[Transplantation of bioengineered corneal epithelium for the treatment of total limbal stem cell deficiency in rabbit].
OBJECTIVE
To study the results of transplantation of cultured autologous limbal stem cells using fibrin gel membrane as substrate for the treatment of limbal stem cell deficiency in rabbits.


METHODS
An ocular surface defect was created in the right eyes of all rabbits by a lamellar keratectomy extending 1 mm outside the limbus. The stem cells were isolated from a small limbal biopsy specimen from the left eyes and cultured. Cells were grown over fibrin gel membrane to construct bioengineered corneal epithelium. After cytologic verification of total limbal stem cell deficiency, rabbits were divided into 4 groups at random. The fibrovascular pannus of each cornea was removed. Groups I - III (n = 24) underwent transplantation of limbus stem cells cultured on fibrin gel membrane and were observed for 3 months (Group I), 1 month (Group II) and 2 weeks (Group III), whereas group IV (n = 8) received only the fibrin gel membrane. Clinical outcome was graded by corneal integrity, opacity and neovascularization. HE staining, impression cytology and immunofluorescence staining with antibodies against keratin-3, MUC5AC and nuclear p63 were performed to assess the phenotype of corneal epithelium.


RESULTS
The cornea in the study groups (group I - III) recovered smoothly, covered with transparent epithelium and without neovascularization. In the control group (group IV), cornea was turbid and irregular, with neovascularization (P = 0.021). Corneal impression cytology indicated PAS (-) in the study groups, while PAS (+) in the control group. HE staining of the cornea in the study groups showed normal corneal phenotypes; whereas the epithelium cells in the control group showed conjunctival phenotypes with vessels and goblet cells. Immunostaining of study groups showed keratin-3 positive and MUC5AC negative corneal epithelium, and p63 was expressed in the basal cell of corneal epithelium. Cells in the control group expressed MUC5AC abundantly.


CONCLUSIONS
Tissue bioengineered cornea epithelium transplantation can successfully reconstruct corneal surface affected by limbal stem cell deficiency. Fibrin gel membrane is a new bioengineered material with the characteristics of absorbability, transparence and good histological compatibility. It is supposed to be an optimum stem cell scaffold.
Lin
Qi
Q
Beijing Tongren Ophthalmic Center, Capital University of Medical Sciences, Beijing 100730, China.
Lü
Lan
L
Han
Bin
B
Jin
Tao
T
D000818:Animals; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D019573:Epithelium, Corneal / Q000166:cytology*; D005260:Female; D005337:Fibrin; D005782:Gels; D016850:Limbus Corneae / Q000473:pathology / Q000601:surgery*; D008297:Male; D011817:Rabbits; D011897:Random Allocation; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering
D005782:Gels; D005337:Fibrin