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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.

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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.

Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG) and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG) to absorb test proteins with different isoelectric points (pI): albumin (~5), hemoglobin (~7), and lysozyme (~11). The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4), all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW) diffusion: lysozyme (14 kDa) > hemoglobin (64 kDa) > albumin (66 kDa). Vascular endothelial growth factor C (VEGF-C), with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.

The possible involvement of orthopedic biomaterial particles such as cobalt-chrome alloy (Co-Cr), ultrahigh molecular weight polyethylene (UHMWPE), titanium alloy (Ti-6Al-4V), and polymethyl methacrylate (PMMA) in the formation of glial and meningeal scars was investigated using an in vitro system. Cell lines were used as models for astrocytes and meningeal fibroblasts. They were incubated with varying concentrations of particle suspensions, after which proliferative and cytotoxic responses were quantified using MTT assay and Live/Dead microscopy. It was determined that relative particulate toxicity (arranged in decreasing order) to astrocytes is Co-Cr > Ti-6Al-4V > PMMA > UHMWPE, and toxicity to fibroblasts is PMMA > Co-Cr > Ti-6Al-4V > UHMWPE. Cell death caused by PMMA was mainly due to necrosis, while the rest of the particles induced apoptosis. Low quantities of Co-Cr and Ti-6Al-4V stimulate increased astrocyte proliferation rate. However, only the cells treated with titanium alloy caused upregulated transcription of reactive astrocyte markers such as glial fibrillary acidic protein, vimentin, nestin, and type IV collagen, suggesting the potential of titanium alloy alone to trigger glial scarring. None of the biomaterials tested promoted proliferation in fibroblasts implying that biomaterial particles are not directly involved in meningeal scar development.