Construction Of Rat Amniotic Epithelial Cells And Collagen Sponge Complex And Effects Of The Complex On Injured Optic Nerve Of Rat

Purpose:Many eye diseases and injuries, such as glaucoma, optic nerveischemia, contusion,optic canal fractures, can make the RGCs irreversible apoptosis, and result in blindness.Until now, there is not effective treatment for such diseases. The optic nerve is a whitematter tract of the diencephalon composed principally of the axons of retinal ganglion cells(RGCs). Optic nerveand retina is the most easily accessible parts of the central nervoussystem (CNS), and lying outside the skull. Therefore, optic nerve injury has been used tomodel brain axonal injury. In this study focusing on tissue engineering, we constructedamniotic epithelial cells (AECs) and collagen sponge complex for optic nerve repair ofanimal model in rat, and observed its effects on optic nerve repair.Methods:1) Rat AECs were collected from late encyesis rats by digestion with trypsin for severaltimes. The third generation of AECs were identified by immunocytochemical staining(CK19, COX10and Nestin) and labeled with PKH26.2) We constructed AECs and the collagen sponge complex and observed AECs in thecomplexes.3) SD rats were randomly divided into four groups: normal group, injury group,collagen sponge group and the complex group. The rat left optic nerve in the injury groupwas completely transected. Epineurium was cut longitudinally about2mm retrobulbar with1ml syringe needle. The optic nerve defect of about0.5mm was made with vessels intact.Collagen sponge group group was based on the injury group, and Gelfoam was transplantedinto the injury part of the nerve. Complex group was based on the injury group also, and thecomplex was transplanted into the injury part of the nerve just after10μl AECs suspension(1×107/ml) was injected at the same site.4) The biocompatibility of Gelfoam or the complex was observed. On7days aftersurgery, the number of AECs in the complex was counted and AECs were identified withNestin antibody by immunocytochemical staining. On28days after surgery, the relationshipof AECs and the regenerative axon was observed.5) Effects of the complex on RGCs were detected by H-E staining, Nissl’s staining,CTB-FITC tracing and MAP-2immunocytochemical staining. 6) Effects of the complex on optic nerve were detected by H-E staining, CTB-FITCtracing and GFAP and GAP-43immunocytochemical staining.7) Pictures were concatenate with Panorama Maker5. Images were measured withPhotoshop CS3and Image Pro plus6.0. Data were analyzed with SPSS18.0.Results:1) In vitro AECs could express CK19, COX10and Nestin. AECs had the potency ofdifferentiation into nerve cell.2) The biological activity of AECs within the complex was well, and surgery was604.3/mm2. Approximately48%of AECs expressed Nestin. A part of AECs still have thepotency of differentiation into nerve cell.3) Both Gelfoam and the complex biocompatibility were well. Their biodegradationhappened on between day28and day56after transplantation. In vivo, the density of AECswithin the complex was604.3/mm2, approximately17%of AECs expressed Nestin. AECscould survival at least28days after transplantation, and migrate toward both sides of thedamaged area. AECs exist around the regenerative axons. The AECs survival rate is heigh.A small part of AECs have the potency of differentiation into nerve cell. This is beneficialto improvement of optic nerve microenvironment.4) Nissl staining of the retina: On day28after surgery, both the number of celldiameter≥12μm’s cell and total cell was increased from injury group to complex group,collagen sponge group in proper order. In each field the number of cell diameter <12μm’scell, both collagen sponge group and the complex group was more than injury group.Both complex and collagen sponge can increase the number of cell in retian, and complexeffectiveness is batter.5) HE staining of the retinal: On day28and day56after surgery, the cell number inretinal ganglion cell layer, the thickness of inner plexiform layer and inner nuclear layer, ineach experimental group was less than that in the normal group. On day28after surgery,the cell number in retinal ganglion cell layer of the complex group was more than that ininjury group and the collagen sponge group and on day56after surgery complex groupwas more than collagen sponge group. On day56after surgery the thickness of innerplexiform layer and the thickness of inner nuclear layer of complex group and collagensponge group is more than that in injury group. Complex can make the cell number inretinal ganglion cell layer, the thickness of inner plexiform layer and inner nuclear layerincrease.6) CTB-FITC tracing RGCs and their axons: On28days and56days after surgery,CTB-FITC positive area rate of central retina, middle retina and peripheral retina in the experimental group was less than the normal group, and CTB-FITC positive area rate ofcentral retina and middle retina in the collagen sponge group was more than the injurygroup. Both on28days after surgery central retina and on56days after surgery middleretina in the complex group were more than the injury group and the collagen spongegroup. On28days and56days aftersurgery, CTB-FITC positive area rate of central retinaand peripheral retina in the complex group were more than the injury group. Both complexand collagen sponge can increase the number of RGCs and their axon in retian, andcomplex effectiveness is batter.7) CTB-FITC tracer optic nerve: On day28and day56after surgery, CTB-FITCpositive area rate of optic nerve both in injury region and in proximal region was increasedfrominjury group to complex group, collagen sponge group in proper order. Both complexand collagen sponge can promote optic nerve regeneration, and complex effectiveness isbatter.8) Immunocytochemical: On day28after surgery, the immune-positive products ofMAP-2in retina were increased from injury group to collagen sponge group, complexgroup and the complex group in proper order. Both complex and collagen sponge canincrease the number of neuron in retian, and complex effectiveness is batter. On day28andday56after surgery, the immunopositive products of GFAP in injury region weredecreased from complex groupto collagen sponge group and injury group in proper order.On day28after surgery, the immune-positive products of GAP-43in injury region wereincreased from injury group to complex group and collagen sponge group in proper order.Complex can make GFAP down-regulation and GAP-43up-regulation, complex has littleeffectiveness.Conclusions:1) Collagen sponge space physics structure is beneficial to AECs adhesion and survival.2) AECs collagen sponge complex transplantation can play AECs and collagen spongesynergistic effect, improve the local microenvironment of injured optic nerve, make thenumber of survival RGCs increase, and promote axon regeneration.3) AECs collagen sponge complex repaired optic nerve injury is feasible.