Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA.
Biomaterials. 2011 Nov;32(32):8205-17. doi: 10.1016/j.biomaterials.2011.07.054. Epub 2011 Aug 11.
The oxidized form of extractable human hair keratin proteins, commonly referred to as keratose, is gaining interest as a biomaterial for multiple tissue engineering studies including those directed toward peripheral nerve, spinal cord, skin, and bone regeneration. Unlike its disulfide cross-linked counterpart, kerateine, keratose does not possess a covalently cross-linked network structure and consequently displays substantially different characteristics. In order to understand its mode(s) of action and potential for clinical translatability, detailed characterization of the composition, physical properties, and biological responses of keratose biomaterials are needed. Keratose was obtained from end-cut human hair fibers by peracetic acid treatment, followed by base extraction, and subsequent dialysis. Analysis of lyophilized keratose powder determined that it contains 99% proteins by mass with amino acid content similar to human hair cortex. Metallic elements were also found in minute quantities. Protein oxidation led to disulfide bond cleavage and drastic reduction of free thiols due to conversion of sulfhydryl to sulfonic acid, chain fragmentation, and amino acid modifications. Mass spectrometry identified the major protein constituents as a heterogeneous mixture of 15 hair keratins (type I: K31-35 and K37-39, and type II: K81-86) with small amounts of epithelial keratins which exist in monomeric, dimeric, multimeric, and even degraded forms. Re-hydration with PBS enabled molecular assembly into an elastic solid-like hydrogel. Highly-porous scaffolds formed by lyophilization of the gel had the compression behavior of a cellular foam material and reverted back to gel upon wetting. Cytotoxicity assays showed that the EC50 for various cell lines were attained at 8-10 mg/mL keratose, indicating the non-toxic nature of the material. Implantation in mouse subcutaneous tissue pockets demonstrated that keratose resorption follows a rectangular hyperbolic regression with 92% degradation by an 8-week time point. Keratose was shown to integrate with the host tissue as evidenced by infiltration of leukocytes and fibroblasts, bulk material angiogenesis, and minimal fibrous encapsulation. Tissue response benchmarks were superior in keratose compared to the control PLGA 90:10 mesh. Finally, the degraded keratose was observed to remodel with the natural collagen extracellular matrix, verifying the benefit of using keratose as a temporary matrix for regenerative medicine applications.
可提取人类毛发角蛋白氧化形式，通常称为角蛋白，作为生物材料越来越受到关注，用于多种组织工程研究，包括周围神经、脊髓、皮肤和骨骼再生。与二硫键交联的角蛋白不同，角蛋白没有共价交联的网络结构，因此表现出截然不同的特性。为了了解其作用模式和临床转化的潜力，需要对角蛋白生物材料的组成、物理性质和生物学反应进行详细的表征。角蛋白是通过过乙酸处理、碱提取和随后的透析从末端切下的人发纤维中获得的。对冻干角蛋白粉末的分析表明，其按质量计含有 99%的蛋白质，其氨基酸含量与人发皮质相似。还发现微量的金属元素。蛋白质氧化导致二硫键断裂，由于巯基转化为磺酸，链断裂和氨基酸修饰，游离巯基急剧减少。质谱鉴定出主要的蛋白质成分为 15 种头发角蛋白（I 型：K31-35 和 K37-39，和 II 型：K81-86）的不均匀混合物，以及少量存在于单体、二聚体、多聚体甚至降解形式的上皮角蛋白。用 PBS 再水合可使分子组装成弹性固体状水凝胶。凝胶冻干形成的高度多孔支架具有细胞泡沫材料的压缩行为，在润湿时恢复为凝胶。细胞毒性试验表明，各种细胞系的 EC50 值在 8-10mg/mL 角蛋白时达到，表明该材料无毒。将角蛋白植入小鼠皮下组织袋中表明，角蛋白的吸收遵循矩形双曲线回归，在 8 周时间点降解 92%。角蛋白与宿主组织整合的证据是白细胞和成纤维细胞的浸润、大块材料的血管生成和最小的纤维包裹。与对照的 PLGA 90:10 网相比，角蛋白的组织反应基准更高。最后，观察到降解的角蛋白与天然胶原蛋白细胞外基质重塑，验证了将角蛋白用作再生医学应用的临时基质的益处。
