| PurposeTo study the features of rabbit conjunctival sac shortening after different injury, and to establish a stable animal model of narrow conjunctival sac. A tissure engineering fornix graft was constructed for the management of severe fornix shortening, and the result was evaluated.Method1. Establishment of the animal model with narrow conjunctival sacThirty rabbits were randomly divided into four groups. In the thermal burn group, the conjunctival sac of9rabbits was respectively burned with400℃electric soldering iron for3s,6s and10s; in the alcli burn group, the conjunctival sac of9rabbits was respectively burned with2M NaOH for60s,120s and180s; in the acid burn group, the conjunctival sac of9rabbits was respectively burned with n-Heptanoic acid for120s,180s and300s; and in the last goup with ocular surface resection, all the bulbar and most of the palpebral conjunctiva were excised, and a superficial keratectomy including the entire corneal surface and the limbal epithelium cells were performed. Postoperatively, the conjunctval sac was monitored under slit-lamp every other day. The degrees of narrow conjunctival sac were judged according to the depth of fonix and the repairement of eipithilium lesions were evaluated by histopathalogical examination4weeks after injury.2. The construction of tissue engineering "conjunctival sac implants" using oral mucosa epithelium cellsPoly(lactic-co-glycolic acid)(PLGA), cross-linked sodium hyaluronate(CLSH) and amniotic membrane(AM) were selected as the alternative scaffolds. After, in vitro proliferation, rabbit oral mucosa epitheliums were seeded onto three scaffolds. And cell attachment and proliferation were investigated. Meanwhile,6rabbits were selected for in vivo tissue compatibility testing. The three scaffolds were implanted surgically into the subconjunctival space of the same rabbit. Clinical evaluation was conducted macroscopically by assessing the inflammatory reaction around the operated area. Histopathological examination was performed to detect inflammatory cell infiltration, fibrous tissue proliferation and scafolds degradation2and4weeks after implantation. A scaffold with good tissue compatibility and high cell attachment and proliferation was selcted for the construction of "conjunctival sac grafts". After seeding oral mucosa epithelium cells, the scaffold was coated on the surface of a prosthetic eye sheet, and incubated in vitro for7～10days. The cell sheet on the gfat was evaluated by histopathological and immunohistochemical examination.3. Preliminary study of "conjunetival sac implants" in treating severe narrow conjunetival sacSix rabbits with severe narrow conjunetival sac were divided into3groups, and received surgery of conjunetival sac reconstruction with implantation of "conjunetival sac implants" in group A, prosthetic eye sheet coated with AM in group B and naked prosthetic eye sheet in group C. The prosthetic eye sheets were removed two weeks after operation, and the depth of conjunetival sac was measured simultaneously and after another two weeks to evaluate the postoperative contraction of fornix. Also, the conjunetival epithelium was examined by histopathological and immunohistochemical examination.Result1. The conjunctiva in thermal burn group displayed a ring like contraction, and moved forward to cover the cornea. And eyelid necrosis accured on those burned for10s. Severe narrow conjunetival sac, but all with blepharocoloboma and ankylobleparon, was obtained in3cases4weeks after ocular surface burn. Histopathological examination revealed disorgnized epithelium and moderate fibroplasia in the outer sac, but relatively healthy epithelium in the inner sac. The ischemia of conjunctiva recovered completely and all the cases result in mild narrow conjunetival sac in the acid burn group. Histopathological examination revealed relatively healthy conjunetival epithelium with mild fibroplasia. All the cases in alkali burn group showed severe conjunctiva ischemia that restored in partial72h after burn. Comeal perforation occurred in2cases2～3weeks after burn. But severe narrow conjunetival sac occurred only in1case. Severe fibroplasia and disorgnised epithelium were dectected under histopathologic examination. Extensive symblepharon and pseudopterygium occurred after ocular surface excision, and all the cases result in severe narrow conjunetival sac4weeks after operation. Histopathological examination revealed morderate fibroplasia.2. Oral mucosal epithelial cells could be harvested with the number about1～2×10b after one amplification passage in vitro. The capacity of cell attachment in different scaffold was CLSH> AM> PLGA, and cell proliferation was AM> PLGA> CLSH. Conjunetival congestion faded away completely within one week in the area of AM implantation and control. And AM could not be distinguished under conjunctiva2weeks after implantation. But for CLSH and PLGA, the fading process of conjunctival congestion lasted for a quite long time, and a subconjunctival dull red nodule was noticed4weeks after implantation. Histopathological examination revealed few inflammatory cells around AM2weeks after implantation, and faded away after4weeks. But large mount of multinuclear giant cells around CLSH and eosinophilic granulocytes around PLGA were detected2weeks after implantation, which reduced accompanied with severe cystic fibroplasia after another2weeks. Oral mucosa membrace cell clone on AM could be seen5d after cell seeding. The cell amplification continued and integrated into monolayer after coating onto the prosthetic eye sheet. Cell monolayer with positive expression of CK3and P63could be detected under histopathological and immunohistochemistory examination.3. Conjunctival sac reconstruction was successfully performed on all the rabbits. The depth of fornix was10～14mm in group A,9.5～13.5mm in group B and8～14.5mm in group C following removal of prosthetic eye sheet2weeks after operation, and was6.5～11mm in group A,5～9mm in group B and3.5～7.5mm in group C6weeks after operation. Cell monolayer could be detected in local areas of conjunctival sac in all the rabbits. Immunohistochemical examination revealed positive expression of CK3in group A, but not in the other2groups.ConclusionIt was concluded that animal model of severe narrow conjunctival sac could be constructed by ocular surface excision. The conjunctival sac implant constructed with AM and oral mucosa epithelium could simplify the process of conjuntival sac reconstruction and alleviate narrow conjunctival sac to some extent. |
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