Effects of Tunable Keratin Hydrogel Erosion on Recombinant Human Bone Morphogenetic Protein 2 Release,Bioactivity,and Bone Induction

Abstract

Keratin proteins derived from human hair are known to contain a large number of cysteine residues. In hydrogels composed of oxidatively extracted keratin (keratose, KOS), sulfonic acid on the cysteine residues prevents disulfide cross-linking. Reductively extracted keratin (kerateine, KTN) has thiol groups on cysteine residues that can form disulfide cross-links. The rates of keratin hydrogel erosion and, in some cases, drug release can be tuned by manipulating disulfide cross-linking levels via the KOS:KTN ratio. To investigate the use of these hydrogel mixtures as carriers for growth factors in tissue engineering applications, we fabricated 15% (w/v) hydrogels with KOS:KTN ratios of 100:0, 70:30, 50:50, 30:70, and 0:100 with and without recombinant human bone morphogenetic protein 2 (rhBMP-2). We compared the keratin rhBMP-2 carriers with the clinical system of resorbable collagen sponges, which are known to elicit problems such as edema, inflammation, and ectopic bone growth. In vitro, hydrogels with increasing levels of KOS eroded more rapidly. However, there was little difference among the various keratin formulations in amounts of rhBMP-2 released after 12 h. Collagen had similar total rhBMP-2 release, although with significantly greater release over the first 12 h compared with all keratin formulations, except for 0:100 KOS:KTN. However, increasing levels of KTN led to increasing release of bioactive rhBMP-2 based on the stimulation of alkaline phosphatase activity in MC3T3-E1 cells by the hydrogel releasate. Micro-CT analysis of heterotopic bone growth in a mouse model indicated that with rhBMP-2, the 30:70 formulation led to significant increases in bone volume compared with KOS and collagen, whereas the 50:50 formulation led to significant bone volume increases compared with all of the formulations except for 30:70 KOS:KTN. Histological analysis of images from the heterotopic mouse model indicated differences in the quality and type of bone among the various carriers of rhBMP-2. KOS:KTN in the ratio of 70:30 with rhBMP-2 elicited greater amounts of cortical bone, whereas the ratio 30:70 resulted in greater amounts of marrow, total bone, and residual material. The results indicate that in addition to the rate of rhBMP-2 release, the carrier itself can have important effects on bone volume, type, distribution, and quality.

Impact Statement

Recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery from collagen sponges for bone formation is an important clinical example of growth factors in tissue engineering. Side effects from rhBMP-2 burst release and rapid collagen resorption have led to investigation of alternative carriers. Here, keratin carriers with tunable erosion rates were formulated by varying disulfide crosslinking via ratios of oxidatively (keratose) to reductively (kerateine) extracted keratin. In vitro rhBMP-2 bioactivity increased with kerateine content, reaching levels greater than with collagen. Heterotopic bone formation in a mouse model depended on the keratin formulation, highlighting the importance of the growth factor carrier.