Natural-Based Biomaterial for Skin Wound Healing (Gelatin vs. Collagen): Expert Review

Collagen (Col) and gelatin are most extensively used in various fields, particularly in pharmaceuticals and therapeutics. Numerous researchers have proven that they are highly biocompatible to human tissues, exhibit low antigenicity and are easy to degrade. Despite their different sources both Col and gelatin have almost the same effects when it comes to wound healing mechanisms. Considering this, the bioactivity and biological effects of both Col and gelatin have been, and are being, constantly investigated through in vitro and in vivo assays to obtain maximum outcomes in the future. With regard to their proven nutritional values as sources of protein, Col and gelatin products exert various possible biological activities on cells in the extracellular matrix (ECM). In addition, a vast number of novel Col and gelatin applications have been discovered. This review compared Col and gelatin in terms of their structures, sources of derivatives, physicochemical properties, results of in vitro and in vivo studies, their roles in wound healing and the current challenges in wound healing. Thus, this review provides the current insights and the latest discoveries on both Col and gelatin in their wound healing mechanisms.


Biomaterials
Biomaterials are substitutes for biological tissues in the human body that have the ability to interact with the body. They can either be natural or synthetic. For more than 50 years, biomaterials have been developed to be used in the therapeutic field [1]. Approximately, the annual growth rate for biomaterials in the global market is 15.97 % and by the year 2027 it is expected to hit up to USD 348.4 billion in value [2]. Being natural biomaterials, both collagen (Col) and gelatin have shown promising results for skin wound healing. With regard to this statement, Col and gelatin are highly biocompatible toward human tissues as they resemble the extracellular matrix [3,4]. This property makes both of these materials the main choices for implantable medical products for in vitro testing [5]. Gelatin is the hydrolysed form of Col and both of them have the same amino acids, but their chemical properties differ.

Collagen
Col is a fibrous protein consisting of more than 1000 amino acids and is a substitute for cell scaffolding in the human body. It is essential for cell signaling, resilience for multicellular organisms and resistance to mechanical stress. In humans, Col is the most predominant protein as it makes up the major component of the extracellular matrix (ECM) [6]. Col can either exist as a homotrimer or a heterotrimer, depending on the composition of the α-chains. To date, there are 29 types of Col which have been identified.

Sources of Collagen and Gelatin
There is a vast range of sources that could be used to extract Col and gelatin. Although the source of derivatives influences the outcome, it still can be modified according to the medical needs. Col is commercially available and can be extracted literally from any sources, even from 75-million-year-old preserved dinosaur tissues [28]. Similarly, since gelatin is a Col derivative, the process of obtaining and the sources of gelatin are also wide. The sources of Col and gelatin are summarized in Table 2 [16,[29][30][31][32][33][34][35][36][37][38][39][40][41][42].

Physicochemical Properties of Collagen and Gelatin for Skin Wound Healing
Collagen and gelatin exhibit properties that are suitable to accelerate skin wound healing. Although both are approved natural biomaterials for skin wound healing, yet their biological properties greatly differ from one another. Yet, both can be ideal biomaterials for skin wound healing. The properties of Col and gelatin are discussed below.

Thermal Resistance
Thermal resistance indicates the ability of the scaffold to be resistant to heat. This is essential to ensure that the elasticity, toughness and strength of the scaffold is maintained upon changes in the temperature. The functionality of the biomaterial must be preserved at the wound site. The transition temperature for acid-soluble Col (115.33 ± 0.97 • C) and pepsin-soluble Col (112.1 ± 0.79 • C) is highest at a heating rate of 2 • C min −1 . The maximum transition temperature was recorded at 39.6 and 110.7 • C for acid-soluble Col, while for pepsin-soluble Col, it was at 38.33 and 109 • C. At the same time, heating for 0.5 min −1 was recorded at 39.6 and 38.33 • C for acid-soluble Col and pepsin-soluble Col, respectively [43]. In contrast, gelatin designed in the form of a gel was proven to be completely thermo reversible. The melting point for gelatin was recorded at 16 • C, while the gelling temperature was at 5 • C [44].

Extraction methods
Alkaline hydrolysis, acid hydrolysis, thermal extraction and high pressure processing.

Composition
The amino acid composition differs depending on the source of derivative.
The extraction method and the source of Col influence the Col physicochemical properties.

Crosslinking
Physical, chemical and enzymatic

Chemical Stability
The chemical stability of any biomaterial scaffold is essential for its functional stability and this is usually confirmed through Fourier transform infrared spectroscopy (FTIR). A uniformity in the major absorption band indicates scaffold stability [45]. FTIR for Col was recorded at 1632 cm −1 , 1548 cm −1 and 1237 cm −1 for amide I, amide II and amide III, respectively. The major absorption band was recorded at 2923 cm −1 , indicating uniform CH 2 stretching [46]. As for gelatin, the major absorption bands were seen at 3433 cm −1 , 1630 cm −1 , 1565 cm −1 and 1240 cm −1 for amide A, amide I, amide II and amide III, respectively. The recorded major absorption band for gelatin ranged from 1460 cm −1 to 1380 cm −1 , which highly corresponded to the presence of the methyl group [32].

Mechanical Strength
An ideal scaffold must be able to withstand force, particularly a scaffold that has been designated for wound healing. In this scenario, mechanical strength determines whether a scaffold is able to withstand the force or not [47]. Ghodbane et al., (2016) stated that the ultimate stress values of ovine, bovine, and porcine were recorded at 15.08 kPa ±15.08 kPa, 12.33 kPa ± 2.37 kPa and 13.91 kPa ± 3.11 kPa, respectively. In addition, the strain percentage and tensile toughness for Col were 50.74% ± 4.02% and 3.32 kJ/m 3 ± 0.81 kJ/m 3 [48]. A study done by Xing et al., (2014) [49] described that unpurified gelatin exhibited better mechanical strength in comparison to purified gelatin. As reported by them, the average storage and loss of modulus of the gelatin were 2.83 ± 0.10 fold and 3.80 ± 0.43 fold, while the ultimate stress was recorded as 107 kPa ± 25 kPa [49].

Oxygen Barrier
An ideal scaffold must be greatly permeable to allow the diffusion of oxygen to enhance the wound healing mechanism [50]. An adequate pore size of the scaffold determines this property of the scaffold. For adult skin healing, the optimal pore size has been determined to be in the range of 20 µm to 125 µm [51]. As stated by Ghodbane et al., (2016) [48], Col derived from ovine, bovine and porcine sources exhibited average mean pore sizes of 73.05 µm ± 10.79 µm, 85.84 µm ± 9.51 µm and 87.32 µm ± 10.69 µm, respectively. All Col scaffolds display heterogenous architectures, despite the sources of the derivative [48], while gelatin scaffolds display open pore structures, highly interconnected and widely tuneable. In addition, the pore size of gelatin can be altered according to the specific need by using ice particulates without affecting its mechanical property [52].

Water Vapor Transmission Rate (WVTR)
The WVTR directly corresponds to the ability of the scaffold to retain moisture in accordance with the need of the skin-healing phase. In relation to this, 2028.3 ± 237.8 g/m 2 /day has proven to be an ideal value to retain the moisture environment at the injury site to promote healing [53]. Col shows a WVTR of s 4750 ± 700.209 g/m 2 /day [54] while for gelatin it is 1040 ± 95 g/m 2 /day [55].

Biocompatibility
The main purpose of designing a scaffold is to promote the migration and proliferation of the cells toward the scaffold in order to promote healing. This can only be achieved if the designed scaffold is compatible to human cells. Mousavi et al., (2019) described that Col enhanced the binding of cells toward the scaffold through integrin binding and the presence of a cell-producing enzyme which was capable of breaking it down directly. This is possible due to the existence of the amino acid sequence in the backbone of Col, arginine-glycine-aspartic acid, which is similar to that in the human body. As a result, cell adhesion and proliferation are accelerated [54]. Meanwhile, gelatin scaffolds have been proven to have no significant effect on cell proliferation and cytotoxicity against human adipose-derived mesenchymal stem cells (hASCs. In view of this, gelatin is also categorized as a compatible biomaterial for human cells [56].

Biodegradability
To ensure a proper remodelling process, the scaffold must be able to degrade in an appropriate time. Since serine protease and collagenase can degrade Col naturally through intracellular degradation, it is thus controlled locally by engineered tissue cells. At the same time, metalloproteinases (MMPs) secreted by the inflammatory cells in the human body can degrade the Col (extracellular degradation) [57]. Meanwhile, gelatin degradation duration ranges from 35 days to 63 days in the presence of lysine diisocyanate ethyl ester [58].

Immune Response
The stimulation of an immune response is a very relevant issue when it comes to a biomaterial. An ideal biomaterial should not trigger any immune reaction by the host tissue. In relation to this, Col induces a very small amount of immune reaction when a scaffold is newly being placed onto the human skin. This happens depending on the source of the extracted Col, crosslinking agent or the presence of remnants of cells [58]. However, this can be overcome by removing the terminal telopeptide from the Col molecule. Upon the removal of that particular structure, the arrangement of the Col fibril pattern is disrupted, leading to an amorphous structure. When this happens, the surface of Col becomes highly soluble due to the positive electrons, eliminating the immunogenicity property of the Col scaffold [59]. Meanwhile, gelatin is proven to stimulate immunogenicity in the host tissue too. However, this is two-fold lower compared to Col. Specifically, when gelatin is crosslinked, a covalent bond is formed. This in turn creates a second network locked in place by chemical bonds [60]. It is widely speculated that biomaterials must be covalently attached to a bigger protein in order to elicit an immune response [61]. A covalent bond can become securely bound to a carrier molecule, most commonly a protein, if it exists. The hapten-carrier complex induces antibody formation, and so becomes immunogenic. The hapten then reacts with the antibodies that have been produced against it, causing an immunological or allergic response [62]. Introducing a covalent net point to gelatin manifested in it suppressing its immunogenicity property [58].

Antibacterial Property
Integrating an antibacterial effect into a biomaterial is an essential component in preventing the entrance and colonisation by microbes at the injury site. Native Col does not possess antibacterial properties. Hence, the incorporation of an antibacterial effect in Col further contributes to its physicochemical characteristics. Col can be integrated with silver nanoparticles (AgNP) [63], collagencin [64], epoxidized safrole [65], polyhexamethylene biguanide [66], titanium dioxide [67], etc. Similarly, native gelatin does not show any antibacterial effect. However, to give it such an effect, gelatin can be easily integrated with Ginkgo biloba extract [68], D-Limonene [69], Finibax [70], black pepper oleoresin [71], etc.

Hemostatic Effect
Hemostasis (stoppage of bleeding) is the primary component in wound healing. In this, native Col plays a vital role as a hemostatic agent as it serves as a major activator for immediate platelet response when there is injury. Furthermore, it allows the platelets to adhere at the wound site, forming a clump or clot together to stop the bleeding [72]. At the same time, this property can be further improved by integrating Col scaffold with oxidized microcrystalline cellulose [73]. On the other hand, gelatin does stimulate the hemostatic process by hastening the development of thrombus and providing it with a structural support. Within two days and up to six weeks, gelatin scaffold has proven to be absorbed or liquefies upon being utilized as a hemostatic agent. In comparison to Col, gelatin has been shown to be a superior hemostatic agent [74].

Cytotoxicity
Cytotoxic is an important parameter to be considered when it comes to a biomaterial. It determines whether the scaffold supports cell viability or not. Col has been proven to not induce cytotoxic effects, particularly in human cells. Instead, Col aids in cell delivery and distribution by creating a microenvironment for the uniform spreading and proliferation of cells [75]. Similarly, native gelatin does not exhibit any form of cytotoxic effect upon being seeded with keratinocytes. However, the method of gelatin extraction can trigger cytotoxic effects in the gelatin scaffold. For instance, >0.5 mg/mL enzymatic extraction could trigger the cytotoxic effect [76].

Cell Proliferation
Cell growth is an essential element in wound healing. With regard to this, the native col scaffold has been proven effective for cell attachment and proliferation, particularly at the wound site. Interestingly, the cells at the wound site appeared to be symmetrical and aligned in accordance with the native Col scaffold [77]. Nonetheless, an in vitro study proved that col scaffolds successfully accelerated healing by enhancing proliferation of fibroblasts [78] by increasing biological and structural integrities which resembled the native extracellular matrix (ECM) at the wound site [79]. In contrast, gelatin has been classified as a biofriendly scaffold which interacts perfectly with adipocytes, keratinocytes, cerebellum stem cells, and pre-osteoblasts, due to its similar structure to ECM. So the gelatin scaffold is able to enhance the vascularization process within the newly engineered tissues at the healing site [56].

Roles of Collagen and Gelatin in Wound Healing Phases
Col makes up the major component of the extracellular matrix (ECM) in humans [4] while gelatin is a Col derivative [12]. Col is a perfectly organized structure as a threedimensional (3D) scaffold that surrounds the cells. Thus, it has a dominant influence in maintaining the structural and biological integrity of the ECM. Being a natural component of the human body, it has been categorized as one of the major biomaterials widely used in wound healing [4]. Nonetheless, the physicochemical properties of Col and gelatin greatly influence the mechanism of skin wound healing.
Col and gelatin can penetrate into lipid free interferences (membranes) and are surfaceactive molecules [80,81]. In addition, they are thermally and chemically stable with high tensile strength, permeable to O 2, highly biocompatible, regardless of the source of the derivative, biodegradable, weakly antigenic and hemostatic agents. They have the ability to form high tensile, stabilized fibers through self-aggregation and crosslinking. These fibers can be modified into any form of scaffolding [63]. Particularly in skin injury, Col primarily, acts as a chemotactic agent by creating a microenvironment for the initiation of the healing mechanism (inflammatory phase) as it forms a protective barrier for the skin [82]. Similarly, gelatin is able to act as a hemostatic agent to initiate the wound healing mechanism and to absorb exudates present at the wound region while creating a suitable microenvironment for the inflammatory phase to take place [83].
In the proliferative phase, the wound bed prepares for the growth of new tissues and the wound undergoes contraction. In this phase, Col aids the growth of new tissues and accelerates the deposition of granulation tissues [84]. At the same time, Col enhances the activity of fibroblasts, leading to a drastic increase in the fibroblast proliferation rate. By means of contraction, Col forms a network that reinforces the adhesion of cells and tissue integrity [85]. As for gelatin, it acts as a porous scaffold to stimulate the migration of cells, specifically fibroblasts to the injury site. It further enhances the formation of new tissues by providing structural and mechanical strength at the wound site [83]. In the maturation phase, the continuity of the skin begins and the process of re-epithelisation continues [84]. In this process, Col supplies adequate nutrition directly to the wound, enhancing the repair mechanism and aiding in scar reduction [86].

Collagen and Gelatin for Skin Wound Healing
To date there is numerous scientific research on the effectiveness of including Col and gelatin in skin wound healing. Being biocompatible, they are beneficial and superior compared to other available natural products. Col is naturally found in the human body while gelatin is a hydrolysed form of Col. Studies done by Dill and Morgelin (2020) [77] and Wiegand et al., (2016) [87] showed that native Col provided a 3D microenvironment that stimulated cell proliferation and aided the migration of keratinocytes and fibroblasts to the physiologic locations during wound healing. Through their in vitro investigations, they noticed that fibroblasts and keratinocytes attached to Col with high affinities. Similarly, Jridi et al., (2015) [88] observed that positive interaction existed between Col and human cells, leading to an increased level of hydroxyproline at the injury site. With regard to this, in vivo experiments carried out by Dang [92] further proved that Col had the capacity to increase cellularity, granulation tissues, expressions of EGF, FGF, and CD31, collagenization, neovascularization and re-epithelization. This thereby resulted in rapid healing at the injury site. Interestingly, there was no immune response observed, despite the varying sources of the Col derivatives used in the in vivo testing. The outcomes clearly indicated that Col derivatives of any source were highly biocompatible to humans, due to their integrin (RGD) components [10]. Table 3 shows the in vitro and in vivo evidence of native collagen for skin wound healing.
Similarly, native gelatin showed good adhesion to fibroblasts and cell viability from 72 h up to 7 days [93,94]. The in vivo studies done by Hsu et al., (2019) [95], Nikpasand et al., (2019) [96], and Jang et al., (2017) [97] indicated the formation of thick granulation tissues, increased re-epithelization and blood vessel formation with an absence of cytotoxic effects confirmed through CCK-8 assays. For both Col and gelatin scaffolds, the analysed physicochemical properties correlated with those of an ideal scaffold for wound healing as per discussed in the physicochemical section above. No contraindication outcome was observed in the usage of Col and gelatin for skin wound healing. Table 4 shows the in vitro and in vivo evidence of native gelatin for skin wound healing.

Collagen as a Drug Carrier in the Pharmaceutical Industry
In the pharmaceutical field, Col modified into nanospheres, nanoparticles or microspheres is used as a device for drug delivery in wound healing which may be formulated either through emulsification, desolvation, coacervation, spray drying, milling technique, fluidization, solvent precipitation method, extrusion, or interfacial polymerization [107]. In this form, they have been proven to be effective in penetrating into the systemic circulation with the aid of Col. This is made possible as the integration of Col has resulted in a larger wound surface area covered due to its small size, high absorption ability and capacity to form a colloidal solution [108]. Apart from this, the crystallite suspension in the gel aggregates emerges as a multiplex chain system in the Col fold configuration. This characteristic eases the formulation of aggregates into colloidal drug delivery carriers [109]. Meanwhile, the formation of nanospheres is regulated by a blend of different electronic and electrostatic forces, with sodium sulphate acting as the liquefying reagent to allow for maximum charge to charge linkages between DNA plasmid and Col, whereas for Col nanoparticles the molecular weight of the Col influences the stability of the Col nanoparticles [109]. Nonetheless, changes in pH and temperature have proven to have significant impact on the molecular weight of the collagen solution and the noncovalent linkage responsible for the collagen's molecular structure during the formation of Col nanoparticles [110].
Col nanoparticle and nanosphere drug delivery carriers are easy to sterilise and, at the same time, they facilitate and accelerate the uptake of exogenous substances. For instance, the uptake of anti-HIV in a variety of cells, specifically from macrophages in antiretroviral therapy in nanotechnology, has always been easy with a Col biomaterial, in comparison to other drug carriers [111]. In addition, Col nanoparticles enhance the delivery of certain drugs such as camptothocin, tetracycline, doxycicline, rolitetracycline, minocycline, amikacin, tobramycin, vancomycin, etc., into the systemic circulation due to its penetration ability into colloidal solution [112]. Conversely, the kinetics in a Col biomaterial are usually influenced by the physical processes, such as swelling to form a gel, polymer hydration via fluid, drug diffusion through the gel formed and eventual erosion of the polymeric gel [113]. For example, the polymer undergoes a relaxation process in an aqueous media, resulting in direct, gradual erodation of the hydrated polymer. It is probable that, like the sponges, it swells and dissolves at the same time, with each of these processes contributing to the overall release mechanism. The diffusion rate of the medium represented by biological fluid in the polymeric sponge, on the other hand, determines the amount of drug released [114]. The main cause of kinetics in this condition is polymeric sponge erosion after water diffusion, as well as the swelling ratio. Aside from that, the drug release kinetics of Col can be affected by various chemical crosslinkings that affect the degradation rate, such as changes in porosity, density, and other factors [115]. Figure 1 shows the kinetics of drug release from the Col matrix [112]. Table 5 shows the current FDA approved skin healing products from native collagen.   1st and 2nd degree burns, full and partial-thickness wounds, pressure ulcer stages II-IV, diabetic vascular and venous insufficiency ulcers, and light to heavily exudating wounds.
-Can be customized to any desired size.

Gelatin as a Drug Carrier in Wound Healing
Gelatin, being a Col derivative, is highly biocompatible to be inculcated as a drug delivery wound dressing. Specifically, gelatin-based antibacterial wound dressing produced via casting has been proven as an effective dressing material. The outcome indicates it has hydrolytically and thermally stable properties, increased mechanical strength, and exhibits the optimum range of hydrophilicity as well as porosity, which accelerate the healing mechanism [116]. In addition, gelatin crosslinked with diosgenin-conjugated nanocellulose loaded with neomycin shows protection against S. aureus and E.coli in a dose dependent manner. Similarly, the pH of the biomaterial does influence the release of drugs in this situation. For instance, 60% of neomycin was released from the gelatin at pH 5.5 while 40% was released at pH 7.4 within 15 min [117]. Nevertheless, another study shows that combining gelatin with poly(ε-caprolactone) and TGF-β1 inhibitor successfully hinders over-proliferation of fibroblasts and wards off scarring. Gelatin nanofibers integrated with silicate particles promoted diabetic wound healing by regulating the release of silicon ions at the wound site [118]. It could be speculated that the nature of the dense and porous novel gelatin-based drug-eluting structures further proves the efficacy of gelatin biomaterial as an ideal drug carrier in wound healing applications.
Nonetheless, this study proves that it is less challenging to modify gelatin into different forms of drug carriers while preserving its natural properties, and particularly the ability to retain moisture at the wound site. These changes can ease the formulation of the microparticle or nanoparticle-based gelatin biomaterial which can be in the form of injection to deliver drugs at the specified location in the body. Along with it, the high waterabsorption capacity of gelatin causes a rapid release profile, which prevents water-soluble medications from being released in a sustained and effective manner. Despite of the different sources of gelatin derivative, initial molecular weight, and degree of crosslinking can all affect water uptake to some extent, which contributes to the healing phase of the wound [119].

Conclusions and Future Prospective
In the healthcare industry, wound healing is the most critical area as any form of infection can result in severe complications. In this review, the authors compiled the existing knowledge of wound treatment and evaluated them critically to help researchers, clinicians and anyone interested in this field to understand the dynamic wound healing phenomenon, particularly focusing on the roles of the natural substances Col and gelatin. The authors introduced the principles of an ideal scaffold for wound management, paying particular attention to recent studies of native Col and gelatin as ideal biomaterials for wound healing. Despite their differing sources of derivative, both Col and gelatin are said to be ideal scaffolds and highly biocompatible to human cells, due to the integrin molecules. Their numerous properties (cost-efficient, biocompatible, swelling index) and the various manners of their introductions into the human system (electrospun fibers, hydrogels, sponge, films, etc.) contribute to their being favorable options for researchers. However, the uses of Col and gelatin for wound treatments are holistic approaches which are still at premature levels and lack large-scale clinical trials. The state-of-the-art technology nowadays is the use of biomaterials for 3D printing which imitates the extracellular matrix. The use of 3D-printed skin on a wound could provide an effective alternative in the field of wound care in the near future. Hence, our assessment of the current knowledge of the wound healing process, together with wound microbiology and the present status of wound dressings would be very helpful in the development of more effective wound dressings for individual patients as well as for the continued progress of the medical and pharmaceutical industries. Funding: This study was funded through the grant provided by GPutra Fast Track: 9594300 in the form of database subscription. The funders did not make any contribution to the decision to publish or the preparation of the manuscript.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.