Browsing by Author "Van Dyke, Mark E."
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- Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2de Guzman, Roche C.; Tsuda, Shanel M.; Ton, Minh-Thi N.; Zhang, Xiao; Esker, Alan R.; Van Dyke, Mark E. (PLOS, 2015-08-28)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..
- Complementary Strategies to Promote Mesenchymal Stem Cell Differentiation for Ligament Tissue EngineeringShaffer, Robyn Denise (Virginia Tech, 2010-11-01)Anterior cruciate ligament (ACL) ruptures and tears are significant orthopedic problems that result in discomfort and limited mobility. Fully functional tissue engineered ligament replacements are promising alternatives to current graft choices for repair of ACL disruptions. The cell-based approach to construct engineered ligament grafts presented herein involves the culture of mesenchymal stem cells (MSC) on biodegradable, fibrous polymeric scaffolds to promote tissue formation. Multipotent MSCs are advantageous because of their in vitro proliferative capacity and ease of harvest; however; the promotion of MSC differentiation into mature fibroblasts and subsequent extracellular matrix (ECM) development is unknown. The proposed studies utilized three complementary methods to promote differentiation of MSCs: scaffold architecture, mechanical stretch and over-expression of the transcription factor, scleraxis. First, elastomeric scaffolds were fabricated by electrospinning a segmented poly(esterurethane urea) with variations in fiber diameter and fiber alignment. Primary mesenchymal stem cells and the mesenchymal stem cell line, C3H10T1/2, were seeded on these scaffolds and assumed spindle-shaped morphologies and oriented with the direction of fiber alignment. Fiber diameter affected cellular responses, including the expression of ECM genes (e.g. collagen type 1 and decorin) which were elevated on smaller mean fiber diameter scaffolds initially. However, scleraxis gene expression was greatest on larger mean fiber diameter scaffolds at the end of two weeks. Second, cyclic stretch was applied to C3H10T1/2 cells on semi-aligned scaffolds using a novel bioreactor. Cell attachment was verified during and after the application of mechanical stress by confocal microscopy. Cyclic stretch induced cells to assume a highly elongated morphology; however ECM gene expression changes were moderate. Third, forced constitutive expression of scleraxis was accomplished by nucleofection of C3H10T1/2 cells. Transient mRNA expression, accumulation of the gene product in the cell nucleus, and cell death were observed. Future work will seek to refine the experimental methods, including the development and testing of an inducible scleraxis transgene and the application of longer periods of mechanical stimulation. Finally, these complementary approaches may be combined to further extend this work in pursuit of directed differentiation of stem cells and the ensuing generation of a robust tissue graft.
- A Decellularized Porcine Xenograft-Derived Bone Scaffold for Clinical Use as a Bone Graft Substitute: A Critical Evaluation of Processing and StructureBracey, Daniel N.; Seyler, Thorsten M.; Jinnah, Alexander H.; Lively, Mark O.; Willey, Jeffrey S.; Smith, Thomas L.; Van Dyke, Mark E.; Whitlock, Patrick W. (MDPI, 2018-07-12)Background: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. Methods: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. Results: The scaffold was devoid of donor cellular material on histology and DNA sampling (p < 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. Conclusion: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.
- Keratin nanomaterials and methods of production(United States Patent and Trademark Office, 2019-09-03)The present disclosure relates to keratin nanomaterials, methods for obtaining keratin nanomaterials, and biomaterials made from keratin nanomaterials. In particular, keratin nanomaterials comprising Type I and Type II monomer pairs are disclosed as well as a method for obtaining keratin nanomaterials comprising obtaining a solution of keratin and processing the solution by ultrafiltration with buffer solution containing phosphate.
- Treatment of a Spinal Cord Hemitransection Injury with Keratin Biomaterial Hydrogel Elicits Recovery and Tissue RepairFearing, Bailey V.; Hartley, Christopher; Dayton, Orrin; Sherwood, Garrett; AbouShwareb, Tamer; Van Dyke, Mark E. (Hindawi, 2014-03-23)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.