Recent advances in preparation and biomedical applications of keratin based biomaterials

Abstract

Keratin has gained increased curiosity from researchers in the last decade for its potential applications in preparation of biomaterials. Most emphasized properties of keratin as a candidate to manufacture biomaterials involves biodegradability, excellent biocompatibility, self – assembling capability, ability to support cell growth and proliferation, water absorption and easy availability as waste. Keratin based biomaterials in the form of fibres, scaffolds, films, hydrogels, nanoparticles are being explored for various biomedical applications including wound healing, drug delivery, oral tissue regeneration, study models as well as nerve regeneration. Methods opted for fabrication of these materials include electrospinning, cross-linking and solution casting among others. In order to improve antimicrobial properties and bioactivity of keratin biomaterials they could also be loaded with drug molecules, antibiotics, growth factors and other functional peptides. Keratin materials have the advantage of high loading capacity as well as controlled and prolonged release of drug, thus maximizing the availability at the target site. This current paper critically reviews the latest developments in the utilization of keratin-based biomaterials in the aforesaid fields.

Graphical Abstract

Introduction

Due to advancements in technologies involving fabrication of biomaterials and their expanded utilizations in various medical applications, the research community in current era is focusing on using the raw materials being derived from livestock and agriculture [47, 85]. Such biomaterials are getting more attention because they tend to be sustainable as well as can deal with the problems of waste accumulation and efficient utilization. Thus, the “Garbage In, Biomaterials Out (GIBO)” concept focuses on the recycling of agricultural waste into biocompatible materials (Sah et al., 2022). Raw materials employed for such purposes involves, plant and animal proteins as well as carbohydrates among others [23, 94, 137]. Keratin based materials holds promising potential owing to their biological and physiochemical properties as well as availability as a cheap source in the form of waste [19, 92]. The keratin could be obtained from feathers, wool, hair, nails and horns and could be fabricated into variety of materials such as films, fibres, scaffolds, sponges and hydrogels [103]. Keratin waste including millions of tons of feathers accounts for a huge fraction among the waste generated worldwide per year [101, 111]. Thus, utilizing keratin waste for biomedical applications is of great interest. This review summarizes the structure, extraction strategies and various biomedical applications of keratin-based biomaterials. Although the review articles published until recently have highlighted the important physical and biochemical properties of keratin as well as their possible biomedical applications, the current article shall provide an exhaustive and updated information on the recent research and studies exploring various biomedical applications of keratin biomaterials including wound healing, drug delivery, oral tissue regeneration, nerve regeneration among others.

Structure, sources and properties of keratin

Keratin is an insoluble fibrous protein that makes up the cytoskeleton and epidermal structures in humans and animals including hair, horns, wool, feathers, claws and nails among others [53]. Based on the source, keratin presents variation in structure and properties but could be broadly classified as hard and soft keratin. The disulphide bridges between the cysteine molecules are mainly responsible for the stability and integrity of the protein structure in keratin. The hard keratins having more sulphur (cysteine) content and thus more disulphide linkages providing toughness to epidermal structures [33, 111]. Whereas the soft keratins have less sulphur content and is responsible for imparting elasticity to the epithelial tissue [20]. The hard keratins from various sources have been mostly employed for the fabrications of biomaterials such as films, hydrogels, fibres and sponges [12, 13, 40, 86]. The polypeptide in keratin could be arranged either in α helix or β- fold. The α helical conformation results in good elasticity whereas the van der waals forces and hydrogen bonds in β- sheets are responsible for high tensile strength. The occurrence of α keratin is predominantly reported in hair, claws and hooves of mammals whereas that of β keratin is seen in feathers, scales and beaks of birds and reptiles. Based on their molecular weight and overall charge, keratins are classified as Type I (acidic and smaller) and Type II (basic-neutral and larger). Type I and Type II keratins interact with each other by forming heterodimers in the initial stage and then assembling into complete intermediate filaments (Fig. 1).

Fig. 1

Human hair keratins and their interactions. a Structure of KRT 85 derived from AlphaFold protein structure database. b Binary interaction of KRT85 with KRT38 drawn with IntAct database. c Binary interaction chart of KRT85 with 25 other proteins involving type 1 hair keratins and other proteins, retrieved from UniProt (ID P78386). d Network showing multiple interactions between different keratins and keratin associated proteins from homo sapiens involved in the formation of hair retrieved from STRING database

The inherent key properties of keratin that makes them usable in biomedical applications includes ability to self-assemble, biocompatibility, biodegradability and support to cellular proliferation [107, 139]. Reports are also available that shows the anti-bacterial and haemostatic property of keratin [60, 108, 131].

Keratin extraction methods

Multiple methods are available for the extraction of keratin from various sources. These extraction methods rely on breaking the disulphide bonds responsible for the stability of the protein structure. These extraction methods could be chemical, physical or biological. Major physical methods include high-pressure hydrolysis method, high- temperature hydrolysis method, high-pressure puffing method and extrusion method. Disadvantages of physical methods of keratin extraction includes destruction of primary structure of the protein as well as high energy input. The chemical extraction of keratin on the other hand can be done by oxidation methods, reduction methods or by acid–base treatments (Alahyaribeik et al. 2020). For the enzymatic isolation of keratin, keratinases from actinomycetes and fungi could be utilized. Reports are also available to extract keratin by using microwave irradiation, ionic liquids as well as steam explosion. Extraction methods of keratin from various sources employing different methods are summarized in Table 1.

Table 1 Recent advances in extraction methods of keratin from various sources

Physical methods

Under physical methods of keratin extraction, high pressure and temperature during hydrolysis has been used. Although it is a convenient method but the extracted keratin is completely degraded into amino acids and peptides thus destroying the primary structure and rendering it unsuitable for biomaterial preparation [84]. Another disadvantage of high pressure or temperature hydrolysis is excess of power consumption. Alternate physical method for keratin extraction is steam explosion in which high pressure steam is enforced into a container with the raw materials. Steam explosion has been studied on wool degradation and it has been observed that almost 62% of wool degradation could be achieved by stem at higher temperatures of about 600 °C [114]. Higher rates of keratin decomposition could be achieved with increasing processing time, temperature and pressure [41].

Chemical methods

Acid–alkali treatment

Employing strong acids such as hydrochloric acid and sulphuric acid for the hydrolysis of keratin involves the treatment of keratinous waste for a given period of time, neutralization and further drying and purification to achieve final dried product [7, 12, 13]. The time employed for hydrolysis dominates the molecular weight composition of the extracted keratin, an increase in hydrolysis time results in lower molecular weight protein chains [87]. As a result of prolonged acid hydrolysis, certain amino acids such as tryptophan are degraded, moreover the leftover acid waste with is cumbersome to handle and dispose.

As far as use of alkali for the hydrolysis of keratin is concerned, the loss of amino acid is not observed [12, 13]. Treatment with alkali weakens the mechanical properties of keratin and thus renders it unsuitable for film formation [21]. Alkali such as Ca(OH)_2, KOH, NaOH have been studied for the hydrolysis of wool keratin. Combination of Acid and alkali for the hydrolysis of keratin have also been explored and found to be more effective [21, 30].

Oxidation

Oxidizing agents such as peracetic acid, performic acid, hydrogen peroxide, peroxyacetic acid, peroxyformic acid have found their use in keratin extraction. These compounds break the disulphide bonds to yield keratoses which predominantly have a crosslinked structure stabilized by noncovalent interactions and depict hygroscopic behaviour [132]. The keratoses are further subdivided into α- keratoses, β-keratoses and γ-keratoses based on their solubility in ammonia and their region of origin from the keratin tissue. α- keratoses could be which are derived from cortex region are soluble in ammonia and could be precipitated at acidic pH. β-keratoses, derived from cuticular region are insoluble in ammonia whereas γ-keratoses are soluble in ammonia but are not precipitated at acidic pH [132]. Disadvantages of oxidation method include loss of certain amino acids such as phenylalanine, tyrosine, tryptophan among others as well as long treatment times [86].

Reduction

This is the most commonly used method of keratin extraction. Reducing agents used for breaking the disulphide linkages are β-mercaptoethanol and other thiols in combination with denaturing agents like urea and thiourea [54, 95]. Upon reduction in alkaline medium soluble protein known as kerateines are formed. Certain protocols also employ the use of sodium dodecyl sulphate and other surfactants along with reducing agents to increase the stability of the keratins in solution. This use of β-mercaptoethanol poses threat as it is toxic in nature thus sodium disulfite could be used as an alternate although it gives lesser yields. Urea in high concentrations disrupts the protein framework in keratin by hindering with the hydrophobic interactions and thus enhancing the action of reducing agents. The reducing methods have been predominantly for keratin extraction with varied concentrations of urea and other components from sources such as feathers, hair, horns and hooves [54, 80].

Ionic liquid treatment

Ionic liquid are salts or cationic/ anionic compounds that exists as liquid at room temperature and possess strong solubilizing properties as they could disrupt the intermolecular hydrogen bonds present in the natural polymers [12, 13, 43]. These liquids have been studied for use in the extraction of keratin from chicken feather and wool. In comparison to acids and alkali, ionic liquids are eco-friendly, non- corrosive and non- flammable. Ionic liquids are often used in combination with chemicals such as sodium bisulfite that could break the disulphide linkages and also reduces the duration of the treatment. Ionic liquids such as BMIM + Cl- and 1-allyl-3-methylimidazolium chloride could be used to extract keratin at high temperatures of up to 130 °C [25] 19% yield of keratin from human hair have been reported with 1-allyl-3-methylimidazolium chloride [133, 135], and reduced solubility have been reported in BMIM + Cl- [112].

Biological/ enzymatic methods

Biological extraction or solubilization of keratin have been reported by the use of micro-organisms as well as purified enzymes. In comparison to chemical method of keratin extraction, biological methods are safer and results in lesser loss of amino acids along with being energy efficient method, as input of energy in the form of higher temperatures or pressure is not desired. But use of microorganisms and purified enzyme preparations make these methods costlier [52]. Bacillus isolated from poultry waste and soil, Amycolatopsis Chryseobacterium, Streptomyces, Staphylococcus, etc., are known to be keratin degrading [2, 5, 115, 116]. In addition to bacteria certain fungal species (Aspergillus flavus, Aphanoascus fulvesence, Microsporum gypseum) have also been studied for this purpose [7, 75]. Use of urea with microorganism have also been reported to achieve higher keratin yields.

Keratinases enzymes from Apergillus, Lysobacter, Bacillus, and Streptomyces genera could be used for keratin extraction [116]. Different molecular weight keratin fractions could be prepared depending upon the pH, temperature and exposure time [22].

Biomedical applications of keratin biomaterials

Wound healing

Wounds can arise from several factors such as severe injuries, major surgeries, diabetes, or vascular illnesses. Wound healing involves different types of cells such as fibroblasts cells, keratinocytes, various immune cells and vascular endothelial cells. Certain wounds do not heal in short time with normal clinical care and may bother the patients for months or even years. The accelerated healing in such challenging wounds could be achieved by application of biomaterials based on protein matrices. Collagen and keratin are the major components of the human skin that have gained interest in recent time to prepare biomaterials capable of accelerating healing in such chronic wounds. These biomaterials generally deliver materials such as growth factors, proteins or other molecules that could expediate the wound healing process. Keratin is present as filament in keratinocytes cells of the epidermal layer of the skin. Apart from providing mechanical strength, it also plays significant role in cell signalling. Keratins undergo post translational modifications and interact with various signalling proteins in order to perform the functions including cell migrations, adhesion and differentiation [104]. According to reports, keratin also plays a vital role in activation of keratinocytes that is an important step in normal wound healing process. Various types of keratin-based biomaterials employed for wound healing involves nanofibers, membranes, hydrogels, scaffolds and dressings. The keratin alone or in combination with polyurethane, PVA and cellulose have recently been reported to form these biomaterials. In a recent study, Ramey et al. [93] prepared human hair keratin matrices and explored their usage in wound healing in diabetic mice. Comparison of these keratin matrices was also made with amniotic membrane, bovine dermis and porcine decellularized small intestinal submucosa for wound healing purposes (Fig. 2). The authors reported these matrices to be thin with smooth and uniform surface morphology. Human epidermal (HEKa) keratinocytes when grown on keratin matrices showed upregulation of Interleukin 6 (IL-6) and Macrophage Inflammatory Protein-1 delta (MIP-1δ), that plays an important role in wound healing by modulating inflammatory response and promoting fibroblast migration. In vivo studies suggested that the wound size was smaller in mice that were treated with keratin matrices then those treated with amniotic membrane after 3, 4 and 5 weeks. Keratin based applications of biomaterial formation and utilization for wound healing has been summarized in Table 2.

Fig. 2

Effect of various biomaterial wound care products on healing in vivo. A (left) Schematic showing four 6 mm diameter full thickness wounds on the backs of db/db mice that were treated with HKM, another biomaterial-based wound care product, or no treatment (control) in randomised locations. Image created with Biorender.com. Representative images of the four wounds at week 0 before application of treatment (middle-left), wounds treated and topped with secondary dressings (middle-right), and wounds after several weekly treatments (right), in this case HKM (i), control (ii), and bovine dermis (iii) at week 3 post-operation. B Bar graph showing average time to complete closure for each treatment applied. *p < 0.05, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparisons. C Healing trajectories of wounds on mice treated with control (black circle, n = 12), amniotic membrane (blue square, n = 12), or HKM (gold triangle, n = 12). D Healing trajectories of wounds on mice treated with control (black circle, n = 12), bovine dermal collagen (blue square, n = 12), or HKM (gold triangle, n = 12). E Healing trajectories of wounds on mice treated with control (black circle, n = 16), porcine small intestinal submucosa (blue square, n = 16), or HKM (gold triangle, n = 16). Symbols indicate statistical significance of HKM compared to other treatments: *p < 0.05 vs. control, **p < 0.01 vs. control, ****p < 0.0001 vs. control, ^#p < 0.05 vs. corresponding comparative advanced wound care product, ^##p < 0.01 vs. corresponding comparative advanced wound care product, ^####p < 0.0001 vs. corresponding comparative advanced wound care product by two-way analysis of variance (ANOVA), paired by mouse, with Tukey's multiple comparisons at each timepoint [93]. Creative Commons Attribution License

Table 2 Wound healing applications of keratin and keratin-based biomaterials

Drug delivery

The term ‘Drug delivery’ defines the administration of any pharmaceutical compound to achieve therapeutic effect in humans or animals [38]. There are various techniques adopted by scientists to deliver these compounds effectively and safely to the target site in the body of human in correct concentration [29]. The aim of the drug delivery system is to enhance the efficacy, safety and bioavailability with minimized side effects to target tissue. This area covered many aspects including route of administration, targeted delivery, formulation technologies and biological barriers. The biocompatible nature of keratin has attracted researchers to exploit it in the applications involving designing of drug delivery systems [31].

Hydrogels and nanogels derived from proteins are lipophilic in nature but they do not dissolve in water instead they swell up after coming in contact with water. They have excessive drug loading capacity and are able to ameliorate cellular uptake efficiency [127]. Keratin biomaterials are loaded with drugs and used as a carrier because they act as a covering shield and protects encapsulated drugs from degradation in the physiological environment, before reaching the target site. Keratins also have the ability to bind effectively with various bioactive compounds, maximizing drug stability and providing controlled release [126, 149].

Keratin naturally possess cysteine-containing residues and ample of thiol groups, these sulfhydryl groups of keratins form a disulfide bond with a desired drug and use it as a carrier for selectively drug release under reducing circumstances. Additionally, it also possesses a lysine and arginine group that can be elicited by a known protease trypsin which is an essential enzyme generally augmented in tumor tissues [149]. Different biomaterials have been formed including nanogels (with hyaluronic acid and sodium alginate), hydrogels, nanofibers, microparticles, nano fibrous mats, nanotubes and nanoparticles like keratin/CHX NPs (keratin/chlorhexidine complex), by using various methods such as nanoprecipitation, self-assembly, de-solvation, iconic gelation and aggregation. Liu (2024) used keratin as an envelope of antitumor drug and used as a drug delivery agent in tumor chemotherapy.

Recent innovations in this area focus on smart drug delivery systems, biologics (i.e. monoclonal antibodies) and nanotechnology. Currently, these advancements are very crucial to treat any disease more effectively with minimizing side effects and improving patients’ health. Recent advancements on the role of keratin in the drug delivery systems are summarized in Table 3.

Table 3 Application of keratin based biomaterials in drug delivery systems

Oral tissue regeneration

Keratin have found a place in various ways pertaining to the production and utilization of biomaterials applicable in oral tissue or bone regeneration. A post operative infection in dentine region, damage to alveolar bone, wound healing and degeneration in pulp dentine are some of the scenarios exploiting remarkable biological properties of keratin to form bio composite materials. Wound repair in oral cavities takes 2–10 days to heal and it requires processes such as epithelial cell migration, proliferation and cell plasticity. Trans-differentiation of epithelial cell resulting from persistent inflammation is called type 2 EMT (Epithelial-Mesenchymal Transition). Vimentin is the biomarker for Type 2 EMT, which indicated that keratin induces EMT in the oral keratinocytes and enhances migration of cells. Thus, human hair keratin could serve as an excellent material to form biomaterials with varied properties and functions. Moreover, the alveolar bone that provides support to the tooth may undergo loss and degeneration as a result of various factors. In order to replace the lost tooth, dental implants need proper dimensions of this alveolar bone with required surface area for implantation. With damaged alveolar edge, the success of implants could be reduced. Keratin biomaterials among others have been reported to promote regeneration of alveolar bone. Another area involves utilization of stem cells including Dental pulp-derived stem cells (DPSCs) to generate pulp-dentine like tissue. Collagen and keratin have been used in form of scaffolds to induce differentiation in DPSCs through cell homing and providing binding sites [110]. Keratin composite membranes could also be employed to release antibacterial agents at a control rate in order to prevent postoperative infections. Latest researches exploring the potential use of keratin biomaterial for various dental applications are summarized in Table 4. In a notable study by Feroz & Dias [34], Scaffolds were prepared from sheep wool keratin, hydroxyapatite and hydroxypropyl methylcellulose which depicted cytocompatibility with osteoblast cells and could be employed for alveolar bone regeneration (Figs. 3 and 4).

Table 4 Advances in the use of keratin biomaterial for oral tissue regeneration

Fig. 3

General Appearance of Keratin/HA/HMPMC scaffolds (diameter: 15 mm, height: 5 mm) (A & B), SEM micrographs of Pure keratin scaffolds (C) and Keratin/HA/MPMC scaffolds (D). (Size bars in Fig. 4 C & D represents 100 μm). Feroz & Dias [34]; Creative Commons CC-BY-NC-ND

Fig. 4

Fluorescence images of keratin (A, B, C) and keratin/HA/HPMC scaffolds (D, E, F) seeded with Saos-2 cells after live/dead viability assay. Images shows Saos-2 cell viability at 24 h, 48 h & 72 h. Bar = 100 μm. [34], Creative Commons CC-BY-NC-ND

Tissue engineering

Tissue engineering comes to safeguard in situations where conventional medicine systems render to be incompetent, such as failure of function or loss of a particular tissue or organ. Success of tissue engineering relies on the fabrications of scaffolds or other forms of biomaterials that could effectively replace the original tissue/ organ. Various biomaterials being explored for in this regard involves nanoparticles, nanofibers, films and hydrogels [37, 69,70,71, 100]. Hydrogels are most commonly being employed for tissue engineering because they could most effectively bio-mimic as well as can be designed into variety of different structures according to specific needs [6]. Owing to their three-dimensional cross-linked network and hydrophilic characteristics, hydrogels have the ability to absorb and retain large amounts of biological fluids [72]. Disulfide bonds in the keratin structure provide it with high mechanical strength, moreover it its non- immunogenicity makes it a suitable candidate for tissue engineering. The amino acid sequences of keratins are known to interact with integrins such as glutamic acid-aspartic acid-serine (EDS), and leucine-aspartic acid-valine (LDV) and others [129]. One more advantage of using keratin for generation of tissue engineering biomaterial is that the animal cells mostly do not contain the keratinase enzymes so the in vivo breakdown of this protein like other does not occurs. The generation of keratin-based hydrogels generally requires a cross-linking agent such as transglutaminase, dialdehyde, formaldehyde, glutaraldehyde, ethylene glycol diglycidyl ether [10, 27, 82, 123, 142].

Application of keratin biomaterial is being studied for the regeneration of skin tissue regeneration, vascular tissue regeneration and skeletal muscles regeneration specifically volumetric muscle loss (VML). Minor injuries as a result of exercise or strain in skeleton muscle could be repaired by the intrinsic mechanism of self – repair involving multiple cell signalling events, but major muscle loss following a trauma or surgical intervention results in disturbances in signalling cascade leading to long term loss of structure and function [89]. Use of allografts, muscle flaps are adopted for volumetric muscle loss treatment but has their own drawbacks. Keratin based scaffolds and other biomaterials constructs are being designed and studied for the purpose of restoring functional loss in VML as well as other tissue engineering applications (Table 5).

Table 5 Tissue engineering applications of keratin-based biomaterials

Peripheral nerve regeneration

Peripheral nervous system (PNS) helps the body to feel sensations and move the muscles. PNS works as a bridge between central nervous system and various tissues or organs [138]. The fundamental units of nervous system i.e. neurons are made up of bundles of axons which forms the peripheral nerves. The types of injuries that can affect the PNS includes neuropraxia, axonotmesis, or neurotmesis. Although the PNS has the capacity to self-repair, but in cases of delayed treatment, severe injury or an injury larger then 3 cm leads to incomplete repair and loss in functionality [64]. In order to regenerate the damaged peripheral nerve, various nerve tissue grafts are being studied including autografts, allografts and xenografts, among which autografts are considered to be the most efficient. Nevertheless, there are certain limitations to nerve grafts including limited availability, surgical complications, immune rejection and diameter mismatch between the donor and recipient nerve to name a few [136]. More recent alternative to nerve grafts includes the artificial nerve conduits made up of biological polymers. Nerve conduits help to fill the nerve gap resulting from nerve injury by guiding the axon regeneration and thus improving the efficiency of the clinical treatment. Different nerve conduits with added functionality of drug and growth factor delivery, capacity to support cell proliferation as well as conductivity with design specific to the particular function are being developed [62, 140, 141]. Similarly other types of biomaterials including membranes have found potential use in regeneration of PNS injuries.

The chitosan/keratin biomimetic composite membrane prepared by [11] depicted potential for angiogenesis and nerve repair efficiency [55]. Fabricated tubular nanofibers with keratin extracted from chicken feather and PVA by using electrospinning, to be used as nerve conduits. These nanofibers had diameter ranging from 170 to 234 nm. The authors also reported a decrease in diameter of the nanofiber with increase in concentration of keratin [39] reported that the human hair keratin can promote the extension of axon in Dorsal root ganglion neurons in vivo. The authors prepared a keratin sponge and also suggested that these could enhance the cell adhesion, proliferation, migration and secretion of neurotrophic factors by Schwann cells in vitro [144] studied spinal cord injury (SCI) in rat models and reported that keratin biomaterials can induce polarization of macrophages and promote functional recovery.

Promoting macrophages to move towards M2 anti-inflammatory phenotype is regarded as a target to treat the SCI [152] studied the anti-inflammatory activities of 17 human hair keratins, the authors have found that recombinant keratins 33A and 35 demonstrated superior anti- inflammatory properties. The authors also established the role of recombinant keratin 33A in nerve regeneration and increasing M2 polarization by working with rat T9 spinal cord lateral hemisection model and utilizing keratin nanofibers.

Qin et al. [90] used activated Schwann cells with human hair keratin to prepare nerve grafts. The nerve grafts thus produced, promoted the nerve conduction function as well as motor function in rats with sciatic nerve injury due to increased expression of nerve growth factors, thus could be applicable in healing peripheral nerve injuries. In yet another more recent research [119], Explored the potential of curcumin to promote peripheral nerve regeneration. The researchers exploited the properties of keratin/ chitosan hydrogel to effectively deliver the curcumin to the target site in appropriate concentration. The hydrogels were found to be capable of delivering the curcumin for 10 days in vitro. In rat studies also, the hydrogel was found to be capable of enhancing nerve regeneration (Fig. 5).

Fig. 5

Schematic diagram of keratin chitosan hydrogel synthesis process loaded with curcumin, cell adhesion in complex hydrogel, complex hydrogel promotes repair of peripheral nerve injury. Sun et al., (2023); Creative Commons CC-BY

Ocular surface reconstruction

Ocular surface reconstruction means repairing the eye’s tissue such as cornea, conjunctiva and limbus and restoring the vision of eyes. Ocular surface reconstruction often become necessary in case of damage caused by various factors including trauma, infections, chemical burns, surgical complications and autoimmune diseases. The ultimate objective of this technique is reconstructing vision, alleviating pain and prevention from further damage. As already stated, keratin is known for its biocompatibility, biodegradability and ability to promote cell proliferation, cell division and cell adhesion it has now gained attention of researchers in the application of ocular surface reconstruction. This can be achieved by creating scaffolds, membranes and fibrous mats to repair and regenerate ocular surface tissue. Keratin-based biomaterials provide a supportive structure to promote cell proliferation and cell migration of corneal and conjunctival epithelial cells. These materials have mechanical properties which are similar to the native ocular surface and facilitate in healing and integration. Owing to its anti-inflammatory effect, keratin can provide a more conducive environment for tissue healing and reduce inflammation in the ocular surface. The research on the exploitation of keratin for ocular surface reconstruction is still evolving with ongoing studies exploring its full potential and optimizing the application processes. However, current results are promising and indicate that keratin and keratin-based biomaterials could become an ideal tool for ocular surface reconstruction. Generally amniotic membrane is applied as an alternative substitute during ocular surface reconstruction. Additionally, dexamethasone eye-drop is continuously required to supress inflammation and fast recovery rate after surgery.

Schwab & Reichl [105] successfully developed keratin films incorporated with dexamethasone drug. They used different concentrations of dexamethasone, and their findings suggest that prepared films with moderate dexamethasone gives satisfactory positive results as they influenced the biochemical properties and transparency of the films whereas highly loaded films showed exact similar result to those of amniotic membranes. The authors also compared these films with amniotic membranes and found that developed films could be a promising alternative to be used in ocular surface reconstruction [9]. Also compared keratin films with amniotic membranes by using ofloxacin and dexamethasone eye-drop externally on the regular intervals instead of incorporating in the membrane. The experiments involved use of amniotic membranes and keratin films separately in white rabbits and recorded the results after a period of 10 days. The eyes of rabbits treated with keratin films were reported to be completely healed without any neovascularization and those treated with amniotic membranes showed neovascularization on seventh day however, it recovered on tenth day.

Haemostatic agent

In case of any injury or cut, the loss of blood from the body is stopped by the formation of blood clot. The sequence of regulated events leading to the formation of blood clot is known as hemostasis and the agents that participate in hemostasis are called hemostatic agents. In case of a major bleeding or accidental situations, hemostasis may not be efficient enough and that could even lead to the death of the patient. Advance and new hemostatic technologies are continuously being developed to tackle uncontrolled hemorrhage in an emergency, battlefield and surgical conditions. Hemostasis involves activation of signaling pathways to clot the blood, including platelets and other proteins like fibrinogen and thrombin.

Although, many hemostatic agents, adhesives, and sealants are available in the market. But developing an ideal hemostatic agent with multiple properties such as effective and immediate management of bleeding, biodegradability, biocompatibility, appropriate mechanical properties, strong adhesion property, antibacterial activity, easily manageable in wet and dynamic conditions and many more still remains a huge leap. Keeping these conditions in mind, researchers have used keratin as a hemostatic agent because it is a versatile compound that has all these characteristics. Keratin activates platelets and other important proteins directly as it promotes platelet adhesion and aggregation. It can be used to produce physical scaffolds that supports the formation of blood clot. Scaffolds trap blood platelets and RBCs which contribute to the formation of a stable clot and can efficiently seal the injury and stop bleeding. Keratin can be isolated from different source material and processed into various forms such as sponges, powders and films which can be applied to wounds and on an injury directly. These materials enhance hemostasis as they can absorb blood immediately, aggregate clotting factors and provide a suitable environment for clot formation.

Goudarzi et al. [42] successfully developed keratin crosslinked sponges with the help of glutaraldehyde by utilizing freeze-drying technique. They performed experiments on human foreskin fibroblasts cells and suggested that developed sponges were able to absorb 91% of water and had good cell viability resulting into blood clotting and major liquid absorption. The authors also observed that prepared sponges were capable to be used in haemostasis [32]. Used freeze-gelation method to prepare composite scaffolds of methylene blue-loaded keratin and alginate. Developed composite scaffolds could absorb over 1600% liquid effectively, had good biodegradability, high biocompatibility and well interconnected pores. The researchers concluded that composite scaffolds of keratin and alginate work synergistically on wound and significantly minimizes haemostasis time. They also reported that the drug loaded into developed scaffolds prevent infection by absorbing wound secretions and increase burst release at the early stages of wound recovery.

Chen et al. [14] worked on keratin polymers (high and low molecular weight keratins) i. e. KIFs and KAPs. They used a combination of both proteins in different ratios to precipitate fibrinogen and reported that equal amount of KIFs and KAPs participate in haemostasis as it yielded highest accumulation of fibrinogen protein [136]. Utilized a novel approach viz. recombinant synthesis for maximizing the performance of keratin in haemostasis. They adopted those α- helical keratin sequences which are responsible for haemostatic activities and noticed that amino acids found on N-terminal of α- helices (such as Tyr, Phe and Gln) residues are very important in fibrin polymerization. The researchers also mutated the Cystine to Serine residues on α-helices and found a positive results in haemostasis. High efficiency keratin biomaterials could be produced by exploring such strategies with improved potential over gelatin sponges. In another study from [140, 141] also reported that keratin/chitosan sponges with porosity 90.12 ± 2.17% have potential to work as haemostatic agent [66]. Successfully developed KAPs nanoparticles from KAPs fragments extracted from human hairs and used these KAPNPs as haemostatic agent. Their researchers reported that KAPNPs have great potential, good biocompatibility and minimum clotting time.

Miscellaneous

Valkov et al. [128] prepared keratin films from human hair with structural similarity to human nail plate. The authors reported that the keratin films could be used as a model for studying onychomycosis. Also after infecting the dermatophytic fungi Trichophyton rubrum, the growth was observed on the surface of the film and the fungi was also able to penetrate inside the films [125] studied the use of chicken feather keratin as a template to produce silver nanoparticles (AgNP) and gold nanoparticles (AuNP). The AuNP and AgNP had spherical shape and a reported diameter of 3-13 nm and 4- 20 nm respectively. The authors proposed the use of synthesized nanoparticles for controlling growth of Klebsiella pneumoniae and Pseudomonas aeruginosa as well as potential urease inhibitor. Keratin materials are also being explored as a substrate or coating material for in vitro culturing of cells [8] utilized keratin from goat hair to prepare biomaterial and use it as coating material for in vitro culturing of mesenchymal stem cells (MSC’s) and primary goat fibroblast cells. The authors reported that the keratin biomaterials hold promising suitability in the area of cell-based tissue engineering and wound healing owing to their biocompatibility.

Conclusion

Keratin from variety of waste sources such as chicken feather, human hair are being utilized for the fabrication of biomaterials and have gained immense interest in various biomedical applications. Interesting physical and biological properties of keratin makes it a suitable candidate for applications such as skin tissue engineering, treating volumetric muscle loss, drug delivery and bone tissue regeneration among others. The use of hydrogels, scaffolds made up of keratin alone or loaded with either growth factors or drug molecules is an emerging option to handle and cure chronic wounds. In a similar fashion, the nanogels, nanoparticles, microfibers based on keratin have also been found to be effective in drug delivery systems that are biocompatible and show prolonged drug delivery in addition to growth promoting capabilities for different human cell lines. For oral tissue regeneration as well, keratin biomaterials have been found to be non- toxic for periodontal fibroblasts or dental keratinocytes as well as have also shown growth promotion for human periodontal ligament fibroblasts among others. At present multiple roles of keratin in tissue engineering and haemostasis are being established and more research could be focused on the detailed role of keratin in these areas.

Future perspectives

Various biomaterials in the form of films, hydrogels, nanoparticles have been utilized and put to diverse biomedical applications. Apart from being a cheap raw material keratin biomaterial have also been found to be biocompatible and biodegradable. Still fraction of keratin based biomaterials in commercial market and actual use in medical field is very less. The major challenges for keratin biomaterials could be summarized into inconsistent source material, complex extraction and purification methods, scalability and structural stability under varied physiological conditions such as pH, moisture and temperature. Detailed studies pertaining to molecular interactions and regarding the mechanical properties of these materials need to be taken up in order to overcome the aforesaid challenges.

Research needs to be focused on the behaviour of the keratin biomaterials with varied concentrations of different keratin components such as keratose, keratein, α keratin, β keratin and γ keratin under diverse physiological conditions as well as their cellular interactions and attachment profiles. Knowledge from these studies would be a great leap towards success in keratin based biomaterials production and application. Further endeavours could be made to fabricate customised biomaterials for specific biomedical roles and additional validation of the usage of keratin-based biomaterials needs to be done in large animal models.