The Successful Culture of Rabbit Oral Mucosal Epithelial Cells

The problem of allograft rejection is the main reason that we decided to develop a new method of autologous oral mucosal epithelium transplantation for OSR.¹ In our laboratory experiment, epithelial cells from the oral mucosa began to form colonies on the denuded AM within 3 days. At 3 weeks, the cultivated oral epithelial cells showed 4–5 layers of stratification, were well differentiated (Fig. 45.1A), and appeared very similar to in vivo normal corneal epithelium (Fig. 45.1B).¹

Cell Biological Characteristics of the Rabbit Cultivated Oral Mucosal Epithelial Sheet

Cytokeratins play an important structural and protective role in maintaining the integrity of the epithelium of the anterior segment of the eye. Defined subsets of individual cytokeratin pairs are characteristically expressed, depending on the type of epithelial cell tissue and level of differentiation. In this study, we used immunohistochemistry to demonstrate that the keratin 1/10 pair, which is involved in the physiological keratinization process in the epidermis, is not expressed in any layers of the cultivated oral epithelial sheet. We also found that the keratin 4/13 pair, which is observed in nonkeratinized, stratified epithelia, is expressed in the superficial and intermediate layers of the cultivated oral epithelial cells. These results led us to believe that the oral epithelial cells cultivated on AM have the characteristics of nonkeratinized mucosa, not keratinized mucosa. Immunohistochemical examination revealed no cornea-specific keratin 12 expression in any layers of the cultivated oral epithelial sheets (Fig. 45.2A), whereas cornea-specific keratin 3 was expressed in all epithelial layers of the cultivated oral epithelial sheet (Fig. 45.2B). Even though the cells of the cultivated oral epithelial sheet are not able to become corneal epithelial cells, we suggest that they might have the potential ability to become cornea-like epithelial cells under proper in vitro culture conditions.

Morphological Characteristics of the Rabbit Cultivated Oral Mucosal Epithelial Sheet

Electron microscopic results of the cultivated oral mucosal epithelial sheet are of particular interest. Examination by use of a scanning electron microscope (SEM) revealed that rabbit oral epithelial cells appeared healthy and well formed with tightly opposed cell junctions (Fig. 45.3). The cultivated oral cells were similar in size and appearance to rabbit corneal epithelial cells. Transmission electron microscopy (TEM) confirmed that the cultivated oral epithelial sheet was very similar in appearance to that of corneal epithelium, and very different from conjunctiva and oral mucosa. Like corneal epithelium, it had 4–5 layers of stratified cells which were differentiated into columnar, wing, and squamous cells (Fig. 45.4). Both our SEM and TEM results show clearly that our oral mucosal cells, cultivated on AM, resemble normal corneal epithelial cells more closely than any other cell type.

In regard to the growth of cultivated oral mucosal epithelial cells, the method of how the basal cells attach to the underlying AM, as well as whether or not the superficial cells develop a normal barrier function, are both important questions. Our TEM results showed that the cultivated oral mucosal epithelial sheet attached to the basement membrane via hemidesmosomal junctions. Adjacent cells in the cultivated oral sheet are also joined with numerous desmosomal junctions, and what appear to be tight junctions are evident between the most superficial cell layers. From these findings, we believe that the cells of the cultivated oral mucosal epithelial sheet have similar junctional specializations to those of in vivo corneal epithelial cells.

The anterior surface of normal corneal epithelium has numerous folds in the anterior epithelial cell membranes in the form of microvilli, together with a glycocalyx layer. Another key point for carrying out successful cultivated oral epithelial sheet transplantation is elucidation of how the most superficial cells contact the tear – ocular surface interface. SEM revealed that the apical surface of the cultivated oral epithelial cells is covered with numerous microvilli, almost identical to those found on corneal epithelial cells. Interestingly, we also found evidence of a cell-surface glycocalyx, similar in appearance to the glycocalyx present on the surface of corneal epithelial cells. These findings encouraged us to carry out the transplantation of cultivated oral mucosal epithelial cells.

Successful Transplantation of the Rabbit Cultivated Oral Mucosal Epithelial Sheet

After the successful culture of rabbit oral mucosal epithelial cells on AM, we tried to reconstruct the damaged corneal surfaces by transplantation of autologous cultivated oral mucosal epithelial cells in order to test the viability of using these cells as a substitute for cultivated corneal epithelial cells. At 48 hours after surgery, most of the area of the transplanted cultivated oral mucosal epithelial sheet possessed intact epithelium. At 10 days after transplantation, the ocular surface covered by the transplanted epithelium was intact and without defects, thus suggesting that the autologous transplantation of cultivated oral mucosal epithelia is a viable procedure for ocular surface reconstruction (Fig. 45.5). Histological examination of the transplanted sheets at 10 days after surgery revealed that the sheets were well adhered to the host corneal stroma, with no evidence of subepithelial cell infiltration or stromal edema. Superficial cells of the transplanted sheets had nuclei, indicating that they were indeed nonkeratinized mucosal epithelial cells.

Therefore, we successfully generated a well-stratified and differentiated rabbit cultivated oral mucosal epithelial sheet. Moreover, we successfully performed autologous transplantation of these cells onto keratectomized rabbit corneas. Hence, we believe that autologous transplantation of rabbit cultivated oral mucosal epithelial sheet is a feasible method for OSR.

The Successful Culture of Human Oral Mucosal Epithelial Cells

We next focused our attention on cultivating human oral mucosal epithelial cells using our previously reported culture methods for rabbit oral mucosal epithelial cells, yet with several modifications. It should be noted that it was quite difficult to cultivate human oral mucosal epithelial cells using the previously reported culture technique for rabbit oral epithelial cells, and therefore, the culture process did require some modification. In the end, and as a result of the modification, we were able to successfully generate a well-stratified and differentiated human cultivated oral mucosal epithelial sheet (Fig. 45.6). Light microscopy revealed that the human cultivated oral mucosal epithelial cells were very similar in appearance to normal corneal epithelial cells. Moreover, immunohistochemistry confirmed the presence of keratins 4/13 and 3 in the human cultivated oral mucosal epithelial cells, similar to those found in the rabbit model.

Successful Xenotransplantation of the Human Cultivated Oral Mucosal Epithelial Sheet

After the successful culture of human oral mucosal epithelial cells, we attempted to reconstruct rabbit keratectomized corneas through the xenotransplantation of a human cultivated oral mucosal epithelial sheet to evaluate the physiological functions of these sheets in the early postoperative stage. Observation of the transplanted sheets was performed only for the first 2 postoperative days, as it is generally thought that after epithelial transplantation, especially xenotransplantation, acute epithelial rejection often occurs, even though intensive postoperative immunosuppression is performed. Rabbit corneas grafted with the human cultivated oral mucosal epithelial cells were clear, and were all epithelialized at 48 hours after surgery. From these results, and although the long-term outcome of such transplantation has yet to be elucidated, we believe that human cultivated oral mucosal epithelial sheets can function as an ocular surface epithelium, and that human COMET is a feasible method of OSR.

We investigated whether it was possible to reconstruct the corneal surface using autologous mucosal epithelium of non-ocular surface origin. To that end, we were successful in generating both rabbit and human cultivated oral mucosal epithelial sheets from biopsy-derived oral mucosal tissues, as well as transplanting these cultivated oral mucosal epithelial cells onto rabbit corneas. In the final analysis, we believe that the transplantation of cultivated oral mucosal epithelial cells represents an effective technique for OSR in patients with severe OSD.

Initial Clinical Results

In the past, several researcher groups investigated the possibility of using oral mucosa for OSR. Ballen reported that oral mucosal grafts which included both epithelium and subepithelial tissues, heavily vascularized with early fibrosis.² In addition, Gipson et al. reported that in vivo oral epithelium, freed of underlying connective tissue, was not maintained in the central avascular corneal regions.³

We investigated the possibility of reconstructing the human ocular surface using autologous mucosal epithelium of non-ocular-surface origin. Using rabbits, we have already established a surgical method for transplanting cultivated autologous oral mucosal epithelial cells.¹ We next applied this method in six eyes of four patients with severe OSD.⁴

Preoperatively, all patients were followed up in regard to their adherence to the requirements for tooth decay treatment, no alcohol or tobacco use, and regular brushing and iodine gargle. The culture process was performed according to the previous method. After removing abnormal conjunctivalized tissues on the corneal surface using surgical scissors, subconjunctival fibroblasts were treated with mitomycin C followed by vigorous repeated washing with saline. The cultivated oral mucosal epithelial sheet was transplanted onto the corneal surface of the damaged eye and secured with 10-0 nylon sutures at the limbus. Postoperatively, ofloxacin and dexamethasone eye drops were instilled four times a day; the doses were tapered to a maintenance dose at 2 to 3 months, depending on the severity of postoperative inflammation. Betamethasone and cyclophosphamide, administered to prevent postoperative inflammation and conjunctival fibrosis, were stopped 1 to 2 months after surgery.

At 48 hours post transplant, the entire corneal surface of all six eyes was free of epithelial defects, indicating complete survival of the transplanted cultivated oral mucosal epithelium. Visual acuity was improved in all eyes. During follow-up (13.8 ± 2.9 months), the ocular surface remained stable, although all eyes showed mild peripheral neovascularization. Therefore, autologous cultivated oral mucosal epithelial sheets can be transplanted to treat severe OSD (Fig. 45.7). This initial clinical study represents a first step toward assessing the feasibility of transplanting autologous cultivated epithelial transplants of non-ocular-surface origin.

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