Abstract As a prominent tool in regenerative medicine, tissue engineering (TE) has been an active field of scientific research for nearly three decades. Clinical application of TE technologies has ...been relatively restricted, however, owing in part to the limited number of biomaterials that are approved for human use. While many excellent biomaterials have been developed in recent years, their translation into clinical practice has been slow. As a consequence, many investigators still employ biodegradable polymers that were first approved for use in humans over 30 years ago. During normal development tissue morphogenesis is heavily influenced by the interaction of cells with the extracellular matrix (ECM). Yet simple polymers, while providing architectural support for neo-tissue development, do not adequately mimic the complex interactions between adult stem and progenitor cells and the ECM that promote functional tissue regeneration. Future advances in TE and regenerative medicine will depend on the development of “smart” biomaterials that actively participate in the formation of functional tissue. Clinical translation of these new classes of biomaterials will be supported by many of the same evaluation tools as those developed and described by Professor David F. Williams and colleagues over the past 30 years.
Advances in the extraction, purification, and characterization of keratin proteins from hair and wool fibers over the past century have led to the development of a keratin-based biomaterials ...platform. Like many naturally-derived biomolecules, keratins have intrinsic biological activity and biocompatibility. In addition, extracted keratins are capable of forming self-assembled structures that regulate cellular recognition and behavior. These qualities have led to the development of keratin biomaterials with applications in wound healing, drug delivery, tissue engineering, trauma and medical devices. This review discusses the history of keratin research and the advancement of keratin biomaterials for biomedical applications.
Treatment of THP-1 derived macrophages with keratin results in a predominance of M2 (anti-inflammatory) macrophages. Display omitted
Macrophage response to biomaterials is emerging as a major focus ...in tissue repair and wound healing. Macrophages are able to differentiate into two distinct states, eliciting divergent effects. The M1 phenotype is considered pro-inflammatory and up-regulates activity related to tissue destruction, whereas the M2 phenotype is considered anti-inflammatory and supports tissue remodeling. Both are necessary but a fine balance must be maintained as dysregulation of naïve macrophages to M1 or M2 polarization has been implicated in several disease and injury models, and has been suggested as a potential cause for poor outcomes. Keratin biomaterials have been shown using different animal models to promote regeneration in several tissues. A potential common mechanism may be the general capability for keratin biomaterials to elicit beneficial inflammatory responses during the early stages of regeneration. In the present study, a keratin biomaterial was utilized in vitro to examine its effects on polarization toward one of these two macrophage phenotypes, and thus its role in inflammation. Exposure of a monocytic cell line to keratin biomaterial substrates was shown to bias macrophages toward an M2 phenotype, while a collagen control surface produced both M1 and M2 macrophages. Furthermore, keratin treatment was similar to the M2 positive control and was similarly effective at down-regulating the M1 response. Keratin biomaterial influenced greater production of anti-inflammatory cytokines and decreased amounts of pro-inflammatory cytokines. The use of a keratin biomaterial in regenerative medicine may therefore provide additional benefit by regulating a positive remodeling response.
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•A coating that mimics the composition of human fingernail was investigated.•Purified human keratin biomaterial coatings demonstrated degradation resistance.•Keratin coatings ...demonstrated skin cell attachment through focal adhesions.•Keratin coatings may serve to improve the skin-implant interface of POP devices.
Percutaneous osseointegrated prosthetics (POP), which consist of a metallic post attached to the bone that extends outward through the skin to connect to an external prosthesis, have become a clinically relevant option to replace the typical socket-residual limb connection. POP devices offer several advantages such as mechanical off-loading of soft tissues, direct force transfer to the musculoskeletal system, greater proprioception, and overall improvement in limb kinesis compared to a socket system. However, POP devices create several challenges including epidermal downgrowth, increased infection risk, and mechanical tearing at the skin-implant interface. To address these issues, biomimetic surfaces and coatings have been developed in an attempt to create an infection-free and cohesive interface between POP devices and skin. The fingernail is a prime example of a natural system with a skin interface that is both mechanically and biologically stable. Exploiting keratins’ previously demonstrated tissue compatibility and creating a biomimetic coating for POP devices that can imitate the human fingernail, and demonstrating its ability to promote a stable interface with skin tissue is the goal of this work. Silane coupling aided in producing a coating on titanium substrates consisting of human keratin proteins. Several combinations of silane and keratin derivatives were investigated, and in general showed a nano-scale coating thickness that supported skin cell (i.e. fibroblast and keratinocyte) adhesion. Initial enzyme-mediated degradation resistance was also demonstrated, but the coatings appeared to degrade at long time periods. Importantly, keratinocytes showed a stable phenotype with no indication of wound healing-like activity. These data provide justification to further explore keratin biomaterials for POP coatings that may stabilize the implant-skin interface.
The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of ...osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as
and
. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.
Abstract The oxidized form of extractable human hair keratin proteins, commonly referred to as keratose, is gaining interest as a biomaterial for multiple tissue engineering studies including those ...directed toward peripheral nerve, spinal cord, skin, and bone regeneration. Unlike its disulfide cross-linked counterpart, kerateine, keratose does not possess a covalently cross-linked network structure and consequently displays substantially different characteristics. In order to understand its mode(s) of action and potential for clinical translatability, detailed characterization of the composition, physical properties, and biological responses of keratose biomaterials are needed. Keratose was obtained from end-cut human hair fibers by peracetic acid treatment, followed by base extraction, and subsequent dialysis. Analysis of lyophilized keratose powder determined that it contains 99% proteins by mass with amino acid content similar to human hair cortex. Metallic elements were also found in minute quantities. Protein oxidation led to disulfide bond cleavage and drastic reduction of free thiols due to conversion of sulfhydryl to sulfonic acid, chain fragmentation, and amino acid modifications. Mass spectrometry identified the major protein constituents as a heterogeneous mixture of 15 hair keratins (type I: K31–35 and K37–39, and type II: K81–86) with small amounts of epithelial keratins which exist in monomeric, dimeric, multimeric, and even degraded forms. Re-hydration with PBS enabled molecular assembly into an elastic solid-like hydrogel. Highly-porous scaffolds formed by lyophilization of the gel had the compression behavior of a cellular foam material and reverted back to gel upon wetting. Cytotoxicity assays showed that the EC50 for various cell lines were attained at 8–10 mg/mL keratose, indicating the non-toxic nature of the material. Implantation in mouse subcutaneous tissue pockets demonstrated that keratose resorption follows a rectangular hyperbolic regression with 92% degradation by an 8-week time point. Keratose was shown to integrate with the host tissue as evidenced by infiltration of leukocytes and fibroblasts, bulk material angiogenesis, and minimal fibrous encapsulation. Tissue response benchmarks were superior in keratose compared to the control PLGA 90:10 mesh. Finally, the degraded keratose was observed to remodel with the natural collagen extracellular matrix, verifying the benefit of using keratose as a temporary matrix for regenerative medicine applications.
Abstract Driven by new discoveries in stem-cell biology and regenerative medicine, there is broad interest in biomaterials that go beyond basic interactions with cells and tissues to actively direct ...and sustain cellular behavior. Keratin biomaterials have the potential to achieve these goals but have been inadequately described in terms of composition, structure, and cell-instructive characteristics. In this manuscript we describe and characterize a keratin-based biomaterial, demonstrate self-assembly of cross-linked hydrogels, investigate a cell-specific interaction that is dependent on the hydrogel structure and mediated by specific biomaterial–receptor interactions, and show one potential medical application that relies on receptor binding - the ability to achieve hemostasis in a lethal liver injury model. Keratin biomaterials represent a significant advance in biotechnology as they combine the compatibility of natural materials with the chemical flexibility of synthetic materials. These characteristics allow for a system that can be formulated into several varieties of cell-instructive biomaterials with potential uses in tissue engineering, regenerative medicine, drug and cell delivery, and trauma.
Abstract Peripheral nerve injuries requiring surgery can be repaired by autograft, the clinical “gold standard”, allograft, or nerve conduits. Most published clinical studies show the effectiveness ...of nerve conduits in small size defects in sensory nerves. Many preclinical studies suggest that peripheral nerve regeneration through conduits can be enhanced and repair lengths increased with the use of a biomaterial filler in the conduit lumen. We have previously shown that a luminal hydrogel filler derived from human hair keratin (HHK) can improve electrophysiological and histological outcomes in mouse, rabbit, and non-human primate nerve injury models, but insight into potential mechanisms has been lacking. Based on the premise that a keratin biomaterial (KOS) hydrogel provides an instantaneous structural matrix within the lumen, the current study compares the cellular behavior elicited by KOS hydrogel to Matrigel (MAT) and saline (SAL) conduit fillers in a 1 cm rat sciatic nerve injury model at early stages of regeneration. While there was little difference in initial cellular influx, the KOS group showed earlier migration of dedifferentiated Schwann cells (SC) from the proximal nerve end compared to the other groups. The KOS group also showed faster SC dedifferentiation and myelin debris clearance, and decreased macrophage infiltration during Wallerian degeneration of the distal nerve tissue.
Purpose The management of peripheral nerve injuries with segmental defects is a challenge to both patient and surgeon. Repairs under tension have a poor prognosis; sensory nerve allografts have donor ...site morbidity and suboptimal motor recovery, but remain the gold standard. The development of conduit-based repair strategies has evolved and these are promising for sensory nerves and short defects; however, no conduit filler is clinically available that improves motor recovery equivalent to sensory autografts. In this study, motor recovery using keratin-based hydrogel filler was compared with that for sensory nerve autografts and empty conduits. Methods Fifty-four mice were randomized into 3 treatment groups: empty conduit, sural nerve autograft, and keratin hydrogel-filled conduit. Animals were followed for 6 weeks, 3 months, and 6 months. Outcomes included compound motor action potential (CMAP), nerve area, myelinated axon number and density, and myelinated axon diameter. Results Neuromuscular recovery with keratin was greater than with empty conduits in most outcome measures. Nerves that regenerated through the keratin hydrogel had lower conduction delays, greater amplitudes, more myelinated axons, and larger axons than nerves that regenerated through empty conduits. Sensory nerve autografts and keratin hydrogel were statistically equivalent in CMAP measurements at 6 months. Moreover, keratin-filled conduits demonstrated greater axon density and larger average axon diameter than both empty conduits and autograft at 6 months. Conclusions In a mouse tibial nerve model, keratin hydrogels significantly improved electrophysiological recovery, compared with empty conduits and sensory nerve autografts, at an early time point of regeneration. Keratin hydrogels also produce long-term electrical and histological results superior to empty conduits and equivalent to sensory nerve autografts.
Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic ...forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10(-4) M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7) M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5) M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.
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