In vitro preparation and biochemical evaluation of oxygen generative keratin/silk fibroin compound biomaterial

doi:10.3969/j.issn.2095-4344.2014.52.021

Abstract

Abstract:

BACKGROUND: Tissue engineering reconstruction materials have gradually entered into so-called intelligent stage, which means that the relevant biological materials will be given more. If biomaterials are capable to release oxygen in vitro and in vivo, the successful rate of the biomaterials in repair of tissue defects can be significantly improved. However, only protein-derived biomaterials have been reported to produce oxygen successfully.

OBJECTIVE: To investigate the preparation of the oxygen generative keratin/silk fibroin scaffold and to evaluate its physicochemical and biological characters.

METHODS: The keratin and silk fibroin solution were extracted from human hair and worm, respectively. Keratin and silk fibroin were blended at a constant rate (60/40) and mixed with various concentrations of calcium peroxide (5%-25%). The optimal concentration of calcium peroxide was confirmed by the oxygen releasing test. The infrared spectroscopy was used to check the structure of proteins in compound materials, and the mechanical test was also used for evaluating their biomechanical properties. The biomaterial was embedded beneath the rabbit back in order to exam its biocompatibility. The smooth muscle cells were seeded onto the oxygen generative scaffold in order to determine its function of supporting cellular growth.

RESULTS AND CONCLUSION: Using extraction and purification technology, human hair keratin and silk fibroin concentration and purity were improved to meet the subsequent experimental requirements. The oxygen releasing test showed that when the optimal concentration of calcium peroxide was 20%, the final biomaterial could generate the oxygen for the longest time (P < 0.05). The mechanical test of oxygen generative films showed similar mechanical characters to those without calcium peroxide, especially in terms of maximum stress and elongation at break (P > 0.05). The infrared spectroscopy examination showed that the structure of proteins kept intact within the final biomaterial. The final biomaterial also demonstrated the good biocompatibility and non-cytotoxcity. And the seeded cells grew better on the oxygen generative scaffold than on the traditional scaffold. With the optimal concentration of calcium peroxide, the keratin/silk fibroin scaffold can release the oxygen constantly in vitro. The physicochemical and biological characteristics of new biomaterials are excellent, which can be suitable for the further tissue engineering research and application.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biomedical engineering, biocompatible materials, biomimetic materials, cell proliferation

CLC Number:

R318

Feng Chao, Li Zhe, Lv Xiang-guo, Xu Yue-min, Fu Qiang. In vitro preparation and biochemical evaluation of oxygen generative keratin/silk fibroin compound biomaterial [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(52): 8480-8486.

Figures/Tables 3

2.1 人发角蛋白和丝素蛋白的定性测定结果通过SDS-PAGE可以看出所提取的人发角蛋白溶液中，其主要蛋白的相对分子质量约为50 000。丝素蛋白溶液的相对分子质量约为25 000(图1)。 2.2 制备的复合材料形态变化最终制备而成的材料，其外观呈褐色半透明片状材料，相对于不含过氧化钙的复合材料，最终混合材料可见有轻度的颗粒感。苏木精-伊红染色见最终材料可被伊红染色，同时其内部成致密样结构，孔隙较少，另在部分区域可见有明显的晶体样结构的存在。而该类结构未发现在不含过氧化钙的复合材料中(图2)。扫描电镜可见材料表面呈现致密样结构，较少见到孔隙，但在材料的表面可见大量残在的颗粒样组织存在，部份颗粒有被周围组织包裹的表现(图3)。 2.3 制备的复合材料生物力学变化生物力学检测显示，当过氧化钙加入到复合材料后，其弹性模量，最大应力及断裂伸长率方面和未加入过氧化钙的复合材料差异无显著性意义(图4)。 2.4 制备的复合材料红外光谱分析结果复合材料的红外光谱分析结果显示当人发角蛋白/丝素蛋白复合材料进一步添加明胶及过氧化钙后，其基本的蛋白分子键结构基本不发生明显的变化，同时最终制备的复合材料又赋予了过氧化钙以及明胶的一些特征(图5)。 2.5 制备的复合材料释氧能力所有制备的材料的释氧能力均观察至19 d，在整个观察其中。所有小瓶中溶液的pH值恒定在7.2-7.6。无论含有何种浓度过氧化钙的复合材料，均可以在9 d的时间内使得溶液的含氧量得以提升。但相比而言，含有20%过氧化钙的复合材料能够使得溶液中的含氧量提升2周之久(图6)。 2.6 制备的复合材料组织相容性评估当含有20%过氧化钙的人发角蛋白/丝素蛋白/明胶复合材料植入兔背部皮下1个月内，植入材料在取出时能够保证基本完整的形态，并且在1个月时仍可以分清材料后周围组织的边界。另可在大体材料的观察过程中见到材料的表面颗粒感增强的现象。同时在组织学检查中基本未发现有周围炎症细胞向材料内部的侵入生长，但材料内部有明显的降解的迹象(图7)。 2.7 制备的复合材料体外细胞增殖情况当平滑肌细胞接种于各类复合材料后，可以在苏木精-伊红染色切片中发现细胞生长的层数明显优于不含20%过氧化钙的复合材料。但两种材料表面的细胞的生长形态基本类似(图8)。"

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