Alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications

Abstract

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or “capping”) of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.

Tunable Keratin Hydrogels for Controlled Erosion and Growth Factor Delivery

“…We also recently found that rhIGF-1 has minimal interactions with keratin proteins, though interactions are greater for KTN than for KOS. 33 This relatively low interaction allowed us to study the effects of only erosion on this molecule with minimal concern for other effects (e.g., molecular interactions) that might affect release. With recent efforts to better mimic processes of development, increasing numbers of exogenously delivered bioactive molecules will be used in tissue engineering constructs.…”

“…Keratins have been reported as stand-alone materials or with controlled release of therapeutic agents for TERM applications including those in nerve, 15, 21, 22, 23 muscle, 24 skin, 25, 26, 27 and bone. 28, 29, 30 Keratins are known to promote attachment of various cell types including osteoblasts, 31, 32, 33 fibroblasts, 34 hepatocytes, 35 and neural cells, 15 though the mechanisms of attachment are not fully known in all cases. The inflammatory response to keratins is minimal.…”

Tailoring degradation rates of silk fibroin scaffolds for tissue engineering

“…The biomaterials should have minimal toxicity and appropriate mechanical properties and provoke minimal inflammatory/immune response that mimics the hierarchical structure of normal tissues . More importantly, the biomaterials should degrade gradually along with the tissue regeneration . Since the optimal degradation rate of scaffold depends on the tissue type to be regenerated, the next generation of scaffolds should demonstrate controlled biodegradation patterns.…”

Biomaterials for the Delivery of Growth Factors and Other Therapeutic Agents in Tissue Engineering Approaches to Bone Regeneration

“…While hydrophobic, van der Waals, and electrostatic interactions between the material and growth factor as well as entrapment in the polymer network are inherently present, additional molecules can be introduced into the biomaterial to retard the rate of release. Examples of these immobilization methods include affinity binding such as heparin-binding (Yang et al, 2010 , 2012 ; Jeon et al, 2011 ; Wang et al, 2013 ), ionic interactions such as those provided by chondroitin sulfate (Wang Y. et al, 2010 ; Bae et al, 2012 ), cyclodexterins (Del Rosario et al, 2015 ), protease degradable tethers (Tokatlian et al, 2010 ), succinylation (Tsujigiwa et al, 2006 ), alkylation (Tachibana et al, 2006 ; Han et al, 2015 ), and even covalent conjugation (Shen et al, 2009 ; Zhang et al, 2010 ).…”