The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3±1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.

Human skin substitutes are needed for implantation and wound repair based on the new concept of tissue engineering in combination with biomaterials and cell biological technology. However, failure sometimes occurs when the wound healing is delayed in vivo due to acute inflammation resulting from the early degradation of the transplanted biomaterials. Accordingly, the current study modified conventional biomaterials to overcome early degradation and strong inflammation. In a conventional skin substitute, the animal origin collagenous materials have a slight antigenicity as xenogenic materials, however, the modified method was able to obtain a low antigenicity and anti-inflammation effect using atelo-collagen and an amniotic component. The tyrosine content in the developed atelo-collagen, representing the antigenicity, was reduced from 0.590% to 0.046% based on an HPLC analysis. In addition, to reduce the inflammation and foreign material reaction, an amniotic component was applied to the atelo-collagen materials. While, to enhance the wound healing, the modified skin substitute was developed as a composite matrix of an atelo-collagen scaffold with an amniotic membrane component. A quantitative analysis of hEGF in the amniotic membrane was also performed using different processing methods. Finally, a tissueengineered skin substitute was constructed by cultivating skin cells in the collagen scaffold attached to an amniotic membrane.

To describe the clinical outcome of a new surgical treatment for the acute stages of severe corneal burn injury and its complications, a prospective study of five acute corneal burn patients with severe limbal damage was performed. Amniotic membrane transplantation (AMT) and conjunctival limbal autograft (CLAU) was performed at the acute stage of corneal burn injury to reconstruct the damaged ocular surface (step I). Three to six months later, the opaque central part of the amniotic membrane containing in vivo grown corneal stem cells were removed and retransplanted to the defect created after the removal of pseudopterygium (step II). All injured eyes were successfully treated, but in one eye with marked stromal lysis, three-layered AMT and penetrating keratoplasty with retransplantation of in vivo grown corneal stem cells was performed. In the former cases, visual acuity was greatly improved more than three lines (ranging from 3 to 12 lines). In short, retransplantation of in vivo grown corneal stem cells after AMT and CLAU is a recommendable modality for restoring a stable corneal epithelium of a severely burned ocular surface in the acute stage and can be considered a preventative measure for avoiding late onset complications.

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대상과 방법 토끼의 각막 간질세포 및 중배엽 줄기세포의 준비