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One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Keratin-based biomaterials represent an attractive opportunity in the fields of wound healing and tissue regeneration, not only for their chemical and physical properties, but also for their ability to act as a delivery system for a variety of payloads. Importantly, keratins are the only natural biomaterial that is not targeted by specific tissue turnover-related enzymes, giving it potential stability advantages and greater control over degradation after implantation. However, in-situ polymerization chemistry in some keratin systems are not compatible with cells, and incorporation within constructs such as hydrogels may lead to hypoxia and cell death. To address these challenges, we envisioned a pre-formed keratin microparticle on which cells could be seeded, while other payloads (e.g. drugs, growth factors or other biologic compounds) could be contained within, although studies investigating the potential partitioning between phases during emulsion polymerization would need to be conducted. This study employs well-established water-in-oil emulsion procedures as well as a suspension culture method to load keratin-based microparticles with bone marrow-derived mesenchymal stem cells. Fabricated microparticles were characterized for size, porosity and surface structure and further analyzed to investigate their ability to form gels upon hydration. The suspension culture technique was validated based on the ability for loaded cells to maintain their viability and express actin and vinculin proteins, which are key indicators of cell attachment and growth. Maintenance of expression of markers associated with cell plasticity was also investigated. As a comparative model, a collagen-coated microparticle (Sigma) of similar size was used. Results showed that an oxidized form of keratin ("keratose" or "KOS") formed unique microparticle structures of various size that appeared to contain a fibrous sub-structure. Cell adhesion and viability was greater on keratin microparticles compared to collagen-coated microparticles, while marker expression was retained on both.

Tissue regeneration following injury is mediated by macrophage recruitment and differentiation in response to environmental signals. In general, macrophages adopt either a classically M1 (M[IFN-γ, LPS]) or alternatively activated M2 (M[IL-4, IL-13] or M[IL-10]) phenotype. Recent studies have highlighted the importance of alternatively activated macrophages in tissue remodeling and repair as well as the contribution of an imbalance of classically and alternatively activated macrophages to tissue degeneration and disease progression. Keratin biomaterials have recently demonstrated their ability to promote alternatively activated macrophage polarization in an in vitro model using a monocytic cell line. In the present study, the ability of extracted human hair keratins to influence alternative activation of human primary monocytes in vitro is assessed by evaluating changes in surface receptor expression, inflammatory cytokine secretion, and phagocytic activity. The impact of keratin molecular weight fractionation on these outcomes was also investigated. High and low molecular weight fractions of the oxidized form of extractable human hair keratins - referred to as keratose (KOS H and KOS P , respectively) - were characterized by size exclusion chromatography, mass spectrometry, and Western blot. Primary macrophages underwent traditional differentiation to the M[IFN-γ, LPS], M[IL-4, IL-13], or M[IL-10]) phenotypes or were plated on different molecular weight keratin coatings (KOS H and KOS P ). Macrophages plated on keratin and analyzed via flow cytometry yielded the largest CD163 + cell populations and CD163 mean fluorescence intensities. Cells in the KOS P group were significantly more phagocytic than all other cell types at the 1.5 and 3 h time points and exhibited behavior and a cytokine production profile most similar to the M[IL-10] treated group. These findings may have important implications for understanding and evaluating the ability of keratin biomaterials to influence inflammation and tissue regeneration in disease and injury models., Statement of Significance: Biomaterials made from human hair keratins have previously been shown to elicit anti-inflammatory responses from naïve macrophages and polarize them toward an M2 phenotype. In this work we show for the first time that primary human cells respond similarly, that it is the M2c phenotype that predominates, that a sub-fraction of hydrolyzed keratin peptides are most likely responsible for the response, and that immobilization of the keratin peptides to a surface is required. Keratin biomaterials have been used to regenerate several tissues such as skin, muscle, bone, nerve, and cornea, in vitro and in animal studies. Our current findings will help guide the development of keratin-based biomaterials that seek to direct responses toward regenerative outcomes by attenuating inflammation., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Ideal material characteristics for tissue engineering or regenerative medicine approaches to volumetric muscle loss (VML) include the ability to deliver cells, growth factors, and molecules that support tissue formation from a system with a tunable degradation profile. Two different types of human hair-derived keratins were tested as options to fulfill these VML design requirements: (1) oxidatively extracted keratin (keratose) characterized by a lack of covalent crosslinking between cysteine residues, and (2) reductively extracted keratin (kerateine) characterized by disulfide crosslinks. Human skeletal muscle myoblasts cultured on coatings of both types of keratin had increased numbers of multinucleated cells compared to collagen or Matrigel(TM) and adhesion levels greater than collagen. Rheology showed elastic moduli from 10(2) to 10(5) Pa and viscous moduli from 10(1) to 10(4) Pa depending on gel concentration and keratin type. Kerateine and keratose showed differing rates of degradation due to the presence or absence of disulfide crosslinks, which likely contributed to observed differences in release profiles of several growth factors. In vivo testing in a subcutaneous mouse model showed that keratose hydrogels can be used to deliver mouse muscle progenitor cells and growth factors. Histological assessment showed minimal inflammatory responses and an increase in markers of muscle formation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 864-879, 2016., (© 2015 Wiley Periodicals, Inc.)

Partial thickness burns can advance to full thickness after initial injury due to inadequate tissue perfusion and increased production of inflammatory cytokines, which has been referred to as burn wound progression. In previous work, we demonstrated that a keratin biomaterial hydrogel appeared to reduce burn wound progression. In the present study, we tested the hypothesis that a modified keratin hydrogel could reduce burn wound progression and speed healing. Standardized burn wounds were created in Yorkshire swine and treated within 30 minutes with keratin hydrogel (modified and unmodified), collagen hydrogel, or silver sulfadiazine (SSD). Digital images of each wound were taken for area measurements immediately prior to cleaning and dressing changes. Wound tissue was collected and assessed histologically at several time points. Wound area showed a significant difference between hydrogels and SSD groups, and rates of reepithelialization at early time points showed an increase when keratin treatment was used compared to both collagen and SSD. A linear regression model predicted a time to wound closure of approximately 25 days for keratin hydrogel while SSD treatment required 35 days. There appeared to be no measurable differences between the modified and unmodified formulations of keratin hydrogels., Competing Interests: At the time of this study, Mark Van Dyke, Ph.D., held stock and was an officer in the company, KeraNetics LLC, which provided partial funding for this research. Wake Forest Health Sciences has a potential financial interest in KeraNetics, LLC, through licensing agreements.

Thermal burns typically display an injury pattern dictated by the transfer of the thermal energy into the skin and underlying tissues and creation of three zones of injury represented by a necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and a distant zone of stressed cells that will recover with proper treatment. The wound healing capabilities of a keratin biomaterial hydrogel were studied in two pilot studies, one using a chemical burn model in mice and the other a thermal burn model in swine. In both studies, keratin was shown to prevent enlargement of the initial wound area and promote faster wound closure. Interestingly, treating thermally stressed dermal fibroblast in culture demonstrated that soluble keratin was able to maintain cell viability and promote proliferation. Separation of so-called alpha and gamma fractions of the keratin biomaterial had differential effects, with the gamma fraction producing more pronounced cell survival and recovery. These results suggest that the gamma fraction, composed essentially of degraded alpha keratin proteins, may facilitate cell rescue after thermal injury. Treatment of burns with gamma keratin may therefore represent a potential therapy for wounds with an intermediate zone of damaged tissue that has the potential to contribute to spontaneous healing., (© The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

A human hair keratin biomaterial hydrogel scaffold was evaluated as a nerve conduit luminal filler following median nerve transection injury in 10 Macaca fascicularis nonhuman primates (NHP). A 1 cm nerve gap was grafted with a NeuraGen® collagen conduit filled with either saline or keratin hydrogel and nerve regeneration was evaluated by electrophysiology for a period of 12 months. The keratin hydrogel-grafted nerves showed significant improvement in return of compound motor action potential (CMAP) latency and recovery of baseline nerve conduction velocity (NCV) compared with the saline-treated nerves. Histological evaluation was performed on retrieved median nerves and abductor pollicis brevis (APB) muscles at 12 months. Nerve histomorphometry showed a significantly larger nerve area in the keratin group compared with the saline group and the keratin APB muscles had a significantly higher myofiber density than the saline group. This is the first published study to show that an acellular biomaterial hydrogel conduit filler can be used to enhance peripheral nerve regeneration and motor recovery in an NHP model.

Death after severe hemorrhage remains an important cause of mortality in people under 50 years of age. Keratin resuscitation fluid (KRF) is a novel resuscitation solution made from keratin protein that may restore cardiovascular stability. This postulate was tested in rats that were exsanguinated to 40% of their blood volume. Test groups received either low or high volume resuscitation with either KRF or lactated Ringer's solution. KRF low volume was more effective than LR in recovering cardiac function, blood pressure and blood chemistry. Furthermore, in contrast to LR-treated rats, KRF-treated rats exhibited vital signs that resembled normal controls at 1-week.

Uncontrolled bleeding continues to be one of the leading causes of death in individuals following traumatic injury. Prognosis is worsened with the onset of acute coagulopathy characterized by metabolic acidosis, hypothermia and hemodilution, which consequently perpetuates blood loss and increases mortality. While there are several limitations to biomaterials employed as hemostatic agents, keratin biomaterials have demonstrated efficacy in mitigating blood loss in an animal model of hemorrhage in prior studies. Here we investigate the hypothesis that keratins actively participate in coagulation and that a potential mechanism of action is independent of temperature and dilution of clotting factors. Data from this study show that keratins appear to contribute to hemostasis by significantly decreasing plasma clotting lag times and are able to maintain activity under simulated conditions of coagulopathy. Moreover, a system of isolated fibrin polymerization provided evidence of increased fibril lateral assembly in the presence of keratin. The data provided here provides a platform for further development of keratin biomaterials as hemostatic agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The structure-property relationships of kerateine materials were studied by separating crude hair extracts into two protein sub-fractions, referred to as α- and γ-kerateines, followed by their de novo recombination into meta-kerateine hydrogels, sponges and films. The kerateine fractions were characterized using electrophoresis and mass spectrometry, which revealed that the α-fraction contained complexes of type I and type II keratins and that the γ-fraction was primarily protein fragments of the α-fraction along with three proteins of the KAP-1 family. Meta-kerateine materials with increased amounts of γ-kerateines showed diminished physical, mechanical and biological characteristics. Most notably, materials with higher γ-content formed less elastic and less solid-like hydrogels and sponges that were less hydrolytically stable. In addition, a model biological assay showed that meta-kerateine films with greater amounts of γ-kerateines were less supportive of hepatocyte attachment. Investigation into the mechanism of attachment revealed that hepatocyte adhesion to meta-kerateines is not mediated by the β1 integrin subunit, despite the presence of LDV binding motifs within the type I α-keratins. This work to define the role of protein composition on biomaterial function is essential for the optimization of keratin biomaterials for biomedical applications., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: Keratin proteins have been utilized as biomaterials for decades, and are currently under investigation for a variety of tissue regeneration and trauma applications. It has been suggested that certain keratins may have the capacity to act as a colloid in fluid resuscitation applications, providing viscosity and oncotic properties that may be beneficial during acute ischemic events. Oxidized keratin derivatives, also known as keratoses, show good blood and cardiovascular compatibility and thus are the subject of this study., Methods: The effects of keratose compounds will be assessed using a topload i.v. infusion model and observation of changes in the microvasculature of the cremaster muscle of rats., Results: Keratose resuscitation fluid (KRF) administration resulted in significant vasodilation in the cremaster muscle. This effect was blocked with pretreatment of l-NA to inhibit NO. Another keratin fraction, alpha-keratose, which is the primary viscosic compound, was not found to induce vasodilation., Conclusions: The apparent mechanism of vasodilation was found to be NO-mediated and isolated to a particular purified fraction, the KAP., (© 2011 John Wiley & Sons Ltd.)

Keratins are naturally derived proteins that can be fabricated into several biomaterial forms including hydrogels. These materials are a potential polymeric system for several tissue engineering and regenerative medicine applications due to their ability to support cell attachment, proliferation, and migration. However, little is known regarding their ability to support sustained release of therapeutic agents. This report describes the use of keratin hydrogels for sustained release of the antibiotic ciprofloxacin, which may prove useful to traumatic injury applications that would benefit from materials promoting tissue regeneration while also preventing acute infection. Hydrogels were formed from keratins obtained by oxidative extraction and known as keratose. We found that keratose hydrogels released ~60% of loaded ciprofloxacin over the first 10 days and that continued release was detectable over the course of 3 weeks. Released ciprofloxacin was bioactive, inhibiting growth of Staphylococcus aureus for 23 days in vitro and for 2 weeks in a mouse subcutaneous model. The rate of ciprofloxacin release was highly correlated with degradation of the keratin hydrogel and not consistent with simple diffusion. Further experiments indicated that ciprofloxacin binds to keratose through electrostatic interactions. These studies demonstrate the specific use of keratose hydrogels for the release of antibiotic and the potential for the more general use of this material in tissue engineering and regenerative medicine applications., (Copyright © 2011 Wiley Periodicals, Inc.)

Entubulation of transected nerves using bioabsorbable conduits is a promising alternative to sural nerve autografting, but full functional recovery is rarely achieved. Numerous studies have suggested that scaffold-based conduit fillers may promote axon regeneration, but no neuroinductive biomaterial filler has been identified. We previously showed that a nerve guide filled with keratin hydrogel actively stimulates regeneration in a mouse model, and results in functional outcomes superior to empty conduits at early time points. The goal of the present study was to develop a peripheral nerve defect model in a rabbit and assess the effectiveness of a keratin hydrogel filler. Although repairs with keratin-filled conduits were not as consistently successful as autograft overall, the use of keratin resulted in a significant improvement in conduction delay compared to both empty conduits and autograft, as well as a significant improvement in amplitude recovery compared to empty conduits when measurable regeneration did occur. Taking into account all study animals (i.e., regenerated and nonregenerated), histological assessment showed that keratin-treated nerves had significantly greater myelin thickness than empty conduits. These data support the findings of our earlier study and suggest that keratin hydrogel fillers have the potential to be used clinically to improve conduit repair.

Keratins are a family of structural proteins that can be isolated from a variety of tissues. "Soft" keratins are cytoskeletal elements found in epithelial tissues while protective tissues such as nails, hooves, and hair are composed of "hard" keratins. Hard keratins have been the subject of biomaterials investigations for more than three decades. Numerous methods exist for denaturing these proteins which are characterized by a high sulfur content and extensive disulfide bonding, under either oxidative or reductive conditions, extracting them from tissue and processing them into various physical states such as gels, films, coatings, and fibers. Kerateines or keratoses (oxidatively or reductively derived, respectively), alone or in combination with other biomaterials, have been tested in a small number of systems to demonstrate feasibility for medical applications such as wound healing, bone regeneration, hemostasis, and peripheral nerve repair. These investigations have shown generally good compatibility with cells and tissues, but the focus of prior investigations has been fairly narrow, and as a result there is relatively little published data on the general behavior of keratin biomaterials in biological systems beyond cell culture assays. The goal of this study was to produce a reduced form of keratin biomaterial, kerateine, using a typical and well-published technique, and characterize several aspects of its behavior that may have implications to its general use as a biomaterial. Kerateines were extracted from human hair, fabricated into gels and porous scaffolds, characterized, and placed into biological systems to determine their interactions with cells and tissue. Initially, the proteins were analyzed for molecular weight and amino acid content, as well as their ability to facilitate cell adhesion and proliferation. Crosslinked hydrogels were investigated for their hydrolytic stability in vitro; the micro-architecture and in vivo tissue response of lyophilized gels was also studied. These experiments both confirmed and expanded earlier findings that kerateines demonstrate excellent compatibility in biological systems.

Effective hemostatic dressings that are compatible with tissues are needed. Keratins are a class of biomaterials that can be derived by extraction of proteins from human hair. We have recently discovered that keratin biomaterials have hemostatic characteristics and hypothesize that a keratin hydrogel having the ability to absorb fluid and bind cells may be an effective hemostat. The goal of this study was to test a keratin hydrogel and evaluate it compared to current hemostats. Thirty-two New Zealand white rabbits received a lethal liver injury. Eight animals each were assigned to negative control, QuickClot, HemCon bandage, and keratin treatment groups. Vital stats and other data were recorded during surgery and all surviving animals were sacrificed after 72 h. Histology was conducted on all surviving animals. Twenty-four-hour survival rates were 0%, 62.5%, 62.5%, and 75% for the negative control, QuickClot, HemCon, and keratin groups, respectively. Other outcomes included blood loss, mean arterial pressure, heart rate, shock index, and liver histology. All of the hemostats were statistically better than the negative control group at late operative time points. The keratin group consistently performed as well as, or better than, the commercial hemostats. Histology showed an interesting healing response at the hemostat-liver interface in the keratin group., ((c) 2008 Wiley Periodicals, Inc.)

The management of trauma-associated nerve defects is difficult because of the absence of autologous donor motor or sensory nerves. Pre-clinical development and clinical experience has shown that damaged nerves can be surgically repaired using a tubular conduit interposed across the defect. Acceptable patient outcomes are achieved so long as the gap distance does not exceed a few centimeters. Although research in animals has demonstrated that nerve repair can be facilitated across slightly larger gaps by introducing a biomaterial filler into the conduit lumen, these biomaterials are not typically "neuroinductive" (i.e. capable of acting directly on regenerative cells to enhance nerve tissue formation beyond clinical limits). Moreover, their use does not often result in functional recovery equivalent to nerve autograft, the clinical gold standard. Here we show that a biomaterial gel made from the proteins found in human hair can mediate a robust nerve regeneration response, in part through activation of Schwann cells. In vitro, keratins extracted from human hair enhance the activity of Schwann cells by a chemotactic mechanism, increase their attachment and proliferation, and up-regulate expression of important genes. Moreover, these characteristics translate to improved functional nerve recovery in an animal model. These results suggest that a biomaterial derived from human hair keratins is neuroinductive and can facilitate an outcome comparable to autograft in a nerve injury model.