into anionic sulfate groups that will aid in stabilizing the dispersion. Phosphoric acid can also be used for CNC production and will, like sulfuric acid, form charged groups on the crystal surface. Hydrolysis from hydrochloric acid or hydrobromic acid will not have any charged surface groups, which makes it harder to form a stable colloidal CNC dispersion. This could, on the other hand, be solved by chemical modification on CNC by esterification reactions or by TEMPO-oxidation. This way, the amount of charged groups on the crystal surface can be controlled.

Chemical modifications on nanocellulose are mainly performed to increase the incorporation of the nanocellulose with a polymer matrix. By improving this incorporation, nanocellulose can be used as reinforcement in biocomposites. It is beneficial to use nanocellulose in compo‐ sites due to their many outstanding properties and a composite prepared from a nanocellulosic material can exhibit a higher tensile strength than, for example, carbon fibers or aramid fibers such as Kevlar.

There could be an interest in finding new ways for chemical modification on CNC during acidic hydrolysis. Two different paths was suggested in the review, either by finding a substitute to the acids used during hydrolysis that could hydrolyze the cellulose and modify the CNC at the same time, or by adding a reagent to the hydrolyze reaction that will modify the CNC during the acidic hydrolysis. Some demands on these types of reagents are the use in acidic conditions and in the presence of water.

An attempt in using an acrylamide sulfate solution consisting of 66 wt% sulfuric acid was described and the aim of this reaction was to attach unsaturated acrylate groups to the CNC, which could be used as a tool in further reactions. AFM pictures of the acrylamide sulfatehydrolyzed pulp showed cellulose whiskers in the nanometer size range, which indicated that the solution could hydrolyze cellulose to CNC. FTIR analysis on the nanocellulose showed unsaturated esters and alkenes, which further indicated a successful reaction. However, more studies on the material need to be performed for further evaluation.

After CNC preparation, the nanocellulose is in an aqueous solution and could display a stabilized colloidal behavior above its critical concentration. Knowledge about the degree of drying of CNC is an important issue because the application of CNC often demands dried CNC that could be re-dispersed at the manufacturing sites, either in water or in organic solvents or polymer matrices for composite manufacturing.

The health and safety aspects of nanocellulose were briefly discussed. The cellulose material itself is classified as non-toxic and approved to be used in food products such as in thickening agents. Also for nanocellulose, the health and safety aspects are approved but since nanopar‐ ticles are quite new in the health and safety area, other tests might be necessary for further understanding the nanoparticle material.

In conclusion, nanocellulose, in its different forms, shows increasing interest in the industry and society due to its many beneficial properties such as being environmentally friendly, its low cost, and high mechanical performance, there is a bright future for these types of materials. Also, other biobased nanomaterials, such as chitin or starch, will play an important role in the nanoarea future.

### **Acknowledgements**

of DMF and the wettability of the tunicin whiskers. These results open up new ways of using

There is a huge interest in nanocomposites from biobased resources in many different industries today due to the many benefits with nanocellulose compared to other conventional filler materials. Some industries interested in nanocomposites from cellulose-based materials

This review has described the properties of nanocellulose and its meaningfulness in the society as a material with extraordinary properties that could be used as reinforcement in paper or composite materials. The addition of few weight percent nanocellulose to a polymer matrix could improve the mechanical properties of the material, compared to pure polymer matrix alone. The use of nanocellulose in the society has also been successful in application areas such as packaging, paper and paperboard, food industry, medical and hygiene products, paints,

The different preparation methods for nanocellulose have been optimized for many years, and good techniques that also work on a larger scale have been developed. For bacterial cellulose, there is an interest to develop a synthesis that could be suitable for larger scale production but the process today is too expensive and takes a long time. The commercialization of nanocel‐ lulose is not yet fully developed, but there are manufacturers as well as pilot plants distributed in different places in the world providing nanocellulose for researchers and early adapters to

Different preparation techniques were briefly described and the techniques used depend on what type of nanocellulose is requested and what cellulose source is used. Cellulose nanofibers (CNFs) are prepared from a mechanical treatment while cellulose nanocrystals (CNCs) are hydrolyzed by acids. Bacterial nanocellulose (BNC), on the other hand, is synthesized to very pure and fine microfibrils from bacteria in a cultivated medium. The main focus in the review

The production of CNC hydrolysis could be made from different acids but it is most common to use a sulfuric acid solution due to a better colloidal suspension and high crystallinity. Sulfuric acid will convert some of the hydroxyl groups on the nanocrystal surface into anionic sulfate groups that will aid in stabilizing the dispersion. Phosphoric acid can also be used for CNC production and will, like sulfuric acid, form charged groups on the crystal surface. Hydrolysis from hydrochloric acid or hydrobromic acid will not have any charged surface groups, which makes it harder to form a stable colloidal CNC dispersion. This could, on the other hand, be solved by chemical modification on CNC by esterification reactions or by TEMPO-oxidation. This way, the amount of charged groups on the crystal surface can be

are the automotive, aerospace, medical and health, packaging and forest industries.

CNC as reinforcements in hydrophobic polymers as matrix.

**8. Summary and outlook**

180 Cellulose - Fundamental Aspects and Current Trends

aid in the commercialization of the products.

was on the acid hydrolysis of cellulose pulp to CNC.

cosmetics, etc.

controlled.

The authors would like to acknowledge Prof. Herbert Sixta for valuable discussions and critical reading of the manuscript. Jari Sundqvist is acknowledged for his creation of some of the illustrations presented in the review.

### **Author details**

Mikaela Börjesson1 and Gunnar Westman1,2\*

\*Address all correspondence to: westman@chalmers.se

1 Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden

2 Wallenberg Wood Science Center, Chalmers University of Technology, Gothenburg, Sweden

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**Author details**

182 Cellulose - Fundamental Aspects and Current Trends

Mikaela Börjesson1

Gothenburg, Sweden

Sweden

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## **Current Trends in the Production of Cellulose Nanoparticles and Nanocomposites for Biomedical Applications**

John Rojas, Mauricio Bedoya and Yhors Ciro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61334

### **Abstract**

The goal of this chapter is to review the most recent trends to produce cellulose nanoparticles and nanocomposites with biomedical applications. These particles could be named as bacterial cellulose, cellulose nanofibers, and cellulose nanocrystals. The production of these nanoparticles with diameters below 100 nm is challenging because of the strong agglomeration tendency which occur upon drying aqueous cellulose suspensions or during the compounding process with hydrophobic polymers. Typically, the physical and mechanical properties of these nanoparticles depend on the source of cellulose and the extraction process employed. Cellulose nanoparticles are obtained by mechanical, chemical, or enzymatic process treatments to open the structure of the cellulose source and facilitate accessibility to its micro‐ structure. Usually, a combination of these processes makes the extraction more efficient.

On the other hand, cellulose and polymer nanocomposites are commonly produced by techniques such as solvent evaporation, melt compounding, compression molding, impregnation, and electrospinning. The most salient nanocellulose applications discussed in this chapter deal with the production of bandages, implants, skins replacements for burnings, face masks, artificial blood vessels, cuffs for nerve surgery, drug delivery, cell carriers, and support matrices for enzyme immobilization, and silver nanoparticles as antimicrobial agents in wound dressing.

**Keywords:** Cellulose nanofibers, cellulose nanocrystals, bacterial cellulose, biomedical composites

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Cellulose is the most abundant, renewable, and sustainable biopolymer on earth. It is present in plants, tunicates, and some bacteria. For instance, it is present in the cell wall of wood fibers along with hemicellulose and lignin. The cellulose fibers are composed of microfibrils that, in turn, are composed of elementary fibrils, which are the basic structur‐ al units. These elementary fibrils or nanofibers are about 2–20 nm in diameter and a few micrometers in length. About 30 to 100 cellulose chains aggregate into an elementary fibril. There are regions within each of these elementary fibrils, where the cellulose chains are arranged in highly ordered (crystalline) structures and regions that are disordered (amor‐ phous) [1]. These elementary fibrils are formed during cellulose biosynthesis. Each microfibril is a flexible hair strand composed of cellulose nanocrystals (CNC) linked along with cellulose nanofibers (CNF) [2] (Fig. 1). The terms nanofibrils, nanofibers, and elemen‐ tary fibrils are usually employed as synonyms. A CNF is a bulky, moderately degraded cellulose with a large surface area with diameter ranging from 20 to 60 nm and length of several micrometers. It presents a weblike structure, and the length/diameter ratio is very high. They are usually extracted by mechanical treatment without using acid hydrolysis. Conversely, when subjected to acid hydrolysis, cellulose microfibrils undergo transverse cleavage along the amorphous regions, and the use of sonication results in CNC also referred as cellulose nanowhiskers, or nanorods. Thus, CNCs are described as the crystal‐ line region of cellulose and exhibits a rodlike shape having a low aspect ratio [2, 3]. Its elastic modulus can be compared to the modulus of crystalline cellulose (up to 140 GPa) due to its high hydrogen bonding capability. Further, cellulose nanoparticles degrade faster than other nanoparticles such as fullerenes and carbon nanotubes, which do not degrade at all [4].

Another type of nanocellulose is bacterial cellulose (BC), which is produced as an extracel‐ lular primary metabolite by bacteria belonging to the genera *Acetobacter* (*Gluconacetobact‐ er*), *Agrobacterium*, *Acanthamoeba*, *Achromobacter*, *Zooglea*, *Aerobacter*, *Azotobacter*, *Rhizobium*, *Sarcina*, *Salmonella*, *Escherichia*, *Pseudomonas*, and *Alcaligenes* [5]. However, the most efficient producer of bacterial cellulose (BC) is *Acetobacter xylinum* (or *Gluconacetobacter xylinus*). BC is secreted as a ribbon-shaped fibril, of less than 100 nm wide, which is composed of nanofibrils of 2–4 nm in diameter. These microfibril bundles have excellent intrinsic properties due to their high crystallinity (84–89%), elastic modulus of ~78 GPa, high water holding capacity, and high degree of polymerization (up to 8,000) [6]. BC, when com‐ pressed into sheets, exhibits a highly planar orientation [7]. BC is degraded by few bacterial strains such as *Trichoderma viride* at pH values ranging from 4.5 to 6.0 [8–12]. BC is highly hydrated and no chemical treatments are needed to remove lignin and hemicellulose [13, 14]. For those reasons, BC is preferred for tissue and bone growth. The goal of this chapter is to describe and discuss the state of the art for the production of cellulose nanoparticles such as cellulose nanofibers, cellulose nanocrystals, bacterial cellulose, and their compo‐ sites intended for biomedical applications.

**Figure 1.** Hierarchical structure of cellulose extracted from plants.

**1. Introduction**

194 Cellulose - Fundamental Aspects and Current Trends

at all [4].

sites intended for biomedical applications.

Cellulose is the most abundant, renewable, and sustainable biopolymer on earth. It is present in plants, tunicates, and some bacteria. For instance, it is present in the cell wall of wood fibers along with hemicellulose and lignin. The cellulose fibers are composed of microfibrils that, in turn, are composed of elementary fibrils, which are the basic structur‐ al units. These elementary fibrils or nanofibers are about 2–20 nm in diameter and a few micrometers in length. About 30 to 100 cellulose chains aggregate into an elementary fibril. There are regions within each of these elementary fibrils, where the cellulose chains are arranged in highly ordered (crystalline) structures and regions that are disordered (amor‐ phous) [1]. These elementary fibrils are formed during cellulose biosynthesis. Each microfibril is a flexible hair strand composed of cellulose nanocrystals (CNC) linked along with cellulose nanofibers (CNF) [2] (Fig. 1). The terms nanofibrils, nanofibers, and elemen‐ tary fibrils are usually employed as synonyms. A CNF is a bulky, moderately degraded cellulose with a large surface area with diameter ranging from 20 to 60 nm and length of several micrometers. It presents a weblike structure, and the length/diameter ratio is very high. They are usually extracted by mechanical treatment without using acid hydrolysis. Conversely, when subjected to acid hydrolysis, cellulose microfibrils undergo transverse cleavage along the amorphous regions, and the use of sonication results in CNC also referred as cellulose nanowhiskers, or nanorods. Thus, CNCs are described as the crystal‐ line region of cellulose and exhibits a rodlike shape having a low aspect ratio [2, 3]. Its elastic modulus can be compared to the modulus of crystalline cellulose (up to 140 GPa) due to its high hydrogen bonding capability. Further, cellulose nanoparticles degrade faster than other nanoparticles such as fullerenes and carbon nanotubes, which do not degrade

Another type of nanocellulose is bacterial cellulose (BC), which is produced as an extracel‐ lular primary metabolite by bacteria belonging to the genera *Acetobacter* (*Gluconacetobact‐ er*), *Agrobacterium*, *Acanthamoeba*, *Achromobacter*, *Zooglea*, *Aerobacter*, *Azotobacter*, *Rhizobium*, *Sarcina*, *Salmonella*, *Escherichia*, *Pseudomonas*, and *Alcaligenes* [5]. However, the most efficient producer of bacterial cellulose (BC) is *Acetobacter xylinum* (or *Gluconacetobacter xylinus*). BC is secreted as a ribbon-shaped fibril, of less than 100 nm wide, which is composed of nanofibrils of 2–4 nm in diameter. These microfibril bundles have excellent intrinsic properties due to their high crystallinity (84–89%), elastic modulus of ~78 GPa, high water holding capacity, and high degree of polymerization (up to 8,000) [6]. BC, when com‐ pressed into sheets, exhibits a highly planar orientation [7]. BC is degraded by few bacterial strains such as *Trichoderma viride* at pH values ranging from 4.5 to 6.0 [8–12]. BC is highly hydrated and no chemical treatments are needed to remove lignin and hemicellulose [13, 14]. For those reasons, BC is preferred for tissue and bone growth. The goal of this chapter is to describe and discuss the state of the art for the production of cellulose nanoparticles such as cellulose nanofibers, cellulose nanocrystals, bacterial cellulose, and their compo‐

### **2. Preparation of cellulose nanofibers**

CNF is mainly extracted from wood. However, it can be extracted from natural resources such as such as sisal [15], flax, hemp, grass [16], sorghum, barley, sugar cane [17], pineapple leaf fibers [18], banana rachis [19], soy hulls [20], algae [21], bacterial cellulose, kenaf stem, swede root, wheat straw [22], carrots, empty fruit bunches, potato pulp, branch bark of mulberry [23], bagasse, rice straw, chardonnay grape skins [24], stems of cacti, coconut husk [25], bamboo, pea hull fiber, cotton and industrial bioresidues [26]. Pineapple leaf and jute fibers are the best sources for its extraction due to the low cost, abundance, and high cellulose content [60–70%) [27]. The size of CNF depends on the source and exhibits an entangled morphology with an aspect ratio over 250. For instance, CNF obtained from wheat straw, soy hull, and soybean stock have diameters ranging from 10 to 80 nm, from 20 to 120 nm, and from 50 to 100 nm, respectively [28, 29]. Nevertheless, other researchers have obtained CNF from sisal, carrots, beet pulp, and Luffa cylindrical with smaller diameters of 20–65 nm, 3–36 nm, 30–100 nm, and 55 nm, respectively. These nonwooden sources contain less lignin and require less processing steps and energy consumption due to the less tightly bound microfibril in the primary cell wall than wood. There are several extraction methods to obtain CNF [30–32] (Fig. 2). They can be performed by mechanical techniques such as grinding, cryocrushing with liquid nitrogen, high-pressure homogenization, etc. In addition, different chemical alkali and enzymatic hydrolyses can be utilized before mechanical processes in order to promote the accessibility of hydroxyl groups, increase the inner surface, alter crystallinity, break cellulose hydrogen bonds, and therefore boost the reactivity of the fibers.

### **2.1. Mechanical treatments**

The mechanical treatments can isolate CNF from the primary and secondary cell wall without severely degrading cellulose. For instance, microfluidization and high-intensity ultrasonic treatments produce a high shear gradients causing transverse cleavage along the longitudinal axis of the cellulose fibers, and as a result, they tend to damage the microfibril structure by reducing the molar mass and degree of crystallinity. Depending on the mechanical force levels and types of mechanical treatment, interfibrillar hydrogen bonding are broken [2, 32, 33]. However, the mechanical methods exhibit high production costs (tools and materials); they are also less efficient and require greater energy than the chemical methods [34].

For this reason, a chemical pretreatment reduces energy consumption and makes the surface more hydrophobic. Further, the mechanical treatment usually reduces the degree of polymer‐ ization (DP) from 1,200 to 1,400 to a DP between 850 and 500. A high cellulose DP is desirable since this is correlated with the nanofiber tensile strength, which can be at least 2 GPa [22, 35]

### *2.1.1. High-Pressure Homogenization (HPH)*

In this process, dilute slurries of cellulose fibers (2–7% w/v) are passed through a spring-loaded valve assembly, at high pressure (8,000 psi), low velocity and exposed to a pressure drop to atmospheric condition while the valve opens and closes in a cyclic motion. This results in high shear and impact forces generated in a minute gap of the valve maintained at a temperature of 70–80°C [30]. As a result, the cell wall is peeled off and the DP is reduced [35, 36]. For instance, the DP is reduced from 2720 to 740 when cotton is used as a source of cellulose. Usually, this method produces fibers with diameters between 20 and 100 nm and lengths of several tens of micrometers. However, this method present some problems such as clogging of the homoge‐ nizer, high energy consumption, and mechanical damage of the crystalline microfibril structure [34, 36, 37]. HPH also decreases the crystallinity of nanofibers by increasing the number of passes.

### *2.1.2. Microfluidization*

It is a process by which a fiber suspension is pumped through thin z-shaped chambers under a high pressure (~30,000 psi). The slurry is accelerated and led into the interaction chamber where it passes through geometrically fixed microchannels at very high velocities. Thin Zshaped chambers with different sizes generate a high shear rate and impact forces against colliding streams. A microfluidizer generates CNFs with several micrometers in length and less than 100 nm in diameter.

### *2.1.3. Grinding*

This is a single process by which the cellulose suspension is passed through an ultrafine grinder where the upper stone is static and the lower stone is rotating at 1400–1500 rpm. As a result, the cell wall structure is broken down by shear forces generating a gel due to the heat generated by friction while evaporating water. However, a mechanical damage of the fiber would occur [38]. This process has been used to extract CNFs from wheat straw and soy hulls [39]. Com‐ pression is a modified grinding system by which delignified fibers of cellulosic materials are placed in a bed of stripes placed between the two plates and subjected to a constant load of 10 tons for several seconds. However, in this process fibers in the micrometer rather in the nanometer size range are obtained [40].

### *2.1.4. Cryocrushing*

of hydroxyl groups, increase the inner surface, alter crystallinity, break cellulose hydrogen

The mechanical treatments can isolate CNF from the primary and secondary cell wall without severely degrading cellulose. For instance, microfluidization and high-intensity ultrasonic treatments produce a high shear gradients causing transverse cleavage along the longitudinal axis of the cellulose fibers, and as a result, they tend to damage the microfibril structure by reducing the molar mass and degree of crystallinity. Depending on the mechanical force levels and types of mechanical treatment, interfibrillar hydrogen bonding are broken [2, 32, 33]. However, the mechanical methods exhibit high production costs (tools and materials); they

For this reason, a chemical pretreatment reduces energy consumption and makes the surface more hydrophobic. Further, the mechanical treatment usually reduces the degree of polymer‐ ization (DP) from 1,200 to 1,400 to a DP between 850 and 500. A high cellulose DP is desirable since this is correlated with the nanofiber tensile strength, which can be at least 2 GPa [22, 35]

In this process, dilute slurries of cellulose fibers (2–7% w/v) are passed through a spring-loaded valve assembly, at high pressure (8,000 psi), low velocity and exposed to a pressure drop to atmospheric condition while the valve opens and closes in a cyclic motion. This results in high shear and impact forces generated in a minute gap of the valve maintained at a temperature of 70–80°C [30]. As a result, the cell wall is peeled off and the DP is reduced [35, 36]. For instance, the DP is reduced from 2720 to 740 when cotton is used as a source of cellulose. Usually, this method produces fibers with diameters between 20 and 100 nm and lengths of several tens of micrometers. However, this method present some problems such as clogging of the homoge‐ nizer, high energy consumption, and mechanical damage of the crystalline microfibril structure [34, 36, 37]. HPH also decreases the crystallinity of nanofibers by increasing the

It is a process by which a fiber suspension is pumped through thin z-shaped chambers under a high pressure (~30,000 psi). The slurry is accelerated and led into the interaction chamber where it passes through geometrically fixed microchannels at very high velocities. Thin Zshaped chambers with different sizes generate a high shear rate and impact forces against colliding streams. A microfluidizer generates CNFs with several micrometers in length and

This is a single process by which the cellulose suspension is passed through an ultrafine grinder where the upper stone is static and the lower stone is rotating at 1400–1500 rpm. As a result, the cell wall structure is broken down by shear forces generating a gel due to the heat generated

are also less efficient and require greater energy than the chemical methods [34].

bonds, and therefore boost the reactivity of the fibers.

**2.1. Mechanical treatments**

196 Cellulose - Fundamental Aspects and Current Trends

*2.1.1. High-Pressure Homogenization (HPH)*

number of passes.

*2.1.3. Grinding*

*2.1.2. Microfluidization*

less than 100 nm in diameter.

In this process, swollen cellulosic fibers are immersed in liquid nitrogen. These brittle fibers are subsequently crushed by high shear and impact forces. As a result, ice crystals exert pressure on the cell walls, causing them to rupture. Usually, this method produces CNFs with diameters ranging from 30 to 80 nm [3].

### *2.1.5. High-intensity ultrasonication*

It is a mechanical process in which oscillating power is used to isolate CNFs by hydrodynamic forces of ultrasound [41]. During the process, cavitation leads to a formation of powerful oscillating high intensive waves. These microscopic gas bubbles expand and implode breaking down cellulose fibers. However, a large feed concentration and a large distance from probe to beaker is not advantageous for fibrillation. A typical treatment requires a cylindrical titanium alloy probe tip of 1.5 cm in diameter, high temperatures, 1000 W power, and 20–25 kHz for ~30 min [42].

### *2.1.6. Steam explosion*

It is a thermomechanical process (200–270°C) that exposes cellulose to a high pressure of steam (14–16 bar). As a result, it penetrates the biomass by diffusion for short periods of time (20 s to 20 min), followed by a sudden decompression (explosion) generating shear forces which hydrolyze the glycosidic and hydrogen bonds between the glucose chains [31].

All the above-described mechanical methods demands a high energy consumption (20,000– 30,000 kWh/tonne), which prevents their successful commercialization. Therefore, by com‐ bining the mechanical treatment with enzymatic or chemical pretreatments, it is possible to decrease the high energy consumption.

### **2.2. Electrospinning**

In this electromechanical method, a cellulose dispersion is extruded and electrospun under the effect of a high electric field. Thus, a charged stream of cellulose dispersion is ejected following a 3D spiral trajectory. Once the solvent evaporates, it leaves behind a randomly oriented nanofibers in the collector [43]. This is a quite simple and cost-effective process. The morphology of the CNFs produced by this technology depends on factors such as the electric field strength, solution feed rate, tip-to-collector distance, etc. [43].

**Figure 2.** Conventional treatments to obtain cellulose nanoparticles.

### **2.3. Enzymatic hydrolysis**

**Figure 2.** Conventional treatments to obtain cellulose nanoparticles.

198 Cellulose - Fundamental Aspects and Current Trends

In this treatment, an enzyme is used to modify and/or degrade the lignin and hemicelluloses, while preventing the cellulose region. These enzymes are produced by cellobiohydrolases, which are A- and B-type cellulases able to attack the crystalline portion of cellulose, and endoglucanases C and D type, which are able to attack the disordered structure (amorphous) of cellulose [12]. Cellobiohydrolases and endoglucanases have strong synergistic effects. Thus, pretreated fibers subjected to the lowest enzyme concentration (0.02%) disintegrate, while molecular weight and fiber length are preserved. Endoglucanases cleave the noncovalent internal bonds, whereas exoglucanases attack the terminal glycosidic bonds [44]. Furthermore, *Trichoderma reesei* and *A. xylinum* produce enzymes which are able to reduce the size of microcrystalline cellulose. Enzymatic methods are highly costly due to the isolation process of the enzymes and the long enzymatic treatment time required for a successful hydrolysis [31].

### **3. Preparation of cellulose nanocrystals**

CNCs are commonly isolated from cellulose fibers by acid hydrolysis. Tunicin is a cellulose extracted from sea animal sources made up of highly crystalline nanofibers and has an helical organization. It has a high modulus, a high aspect ratio, and good compatibility with matrix materials [45]. This cellulose is obtained by cutting into small fragments followed by bleaching. Subsequently, CNC is extracted from bleached samples by acid hydrolysis with 64 v/v% H2SO4, for 5 h, at 50°C. Typically, the diameter, the length, and the aspect ratio of CNC are 4– 25 nm, 100–500 nm, and 15–50, respectively. Among the many cellulosic sources used for its isolation, cotton constitutes the main source. It exhibits an elongated crystalline rodlike shape and has a limited flexibility since it has no amorphous regions. These CNC have a degree of crystallinity from 55% to 90%. Moreover, the degree of crystallinity, aspect ratio, and mor‐ phology depends on the source of cellulosic material and preparation conditions [46].

A typical production process involves acid hydrolysis, washing, centrifugation, dialysis, and sonication to form a suspension followed by drying [47]. The main process for the preparation of CNCs is based on strong acid hydrolysis under strictly controlled conditions of concentra‐ tion, temperature, agitation, and time. The mineral acid breaks the *β*-1,4 glycosidic bonds in cellulose. During hydrolysis, the amorphous regions are attacked, leaving the crystalline regions intact [48]. The resulting suspension is washed and centrifuged, and dialysis is performed to remove any free acid molecules. Microbial hydrolysis has also been utilized to produce CNCs. This microbial hydrolysis is eco-friendly and does not require any surface modification [44]. The charge and colloidal behavior of CNCs depends on the acid used for the production. Sulfuric and hydrochloric acids are the most commonly used, but phosphoric and hydrobromic acids have also been used [7].

For instance, hydrolysis with 63.5 v/v% H2SO4 for 2 h leads to a 30% yield, a width narrower than 10 nm and length ranging between 200 and 400 nm [3]. Smaller CNCs are obtained by increasing hydrolysis time and acid concentration [15]. Another treatment employs wood pulp boiled with 2.5 N sulfuric acid for 12 h, generating CNCs with lengths between 50 and 60 nm and widths between 5 and 10 nm [49].

The physical characteristics of CNCs depend on the origin of cellulose sources, concentration of acid, types of acid, reaction time, and temperature [3]. If sulfuric acid (50–70% w/v), temperature of 20–70°C, rate of 500 rpm, and time of 0.5–6 h are employed, esterification also occurs forming "cellulose sulfate," resulting in a negatively charged surface on the cellulose crystallites. Longer hydrolysis time and higher temperature generate shorter nanocrystals with higher surface charge, high crystallinity (~80%), and narrower polydispersity [50–53]. How‐ ever, the very limited commercial availability of CNC is due to the time consuming production process and the low yield produced, especially if the initial amount of amorphous cellulose is very high. The aggregation of CNC occurs with HCl, but sulfuric acid creates charged sulfate esters promoting the dispersion of the CNCs in water preventing aggregation. The combina‐ tion of both sulfuric and hydrochloric acids during hydrolysis generates spherical nanoparti‐ cles with improved thermal stability due to the reduced presence of sulfate groups on their surface. The negative surface charge of CNCs stabilizes the aqueous suspension against flocculation, but this charge also compromises the thermostability of nanocrystals. Therefore, the increase in the sulfate group content decreases the temperature at which thermal degra‐ dation takes place [54]. CNCs also exhibit chiral nematic liquid crystalline alignments, which are seen as a flow of birefringence between two crossed polarizing films.

### **4. Pretreatment processes**

The aim of the pretreatment process is to remove ashes, waxes, lignin, hemicellulose, and other noncellulosic compounds, which are crucial to produce pure cellulosic products such as CNFs and CNCs [30–32]. A pretreatment also reduces the energy demand of mechanical processes from 20,000 to 30,000 kWh/tonne to 1000 kWh/tonne. The types of pretreatments applied on different raw materials such as tunicate, algae, and bacteria cellulose have been reported previously [21, 55]. The alkaline delignification and organosolvation with acetic acid, aqueous methanol, or ethanol are also considered as pretreatment processes [56, 57].

### **4.1. Alkaline hydrolysis**

Alkaline treatments are conducted when a more effective lignin, hemicellulose, and pectin solubilization and removal is needed. Alkaline extraction needs to be controlled to avoid cellulose degradation [3]. A typical treatment involves dipping of fibers in a 5% sodium hydroxide solution for ~48 h at 30°C. At pH >12, NaOH reduces super oxide radicals (-O2), wherein lignin and hemicellulose are hydrolyzed [15]. However, if lignin content is high in the cellulosic source, the nanocellulose yield is low [15, 58].

### **4.2. Bleaching**

Pulp can be bleached to improve ageing resistance avoiding yellowing and brittleness. These two defects are mainly related to the presence of lignin. Different compounds are commonly used for bleaching. These include hydrogen peroxide (H2O2), chlorine dioxide (ClO2), ozone (O3), peracetic acid, and NaClO2. Sulfite pulps are more readily bleached and results in high yields [59]*.*

### **4.3. Oxidation**

boiled with 2.5 N sulfuric acid for 12 h, generating CNCs with lengths between 50 and 60 nm

The physical characteristics of CNCs depend on the origin of cellulose sources, concentration of acid, types of acid, reaction time, and temperature [3]. If sulfuric acid (50–70% w/v), temperature of 20–70°C, rate of 500 rpm, and time of 0.5–6 h are employed, esterification also occurs forming "cellulose sulfate," resulting in a negatively charged surface on the cellulose crystallites. Longer hydrolysis time and higher temperature generate shorter nanocrystals with higher surface charge, high crystallinity (~80%), and narrower polydispersity [50–53]. How‐ ever, the very limited commercial availability of CNC is due to the time consuming production process and the low yield produced, especially if the initial amount of amorphous cellulose is very high. The aggregation of CNC occurs with HCl, but sulfuric acid creates charged sulfate esters promoting the dispersion of the CNCs in water preventing aggregation. The combina‐ tion of both sulfuric and hydrochloric acids during hydrolysis generates spherical nanoparti‐ cles with improved thermal stability due to the reduced presence of sulfate groups on their surface. The negative surface charge of CNCs stabilizes the aqueous suspension against flocculation, but this charge also compromises the thermostability of nanocrystals. Therefore, the increase in the sulfate group content decreases the temperature at which thermal degra‐ dation takes place [54]. CNCs also exhibit chiral nematic liquid crystalline alignments, which

The aim of the pretreatment process is to remove ashes, waxes, lignin, hemicellulose, and other noncellulosic compounds, which are crucial to produce pure cellulosic products such as CNFs and CNCs [30–32]. A pretreatment also reduces the energy demand of mechanical processes from 20,000 to 30,000 kWh/tonne to 1000 kWh/tonne. The types of pretreatments applied on different raw materials such as tunicate, algae, and bacteria cellulose have been reported previously [21, 55]. The alkaline delignification and organosolvation with acetic acid, aqueous

Alkaline treatments are conducted when a more effective lignin, hemicellulose, and pectin solubilization and removal is needed. Alkaline extraction needs to be controlled to avoid cellulose degradation [3]. A typical treatment involves dipping of fibers in a 5% sodium hydroxide solution for ~48 h at 30°C. At pH >12, NaOH reduces super oxide radicals (-O2), wherein lignin and hemicellulose are hydrolyzed [15]. However, if lignin content is high in

Pulp can be bleached to improve ageing resistance avoiding yellowing and brittleness. These two defects are mainly related to the presence of lignin. Different compounds are commonly

are seen as a flow of birefringence between two crossed polarizing films.

methanol, or ethanol are also considered as pretreatment processes [56, 57].

the cellulosic source, the nanocellulose yield is low [15, 58].

and widths between 5 and 10 nm [49].

200 Cellulose - Fundamental Aspects and Current Trends

**4. Pretreatment processes**

**4.1. Alkaline hydrolysis**

**4.2. Bleaching**

The TEMPO-mediated surface oxidation is the most commonly used chemical pretreatment conducted under aqueous and mild conditions. It converts the primary hydroxyl group (C6) to a charged aldehyde or carboxylate functional group, whereas the secondary hydroxyl moieties present in the cellulose molecule remain unaffected [1, 12]. The oxidation of cellulose fibers occurs in the presence of NaClO and catalytic amounts of 2,2,6,6 tetramethyl-1 piperi‐ dinyloxy radical (TEMPO) and NaBr as catalyst at a pH between 9 and 11 and room temper‐ ature. The higher the amount of NaClO in the reaction medium, the larger is the number of carboxylic groups formed at the surface of the CNFs and the stronger is the decrease in DP [44]. This oxidation creates negative charges on the surface of CNC [31] without changing the original fibrous morphologies [61–66]. The reaction by-product is only sodium chloride. Other N-oxyl compounds, such as the 4-hydroxy TEMPO derivative (less expensive than TEMPO), have been proposed. The residual aldehyde groups causes discoloration. In order to avoid depolymerization or discoloration of the oxidized cellulose, a TEMPO/NaClO/NaClO2 system is employed under neutral or slightly acidic conditions [60]. This treatment also prevents the postaggregation of nanoparticles during the drying step.

A variation of this condition oxidizes wood cellulose rendering CNF with a higher molecular weight and with no aldehyde groups using a TEMPO/NaClO/NaClO2 system at pHs ranging from 5 to 7 [67]. The TEMPO pretreatment eases the separation of the nanofibrils from each other due to the repulsive forces of the ionized carboxylate groups, which overwhelm the hydrogen bonds. The TEMPO oxidation pretreatment is usually followed by a mechanical treatment. Other less commonly used processes include oxidation at 60°C with ammonium persulfate and the sequential periodate and chlorite oxidation [68].

### **4.4. Ionic liquids**

Ionic liquids are organic salts having no corrosive properties, no flammability, a melting point below 100°C, low vapor pressure, and low viscosities [34]. Ionic liquids dissolve cellulose and render a wide range of particle morphologies after precipitation. The ionic liquid breaks intramolecular hydrogen bonds, whereas the cations attack the O atom of the –OH, and anions attack the hydrogen atoms of the -OH group [34]. PF6 - , BF4-, (CF3CO2) - , (SbF6) - , (OTS)- , (ClO4) - , (GeCl3) - , (Al2Cl7) - , and (AlCl4) are the most common anions employed [69, 70].

### **5. Nanocellulose derivatization**

This chemical treatment is performed on the surface of BC, CNC, or CNF for making them more hydrophobic reducing the agglomeration tendency of these materials. The goal of the derivatization is to endow nanocellulose with a hydrophobic character in order to improve its compatibility with nonpolar polymers [60, 71–73].

### **5.1. Carboxymethylation**

This process makes the surface negatively charged, promotes the formation of a stable suspension, and increases the breakup of lignocellulosic fibers [2]. If carboxymethylation is conducted before mechanical treatment, the fibers become more dispersible having a lower degree of crystallinity [72].

### **5.2. Acetylation**

In this reaction, the C6 hydroxyl groups of cellulose are selectively converted to carboxylate groups and only NaClO and NaOH are consumed. The amount of carboxylate groups formed increases with the amount of NaClO and by employing long reaction times [74]. This reaction causes plasticization of lignocellulosic fibers [31] [54]. Further, a reaction of CNF with acetic anhydride at 105°C for 30 min causes a degree of substitution (DS) of 0.43. As a result, the contact angle increases from 33° for nonacetylated nanofibers to 115° for acetylated ones. The acetylated fibers have a lower crystallinity due to degradation of crystalline regions during the reaction.

### **5.3. Isocyanate**

Isocyanates, in particular, octadecyl isocyanate, can generate covalent bonds with hydroxyl groups on the particle surface, rendering a degree of substitution of 0.07 and 0.09 for CNC and NFC, respectively [75].

### **5.4. Silylation**

The reaction between silanol and OH groups of cellulose at high temperature is initiated by water. Surface silylation of CNFs from bleached softwood pulp using chlorodimethyl isopro‐ pylsilane renders a degree of surface substitution from 0.6 to 1. Conversely, silylation of CNFs by isopropyl dimethylchlorosilane renders a CNF that forms suspensions with a shearthinning behavior [54].

### **6. Physical properties**

### **6.1. Morphology**

Depending upon the source of the cellulose and the method of production, a CNF displays similar morphologies but several dimensions. Typically, a CNF and a CNC have typical diameters of 2–100 nm and between 2 and 30 nm, respectively. For instance, CNFs from wheat straw have diameters from 10 to 80 nm [22] as compared to soy hulls (20–120 nm) [20], kenaf bast (2–6 nm), wood (15 nm), bagasse (5–15 nm), rice straw (4–13 nm), soybean stock-based (50–100 nm), cotton (10– 25 nm), and empty fruit bunch (10–30 nm) [31, 76–85].

### **6.2. Crystallinity**

derivatization is to endow nanocellulose with a hydrophobic character in order to improve its

This process makes the surface negatively charged, promotes the formation of a stable suspension, and increases the breakup of lignocellulosic fibers [2]. If carboxymethylation is conducted before mechanical treatment, the fibers become more dispersible having a lower

In this reaction, the C6 hydroxyl groups of cellulose are selectively converted to carboxylate groups and only NaClO and NaOH are consumed. The amount of carboxylate groups formed increases with the amount of NaClO and by employing long reaction times [74]. This reaction causes plasticization of lignocellulosic fibers [31] [54]. Further, a reaction of CNF with acetic anhydride at 105°C for 30 min causes a degree of substitution (DS) of 0.43. As a result, the contact angle increases from 33° for nonacetylated nanofibers to 115° for acetylated ones. The acetylated fibers have a lower crystallinity due to degradation of crystalline regions during

Isocyanates, in particular, octadecyl isocyanate, can generate covalent bonds with hydroxyl groups on the particle surface, rendering a degree of substitution of 0.07 and 0.09 for CNC and

The reaction between silanol and OH groups of cellulose at high temperature is initiated by water. Surface silylation of CNFs from bleached softwood pulp using chlorodimethyl isopro‐ pylsilane renders a degree of surface substitution from 0.6 to 1. Conversely, silylation of CNFs by isopropyl dimethylchlorosilane renders a CNF that forms suspensions with a shear-

Depending upon the source of the cellulose and the method of production, a CNF displays similar morphologies but several dimensions. Typically, a CNF and a CNC have typical diameters of 2–100 nm and between 2 and 30 nm, respectively. For instance, CNFs from wheat straw have diameters from 10 to 80 nm [22] as compared to soy hulls (20–120 nm) [20], kenaf bast (2–6 nm), wood (15 nm), bagasse (5–15 nm), rice straw (4–13 nm), soybean stock-based

(50–100 nm), cotton (10– 25 nm), and empty fruit bunch (10–30 nm) [31, 76–85].

compatibility with nonpolar polymers [60, 71–73].

**5.1. Carboxymethylation**

202 Cellulose - Fundamental Aspects and Current Trends

degree of crystallinity [72].

**5.2. Acetylation**

the reaction.

**5.3. Isocyanate**

**5.4. Silylation**

NFC, respectively [75].

thinning behavior [54].

**6.1. Morphology**

**6. Physical properties**

Crystallinity is highly dependent on the lignocellulosic source. For instance, crystallinity of flax, rutabaga, and wood CNFs are 59%, 64%, and 54%, respectively, whereas a crystallinity of 85.9%, 76%, 84.9%, 94%, 80.6%, and 81.7% has been found for CNCs obtained from sisal, rice husk, flax, cotton, corn stover, and commercial MCC, respectively. On the other hand, a DC of 78% and 70% has been obtained for wheat straw [22] and soy hull CNFs, respectively. Further, very low values have been obtained for beet pulp ~30–40% [20]. The degree of crystallinity ranges in the order: pineapple > banana > jute, and this order agrees with the values of cellulose content determined in these samples. Usually, CNCs prepared from H2SO4 have lower crystalline values than those prepared from HCl. In addition, the increase in hydrolysis time also increases crystallinity due to the elimination of amorphous regions [15].

### **6.3. Thermal properties**

The thermal degradation of lignocellulosic materials begins with an early decomposition of hemicelluloses, followed by an early stage of pyrolysis of lignin, depolymerization, active flaming combustion, and char oxidation. Further, CNF has a high degradation temperature onset (350°C) and better thermal behavior than hemicellulose, pectin, and lignin. On the contrary, the onset of the thermal degradation of CNC typically occurs at 200–300°C[86]. CNC with lower sulfate content have better thermal stability [53]. On the other hand, banana CNF exhibited three main weight loss regions. The initial weight is mainly due to moisture evaporation followed by thermal depolymerization of hemicellulose and the cleavage of glycosidic linkages of cellulose. The broad peak in the region from 200°C to 500°C is due to residual lignin components. A convection drying of a CNF removes water slowly causing the formation of aggregates. Therefore, the dried CNF presents a lower degree of thermal stability than that of the original fibers. [19].

### **6.4. Degree of Polymerization (DP) and mechanical properties**

DP is strongly correlated with the aspect ratio of the nanofibers. As explained previously, any pretreatment process also reduces the DP. On the other hand, the mechanical properties of cellulose nanoparticles depend on morphology, geometrical dimensions, crystal structure, crystallinity, and the process used to produce CNCs and CNFs. For instance, the DP of softwood is 2249, but the DP of sulfated CNF is 825. Further, the tensile strength of native CNCs ranges from 7.5 to 7.7 GPa. Further, the Young's modulus of CNCs is estimated to lie between 130 and 250 GPa.

### **6.5. Hornification**

Drying of individual cellulose nanoparticles creates irreversible agglomeration affecting their dimension and, therefore, their unique properties [87]. This irreversible agglomeration is known as hornification and is related to the hydrogen bonds formed [81]. If freeze-drying or supercritical dying with CO2 is used, agglomeration is avoided [87–89].

### **6.6. Film properties**

CNF gels can be diluted and either cast or vacuum filtered followed by drying to form stiff films due to the formation of an interfibrillar hydrogen bonding network [63, 90–93]. For instance, CNF films obtained from sugar beet have values of tensile strength and modulus of 104 MPa and 3.5 GPa, respectively [37, 61, 94].

### **6.7. Surface area**

Cellulose nanoparticles have a high specific surface area (SSA). Typically, cellulose nanopar‐ ticles have SSA ranging from 50 to 200 g/m2 . Conversely, the SSA of nanocellulose aerogels ranges higher from 250 to 350 m2 /g and have a very low density (0.02 g/cm3 ) and a high porosity of 98% [61, 63, 94].

### **6.8. Rheological properties**

CNF suspensions exhibit a shear-thinning behavior and pseudoplasticity, which in turn depends on the pH medium [95]. Further, sulfate cellulose shows a pH-dependent viscosity profile due to electrostatic interactions. Moreover, a CNF suspension has a decreasing viscosity with increasing shear rates. Further, CNF also has a high elastic modulus due to the entangled network structure [96].

### **6.9. Water sorption and permeability**

CNFs are able to form films with a low moisture diffusivity due to a rigid fiber network [90] and, thus, have excellent barrier properties [90]. The water vapor transfer rate (WVTR) of CNFs films are 20% smaller than those made of macrofibers (from 20% to 30%). CNC films are expected to provide a better barrier to water since CNC films have a more crystalline nature than CNF [90].

### **6.10. Oxygen barrier**

The oxygen transmittance rate of CNF at 0% RH is in the range 17–18 mL/m2 /day. The increase of RH increases the oxygen permeability due to the limited hydrogen bonding and loose network caused by the incoming water molecules. The water and oxygen permeability decreased with increasing film thickness due to the lack of interconnectivity of pores [63].

### **7. Toxicity**

CNCs have low toxicity and low environmental risk according to ecotoxicological tests with several aquatic species (e.g., Daphnia, rainbow trout and fathead minnow). Further, cytotox‐ icity (intracellular toxic effect) and proinflammatory response are significantly lower than those for MWCNT (multiwalled carbon nanotubes) and CAF (crocidolite asbestos fibers). CNF shows no toxicity and genotoxicity in vitro [97]. The toxicity of BC nanofibers has been successfully evaluated in vitro through cell viability and flow cytometric assays and in vivo using C57/B16 mice surgeries. Further, BC shows no toxicity in human umbilical vein endo‐ thelial cell culture, fibroblasts, and chondrocytes. The in vitro evaluation also shows that 95% of the mesenchymal stem cells aggregate to cellulose membrane [98, 99] (Fig. 3).

### **8. Biomedical applications**

### **8.1. Biological benefits**

**6.6. Film properties**

204 Cellulose - Fundamental Aspects and Current Trends

**6.7. Surface area**

of 98% [61, 63, 94].

104 MPa and 3.5 GPa, respectively [37, 61, 94].

ticles have SSA ranging from 50 to 200 g/m2

ranges higher from 250 to 350 m2

**6.8. Rheological properties**

network structure [96].

than CNF [90].

**7. Toxicity**

**6.10. Oxygen barrier**

**6.9. Water sorption and permeability**

CNF gels can be diluted and either cast or vacuum filtered followed by drying to form stiff films due to the formation of an interfibrillar hydrogen bonding network [63, 90–93]. For instance, CNF films obtained from sugar beet have values of tensile strength and modulus of

Cellulose nanoparticles have a high specific surface area (SSA). Typically, cellulose nanopar‐

CNF suspensions exhibit a shear-thinning behavior and pseudoplasticity, which in turn depends on the pH medium [95]. Further, sulfate cellulose shows a pH-dependent viscosity profile due to electrostatic interactions. Moreover, a CNF suspension has a decreasing viscosity with increasing shear rates. Further, CNF also has a high elastic modulus due to the entangled

CNFs are able to form films with a low moisture diffusivity due to a rigid fiber network [90] and, thus, have excellent barrier properties [90]. The water vapor transfer rate (WVTR) of CNFs films are 20% smaller than those made of macrofibers (from 20% to 30%). CNC films are expected to provide a better barrier to water since CNC films have a more crystalline nature

of RH increases the oxygen permeability due to the limited hydrogen bonding and loose network caused by the incoming water molecules. The water and oxygen permeability decreased with increasing film thickness due to the lack of interconnectivity of pores [63].

CNCs have low toxicity and low environmental risk according to ecotoxicological tests with several aquatic species (e.g., Daphnia, rainbow trout and fathead minnow). Further, cytotox‐ icity (intracellular toxic effect) and proinflammatory response are significantly lower than those for MWCNT (multiwalled carbon nanotubes) and CAF (crocidolite asbestos fibers). CNF shows no toxicity and genotoxicity in vitro [97]. The toxicity of BC nanofibers has been

The oxygen transmittance rate of CNF at 0% RH is in the range 17–18 mL/m2

/g and have a very low density (0.02 g/cm3

. Conversely, the SSA of nanocellulose aerogels

) and a high porosity

/day. The increase

Nanocellulose has been mainly used as a filler in nanocomposites because of its good me‐ chanical properties due to their biodegradability, renewability, availability, sustainability, lower cost, lower weight, higher mechanical strength, biocompatibility, high hydrophilicity, and high surface area [94, 100, 101–105]. It evades adverse tissue reactions, and unlike proteins, its polysaccharide nature makes it less immunogenic and nonhemolytic. It also promotes cellular interaction and tissue development. It is a slow/nondegrading material in vivo and in vitro, which makes it suitable for use as a scaffold providing a long-term support, sustains high loads, and has a high wear resistance. The biomedical industry includes skin replacements for burnings and wounds; drug releasing system; blood vessel growth; nerves, gum, and dura mater reconstruction; scaffolds for tissue engineering; stent covering; and bone reconstruction. The cellulose nanoparticle surface dictates cellular response by interfering with cellular adhesion, proliferation, migration, and functioning. On the other hand, cells support, hold, synthesize the matrix for the new tissues, and keep the proper growth ambient, whereas the growth factors promote the cell regeneration [97, 101, 106, 107].

In one study, a charged CNC–FITC and newly synthesized CNC–rhodamine B isothiocyanate (RBITC) were synthesized, and in vitro cellular uptake studies showed that the positively charged CNC-RBITC was taken up by human embryonic kidney (HEK) and *Spodoptera frugiperda* (Sf9) cells without any noticeable cytotoxic effect on the two cell lines [108].

### **8.2. Biosensors and diagnostics**

CNFs serve as a suitable platform for immobilization of bioactive molecules (e.g., enzymes, antibodies, etc.), which is useful in biosensors and diagnostics. For example, novel gold bacterial cellulose (Au-BC) nanocomposites have been prepared by a one-step biotemplated method in aqueous suspensions. This material shows excellent biocompatibility, good conductivity, and ultrafine nanofiber network structure, which makes it able to entrap horseradish peroxidase (HRP), maintaining enzyme bioactivity. HRP biosensors allow detection of H2O2 with a detection limit lower than 1 µM [109]. CNF films carboxlylated with TEMPO and activated via EDC/NHS coupling have been used to immobilize the antibody (antihuman immunoglobulin G (anti-IgG)) by physical adsorption. This surface can detect positively charged molecules. A TEMPO-activated film has also been used to conjugate Avidin for selectively capturing biotinylated molecules (anti-IgG) [109, 110].

**Figure 3.** Biomedical applications of cellulose nanoparticles.

Another study showed CNF to prepare support films with carboxyl groups, which are then converted to amine-reactive species. These substrates were then used to bind polyclonal anti-IgG. The CNF surface can also be activated by copolymer grafting. Thus, a peptide with specific affinity to human IgG is conjugated to the grafted polymer having a high selectivity.

Proteins such as collagen, elastin, hyaluronan, and growth factors such as the basic fibroblast growth factor (B-FGF), human epidermal growth factor (H-EGF), and keratinocyte growth factor (KGF) have been immobilized on macroporous BC to improve biocompatibility. The attachment of cells can be improved by utilizing adhesive amino acid sequences, such as Arg-Gly-Asp (RGD) found in several extracellular matrix proteins [111, 112].

Peptides such as the HWRGWV peptide can be immobilized on the TEMPO-activated film to detect human IgG. Thus, the acetylated peptide is covalently immobilized to the spacers on CNF via amide reaction. For instance, chitosan can be physically adsorbed onto the CNF and used as a spacer. The resulting biosensor has a very high specific binding capability for IgG and exhibits excellent resistance for nonspecific protein adsorption [113]. In another study, Edwards et al. created biosensors based on CNC by peptide conjugation for detection of human neutrophil elastase [114].

On the other hand, CNCs have been used as electrochemical sensors to selectively detect DNA hybridization. DNA oligomers were grafted onto TEMPO-oxidized CNC produced from cotton. This DNA-grafted CNC is able to self-assembling into larger aggregates as compared to the unmodified CNC. In another study, silver nanoparticles were obtained onto TEMPOmediated oxidized CNC by using NaBH4 as a reducing agent. The presence of CNC prevented the aggregation of the nanoparticles [115].

Moreover, the layer by layer (LbL) assembly technique has been used to adsorb collagen onto the surface of CNF. The LbL technique has also been used to prepare a luminescent singlewalled carbon nanotube–CNC films enhancing their water dispersibility [116].

Dong et al. synthesized folic acid (FA)-grafted CNCs and explored their folate-receptormediated uptake by human and rat brain tumor cells. First, CNCs were labeled with fluores‐ cein isothiocyanate (FITC) for detection in the cells and were then conjugated with FA. In vitro studies showed that the cellular binding of the FITC–CNC–FA by the folate receptor (which is overexpressed by cancer cells) was higher than that of the free FA [117].

### **8.3. Skin tissue repair**

Another study showed CNF to prepare support films with carboxyl groups, which are then converted to amine-reactive species. These substrates were then used to bind polyclonal anti-IgG. The CNF surface can also be activated by copolymer grafting. Thus, a peptide with specific

Proteins such as collagen, elastin, hyaluronan, and growth factors such as the basic fibroblast growth factor (B-FGF), human epidermal growth factor (H-EGF), and keratinocyte growth factor (KGF) have been immobilized on macroporous BC to improve biocompatibility. The attachment of cells can be improved by utilizing adhesive amino acid sequences, such as Arg-

Peptides such as the HWRGWV peptide can be immobilized on the TEMPO-activated film to detect human IgG. Thus, the acetylated peptide is covalently immobilized to the spacers on CNF via amide reaction. For instance, chitosan can be physically adsorbed onto the CNF and used as a spacer. The resulting biosensor has a very high specific binding capability for IgG and exhibits excellent resistance for nonspecific protein adsorption [113]. In another study, Edwards et al. created biosensors based on CNC by peptide conjugation for detection of human

affinity to human IgG is conjugated to the grafted polymer having a high selectivity.

Gly-Asp (RGD) found in several extracellular matrix proteins [111, 112].

**Figure 3.** Biomedical applications of cellulose nanoparticles.

206 Cellulose - Fundamental Aspects and Current Trends

neutrophil elastase [114].

Nanocellulose membranes could serve as an infection barrier, prevent loss of fluids, have a painkiller effect, allow drugs to be easily applied, and also absorb the purulent fluids during all inflammatory stages, expelling them later on in a controlled and painless manner [118].

Properties such as biocompatibility, high superficial area, high water absorption capacity, high elastic modulus, low thermal expansion coefficient, optical transparency, anisotropy, and flexible surface chemistry endow nanocellulose with suitable wound dressing applications. For instance, a membrane has been developed with BC and propolis extract, rendering antimicrobial and anti-inflammatory activities in chronic wounds absorbing purulent exu‐ dates. Further, it eases the BC removal from a wound surface after recovery [97].

Traditionally, skin tissue repair materials have been absorbent, permeable materials such as gauze, which can adhere to desiccated wound surfaces inducing trauma upon removal [119, 120]. BC controls wound dressing since it can control wound exudates and can provide a moist environment to a wound resulting in better wound healing. For instance, Biofill® is a com‐ mercial product of BC used as a temporary substitute for human skin in cases of second and third degree burns.

Czaja et al. showed that the skin of the patients whose burns were covered with a BC membrane healed faster than the wounds of patients who received conventional wound dressings. This is explained by the faster tissue regeneration, capillary formation and cell proliferation [121].

In a different study, BC wound dressing materials were compared to two different commercial dressings, Vaseline gauze, and Algisite M in a rat model. This study showed that BC-dressed animals had more rapid wound healing within 14 days without any evidence of toxicity [122]. Further, BC, gelatin, and alginate composite membranes showed the successful growth of NIH/ 3T3-type cells and, hence, proved potential as a skin tissue regeneration template [123].

The addition of chitosan to a tempo-oxidized BC renders a composite with high superior mechanical properties, water holding capacity, and water release rate, and thus, these composites can be used for wound dressing [124].

BC has also been used in a surgery of the lateral wall of the nose preventing nasal bleeding, surgical wound infections, local pain, and clotting [125]. Therefore, it provides a more rapid healing without the formation of crusts and prevent infection without the need for removal as compared to commercial nasal packing which causes a great discomfort upon removal [125].

A nanocellulose membrane has also been implanted into the subcutaneous tissue of diabetic rats for 12 weeks. Rats showed no macroscopic signs of inflammation around the implants, no formation of fibrotic capsule or giant cells, and fibroblasts were fully integrated to the cellulosic membrane and started to synthesize collagen [126].

### **8.4. Cortical implants**

Neural interfaces are able to record neural signals from individual neurons or small groups of neurons in the brain. The most common neural interfaces are made of iridium oxide, silicon, platinum, titanium, glassy carbon, gold, and stainless steel. However, they cause glia encap‐ sulation at the electrode interface, leading to neuron death near the surface of the implanted electrode. For this reason, adaptive cellulose nanoparticles interfaces should be stiff enough to be easily implanted into the brain but subsequently soften under in vivo conditions to closely match the stiffness of the brain tissue.

This nanocellulosic interface relies on stiff collagen fibers dispersed throughout a soft fibrillin matrix. Because of the abundance of surface hydroxyl groups, CNCs strongly interact with each other through hydrogen bonding and/or van der Waals' forces, but exposure to water reduces CNC–CNC interactions because of competitive hydrogen bonding or interfacial interactions with intermolecular van der Waal's forces. For instance, CNCs isolated from tunicate sea creatures have been successfully integrated into a rubbery ethylene oxideepichlorohydrin copolymer matrix having a lower stiffness than conventional electrodes [127].

Another study reported the development of poly(vinyl acetate) (PVAc) and CNCs composites showing a dual responsive behavior. Upon exposure to physiological conditions, the compo‐ sites are plasticized and the CNC network loses its stiffness capabilities due to the loss of hydrogen bonding. Once these composites were implanted in the pia mater of the cerebral cortex of a rat, the initial stiffness rapidly decreased matching that of the brain tissue [128, 129]. These adaptive microelectrodes implanted into a rat cortex for 8 weeks increased the cell density at the electrode–tissue interface. After 16 weeks of implantation, there was no neuron death surrounding PVAc/CNC implants as compared to PVAc-coated microelectrodes [130]. Another cortical implant in rats containing CNC/PVA and curcumin showed that after 4 weeks, curcumin promoted higher neuron survival and a more stable blood–brain barrier than the neat PVA controls [131].

### **8.5. Vascular grafts**

BC-based implants have been developed to replace synthetic by-pass implants made of polytetrafluoroethylene, poly(ethylene terephthalate), polyethylene, and polyurethane. Further, BC tubes have been successfully used to replace carotid arteries in rats, pigs, and sheep without any rejection after 4 weeks [127].

One study showed that the mechanical properties of CNC-PVA composites are similar to that of cardiovascular tissues, such as aorta and heart valve leaflets. For instance, the stress–strain properties for porcine aorta and heart valve tissue matches those of PVA-nanocellulose composites in the circumferential and the axial tissue directions [132].

The nanocellulose implants have been attached in the carotid artery of rats for 1 year, resulting in incorporation nanocellulose forming neointima and ingrowth of active fibroblasts. In another study, the grafts were used to successfully replace the carotid arteries of pigs [133]. Further, cellulose and chitosan composites have been successfully used to produce hollow tubes with a compliance compared to that of human coronary arteries showing potential for coronary artery bypass graft applications [134].

In one study, part of the carotid artery (4–6 mm) of a rat was replaced using BC and after 4 weeks, the BC complex was wrapped up with connective tissue and was infused with small vessels [28, 135]. BC porous surfaces have also been reported to maintain fiber network arrangement viable in endothelial cells for 20 days [136]. Further, the surface modification of BC by nitrogen-containing plasma improved cell adhesion and proliferation of the endothelial and neuroblast cells [137].

### **8.6. Medical implants**

The addition of chitosan to a tempo-oxidized BC renders a composite with high superior mechanical properties, water holding capacity, and water release rate, and thus, these

BC has also been used in a surgery of the lateral wall of the nose preventing nasal bleeding, surgical wound infections, local pain, and clotting [125]. Therefore, it provides a more rapid healing without the formation of crusts and prevent infection without the need for removal as compared to commercial nasal packing which causes a great discomfort upon removal [125]. A nanocellulose membrane has also been implanted into the subcutaneous tissue of diabetic rats for 12 weeks. Rats showed no macroscopic signs of inflammation around the implants, no formation of fibrotic capsule or giant cells, and fibroblasts were fully integrated to the cellulosic

Neural interfaces are able to record neural signals from individual neurons or small groups of neurons in the brain. The most common neural interfaces are made of iridium oxide, silicon, platinum, titanium, glassy carbon, gold, and stainless steel. However, they cause glia encap‐ sulation at the electrode interface, leading to neuron death near the surface of the implanted electrode. For this reason, adaptive cellulose nanoparticles interfaces should be stiff enough to be easily implanted into the brain but subsequently soften under in vivo conditions to closely

This nanocellulosic interface relies on stiff collagen fibers dispersed throughout a soft fibrillin matrix. Because of the abundance of surface hydroxyl groups, CNCs strongly interact with each other through hydrogen bonding and/or van der Waals' forces, but exposure to water reduces CNC–CNC interactions because of competitive hydrogen bonding or interfacial interactions with intermolecular van der Waal's forces. For instance, CNCs isolated from tunicate sea creatures have been successfully integrated into a rubbery ethylene oxideepichlorohydrin copolymer matrix having a lower stiffness than conventional electrodes [127]. Another study reported the development of poly(vinyl acetate) (PVAc) and CNCs composites showing a dual responsive behavior. Upon exposure to physiological conditions, the compo‐ sites are plasticized and the CNC network loses its stiffness capabilities due to the loss of hydrogen bonding. Once these composites were implanted in the pia mater of the cerebral cortex of a rat, the initial stiffness rapidly decreased matching that of the brain tissue [128, 129]. These adaptive microelectrodes implanted into a rat cortex for 8 weeks increased the cell density at the electrode–tissue interface. After 16 weeks of implantation, there was no neuron death surrounding PVAc/CNC implants as compared to PVAc-coated microelectrodes [130]. Another cortical implant in rats containing CNC/PVA and curcumin showed that after 4 weeks, curcumin promoted higher neuron survival and a more stable blood–brain barrier than the

BC-based implants have been developed to replace synthetic by-pass implants made of polytetrafluoroethylene, poly(ethylene terephthalate), polyethylene, and polyurethane.

composites can be used for wound dressing [124].

208 Cellulose - Fundamental Aspects and Current Trends

membrane and started to synthesize collagen [126].

**8.4. Cortical implants**

neat PVA controls [131].

**8.5. Vascular grafts**

match the stiffness of the brain tissue.

Medical implants must have mechanical characteristics as the tissue it replaces. It must also show nonthrombogenicity, sterilizability, durability, lesser degree of calcification, and good processability. The implant should be biocompatible with the host tissues in terms of chemical, mechanical, surface chemistry, and pharmacological properties.

BC is a good candidate for use as medical implants since it is nondegradable under physio‐ logical conditions and provides durable mechanical properties and chemical stability. One study showed that the mechanical properties of the BC gel with collagen meniscal implants were similar in magnitude to the ones of pig menisci [138].

Further, an ear-shaped BC prototype material showed suitable mechanical properties for ear cartilage replacement for a customized patient-specific ear shapes [139].

A nanocellulose membrane has also been implanted in the nasal dorsum of 22 rabbits as an excellent substitute of bone cartilage. After 6 months, residual inflammation was attributed to the surgical procedure itself and not to the cellulosic membrane [140].

The cartilage that covers the trochlear groove in dogs is composed of chondrocytes. Further, BC was utilized successfully in experimental trochleoplasty in dogs, showing advantage in respect to conventional treatment for osteochondral injuries [141]. Moreover, BC membrane was applied in the tissue formation of fibrocartilage ripe, resulting in a good integration of the newly formed tissue.

Other researchers have successfully incorporated BC and polytetrafluoroethylene (PTFE) membranes in rats to correct abdominal wall defects [142]. Further, BC implanted in the peritoneum of dogs formed an integrated net along the conjunctive tissue after 6 months [143].

On the other hand, the biggest challenge in dental applications is the loss of alveolar bone. For this reason, synthetic hydroxyapatite (HAP-91) was implanted in the dental cavities and covered by nanocellulose membranes. Nanocellulose promoted faster bone regeneration and resembled those of the original tissue after 50 days [144].

A bandage product derived from BC (Gengiflex®) restores the osseous defects. It consists of an inner layer of microbial cellulose, which offers rigidity to the membrane, and an outer layer of alkali cellulose. A greater amount of bone formation was present in bone defects protected by the BC membrane, when compared to the control sites [145, 146].

BC membranes were tested as physical barriers used to treat bone defects. In this scenario, two osseous defects (8 mm in diameter) were performed in each hind foot of four adult rabbits. After 3 months, the bone defects showed lamellar bone formation resulting in partial bone deposition [147].

### **8.7. Cell culture and scaffolds**

The asymmetric structure of a scaffold is composed of a fine network of nanofibrils, which is similar to a collagen network, which promotes the adhesion and proliferation of muscle cells [97, 101]. BC has shown significantly higher levels of chondrocyte growth, suggesting the potential application of scaffolds for cartilage tissue engineering [148]. The beneficial proper‐ ties of CNF are based on its unique nanofibrillar structure, mimicking properties of the extracellular matrix, and thus, a CNF scaffold promotes hepatocyte 3D cell culture without added bioactive components.

Nanocellulose and hydroxyapatite (HA) are both capable of bone replacement because of their properties, including biocompatibility with the human body, bioactivity, osteoconductivity, and noninflammatory properties [74]. Further, nanocellulose has also been soaked into HA to develop a composite scaffold for bone regeneration. Thus, CNC/HA scaffolds were prepared by absorption of HA onto the BC surface to induce nucleation of calcium-deficient HA. The presence of calcium-deficient HA crystals on the BC surface increased cell attachment and alkaline phosphatase activity on bone cells. Further, nanocellulose has been combined with polyacrylamide and gelatin, yielding hydrogels with improved toughness [149].

Moreover, enzymatically modified gelatin (EMG) and nanocellulose composites have been prepared to improve the rehydration properties and, thus, can be used as scaffold for the cornea tissue since the stromal cells are able to grow into the scaffold [150].

In one study, BC and poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB) composite scaffolds showed excellent biocompatibility in Chinese hamster lung (CHL) fibroblast cells [151]. Further, the BC and alginate composite (80:20 w/w) dried by supercritical carbon dioxide formed a nanoporous structure, which supports the proliferation of keratino‐ cytes and gingival fibroblasts [152, 153].

In another study, nanocellulose/PEG composite scaffolds were prepared by soaking a nano‐ cellulose hydrogel with a PEG solution forming scaffold with improved thermal stability. Results indicated that the Young's modulus and tensile strength tended to decrease while the elongation at break had a slight increase. Thus, the prepared nanocellulose/PEG composite scaffolds were suitable for cell attachment [154].

The functionalization of the BC surface with recombinant proteins containing a bioactive peptide (IKVAV) and a carbohydrate-binding module (CBM3) has improved their biocom‐ patibility with neuronal and mesenchymal cells [155]. BC cross-linked with heparin is able to prevent the formation of blood clots [156]. A natural peptide called as polylysine (PLL) has been cross-linked to the surface of BC resembling the collagen fibers and composition of natural bone [157].

CNCs have been coated with 2-hydroxyethylmethacrylate and methacrylic functional groups forming hydrogels with excellent mechanical properties comparable to articular cartilage with hydrogel-like properties [158].

One study reported the creation composites based on BC and type I collagen (COL) for potential bone tissue engineering, in which collagen was covalently introduced into the BC network. Further, cell culture with osteogenic cells revealed that collagen did not affect cell adhesion and proliferation or its morphology [159]. Heparin and nanocellulose scaffolds have been prepared with anticoagulant properties for potential use in vascular tissue engineering [160].

Further, grafted zwitterionic carboxybetaine improved CNF membrane blood compatibility. In another study, dialdehyde BC membranes supported the epidermal cell adhesion and proliferation [161].

BC has also been used to treat wounds in diabetic foot ulcers. The mean time for 75% epithe‐ lization was achieved in 79 days, and BC shortened the epithelization time as compared to XeroformTM® Petrolatum gauze [162]. Another study showed a complete closure of the facial wound within 44 days with no significant signs of extensive scarring [121]. Further, the release of BC dressing from the wound is a painless operation due to the moisture still present in the cellulose structure [163].

A nanogel made of poly(*N*-isopropyl acrylamide-co-butyl methacrylate), BC, and a surfactant has been produced by emulsion polymerization. This nanogel showed a thermal responsive behavior from a swollen to shrunken gel with increasing temperature. This is explained by the decrease of hydrogen bonding interactions with temperature [164].

### **8.8. Antimicrobial activity**

Other researchers have successfully incorporated BC and polytetrafluoroethylene (PTFE) membranes in rats to correct abdominal wall defects [142]. Further, BC implanted in the peritoneum of dogs formed an integrated net along the conjunctive tissue after 6 months [143].

On the other hand, the biggest challenge in dental applications is the loss of alveolar bone. For this reason, synthetic hydroxyapatite (HAP-91) was implanted in the dental cavities and covered by nanocellulose membranes. Nanocellulose promoted faster bone regeneration and

A bandage product derived from BC (Gengiflex®) restores the osseous defects. It consists of an inner layer of microbial cellulose, which offers rigidity to the membrane, and an outer layer of alkali cellulose. A greater amount of bone formation was present in bone defects protected

BC membranes were tested as physical barriers used to treat bone defects. In this scenario, two osseous defects (8 mm in diameter) were performed in each hind foot of four adult rabbits. After 3 months, the bone defects showed lamellar bone formation resulting in partial bone

The asymmetric structure of a scaffold is composed of a fine network of nanofibrils, which is similar to a collagen network, which promotes the adhesion and proliferation of muscle cells [97, 101]. BC has shown significantly higher levels of chondrocyte growth, suggesting the potential application of scaffolds for cartilage tissue engineering [148]. The beneficial proper‐ ties of CNF are based on its unique nanofibrillar structure, mimicking properties of the extracellular matrix, and thus, a CNF scaffold promotes hepatocyte 3D cell culture without

Nanocellulose and hydroxyapatite (HA) are both capable of bone replacement because of their properties, including biocompatibility with the human body, bioactivity, osteoconductivity, and noninflammatory properties [74]. Further, nanocellulose has also been soaked into HA to develop a composite scaffold for bone regeneration. Thus, CNC/HA scaffolds were prepared by absorption of HA onto the BC surface to induce nucleation of calcium-deficient HA. The presence of calcium-deficient HA crystals on the BC surface increased cell attachment and alkaline phosphatase activity on bone cells. Further, nanocellulose has been combined with

Moreover, enzymatically modified gelatin (EMG) and nanocellulose composites have been prepared to improve the rehydration properties and, thus, can be used as scaffold for the

In one study, BC and poly(3-hydroxubutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB) composite scaffolds showed excellent biocompatibility in Chinese hamster lung (CHL) fibroblast cells [151]. Further, the BC and alginate composite (80:20 w/w) dried by supercritical carbon dioxide formed a nanoporous structure, which supports the proliferation of keratino‐

polyacrylamide and gelatin, yielding hydrogels with improved toughness [149].

cornea tissue since the stromal cells are able to grow into the scaffold [150].

resembled those of the original tissue after 50 days [144].

deposition [147].

**8.7. Cell culture and scaffolds**

210 Cellulose - Fundamental Aspects and Current Trends

added bioactive components.

cytes and gingival fibroblasts [152, 153].

by the BC membrane, when compared to the control sites [145, 146].

Nanocellulose intrinsically does not possess any antimicrobial property. Therefore, it needs to be functionalized with antimicrobial agents. For instance, chemical grafting of aminoalkyl groups [165], 2-benzyl-4- chlorophenol [166], and L-cysteine [167], onto the surface of the cellulose backbone has been reported [168]. In one study, a BC film was soaked in a benzal‐ konium chloride solution, resulting in antimicrobial activity against *Staphylococcus aureus* and *Bacillus subtilis*, which are bacteria generally found on contaminated wounds [169].

The electrospun composite nanofiber membrane containing bis(*N*-chloro-2,2,6,6-tetrameth‐ yl-4-piperidinyl) sebacate (Cl-BTMP) showed significant antimicrobial activity against *S. aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa* attributed to the aggregation of Cl-BTMP. Further, nanocomposites with curcumin rendered antimicrobial activity against *E. coli* and *S. aureus* over a period of 24 h [170].

The immersion of BC in a silver nitrate solution, followed by reduction of absorbed silver ions (Ag+) with sodium borohydride, formed metallic silver nanoparticles with antimicrobial activity against *E. coli* and *S. aureus* [169]. Further, aminoalkyl-grafted bacterial nanocellulose (BC-NH2) membranes were prepared by hydrolysis of the silane derivative, adsorption of the hydrolyzed species onto BC nanofibrils, followed by a chemical condensation reaction. These BC–NH2 membranes showed antimicrobial activity against *E. coli* and *S. aureus* after 24 h [167].

In another study, composites of CNF and chitin nanocrystals formed a 3D network with bactericidal activity against *E. coli*. On the other hand, the in situ sol-gel formation of silver and gold particles within CNF endowed it with antibacterial activity against *E. coli*, *S. aureus*, and *Klebsiella pneumonia* strains. The assembly of the CNFs and silver nanoparticle (AgNP) composites occurs through electrostatic interactions. Another strategy of incorporation of silver is through magnetic interaction where the 3D structure of cellulose provides plenty of sites for heterogeneous nucleation of magnetite rendering a high antimicrobial activity against *E. coli* and *B. subtilis* [2]. Further, nanocomposite films prepared by the addition of cellulose nanocrystals, with silver nanoparticles, in a PLA matrix generate an antibacterial film against *S. aureus* and *E. coli* [171]. Silver ions interfere with the respiratory chain causing a decrease in bacterial viability [172–174]. Moreover, Ag and Au nanoparticles have been synthesized on CNC using cationic surfactants such a CTABr [175, 176].

In one study, BC was first homogenized with a ferric and ferrous salt mixture followed by soaking in dopamine and silver nitrate solution. The resulting magnetic silver/BC nanocom‐ posites had antimicrobial activity against *E. coli* and *B. subtilis* were developed.

### **8.9. Drug delivery**

The abundant surface hydroxyl groups in nanocellulose provide a site for the surface modifi‐ cation by a variety of methods. Surface modification modulates the loading and release of nonionized or hydrophobic drugs that would not normally bind to nanocellulose. For instance, poly(caprolactone) chains might be conjugated onto CNC for drug release [31].

PVA and methyl cellulose form aninterprenetrating polymer network through cross-linking with epichlorohydrin and serve as a carrier to load a drug for controlled release [31]. Further, coating of CNC with a cationic surfactant such as cetyltrimethylammonium bromide (CTABr) has been useful to load significant quantities of anticancer agents for controlled released [177– 179]. In one study, nanoparticles of itraconazole were stabilized by the nanostructured cellulose matrix during freeze-drying and storage increasing its dissolution rate and in vivo performance [177].

On the other hand, spray-dried CNFs were produced in order to increase the long-term stability of drugs due to a better ability to pack having a low porosity and forming fast disintegrating tablets [179]. Further, BC membranes loaded with lidocaine rendered lower permeation rates in the skin than traditional drug delivery systems. The greatest advantage of the BC membrane is the combination of its wound healing capacity and the ability to absorb exudates with the release of antimicrobial and anti-inflammatory drugs [180].

In another study, freeze-dried BC and serum albumin composites were investigated as potential drug delivery systems for proteins [110]. In one report, CNC was oxidized with periodic acid to graft a spacer molecule (aminobutyric acid), and then syringyl alcohol was attached. In another investigation, calcium peroxide (CPO) was embedded into highly porous CNCs to produce H2O2, whereas catalase was added to convert the generated H2O2 to O2, increasing cell survival up to 5 days [181].

Weng et al. created biodegradables cellulose microspheres loaded with doxorubicin for arterial embolization applications. They showed a burst release profile within 8 h followed by a release plateau over a 24-h period in rabbits [182].

Further, docetaxel-loaded CMC-based nanoparticles have been produced for enhanced cytotoxicity against cancer cells releasing 100% of drug within 3 weeks inhibiting 90% of tumor growth [183]. In another study, CMC gels were produced by polymerization of oligo(ethylene oxide)-methacrylate (OEOMA) in the presence of CNC. These gels have a dual drug release in response to acidic pH and thiol-reducing agents [184].

Zoppe and collaborators applied CNC-based systems as viral inhibitors (alphavirus infectiv‐ ity) and suggested that CNC can be used for inhibition of HIV [185].

### **9. Conclusion**

konium chloride solution, resulting in antimicrobial activity against *Staphylococcus aureus* and

The electrospun composite nanofiber membrane containing bis(*N*-chloro-2,2,6,6-tetrameth‐ yl-4-piperidinyl) sebacate (Cl-BTMP) showed significant antimicrobial activity against *S. aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa* attributed to the aggregation of Cl-BTMP. Further, nanocomposites with curcumin rendered antimicrobial activity against *E. coli* and *S.*

The immersion of BC in a silver nitrate solution, followed by reduction of absorbed silver ions (Ag+) with sodium borohydride, formed metallic silver nanoparticles with antimicrobial activity against *E. coli* and *S. aureus* [169]. Further, aminoalkyl-grafted bacterial nanocellulose (BC-NH2) membranes were prepared by hydrolysis of the silane derivative, adsorption of the hydrolyzed species onto BC nanofibrils, followed by a chemical condensation reaction. These BC–NH2 membranes showed antimicrobial activity against *E. coli* and *S. aureus* after 24 h [167]. In another study, composites of CNF and chitin nanocrystals formed a 3D network with bactericidal activity against *E. coli*. On the other hand, the in situ sol-gel formation of silver and gold particles within CNF endowed it with antibacterial activity against *E. coli*, *S. aureus*, and *Klebsiella pneumonia* strains. The assembly of the CNFs and silver nanoparticle (AgNP) composites occurs through electrostatic interactions. Another strategy of incorporation of silver is through magnetic interaction where the 3D structure of cellulose provides plenty of sites for heterogeneous nucleation of magnetite rendering a high antimicrobial activity against *E. coli* and *B. subtilis* [2]. Further, nanocomposite films prepared by the addition of cellulose nanocrystals, with silver nanoparticles, in a PLA matrix generate an antibacterial film against *S. aureus* and *E. coli* [171]. Silver ions interfere with the respiratory chain causing a decrease in bacterial viability [172–174]. Moreover, Ag and Au nanoparticles have been synthesized on

In one study, BC was first homogenized with a ferric and ferrous salt mixture followed by soaking in dopamine and silver nitrate solution. The resulting magnetic silver/BC nanocom‐

The abundant surface hydroxyl groups in nanocellulose provide a site for the surface modifi‐ cation by a variety of methods. Surface modification modulates the loading and release of nonionized or hydrophobic drugs that would not normally bind to nanocellulose. For instance,

PVA and methyl cellulose form aninterprenetrating polymer network through cross-linking with epichlorohydrin and serve as a carrier to load a drug for controlled release [31]. Further, coating of CNC with a cationic surfactant such as cetyltrimethylammonium bromide (CTABr) has been useful to load significant quantities of anticancer agents for controlled released [177– 179]. In one study, nanoparticles of itraconazole were stabilized by the nanostructured cellulose matrix during freeze-drying and storage increasing its dissolution rate and in vivo

posites had antimicrobial activity against *E. coli* and *B. subtilis* were developed.

poly(caprolactone) chains might be conjugated onto CNC for drug release [31].

*Bacillus subtilis*, which are bacteria generally found on contaminated wounds [169].

*aureus* over a period of 24 h [170].

212 Cellulose - Fundamental Aspects and Current Trends

CNC using cationic surfactants such a CTABr [175, 176].

**8.9. Drug delivery**

performance [177].

The attractive properties of nanocellulosic materials such as biodegradability, biocompacti‐ bility, renewability, low density, high strength, good stiffness, low thermal expansion, and high aspect ratio make them suitable for biomedical applications. For this reason, the number of publications and patents related to these applications has skyrocketed in the last 5 years. However, a great effort has still to be made to reduce the high cost involved with the production process of nanocellulose intended for biomedical used.

### **Author details**

John Rojas\* , Mauricio Bedoya and Yhors Ciro

\*Address all correspondence to: jrojasca@gmail.com

Department of Pharmacy, School of Pharmaceutical Chemistry, University of Antioquia, Medellín, Columbia

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### **Cellulose - Chitosan Nanocomposites - Evaluation of Physical, Mechanical and Biological Properties**

Guillermo H. Riva, Joaquín García-Estrada, Brenda Vega, Fernando López-Dellamary, María E. Hérnandez and José A. Silva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61727

#### **Abstract**

el for enhanced cytotoxicity against cancer cells. Bioconjugate Chem. 2011;22:2474–

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86.

228 Cellulose - Fundamental Aspects and Current Trends

This research describes the preparation of membranes with chitosan (CS) as the polymer‐ ic matrix and cellulose nanocrystals (CNC) as reinforcement. The aim was to evaluate their physical, mechanical and biological properties, and to determine their potential for biomedical use. Membranes were prepared via casting CNC suspensions in CS solution, at CNC concentrations of 0.5%, 1.0% and 2.0% (w/w) with pure chitosan as a reference. Analysis of membrane properties was performed using several techniques, such as ATR – FTIR, SEM, swelling test, maximum water absorption, dynamical mechanical analysis and *in vivo* (Winstar rats) biocompatibility and biodegradability assays for biological evaluation. Experimental results established that CNC reduced swelling rates and in‐ creased the maximum water absorption when CNC concentration was higher. Therefore, the presence of CNC in the matrix reduced Young's modulus by approximately 50% in comparison with pure chitosan. All formulations demonstrated biocompatibility and bio‐ degradability values ranged between 4% and 21% in the 30 days after implantation. Based on these results, these membranes may be of use for biomedical applications.

**Keywords:** Cellulose nanocrystals, chitosan, biomedical, biocompatibility, nanocompo‐ sites

### **1. Introduction**

### **1.1. Nanotechnology in actual context**

The advances in nanotechnology for biomedical use are increasing and are at the forefront of scientific research. In recent years, hot spot areas such as drug transportation, tissue regener‐

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ation or nanomaterial development for cell-growth scaffolds have been constantly advancing. Biomaterials that are considered for biomedical applications must confirm to strict biological, physical and mechanical characteristics. Some biopolymers in particular, offer advantages in terms of sustainability and low environment impact compared to ceramics and metals. These attributes are the *biocompatibility* (an absence of inflammatory, cytotoxicity or invasive response in native cells, tissues or organs in vivo), *biodegradability* and *bioabsorbability* (the material and its by-products will degrade and/or be absorbed or safety eliminated from the body). More suitable properties may be the *degradation rate* (this rate must match the regener‐ ation time of tissue in damaged zones, as well as transfer the mechanical efforts to new tissue in a timely manner), *porosity* (directly linked to mass transport and efficient tissue regeneration) and *surface morphology*. As drawbacks, the physical and mechanical properties of these materials make them less suitable than petroleum based plastics and other materials (metals, alloys and clays). As a consequence, reinforcement of the matrix is an option to counterbalance some of those drawbacks.

### **1.2. Biopolymers**

### *1.2.1. Cellulose*

Cellulose — the most abundant biopolymer on Earth — has an annual production of 7.5 x 1010 tons. This biopolymer is widely distributed in higher plants, sea animals (tunicates), and to a lesser degree in algae, fungi, bacteria, invertebrates, and is even found in protozoans such as *Dictyostelium discoideum*. In general, cellulose is a hard, fibrous, and water insoluble substance that plays an essential function in keeping the structure of cell walls in plants [1]. Cellulose can be found in its purest form in plants (i.e. cotton fibers). However, in wood, leaves and plant stalks, it is found mixed with other materials such as lignin and hemicelluloses. Cellulose nanofibers have the potential to be used in multiple ways, notably as a reinforcement material in the development of nanocomposites [2].

Thus, the preparation of biocompatible nanocomposites employing cellulose nanocrystals (CNC) as a reinforcement is a natural choice based on them being inert, biocompatible, biodegradable [3], and non-cytotoxic. They also contribute to the regeneration of damaged tissues or organs [4] and have mechanically desirable properties [5].

### *1.2.1.1. Types of processes used to obtain CNC*

Cellulose nanocrystals (CNC) can be obtained by different techniques and processes:

**a.** Mechanical processes [6], i.e. using used bleached pulp of softwoods and hardwoods as a material raw to obtain nanocrystals from. The process begins with the soaking and grinding of fibers, followed by sieving and refining (for hardwoods this process is repeated several times). Finally, fibers are submitted to high pressure and homogenization processes (1000 Bar, 180 min), which are repeated until CNC is obtained. Energy con‐ sumption and Young's modulus (YM) are higher in hardwoods than softwoods. The tensile resistance in softwoods is better (75 MPa versus 63 MPa respectively).


### *1.2.1.2. Nanocellulose for biomedical use*

ation or nanomaterial development for cell-growth scaffolds have been constantly advancing. Biomaterials that are considered for biomedical applications must confirm to strict biological, physical and mechanical characteristics. Some biopolymers in particular, offer advantages in terms of sustainability and low environment impact compared to ceramics and metals. These attributes are the *biocompatibility* (an absence of inflammatory, cytotoxicity or invasive response in native cells, tissues or organs in vivo), *biodegradability* and *bioabsorbability* (the material and its by-products will degrade and/or be absorbed or safety eliminated from the body). More suitable properties may be the *degradation rate* (this rate must match the regener‐ ation time of tissue in damaged zones, as well as transfer the mechanical efforts to new tissue in a timely manner), *porosity* (directly linked to mass transport and efficient tissue regeneration) and *surface morphology*. As drawbacks, the physical and mechanical properties of these materials make them less suitable than petroleum based plastics and other materials (metals, alloys and clays). As a consequence, reinforcement of the matrix is an option to counterbalance

Cellulose — the most abundant biopolymer on Earth — has an annual production of 7.5 x 1010 tons. This biopolymer is widely distributed in higher plants, sea animals (tunicates), and to a lesser degree in algae, fungi, bacteria, invertebrates, and is even found in protozoans such as *Dictyostelium discoideum*. In general, cellulose is a hard, fibrous, and water insoluble substance that plays an essential function in keeping the structure of cell walls in plants [1]. Cellulose can be found in its purest form in plants (i.e. cotton fibers). However, in wood, leaves and plant stalks, it is found mixed with other materials such as lignin and hemicelluloses. Cellulose nanofibers have the potential to be used in multiple ways, notably as a reinforcement

Thus, the preparation of biocompatible nanocomposites employing cellulose nanocrystals (CNC) as a reinforcement is a natural choice based on them being inert, biocompatible, biodegradable [3], and non-cytotoxic. They also contribute to the regeneration of damaged

Cellulose nanocrystals (CNC) can be obtained by different techniques and processes:

tensile resistance in softwoods is better (75 MPa versus 63 MPa respectively).

**a.** Mechanical processes [6], i.e. using used bleached pulp of softwoods and hardwoods as a material raw to obtain nanocrystals from. The process begins with the soaking and grinding of fibers, followed by sieving and refining (for hardwoods this process is repeated several times). Finally, fibers are submitted to high pressure and homogenization processes (1000 Bar, 180 min), which are repeated until CNC is obtained. Energy con‐ sumption and Young's modulus (YM) are higher in hardwoods than softwoods. The

some of those drawbacks.

230 Cellulose - Fundamental Aspects and Current Trends

material in the development of nanocomposites [2].

*1.2.1.1. Types of processes used to obtain CNC*

tissues or organs [4] and have mechanically desirable properties [5].

**1.2. Biopolymers**

*1.2.1. Cellulose*

Nanocellulose has been called "biomaterials´ eyes" due to its potential for numerous applica‐ tions in the biomedical field, including skin grafts to burn damage and wounds, growing of blood vessels, nerve reconstruction, brain membranes, and scaffolds in tissue engineering and bone reconstruction. Tissue engineering (TE) involves searching for new materials and artefacts to interact in positive ways with biological tissues. Furthermore, TI is seeking a primal artefact to cellular development *in vitro*, rearrangement and development of tissue when it will be implanted. The main attribute wanted in biopolymers with a potential biomedical use is a controllable and specific activity, to be used mainly in cellular scaffolds. Recently, many of these kinds of materials have been developed, having the required properties (physical/ chemical and mechanical) dependent mostly on the final application (tissue regeneration, drug releasing, scaffolding, etc.). The success of scaffolds depends mainly on cellular adhesion and surface growth. The chemical surface of a biopolymer can cause the cellular response to interfere with the adhesion, proliferation, migration and cellular functionalization. The interaction in cell surface it's whole important in the graft, including its rejection. For the regeneration of tissues, three fundamental aspects are important: the cells, and the bearing and growing factors. The cells synthetize the matrix to new tissues, the bearing creates a suitable environment for cell development, and growing factors promote cell regeneration. Further‐ more, regeneration must be promoted and if it is necessary, the new material must be absorbed or biodegraded. Studies of the interactions of cell bearing are crucial for the feasibility of grafts. Different responses of cells can be observed from several materials, based on the ability of cells to distinguish and/or adapt to the surface of the material. This last factor is crucial, because it drives different responses such as cell proliferation, cell migration or feasibility. With issues regarding the skin, several laboratories have shown an interest in developing products that offer advantages such as the immediate mitigation of pain, close adhesion at wound surfaces and the reduction of infection rates. Nanocellulose has a large surface area that brings a better capability for water absorption and elasticity, these being the best characteristics for a recov‐ ering bandage, as microbial activity is stopped. Indeed, nanocellulose is very effective in reducing pain and promoting the granulation suitable for wound bandages. Another great advantage of nanocellulose consists in the capability to be built in any shape and size, making it ideal for covering extensive and difficult areas of the human body [10].

Hence, cellulose and CNC have been used in biomedicals. Past research has shown them to be ideal for tissue engineering, producing favorable results [3, 6]. CS membranes with nanorein‐ forcement must show a Young's modulus of 1,500—2,300 MPa to be suitable for biomedical use [11].

Furthermore, applications with excellent, proven results have been reported as follows [10]:


active fibroblasts over a long period. In a second study, the implants were used to replace the carotid artery of pigs. After three months, the implants were retired and analyzed at both macro and microscopic levels. Seven implants (87.5%) were found in use and just one of them was blocked. This data showed that the innovative techniques of nanocellu‐ lose engineering have allowed the production of stable vascular conduits and confirmed the very notable achievement of the use of tissues for blood vessels in vivo as a part of cardiovascular programs.

**e.** Reconstructive / aesthetic surgery [14] Ideal for nasal reconstruction. The response of tissue in the presence of nanocellulose in nose bone was evaluated. In the study, 22 rabbits were used and in 20 of them a cellulose film was added to the nasal dorsum, with the remaining two acting as a control. After three and six months, the new bone was extracted for histopathology studies. Parameters such as blocking of blood flow, inflammation intensity and inflammation by the presence of purulent liquids were found to be stable, probably due to the surgical process itself rather than the presence of cellulose. For the other parameters, the statistical response was not significant. The nanocellulose coverage showed good compatibility and remained unchanged over time, making this material an excellent option for rebuilding new bone.

### *1.2.2. Chitosan*

or biodegraded. Studies of the interactions of cell bearing are crucial for the feasibility of grafts. Different responses of cells can be observed from several materials, based on the ability of cells to distinguish and/or adapt to the surface of the material. This last factor is crucial, because it drives different responses such as cell proliferation, cell migration or feasibility. With issues regarding the skin, several laboratories have shown an interest in developing products that offer advantages such as the immediate mitigation of pain, close adhesion at wound surfaces and the reduction of infection rates. Nanocellulose has a large surface area that brings a better capability for water absorption and elasticity, these being the best characteristics for a recov‐ ering bandage, as microbial activity is stopped. Indeed, nanocellulose is very effective in reducing pain and promoting the granulation suitable for wound bandages. Another great advantage of nanocellulose consists in the capability to be built in any shape and size, making

Hence, cellulose and CNC have been used in biomedicals. Past research has shown them to be ideal for tissue engineering, producing favorable results [3, 6]. CS membranes with nanorein‐ forcement must show a Young's modulus of 1,500—2,300 MPa to be suitable for biomedical

Furthermore, applications with excellent, proven results have been reported as follows [10]: **a.** Pharmaceutical. Cellulose has excellent properties of compaction when it is mixed with other pharmaceutical excipients, forming dense matrices that make the administration of therapeutic drugs easy. Nanocellulose offers potential advantages as an excipient in drug release. Its large surface area and negative charge suggest that higher quantities of therapeutical drugs can be added to the surface of this material, showing the potential for a large quantity of charge and the optimal control of dosification. The proven biocom‐ patibility of cellulose supports the use of nanocellulose for similar purposes. The hydrox‐ ide groups on the surface offer a site for surface modification to a broad range of chemical groups, using different methods. The surface modification can be used to tune the charge and drug release that are not normally linked with nanocellulose such as hydrophobic

**b.** Odontology. Nanocellulose can be used as biological barrier due to its porosity. This makes it ideal for use with infections, loss of fluids and it has an analgesic effect that allows therapeutic drugs be used easily and absorb the residual fluids during inflammatory

**c.** Ophthalmology [12]. Researchers explored the potential of nanocellulose as a scaffold and found it suitable for use in the development of tissue engineering for the cornea. They studied the growth of human stem cells in nanocellulose. The growth of corneal stem cells inside the scaffold was verified with a scanning laser microscope. The results suggested the potential of this biomaterial as a scaffold for tissue engineering of artificial corneas. **d.** Vascular surgery [13]. Researchers studied artificial vascular implants of nanocellulose in two cases: The first was a microsurgery study, where nanocellulose implants were used as an artificial part of the carotid artery of rats for a year. These results showed the incorporation of nanocellulose under the formation of tissues and internal growth of

stages, can be rejected in a controlled and painless way.

it ideal for covering extensive and difficult areas of the human body [10].

use [11].

and non-ionized drugs.

232 Cellulose - Fundamental Aspects and Current Trends

Chitosan (CS) is a biomaterial of proven use in the biomedical field due its biocompatibility, biodegradability and antibacterial activity, making it ideal for drug transportation, tissue engineering, wound healing, and antibacterial uses [15]. Furthermore, chitosan is bioactive and nontoxic. This biopolymer has a wide range of uses such as substance separation due to its barrier property and sensors, as well as food packing. Other authors describe the prepara‐ tion of bionanocomposites using chitosan and different nanoreinforcements with the goal of obtaining a better mechanical performance, as well as the barrier properties and sensing detectors [16]. Chitosan is commonly amorphous and can be processed in flexible films. It is bioactive, non-toxic, and suited to biomedical applications such as pharmaceutical products like films, pearls or spheres, and gels, powders, etc. Previous works in cellulose-chitosan nanocomposites showed good results in physical, mechanical and biological tests [17, 18].

Chitosan can form flexible, clean and hard films [19] with a good oxygen barrier [20]. Further‐ more, it can be used as packing material, mainly as a covering and edible film [21] extending the average life of foods [22, 23]. Chitosan can also form a semipermeable covering to modify the inner atmosphere, thereby reducing the transpiration rate of the product in the packaging [24]. Despite good results with respect to their mechanical properties, chitosan films can be brittle, making it necessary to use plasticizers to increase their flexibility [25]. Plasticizers such as glycerol can improve the processability, as well as the mechanical properties of chitosan [26]. In another study, researchers reported a concentration of 20% (w/w) of glycerol as the appropriate concentration to improve the flexibility of chitosan films [27]. To prevent this drawback of rigidity and brittleness of chitosan, the addition of reinforcement has showed to be useful in enhancing its mechanical, thermal and barrier properties. When the particles are smaller, the interaction with the matrix is better [28], with the low cost a sign of efficiency [29]. Fillers on a nanometric scale (called nanoparticles or nanoreinforcements) with good disper‐ sion drive an interface matrix / filler, changing the molecular mobility, relaxation behavior and thermal and mechanical properties of the material [30].

A positive result in the elaboration of chitosan – CNC nanocomposites was obtained, using electrospinning for fibers of a derivative of chitosan / cellulose in an ionic liquid (IL). The chitosan / cellulose composite were electrospun in the ethanol co-solvent, using the IL to dissolve the chitosan and cellulose at the same time. Furthermore, the IL was capable of building fibers of pure chitosan / cellulose composite after the IL was removed by the ethanol. The fibers of this composite were manufactured as a three-dimensional shape, offering antibacterial activity to treat burns, bedsores and skin ulcers [31].

Nanofibers were obtained from chitosan and cellulose, with chitin used as a reinforcement material at different concentrations (from 1.25% to 5.0% w/w). This allowed the optimizing of the process conditions to obtain homogeneous and porous nanofibers. This material has a potential for use in wound bandages and skin burns [32].

Layer-by-Layer technique (LbL) is a technique to elaborate nanocomposites of chitosan and cellulose whiskers. The interactions between amine groups (chitosan) and sulfate groups (cellulose whiskers) ensure the linkages between matrix and nanoreinforcement to elaborate the films. The average thickness of each bi-layer (whiskers / chitosan) was 7 nm and each film was formed by 30 bi-layers. These materials have a wide range of uses such as packaging and biomedicals [15].

Chitosan films used for cell scaffolding in the regeneration of the tympanic membrane (type I experimental tympanoplasty) were elaborated. These films were grafted in New Zealand rabbits with successful results of tympanic tissue regeneration [33].

### **2. Objective**

Demonstrate that CNC can improve the mechanical and physical properties of a chitosan matrix. Furthermore, determine the biocompatibility and biodegradability of CS and CNC nanocomposites via biological tests and then based on the results obtained, determine a potential use of these nanocomposites in the biomedical area.

### **3. Experimental**

### **3.1. Materials**

Materials used for this study were Biomedical Grade chitosan from Sigma Aldrich (Deacety‐ lation grade 75–85%), acetic acid, alpha cellulose (Neucel Cellulose Ltd.), male laboratory rats (Wistar), cellulose acetate membranes for dialysis, vacuum oven, high resolution microscopy, dynamical-mechanical and chemical analysis. The formulations employed in this research are presented in Table 1.


**Table 1.** Formulations of CS + CNC films

### **3.2. Methodology**

Fillers on a nanometric scale (called nanoparticles or nanoreinforcements) with good disper‐ sion drive an interface matrix / filler, changing the molecular mobility, relaxation behavior and

A positive result in the elaboration of chitosan – CNC nanocomposites was obtained, using electrospinning for fibers of a derivative of chitosan / cellulose in an ionic liquid (IL). The chitosan / cellulose composite were electrospun in the ethanol co-solvent, using the IL to dissolve the chitosan and cellulose at the same time. Furthermore, the IL was capable of building fibers of pure chitosan / cellulose composite after the IL was removed by the ethanol. The fibers of this composite were manufactured as a three-dimensional shape, offering

Nanofibers were obtained from chitosan and cellulose, with chitin used as a reinforcement material at different concentrations (from 1.25% to 5.0% w/w). This allowed the optimizing of the process conditions to obtain homogeneous and porous nanofibers. This material has a

Layer-by-Layer technique (LbL) is a technique to elaborate nanocomposites of chitosan and cellulose whiskers. The interactions between amine groups (chitosan) and sulfate groups (cellulose whiskers) ensure the linkages between matrix and nanoreinforcement to elaborate the films. The average thickness of each bi-layer (whiskers / chitosan) was 7 nm and each film was formed by 30 bi-layers. These materials have a wide range of uses such as packaging and

Chitosan films used for cell scaffolding in the regeneration of the tympanic membrane (type I experimental tympanoplasty) were elaborated. These films were grafted in New Zealand

Demonstrate that CNC can improve the mechanical and physical properties of a chitosan matrix. Furthermore, determine the biocompatibility and biodegradability of CS and CNC nanocomposites via biological tests and then based on the results obtained, determine a

Materials used for this study were Biomedical Grade chitosan from Sigma Aldrich (Deacety‐ lation grade 75–85%), acetic acid, alpha cellulose (Neucel Cellulose Ltd.), male laboratory rats (Wistar), cellulose acetate membranes for dialysis, vacuum oven, high resolution microscopy, dynamical-mechanical and chemical analysis. The formulations employed in this research are

thermal and mechanical properties of the material [30].

234 Cellulose - Fundamental Aspects and Current Trends

antibacterial activity to treat burns, bedsores and skin ulcers [31].

rabbits with successful results of tympanic tissue regeneration [33].

potential use of these nanocomposites in the biomedical area.

potential for use in wound bandages and skin burns [32].

biomedicals [15].

**2. Objective**

**3. Experimental**

presented in Table 1.

**3.1. Materials**

### **•** Preparation of CNC

Alpha cellulose was ground and mixed with sulfuric acid (64% concentration) for one hour under constant stirring and at a controlled temperature (approximately 50° C in a warm bath). After that, the liquor was added to deionized water, cooled at 8° C 1:10 (v/v) to stop the reaction. The liquor was centrifuged at 4000 RPM for 5 minutes, separating the liquor into two phases: solid (cellulose gel in the bottom of recipient) and liquid (with acid remainders). The liquid phase was disposed of and deionized water was added to the recipient to remove excess acid from the gel (containing CNC), prior to centrifugations (three in total). The washed gel was put in dialysis membranes in deionized water under stirring until it reached a pH of 5. After that, the CNC were submitted to ultrasound treatment for 2 minutes and finally vacuum filtered using 0.45 micron Wharton paper, and kept cooled.

**•** Chitosan – CNC films elaboration

Chitosan (4 g) was dissolved in acetic acid at 2% (v/v) per each formulation, under constant stirring for 2 hours. Next, CNC in different concentrations were added (0.5%, 1.0% and 2.0%) and then stirred for two more hours. The substance was cast on Petri plates affording a concentration of 0.4 mL/cm2 to obtain the same quantity of nanocomposite on the plates. The plates were then put into a vacuum oven (28°C–70 MPa) for 96 hours. After drying, NaOH (1.0 N) was added to the Petri dishes to precipitate the films and then the CS + CNC films were washed with deionized water until they reached a pH of 7. Finally, films were dried at room temperature for 48 hours. The thickness of films was measured with a micrometer and dimensioned for physical and mechanical testing.

**•** Physical evaluation

When performing swelling tests, modifications in the dimensions of specimens were made to evaluate swelling changes over time. The dry weight (Wi ) of each specimen was taken and then films of each formulation were put in deionized water to control the weight each minute until a constant value was achieved [34]. For maximum water retention (MWR), the dry weight (Wi ) of each specimen (8 per formulation) was taken and it was then added to deionized water. Weights were controlled at 30, 60, and 120 minutes, and then 24 hours, before the final weight was obtained (Wf ) [34]. Finally, the MWR was determined by the following formula:

$$\text{MWR} \left( \% \right) = \left( \text{W}\_{\text{f}} - \text{W}\_{\text{i}} \right) / \left( \text{W}\_{\text{i}} \right) \times 100 \text{ J}$$

**•** Mechanical evaluation

Dynamical mechanical analysis was used in static mode to evaluate Young's modulus for all formulations, with the stress-strain test operating in the controlled force mode. Typical testing conditions were 0.1 N preload, 1 N/min ramp and a gauge length of ca. 10 mm. Strips of 0.075 mm x 5 mm x 20 mm were used. The temperature range was 37.05 ± 0.05°C and the moisture content was 98%, determined by a TA Instruments DMA 800 used in wet conditions.

**•** Biological evaluation

The best way to test our material was in living specimens. Biocompatibility and biodegradabil‐ ity tests were carried out. For these, the dry weight (Wi ) of films was controlled. The biologi‐ cal subjects for testing were 16 Lab rats (of the Wistar breed). The animals were submitted to a surgical procedure involving grafting two portions of films for each formulation (4 rats per formulation). Each animal had two sub-cutaneous cuts (in the middle of back, on the right side for biocompatibility and on the left side for biodegradability). Every three days, the rats were controlled to prevent any infection or adverse reaction to the nanomaterial. Specimens were euthanized 30 days after surgery and the two portions of membrane were retired, lyophilized and controlled for dry weight (Wf ) in order to obtain the biodegradability value:

Biodegradability (%) = (Wi – Wf ) / (Wi ) x 100

Biocompatibility was proved by SEM images at the moment the grafts were retired, the mortality rate of specimens and the non-presence of encapsulation, fibrillation or any rotten portion of membrane after 30 days.

**•** Characterization of nanocomposites

To characterize the morphology of films, the response and the different levels of biocompati‐ bility and biodegradability, a JEOL JM 6300 with a double gold layer to avoid the electrical charge of the sample was used. The magnification was from around 500x up to 35,000x and the voltage employed was between 7kV and 20kV.

**•** IR spectroscopy

This technique was used to determine changes in functional groups of nanocomposite as a consequence of the presence of CNC in the chitosan matrix, or previous chemical treatment. Portions of thin films of all formulations were analyzed in an IP Spectrometer in transmittance mode (4,000 to 500 cm-1). Other parameters were: 16 scans per spectrum, ATR mode and a resolution of 0.4 cm-1. A Perkin Elmer Spectrum GX FT-IR System was used.

### **4. Results**

### **4.1. CNC + chitosan films**

The films obtained from the combination of chitosan and CNC showed a transparent aspect with a slight yellow color. Transparence in the films suggests a good distribution of CNC in the CS matrix. At the same time, the slight yellow color is due to the natural presence of Biocompatibility was proved by SEM images at the moment the grafts were retired, the mortality rate of specimens and the non‐presence of encapsulation, fibrillation or any rotten portion of

To characterize the morphology of films, the response and the different levels of biocompatibility and biodegradability, a JEOL JM 6300 with a double gold layer to avoid the electrical charge of the sample was used. The magnification was from around 500x up to 35,000x and the voltage

This technique was used to determine changes in functional groups of nanocomposite as a consequence of the presence of CNC in the chitosan matrix, or previous chemical treatment. Portions of thin films of all formulations were analyzed in an IP Spectrometer in transmittance mode (4,000 to 500 cm‐1). Other parameters were: 16 scans per spectrum, ATR mode and a

impurities in this biopolymer [35]. The average thickness of films was 30 microns and the surface was smooth. Visible CNC agglomerates and dark points were absent from these films as can be seen in Figure 1. The films obtained from the combination of chitosan and CNC showed a transparent aspect with a slight yellow color. Transparence in the films suggests a good distribution of CNC in the CS matrix. At the same time, the slight yellow color is due to the natural presence of impurities in this biopolymer [35]. The

resolution of 0.4 cm‐1. A Perkin Elmer Spectrum GX FT‐IR System was used.

These characteristics in all formulations of pure CS and CS + CNC were the same, and accorded with findings of other authors [19, 21, 35, 36]. Even with CNC concentrations over 20% (w/w), transparent films were reported, although with CNC concentrations over 60% the films showed a translucent appearance (a greyish color) and a rigid but brittle consistency [35]. Chitosan films and polyethylene oxide (PEO) employing electrospinning produced good results in transparence and homogeneity [32]. average thickness of films was 30 microns and the surface was smooth. Visible CNC agglomerates and dark points were absent from these films as can be seen in Figure 1. These characteristics in all formulations of pure CS and CS + CNC were the same, and accorded with findings of other authors [19, 21, 35, 36]. Even with CNC concentrations over 20% (w/w), transparent films were reported, although with CNC concentrations over 60% the films showed a translucent appearance (a greyish color) and a rigid but brittle consistency [35]. Chitosan films and polyethylene oxide (PEO)

employing electrospinning produced good results in transparence and homogeneity [32].

Fig. 1a. Film of CS + CNC (0.5%). 1b. Film of CS + CNC (1.0%) and 1c. Film of CS + CNC (2.0%)

SEM images showed a good dispersion of CNC in the nanocomposites due to the affinity between the

**Figure 1.** a. Film of CS + CNC (0.5%). b. Film of CS + CNC (1.0%) and c. Film of CS + CNC (2.0%)

#### **3.2 Morphology of CS + CNC films 4.2. Morphology of CS + CNC films**

membrane after 30 days.


**3.1 CNC + chitosan films**


employed was between 7kV and 20kV.

**•** Mechanical evaluation

236 Cellulose - Fundamental Aspects and Current Trends

**•** Biological evaluation

and controlled for dry weight (Wf

portion of membrane after 30 days.

**•** Characterization of nanocomposites

the voltage employed was between 7kV and 20kV.

Biodegradability (%) = (Wi

**•** IR spectroscopy

**4. Results**

**4.1. CNC + chitosan films**

Dynamical mechanical analysis was used in static mode to evaluate Young's modulus for all formulations, with the stress-strain test operating in the controlled force mode. Typical testing conditions were 0.1 N preload, 1 N/min ramp and a gauge length of ca. 10 mm. Strips of 0.075 mm x 5 mm x 20 mm were used. The temperature range was 37.05 ± 0.05°C and the moisture

The best way to test our material was in living specimens. Biocompatibility and biodegradabil‐

cal subjects for testing were 16 Lab rats (of the Wistar breed). The animals were submitted to a surgical procedure involving grafting two portions of films for each formulation (4 rats per formulation). Each animal had two sub-cutaneous cuts (in the middle of back, on the right side for biocompatibility and on the left side for biodegradability). Every three days, the rats were controlled to prevent any infection or adverse reaction to the nanomaterial. Specimens were euthanized 30 days after surgery and the two portions of membrane were retired, lyophilized

Biocompatibility was proved by SEM images at the moment the grafts were retired, the mortality rate of specimens and the non-presence of encapsulation, fibrillation or any rotten

To characterize the morphology of films, the response and the different levels of biocompati‐ bility and biodegradability, a JEOL JM 6300 with a double gold layer to avoid the electrical charge of the sample was used. The magnification was from around 500x up to 35,000x and

This technique was used to determine changes in functional groups of nanocomposite as a consequence of the presence of CNC in the chitosan matrix, or previous chemical treatment. Portions of thin films of all formulations were analyzed in an IP Spectrometer in transmittance mode (4,000 to 500 cm-1). Other parameters were: 16 scans per spectrum, ATR mode and a

The films obtained from the combination of chitosan and CNC showed a transparent aspect with a slight yellow color. Transparence in the films suggests a good distribution of CNC in the CS matrix. At the same time, the slight yellow color is due to the natural presence of

resolution of 0.4 cm-1. A Perkin Elmer Spectrum GX FT-IR System was used.

) in order to obtain the biodegradability value:

) of films was controlled. The biologi‐

content was 98%, determined by a TA Instruments DMA 800 used in wet conditions.

ity tests were carried out. For these, the dry weight (Wi

– Wf

) / (Wi

) x 100

nanoreinforcement and the matrix (having similar chemical structure and hydrophilic nature), as well as a good interaction of negative charges of the sulfate groups of CNC with the amine groups of CS [35]. The SEM images showed a good dispersion of CNC in the nanocomposites due to the affinity between the nanoreinforcement and the matrix (having similar chemical structure and hydrophilic nature), as well as a good interaction of negative charges of the sulfate groups of CNC with the amine groups of CS [35]. The presence of nanoparticles was observed in different formulations of nanocomposites, as can be seen in Figure 2. These bundles formed by several tens of CNC, with dimensions of between 150–200 nm up to 500 nm on average, and in some cases up to 1 or 2 microns, mainly in CS + CNC (2.0%). The phenomenon was common when the CNC concentration was higher (over 5% and 10%) and even when working with concen‐ trations of 5% and 10% it was possible to see white punctuations in the matrix surface via SEM images, seen in the cross section of CNC [36]. Another possible phenomenon linked to a higher concentration of CNC in the matrix, is the formation of a polyelectrolyte macroion complex between CS and CNC. The size of these complexes could reach several microns in length. These particles are formed by CNC and surrounded by CS chains. The shape of these complexes depends on the quantity of NH2 in CS. When the concentration of NH2 is high, the shape of the complex tends to be spherical [37]. These particles could have potential in biomedicals for drug transportation and controlled release due to the charges on cellulose and the linkage that can be achieved with other substances and active composites.

**3.3 Physical Properties**

CNC and surrounded by CS chains. The shape of these complexes depends on the quantity of NH2 in CS.

could have potential in biomedicals for drug transportation and controlled release due to the charges on

cellulose and the linkage that can be achieved with other substances and active composites.

clusters of CS + CNC (0.5%) film at 8,500x (circled in red), 2d. SEM image of clusters of CS + CNC (0.5%) at 35,000x (circled in red). 2e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC (2.0%) at 15,000x. **Figure 2.** a SEM image of a cross section of CS + CNC (1.0%) film at 950x. b. SEM image of CS film at 4,300x. c. SEM image of clusters of CS + CNC (0.5%) film at 8,500x (circled in red), d. SEM image of clusters of CS + CNC (0.5%) at 35,000x (circled in red). e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC (2.0%) at 15,000x.

Fig. 2a. SEM image of a cross section of CS + CNC (1.0%) film at 950x. 2b. SEM image of CS film at 4,300x. 2c. SEM image of

Swelling capability is presented in Figure 3. Swelling capability showed an evident change with the presence of CNC in the matrix. For water swelling, pure CS films showed saturation times of 3–4 minutes. With formulations of CS + CNC (0.5%) saturation times reduced to 1–2 minutes, while for formulations with CNC of 1% and 2%, the saturation time was 1 minute. These results show that CNC allow water entrance between CS chains as an unstructuring element, making the material more hydrophilic. This

### **4.3. Physical properties**

as presented in Table 2.

 **b)**

**d)**

CNC and surrounded by CS chains. The shape of these complexes depends on the quantity of NH2 in CS. When the concentration of NH2 is high, the shape of the complex tends to be spherical [37]. These particles could have potential in biomedicals for drug transportation and controlled release due to the charges on

Fig. 2a. SEM image of a cross section of CS + CNC (1.0%) film at 950x. 2b. SEM image of CS film at 4,300x. 2c. SEM image of clusters of CS + CNC (0.5%) film at 8,500x (circled in red), 2d. SEM image of clusters of CS + CNC (0.5%) at 35,000x (circled in red). 2e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC (2.0%) at 15,000x.

**Figure 2.** a SEM image of a cross section of CS + CNC (1.0%) film at 950x. b. SEM image of CS film at 4,300x. c. SEM image of clusters of CS + CNC (0.5%) film at 8,500x (circled in red), d. SEM image of clusters of CS + CNC (0.5%) at

35,000x (circled in red). e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC (2.0%) at 15,000x.

**e) f)**

Swelling capability is presented in Figure 3. Swelling capability showed an evident change with the presence of CNC in the matrix. For water swelling, pure CS films showed saturation times of 3–4 minutes. With formulations of CS + CNC (0.5%) saturation times reduced to 1–2 minutes, while for formulations with CNC of 1% and 2%, the saturation time was 1 minute. These results show that CNC allow water entrance between CS chains as an unstructuring element, making the material more hydrophilic. This

**3.3 Physical Properties**

cellulose and the linkage that can be achieved with other substances and active composites.

238 Cellulose - Fundamental Aspects and Current Trends

**a)**

**c)**

Swelling capability is presented in Figure 3. Swelling capability showed an evident change with the presence of CNC in the matrix. For water swelling, pure CS films showed saturation times of 3–4 minutes. With formulations of CS + CNC (0.5%) saturation times reduced to 1–2 minutes, while for formulations with CNC of 1% and 2%, the saturation time was 1 minute. These results show that CNC allow water entrance between CS chains as an unstructuring element, making the material more hydrophilic. This behavior was opposite to that observed in another study, which used higher concentrations of CNC (from 5% up to 60%) making the CS films less permeable, thereby reducing the swelling capability significantly [35, 36, 38]. The maximum water retention (MWR) showed a similar response with the presence of CNC, as presented in Table 2. Fig. 2a. SEM image of a cross section of CS + CNC (1.0%) film at 950x. 2b. SEM image of CS film at 4,300x. 2c. SEM image of clusters of CS + CNC (0.5%) film at 8,500x (circled in red), 2d. SEM image of clusters of CS + CNC (0.5%) at 35,000x (circled in red). 2e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC (2.0%) at 15,000x. **3.3 Physical Properties** Swelling capability is presented in Figure 3. Swelling capability showed an evident change with the presence of CNC in the matrix. For water swelling, pure CS films showed saturation times of 3–4 minutes. With formulations of CS + CNC (0.5%) saturation times reduced to 1–2 minutes, while for formulations with CNC of 1% and 2%, the saturation time was 1 minute. These results show that CNC allow water entrance between CS chains as an unstructuring element, making the material more hydrophilic. This behavior was opposite to that observed in another study, which used higher concentrations of CNC (from 5% up to 60%) making the CS films less permeable, thereby reducing the swelling capability significantly [35, 36, 38]. The maximum water retention (MWR) showed a similar response with the presence of CNC,

CNC and surrounded by CS chains. The shape of these complexes depends on the quantity of NH2 in CS. When the concentration of NH2 is high, the shape of the complex tends to be spherical [37]. These particles could have potential in biomedicals for drug transportation and controlled release due to the charges on

cellulose and the linkage that can be achieved with other substances and active composites.

Fig. 3 Swelling capability for CS + CNC nanocomposites **Figure 3.** Swelling capability for CS + CNC nanocomposites


Table 2 Values of MWR for CS + CNC nanocomposites

**Table 2.** Values of MWR for CS + CNC nanocomposites

For pure CS films, the MWR was 187% of its own weight in water, while CS + CNC (0.5%) films obtained values of 226%, CS + CNC (1.0%) values of 258% and CS + CNC (2.0%) values of 214%, as can be seen in Figure 4. In these cases, the low concentrations of CNC improve the water retention properties of films in comparison with results of other authors [15, 35] which showed a remarkable reduction in the physical properties of CS + CNC films, using concentrations from 5% up to 60%. This phenomenon can be explained by the affinity of CNC to the water, due their hydrophilic nature (and O–H linkages). Another reason is the plasticizing effect of CNC at low concentrations of CS matrix, contributing to the unstructuring effect in CS chains allowing the passage of water inside the polymer. This effect makes the film more flexible, facilitating the entry of water into the polymer chains. A similar effect was reported when adding different concentrations of glycerol (from 2% to 6%), used as a plasticizer in starch films with CS covering. Even at the highest CS concentration, the presence of glycerol increased this parameter, with values from 71.8% to 125.4% in comparison with pure CS (35.7% to 74.8%) [21]. CS covering. Even at the highest CS concentration, the presence of glycerol increased this parameter, with values from 71.8% to 125.4% in comparison with pure CS (35.7% to 74.8%) [21].

Fig. 4 Values (%) of maximum water retention for pure CS and CS + CNC films

The values obtained from formulations with CNC suggest that these modified the mechanical properties

**Figure 4.** Values (%) of maximum water retention for pure CS and CS + CNC films

#### **3.4 Mechanical properties 4.4. Mechanical properties**

deformation or rupture of films.

in comparison with pure CS. Young's modulus (YM) reached by pure CS was 2,916 ± 933 MPa, similar to that observed in another study [35], while for formulations with CNC the values were CS + CNC (0.5%) 1,575 ± 82 MPa, CS + CNC (1.0%) 1690 ± 433 MPa and for CS + CNC (2.0%) the value was 1657 ± 4 MPa, as can be seen in Figure 5. There was a large variation in the results for YM in pure CS, with values from 1,080 MPa up to 2,852 MPa. The average for pure CS ranged between 2,300 and 2,500 MPa. This variation is because the matrix polymer has crystalline and amorphous zones, which will exhibit different resistances (high and low values). Furthermore, residual efforts stored in films as a consequence of some conditions in their elaboration process (casting solution), appears in the first stages of assay, when the machine break these efforts first before apply all the force in the film in a homogeneous way. The graphs showed an inflexion point in effort–strain curves. This phenomenon was present in some films of CS + CNC (1.0%) and CS + CNC (2.0%). Some explanations for the decrease of Young's Modulus in the films with CNC suggest the cause as the formation of aggregates and CS–CNC complexes due to the agglomeration of nanoparticles in one single The values obtained from formulations with CNC suggest that these modified the mechanical properties in comparison with pure CS. Young's modulus (YM) reached by pure CS was 2,916 ± 933 MPa, similar to that observed in another study [35], while for formulations with CNC the values were CS + CNC (0.5%) 1,575 ± 82 MPa, CS + CNC (1.0%) 1690 ± 433 MPa and for CS + CNC (2.0%) the value was 1657 ± 4 MPa, as can be seen in Figure 5. There was a large variation in the results for YM in pure CS, with values from 1,080 MPa up to 2,852 MPa. The average for pure CS ranged between 2,300 and 2,500 MPa. This variation is because the matrix polymer has crystalline and amorphous zones, which will exhibit different resistances (high and low values). Furthermore, residual efforts stored in films as a consequence of some conditions in their elaboration process (casting solution), appears in the first stages of assay, when the machine break these efforts first before apply all the force in the film in a homogeneous way.

place, generating stresses around them and becoming a potential weak point in the film. It could be deduced that CNC acted as a plasticizer of the matrix, making it less rigid. However, CS + CNC formulations showed values lower than those obtained for pure CS. However, these values were less variable than pure CS values and a further detail was observed: CNC aggregation didn't contribute to the

Another possible cause of the observed decrease in YM could be the low concentrations of CNC employed in this study (0.5%, 1.0% and 2.0%) in comparison with other studies that used concentrations above 5%, 10% until 60% [15, 35] that saw improvements in YM of 78% to 150% and 230% to 320% respectively. The decrease in YM must be understood as a loss of stiffness, making a more flexible material, but with low strain values (1–2%). This last can be improved if plasticizers (i.e. glycerol) are included in the formulations. The values obtained are similar with CS films with chitin concentrations of 2.7% to 37.5%,

The graphs showed an inflexion point in effort–strain curves. This phenomenon was present in some films of CS + CNC (1.0%) and CS + CNC (2.0%).

Some explanations for the decrease of Young's Modulus in the films with CNC suggest the cause as the formation of aggregates and CS–CNC complexes due to the agglomeration of nanoparticles in one single place, generating stresses around them and becoming a potential weak point in the film. It could be deduced that CNC acted as a plasticizer of the matrix, making it less rigid. However, CS + CNC formulations showed values lower than those obtained for pure CS. However, these values were less variable than pure CS values and a further detail was observed: CNC aggregation didn't contribute to the deformation or rupture of films.

Another possible cause of the observed decrease in YM could be the low concentrations of CNC employed in this study (0.5%, 1.0% and 2.0%) in comparison with other studies that used concentrations above 5%, 10% until 60% [15, 35] that saw improvements in YM of 78% to 150% and 230% to 320% respectively. The decrease in YM must be understood as a loss of stiffness, making a more flexible material, but with low strain values (1–2%). This last can be improved if plasticizers (i.e. glycerol) are included in the formulations. The values obtained are similar with CS films with chitin concentrations of 2.7% to 37.5%, having values from 1,622 ± 377 MPa to 2,318 ±619 MPa respectively [11]. Based on these values, a potential use of these films could be in healing and wound recovery, as well as scar prevention.

**Figure 5.** Young's Modulus values of pure CS and CS + CNC films

3360 3295

#### **3.5 IR Spectroscopy of CS + CNC films** As can be seen in Figure 6, for pure CS the IR spectrum showed bands at 3,360 and 3,265 cm‐<sup>1</sup> typical of O– **4.5. IR Spectroscopy of CS + CNC films**

retention properties of films in comparison with results of other authors [15, 35] which showed a remarkable reduction in the physical properties of CS + CNC films, using concentrations from 5% up to 60%. This phenomenon can be explained by the affinity of CNC to the water, due their hydrophilic nature (and O–H linkages). Another reason is the plasticizing effect of CNC at low concentrations of CS matrix, contributing to the unstructuring effect in CS chains allowing the passage of water inside the polymer. This effect makes the film more flexible, facilitating the entry of water into the polymer chains. A similar effect was reported when adding different concentrations of glycerol (from 2% to 6%), used as a plasticizer in starch films with CS covering. Even at the highest CS concentration, the presence of glycerol increased this parameter, with values from 71.8% to 125.4% in comparison with pure CS (35.7% to 74.8%) [21]. CS covering. Even at the highest CS concentration, the presence of glycerol increased this parameter, with

Fig. 4 Values (%) of maximum water retention for pure CS and CS + CNC films

S.D. ± 7.78

Variation in time W30min Variation in time W120min Variation in time W24h

Pure CS CS + CNC (0.5%) CS + CNC (1.0%) CS + CNC (2.0%)

S.D. ± 46.38

S.D. ± 12.18

The values obtained from formulations with CNC suggest that these modified the mechanical properties in comparison with pure CS. Young's modulus (YM) reached by pure CS was 2,916 ± 933 MPa, similar to that observed in another study [35], while for formulations with CNC the values were CS + CNC (0.5%) 1,575 ± 82 MPa, CS + CNC (1.0%) 1690 ± 433 MPa and for CS + CNC (2.0%) the value was 1657 ± 4 MPa, as can be seen in Figure 5. There was a large variation in the results for YM in pure CS, with values from 1,080 MPa up to 2,852 MPa. The average for pure CS ranged between 2,300 and 2,500 MPa. This variation is because the matrix polymer has crystalline and amorphous zones, which will exhibit different resistances (high and low values). Furthermore, residual efforts stored in films as a consequence of some conditions in their elaboration process (casting solution), appears in the first stages of assay, when the machine break these efforts first before apply all the force in the film in a homogeneous way. The graphs showed an inflexion point in effort–strain curves. This phenomenon was present in some films of CS +

The values obtained from formulations with CNC suggest that these modified the mechanical properties in comparison with pure CS. Young's modulus (YM) reached by pure CS was 2,916 ± 933 MPa, similar to that observed in another study [35], while for formulations with CNC the values were CS + CNC (0.5%) 1,575 ± 82 MPa, CS + CNC (1.0%) 1690 ± 433 MPa and for CS + CNC (2.0%) the value was 1657 ± 4 MPa, as can be seen in Figure 5. There was a large variation in the results for YM in pure CS, with values from 1,080 MPa up to 2,852 MPa. The average for pure CS ranged between 2,300 and 2,500 MPa. This variation is because the matrix polymer has crystalline and amorphous zones, which will exhibit different resistances (high and low values). Furthermore, residual efforts stored in films as a consequence of some conditions in their elaboration process (casting solution), appears in the first stages of assay, when the machine break these efforts first before apply all the force in the film in a homogeneous way.

Some explanations for the decrease of Young's Modulus in the films with CNC suggest the cause as the formation of aggregates and CS–CNC complexes due to the agglomeration of nanoparticles in one single place, generating stresses around them and becoming a potential weak point in the film. It could be deduced that CNC acted as a plasticizer of the matrix, making it less rigid. However, CS + CNC formulations showed values lower than those obtained for pure CS. However, these values were less variable than pure CS values and a further detail was observed: CNC aggregation didn't contribute to the

Another possible cause of the observed decrease in YM could be the low concentrations of CNC employed in this study (0.5%, 1.0% and 2.0%) in comparison with other studies that used concentrations above 5%, 10% until 60% [15, 35] that saw improvements in YM of 78% to 150% and 230% to 320% respectively. The decrease in YM must be understood as a loss of stiffness, making a more flexible material, but with low strain values (1–2%). This last can be improved if plasticizers (i.e. glycerol) are included in the formulations. The values obtained are similar with CS films with chitin concentrations of 2.7% to 37.5%,

**3.4 Mechanical properties**

**4.4. Mechanical properties**

0.00

50.00

100.00

Max water retention (%)

150.00

200.00

S.D. ± 6.02

**Figure 4.** Values (%) of maximum water retention for pure CS and CS + CNC films

250.00

300.00

350.00

400.00

240 Cellulose - Fundamental Aspects and Current Trends

CNC (1.0%) and CS + CNC (2.0%).

deformation or rupture of films.

values from 71.8% to 125.4% in comparison with pure CS (35.7% to 74.8%) [21].

cm‐<sup>1</sup> is commonly seen with type I amides and C=O bonds linked to acetyl and amine groups, and 1,560 cm‐<sup>1</sup> corresponds to amide groups (N–H). Bands of 1,420 and 1,375 cm‐<sup>1</sup> correspond to residual CH3N– acetyl glucosamine and –CH2 groups respectively. Finally, bands of 1,060 and 1,030 cm‐<sup>1</sup> confirm the presence of C–O bonds. Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the As can be seen in Figure 6, for pure CS the IR spectrum showed bands at 3,360 and 3,265 cm-1 typical of O–H and N–H bonds respectively. The band at 2,870 cm-1 corresponds to C–H bonds and the signal at 1,650 cm-1 is commonly seen with type I amides and C=O bonds linked to acetyl and amine groups, and 1,560 cm-1 corresponds to amide groups (N–H). Bands of 1,420

similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar

<sup>1560</sup> <sup>1420</sup>

1375

1060

1030

phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38].

2870

H and N–H bonds respectively. The band at 2,870 cm‐<sup>1</sup> corresponds to C–H bonds and the signal at 1,650

Fig. 5 Young's Modulus values of pure CS and CS + CNC films

and 1,375 cm-1 correspond to residual CH3N–acetyl glucosamine and –CH2 groups respective‐ ly. Finally, bands of 1,060 and 1,030 cm-1 confirm the presence of C–O bonds.

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38]. Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38]. Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar

phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38].

Fig. 6 IR spectrum of pure CS film

Fig. 6 IR spectrum of pure CS film

**Figure 6.** IR spectrum of pure CS film

Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point

Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point

Fig. 7 IR spectrum of CS + CNC (2.0%) film

**3.6 Biological Testing 3.6 Biological Testing Figure 7.** IR spectrum of CS + CNC (2.0%) film

### **4.6. Biological testing**

prevent a different reaction or influence over the grafts.

and 1,375 cm-1 correspond to residual CH3N–acetyl glucosamine and –CH2 groups respective‐

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar phenomenon has been observed in chitin,

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar

Fig. 6 IR spectrum of pure CS film

Fig. 6 IR spectrum of pure CS film

1647

1647

1570 1380

1570 1380

1029

1029

<sup>1560</sup> <sup>1420</sup>

<sup>1560</sup> <sup>1420</sup>

1375

1375

1060

1060

1030

1030

Fig. 7 IR spectrum of CS + CNC (2.0%) film

Fig. 7 IR spectrum of CS + CNC (2.0%) film

Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point

Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point

ly. Finally, bands of 1,060 and 1,030 cm-1 confirm the presence of C–O bonds.

phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38].

phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38].

2870

2870

2874

2874

chitosan and glycerol nanocomposites [38].

242 Cellulose - Fundamental Aspects and Current Trends

3360 3295

3360 3295

3362

3362

**3.6 Biological Testing**

**Figure 7.** IR spectrum of CS + CNC (2.0%) film

**3.6 Biological Testing**

**Figure 6.** IR spectrum of pure CS film

Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point the grafts were retired, no negative influence was observed over the surface of muscles or sub-cutaneous tissues (no evidence of coagulation, fibrosis, and/or rotten or dead tissues), as can be seen in Figure 8. A further typical sign of CS in living tissues is the slight yellow color over the muscle or tissue. In a few cases rolled grafts were found, due mainly to movement of the specimen during the 30-day period. A further possible cause could be that the graft was not fixed with sutures or staples in the biological test, in order to prevent a different reaction or influence over the grafts. **3.6 Biological Testing** Biological results showed 100% biocompatibility for all formulations in 16 rats. After 30 days, the specimens had not shown an adverse reaction, septicemia or death in the presence of CNC. At the point the grafts were retired, no negative influence was observed over the surface of muscles or sub‐cutaneous tissues (no evidence of coagulation, fibrosis, and/or rotten or dead tissues), as can be seen in Figure 8. A further typical sign of CS in living tissues is the slight yellow color over the muscle or tissue. In a few cases rolled grafts were found, due mainly to movement of the specimen during the 30‐day period. A further possible cause could be that the graft was not fixed with sutures or staples in the biological test, in order to

Fig. 7 IR spectrum of CS + CNC (2.0%) film

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0% and 2.0% didn't show variations in their spectrums when compared with a pure CS spectrum, as shown in Figure 7. This is due to the similarity between cellulose and chitosan in terms of their chemical structure. For that reason, the bands for both polysaccharides are the same, except for the presence of an amine group with chitosan [15]. Another cause is the low concentration of CNC, which could be undetected by FTIR. A similar

phenomenon has been observed in chitin, chitosan and glycerol nanocomposites [38].

biodegradability in the specimen. The biodegradability values were partial but positive for all formulations, as presented in Table 3. In pure **Figure 8.** Surgical stage of retiring the portions of CS + CNC (0.5 %) (circled in blue), to evaluate biocompatibility and biodegradability in the specimen.

Fig. 8 Surgical stage of retiring the portions of CS + CNC (0.5 %) (circled in blue), to evaluate biocompatibility and

CS, the values reached between 6.21% and 8.55% in parallel. One sample had 100% biodegradability and another sample had more weight than the initial weight, suggesting the presence of new tissue as fascia and/or loose connective tissue over the graft (as presented in Figure 9), or cells or blood inside the membrane (biodegradability value: ‐12.89%). The CS + CNC (0.5%) had the most weight gain in their films in comparison with other formulations (‐13.63%, ‐12.64% and ‐55.83% respectively). Just one sample of this group had a low biodegradability value (7.82%). For CS + CNC (1.0%) the values reached 0.71% to 21.02%. The biodegradability values were partial but positive for all formulations, as presented in Table 3. In pure CS, the values reached between 6.21% and 8.55% in parallel. One sample had 100% biodegradability and another sample had more weight than the initial weight, suggesting the presence of new tissue as fascia and/or loose connective tissue over the graft (as presented in Figure 9), or cells or blood inside the membrane (biodegradability value: -12.89%). The CS + CNC (0.5%) had the most weight gain in their films in comparison with other formulations (-13.63%, -12.64% and -55.83% respectively). Just one sample of this group had a low biode‐ gradability value (7.82%). For CS + CNC (1.0%) the values reached 0.71% to 21.02%. The highest values of biodegradability were shown by CS + CNC (2.0%) being 19.59% to 60.37% respec‐ tively) with just one specimen that gained weight.

Fig. 9 Presence of loose connective tissue and blood vessels (circled in blue) formed over the portion of CS + CNC (1.0%) **Figure 9.** Presence of loose connective tissue and blood vessels (circled in blue) formed over the portion of CS + CNC (1.0%)


Specimen 3 100.00 7.82 21.02 **‐4.92**

Specimen 4 6.21 **‐55.83** 5.52 40.52 **Table 3.** Biodegradability values for CS + CNC nanocomposites

Indeed, a relationship between the concentration of CNC and higher values of biodegradability can be deduced. The unstructuring effect of CNC in CS chains allows the inflow of water, cells and living fluids in the membrane. This relationship is important because it informs predictions and design of future research into different presentations of nanocomposites with higher concentrations of CNC, in order to study control of the rate of biodegradation for specific biomedical uses (depending on the part or organ in the body to be used). Indeed, a relationship between the concentration of CNC and higher values of biodegradability can be deduced. The unstructuring effect of CNC in CS chains allows the inflow of water, cells and living fluids in the membrane. This relationship is important because it informs predic‐ tions and design of future research into different presentations of nanocomposites with higher concentrations of CNC, in order to study control of the rate of biodegradation for specific biomedical uses (depending on the part or organ in the body to be used).

The presence of loose connective tissue and fascia were evident for all formulations, as well as the formation of small blood vessels surrounding the graft. Based on the principle of time of biodegradability of new tissue, these films presented different values after 30 days. For example, CS + CNC (0.5%) showed the best results for new scaffold tissue in three formulations. In the other cases, we can estimate the total time for biodegradation to be 3–6 months (less time with higher concentrations of CNC based on these results). This could be useful in the future development of possible biomedical uses of these films. Another aspect to consider with respect to biodegradation values is the type of films. In this study, non The presence of loose connective tissue and fascia were evident for all formulations, as well as the formation of small blood vessels surrounding the graft. Based on the principle of time of biodegradability of new tissue, these films presented different values after 30 days. For example, CS + CNC (0.5%) showed the best results for new scaffold tissue in three formulations. In the other cases, we can estimate the total time for biodegradation to be 3–6 months (less time with higher concentrations of CNC based on these results). This could be useful in the future development of possible biomedical uses of these films.

porous films were elaborated. The porosity rate and the size of pores directly affect the biodegradability of films, because they allow the easy passage of water, blood, cells and other fluids. From their condition, these films could be useful for tissue scaffolding as can be seen in Figures 10 and 11 (SEM images). A typical pattern of degradation of chitosan shown as hexagonal borders was observed in all formulations, at

different stages. The formation of fascia in different stages was evident.

Biodegradability values (%) Pure CS CS+CNC (0.5%) CS+CNC (1.0%) CS+CNC (2.0%)

Specimen 1 **‐12.89 ‐13.48** 8.33 19.59 Specimen 2 8.55 **‐12.64** 0.71 60.37 Specimen 3 100.00 7.82 21.02 **‐4.92** Specimen 4 6.21 **‐55.83** 5.52 40.52

Indeed, a relationship between the concentration of CNC and higher values of biodegradability can be deduced. The unstructuring effect of CNC in CS chains allows the inflow of water, cells and living fluids in the membrane. This relationship is important because it informs predictions and design of future research into different presentations of nanocomposites with higher concentrations of CNC, in order to study control of the rate of biodegradation for specific biomedical uses (depending on the part or organ in

The presence of loose connective tissue and fascia were evident for all formulations, as well as the formation of small blood vessels surrounding the graft. Based on the principle of time of biodegradability

time for biodegradation to be 3–6 months (less time with higher concentrations of CNC based on these

Another aspect to consider with respect to biodegradation values is the type of films. In this study, non porous films were elaborated. The porosity rate and the size of pores directly affect the biodegradability of films, because they allow the easy passage of water, blood, cells and other fluids. From their condition, these films could be useful for tissue scaffolding as can be seen in Figures 10 and 11 (SEM images). A typical pattern of degradation of chitosan shown as hexagonal borders was observed in all formulations, at different stages. The formation of fascia in different stages was evident. results). This could be useful in the future development of possible biomedical uses of these films. Another aspect to consider with respect to biodegradation values is the type of films. In this study, non porous films were elaborated. The porosity rate and the size of pores directly affect the biodegradability of films, because they allow the easy passage of water, blood, cells and other fluids. From their condition, these films could be useful for tissue scaffolding as can be seen in Figures 10 and 11 (SEM images). A typical pattern of degradation of chitosan shown as hexagonal borders was observed in all formulations, at different stages. The formation of fascia in different stages was evident.

the body to be used).

Fig. 9 Presence of loose connective tissue and blood vessels (circled in blue) formed over the portion of CS + CNC (1.0%)

**Figure 9.** Presence of loose connective tissue and blood vessels (circled in blue) formed over the portion of CS + CNC

Table 3 Biodegradability values for CS + CNC nanocomposites

Specimen 1 **-12.89 -13.48** 8.33 19.59 Specimen 2 8.55 **-12.64** 0.71 60.37 Specimen 3 100.00 7.82 21.02 **-4.92** Specimen 4 6.21 **-55.83** 5.52 40.52

Specimen 1 **‐12.89 ‐13.48** 8.33 19.59 Specimen 2 8.55 **‐12.64** 0.71 60.37 Specimen 3 100.00 7.82 21.02 **‐4.92** Specimen 4 6.21 **‐55.83** 5.52 40.52

Indeed, a relationship between the concentration of CNC and higher values of biodegradability can be deduced. The unstructuring effect of CNC in CS chains allows the inflow of water, cells and living fluids in the membrane. This relationship is important because it informs predictions and design of future research into different presentations of nanocomposites with higher concentrations of CNC, in order to study control of the rate of biodegradation for specific biomedical uses (depending on the part or organ in

Indeed, a relationship between the concentration of CNC and higher values of biodegradability can be deduced. The unstructuring effect of CNC in CS chains allows the inflow of water, cells and living fluids in the membrane. This relationship is important because it informs predic‐ tions and design of future research into different presentations of nanocomposites with higher concentrations of CNC, in order to study control of the rate of biodegradation for specific

The presence of loose connective tissue and fascia were evident for all formulations, as well as the formation of small blood vessels surrounding the graft. Based on the principle of time of biodegradability of new tissue, these films presented different values after 30 days. For example, CS + CNC (0.5%) showed the best results for new scaffold tissue in three formulations. In the other cases, we can estimate the total time for biodegradation to be 3–6 months (less time with higher concentrations of CNC based on these results). This could be useful in the future development of possible biomedical uses of these films.

The presence of loose connective tissue and fascia were evident for all formulations, as well as the formation of small blood vessels surrounding the graft. Based on the principle of time of biodegradability of new tissue, these films presented different values after 30 days. For example, CS + CNC (0.5%) showed the best results for new scaffold tissue in three formulations. In the other cases, we can estimate the total time for biodegradation to be 3–6 months (less time with higher concentrations of CNC based on these results). This could be useful in the future

biomedical uses (depending on the part or organ in the body to be used).

Another aspect to consider with respect to biodegradation values is the type of films. In this study, non porous films were elaborated. The porosity rate and the size of pores directly affect the biodegradability of films, because they allow the easy passage of water, blood, cells and other fluids. From their condition, these films could be useful for tissue scaffolding as can be seen in Figures 10 and 11 (SEM images). A typical pattern of degradation of chitosan shown as hexagonal borders was observed in all formulations, at

different stages. The formation of fascia in different stages was evident.

development of possible biomedical uses of these films.

Biodegradability values (%) Pure CS CS+CNC (0.5%) CS+CNC (1.0%) CS+CNC (2.0%)

**Biodegradability values (%) Pure CS CS+CNC (0.5%) CS+CNC (1.0%) CS+CNC (2.0%)**

244 Cellulose - Fundamental Aspects and Current Trends

**Table 3.** Biodegradability values for CS + CNC nanocomposites

the body to be used).

(1.0%)

degradation in CS + CNC (0.5%) at 500x. **Figure 10.** a. Presence of loose connective tissue in CS + CNC (0.5%) films at 100x and Fig. b. Hexagonal pattern of chitosan degradation in CS + CNC (0.5%) at 500x.

A pattern of biodegradation of CS films is supported by the fact that the biodegradation rate of CS has a linear relationship with the Degree of Deacetylation (DD). When the DD value (from 0 to 100) is closer to 100, the material shows a slow rate of biodegradation [39]. Other aspects to consider are that CS is degraded by proteases (mainly lysozymes) that attack Nacetyl glucosamine linkages making the process faster, but these linkages have less presence in CS chains when DD values are higher. Also, in this state, lysozyme activity is low and the biodegradation rate is slow. Furthermore, CS is a semi crystalline polymer. In amorphous zones, lysozyme activity is intense and when the positive charges increase, the interactions between cells with CS are better, thereby improving biocompatibility [39].

CS scaffolds with a low Degree of Acetylation (DA) and low molecular weight have a higher rate of biodegradation. Indeed, CS scaffolds with a low DA present lower biodegradation times, smaller pores, better mechanical properties, moderate water absorption and more intense cell activity than CS scaffolds with a higher DA [40]. A pore size of between 60 and 90 microns is suitable to allow lysozymes inside the structure and to act in polymer chains.

The CS type used in this research has a DD between 75–85% and a medium molecular weight related to low biodegradation values. In CS + CNC films, the CNC had an unstructuring effect, creating spaces between CS chains, thereby allowing water (and lysozymes) access into the scaffold structure, and showing larger values of biodegradation when CNC concentration was higher.

degradation of chitosan 11d. Presence of fascia (circled in red) in CS + CNC (1.0%) films at 500x **Figure 11.** a. Presence of loose connective tissue (circled in red) in CS + CNC (1.0%) films at 100x and b. 300x. Fig. 11c First steps of degradation of chitosan d. Presence of fascia (circled in red) in CS + CNC (1.0%) films at 500x

11a. Presence of loose connective tissue (circled in red) in CS + CNC (1.0%) films at 100x and Fig. 11b. 300x. Fig. 11c First steps of

A pattern of biodegradation of CS films is supported by the fact that the biodegradation rate of CS has a

The attachment, morphology and proliferation of cells on CS scaffolds is highly dependent on the type of cell lines, the source and characteristics of CS, the methods of CS scaffold prepa‐ ration, and the characteristics of the chitosan scaffolds. In addition, chitosan with a low molecular weight and low DA has excellent potential as a scaffolding material for a variety of tissue regeneration systems [40]. linear relationship with the Degree of Deacetylation (DD). When the DD value (from 0 to 100) is closer to 100, the material shows a slow rate of biodegradation [39]. Other aspects to consider are that CS is degraded by proteases (mainly lysozymes) that attack N‐acetyl glucosamine linkages making the process faster, but these linkages have less presence in CS chains when DD values are higher. Also, in this state, lysozyme activity is low and the biodegradation rate is slow. Furthermore, CS is a semi crystalline polymer. In amorphous zones, lysozyme activity is intense and when the positive charges increase, the

interactions between cells with CS are better, thereby improving biocompatibility [39].

showing larger values of biodegradation when CNC concentration was higher.

excellent potential as a scaffolding material for a variety of tissue regeneration systems [40].

#### **5. Conclusions** CS scaffolds with a low Degree of Acetylation (DA) and low molecular weight have a higher rate of biodegradation. Indeed, CS scaffolds with a low DA present lower biodegradation times, smaller pores,

**•** The addition of CNC improves physical properties, increasing their capability of swelling and water capitation. Mechanically, the main factor was the unstructuring effects over the chitosan chains. In biological tests, CNC show positive results for biocompatibility and biodegradability. with a higher DA [40]. A pore size of between 60 and 90 microns is suitable to allow lysozymes inside the structure and to act in polymer chains. The CS type used in this research has a DD between 75–85% and a medium molecular weight related to low biodegradation values. In CS + CNC films, the CNC had an unstructuring effect, creating spaces

between CS chains, thereby allowing water (and lysozymes) access into the scaffold structure, and

The attachment, morphology and proliferation of cells on CS scaffolds is highly dependent on the type of cell lines, the source and characteristics of CS, the methods of CS scaffold preparation, and the characteristics of the chitosan scaffolds. In addition, chitosan with a low molecular weight and low DA has

better mechanical properties, moderate water absorption and more intense cell activity than CS scaffolds


### **Acknowledgements**

Guillermo H. Riva, wishes to express his appreciation to the following institutions: German Exchange Students Agency (DAAD) for the scholarship and kind support during his M.Sc. Studies in Mexico; to the University of Guadalajara (UdG) for his formation as a M.Sc., for sponsorship and the financial support of this study, and Neucel Specialty Cellulose Limited (BC, Canada) for donating the alpha cellulose used in this study.

### **Author details**

Fig.

Guillermo H. Riva1 , Joaquín García-Estrada2 , Brenda Vega2 , Fernando López-Dellamary1 , María E. Hérnandez3 and José A. Silva1\*

\*Address all correspondence to: jasilva@dmcyp.cucei.udg.mx

1 University of Guadalajara, Wood, Cellulose and Paper Research Department "Karl Augus‐ tinGrellmann" Carretera Guadalajara, Jalisco, C.P., México

2 Biomedical Research Center of Occidente (CIBO), Guadalajara, Jalisco, C.P., México

3 University of Guadalajara, Exact Sciences and Engineering Faculty, Colonia Olímpica, Guadalajara, Jalisco, C.P., México

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The attachment, morphology and proliferation of cells on CS scaffolds is highly dependent on the type of cell lines, the source and characteristics of CS, the methods of CS scaffold prepa‐ ration, and the characteristics of the chitosan scaffolds. In addition, chitosan with a low molecular weight and low DA has excellent potential as a scaffolding material for a variety of

**Figure 11.** a. Presence of loose connective tissue (circled in red) in CS + CNC (1.0%) films at 100x and b. 300x. Fig. 11c

First steps of degradation of chitosan d. Presence of fascia (circled in red) in CS + CNC (1.0%) films at 500x

11a. Presence of loose connective tissue (circled in red) in CS + CNC (1.0%) films at 100x and Fig. 11b. 300x. Fig. 11c First steps of degradation of chitosan 11d. Presence of fascia (circled in red) in CS + CNC (1.0%) films at 500x

A pattern of biodegradation of CS films is supported by the fact that the biodegradation rate of CS has a linear relationship with the Degree of Deacetylation (DD). When the DD value (from 0 to 100) is closer to 100, the material shows a slow rate of biodegradation [39]. Other aspects to consider are that CS is degraded by proteases (mainly lysozymes) that attack N‐acetyl glucosamine linkages making the process faster, but these linkages have less presence in CS chains when DD values are higher. Also, in this state, lysozyme activity is low and the biodegradation rate is slow. Furthermore, CS is a semi crystalline polymer. In amorphous zones, lysozyme activity is intense and when the positive charges increase, the

**•** The addition of CNC improves physical properties, increasing their capability of swelling and water capitation. Mechanically, the main factor was the unstructuring effects over the chitosan chains. In biological tests, CNC show positive results for biocompatibility and

The CS type used in this research has a DD between 75–85% and a medium molecular weight related to low biodegradation values. In CS + CNC films, the CNC had an unstructuring effect, creating spaces between CS chains, thereby allowing water (and lysozymes) access into the scaffold structure, and

The attachment, morphology and proliferation of cells on CS scaffolds is highly dependent on the type of cell lines, the source and characteristics of CS, the methods of CS scaffold preparation, and the characteristics of the chitosan scaffolds. In addition, chitosan with a low molecular weight and low DA has

CS scaffolds with a low Degree of Acetylation (DA) and low molecular weight have a higher rate of biodegradation. Indeed, CS scaffolds with a low DA present lower biodegradation times, smaller pores, better mechanical properties, moderate water absorption and more intense cell activity than CS scaffolds with a higher DA [40]. A pore size of between 60 and 90 microns is suitable to allow lysozymes inside the

interactions between cells with CS are better, thereby improving biocompatibility [39].

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**c) d)**

**a) b)**

246 Cellulose - Fundamental Aspects and Current Trends

**5. Conclusions**

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Tamilselvan Mohan, Silvo Hribernik, Rupert Kargl and Karin Stana-Kleinschek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61344

#### **Abstract**

This chapter deals with an overview of design and fabrication of three-dimensional (3D) scaffolds for tissue engineering (TE) applications using the electrospinning technique. A general introduction to cellulose, a short overview of sources and methodology for the production of cellulose nanocrystals (CNCs), and principles of tissue engineering and the electrospinning technique will be given. Applications for CNCs are manifold and range from super water absorbent, drug delivery, packaging, personal care to pharmaceuticals. However, in this chapter the application in tissue engineering will be discussed in detail.

**Keywords:** Cellulose nanocrystals, tissue engineering, electrospinning, three-dimen‐ sional scaffold

### **1. Introduction**

Cellulose, the most ubiquitous natural biopolymer, is considered as a virtually inexhaustible source of raw material for the increasing demand of environmentally friendly, sustainable and biocompatible products [1, 2]. Despite its ubiquity in essentially every traditional and longestablished scientific and industrial field, cellulose permanently occupies prominent positions in the most advanced and emerging technologies, as well [3]. It has been used either in native or regenerated form in several applications including paper, board, fibers, films, textiles,

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packaging materials, hygienic products, and serves as a support material in chromatography and many other life science applications[1]. The most common attributes when describing the importance of cellulose are hydrophilicity, high reactivity, low fouling properties, renewabil‐ ity, nontoxicity, and the compatibility with many other organic and inorganic materials. This facilitates the creation of hybrid materials and nanocomposites with unique properties such as conductive fibers, papers with printed circuits, flame retardancy, and wound dressing materials [1].

Cellulose is the major component in the rigid cell walls of wood, other bio-plants and certain bacteria, algae, fungi, and some marine animals (e.g., tunicates). Cellulose is a linear syndio‐ tactic homopolymer consisting of D-anhydroglucopyranose units (AGU), which are covalently connected through β-(1 → 4)-glycosidic bonds between the carbon atoms C(1) and C'(4) of adjacent glucose units, resulting in a cellobiose unit (see Figure 1), which is the main building block of the cellulose polymer [4, 5]. The elemental composition of cellulose (carbon: 44-45%, hydrogen: 6.0-6.5%, and the rest being oxygen) was first revealed by the French chemist, Anselme Payen in 1838 [6]. Each of the AGU units consists of three free hydroxyl groups (at C-2, C-3 and C-6), which usually determine the reactivity of the cellulose polymer. The terminal hydroxyl groups at both ends of the cellulose chains (C-1 and C-4) are chemically different in nature. The C-1 at the right end of the chain is an aliphatic aldehyde group with reducing property, whereas C-4 at the left end is an alcoholic hydroxyl group with nonreducing property (see Figure 1). Cellulose exists in six different polymorphs, namely cellulose I, II, III, and IV. Among these, cellulose I occurs in two allomorphs, that is, Iα and Iβ. Cellulose II or regenerated cellulose is formed by treating native cellulose I with either strong alkali solution or by precipitating cellulose I with Schweitzer's reagent (tetraamminediaquacopper dihydroxide [Cu(NH3)4(H2O)2](OH)2). Cellulose III (IIII and IIIII) and cellulose IV (IVI and IVII) are obtained from either cellulose I or cellulose II with suitable modification [7-9]. Hearle et al. developed a two-phase model or fringed fibril model assuming that cellulose I contains low-ordered socalled amorphous and highly ordered, that is, crystalline regions [10]. This means that cellulose I is not perfectly crystalline. The intramolecular hydrogen bonds are formed between O-3-H and O-5´and also between O-2-H and O-6´ of one AGU unit and the adjacent AGU unit of the same polymer chain. The intermolecular hydrogen bonds are formed between O-6-H and O-3 ´of the AGU units of one polymer chain and the neighboring chain. Eventually, the different hydrogen bonding modes are responsible for the formation of stiff and rigid linear chains, which can further assemble into microfibrils with different chain lengths and orientation. When subjected to proper combinations of mechanical, chemical, and/or enzymatic treatments, these highly ordered crystalline regions present within the microfibrils can be isolated [11]. The isolated crystalline part is referred to as cellulose nanocrystals (CNCs). Owing to its nanoscale dimension and intrinsic physicochemical properties such as excellent biodegrada‐ bility, a low eco-toxicity, biocompatibility, high specific strength and modulus, high surface area, and unique optical properties, CNCs are promising renewable biomaterials that can be used as a reinforcing component in the preparation of high-performance nanocomposite scaffold materials. Many new highly porous 3D scaffold materials with attractive properties can be prepared by the physical incorporation of CNC into a natural or synthetic polymer matrix, which will be discussed in section 5.

**Figure 1.** Molecular structure of the cellulose macromolecule.

packaging materials, hygienic products, and serves as a support material in chromatography and many other life science applications[1]. The most common attributes when describing the importance of cellulose are hydrophilicity, high reactivity, low fouling properties, renewabil‐ ity, nontoxicity, and the compatibility with many other organic and inorganic materials. This facilitates the creation of hybrid materials and nanocomposites with unique properties such as conductive fibers, papers with printed circuits, flame retardancy, and wound dressing

Cellulose is the major component in the rigid cell walls of wood, other bio-plants and certain bacteria, algae, fungi, and some marine animals (e.g., tunicates). Cellulose is a linear syndio‐ tactic homopolymer consisting of D-anhydroglucopyranose units (AGU), which are covalently connected through β-(1 → 4)-glycosidic bonds between the carbon atoms C(1) and C'(4) of adjacent glucose units, resulting in a cellobiose unit (see Figure 1), which is the main building block of the cellulose polymer [4, 5]. The elemental composition of cellulose (carbon: 44-45%, hydrogen: 6.0-6.5%, and the rest being oxygen) was first revealed by the French chemist, Anselme Payen in 1838 [6]. Each of the AGU units consists of three free hydroxyl groups (at C-2, C-3 and C-6), which usually determine the reactivity of the cellulose polymer. The terminal hydroxyl groups at both ends of the cellulose chains (C-1 and C-4) are chemically different in nature. The C-1 at the right end of the chain is an aliphatic aldehyde group with reducing property, whereas C-4 at the left end is an alcoholic hydroxyl group with nonreducing property (see Figure 1). Cellulose exists in six different polymorphs, namely cellulose I, II, III, and IV. Among these, cellulose I occurs in two allomorphs, that is, Iα and Iβ. Cellulose II or regenerated cellulose is formed by treating native cellulose I with either strong alkali solution or by precipitating cellulose I with Schweitzer's reagent (tetraamminediaquacopper dihydroxide [Cu(NH3)4(H2O)2](OH)2). Cellulose III (IIII and IIIII) and cellulose IV (IVI and IVII) are obtained from either cellulose I or cellulose II with suitable modification [7-9]. Hearle et al. developed a two-phase model or fringed fibril model assuming that cellulose I contains low-ordered socalled amorphous and highly ordered, that is, crystalline regions [10]. This means that cellulose I is not perfectly crystalline. The intramolecular hydrogen bonds are formed between O-3-H and O-5´and also between O-2-H and O-6´ of one AGU unit and the adjacent AGU unit of the same polymer chain. The intermolecular hydrogen bonds are formed between O-6-H and O-3 ´of the AGU units of one polymer chain and the neighboring chain. Eventually, the different hydrogen bonding modes are responsible for the formation of stiff and rigid linear chains, which can further assemble into microfibrils with different chain lengths and orientation. When subjected to proper combinations of mechanical, chemical, and/or enzymatic treatments, these highly ordered crystalline regions present within the microfibrils can be isolated [11]. The isolated crystalline part is referred to as cellulose nanocrystals (CNCs). Owing to its nanoscale dimension and intrinsic physicochemical properties such as excellent biodegrada‐ bility, a low eco-toxicity, biocompatibility, high specific strength and modulus, high surface area, and unique optical properties, CNCs are promising renewable biomaterials that can be used as a reinforcing component in the preparation of high-performance nanocomposite scaffold materials. Many new highly porous 3D scaffold materials with attractive properties can be prepared by the physical incorporation of CNC into a natural or synthetic polymer

materials [1].

252 Cellulose - Fundamental Aspects and Current Trends

matrix, which will be discussed in section 5.

### **2. Cellulose nanocrystals – Sources and methodology of preparation**

Cellulose is at the forefront in scientific development in the areas of medicine, drug delivery, and sensoric applications, to name just a few, with a pronounced shift of employing cellulose substrates in nanometric dimensions, especially cellulose nanocrystals (CNCs). An array of attractive properties in combination with above-mentioned attributes are the main reason for an immense body of work, conducted with CNCs can be obtained following either of the two approaches: bottom-up by biosynthesis or top-down by disintegration of plant materials [11, 12]. Acid hydrolysis of cellulose substrates is the most widely used procedure for obtaining CNCs. The driving force of CNC formation in acidic media is the difference in solubility of structural regions in bulk cellulose, namely amorphous and crystalline entities. While amor‐ phous regions of the cellulose substrates dissolve and hydrolyze in acidic solutions, crystalline domains readily reassemble to form nanocrystals, nanorods, or nanowhiskers (see Figure 2) [13]. Sulfuric acid (H2SO4) is a common hydrolyzing agent, which also imparts the resultant nanocrystals with surface sulfate groups, making their colloidal dispersions very stable due to present charges (accordingly, hydrochloric acid, for instance, yields CNC particles with a weaker charge with poor colloidal stability).

A wide variety of suitable cellulose raw materials can be employed for preparation of CNCs; consequently, different physical properties can be expected. An interesting and, so far to a certain extent, underutilized source for production of cellulose nanocrystals are fibers derived from oil palm trunks. Processed using acid hydrolysis with H2SO4, prepared CNCs possess an increase in crystallinity and this feature, coupled with removal of lignin and hemicelluloses contributed to enhanced thermal stability [18]. Despite its abundance, corn husks are also representatives of a class of natural materials which have not been extensively appropriated for the production of cellulose nanocrystals. Isolation of CNCs with acid hydrolysis of pretreated corn husk verified the suitability of the material as a precursor for cellulose nanocrystals, with emphasis on treatment time, which affects the degree of crystallinity, particle size, and thermal stability of the product [19]. Another environmentally sustainable approach for the production of cellulose nanocrystals is the use of wastepaper with its abundance and inexpensiveness. Acid hydrolysis with H2SO4 of alkali-treated and bleached cellulose particles yielded CNCs with 100-300 nm in length and 3-10 nm in diameter and

**Figure 2.** A) Schematics of cellulose fibers depicting crystalline and amorphous regions, and cellulose nanocrystals (with hydroxyl and sulfate groups) after sulfuric acid hydrolysis of the disordered amorphous regions. Reprinted with permission from ref [14]. Copyright 2011 Royal Society of Chemistry. (B) TEM micrographs of dispersion of cellulose nanocrystals derived from different sources: (b1) microcrystalline cellulose (Avicel), (b2) tunicate[15], (b3) green algae (*Cladophora sp*.) [16], and (b4) ramie [17]. (A) Reprinted with permission from ref [17]. Copyright 2008 Royal Society of Chemistry. (B) Reprinted with permission from refs [15] and [16]. Copyright 2012 and 2008 American Chemical Soci‐ ety.

crystallinity degree of approx. 76% [20]. An alternative source for production of cellulose nanocrystals is also bacterial cellulose, which is produced by acetic acid bacteria in either synthetic or nonsynthetic media via oxidative fermentation [21]. Preparation of bacterial cellulose-derived nanocrystals with different sulfate contents and, consequently, widely different colloidal properties was reported by varying the type of acid used as hydrolytic agent [22]. Whether hydrochloric acid (HCl), its mixture with H2SO4 or H2SO4 alone were used, prepared crystals exhibited zeta potential values of -5, -40, and -46 mV; in return, surface character of particles influenced their colloidal stability and interactions with other materials (e.g., xyloglucan). Kenaf bast fibers were also employed for the preparation of CNC colloidal suspensions, with cellulose extraction (a combination of alkaline treatment and bleaching) followed by acid hydrolysis [23]. Sugarcane bagasse served as a source for CNCs, which were prepared with a sulfuric acid treatment [24]. Softwood and hardwood were used in addition to nonwood sources (cotton linters and cattail, i.e., Typha) and red algae as a representative of marine pulp for the production of CNCs by H2SO4 hydrolysis. Dimensions of the obtained CNCs can be grouped according to the material class, with wood-source-derived particles having length from approx. 170 nm to approx. 180 nm, cattail and cotton-based particles from approx. 250 nm to approx. 278 nm, while red-algae-based CNCs exhibit the longest particles, i.e., approx. 432 nm. Nonwood CNCs exhibit superior thermal stability to wood-derived ones. Behavior, performance, and interactions with host material of CNC-embedded products rely on particles' geometry, i.e., 3D size, their aspect ratio, crystallinity, amount and type of surface groups, their distribution in a host matrix, and rheological properties [25]. Besides its abovementioned intrinsic properties, CNCs also have superior mechanical properties, and a surface covered with numerous hydroxyl groups that enables different chemical modifications like oxidation esterification, silyation, and polymer grafting. These amazing physicochemical properties and widespread application prospect of CNCs (in personal care, foods, pharma‐ ceuticals, as drug carriers, anticoagulant materials, and in TE) have attracted significant interest from both research scientists and industrialists.

### **3. Tissue engineering – Introduction to basic principles**

crystallinity degree of approx. 76% [20]. An alternative source for production of cellulose nanocrystals is also bacterial cellulose, which is produced by acetic acid bacteria in either synthetic or nonsynthetic media via oxidative fermentation [21]. Preparation of bacterial cellulose-derived nanocrystals with different sulfate contents and, consequently, widely different colloidal properties was reported by varying the type of acid used as hydrolytic agent [22]. Whether hydrochloric acid (HCl), its mixture with H2SO4 or H2SO4 alone were used, prepared crystals exhibited zeta potential values of -5, -40, and -46 mV; in return, surface character of particles influenced their colloidal stability and interactions with other materials (e.g., xyloglucan). Kenaf bast fibers were also employed for the preparation of CNC colloidal

ety.

254 Cellulose - Fundamental Aspects and Current Trends

**Figure 2.** A) Schematics of cellulose fibers depicting crystalline and amorphous regions, and cellulose nanocrystals (with hydroxyl and sulfate groups) after sulfuric acid hydrolysis of the disordered amorphous regions. Reprinted with permission from ref [14]. Copyright 2011 Royal Society of Chemistry. (B) TEM micrographs of dispersion of cellulose nanocrystals derived from different sources: (b1) microcrystalline cellulose (Avicel), (b2) tunicate[15], (b3) green algae (*Cladophora sp*.) [16], and (b4) ramie [17]. (A) Reprinted with permission from ref [17]. Copyright 2008 Royal Society of Chemistry. (B) Reprinted with permission from refs [15] and [16]. Copyright 2012 and 2008 American Chemical Soci‐ Tissue engineering (TE) is an interdisciplinary field that applies the principles of engineering and life science toward the development of smart biological substitutes (e.g., 3D scaffolds), which potentially restore, maintain, and improve tissue functions that are malfunctioned or have been lost by different pathological conditions or as a whole organ of the human body [26]. The field of TE still remains a fast-growing area with high-potential treatments for many kinds of disease states. In TE, the cells are usually cultured/seeded onto biodegradable scaffolds that mimic the extracellular matrix (ECM), which exhibits a hierarchical architecture with structural features ranging from nano- to macrometer scale [27]. In general, cell microenvironment is very complex, and it constitutes ECM proteins, bioactive growth factors, receptors, and neighboring cells (see Figure 3). Behavior, function, and fate of cells are determined by their interaction with biochemical and biophysical cues within their surrounding microenviron‐ ment [28]. In particular, the interaction between biochemical cues, for example, receptor coupling to ECM proteins or cytokines, and biophysical cues such as modulus and fibrillar structure play a decisive role in cell fate decision [28, 29]. These cell–ECM interactions are very dynamic, since cells interact with and respond to ECM signals and accordingly can change their microenvironments. Therefore, the understanding and interlinking of bidirectional cross talk between the microenvironment and resident cells are highly important for developing efficient strategies to regenerate tissues [28]. Generally, ECM differs in composition and spatial assembly of their protein components such as collagen, elastin, proteoglycans, and adhesion molecules depending on the types of body tissues. It further inherently assembles to keep a specific tissue morphology and to supply specific instructive cues for the cells of the different organs [30].

**Figure 3.** Schematic of the extracellular matrix. Fibrous matrix proteins (e.g., collagen, fibrin, elastin) provide structural and mechanical cues to direct cell behavior; soluble signals are sequestered by proteoglycans (proteins with polysac‐ charide moieties) and interact with cell surface receptors to direct cell migration, proliferation, and differentiation; in‐ tegrins (transmembrane receptors) bind to matrix proteins for cell adhesion; ECM degradation enzymes (e.g., matrix metalloproteinases, serine proteinases, plasmin) cleave matrix components during cell motility and matrix remodeling. Reprinted with permission from ref [31]. Copyright 2012 Elsevier.

A unique but the simplest approach in TE and its related field regenerative medicine comprises the implantation of a scaffold that possesses inherent properties to actively promote the body's inborn capacity of healing and self-repair/growth of cells and tissue regeneration [27]. Scaffolds in general exhibit several attractive features that can influence their performance, and therefore the scaffolds should be carefully designed with all essential physicochemical properties for each targeted tissue. The scaffold materials, in general, should have the capacity to mimic the form and function of ECM in order to promote the cell–biomaterial interactions, cellular invasion, attachment, infiltration or migration, differentiation, and proliferation, thus render‐ ing the lost tissue regeneration [27]. They should also permit sufficient transport of gases and nutrients, and allow regulatory factors to achieve cell survival [27]. Physiochemical properties of the scaffold materials, for instance, the roughness, topography, functional groups, porosity, pore size, pore interconnectivity, and surface area to volume ratio are the key aspects that should be considered in the design of scaffolds, because they play a pivotal role in tissue growth, vascularization, and nutrient supply [28, 32-38]. Furthermore, the scaffolds should be mechanically strong and easily biodegradable at a controlled rate under the condition of interest, and provide a low degree of inflammation and cytotoxicity [39].

Biodegradability of a scaffold under a given condition is an important issue in TE, and thus materials for scaffolds design should be carefully chosen. The material should be absorbable and subjectable to degradation. The rate of degradation should match the time of tissue formation,thatis, untilthe scaffold material is mechanically stable and possesses all its intrinsic physicochemical properties and the injured tissue is completely replaced by healthy tissue and its functions are restored [35, 39]. Taking these points into consideration, the selection of the scaffold materials and the preparation methodology is highly dependent on the requirements of the desired applications and given properties of the scaffold. Further, the most difficult challenge inTEis tocreatea scaffoldwithspecificphysical, chemical,mechanical,andbiological properties. As stated above, the mechanical (dimensional) stability of the scaffold is impera‐ tive in TE, and it should yield similar performance to the tissues to be repaired or regenerat‐ ed. In this way, the scaffold would give a better mechanical integration of the construct in the biological tissue.In addition, the mechanical signals (i.e., the performance of the scaffold under a given condition) are known to create impact on the "cell fate" [40] and therefore a considera‐ tion is usually given to the role of mechanotransduction during cell expansion and differentia‐ tion [41, 42]. Overall, a precise modulation of these mentioned properties is highly challenging as biological tissues present a vast spectrum of mechanical properties, which are governed by their anatomical functions. For example, many tissues, in general, are viscoelastic with nonlinear, anisotropic, and heterogeneous mechanical properties [43].

The materials (synthetic and/or from natural origin) and fabrication methods that enable the manufacturing of scaffolds with defined micro- and nanostructure length scales for different TE applications are manifold. As natural and derivatized biopolymers starch, carrageenan, alginate, chitin, chitosan, gellan gum, hyaluronic acid, chondroitin sulfate, lingocellulose, cellulose acetate [44], carboxymethyl cellulose [45], cellulose acetate propionate [46], hydroxy‐ proply cellulose [47] are used for creating scaffolds and cell culturing owing to their inherent bioactivity,low toxicity,highbiocompatibility andinmost cases biodegradability [48].Proteins such as collagen, gelatin, elastin, silk fibroin, and fibrin are also employed forthe same purpose [48-50]. In the case of the synthetically based category, aliphatic polyesters such as poly(Llactic acid),polycaprolactone,poly(glycolide), andpoly-[D,L-(lactide-co-glycolide)],polyethy‐ lene oxide, polyvinyl alcohol, poly(N-isopropylacrylamide), polyacrylic acid, and poly(2 hydroxyethylmethacrylate) are widely employed for preparing TE scaffolds since they exhibit excellent biocompatibility, biodegradability, and considerable mechanical stability [34].

**Figure 3.** Schematic of the extracellular matrix. Fibrous matrix proteins (e.g., collagen, fibrin, elastin) provide structural and mechanical cues to direct cell behavior; soluble signals are sequestered by proteoglycans (proteins with polysac‐ charide moieties) and interact with cell surface receptors to direct cell migration, proliferation, and differentiation; in‐ tegrins (transmembrane receptors) bind to matrix proteins for cell adhesion; ECM degradation enzymes (e.g., matrix metalloproteinases, serine proteinases, plasmin) cleave matrix components during cell motility and matrix remodeling.

A unique but the simplest approach in TE and its related field regenerative medicine comprises the implantation of a scaffold that possesses inherent properties to actively promote the body's inborn capacity of healing and self-repair/growth of cells and tissue regeneration [27]. Scaffolds in general exhibit several attractive features that can influence their performance, and therefore the scaffolds should be carefully designed with all essential physicochemical properties for each targeted tissue. The scaffold materials, in general, should have the capacity to mimic the form and function of ECM in order to promote the cell–biomaterial interactions, cellular invasion, attachment, infiltration or migration, differentiation, and proliferation, thus render‐ ing the lost tissue regeneration [27]. They should also permit sufficient transport of gases and nutrients, and allow regulatory factors to achieve cell survival [27]. Physiochemical properties of the scaffold materials, for instance, the roughness, topography, functional groups, porosity,

Reprinted with permission from ref [31]. Copyright 2012 Elsevier.

256 Cellulose - Fundamental Aspects and Current Trends

While significant advances were made over the years, it is still impossible to design and engineer TE scaffolds having all desired properties from a conventional single polymeric material. In this regard, in order to better mimic the ECM hierarchical structure and for efficient growth of cells, electrospun nanocomposite fiber mats generated from multicomponent systems have gained a significant interest for different TE applications in the recent years, due to their good mechanical properties, nanotopography, and well-defined porosity [28, 32-34]. In order to overcome some limitations such as weak mechanical properties, lack of electrical conductivity, the absence of adhesive and microenvironment-defining moieties, or the incompetence to enable cells to self-assemble to 3D tissues [51], incorporation of nanofillers as reinforcing agents have become a popular approach in the past years. Several inorganic and organic nanofillers have been used together with polymeric materials for the preparation of TE scaffolds targeting at regeneration of different tissues of interest. As nanofillers, hydrox‐ yapatite, carbon nanotubes, metal nanoparticles (e.g., silver, gold, and iron oxide), etc. have been used for the controlled immobilization of several biologically active molecules as well as to construct complex 3D tissues of different cell types [51-53]. Despite the number of advan‐ tages of metal or carbon-based nanofillers, there are some challenges and issues that remain to be addressed, including processability, toxicity, biocompatibility, and biodegradability. In this context, CNCs in particular, are highly attractive and important. They have emerged as potential nanofillers for preparation of biobased nanostructured composite materials, due to their aforementioned intrinsic properties [54]. Moreover, as specified before, CNCs exhibit low elongation at break, high aspect ratio, high surface area, and high crystallinity, which make them excellent candidates as load-bearing components for obtaining high-strength scaffold materials [55, 56]. For fabricating 3D scaffolds with nanofibrillar structures and with nanoto‐ pography to better mimic ECM dimensions, the electrospinning technique has been used extensively during the last few years, which will be discussed in the next section.

### **4. Electrospinning technique – Introduction to basic principles**

Electrospinning is a straightforward, cost-effective, and robust technique, which is nowadays frequently applied in engineering well-defined nanocomposites fiber mats/scaffolds that hold promise in serving as a synthetic ECM. It utilizes electrical forces to create fine polymer fibers with diameters ranging from a few micrometers down to a few nanometers using polymer solutions of both natural and synthetic polymers [57]. This technique has been known for over 60 years in the textile industry for manufacturing nonwoven fiber fabrics, but has received a great importance only in the past decade. This is not only because of its versatility in spinning a wide range of polymeric fibers with controlled pore structure, but also due to its ability to consistently produce fibers in the submicron range that is otherwise difficult to achieve by using standard mechanical fiber spinning techniques [58-62]. Among several other advantages provided by this technique, it also gives the possibility to manipulate the spun fiber compo‐ sition and produce highly reproducible fiber mats with micro- or nanotopography while enabling to tailor the fiber diameter, porosity, orientation, density, and high surface-area-tovolume ratio. Owing to these advantages, electrospun nanofibers have been widely examined for their use in numerous fields including nanocatalysis, protective clothing, filtration, biomedical, pharmaceutical, optical electronics, healthcare, as well as in tissue engineering [63-68]. Techniques such as electrostatic precipitators and pesticide sprayers work similarly to the electrospinning process; however, the latter process is mainly based on the principle that the applied strong mutual electrical repulsive forces overcome weaker forces of surface tension in the charged polymer liquid. There are, currently, two standard electrospinning setups – vertical and horizontal. In general, the electrospinning process is conducted at ambient temperature and pressure. The typical electrospinning apparatus setup is depicted in Figure 4.

to their good mechanical properties, nanotopography, and well-defined porosity [28, 32-34]. In order to overcome some limitations such as weak mechanical properties, lack of electrical conductivity, the absence of adhesive and microenvironment-defining moieties, or the incompetence to enable cells to self-assemble to 3D tissues [51], incorporation of nanofillers as reinforcing agents have become a popular approach in the past years. Several inorganic and organic nanofillers have been used together with polymeric materials for the preparation of TE scaffolds targeting at regeneration of different tissues of interest. As nanofillers, hydrox‐ yapatite, carbon nanotubes, metal nanoparticles (e.g., silver, gold, and iron oxide), etc. have been used for the controlled immobilization of several biologically active molecules as well as to construct complex 3D tissues of different cell types [51-53]. Despite the number of advan‐ tages of metal or carbon-based nanofillers, there are some challenges and issues that remain to be addressed, including processability, toxicity, biocompatibility, and biodegradability. In this context, CNCs in particular, are highly attractive and important. They have emerged as potential nanofillers for preparation of biobased nanostructured composite materials, due to their aforementioned intrinsic properties [54]. Moreover, as specified before, CNCs exhibit low elongation at break, high aspect ratio, high surface area, and high crystallinity, which make them excellent candidates as load-bearing components for obtaining high-strength scaffold materials [55, 56]. For fabricating 3D scaffolds with nanofibrillar structures and with nanoto‐ pography to better mimic ECM dimensions, the electrospinning technique has been used

258 Cellulose - Fundamental Aspects and Current Trends

extensively during the last few years, which will be discussed in the next section.

**4. Electrospinning technique – Introduction to basic principles**

Electrospinning is a straightforward, cost-effective, and robust technique, which is nowadays frequently applied in engineering well-defined nanocomposites fiber mats/scaffolds that hold promise in serving as a synthetic ECM. It utilizes electrical forces to create fine polymer fibers with diameters ranging from a few micrometers down to a few nanometers using polymer solutions of both natural and synthetic polymers [57]. This technique has been known for over 60 years in the textile industry for manufacturing nonwoven fiber fabrics, but has received a great importance only in the past decade. This is not only because of its versatility in spinning a wide range of polymeric fibers with controlled pore structure, but also due to its ability to consistently produce fibers in the submicron range that is otherwise difficult to achieve by using standard mechanical fiber spinning techniques [58-62]. Among several other advantages provided by this technique, it also gives the possibility to manipulate the spun fiber compo‐ sition and produce highly reproducible fiber mats with micro- or nanotopography while enabling to tailor the fiber diameter, porosity, orientation, density, and high surface-area-tovolume ratio. Owing to these advantages, electrospun nanofibers have been widely examined for their use in numerous fields including nanocatalysis, protective clothing, filtration, biomedical, pharmaceutical, optical electronics, healthcare, as well as in tissue engineering [63-68]. Techniques such as electrostatic precipitators and pesticide sprayers work similarly to the electrospinning process; however, the latter process is mainly based on the principle that the applied strong mutual electrical repulsive forces overcome weaker forces of surface tension

**Figure 4.** Schematic representation illustrating the set up of an electrospinning apparatus. A) typical vertical set up, b) horizontal setup. Reprinted with permission from ref [59]. Copyright 2010 Elsevier.

A basic electrospinning setup consists of three major components such as a high-voltage power supply; a spinneret, i.e., a pipette tip or a plastic syringe; and a grounded collecting plate that is usually a metal screen, plate, or rotating mandrel, and the technique uses electrical forces for stretching the solubilized polymer as it falls and solvent evaporates out. In this technique, prior to the spinning the dissolved polymer solution or melt of interest is introduced into the capillary tube and then a high-voltage electrical field is applied between a metallic nozzle of a syringe and a metallic collector. As a result, the polymer solution or melt, held by its surface tension at the end of the capillary tube is subjected to a high electrical field, and this highvoltage source induces charge of a certain polarity on the liquid surface of the polymer solution or melt. At end of the needle tip, the polymer solution deforms from a pendant droplet to a conical shape (Taylor cone). When the strength of the electrical force is stronger than the surface tension of the polymer solution, a jet with polymer solution is formed and ejected subsequently from the cone surface and travels to the opposite polarity, i.e., metallic collector. As soon as the polymer jet solution travels, the solvent evaporates in the air, together with stretching and acceleration of the polymer jet, leading to the deposition of an extremely thin polymer (nano-)fiber on the collector. The polymer jet is only stable at the end of the capillary tube and after that the instability begins, and thus electrospinning process offers a simplified method for the manufacturing of ultrathin nanofibers [59-62].

### **5. Cellulose-based scaffold – Materials' characterization and role in cell growth**

Despite several advantages mentioned above in different fields, the application of electro‐ spinning and spun nanofibers in the field of TE is as yet not realized up to its full potential, and there are still some challenges that need proper attention. The diameters of electrospun nanofibers are usually orders of magnitude smaller when compared to the sizes of cells. As a result, the cells are able to organize over the fibers of the scaffold or spread and attach to the initially adsorbed proteins or cells at multiple focal points [62]. Regardless of its numerous attractive properties, scaffolds pose some other limitations such as nonuniform cellular distribution and lack of cellular migration/infiltration with increasing depth under normal passive seeding conditions. This is because the electrospun scaffolds or mats are often characterized by entangled fibers and a densely packed membrane structure that give obvious limitation to cellular migration/infiltration and populate in the interior of the scaffolds. These drawbacks could potentially hamper the development and potential applicability of electro‐ spun fibers, in particular for 3D tissue or organs. In this context, the issue of cellular infiltration into the fiber architecture is gaining more and more attention owing to its potential in stagnating further applications of electrospun scaffolds materials in various TE applications. To overcome these limitations, an extensive set of fabrication techniques, such as wet electro‐ spinning, laser or UV photolithography, salt leaching, polymer blending, solvent casting or a combination of nanofibers and microfibers, have been used to obtain scaffolds having enlarged pore size, and interconnectivity, and subsequently promote cellular migration/infiltration [69-71]. Up to now, the electrospinning technique has been increasingly exploited in the production of nanofibrous scaffolds or mats from the widest range of polymeric materials (natural and synthetic) for tissue engineering applications (see section 3). Besides the limitation to cellular migration/infiltration, another important major concern with spun 3D scaffolds, which are made from either mono or bicomponent system, is mechanical stability, since they are often not strong enough for many tissue engineering applications, as described above. One way to improve the mechanical properties of the electrospun scaffold is to integrate some reinforcing agents or fillers, and for such purpose a range of materials is used, which can be found elsewhere.

Among other materials, incorporation of stiff and rodlike CNCs with high aspect ratios as reinforcing agent into the electrospun fiber mats have been investigated as an effective and alternative method in numerous studies in fabricating a high-strength and highly porous composite scaffold with multiscaled features, which would better mimic the natural hierarch‐ ical organization of ECM [72-74]. 3D porous scaffolds consisting of naturally occurring CNCs as reinforcing agents are important and attractive for many TE applications due to their vast number of above-mentioned advantages [73,74]. Electrospinning technique allows CNCs to orient along the fiber axis, thus yielding unidirectional fiber-reinforced composites for applications that require high-fiber stiffness, uniform morphology, and reduced fiber diameter [72-74]. Until now, numerous electrospun composite materials incorporated with CNCs have been produced from natural and synthetic polymers, and employed in the production of 3D tissues for implantation. Jia et al. investigated the potential applicability of CNCs in combi‐ nation with microcrystalline cellulose (MC) as fillers in electrospun cellulose acetate (CA) nanofiber mats for vascular tissue scaffolds [75]. It has been found that the electrospun fibers are in the range of submicron and the incorporation of CNC and MC increased the porosity of the scaffolds, for example, a mean diameter of 1016 ± 572 nm and porosity of 80±9% are obtained at a ratio of 1:1 (MC:CNC, 10 wt.%). To understand the impact of CNC and MC on the viability of rat aortic vascular smooth muscle cells (VSMC) within the generated scaffolds were tested in vitro. It turned out that the cell viability, adhesion, migration, and proliferation with the scaffold integrated with CNC and MC are significantly improved compared with those of CA alone, confirming the synergistic enhancement by the micro- and nanoscaled features within the 3D fibrous mesh structure. In the recent work, He et al. fabricated the electrospun all-cellulose nanocomposite materials reinforced with 20% CNC, which are uniaxially aligned using a rotating drum as collector [76]. The obtained scaffold material showed well-dispersed and substantially orientated CNCs along the fiber axis, and, further, the inclusion of CNCs induced a positive effect in creating scaffolds with uniform morphology and ordered distribution of nanofibers with diameter ranging from 212 to 221 nm. The resulting final material with ordered microstructure demonstrated a remarkable improvement in the tensile properties; for instance, the tensile strength and the elastic modulus are increased by 102% and 172% in the fiber alignment direction, due to enhanced interfacial bonding between CNCs and the regenerated cellulose matrix employed in the production of the electrospun scaffold. The applicability of the material is tested for the growth of human dental follicle cells (cDFCs), and the results illustrated that the fiber mats are nontoxic and the cells are able to attach, infiltrate, and subsequently proliferate in the entire scaffold, thereby inducing ordered organization of cDFCs along the fiber alignment direction (Figure 5). The authors have further proposed that these microstructured electrospun scaffolds can be used in several other TE strategies, for example, in the development of blood vessel, tendon, or nerves, where the mechanical performance and cell orientation are critical issues [77].

acceleration of the polymer jet, leading to the deposition of an extremely thin polymer (nano-)fiber on the collector. The polymer jet is only stable at the end of the capillary tube and after that the instability begins, and thus electrospinning process offers a simplified method

**5. Cellulose-based scaffold – Materials' characterization and role in cell**

Despite several advantages mentioned above in different fields, the application of electro‐ spinning and spun nanofibers in the field of TE is as yet not realized up to its full potential, and there are still some challenges that need proper attention. The diameters of electrospun nanofibers are usually orders of magnitude smaller when compared to the sizes of cells. As a result, the cells are able to organize over the fibers of the scaffold or spread and attach to the initially adsorbed proteins or cells at multiple focal points [62]. Regardless of its numerous attractive properties, scaffolds pose some other limitations such as nonuniform cellular distribution and lack of cellular migration/infiltration with increasing depth under normal passive seeding conditions. This is because the electrospun scaffolds or mats are often characterized by entangled fibers and a densely packed membrane structure that give obvious limitation to cellular migration/infiltration and populate in the interior of the scaffolds. These drawbacks could potentially hamper the development and potential applicability of electro‐ spun fibers, in particular for 3D tissue or organs. In this context, the issue of cellular infiltration into the fiber architecture is gaining more and more attention owing to its potential in stagnating further applications of electrospun scaffolds materials in various TE applications. To overcome these limitations, an extensive set of fabrication techniques, such as wet electro‐ spinning, laser or UV photolithography, salt leaching, polymer blending, solvent casting or a combination of nanofibers and microfibers, have been used to obtain scaffolds having enlarged pore size, and interconnectivity, and subsequently promote cellular migration/infiltration [69-71]. Up to now, the electrospinning technique has been increasingly exploited in the production of nanofibrous scaffolds or mats from the widest range of polymeric materials (natural and synthetic) for tissue engineering applications (see section 3). Besides the limitation to cellular migration/infiltration, another important major concern with spun 3D scaffolds, which are made from either mono or bicomponent system, is mechanical stability, since they are often not strong enough for many tissue engineering applications, as described above. One way to improve the mechanical properties of the electrospun scaffold is to integrate some reinforcing agents or fillers, and for such purpose a range of materials is used, which can be

Among other materials, incorporation of stiff and rodlike CNCs with high aspect ratios as reinforcing agent into the electrospun fiber mats have been investigated as an effective and alternative method in numerous studies in fabricating a high-strength and highly porous composite scaffold with multiscaled features, which would better mimic the natural hierarch‐ ical organization of ECM [72-74]. 3D porous scaffolds consisting of naturally occurring CNCs as reinforcing agents are important and attractive for many TE applications due to their vast

for the manufacturing of ultrathin nanofibers [59-62].

260 Cellulose - Fundamental Aspects and Current Trends

**growth**

found elsewhere.

Huang et al. manufactured mechanically highly stable electrospun scaffold composed of nanofibers from silk protein such as fibroin and CNCs (diameter: 20–40 nm, length: 400–500 nm) as nanofillers that are extracted from *Morus alba L*. branch bark [73]. Results showed that in the resulting material, the nanoparticles CNCs are well dispersed and considerably oriented along the fiber axis in the silk fibroin matrix, as reported by the author. The electrospun fiber mats reinforced with CNCs (2%) showed almost twofold increase in the mechanical properties such as the tensile strength and the Young's modulus compared to virgin (unreinforced) silk fibroin nanofiber. Interestingly, the electrospun nanofibers that were obtained using bacterial cellulose nanocrystals as nanofiller and silk fibroin showed similar increase in the mechanical properties [73, 78]. Unfortunately, the importance of electrospun scaffolds derived from fibroin/CNCs is not recognized as cell growth material in TE to date.

**Figure 5.** Confocal laser scanning microscopy images of hDFCs loaded in electrospun cellulose/CNCs nanocomposite nanofibers: (a) cultured for 3 days, (b) cultured for 7 days, and (c) the 3D view of electrospun nanofibers with cells. Scale bar: 50 µm. Reprinted with permission from ref [76]. Copyright 2014 American Chemical Society.

While the scaffolds manufactured from fully renewable cellulose are highly captivating and important for TE applications, the electrospinning of these natural origins is always demand‐ ing and needs special care in choosing the right solvent and viscosity of the polymer solution. It can be resolved, however, by blending CNCs with synthetic biopolymers to meet the right solution viscosity and consistency in electrospinning. In this regard, in the recent years, synthetic biopolymers, in particular, PLA in combination with CNCs as nanofiller, have received a vast interest in the production of electrospun nanocomposite fibers [79]. For example, in the work of Xian et al., PLA electrospun nanofiber mats incorporated with CNCs prepared from avicel (microcrystalline) cellulose are fabricated [79]. It has been found that the strength of the nanofibers is improved by 30% with loading of 1 wt.% CNCs. The latter acted as nucleating agent of PLA crystallization leading to increased crystallinity of PLA in the resulting nanocomposite fibers. In another work by Shi et al., PLA/CNCs electrospun fiber mats from a solvent mixture consisting of N,N'-dimethylformamide (DMA) and chloroform were prepared and characterized in terms of in vitro degradation properties [80]. The obtained nanocomposite mats showed 5-fold and 22-fold increase in tensile stress and Young's modulus upon the addition of 5 wt.% CNCs, and they undergo faster degradation in phosphate-buffered saline (PBS) solution than that in neat PLA mats. These types of materials exhibiting superior mechanical and fast degradation properties are particularly interesting for making TE scaffolds targeted at short-term application CNCs.

Owing to the fact that CNCs are highly polar and show high tendency for the formation of hydrogen bonding promoting agglomeration, dispersing aqueous or nonpolar-based CNCs in a PLA matrix is highly tedious and time consuming. To overcome this hurdle, Li et al. proposed an alternative approach based on a water-in-oil (W/O) emulsion system consisting of a dispersed phase of CNCs aqueous suspension and an immiscible continuous phase of PLA solution [81]. The electrospun fiber mats prepared from these system showed either a coreshell or hollow structure depending on the emulsion droplet size, and the CNCs are aligned along the core or on the wall of the hollow cylinder (Figure 6). Upon the addition of 5 wt.% CNCs, a drastic increase in stiffness (Young's modulus), i.e., up to 549% and maximum increase of tensile strength of 90% are obtained for the electrospun composite fiber mats. As described by the authors, besides the reinforcement offered by the CNCs, these hollow structures, as multifunctional system, can be potentially employed in several applications such as for the attachment of biologically active molecules, controlled drug delivery, and as scaffolds.

While the scaffolds manufactured from fully renewable cellulose are highly captivating and important for TE applications, the electrospinning of these natural origins is always demand‐ ing and needs special care in choosing the right solvent and viscosity of the polymer solution. It can be resolved, however, by blending CNCs with synthetic biopolymers to meet the right solution viscosity and consistency in electrospinning. In this regard, in the recent years, synthetic biopolymers, in particular, PLA in combination with CNCs as nanofiller, have received a vast interest in the production of electrospun nanocomposite fibers [79]. For example, in the work of Xian et al., PLA electrospun nanofiber mats incorporated with CNCs prepared from avicel (microcrystalline) cellulose are fabricated [79]. It has been found that the strength of the nanofibers is improved by 30% with loading of 1 wt.% CNCs. The latter acted as nucleating agent of PLA crystallization leading to increased crystallinity of PLA in the resulting nanocomposite fibers. In another work by Shi et al., PLA/CNCs electrospun fiber mats from a solvent mixture consisting of N,N'-dimethylformamide (DMA) and chloroform were prepared and characterized in terms of in vitro degradation properties [80]. The obtained nanocomposite mats showed 5-fold and 22-fold increase in tensile stress and Young's modulus upon the addition of 5 wt.% CNCs, and they undergo faster degradation in phosphate-buffered saline (PBS) solution than that in neat PLA mats. These types of materials exhibiting superior mechanical and fast degradation properties are particularly interesting for making TE scaffolds

**Figure 5.** Confocal laser scanning microscopy images of hDFCs loaded in electrospun cellulose/CNCs nanocomposite nanofibers: (a) cultured for 3 days, (b) cultured for 7 days, and (c) the 3D view of electrospun nanofibers with cells.

Scale bar: 50 µm. Reprinted with permission from ref [76]. Copyright 2014 American Chemical Society.

Owing to the fact that CNCs are highly polar and show high tendency for the formation of hydrogen bonding promoting agglomeration, dispersing aqueous or nonpolar-based CNCs in a PLA matrix is highly tedious and time consuming. To overcome this hurdle, Li et al. proposed an alternative approach based on a water-in-oil (W/O) emulsion system consisting of a dispersed phase of CNCs aqueous suspension and an immiscible continuous phase of PLA solution [81]. The electrospun fiber mats prepared from these system showed either a coreshell or hollow structure depending on the emulsion droplet size, and the CNCs are aligned along the core or on the wall of the hollow cylinder (Figure 6). Upon the addition of 5 wt.% CNCs, a drastic increase in stiffness (Young's modulus), i.e., up to 549% and maximum increase

targeted at short-term application CNCs.

262 Cellulose - Fundamental Aspects and Current Trends

**Figure 6.** Correlations between emulsion droplet size (a ~6 µm, b ~3 µm, and c <1 µm) and electrospun fiber structure (d and g represent core-shell; e and f, hollow cylinder; and h, CNC aggregation). Reprinted with permission from ref [81]. Copyright 2013 American Chemical Society.

Zhou et al. used PLA grafted with maleic anhydride (MPLA) in order to advance the interfacial tension between the hydrophobic PLA matrix and the hydrophilic CNCs [82]. The fiber mats electrospun from MPLA and CNCs (5 wt.%) showed reduced diameter and polydispersity with increased CNCs content, and improved thermal and mechanical properties (e.g., tensile strength increased up to more than 10 MPa). The suitability of scaffolds for in vitro degradation and cytocompatability are tested using human adult adipose (derived from mesenchymal stem cells, hASCs; see Figure 7). Furthermore, it has been demonstrated that the cells could attach and proliferate in the entire scaffold (not only on the surface but also deep inside the fiber mats) and induce ordered cellular organization in fiber alignment direction. Results showed that the scaffolds are highly biocompatible, biodegradable, and cytocompatible, demonstrat‐ ing that they have high potential to be used in bone tissue engineering [82].

**Figure 7.** Response of hASCss to PLA/CNC and MPLA/CNC nanofibrous scaffolds after 7 days of culture. Fluores‐ cence micrographs of stained cells consisting of live (green) and dead (red) cells for PLA (a); PLA/CNC-5 (b); MPLA (c); and MPLA/CNC-5 (d) scaffolds, scale bar represents 75 µm; proliferation viability of cells (e). Reproduced with permission from ref [82]. Copyright 2013 American Chemical Society.

Nanocomposite fiber mats based on biodegradable poly(ε-caprolactone), PCL, widely used in TE, is also prepared by means of electrospinning by incorporating with CNCs [83]. The inclusion of CNCs with increasing concentrations into the PCL matrix induced considerably positive effects on the mechanical, thermal, and surface properties. The tensile strength and modulus are increased to 36% (with 10 wt.% CNCs) and 167% (with 15 wt.% CNCs). In another study, electrospun PCL nanofibers are reinforced with CNCs, which are obtained from ramie cellulose fibers. In this case, in order to improve the interfacial tension with the PCL matrix, the surface of CNCs is chemically modified with low molecular weight PCL diol [84]. Although this approach yielded unexpected results on the morphology of the nonwoven mats in which the individual nanofibers became annealed during the electrospinning process, the mechanical properties of the nanofibers are significantly improved after reinforcing with 2.5 wt.% CNCs. For example, the Young's modulus and the tensile strength are increased up to 1.5-fold and many folds compared to those of pure PCL mats.

As CNCs are dispersible in water, a direct mixing of CNCs with several water soluble polymers is feasible. As a consequence, several research groups have attempted to develop multifunc‐ tional electrospun scaffolds based on water-soluble polymer matrices applicable for TE applications, such as polyethylene oxide (PEO) [85, 86] and polyvinyl alcohol (PVA) [74, 84]. In the case of nanofiber mats electrospun from PVA and CNCs aqueous dispersion, the incorporation of CNCs displayed a considerable reinforcement effect, in particular, on the aligned electrospun PVA fiber mats compared with the isotropic ones. For instance, the aligned mats demonstrated a 35% and 45% increase in Young's modulus and tensile strength compared to the isotropic mats. Moreover, aligned PVA/CNCs fibers yielded a lower diameter compared to the fibers that are produced from PVA alone. With addition of 15 wt.% CNCs the tensile strength and Young's modulus are further increased to 95% and 118% for the aligned PVA/ CNCs, clearly demonstrating that the CNCs with high aspect ratio can align inside the polymer matrix during electrospinning because of the higher shear force of the polymer jet, thereby causing tremendous increase in the physical properties of the material [74]. In a similar work carried out by Peresin et al., the reinforcement effect of CNCs on electrospun PVA with different concentrations of acetyl groups and CNCs is studied, and the results showed a significant improvement in the mechanical properties [84]. It is suggested that the higher hydrolysis degree of PVA caused a strong interaction between PVA and CNCs, led to effective reinforcement of the fiber mats, which can be related to the reinforcing effect of the dispersed phase of CNCs, via the percolation network held by hydrogen bonds. The PEO/CNCs electrospun nanocomposite fiber mats are also prepared by using different concentration of wood-based CNCs (up to 20 wt.%). In this case, upon increasing the CNCs concentration in electrospinning solution, more uniform and finer nanofibers are formed [85], due to an enhanced electrical conductivity of the spinning solution. Furthermore, the tensile strength and Young's modulus are substantially improved 152% and 180% upon the addition of nanofiller CNCs. This enhanced mechanical property is due to the efficient stress transfer from PEO to CNCs stemming from their strong hydrogen bonding interactions and uniform dispersion, as well as high alignment of CNCs in the electrospun nanocomposite fibers.

Generally, the incorporation of CNCs can decrease the fiber diameter of the electrospun nanocomposite fiber mats, and this can impose some limitations of these system in TE applications. One such example is cellular infiltration which can be hindered by decreasing the scaffolds pore size/fiber diameter. Zhou et al. reported that 24% decrease in fiber diameter for the electrospun nancomposite PEO/CNCs when incorporated with 20 wt.% [85]. Whereas only a 19% reduction in fiber diameter is observed for the nanocomposite fiber mats electro‐ spun from MPLA/CNCs upon incorporation of 5 wt.% CNCs. This limitation can be alleviated by adopting the electrospinning technique to increase the pore size of the scaffolds [82]. It is known that the mechanical stability of the electrospun scaffolds is weakened by the increased porosity and pore size. In this case, incorporation of nanofiller CNCs to reinforce or impart higher mechanical strength of the nanofibrous scaffolds is highly useful strategy, while offering effective and appropriate 3D scaffolds for the accommodation and sufficient migration or infiltration of cells.

### **6. Summary and outlook**

**Figure 7.** Response of hASCss to PLA/CNC and MPLA/CNC nanofibrous scaffolds after 7 days of culture. Fluores‐ cence micrographs of stained cells consisting of live (green) and dead (red) cells for PLA (a); PLA/CNC-5 (b); MPLA (c); and MPLA/CNC-5 (d) scaffolds, scale bar represents 75 µm; proliferation viability of cells (e). Reproduced with

Nanocomposite fiber mats based on biodegradable poly(ε-caprolactone), PCL, widely used in TE, is also prepared by means of electrospinning by incorporating with CNCs [83]. The inclusion of CNCs with increasing concentrations into the PCL matrix induced considerably positive effects on the mechanical, thermal, and surface properties. The tensile strength and modulus are increased to 36% (with 10 wt.% CNCs) and 167% (with 15 wt.% CNCs). In another study, electrospun PCL nanofibers are reinforced with CNCs, which are obtained from ramie cellulose fibers. In this case, in order to improve the interfacial tension with the PCL matrix, the surface of CNCs is chemically modified with low molecular weight PCL diol [84]. Although this approach yielded unexpected results on the morphology of the nonwoven mats in which the individual nanofibers became annealed during the electrospinning process, the mechanical properties of the nanofibers are significantly improved after reinforcing with 2.5 wt.% CNCs. For example, the Young's modulus and the tensile strength are increased up to 1.5-fold and

As CNCs are dispersible in water, a direct mixing of CNCs with several water soluble polymers is feasible. As a consequence, several research groups have attempted to develop multifunc‐ tional electrospun scaffolds based on water-soluble polymer matrices applicable for TE applications, such as polyethylene oxide (PEO) [85, 86] and polyvinyl alcohol (PVA) [74, 84].

permission from ref [82]. Copyright 2013 American Chemical Society.

264 Cellulose - Fundamental Aspects and Current Trends

many folds compared to those of pure PCL mats.

During the past few years, the need to utilize biomaterials such as cellulose nanocrystals for tissue engineering application has increased dramatically. CNCs as a potential nanofiller and reinforcement material have created a significant interest especially in the design and fabri‐ cation of three-dimensional porous scaffold materials for the growth of 3D tissue. Scaffolds prepared from the CNC-based nanocomposites using electrospinning technique have dem‐ onstrated to be highly suitable to better mimic the extracellular matrix. Regardless of the considerable numbers of different polymeric (natural and synthetic) materials used for scaffold preparation, incorporation of CNCs has significantly improved the mechanical properties, porosity, and thus the adhesion, migration, and proliferation of the cells. Even though only a very few and no in vivo biological tests have been carried out in order to access the performance of scaffolds for practical applications, several studies are in progress to develop novel multi‐ functional biomaterials with tailored properties for tissue engineering applications.

### **Author details**

Tamilselvan Mohan1\*, Silvo Hribernik2 , Rupert Kargl2 and Karin Stana-Kleinschek2

\*Address all correspondence to: tamilselvan.mohan@uni-graz.at; tamilselvan.mo‐ han@gmail.com

1 Institute of Chemistry, University of Graz, Graz, Austria

2 Institute of Materials and Design, University of Maribor, Maribor, Slovenia

### **References**


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reinforcement material have created a significant interest especially in the design and fabri‐ cation of three-dimensional porous scaffold materials for the growth of 3D tissue. Scaffolds prepared from the CNC-based nanocomposites using electrospinning technique have dem‐ onstrated to be highly suitable to better mimic the extracellular matrix. Regardless of the considerable numbers of different polymeric (natural and synthetic) materials used for scaffold preparation, incorporation of CNCs has significantly improved the mechanical properties, porosity, and thus the adhesion, migration, and proliferation of the cells. Even though only a very few and no in vivo biological tests have been carried out in order to access the performance of scaffolds for practical applications, several studies are in progress to develop novel multi‐

functional biomaterials with tailored properties for tissue engineering applications.

, Rupert Kargl2

\*Address all correspondence to: tamilselvan.mohan@uni-graz.at; tamilselvan.mo‐

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