Corneal Reconstruction by tissue-engineering

供体年龄越大，传代后的HCEC中出现大的衰老细胞的频率越高。用成人血清代替牛血清培养HCEC，以除去可被朊病毒感染的牛材料。用成人血清可以进行肝细胞内皮细胞的原代和传代培养。我们使用培养的HCEC和人角膜基质重建角膜。将其上倾倒有HCEC细胞悬液的角膜基质温和离心以增强HCEC与基质的附着。角膜上皮细胞密度达2500个/mm^2。用Ussing室测量重建角膜的泵功能，重建角膜与正常角膜的电位差分别为0.30 mV和0.40 mV，表明重建角膜的泵功能为正常角膜的75%。将重建的角膜移植到兔眼，术后6个月保持透明。移植后1个月，荧光素标记的培养的HCEC仍保留在移植物上，表明移植的HCEC有助于移植物的透明化。碱处理的胶原蛋白制成的人工基质不能缝合，但显示出良好的透明性，生物相容性和细胞附着性。猪角膜基质仅表达少量异种糖抗原α-gal表位，移植于具有α-gal表位天然抗体的动物角膜后，无超急性排斥反应，仅引起轻度细胞排斥反应。与正常角膜相比，用猪角膜基质和HCEC重建的角膜的平均细胞密度为1721/mm^2，泵功能约为60%。这些结果表明，临床应用的可能性，角膜重建的HCEC。

The older the donor was, the more frequently large senescent cells appeared in the passaged HCEC. HCEC was cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prion. Primary and passage culture of HCEC was possible using adult human serum. We reconstructed the cornea using cultured HCEC and human corneal stroma. The corneal stroma, on which cell suspension of HCEC was poured, was mildly centrifuged to enhance the HCEC attachment to the stroma. The cell density of HCEC on the reconstructed cornea reached to 2500 cells/mm^2. The pump function of the reconstructed cornea was measured with Ussing chamber The potential difference in the reconstructed cornea and normal cornea was 0.30 mV and 0.40 mV, respectively ; indicating the pump function of the reconstructed cornea is 75% of that of the normal cornea. The reconstructed cornea was transplanted to rabbit eye and stayed transparent during postoperative 6 months. Fluorescein labeled cultured HCEC remained on the graft 1 month after the transplantation, indicating transplanted HCEC contributed to the transparency of the graft. The artificial stroma made of alkaline-treated collagen could not be sutured but showed good transparency, biocompatibility, and cell-attachability. Porcine corneal stroma, expressing little xeno-sugar antigen α-gal epitope, induced no super acute rejection but mild cellular rejection when transplanted in the cornea of animals possessing natural antibody to a-gal epitope. The cornea reconstructed with porcine corneal stroma and HCEC had average cell density of 1721/mm^2 and had approximately 60% pump function compared with the normal cornea. These results indicated the possibility of clinical use of corneas reconstructed using HCEC.