**3.1. Tissue engineering**

Tissue engineering and regenerative medicine have been important research areas that aim to repair and replace malfunction tissues or organs [23]. Assistive material or system is produced in order to support tissue generation which then can continue growing and functioning like original tissue in the body. Ideal three‐dimensional (3D) tissue scaffolds must have certain characters to promote new tissue formation. Polymer‐based material for 3D tissue scaffolds needs to possess high porosity, high surface area, suitable biodegradability, and good structural integrity. Human bodies are made up of complex and sensitive biological systems. Therefore, thorough attention needs to be taken in developing materials for tissue regeneration. Polysac‐ charide‐based materials have since been of interest because of their important properties for biomedical application such as biocompatibility, biodegradability, and low cost [24]. They have acceptable response to the host and have ability to promote cell proliferation and adhesion. The emerging technology has investigated and explored many potential new forms of biomaterials for this purpose.

Halloysite nanotubes have been of interest since decades as biomaterials and fillers in com‐ posite scaffolds. A study by Liu et al. used this clay in alginate matrix to construct porous tissue engineered scaffolds [25]. They have successfully produced scaffold with 96% porosity. The composite scaffolds showed increased mechanical properties of alginate where higher compressive strength and modulus than pure alginate scaffold were obtained in dry and wet states. Halloysite nanotubes assisted in cell attachment and improved the stability against enzymatic degradation. Another attempt reported that chitosan/alginate/halloysite nanotube undergo amine treatment which later showed better cell growth and adherence than nonami‐ nated composite scaffold [26]. Biomimetic synthetic scaffold was fabricated with inclusion of amorphous silica into alginate hydrogel [27]. They embedded bone cells, osteoblast‐related SaOS‐2 cells, and osteoclast‐like RAW 264.7 cell into the hydrogel beads. The bead encapsu‐ lation of bone cells is a useful technique to produce bioactive programmable hydrogels. It is observed that it does not impair the viability of the encapsulated cells. Furthermore, incorpo‐ ration of nanoceramic may improve the capability of polymeric scaffold for tissue regeneration. A study found that as‐fabricated alginate/nano‐TiO2 needles nanocomposites by lyophiliza‐ tion technique contain well controlled swelling and degradation compared to neat alginate scaffold [28].

A unique honeycomb composite of mollusca shell matrix and calcium alginate was fabricated to carry cells for soft tissue, skin, bone, and cartilage tissue regenerative therapies [29]. The composite was produced by frozen and treated mixture of *Anodonta woodiana* shell powder and sodium alginate with hydrochloric acid. It was transplanted into rats for 7, 14, 42, and 70 days. The composite displayed honeycomb structure under laser confocal microscope. This composite has significant mechanical properties, good biological safety over 70‐day period, and lower degradation rate compared to the calcium carbonate (control). The regeneration of soft tissue requires substitutes that exhibit mechanical properties similar to native tissue. Thin saloplastic membranes from chitosan/alginate polyelectrolyte complexes containing different concentration of sodium chloride were prepared [30]. The membranes are resistant to degra‐ dation by lysozyme and stale at different pH. With high salt concentration, the water uptake and tensile moduli were increased, but decreasing the ultimate strength. High proliferation rates and viability of L929 fibroblasts were demonstrated. Structural modification to bacterial cellulose/alginate scaffold was constructed by two procedures, first is producing composite sponge bacterial cellulose/alginate (BCA) by crosslinking and freeze drying, and second is by reversing the previous procedure [31]. These procedures resulted in open and interconnected porous structure and thus lift up the problem of limited *in vivo* application due to dense outer layer of scaffolds.

#### *3.1.1. Bone tissue engineering*

Being the largest natural polymer available is the most advantageous character for material sustainability and renewability. Cellulose has been tremendously applied in the production of cardboard and paper [19]. Current development of cellulose shows its potential in biomedical and biotechnological implementation. It is used in bioseparation, adsorbent for sewage

Fabrications of polysaccharide‐based composites containing different kind of reinforcements

Tissue engineering and regenerative medicine have been important research areas that aim to repair and replace malfunction tissues or organs [23]. Assistive material or system is produced in order to support tissue generation which then can continue growing and functioning like original tissue in the body. Ideal three‐dimensional (3D) tissue scaffolds must have certain characters to promote new tissue formation. Polymer‐based material for 3D tissue scaffolds needs to possess high porosity, high surface area, suitable biodegradability, and good structural integrity. Human bodies are made up of complex and sensitive biological systems. Therefore, thorough attention needs to be taken in developing materials for tissue regeneration. Polysac‐ charide‐based materials have since been of interest because of their important properties for biomedical application such as biocompatibility, biodegradability, and low cost [24]. They have acceptable response to the host and have ability to promote cell proliferation and adhesion. The emerging technology has investigated and explored many potential new forms of

Halloysite nanotubes have been of interest since decades as biomaterials and fillers in com‐ posite scaffolds. A study by Liu et al. used this clay in alginate matrix to construct porous tissue engineered scaffolds [25]. They have successfully produced scaffold with 96% porosity. The composite scaffolds showed increased mechanical properties of alginate where higher compressive strength and modulus than pure alginate scaffold were obtained in dry and wet states. Halloysite nanotubes assisted in cell attachment and improved the stability against enzymatic degradation. Another attempt reported that chitosan/alginate/halloysite nanotube undergo amine treatment which later showed better cell growth and adherence than nonami‐ nated composite scaffold [26]. Biomimetic synthetic scaffold was fabricated with inclusion of amorphous silica into alginate hydrogel [27]. They embedded bone cells, osteoblast‐related SaOS‐2 cells, and osteoclast‐like RAW 264.7 cell into the hydrogel beads. The bead encapsu‐ lation of bone cells is a useful technique to produce bioactive programmable hydrogels. It is observed that it does not impair the viability of the encapsulated cells. Furthermore, incorpo‐ ration of nanoceramic may improve the capability of polymeric scaffold for tissue regeneration. A study found that as‐fabricated alginate/nano‐TiO2 needles nanocomposites by lyophiliza‐

treatment, cell suspension culture, and wound healing system.

were subjected to many applications.

70 Composites from Renewable and Sustainable Materials

**3.1. Tissue engineering**

biomaterials for this purpose.

**3. Applications of polysaccharide composite biomaterials**

Scaffolds fabrication in bone tissue engineering becomes preferable alternative to autografts and allografts which require surgical transplant of tissue or bone whether from the patient's own body or from a donor, respectively. These procedures often suffer from limited availability and risks of immunogenicity [32]. The performance of scaffolds for hard tissue critically depends on their mechanical and biological properties. Reinforcement of nanomaterials inside polysaccharide matrix is always proposed to increase the material surface area for enhanced cell adhesion and proliferation.

A blend of alginate and chitosan was added with nanosized bioactive silica (SiO2) particles to provide biomineralization capability and polymer stiffness [33]. The composite scaffolds showed increased protein adsorption, controlled swelling ability, and improved apatite deposition without significant cytotoxicity toward osteolineage cells. Nanoscale fibers have been suggested to be effective reinforcing agents because of their resemblance to the fibrous structures of bone tissue bone extracellular matrix (ECM). A composite was developed by unique combination of wet electrospinning, particulate leaching, and freeze drying of starch/ silk fibroin [34]. Silk fibroin has slow degradation rate with high oxygen permeability and thus is suitable for slow regeneration of tissue. Hadisi et al. fabricated the silk fibroin nanofibers by wet spinning directly via wet electrospinning using methanol coagulation bath before incorporating the chopped electrospun nanofibers into the starch matrix, followed by partic‐ ulate leaching and freeze drying. The silk fibroin‐containing starch hydrogel was further coated with calcium phosphates for better compatibility with the surrounding tissues. The viability of osteoblast‐like cells (MG63) exposed to the composites' extracts was significantly higher than that of the pure starch.

Hydroxyapatite (HAp) is the main inorganic component of natural bone that has been extensively used in many biocomposites to boost osteoconductivity and reinforce the structure of polymer‐based bone scaffolds [35, 36]. The formation of bone‐like apatite on scaffolds can be seen through the detection of calcium phosphates on the material surfaces. Incorporation of HAp nanoparticles in carrageenan [37], alginate [38, 39], cellulose [40, 41], and chitosan [42] displayed favorable site for bone cell adhesion and tissue regeneration compared to the neat polysaccharide scaffolds. The preparation of HAp‐containing composites can be carried out either by using conventional mixing technique or by precipitating HAp crystals on the polymer matrices [36]. Mixture of two or more types of polysaccharides with HA like in Sharma et al. were believed to generate more synergistic effect to better mimic to the bone extracellular matrix, which comprises a variety of components [43].

#### *3.1.2. Skin tissue engineering*

Skin is the largest organ of human body. It serves as the first protection to human from environmental and surrounding threat. Fabrication of quaternary composite scaffold using chitosan, alginate, gelatin, and silk fibroin has successfully produced scaffold of 88% porosity with good mechanical stability [44]. L929 fibroblast cell cultured onto this quaternary compo‐ site scaffold showed good viability, adhesion, and proliferation, thus indicating the great prospect of the scaffold for skin tissue engineering. Boateng et al. studied two different methods for wound dressing to test their adhesive properties [45]. Solvent cast films and freeze‐ dried wafers containing polyethylene oxide (polyox) and carrageenan or sodium alginate. Wafers and films produced demonstrated high detachment force indicating strong interactions between polymers and the model wound surface. The adhesive properties were evaluated using attenuated reflectance Fourier transform infrared spectroscopy by monitoring the diffusion of mucin solution. The diffusion of mucin solution as model protein was faster for the wafer form than the film.

Wound dressings with antimicrobial and antiinflammatory properties are favorable besides the general noncytotoxic requirement. The gel‐forming ability of polysaccharide materials helps in dressing application and removal without much pain to the skin. Incorporation of certain fillers to the dressing can provide additional function to the wound dressing to meet patients' needs. Encapsulation of antimicrobial and antiinflammatory drugs into wound dressing is the most common attempt for this purpose. The previous polyox/carrageenan composite has been loaded with diclorofenac and streptomycin to enhance the healing effect of wound [46]. The dressing showed higher zones of inhibition against three microbes compared to the individual drugs zones of inhibition. The insertion of diclorofenac can prevent inflammation while streptomycin can prevent the wound infections. However, adding multiple drugs into wound dressing without disturbing the healing function of the dressing is quite challenging. Thus, several studies have been done to incorporate other materials as antimicrobial agents, such as essential oil [47] and metal oxide [48], inside wound dressing to support its purpose.

#### *3.1.3. Neural tissue engineering*

viability of osteoblast‐like cells (MG63) exposed to the composites' extracts was significantly

Hydroxyapatite (HAp) is the main inorganic component of natural bone that has been extensively used in many biocomposites to boost osteoconductivity and reinforce the structure of polymer‐based bone scaffolds [35, 36]. The formation of bone‐like apatite on scaffolds can be seen through the detection of calcium phosphates on the material surfaces. Incorporation of HAp nanoparticles in carrageenan [37], alginate [38, 39], cellulose [40, 41], and chitosan [42] displayed favorable site for bone cell adhesion and tissue regeneration compared to the neat polysaccharide scaffolds. The preparation of HAp‐containing composites can be carried out either by using conventional mixing technique or by precipitating HAp crystals on the polymer matrices [36]. Mixture of two or more types of polysaccharides with HA like in Sharma et al. were believed to generate more synergistic effect to better mimic to the bone extracellular

Skin is the largest organ of human body. It serves as the first protection to human from environmental and surrounding threat. Fabrication of quaternary composite scaffold using chitosan, alginate, gelatin, and silk fibroin has successfully produced scaffold of 88% porosity with good mechanical stability [44]. L929 fibroblast cell cultured onto this quaternary compo‐ site scaffold showed good viability, adhesion, and proliferation, thus indicating the great prospect of the scaffold for skin tissue engineering. Boateng et al. studied two different methods for wound dressing to test their adhesive properties [45]. Solvent cast films and freeze‐ dried wafers containing polyethylene oxide (polyox) and carrageenan or sodium alginate. Wafers and films produced demonstrated high detachment force indicating strong interactions between polymers and the model wound surface. The adhesive properties were evaluated using attenuated reflectance Fourier transform infrared spectroscopy by monitoring the diffusion of mucin solution. The diffusion of mucin solution as model protein was faster for

Wound dressings with antimicrobial and antiinflammatory properties are favorable besides the general noncytotoxic requirement. The gel‐forming ability of polysaccharide materials helps in dressing application and removal without much pain to the skin. Incorporation of certain fillers to the dressing can provide additional function to the wound dressing to meet patients' needs. Encapsulation of antimicrobial and antiinflammatory drugs into wound dressing is the most common attempt for this purpose. The previous polyox/carrageenan composite has been loaded with diclorofenac and streptomycin to enhance the healing effect of wound [46]. The dressing showed higher zones of inhibition against three microbes compared to the individual drugs zones of inhibition. The insertion of diclorofenac can prevent inflammation while streptomycin can prevent the wound infections. However, adding multiple drugs into wound dressing without disturbing the healing function of the dressing is quite challenging. Thus, several studies have been done to incorporate other materials as antimicrobial agents, such as essential oil [47] and metal oxide [48], inside wound dressing to

higher than that of the pure starch.

72 Composites from Renewable and Sustainable Materials

*3.1.2. Skin tissue engineering*

the wafer form than the film.

support its purpose.

matrix, which comprises a variety of components [43].

Central nervous system diseases are usually caused by the death of neurons and progressive loss of its function. Current developments in neural technology have opened up possibilities of nerve tissue regeneration. Two potential natural polymers for nerve tissue engineering were combined with hyaluronic acid and heparan sulfate via freeze‐drying technique [49]. The composite scaffolds demonstrated highly homogenous and interconnected pores with porosity above 96%. The presence of hyaluronic acid and heparan sulfate has promoted the adhesion of initial neural stem and progenitor cells. Nanofiber‐hydrogel of polycaprolactone (PCL) and sodium alginate composite was prepared by electrospinning [50]. The fibrous form of this scaffold is to provide suitable environment for regeneration of the peripheral nerve injury. This kind of combination of natural and synthetic polymers has long been worked on to utilize the mechanical properties of PCL while preserving alginate hydrophilicity to support cell adhesion. The composite displayed that a good suture pulled out strength and assists the human mesenchymal stem cells (hMSCs) viability, adhesion, proliferation, and neurogenic differentiation in neural induction media.
