含硅磷灰石活性涂层促进人工角膜生物愈合的机理研究

Corneal blindness is the second most common cause of visual impairment in worldwide populations. To the severe corneal blindness with ocular surface disorders such as immunologically mediated cicatrizing conjunctivitis, chemical or thermal burns or after multiple transplant rejections, traditional penetrating keratoplasty has no chance of survival, and keratoprosthesis (KPro) offers the last approach to restore vision. Keratoprosthesis consist of a clear central optic and a surround skirt. The skirt element is made of titanium, and designed to integrate with the host. Clinical data demonstrated that stromal melting around skirt is the common complication, which has been threatening the long-term anatomical safety of KPro. Thus, there is a need to improve the biointegration of titanium frame. In our previous experiments, hydroxyapatite (HA) has been approved to be able to induce a faster and more efficient cell adhesion and Si can increase the biosynthesis of collagen and extracellular matrix. Therefore, in this work, we prepare the nano silicate doped hydroxyapatite (Si-HA) by the technique of ion beam assisted deposition to modify the scaffold of KPro. The characterization and fine structure of Si-HA will be analyzed, and the effect of Si-HA improving tissue healing and mechanism of Si-HA promoting the extracellular matrix secretion will be studied in the animal model. Furthermore, to study the cell-biomaterial interaction, we will investigate the cell biofunctions on Si-HA by live cell station and microRNA microarray techniques in cell line. As a promising material, Si-HA-Ti based KPro could be utilized for the corneal blindness treatment.

对于眼部烧伤、重症眼表疾病以及由于感染、外伤、重复移植失败等原因造成的复杂角膜盲，角膜移植难以成功，人工角膜(keratoprosthesis，KPro)是这些患者复明的唯一手段。人工角膜由周边支架和中央光学部组成，临床发现KPro支架周围组织溶解是危及术后长期安全的最主要原因。进一步改进支架材料，提高材料与组织的生物整合是人工角膜研究中急需解决的关键问题。研究发现元素硅具有促进胶原合成功能，因此本项目将硅与羟基磷灰石结合,采用离子束辅助沉积方法，在钛表面制备含硅磷灰石(Si-HA)活性涂层，对材料进行表征分析。采用体外细胞培养体系，应用活细胞工作站、microRNA芯片等技术研究Si-HA对角膜细胞生物合成功能的影响及材料与细胞相互作用机制，并通过体内植入试验研究Si-HA在体内对角膜胶原合成的影响及作用机制，旨在研制具有良好生物愈合功能的人工角膜, 为支架材料的改进提供理论和实践依据。

研发优化具有良好生物相容性的医用材料对降低人工角膜手术并发症具有重要意义。本项目已按计划完成了功能化含硅羟基磷灰石活性涂层的制备和表面优化,通过比较不同制备工艺，筛选了适合人工角膜支架材料表面修饰的等离子喷涂方法。应用体外培养原代角膜成纤维细胞，建立了人工角膜支架材料体外评价体系，通过细胞生物学试验，包括细胞活力试验、细胞增殖试验、角膜基质细胞生物合成功能检测等方法研究涂层材料对细胞生物相容性的影响。进行系统的体内评价试验，应用肌肉内植入材料，观察材料组织相容性；眼内植入试验，观察材料在角膜层间存留情况，进一步研究涂层材料在角膜组织的相容性。材料生物学研究结果主要包括以下三方面：（1）含硅羟基磷灰石活性涂层有助于提高材料的组织结合能力。肌肉内植入试验材料后，功能化生物活性磷灰石涂层组材料周围胶原纤维较对照组明显增厚。涂层组支架材料角膜原位存留情况明显优于钛对照组。（2）生物活性磷灰石涂层能够提高细胞生物合成功能。体外机理研究发现在生物活性磷灰石涂层表面，血浆蛋白吸附量明显高于钛对照组。角膜基质细胞的生物合成功能大大增加，胶原蛋白的含量明显高于钛对照组和PMMA对照组。（3）在探讨材料-细胞界面交互作用机理方面，利用胶原包被方法进行材料表面修饰，研究材料表面性质对细胞骨架和细胞粘附信号蛋白的影响，证明胶原包被材料组细胞粘附作用优于钛对照组。这一系列关键数据回答了长期以来困扰钛支架人工角膜生物整合程度差、长期随访支架暴露率高的临床难题，为优化钛支架人工角膜提供了理论依据和现实方向。