[Show abstract][Hide abstract] ABSTRACT: 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 (K<sub>D</sub>) of 1.8 × 10<sup>−4</sup> 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 (K<sub>D</
PLoS ONE 08/2015; 10(8-8):e0137233. DOI:10.1371/journal.pone.0137233 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophage response to biomaterials is emerging as a major focus in tissue repair and wound healing. Macrophages are able to differentiate to two distinct states eliciting divergent effects. The M1 phenotype is considered pro-inflammatory and up-regulates activity related to tissue destruction, while 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, which has been suggested as a potential cause for poor outcomes. Keratin biomaterials have been shown to promote regeneration in several tissues using different animal models. 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, a 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.
[Show abstract][Hide abstract] ABSTRACT: The Industry Committee of the Tissue Engineering Regenerative Medicine International Society, Americas Chapter (TERMIS-AM) administered a survey to its membership in 2013 to assess the awareness of science requirements in the FDA regulatory process. 144 members responded to the survey. Their occupational and geographical representation was representative of the TERMIS-AM membership as a whole. The survey elicited basic demographic information, the degree to which members were involved in tissue engineering technology development and their plans for future involvement in such development. The survey then assessed awareness of general FDA scientific practices as well as specific science requirements for regulatory submissions to the Center for Biologics Evaluation and Research (CBER), the Center for Drug Evaluation and Research (CDER), the Center for Devices and Radiological Health (CDRH) and the Office of Combination Projects (OCP). The FDA-specific questions in the survey were culled from guidance documents posted on the FDA web site (www.fda.gov). One of the answer options was an opt-out clause that enabled the survey respondent to claim a lack of sufficient awareness of the topic to answer the question. This enabled the stratification of respondents on the basis of confidence in the topic. Results indicate that across all occupational groups (academic-business-government) that are represented in the TERMIS-AM membership, awareness of FDA science requirements varies markedly. Those who performed best represented for-profit company employees, consultants and government employees while students, professors and respondents from outside the USA performed least well. Confidence in question topics substantially increased correctness in responses across all groups, though the association between confidence and the ability to answer correctly was poorest among students and professors. Though 80% of respondents claimed involvement in development of a tissue engineering technology (12% Biologic, Non-Cellular; 34% Biologic, Cellular; 14% Medical Device and 40% Combination Product), their responses were no more correct than those who were not. The data suggest that early exposure to regulatory experts would be of value for entrepreneurs seeking to bring their technology to market. For students and professors, formal education in Regulatory Science in academia should be considered to best support translational tissue engineering research and development.
Tissue Engineering Part A 03/2014; 20(11-12). DOI:10.1089/ten.TEA.2014.0112 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When skin is thermally burned, transfer of heat energy into the skin results in the destruction of cells. Some of these cells are damaged but may be capable of self-repair and survival, thereby contributing to spontaneous healing of the wound. Keratin protein-based biomaterials have been suggested as potential treatments for burn injury. Isolation of cortical proteins from hair fibers results in an acid soluble fraction of keratin proteins referred to as "gamma" keratose. In the present study, treatment with this fraction dissolved in media was able to maintain cell viability after thermal stress in an in vitro model using primary mouse dermal fibroblasts. PCR array analysis demonstrated that gamma keratose treatment may assist in the survival and salvage of thermally stressed cells by maintaining their viability through regulation of cell death pathway-related genes. Gamma keratose may be a promising biomaterial for burn treatment that aids in spontaneous wound healing from viable tissue surrounding the burn.
[Show abstract][Hide abstract] ABSTRACT: Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.
[Show abstract][Hide abstract] ABSTRACT: Medical care costs can reach an estimated value of $4 billion for spinal cord injuries (SCI) each year in the USA alone. With no viable treatment options available, care remains palliative and aims to minimize lifelong disabilities and complications, such as immobility, bladder and bowel dysfunction, breathing problems, and blood clots. Human hair keratin biomaterials have demonstrated efficacy in peripheral nerve injury models and were shown to improve conduction delay and increase axon number and density. In this study, a keratin hydrogel was tested in a central nervous system (CNS) application of spinal cord hemisection injury. Keratin-treated rats showed increased survival rates as well as a better functional recovery of gait properties and bladder function. Histological results demonstrated reduced glial scar formation with keratin treatment and suggested a greater degree of beneficial remodeling and cellular influx. The data provided in this pilot study suggest the possibility of using a keratin-based treatment for SCI and warrant further investigation.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Naturally derived tendon scaffolds have the potential to improve the treatment of flexor tendon injuries. Seeded and unseeded tendon scaffolds were maintained in the presence or absence of physiologic strain for 7 days. After 7 days, the tensile properties and associated messenger RNA expression were compared. Seeded scaffolds maintained in the absence of strain had significantly lower tensile properties than unseeded tendons and fresh-frozen tendons. The loss of tensile properties was associated with elevated matrix metalloproteinase-2 and collagen III expression. Tensile properties of seeded scaffolds maintained in the presence of strain for 7 days after seeding did not differ from those of fresh-frozen tendons. This study demonstrates that the tensile properties of seeded, naturally derived tendon scaffolds will degrade rapidly in the absence of cyclic strain. Seeded scaffolds used for tendon reconstruction should be maintained under cyclic strain to maintain essential tensile properties.
Journal of surgical orthopaedic advances 09/2013; 22(3):224-32. DOI:10.3113/JSOA.2013.0224
[Show abstract][Hide abstract] ABSTRACT: In response to the Bayh-Dole Act, synergies between the academic and business sectors have been contemplated for the development of biotechnology companies for the past 30 years. However, the failure rate for biotech startups has been notably high, albeit variable depending on the source and more importantly, the definition of failure that is applied. Shikhar Ghosh from the Harvard Business School stated "If failure means liquidating all assets,… then the failure rate for start-ups is 30-40%. If failure refers to failing to see the projected return on investment (ROI,) then the failure rate is 70-80%. If failure is defined as declaring a projection and then falling short of meeting it, then the failure rate is a whopping 90-95% (1)." The risk of failure as well as the decrease in available funding from both public and private sectors has led to more forethought given to the development of translational technologies, i.e. technologies developed with the goal of moving from bench to bedside. The Tissue Engineering and Regenerative Medicine International Society - Americas (TERMIS-AM) Industry Committee seeks to guide academic-entrepreneurs on approaches to successfully assess their technologies and to make strategically-focused business and development decisions. Early evaluation of a technology's potential allows for optimization of a product in a pre-market setting and more importantly, mitigation of risk. Academics most often consider the "science risk," or risk associated with the intital R&D phase, but a more long-range consideration of commercialization, regulatory and investment risks, as well as potential exit strategies would most likely result in a greater likelhood of success. The TERMIS-AM IC established a vision to identify barriers to commercialization and to work toward offering insights and understanding of these barriers, as well as approaches to potential business solutions (2-4). A workshop focused on key risks has been developed with experts who will share their insights on the successful navigation of these risks and advancement of their products toward commercial reality. One approach that may be helpful is to illustrate this process with respect to the risks that are within an academic's control versus those that are outside.
Tissue Engineering Part A 09/2013; 19(21-22). DOI:10.1089/ten.TEA.2013.0474 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TERMIS-AM Industry Committee (TERMIS-AM/IC), in collaboration with the TERMIS-Europe (EU)/IC, conducted a symposium involving the European Medicines Agency and the U.S. Food and Drug Administration (FDA) toward building an understanding of the rational basis for regulatory decision-making and providing a framework for decisions made during the evaluation of safety and efficacy of TE/RM technologies. This symposium was held in August 2012 during the TERMIS-WC in Vienna, Austria. Emerging from this international initiative by the European Union and the United States, representatives from the respective agencies demonstrated that there are ongoing interagency efforts for developing common national practices toward harmonization of regulatory requirements for the TE/RM products. To extend a broad-based understanding of the role of science in regulatory decision-making, TERMIS-AM/IC, in cooperation with the FDA, organized a symposium at the 2014 TERMIS-AM Annual Meeting, which was held in Washington, DC. This event provided insights from leaders in the FDA and TERMIS on the current status of regulatory approaches for the approved TE/RM products, the use of science in making regulatory decisions, and TE/RM technologies that are in the development pipeline to address unmet medical needs. A far-ranging discussion with FDA representatives, industrialists, physicians, regenerative medicine biologists, and tissue engineers considered the gaps in today's scientific and regulatory understanding of TE/RM technologies. The identified gaps represent significant opportunities to advance TE/RM technologies toward commercialization.
Tissue Engineering Part A 09/2013; 19(21-22). DOI:10.1089/ten.TEA.2013.0536 · 4.64 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Traumatic injury is the leading cause of death in people aged 44 or less in the US. It is also estimated that 82% of deaths from battlefield hemorrhage may be survivable with better treatment options. In this study, two biomaterial hemostats having disparate mechanisms were evaluated in a large animal lethal hemorrhage model and compared to a commercial product and standard cotton gauze. We hypothesized that the biomaterial with a biologically active mechanism, as opposed to a mechanical mechanism, would be the most effective in this model. Using a published study protocol, the femoral artery in swine was punctured and treated. KeraStat™ (KeraNetics) and Nanosan®-Sorb (SNS Nano) hemostats were compared to a commercial chitosan dressing (second generation Hemcon®) and cotton gauze. Both KeraStat and Nanosan increased survival, significantly increased mean arterial pressure (MAP), and significantly decreased shock index compared to both controls. The Hemcon dressing was no different than gauze. Platelet adhesion assays suggested that the KeraStat mechanism of action involves β1 integrin mediated platelet adhesion while Nanosan-Sorb operates similar to one reported mechanism for Hemcon, absorbing fluid and concentrating clotting components. The Nanosan also swelled considerably and created pressure within the wound site even after direct pressure was removed.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Infuse(®) is used clinically to promote bone repair. Its efficacy is dependent on a crosslinked collagen carrier/scaffold system that has come under scrutiny due to an inability to control BMP-2 release, which may result in unwanted outcomes such as heterotopic ossification. In this study, keratose biomaterial was evaluated as a new carrier/scaffold. Keratose was mixed with BMP-2, fabricated into a scaffold, and implanted into a critical-size rat femoral defect. This construct showed bridging as early as 4 weeks and induced trabecular morphology characteristic of a remodeling hard fracture callus at 16 weeks. Compared to the normal cortical bone, the regenerated tissue had greater volume and mineral content but less density and ultimate shear stress values. Moreover, μ-CT, biomechanics, FTIR-ATR spectroscopy, and polarized light microscopy data showed regeneration using keratose was similar to an Infuse control. However, unlike Infuse's collagen carrier system, in vitro analysis showed that BMP-2 release correlated with keratose scaffold degradation. Surprisingly, treatment with keratose only led to deposition of more bone outgrowth than the untreated negative control at the 8-week time point. The application of keratose also demonstrated a notable reduction of adipose tissues within the gap. While not able to induce osteogenesis on its own, keratose may be the first biomaterial capable of suppressing adipose tissue formation, thereby indirectly enhancing bone regeneration.
[Show abstract][Hide abstract] ABSTRACT: Natural extracellular matrix (ECM) proteins possess critical biological characteristics that provide a platform for cellular adhesion and activation of highly regulated signaling pathways. However, ECM based biomaterials can have several limitations including poor mechanical properties and risk of immunogenicity. Synthetic biomaterials alleviate the risks associated with natural biomaterials but often lack the robust biological activity necessary to direct cell function beyond initial adhesion. A thorough understanding of receptor mediated cellular adhesion to the ECM and subsequent signaling activation has facilitated development of techniques that functionalize inert biomaterials providing a biologically active surface. Here we review a range of approaches used to modify biomaterial surfaces for optimal receptor mediated cell interactions as well as provide insights into specific mechanisms of downstream signaling activation. In addition to a brief overview of integrin receptor-mediated cell function, so-called "biomimetic" techniques reviewed here include (1) surface modification of biomaterials with bioadhesive ECM macromolecules or specific binding motifs, (2) nanoscale patterning of the materials, and (3) use of "natural-like" biomaterials.
[Show abstract][Hide abstract] ABSTRACT: Swine are the preferred animal models to study the effects of burns on dermal wound healing. Various studies have been published in which little emphasis was placed on minimizing burn variability and inconsistency. We developed a novel method to create deep partial thickness burns that are highly consistent. A custom-made burn device was fabricated to control the pressure applied on the swine skin during burn creation. Cylindrical brass blocks, measuring 3cm in diameter, are used to create the burns. A stainless steel post extends from the block for insertion into the device holder. In this study, burns were created in four female Yorkshire swine. Heating of the brass blocks was conducted using a boiling azeotropic solution of 80% polyethylene glycol (PEG) and 20% water and boiling water alone. Contact times ranging from 12 to 20s were used. At 24h and 7d post-injury, two swine were euthanized and tissues collected for digital image evaluation and histological assessment using Gomori trichrome staining. Digital image analysis showed inconsistent healing in burns created using boiling water as compared to the boiling PEG:H(2)O solution. Additionally, histological analyses showed that burns created using boiling water were superficial and more variable compared to those created using the boiling PEG:H(2)O solution. With a burn contact time of 20s, 48.5±5.7% tissue damage was demonstrated at 24h when the PEG:H(2)O solution was used, whereas only 11.9±1.3% was observed with boiling water.
Burns: journal of the International Society for Burn Injuries 09/2012; 39(2). DOI:10.1016/j.burns.2012.06.011 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Any faculty member wishing to commercialize a discovery or technology should contemplate the pros and cons of pursuing the entrepreneurial route or licensing to an outside entity.