京尼平交联脱细胞角膜基质制备美容性彩色角膜镜片和屈光性角膜透镜

It is necessary to consider cosmetic repair for corneal opacities in some carefully selected cases – especially for children – to improve one’s social relationships and personal sense of well-being. Cosmetic contact lens and cosmetic keratopigmentation, as the main treatments for corneal imperfections at present, are limited in clinical applications due to a number of crucial flaws. The surgical techniques of keratophakia and epikeratophakia have been fully validated and affirmed. If refractive corneal lens implant (RCLI) is replaced by cosmetic corneal lens implant (CCLI), the implementation of which can fully meet the coverage requirements of corneal leukoma, the appearance of the affected eyes can thereby be improved. Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. Genipin crosslinking is a bio-safe, powerful and stable process. Nevertheless, the dark-blue pigment formed during genipin crosslinking is the main shortcoming of the various applications, thereby limiting its clinical application, but it is exactly what we need for the fabrication of CCLI. This study aims to develop a method to prepare CCLI for leukoma using genipin-crosslinked APCS (Gc-APCS). The color and crosslinking degree of Gc-APCS will be monitored, while the characteristic absorption peak of genipin aqueous solution is tracked during the crosslinking reaction. In vitro, the residual amino acid, microstructure, specific surface area and porosity will be examined. Swelling behavior, biomechanical test, resistance to collagenase and light degradation, stability analysis in different pH, and extract cytotoxicity assay will be evaluated. Keratoplasty in the New Zealand rabbits will be conducted to evaluate the color and physiological stability and healing process of Gc-APCS graft, and refractive stability of presbyopia correction. The project is expected to get CCLIs with various colorations for the wide variety of iris colors in different ethnic populations, therefore a new cosmetic repair program for leukoma and presbyopia.

角膜白斑的美容治疗举足轻重，儿童尤甚。目前，美容性角膜接触镜和美容性角膜层间染色术仍因较多问题限制其临床应用。既往角膜镜片手术技巧已经充分验证和肯定，将屈光性角膜镜片更换为美容性角膜镜片即可满足美容需求。脱细胞角膜基质被证实较好的保留基质微环境，适用于修复和重建角膜基质。京尼平是良好的天然交联剂，形成的交联安全、强效、稳定持久，然而交联衍生的深蓝色素是目前应用中不需要的缺点并因此限制其应用；而此交联衍生色素的特点恰恰可用作生物染剂，是制备美容性角膜镜片所需要的。本项目拟用京尼平交联脱细胞角膜基质，使其获得满足临床需求的染色，旨在制作美容性角膜镜片，并体外评估所得镜片的孔结构、理化性能及染色稳定性，进而通过细胞毒性分析后，进行新西兰大白兔移植实验，评价和证实其修复角膜和美容白斑的双重作用，以及矫正老视的屈光稳定性。本项目有望获得颜色丰富的角膜镜片，为角膜白斑美容治疗和老视矫正提供新方案。

目的：探索京尼平交联脱细胞猪角膜基质（Gc-APCS）使之染色进而用作美容性角膜镜片的可行性。.方法：在适宜的交联反应条件（37 ℃，pH值为 5.5）下，使用1.0 %京尼平水溶液处理脱细胞猪角膜基质（APCS）4 h，并行-10 ℃冷冻干燥以制备美容性角膜镜片，并对制备所得Gc-APCS的理化性能和生物学特性进行详细评价。制备过程中监测交联反应进行时Gc-APCS颜色变化情况，并同时跟踪京尼平水溶液的特征性光吸收峰和Gc-APCS交联度的变化情况。体外条件下，检测了Gc-APCS的氨基酸组成变化情况，孔结构形态学参数（孔隙率和比表面积），以及溶胀性能、抗降解性能、力学强度和抵抗不同pH值和光照度的稳定性情况。Gc-APCS浸提液对兔角膜基质细胞增殖活性的影响测试分析后，行新西兰兔角膜基质层间移植术进而评价其整体功能：颜色稳定性、生物相容性、术后基质愈合情况。.结果：随着交联反应的进行，APCS（10 mm直径）由白色渐渐变为深蓝色，京尼平水溶液240 nm处的特征性光吸收峰逐渐下降，反应结束时，京尼平消耗约为2 mg，Gc-APCS交联度为45.7 ± 4.6 %，经氨基酸分析发现，Gc-APCS仅有碱性氨基酸和羟基氨基酸含量相比交联前减少。交联对材料的孔隙结构无影响，Gc-APCS胶原板层结构存在，孔隙率为72.7 ± 4.6 %，比表面积为23.01 ± 3.45 m2/g；溶胀性能得以提升，恢复生理含水量仅需约5 min；抗消化能力提升，胶原酶消化56天后样本残留率仍为93.9 ± 2.3 %；生物力学强度得以加强，不同压力下的面膜量和曲率变化，与天然猪角膜无差异；稳定性良好，在不同pH值（5.5、7.0、9.0）和光照条件下，颜色无变化，活体移植术后，植片无褪色；生物相容性好，Gc-APCS浸提液对兔角膜基质细胞增殖活性未见影响，新西兰兔角膜基质层间移植术后，植床角膜组织一直保持透明，植片和植床未见新生血管，未出现植片溶解、角膜钙化和免疫排斥现象。.结论：京尼平交联脱细胞猪角膜基质制备了美容性角膜镜片。京尼平是良好的天然交联剂，交联APCS后：未影响孔隙结构和微环境，提升了稳定的力学性能，保持了较好的溶胀性能，具备了良好的抗降解性能；Gc-APCS植入体内后：生物相容性良好，染色稳定，基质愈合良好，可用于移植治疗角膜白斑。