Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering

*Periasamy Srinivasan and Dar-Jen Hsieh*

## **Abstract**

The rise of tissue engineering and regenerative medicine (TERM) is a developing field that focuses on the advancement of alternative therapies for tissue and organ restoration. Collagen scaffold biomaterials play a vital role as a scaffold to promote cell growth and differentiation to promote the repair and regenerate the tissue lesion. The goal of this chapter will be to evaluate the role of supercritical carbon dioxide extraction technology in the production of collagen scaffold biomaterials from various tissues and organs and relate it to the traditional decellularization techniques in the production of collagen biomaterials for TERM. Therefore, we will study the collagen scaffold biomaterials produced using supercritical carbon dioxide extraction technology and their characteristics, such as chemical-physical properties, toxicity, biocompatibility, *in vitro* and *in vivo* bioactivity that could affect the interaction with cells and living system, relative to traditional decellularization technique-mediated collagen scaffolds. Furthermore, the chapter will focus on supercritical carbon dioxide extraction technology for the production of collagen scaffolds biomaterial appropriate for TERM.

**Keywords:** supercritical carbon dioxide extraction technology, tissue engineering, regenerative medicine, biomaterial, collagen scaffold, biocompatibility

## **1. Introduction**

Tissue engineering advanced from the field of biomaterials development and denotes the practice of combining cells, tissue scaffolds, and bioactive signal molecules. These tissue scaffolds are produced by various decellularization processes, such as chemical and physical methods. Tissue scaffolds, cells, and biologically active signal molecules are the three key elements for tissue and organ reparation. Tissue engineering is defined as "*an interdisciplinary field of research that applies the principles of engineering and the life science toward the development of biological substitutes that restore, maintain or improve tissue function*" [1]. Regenerative medicine is a wide field that comprises tissue engineering but also integrates research on self-healing in which the body uses its systems, sometimes with help of foreign biological material to reconstruct and rebuild tissues and organs. The terms tissue engineering and "regenerative medicine" have become largely interchangeable, as the field hopes to focus on cures as an alternative for the treatment of complex, mainly chronic diseases (e.g. Diabetic wound healing, burn wounds).

## **2. Tissue engineering and regenerative medicine**

Tissue engineering and regenerative medicine (TERM) have been projected and established for almost 30 years. Though many fruitful challenges in tissue regeneration have been attained, TERM is still in its infancy stage and most of the vital questions remain to be answered, including the selection of cell sources, development of tissue-specific materials, and construction of complex organs. The most important is the *in vivo* mechanism of the formation of new tissue and organ employing the tissue-engineered biomaterials, and the process to resemble and transform to native tissue and organ. The subsequent transformation and final destination of the biomaterials remain to be the serious apprehensions in this dynamically emerging field. Addressing these queries is significant to the effectiveness, stability, and security of the clinical application of tissue-engineered biomaterials [2].

Tissue and organ repair remain a clinical issue and challenge. Entirely restoring or regenerating damaged tissues and organs and reestablishing their functions have been a vision of medical society. The emergence of tissue engineering and regenerative medicine (TERM) makes it possible. TERM is a developing field that focuses on the advancement of alternative therapies for tissue and organ restoration [2, 3]. TERM is an extremely multidisciplinary arena, in which bioengineering and medicine unite. It is constructed on integrative approaches using scaffolds, cells, growth factors, nanomedicine, and other techniques to pass on the restrictions that presently exist in the hospitals. Certainly, TERM overall aim is to encourage the formation of new functional tissues, rather than just implanting tissue and bone parts [2]. TERM is a multifaceted science and associates basic sciences such as materials science, biomechanics, cell biology, and medical sciences to comprehend functional tissue and organ restoration and reconstruction. The world's population is aging and the trend is escalating. There is a severe global shortage of tissues and organs for transplantation. TERM has the potential to meet the requirements of the forthcoming needs of patients [2, 4]. TERM aims to create a three-dimensional (3D) cell-biomaterial composite, that possesses a comparable function as living tissue and organ and is employed to restore or regenerate damaged tissue and organ. The basic condition for the 3D composite is to support cell growth, nutrition and waste transport and gas exchange. TERM typically employs the following strategies, cell-biomaterial composite, in which cell-seeded biomaterials are implanted into the body to restore and regenerate tissues and organs; stem cell transplantation; and biomaterial implanted into the body and undertake the process of tissue integration [3]. Scaffolds are vital for tissue engineering approaches for several reasons; as a three-dimensional structure, they offer volume fill, mechanical integrity and a surface that can afford chemical and architectural guidance for regenerating tissues [5]. The three vital elements in TERM are cells, scaffolds and signals (**Figure 1**). Several decellularization techniques had been used for the production of collagen scaffolds for TERM application, including the supercritical carbon dioxide (SCCO2) extraction technology to be discussed here in this article.

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

## **3. Collagen scaffolds-biomaterial for TERM**

Collagen-based biomaterial application in the field of TERM has been significantly increasing over the past decades. Collagen owns the main advantages as it is biodegradable, biocompatible, easily available and highly versatile. However, collagen is a protein, therefore it is problematic to sterilize without altering its native structure. Collagen-based biomaterials developed for TERM were intended to provide a functional biomaterial for use in TERM from the laboratory bench to the patient bedside [6]. Collagen is present in all connective tissue and makes it one of the most studied biomolecules of the extracellular matrix (ECM). It is the major component of skin and bone and constitutes approximately 25–35% of mammalian total dry weight [7]. Until


**Table 1.** *Collagen types, forms and distribution [6].* now, 29 diverse collagen genotypes have been characterized and all depict a typical triple helix structure. Fiber form of collagens are types I, II, III, V and XI. Collagen molecules are made up of three α chains that assemble due to their molecular structure. Each α chain is made up of more than a 1000 amino acids based on the repeated sequence -Gly-X-Y-. The vital part is the presence of glycine at every third amino acid position to permit for a tight triple-helical packaging of the three α polypeptide chains. In the tropocollagen molecule the X and Y positions are mostly filled by proline and 4-hydroxyproline [6, 7]. Though numerous types of collagens (**Table 1**) have been defined, only a few types are used to yield collagen-based biomaterials. Currently, type I collagen is the gold standard in the field of TERM.

## **4. Collagen immunogenicity and biocompatibility**

Medical application of collagen biomaterial needs to make a clear difference between immunogenicity and antigenicity. Immunogenicity is triggering an immune response; however, antigenicity denotes the interaction between the antibodies and the antigenic epitopes. Collagen mediated immune response primarily targets epitopes in the telopeptide region at each end of the tropocollagen molecule. The polymerized collagen fibrils conformity of the helical part and the amino acid sequence on the surface can influence the immunologic profile of the collagen molecule [7]. Type I collagen is an appropriate biomaterial for implantation meanwhile only an insignificant number of people have humoral immunity against type I collagen. In addition, a simple serologic test can validate an allergic reaction in response to type I collagenbased biomaterial. It is most crucial to discuss that collagen immunogenicity which is relevant to collagen molecules that are made up of an acellular ECM and the utmost adverse immune responses that have been come across with an acellular scaffold are not necessarily initiated from the collagen molecule itself. Incomplete decellularization with the presence of remaining oligosaccharide α-Gal and DNA is the common reason for acute immune responses and subsequent acellular ECM rejection [7, 8].

## **5. Traditional decellularization of tissues and organs for collagen biomaterial**

The traditional decellularization techniques involve long duration and increased cost as well as long-term washing of the tissue material from the residual and traces of the chemicals used. Despite the numerous decellularization process that exists, it is necessary to go through a lot of parameters for multiple reasons in the decellularization process (**Table 2**). The decellularization process aims to remove the cellular material of the donor, antigens, and potential pathogens. In addition, the most critical issue is to offer the conservation of the structural organization of an ECM with the set of functions inherent in it. Therefore, the optimization of these decellularization methods and the pursuit of improved methods are still ongoing [9]. At present, numerous procedures for decellularization of tissues were employed that include the treatment by detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100, etc., and treatment by enzymes such as trypsin, deoxyribonuclease (DNase), and ribonuclease (RNase). Other methods include alkali treatment, as well as cyclic freezing-thawing and high-pressure action up to 1 GPa, which have been tried (**Table 3**) [9, 23].

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*


#### **Table 2.**

*Decellularization techniques used for tissues, organs and their advantages and disadvantages.*



#### **Table 3.**

*Porcine tissues and organs had been decellularized by the SCCO2 process applied in different medical applications.*

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

#### **5.1 Tissues and organs**

Currently, the most frequently employed decellularization technique for tissue and organ to manufacture scaffolds employing detergents are sodium dodecyl sulfate, Triton X-100, and CHAPS, branded as ionic, non-ionic, and zwitterionic detergents, respectively. Detergents were found to be effective in the decellularization of the tissues and organs, including the removal of lipids [24, 25]. Enzymes such as nucleases are also employed in limited decellularization protocols to eliminate the DNA from the tissues and organs [25, 26]. However, detergent-employed decellularization often disrupts the ECM by changing tertiary and quaternary structures of the proteins. SDS is known to proficiently eliminate glycosaminoglycans, thereby destructing the collagen structure [27]. Detergent decellularization is known to reduce the number of valuable growth factors that are vital for the recellularization of tissues. Moreover, residual surfactants and chemicals often cause cytotoxicity [28] inducing adverse effects in the recellularization of tissue and organ scaffolds (**Table 3**) [13, 26, 27].

## **5.2 Adipose tissue**

Common traditional decellularization methods for adipose tissue include numerous freezing-thawing cycles, extraction of lipids with isopropanol, and enzymatic treatment. Developing a protocol for the preparation of ECM from adipose tissue in an accessible and eco-friendly manner will promote the upgrading of the methods of tissue engineering with the use of autologous material [9, 27, 29–31].

#### **5.3 Pericardium**

The existing techniques for pericardium decellularization include the treatment by non-ionic detergents such as Triton X-100, 3-3-chloroamidopropyl-dimethylammonio-1-propanesulfonate (CHAPS), ionic detergents (SDS), sodium deoxycholate, alkalis, and enzymes such as trypsin with EDTA. However, the adverse effects are commonly occurred by the above-mentioned procedures on the ECM structure and composition. The detergents such as SDS and Triton X-100 were found to denature the collagen of the ECM which was elucidated by staining fluorescently labeled collagen hybridizing peptide. CHAPS and sodium deoxycholate altered the structural organization of collagen established by the recording of the second harmonic signal and transmission electron microscopy. Decellularization of bovine pericardium tissue using Triton X-100 reduces the concentration of glycosaminoglycans by ~62–66%, and in an alkaline solution, by ~88.6%, at the initial concentration of ~0.6 mg/g [9, 27, 29–31].

#### **5.4 Bone**

The current standard method employed for bone decellularization is by hightemperature sintering at 300–1300°C. Moreover, this procedure completely removes any possible zoonotic infectious agents, in addition to the immunogenic components that existed in the animal bone tissues [32]. However, the high-temperature sintering damages the intrinsic collagen and alters the porous ECM structures of the animal bones. Bone decellularization can also be carried out by various chemical agents and techniques. The chemical process includes processing the bone with acidic and alkaline solutions and organic agents, as well as detergents and enzymes, that unavoidably alter the ECM structure. Delipidation is the key factor in decellularization processing because indisputably, the residual lipids in the bone act as a barrier to cell removal, in addition to altering its biocompatibility and osseointegration [33]. Moreover, it encourages adverse reactions which can give rise to bone resorption and encapsulate fibrosis [10, 34].

## **5.5 Cartilage**

Decellularization of cartilage is challenging, due to its dense structure with lacunae. Generally, decellularization of cartilage is performed by the perfusion of detergents into the lacunae to break down the chondrocytes. In continuation, the detergents were washed out of the residual cellular fragments and nucleic acids. In another case, decellularization of cartilage was performed by treating with 0.05% Trypsin/EDTA for 1 day followed by 3% SDS for 2 days and 3% Triton X-100 for another 2 days [35]. Decellularization of the cartilage process includes a mixture of physical, chemical, and enzymatic steps [35]. Decellularization of cartilage by SDS and Triton X-10 resulted in only a 77% decrease in DNA content (262 ± 42 ng/mg) relative to the untreated cartilage. But, the key norms for medical devices, the decellularized tissue residual DNA content should be less than 50 ng/mg in decellularized materials. However, the dense nature of the cartilages reticular network of fibrous ECM is a substantial barrier for the detergents to penetrate. It is the key limitation of SDS and Triton X-100 in cartilage decellularization [35, 36]. Cartilage complete decellularization by SDS (2%) treatment for 4 or 8 h; however, 60% of the DNA remained in the decellularized cartilage [20, 37]. Decellularization of cartilage by using 1% SDS for 24 h and 2% Triton X-100 for 48 h preserved most of the ECM components with a complete chondrocyte's removal. The complete decellularization of chondrocytes and the movement of seeded cells into the scaffolds during recellularization is challenging. The decellularization process in the SDS process caused the denaturation of proteins in ECM structures, which may also destroy the protein function [20, 38]. Cartilage decellularization methods such as chemical and enzymatic methods lead to disadvantages including traces of impurities and loss of ECM scaffold structure caused by the degradation of native collagen ECM structure leading to difficulty in the recellularization of the cartilage scaffold. Porcine articular cartilage decellularized by a succession of freeze-thaw cycles and 0.1–0.5% (w/v) sodium dodecyl sulfate detergent cycles with chondroitinase ABC and hyaluronidase were employed to breakdown glycosaminoglycans, resulting in the removal of 80–90% of the DNA [22, 39].

## **6. Supercritical carbon dioxide extraction technology, an innovative and efficient approach for collagen biomaterial production**

The conditions necessary for the decellularization processing of biomaterials frequently reject the use of traditional approaches involving destructive action on the biomaterial such as high-temperature treatment, acid, and alkali, etc. A result of the search for an alternative process leads to novel processing technologies and approaches concentrating on the direction of green technology in the first place. Supercritical carbon dioxide extraction technology comes in the first place in green technology. Supercritical carbon dioxide extraction technology owns exceptional advantages that can be employed in the production of biomaterials efficiently and cost-effectively. The most vital and important advantage of SCCO2 is the option of conducting processes at low temperatures, which offers the opportunity to work with *Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

a variety of biomaterials and thermally sensitive components such as collagen [40]. In the SCCO2 process, the low surface tension encourages the penetration of CO2 into solid and colloidal structures, which makes it competently decellularize and sterilize biomaterial and medical devices with the preservation of the structure and physicochemical properties (**Table 4**) [41].

In the supercritical process, the carbon dioxide gas above a critical temperature, Tc = 31.1°C, and pressure, Pc = 73.8 bar is said to be supercritical (**Figure 2**). In this state, carbon dioxide is neither a gas nor a liquid but possesses properties of both. The critical state of carbon dioxide is established by the phase diagram in **Figure 2**; varying the temperature and pressure changes the phase from solid to liquid to gas. However, at the critical point (the intersection of Tc and Pc), the difference between the liquid and gas phases disappears. The single fluid phase of carbon dioxide at this point is supposed to be "supercritical". The decellularization of mammalian tissues was successfully carried out using the extractive properties of SCCO2 technology (**Figure 3**). To eliminate the immunogenicity of xenogeneic and allogeneic tissues requires decellularization. The decellularization process of the tissues to ECM scaffolds is to remove cells and antigens from the source tissue material. The ECM scaffold developed as an outcome of the decellularization process is the ECM consisting of proteins such as collagen, laminin, elastin, proteoglycans, and glycoproteins, as well as essential growth factors, angiogenesis factors [24]. Many porcine tissues and organs had been decellularized by the SCCO2 process (**Figures 3** and **4**) and had been applied in several different medical applications by our team as listed in **Table 5**.

#### **6.1 Aorta**


The first effort for the decellularization of the porcine aorta employing SCCO2 with the cosolvent as absolute ethanol was reported in 2008 [42]. The structural

#### **Table 4.**

*Decellularization techniques are used for tissues and organs.*

**Figure 2.** *Phase diagram of CO2.*

**Figure 3.**

*Production of collagen scaffolds by SCCO2 technology.*

analysis depicted that the addition of ethanol encourages the removal of cellular material such as nuclei and phospholipids, which was unattainable without the use of SCCO2 as a cosolvent. The results showed a decrease in the amount of phospholipids which depends on the time of processing, pressure, and rate of venting in the reactor. Altering the conditions, the lowest residual amount of phospholipids was 20%, which was attained as a result of 20 min at 15 MPa and 37°C. During the progression SCCO2 process with the ethanol system, the aorta obtains rigidity, which reflects upon the character of the stress-strain diagrams. It is related to the dehydration of the aorta tissue due to the hygroscopic nature of ethanol and SCCO2 dissolves up to 0.5% water [43]. The insignificant changes in mechanical properties and the deviations are not functionally significant [42]. This is the basics in the field of SCCO2 decellularization; however, this process was not continued, due to the fast progress of methods of decellularization using detergents, enzymes, and other physicochemical methods. The preparation of biomaterials using SCCO2 was resumed due to renovating the interest

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*


#### **Table 5.**

*Supercritical carbon dioxides principle, advantages, disadvantages and applications.*

in solving the problems of decellularization and the factors such as the growth of new instrumentation, transition to green chemistry.

In the SCCO2 decellularization process, the native collagen scaffold remains completely intact, even the smallest of the collagen strand (**Figure 5**, dermis ECM) as shown in the scanning electron microscopic photos of several different porcine tissues and organs. Therefore, we believe SCCO2 decellularization is superior to other decellularization processes and thus the holy grail technology for the preparation of

**Figure 5.** *Porcine bone derived products.*

collagen scaffolds for tissue engineering and regenerative medicine. The process of decellularization of the aorta by SCCO2 was continued in 2017 by altering the protocol using 70% ethanol and the processing was executed for 1 h at 37°C in addition 17.2 and 31 Mpa [44]. The results of histological studies and residual DNA exhibited complete elimination of the cellular debris from the aorta tissue is accomplished at 31 MPa. However, the ECM structure of the aorta is significantly altered at higher pressure, and the organization of the layers of the aorta external and internal layers is altered. These alterations in the aorta are capable of encouraging the development of embolism and aneurism in the case of grafting, which is a severe constraint for the clinical use of the aorta graft. In addition, these alterations of the aorta structure change the mechanical properties of an ECM.

To treat ischemic diseases, cardiac tissues were decellularized using SCCO2with a cosolvent of absolute ethanol, leading to the formation of a hydrogel-based on an ECM, a source of glycosaminoglycans, proteins, and growth factors [26]. To attain the determined effect, the pressure was elevated to 35 MPa, and the time of the processing was extended to 6 h. The cardiac tissues were then rinsed in a solution of DNase I for 5 days. ECM components responsible for angiogenesis are preserved in the SCCO2 decellularization; however, 1% SDS altered the ECM. Upon subcutaneous implantation of the hydrogel to mice induced angiogenesis. Subsequently encouraged the development of vessels to a significantly superior extent in comparison with the SDS treated and control gel based on type I collagen. Therefore, decellularization using the SCCO2 opens up projections for the progression of bioinks for bioprinters and the formation of three-dimensional structures based on hydrogels [26].

#### **6.2 Cornea**

The porcine cornea was decellularized by SCCO2 [45], the cornea tissue was initially subjected to osmotic shock by changing 2 M NaCl solution and deionized water. The process of SCCO2 was done with the cosolvent addition of 60% ethanol at 35 MPa and 45°C for 80 min. In this process, it is likely to eliminate cellular components from the corneal tissue with the conservation of the suitable optical properties of the cornea. However, the decrease in the quantity of glycosaminoglycans and structural proteins during the processing in SCCO2 directed to the alterations of the structural

#### *Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

organization of the corneal ECM. In the traditional procedure decellularization by Triton X-100, the effect was less noticeable. The transplantation of the SCCO2 decellularized cornea to rabbits showed regeneration of the cornea in 2 months, which confirmed the migration of keratocytes and corneal epithelial cells to the implanted cornea. In addition, no adverse rejections, inflammation, or angiopoiesis was observed in the implanted cornea. For the first time, the results of the regeneration of corneal tissues with the use of SCCO2 decellularized transplants over the long term were described. The physical decellularization method of the cornea was established previously by the destruction of cells under the action of high pressure up to 100 MPa. However, the high-pressure method involves complex and costly hardware.

The SCCO2-decellularized corneas displayed intact stromal structures and appropriate mechanical properties and had biocompatibility. Additionally, no immunological reactions and neovascularization were observed after lamellar keratoplasty in rabbits without complications. The transplanted decellularized corneas became transparent within 2 weeks of surgery. The decellularized corneas were completely re-epithelialized within 4 weeks. In conclusion, SCCO2 decellularized corneas could be an ideal and useful scaffold for corneal tissue engineering [16]. The SCCO2 technology-mediated production of the acellular porcine cornea (APC) depicted complete cells and non-collagenous protein removal relative to the Triton-sodium dodecyl sulfate decellularization process. APC presented excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future [18].

#### **6.3 Bone**

Decellularized bone tissue matrix produced by SCCO2 [46], bovine cancellous bone was treated at 35 MPa and 50°C for 30 min with 25 min in a dynamic mode at a rate of the flow of SCCO2 of 16.9 g/min and 5 min in a static mode of supercritical process. Subsequently, bovine cancellous bone was treated with a 7% solution of NaCl for 12 h first and then in a 0.1% solution of H2O2 for 48 h. On comparing lipid removal in bovine cancellous bone by SCCO2 with traditional extraction with n-hexane in a Soxhlet apparatus, the SCCO2 removed lipids 14% more efficiently. The biocompatibility of the SCCO2 decellularized bone was proved by seeding and culturing with mesenchymal stem cells. However, mechanical properties and immunogenicity of the SCCO2 decellularized bone were not determined. Similarly, xenogeneic bone decellularization [47] by SCCO2 was done by rinsing with a 3% H2O2 solution and processing in the subcritical water, and final processing in SCCO2.

The SCCO2 technology was used to produce a series of novel decellularized porcine collagen bone grafts (DPB) in an assortment of shapes and sizes (**Figure 5**, cancellous bone). The native intact collagen was preserved in the SCCO2 processed DPB was confirmed by Masson trichrome staining. The cytotoxicity and biocompatibility tests according to ISO10993 and their efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was nontoxic. *In vitro* and *in vivo* biocompatibility tests of the DPB did not show any toxic or mutagenic effects. *in vitro* cytotoxicity test, *in vivo* pyrogen study, *in vitro* mammalian cell gene mutation test, and systemic toxicity study in SD rats. The DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits. Therefore, the potential application of the SCCO2 extraction technique to generate a native decellularized bone scaffold for regeneration in human clinical trials [10]. The DPB produced by SCCO2 on alveolar socket healing after tooth extraction had promising bone regeneration properties similar to that of Bio-Oss® in a canine tooth extraction socket model [15].

The DPB produced by SCCO2 ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework in humans. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. All subjects showed improvement of enophthalmos on computerized tomography at week 8 follow-up. No replacement of implants was needed during follow-ups. Thus, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate, and low complication rate [17]. The DPB produced by SCCO2 seeded with adipose-derived stem cells (ASCs) boosted callus formation in a segmental femoral defect. The mechanism of DPB might be modulation in the expression of BMP 2 and osteocalcin, thus leading to enhanced bone regeneration and new bone formation in a rat segmental femoral defect model. Thus the DPB scaffold is an excellent biomaterial for bone tissue repair. Implantation of the DPB seeded ASCs stimulated endochondral ossification for substantial bone regeneration. The DPB seeded ASCs system is of clinical relevance for segmental defect bone regeneration [19].

#### **6.4 Acellular dermal matrix**

The SCCO2 decellularized porcine acellular dermal matrix (ADM) seeded with autologous adipose-derived stem cells (ASCs) in streptozotocin (STZ)-induced diabetes mellitus rats showed the wound healing rate increased in diabetes mellitus. Diabetes mellitus wound treated with ADM-ASCs showed a significantly higher wound healing. ADM-ASC-treated rats showed significantly increased epidermal growth factor, Ki67, and prolyl 4-hydroxylase and significantly decreased CD45. The intervention comprising ADM decellularized from porcine skin by using SCCO2 and ASCs was proven to improve diabetic wound healing. The SCCO2 produced ADM-ASCs had a positive effect on epidermal regeneration, anti-inflammation, collagen production and processing, and cell proliferation; thus, it accelerated wound healing [14].

#### **6.5 Cartilage**

Cartilage tissue engineering that combines the triads of decellularized porcine cartilage graft as a scaffold, plasma rich platelet (PRP) as signal, and chondrocytes as the cell to attenuate anterior cruciate ligament transection (ACLT)-induced OA progression and regenerate the knee cartilage in rats. The SCCO2 decellularized porcine cartilage graft (dPCG) significantly reduced the ACLT-induced OA symptoms and attenuated the OA progression. The histological analysis depicted cartilage protection by dPCG. The repair and attenuation effect were proved by dPCG in the articular knee cartilage damage as evidenced by safranin-O, type II collagen, aggrecan, and SOX-9 immuno-staining. To conclude, intra-articular administration of dPCG with or without PRP is efficient in repairing the damaged cartilage in the experimental OA model [21]. A 3D composite was constructed using SCCO2-dPCG that promotes chondrogenic marker expression *in vitro*. The *in vivo* implantation of 3D composite to cartilage defect exhibited significant regeneration by increasing the expression of Collagen type II and aggrecan. The bioengineered 3D composite by combining dPCG scaffold, chondrocytes, and PRP facilitated the chondrogenic marker expression in both *in vitro* and *in vivo* models with accelerated cartilage regeneration. This might serve the purpose of clinical treatment of large focal articular cartilage defects in humans in the near future [22].

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

#### **6.6 Nasal cartilage**

A bioactive 3D histotypic SCCO2 decellularized nasal cartilage (dPNCG) construct was engineered with adipose-derived stem cells (ADSC) and chondrocytes and cultured for 21 days. The 3D histotypic constructs produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. The SCCO2-dPNCG granules are bound to one another by extracellular matrix and proteoglycan, to form a 3D structure expressed chondrogenic markers such, as type II collagen, aggrecan, and SOX-9. The SCCO2-dPNCG substrate enabled the synthesis of type II collagen along with ECM to yield 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty [20].

#### **6.7 Atelocollagen**

Atelocollagen was prepared by using SCCO2 technology. To our knowledge, we are the first to use SCCO2 technology to produce atelocollagen. The sliced porcine skin was subjected to a proprietary SCCO2 for decellularization. The decellularized porcine skin scaffold was freeze-dried and freeze-milled to granules and subjected to enzymatic hydrolysis using pepsin in acidic conditions, then subjected to ultrafiltration for pepsin and telopeptide removal. The atelocollagen solution was filtered through a 0.2-μm filter for sterilization. The acidic atelocollagen solution was subjected to fibrillogenesis by bringing the pH to 7, then centrifuged to obtain the atelocollagen slurry. This slurry was then freeze-dried to obtain atelocollagen dry powder [12]. The whole process saves a lot of time and cost as compared to the traditional collagen purification process. Atelocollagen prepared by SCCO2 followed by pepsin digestion of the telo-peptides process showed complete removal of the telo-peptides as compared to the traditional purification process [12].

## **6.8 Skin**

The SCCO2 technology was employed to decellularize porcine skin to produce a collagen matrix (**Figure 6**). This novel collagen matrix was developed to accelerate wound healing for hard-to-heal or delayed wound healing clinical conditions. The collagen matrix produced by SCCO2 technology from porcine skin is chemically comparable and biocompatible to human skin. The SCCO2 produced collagen matrix showed complete decellularization, the chemical content was found to be type I collagen and characteristic features were similar to that of humans. The collagen matrix proved to be non-toxic in *in vitro* cytotoxicity-agar diffusion test, *in vivo* pyrogen study, *in vitro* mammalian cell gene mutation test, acute systemic toxicity study in mice, systemic toxicity study in SD rats, intracutaneous irritation test, skin sensitization study (maximization test), and muscle implant study. In the porcine excision full-thickness skin wound healing model, the collagen matrix cocultured with fibroblast and keratinocytes exhibited decreased inflammation, complete epithelization, and enhanced wound healing [11].

#### **6.9 Adipose tissue**

The SCCO2 process was used for the decellularization of adipose tissue extracellular matrix [48]. The adipose tissue was subjected to the SCCO2 process for 3 h at 18 MPa and 37°C with the addition of ethanol as the cosolvent. The decellularized adipose

#### **Figure 6.** *Porcine skin derived products.*

#### **Figure 7.** *SCCO2 decellularized biomaterials for TERM.*

tissue consisted of the extracellular matrix components and was free from lipids. The decellularized adipose tissue extracellular matrix can help the widespread coating progress the adhesion of cells due to the presence of active components such as collagen, laminin, elastin, fibronectin, and glycosaminoglycans. The coating of the decellularized adipose tissue extracellular matrix increases the proliferation of human endothelial cells isolated from umbilical vein, human adipose tissue-derived mesenchymal stem cells, human monocytic leukemia cells, and immortalized human keratinocytes on a plastic culture plate and does not induce the production of the proinflammatory

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

phenotype of macrophages. The decellularized adipose tissue extracellular matrix was used as a model for the investigation of the action of anticancer drugs on cells for breast cancer, which is similar to the native condition [49]. The SCCO2 decellularization contrasts with the prevailing methods in the rapidity and cost-effective nature. Traditional methods of decellularization of adipose tissue include several freezing-thawing cycles, extraction of lipids with isopropanol, and enzymatic treatment [49, 50]. The development of the SCCO2 decellularization for the preparation of an extracellular matrix from adipose tissue is an environmentally friendly approach that will endorse the development of the methods of tissue engineering with the use of autologous material.

We produce tissue and organ scaffold using SCCO2 extraction technology, such as liver, kidney, heart, pancreas, artery, skin, bone, cartilage, and cornea [13]. **Table 5** listed our works on the various tissue and organ scaffolds extracted by SCCO2 technology for tissue engineering applications [13]. The ultimate goal of TERM is to use the tissues and organs produced by SCCO2 from the porcine or bovine to regenerate the human tissues and organs (**Figure 7**). We hope to develop the whole animal application without any waste materials, which suits the purpose of the circular economy. Eventually, we intend to regenerate any human tissue and organ by its animal counterpart.

### **7. Conclusions**

Substantial progression in the field of TERM and scaffold biomaterials engineering by SCCO2 proposes extended potentials to acquire novel, effective achievements, which may be applied in biomedical applications. Recently, the interest in natural biomaterials produced by SCCO2 technology for medical devices production has increased, and a greater number of in-depth studies are done to better detect their likely applications related to chemical and physical characteristics and the extraction procedures, which do not modify their structural properties and biocompatibility. Tissue engineering approaches have become a valid alternative for body structure and function restoring, natural scaffold biomaterials produced by SCCO2 technology are also used as biomimetic scaffolds with controlled degradation rate *in vivo* and regeneration. *In vitro* and *in vivo* studies have shown the advantages related to natural scaffold biomaterials produced by SCCO2 technology use in the regenerative medicine field.

The SCCO2 decellularization technology as compared to other traditional processes is a minimally manipulated process and thus cost-effective, and gentle to the natural collagen scaffold ECM structure. Therefore, SCCO2 decellularized scaffolds might contain unaltered signals that are indispensable for stem cell adhesion, migration, homing, proliferation, and differentiation. No chemicals and solvents were involved in the process, therefore it is eco-friendly. It destroys bacteria and inactivates viruses during the process. SCCO2 technology costs only about 1/10th of the traditional process. Different tissues and organs from animals such as pigs, cows, horses, sheep can be used to produce decellularized scaffolds. The most important and key point is SCCO2 process drastically reduces immune rejection.

Our study indicated that the natural collagen scaffolds prepared by the SCCO2 process might be able to induce stem cell differentiation *in vivo*, with the help of the growth factors and cytokines in the microenvironment. The signal for stem cell differentiation could be pre-built by the combination of various genotypes of 29 collagen polypeptides during scaffold synthesis, which exhibits different signals in different tissues and organs that guide the stem cells to differentiate into the right cell types.

The revelation of this intrinsic signal will be our future research focus. Before that, we boldly hypothesize that any organ decellularized by the SCCO2, with the intact scaffold structure, can be reconstructed *in vivo* when implanted back into the live animal with the proper connection of blood circulation to bring in the stem cells required for the organ regeneration. We are testing this hypothesis and hope to find out soon. The application of biomaterials produced by SCCO2 technology to tissue engineering, in modern-day science is using the natural biomaterial with the most suitable performance *in vivo*, able to promote cell proliferation and differentiation in damaged tissue to restore the normal architecture of ECM. To conclude, TERM strategies particularly in the orthopedic and plastic surgery clinical field epitomize an effective and sophisticated alternative for the future, but their success firmly rests on an ever in-depth knowledge regarding the features of the scaffold biomaterial.

## **Acknowledgements**

This research was financially supported by the Southern Taiwan Science Park Bureau, (107SMIC-RC02; BX-01-03-05-108 and 108CB01), Taiwan, R.O.C.

## **Author details**

Periasamy Srinivasan and Dar-Jen Hsieh\* R&D Center, ACRO Biomedical Co., Ltd., Kaohsiung City, Taiwan

\*Address all correspondence to: dj@acrobimomedical.com

© 2022 The Author(s). Licensee IntechOpen. 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.

*Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.102438*

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## **Chapter 3**

## Nanoparticle Based Collagen Biomaterials for Wound Healing

*Kausalya Neelavara Makkithaya, Sharmila Nadumane, Guan-Yu Zhuo, Sanjiban Chakrabarty and Nirmal Mazumder*

## **Abstract**

Wounds and infections are extremely common cases that are dealt with in the medical field. Their effective and timely treatment ensures the overall well-being of patients in general. Current treatments include the use of collagen scaffolds and other biomaterials for tissue regeneration. Although the use of collagenous biomaterials has been tested, the incorporation of nanoparticles into these collagenous biomaterials is a fairly new field, whose possibilities are yet to be explored and discovered. The current chapter explores the applications of the amalgamation of collagenous biomaterials with nanoparticles, which themselves are known to be effective in the treatment and prevention of infections.

**Keywords:** wound healing, nanoparticles, nanotechnology, collagen, biomaterials

### **1. Introduction**

An injury that occurs in a quick manner, which often leaves the skin torn, cut, or punctured, or wherein the skin or any other tissues of the body undergoes acute trauma resulting in a contusion, is defined as a 'wound'. This is when the body's repair mechanism works to repair the damage by replacing the damaged tissue with newly synthesized tissue. This is characterized by a cascade of highly coordinated reactions that occur at the tissue damage region, working to restore normal tissue, which is called wound healing mechanism. This process requires nutrients and amino acids in adequate amounts to ensure the smooth repair of damaged cells, the supplementation of which has been viewed as a possible solution to augment the process and provide better strength and elasticity to the newly developing tissue [1].

It is known that collagen, being an integral part of most tissues in the body, plays an important role in the structural stability, elasticity, and tensile strength. It is therefore unsurprising that collagen is vital for restoring the structural integrity of the wounded tissue. It has been observed that, formation of scar tissue is an integral part of wound healing in most cases, with epidermal wounds being the exception. This scar tissue is composed primarily of collagen. This makes collagen synthesis an extremely crucial part of the wound healing process [2]. It is therefore practical to employ collagen supplements to augment and speed up the process of hound healing, and even enhance the tensile strength and other innate properties of the tissue. Through a study conducted by Felician et al., it was proven that collagen obtained from a species of jelly fish was indeed effective in escalating the pace of wound healing, making it a potential product that could be used in treating major wounds [3]. There is growing interest in the applications of collagen powder derived from marine sources to treat wounds effectively and reducing the possibility of a scar on the skin along with many other biomedical applications [4]. However, it must be understood that collagen powder is not the only form of collagen supplement for treatment of wounds and other tissue replacement procedures. There are a variety of forms, in which collagen is used as a biomaterial, for wound treatment [5].

Collagen derived from various sources is fabricated into various scaffolds, which can be implanted or grafted into the region of tissue damage, to act as an effective substrate for the attachment of precursor cells and allow their proliferation, thereby increasing the chances of tissue repair effectively. These precursor cells are multipotent adult stem cells which have the ability to differentiate to form various cells depending on the environment they are in, or the stimuli they receive for differentiation. These scaffolds can also be in the form of hydrogels, or fibers, and not just solid in nature. The use of collagen has proven to be effective for wound healing, due to the fact that it is an integral part of the extracellular matrix (ECM) on which most tissues are constructed [6]. Nanotechnology is a field of science that has been explored for its possible applications in the biomedical sector. Many nanomaterials such as nanoparticles and fibers are known to possess antimicrobial activities, which could be effective in the wound healing mechanism for the prevention of further infection. It is thereby prudent that the nanomaterials should be tried and tested along with those of collagen in order to come up with innovative methods to treat major wounds effectively. This chapter aims to summarize the importance of collagen and nanoparticles, synthesis of nano collagen in order to benefit from the wound healing properties of both nanoparticles and collagen, along with the areas of wound healing in which nano collagen is currently being used.

## **2. Nanotechnology**

Nanotechnology is the branch of science and engineering that involves design, construction, and characterization of materials by restructuring the atoms and molecules with the size range of 1–100 nm in one or more dimensions [7, 8]. The engineered materials are nanomaterials that show distinct chemical and physical properties compared to the bulk materials due to the synthesis and assembly at the molecular level that can be exploited for commercial use [9]. Nanomaterials can be of different shapes mainly based on their dimensions i.e., nanoparticles of zero dimension, nanorods of one dimension, and nanosheets of two dimensions [10]. Nanoparticles, due to their small size have the ability to penetrate the bacterial cell wall, and though the cells metabolic pathway cause changes to the cell structure and function. Nanoparticles are also known to interact various components of the bacterial cell, such as lysosomes, enzymes, and ribosomes, thereby leading to oxidative stress, altered permeability of the cell membrane, protein deactivation, and altered gene expression, eventually causing cell death among the bacteria. Thus, it can be said that the Nanoparticles have antibacterial properties, which can be exploited for sterilization of larger wounds, thereby preventing infections from occurring during the wound healing process. When compared to the conventional wound healing drugs certain nanoparticles exhibit greater penetration of cell membrane [9]. Nanoparticles, nanocomposites, coatings, and scaffolds are the main nanomaterials used for wound healing (as shown in **Figure 1**). Nanoparticles can be (i) inorganic metal or non-metal (ii) organic non-polymeric or polymeric. Nanocomposites are made of porous materials, colloids, copolymers, or gels. Coating and scaffolds include hydrogels, nanofibers, films, and coatings [11]. Different classes of nanoparticles are involved for the treatment of wounds. They are discussed below:

## **2.1 Metallic nanoparticles**

The antimicrobial property of metallic nanoparticles is exploited in wound management and can be used as a nanocarrier. The surface area to volume ratio of metallic nanoparticles is high. The small size enables them to cross barriers and penetrate the underlying layers of thick tissues like skin. These features make them ideal for drug delivery and to treat wounds. Some of the widely used metallic nanoparticles includes—silver nanoparticles (Ag NPs), gold nanoparticles (AuNPs), zinc oxide nanoparticles (ZnO NPs), iron oxide nanoparticles (IONPs), and titanium dioxide nanoparticles (TiO2 NPs) [12].

## **2.2 Polymeric nanoparticles (PNPs)**

Polymeric nanoparticles include polymer nanospheres and polymer nano capsules. Biologically active molecules such as drugs, genes, and fluorophores are absorbed on the surface of polymer nanospheres forming antibiotic incorporated nanoparticles (NPs). Griseofluvin (GF), one such NP, is known to function as an effective carrier of biologically active entities [12, 13]. The polymer nano capsules are vesicles where the core contains bioactive agents surrounded by polymeric shell. The polymers used in the preparation can be natural polymers like starch, polypeptides, albumin, sodium alginate, chitin, cellulose, gelatin, polyhydroxy alkanoates (PHAs) or artificial polymers like polyethylene glycol (PEG), poly lactic acid co-glycolic acid (PLGA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene etc. They show higher encapsulation efficiency and high stability of encapsulated active substance that helps them in the effective delivery of drugs to targeted sites [13].

#### **Figure 1.**

*Types of nanomaterials used for treatment of wounds. The figure is reproduced with permission from [11].*

#### **2.3 Nano emulsions**

Nano emulsions shows small droplet size and high surface area that makes them a suitable vehicle for drug delivery to treat wounds. A unique feature of these nano emulsions is their ability to deliver hydrophobic drugs [14]. They also have long shelf life, and are easily formulated [12]. The components of nano emulsions include different oil types, emulsifying agents like sodium deoxycholate, sodium dodecyl sulphate, antioxidants, chelating agents, preservatives etc. [15].

#### **2.4 Solid-lipid nanoparticles (SLNs)**

Solid-lipid nanoparticles are used as drug vehicles in case of inflamed or damaged skin. They are efficient and non-toxic carriers of both lipophilic and hydrophilic drugs. The structure is made up of long-fatty acid chains of palmitic acid, stearic acid or arachidic acid taurocholate, emulsifiers, and water.

#### **2.5 Nanofiber scaffolds/mats**

Nanofiber scaffolds/mats, considered as a substitute to damaged ECM, are mainly used in the wound dressing due to its healing power and unique structure. As the scaffolds are applied on the wound there will be attachment of fibroblasts and formation of matrix that acts as ground substrate and aid in faster wound recovery. Manufacturing of nanofibrous scaffolds involves electrospinning that produces uniform nanofibers [16].

### **2.6 Nanogels**

Hydrogels are used as delivery vehicles for wound treatment due to their properties such as high porosity which keeps the wound environment moist, and the presence of 3D polymeric matrix that absorbs the wound exudates allowing for proper permeation of oxygen [12]. While nanogels demonstrates some advanced features compared to those of hydrogels such as stability, ease of synthesis, quick response to stimulus, an adjustable size that can be exploited for drug delivery, controlled release of drugs, and tumor imaging. Nanogels are made up of chemical polymers and biomolecules. The nanogels of amino acids and polypeptides are easy to synthesize and modify and show higher biocompatibility [17].

## **3. Collagen**

The word 'collagen' is derived from a Greek term 'kolla', which means 'Glue'. Collagen is essentially a matrix, which holds the connective tissue together, making it a major component of the ECM, and connective tissues, and is rightfully called the most abundant protein in the animal kingdom [18]. Collagen is a major component of the ECM, which provides mechanical support for cell growth and their integrity. Collagen represents an entire superfamily of glycoproteins, having, a polypeptide sequence signature with [Gly-X-Y]n as the repeating amino acid unit, wherein X and Y are proline and hydroxyproline respectively. Another salient feature of these glycoproteins is their noteworthy quaternary structure with the right-handed triple helix structure composed of three left-handed polyproline chains of uniform length.

#### *Nanoparticle Based Collagen Biomaterials for Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.104851*

The chains in the triple helix can either be identical, forming homotrimers as seen in collagen II, or be different from each other, forming heterotrimers, as seen in collagen IX. Presence of glycine is invariant in collagen and is known to stabilize the collagen structure. It has been found that the absence of glycine or any mutations to the same is known to cause disruption in the hydrogen bonds formed in collagen and distort the structure [19].

The presence of collagen and collagenous structures throughout the animal kingdom indicates its importance in biological structures. Collagen is expressed in all life forms classified under the animal kingdom. Right from sponges, the simplest multicellular animal which expresses genes for the formation of at least two types of collagens, to the various vertebrates, in which collagen is a major component of various connective tissues, thereby accounting for roughly a quarter of the whole-body protein in humans [20]. The basic triple helical pattern has been partially carried over into the architectures of other complex molecules in higher organisms, with complex physiologies. Evolutionary branching which was partially driven by chromosomal duplication has resulted in a plethora of collagen types, which are genetically distinct. There are 29 types of collagens that have been identified so far [21]. Although the exact function of many types of collagens is yet to be confirmed, the role and presence of collagen throughout the body is unmistakable. However, it is known that collagen types I, II and III represent the majority (approx. 80–90%) of the total body collagen. They are known to provide mechanical and tensile strength to the skin and various other organs. The ability of fully developed collagen to integrate hydroxyapatites and undergo mineralization to amalgamate with solid structures such as bones and teeth, combined with its nature of elasticity and strength makes it a very desirable candidate to be used as a primary component of biomaterials with various applications [22]. Biomaterials are defined as synthetic components that may be transplanted into body tissue as a part of a medical device. Biomaterials can also be employed to replace an organ or a part of it, thereby aiding it in its physiological and mechanical functions [23].

Despite the wide range in the types of collagens, only a handful of them are actually utilized for the production of collagen-based biomaterials. Fibril forming collagens, such as type I, which also happens to be the most abundant collagen in mammals, is often employed for construction of collagen-based biomaterials for various purposes such as wound healing and tissue engineering, and even 3D bioprinting of collagen-based structures or scaffolds [24]. Collagen can be extracted from any animal's tissue including vertebrate's skin and tendons, porcine skin, gut, bladder mucosa, rat tails, as well as invertebrates' sponges and corals. The extracted collagen can show a slight difference in some characteristics, depending on the source of the animal, and the tissue. It has been found that the use of collagen from marine sources [25–28] has advantages over those obtained from terrestrial organisms, such as being environmentally sustainable, high production of collagen, non-toxicity, and ease of absorption thanks to its lower molecular weight. However, occurrence of allergies and transmission of disease can hamper the use of collagen obtained from animal sources, thereby the application of recombination technology was duly suggested, wherein yeast and *Escherichia coli* were transfected to produce recombinant collagens [29].

#### **3.1 Collagen and biomaterials**

Biomedicine is currently seeing an increase in the use and integration of collagenbased scaffold and biomaterials in its applications. The technology aids the creation

of biomaterials which exhibit biomimicry of the complex native tissues and organs. Decellularized collagen and refined scaffolds are the two categories into which collagen-based biomaterials are categorized. While the decellularized collagen structures retain most of the structural and functional properties of the tissue from which it is derived from, refined scaffolds are mostly obtained from the purification and polymerization of collagen. Decellularized collagen exhibits biomimicry the best [30]. Tissue grafts for tissue engineering, self-assembled hydrogels, freeze dried sponges, collagen films and tubes are some commonly used collagen-based biomaterials.

Tissue grafts are one of the most commonly used collagen scaffolds. Due to their resemblance to the native tissues, along with the ability to promote cell attachment and spatiotemporal organization of the cells, tissue grafts have been demonstrated as the most convenient and effective implantable devices [31]. Self-assembled hydrogels are generally used in the form of cell carriers, and injectables. They are often reliable for soft tissue treatment, for they resemble the structures on polymerization to form a fibrillar hydrogel structure, which is held together by ionic and hydrophobic bonds, thus aiding the entrapment of fluids, making it conducive for the exchange of ions and metabolites in the environment created [32]. Collagen type I hydrogels in combination with the appropriate precursor cells have been extensively used for the repair and as a structural and mechanical support for the attachment and stable growth of tissues such as skin to treat burns [33], cardiac myocytes [34], neurons [35], ocular tissues [36], etc. Collagen type I and type II hydrogels have often been used in combination for the treatment and repair of osteochondral tissues, and cartilage [37, 38]. Collagen scaffolds that can be easily used as grafts for various clinical purposes are created by the freeze-drying technique, wherein, collagen on undergoing freezing in a controlled environment, is trapped within the ice crystals formed, and is porous enough to facilitate cell migration, attachment, and growth [39]. So far, a variety of cell populations have been used to improve the bioactivity of the collagen sponge, and the experiments performed have shown encouraging results both *in vivo* and *in vitro*. Collagen scaffolds, integrated with glycosaminoglycan and fibrin networks have demonstrated their ability to enhance osteogenesis, and induce osteogenic and chondrogenic differentiation [40]. It was also established that these scaffolds are also used to aid in bone regeneration [41], vascularisation [42], and skin wound healing [43]. As discussed earlier, collagen and its biomaterials have already been well established in the biomedical field for their potential bioactivities. The integration of such collagen, with nanoparticles, which in itself has found extensive applications in the field of treatment and drug delivery, has piqued the interest of the scientific community for their potential synergistic activity to enhance wound healing. The synthesis and current application of this amalgamation is discussed further.

## **4. Nano collagen synthesis**

Nano collagen is the term used to describe collagen brought down to the nanoscale range. This substance has the desirable properties of both nanoparticles, such as a high ratio between the surface to volume of the particle, and collagen, with its wound healing properties of biomaterials, and their functions simultaneously. The downscaling of the size of the collagen fibers, is beneficial in terms of the penetration, and wound accessibility to initiate wound healing [44]. Nano collagen is produced through various chemical, physical, and self-assembly methods, such as emulsification, complex coacervation, phase separation, nano spray drying, desolvation and

many other techniques. The following section explains briefly the most popular techniques employed. Nano collagen fibers are produced through the following techniques: (a) electrospinning (b) nano emulsion (c) electrospray deposition (d) milling (as shown in **Figure 2**, **Table 1**).

## **4.1 Electrospinning**

Electrospinning is one of the methods used to create nano collagen fibers, wherein nanofibers are created from polymeric solutions in the presence of an electrostatic field. Electrospinning is achieved by charging a spinneret to high voltages and low current, and then adding droplets of the polymeric solution. As a result, the surface becomes highly charged, and elongates to form a conical shape, which is called the Taylor cone. The conical form is a result of the electrostatic repulsion between the charged droplet surface and columbic forces from the spinneret. At a specific threshold of the electric field, the electrostatic forces are strong enough to overcome the surface tension holding the Taylor cone, thus creating the fibers by stretching the cone, whipping it. This process is generally preferred to create nano fibers, because it is cost effective, and can produce nano collagen scaffolds for various purposes including tissues engineering, tissue repair and regeneration [47], and matrices that mimic the native ECM. The fibers produced through electrospinning are dry, and devoid of any solvent molecules, which are then collected in a metallic collector, which also determines the shape [51]. Over a period of time, electrospun collagen nanofibers have been endowed with certain 'smart' abilities, to improve their applications. Some smart abilities include response to external stimuli such as change in pH, exposure to light, and magnetic fields, etc., retaining a shape memory, self-cleaning, and some more [46].

#### **Figure 2.**

*(A) Electrospraying—after applying a high voltage to the protein solution, a liquid jet stream is released via a nozzle (coaxial needle), generating an aerosolized droplet. To ensure that the polymer solution comes out of the syringe as NP, a high voltage is provided to it. (B) Electrospinning—at a high voltage and low current in the spinneret, collagen polymer solution added dropwise. The Taylor cone is formed at such conditions. The columbic forces also cause the dehydration of the ejected polymer thereby resulting in thin and dry fibers of nano collagen. (C) Milling—the application of mechanical energy through the spinning of a milling bowl breaks down a polymer substance into finer NPs. Milling balls are used to conduct high-energy mechanical impacts to break down polymers utilizing centrifugal force. (D) Nanoemulsion—the emulsion is formed by the mechanical agitation of two immiscible liquid phases, one of which has the protein, and the other in which the drug is dissolved. Figures A, C, and D are reproduced with permission from [11]. Figure B is reproduced with permission from [45].*


#### **Table 1.**

*Collagen nanoparticle preparation methods, their principles, advantages and limitations.*

#### **4.2 Electrospray deposition**

As the name suggests, electrospray deposition is a process which involves the spraying of nano collagen solution as a fine mist onto a specific target. This method is mostly used for the applications of nanoparticles in the biomedical field for pharmaceutical application. This is mostly because, in this technique, collagen is used in its particle form. It is then sprayed through a nozzle onto a target with a high negative voltage, in the form of a fine mist. The solvent of the collagen particles generally evaporates on deposition onto the target surface, leaving an even spread of nano collagen particles, making it ideal for drug delivery purposes. This evaporation prevents the aggregation of molecules, and thus reduces the risk of contamination [52].

#### **4.3 Milling**

Milling is a process in which nano collagen is produced by the application of great amounts of mechanical stress onto a polymeric solution of collagen, to form particles of the nano scale range. This process is one of the most inexpensive methods for the large-scale production of nano collagen [53]. The mechanical energy along with the kinetic energy in the milling container also produces large amounts of heat, which can lead to the denaturation of collagen [54]. Therefore, this generation of heat is contained by performing this process at cryogenic temperatures, with the use of liquid nitrogen, thereby preserving the integrity of collagen.

#### **4.4 Nanoemulsion**

Nanoemulsion is a method used to integrate collagen with nanoparticles in a droplet form. Two immiscible liquids in different phases, i.e., oil-in-water-phase (oil is dispersed in water) and water-in-oil phase (water is dispersed in oil) when combined, form a concoction called an emulsion. Nanoemulsions differ from emulsions in their size ranges. The size of a nanoemulsion droplet ranges from 20 to 200 nm, while a normal emulsion droplet size is around 1 μm [55]. An aqueous phase with collagen, and a hydrophilic surfactant in water, is mixed with an organic phase with a lipophilic surfactant in a solvent that is immiscible in water and is continuously agitated under room temperature conditions to produce a uniform emulsion system. Nano collagen emulsion particles are then obtained by combining this emulsion system with a heated oil in a drop-by-drop manner [56]. Nanoemulsions naturally tend to penetrate deep into the tissue to deposit active compounds. This property has been exploited for purposes such as drug delivery in pharmaceutical, food and cosmetic industries. The same properties can be attributed to the collagen Nanoemulsion droplets to enhance the wound healing mechanism and speed up the process. The production of collagen nanoemulsions has increased greatly along with their application mainly in the field of cosmetics and drug delivery due to the technological advantages it offers for the manufacturers [57].

## **5. Applications**

#### **5.1 Bone grafting**

Collagen is a major component of the bone matrix. Bone formation is facilitated by the osteoblasts, which are involved in the production of collagen type I protein. The ECM supports the collagen fibers (50–500 nm) synthesized by the osteoblasts. The hydroxyapatite crystals are then deposited on these collagen fibers, leading to the hardening and maturation of the bone [58]. This mechanism can be exploited for the purposes of bone remodeling, in the case of a grave bone injury such as a compound fracture. A collagen scaffold can be grafted onto the damaged tissue area, to provide a solid support onto which the apatite crystals can be deposited, to increase the speed and efficiency of new bone formation. It is thus prudent that the collagen scaffold mimics native collagen fibers to achieve successful bone grafting and promote optimal bone regrowth.

It is well known that bone related tissue trauma is difficult to treat and is a timeconsuming process, due to the complexity of the bone healing process itself, and the loss of bone from non-sterile wounds, creating a high risk and susceptibility for infections. Cardoso et al., proposed the use of silver nanoparticles stabilized with type I collagen to form nano collagen biomaterials (AgNPcol) for the collagen scaffold to support rapid bone remodeling. This was an optimal solution for the problem of infections caused due to the non-sterility of the bone wounds. The silver nanoparticles in the collagen also showed anti-microbial activity against a number of microorganisms. Thereby proving to be effective in wound healing. The developed cells also showed no signs of cell toxicity [59]. In another study by Sun et al., collagen scaffolds

were infused with AgNPs along with BMP2, a bone morphogenic protein to improve the bone healing process effectively. The role of silver nanoparticles in antibacterial property was already established. However, the incorporation of the bone morphogenic protein induced an increase in the expression of runt related transcription factor 2, osteopontin and osteonectin, which are known to accelerate the differentiation of the bone marrow derived mesenchymal stromal cells, thereby proving the therapeutic potential of nano collagen in bone grafting, and healing [60].

Poor development of alveolar ridge after tooth extraction is an issue faced by most dental patients due to the lack of oral hygiene or knowledge about it. Wang et al., in their research, proposed the usage of artificial nano collagen bone implants. This was done to support the alveolar ridge post extraction of tooth. The implantation was followed by a CT scan to track the bone mineral density progressively. It was found that the implanted nano collagen bone has successfully fused with the native alveolar bridge. It also showed an increase in the overall bone mineral density [61].

#### **5.2 Nerve tissue**

Treatment of damaged nerve tissues has been a topic of interest for many researchers. This can be attributed to the inability of terminally differentiated neurons to undergo further cell division and also the fact that the nervous system controls and coordinates most of our body's processes. Damage or injury caused to the nerve tissue can seriously impair many functions of the body. Autografts of the nerve tissue has been performed in some cases. However, this has proven to be more challenging, due to the shortage of the donor sites, or occurrence of deformities. This has fuelled the search for alternative methods or materials to treat nerve damages effectively. The extensive study on collagen and nano collagen has tested the ability of collagen to act as an effective scaffold and promote cell attachment and growth [62]. Collagen has been used in the manufacture of nerve guidance conduits to aid the nerve regeneration in small nerve gaps of 2–3 cm across the peripheral nerve tissue. The use of collagen hydrogels for the treatment of lesions in the central nervous system effectively has been demonstrated by Orive et al. [63]. Further degradation of the nerve tissues can be prevented on injection of collagen nanospheres, which have the potential to deliver therapeutic drugs, and other stem cells for structural support as well [64]. Zhang et al., illustrated the application of collagen—nano size β tricalcium phosphate, together with growth factors of nerves and some collagen fibers, for the treatment of facial nerve repair and regeneration. Improved action potential was seen in the muscles, along with the formation of thicker myelin sheath, making it a highly promising avenue for further innovation and studies in nerve regeneration [65].

#### **5.3 Articular cartilage**

Articular cartilage covers the edge of a bone, and it is a connective tissue which forms a synovial joint that provides low frictional surface and enables the smooth movement of the joint. So, any damage to the articular cartilage results in acute pain during the movement of the joint. However, unlike most tissues in the body, articular cartilage lacks the potential to heal itself by replacing damaged areas in the tissue with new cells. This is mainly due to its avascular nature, i.e., there is no direct blood supply to the cartilage, thereby making it a difficult to heal by targeting therapeutic drugs. Treatment for articular cartilage necessitates surgical intervention techniques

*Nanoparticle Based Collagen Biomaterials for Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.104851*

such as chondrocytes implantation and osteochondral transplant. However, the high cost and numerous other risk factors of patients has given rise to much needed research in the field of cartilage tissue engineering [66].

Cartilage tissue engineering employs the use of 3D bioprinting for the creation of collagen 3D scaffolds, which are then treated *in vitro* to make them suitable for implantation. The application of 3D bioprinting techniques along with the nano collagen scaffolds effectively reduces the requirement of a cartilage transplantation from a donor, along with the need for other less effective surgical options. A collagenhydroxyapatite hydrogel nanocomposite was developed and effectively used in an investigation which showed promising results. Hydrogel composite was found to be suitable to facilitate fluid transport, and also thermally stable up to a temperature of 90°C [67]. Jiang et al., illustrated a different approach to stimulate the differentiation of the chondrocytes in the articular cartilage in order to initiate the repair mechanisms. The inhibition of chondrocyte dedifferentiation was achieved by the use of nano hydroxyapatite collagen scaffolds [68].

#### **5.4 Skin wound healing**

The process of wound healing involves four steps viz., hemostasis, inflammation, proliferation, and remodeling which occur in a sequential order [69]. Disruption of any of these steps will make the process lengthy. The main issue involved in wound healing is infection by pathogens that results in inflammation, interrupting the healing process [45]. Schimek et al., developed full-thickness skin equivalents (ftSEs) to hold the 96-well cell culture [70]. Collagen powder can be used as the dermal substitute as they are part of the ECM that shows slow biodegradation and accelerates wound healing [45]. Collagen with nanoparticles is widely used in therapy. Munish et al., used collagen granules for the diabetic foot ulcer treatment and the results were compared with the saline dressing. The study demonstrated that the wound, when treated with collagen showed a speedy recovery [71]. In another study, Akturk et al., developed gold nanoparticles (AuNPs) based collagen scaffold, and they were incorporated into the cross- linked collagen scaffolds. It was found that it helps in enhancing the stability against enzymatic degradation and increases the tensile strength [72]. The main advantage includes the absence of rejection and the fact that they can reduce the inflammation in and around the wound. Apart from gold nanoparticles, the use of silver as an antimicrobial agent has also been of great interest recently. Silver nanoparticles (AgNPs) are usually used in the treatment of burns and infection as they are known to demonstrate antibacterial property. There is sufficient evidence to prove that the bacterial resistance against AgNPs may not be a matter of concern, for AgNPs are known to hinder quorum sensing mechanisms in bacteria [45].

Collagen-based dermal scaffolds are coated with silver nanoparticles that act as antimicrobial dressing without having any toxic side effects. Nano silver reacts with gram-negative and gram-positive bacteria, causing damage to the intracellular structure. The positively charged silver nanoparticles react with negatively charged bacterial surfaces leading to the disruption of the inner membrane. During electrospinning, the synthesized silver nano particles are incorporated into the collagen nano fibers. The *in vitro* results prove that the AuNPs and AgNPs can provide the antimicrobial conditions for wound healing. The rate of wound healing in case of collagen composite nanofiber mat was significantly higher compared to the regular nanofiber collagen [44].

### **5.5 Drug delivery**

Collagen nanoparticles have shown promise as treatment carriers [73]. The recent trends in nanotechnology research and development aims to create collagen scaffolds that deliver the drug to the specific site and are released in a controlled manner [74]. Gold nanoparticles with different concentrations of gold (Au) was synthesized and coated onto collagen to form an amalgamation of nanoparticles and collagen (Au-Hp-Col). This amalgamation was found to be effective in the delivery of the drug Doxorubicin [70]. Poloxamer 407 (PM) is a polymer soluble in water used in the delivery of ophthalmic drugs like Ketorolac Tromethamine (KT). The PM is incorporated into the cellulose nano collagen particles that showed controlled release of the drug *in vitro* [73]. One of the case studies demonstrated the use of collagen nanoparticles in drug delivery to treat tumors. Collagen is a major component of the tumor microenvironment. The study involved the development of tumor spheroids based on collagen that are optimized using cell lines like 95-D, U87, HCT116 [75]. It was observed that the conjugated nanoparticles showed greater penetration into the gel matrix and were able to gain access to the tumor cells [76].

#### **5.6 Vascular grafting**

Cardiovascular disease is the major cause of death worldwide [77]. These disorders are caused by reduced blood flow by blockage of blood vessels [78, 79]. Presently, the saphenous vein, the internal thoracic artery, and autologous vessels are used as grafts which are known to perform better than the synthetic alternative [80]. However, their limited availability and invasive harvest make them unsuitable for use. Tissue-engineered vascular grafts (TEVG) are currently used in order to overcome these limitations [48]. TEVG makes use of modern technology for the construction of vascular medical implants. The collagen along with the other components are used as a scaffold in the preparation of the TEVGs. In a previous study, Park et al., described a poly-epsilon-caprolactone (PCL) vascular graft, and its suitability for healing process. It was observed that the graft undergoes gradual degradation replaced by natural blood vessels. Collagen is also incorporated on to the inner layer and silica (sol-gel-derived ceramic) into the outer layer of PCL to improve the vascular response [49].

## **6. Conclusion**

This chapter conclusively describes the importance and role of nanoparticlesbased collagen biomaterials in the treatment of various wounds. The ECM is mainly comprised of collagen, which provides support and elasticity against mechanical stress. While collagen in itself is useful in the form of various biomaterials like scaffold s and hydrogels, the introduction of nanotechnology to it comes with its own set of challenges as well as advantages. The reduction of collagen to the nano particle's sizes, giving it a large surface-to-volume ratio, is known to increase its efficiency of dealing with mechanical stress, thereby making it a viable option for treatment of wounds. Multiple research studies are conducted on wound healing using various materials and methods to reduce risk infection and aid in speedy recovery of the patient. The antimicrobial properties of nanoparticles of various elements such as gold and silver has already been proven, which can be further exploited in the

effective treatment of wounds and injuries, in combination with collagen. The current challenge lies in the effective incorporation of nanoparticles and collagen in the production of nano collagen biomaterials, upscaling the production of nano collagen and making it affordable to the general public.

## **Acknowledgements**

NM thank Global Innovation and Technology Alliance (GITA), Department of Science and Technology (DST), India [Project Number-GITA/DST/TWN/P-95/2021], and Indian Council of Medical Research (ICMR), (Project Number-ITR/ Ad-hoc/43/2020-21, ID No. 2020-3286) Government of India, India for financial support.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Kausalya Neelavara Makkithaya1† , Sharmila Nadumane1† , Guan-Yu Zhuo2 , Sanjiban Chakrabarty3 \* and Nirmal Mazumder1 \*

1 Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

2 Institute of New Drug Development, China Medical University, Taichung, Taiwan

3 Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

\*Address all correspondence to: sanjiban.c@manipal.edu and nirmaluva@gmail.com

† Equal contribution.

© 2022 The Author(s). Licensee IntechOpen. 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.

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## **Chapter 4**
