The Effects Of SPIONs-labeled Amniotic Membrane-derived Mesenchymal Stem Cell Transplantation On Alkali Burn Model In Rabbit

Background and ObjectiveAfter alkali burn of cornea, alkaline substance can penetrate ocular surface, and lead to severe characteristics of inflammatory reaction through the cell permeability or the secretion of proteolytic enzymes and cytokines. A variety of methods of treatment have been used to protect the integrity of corneal epithelial, and prevent secondum stromal ulcers, but the optimal therapy is still controversial. In this study, the effects of SPIONs-labeled amniotic membrane-derived mesenchymal stem cell (hAM-dMSCs) transplantation on corneal alkali burn in rabbits have been researched through the following four stages.1. Mesenchymal stem cells derived from amnion have been identified by cell morphology, MTT and flow cytometry, which then provide stem cells for the post-study.2. Using different ways to mark primary cultured hAM-dMSCs, and explore the most appropriate cell markers.3. Transplanting amniotic membrane-derived mesenchymal stem cells labeled by superparamagnetic iron oxide nanoparticles (SPIONs) into alkali burned eye through localized subconjunctival injection, then observe survival and migration of cells in host. Combined with traditional amniotic membrane transplantation, investigate the role of tissue regeneration stem cells in corneal alkali burns through clinical examination and detection of TNF-a and IL-1β to assess treatment effects. Collecting tears of normal rabbits and alkali burns undergoing treatment.4. Then using radioimmunoassay methods to measure concentration of lactoferrin (LF) to investigate the relationship and significance between alkali burn and LF levels in tears. Materials and methods1. Primary culture and appraisement of amniotic membrane-derived mesenchymal stem cellsObtain amniotic cells by Enzyme digestion and adherent tissue culture method and identify amniotic membrane-derived mesenchymal stem cells through cell morphology, repeated passage cell in order to carry out amplification and purification.To identify the mesenchymal stem cells derived from amniotic, we used the cell morphology, flow cytometry, and MTT cell activity detection. Observe the generation of cell morphology and arrangement by inverted microscope.2. Cell cytotoxicity of human amniotic membrane-derived mesenchymal stem cells labeled by SPIONs in vitroSuperparamagnetic iron oxide nanoparticles (SPIONs), which the concentration being3.5、7、14and28μg/ml respectively, was used in this study. Cells were labeled with solo SPIONs marker method. The cell culture medium was replaced with SPIONs for the first time (0d), and that replaced without SPIONs1d、2d and4d later. The cultured cells were harvested in1、2、4and7d. Cells gained at each time point were divided into five groups for the performance of MTT method, Prussian blue staining and flow cytometry; no SPIONs labeled cell as control group.3. Marking human amniotic membrane-derived mesenchymal stem cellsTo determine the better marked way and method, we compared three different marked methods respectively such as Brdu, DAPI and SPIONs. Thus showing track preparation of hAM-dMSCs transplantation treatment of corneal alkali burn.4. Effects of Amniotic membrane-derived mesenchymal stem cell trans-plantation on severe corneal alkali burn in rabbits Experimental animal were divided into two groups:1. alkali burned eye was treated with hAM-dMSCs suspension, which had been labeled by SPION;2.14mesenchμg/ml SPIONs transplanted into animal. Preparation of transplantation cell: ymal stem cells derived from amniotic was marked by14μg/ml SPIONs (1days), then harvested5μ1cell suspension (content1×106transplanted cell) as backup. Amniotic membrane transplantation methods:after alkali burn, washing conjunctival by1:1000gentamicins liquid. Cutting off bulbar conjunctiva circularly along limbal, separation, curettage of necrotic tissue on the corneal surface. Using amnion which epithelial side was backward to the cornea.covered the entire cornea and limbus, interrupted suturing it to the limbus with10nylon suture line. And fixing it in the superficial layer of the sclera. Suturing the free edge of bulbar conjunctiva to the amnion, besides, snipping spare amniotic tissue. After the operation, giving subconjunctival injection of gentamicin20000u and2mg dexamethasone mixed liquor, erythromycin eye ointment to prevent injury, and suturing eyelid to protect operation wound of rabbits. Using local conventional antibiotics and hormone postop erative.Cell transplantation methods:animal model were randomly divided into three groups. The first study group (hAM-dMSCs group, n=12) was treated with a subconjunctival injection in the injured eye using a Hamilton syringe, and into the temporal quadrant of the bulbar conjunctiva. One hundred microliters of hAM-dMSCs suspension, containing1×106hAM-dMSCs, which had been labeled by SPION, was used. The second study group (AM group, n=12) was treated with AM grafting, and the third group (association group, n=12) was treated with labeled hAM-dMSCs in combination with AM grafting. The situs of subconjunctival injection was conjunctiva temporal parts; injection volume was100microliter, which containing about1×106hAM-dMSCs. The first and third study group, D group eye were injected with equivalent volume containing7μg/mlSPIONs saline as control in the contra lateral.5. Lactoferrin concentration of rabbit tears after corneal alkali burn undergoing treatment Amniotic mesenchymal stem cells combined with amniotic membrane transplantation was put into the treatment of alkali burns.30rabbits were randomly divided into three groups:The first group (blank group, n=10):without any treatment; The second group (control group, n=10):making rabbit cornea alkali burn model, without any transplantation treatment; The third group (study group, n=10):subconjunctival injection of amniotic mesenchymal stem cells suspension combined with amniotic membrane transplantation after making model. Modeling animal accepted local conventional medication. Assay began after stopping eye drops for two weeks. A volume of80～100ul tears was collected from the conjunctival sac using microcapillary tubes, and separately placed in0.5ml aseptic intravenous transfusion tubes. The intravenous tubes were sealed by heating their opening, and stored at-30℃until analysis. LF levels in the tears of modeling animal with and healthy controls were measured by radial immunodiffusion assay.Results1. Fresh amniotic membrane was digested by collagenase and trypsin, and the cell suspension collected was primarily inoculated to the culture flask. A small amount of cell adhesion appeared in P0after a24-hour culture; The number of adherent cells increased at48-hour; When cultured for72hours, the number of adherent cells increased significantly; for7days, adherent cells covered the bottom of the cell culture flask, close to50%-60%of fusion approximately, and the adherent cells looked like fibroblast.2. Amniotic membrane-derived mesenchymal stem cells detection by flow cytometry analysis:P3cells of primary cultures were in the expression of surface molecules CD29, CD44, CD90, CD105, and negative expression of CD31, CD34, CD45and CD106.3. After different SPIONs-labeling concentration to label mesenchymal stem cells, Prussian blue staining showed that:the cells were labeled at100%, and the cytoplasms contain blue dye particles in varying amounts. MTT results showed that the safe concentration of SPIONs to label amniotic membrance-derived mesenchymal stem cells is less than or equal to14μg/ml, meanwhile cell activity is more than90%. Blue staining particles in the cytoplasm aggregate with the extension of labeled time when SPIONs concentration is less than or equal to14μg/ml; While no aggregation of blue staining particles happens in the cytoplasm when SPIONs concentration is higher than14p,g/ml.4. Cell number reduced obviously after labeled by three different kind of marker. While cell number began to increase3d after labeled by SPIONs and DAPI, which cell of the SPIONs group growed better, and reached a peak in the six day. On the contrary, cell number of the BrdU labeled group always maintained at about5×104.5. Using subconjunctival injections,14μg/ml SPIONs labeled hAM-dMSCs were transplanted into subconjunctival of rabbit. At day28, the cornea was isolated for Prussian blue staining, and dark blue stained particles were detected in the limbus corneae, which indicated that hAM-dMSCs had survived at this site. Therabbit was in good condition, and without partial eye rejection. There was no survival of hAM-dMSCs in control group of7μg/ml SPIONs PBS fluid in transplantation at4weeks.6. Curative effect of corneal alkali burns in hAM-dMSCs of AM transplantation: compared with control group, the healing rate in the experimental group was significantly higher than that in control group (P<0.01), whereas there,had no significant difference in the three experimental groups (χ2=1.2, P>0.05).4weeks after transplantation, corneal neovascularization of transplantation group reduced (P<0.05); The control group exhibited corneal opacity scores that were significantly higher than those observed in the other three groups (P<0.01), and scores of the hAM-dMSCs transplantation and AM grafting groups were significantly higher than those of the association group (P<0.01), while the difference between the hAM-dMSCs transplantation and AM grafting groups was not statistically significant. There were significant differences of the scores for corneal neovascularization area of study group and control group (P<O.Olor P<0.05), besides, among experimental groups, compared with amniotic membrane transplantation combined with hAM-dMSCs transplantation group, there was a significant difference in the other two experimental groups (P<0.05). 7. Light microscopic findings28days after the alkali burn that the corneal thickness was greatly increased and many polymorphonuclear leukocytes had infiltrated into the corneal stroma with extensive NV in the control group. However, relatively few polymorphonuclear leukocytes had infiltrated, and less NV had occurred in the cornea in the hAM-dMSCs transplantation, AM grafting and association groups. In addition, the corneal thickness was slightly increased in the hAM-dMSCs transplantation, AM grafting, and association groups, compared to the control group.8. Using subconjunctival injections,14μg/ml SPIONs labeled hAM-dMSCs were transplanted. At day28, the cornea was isolated for Prussian blue staining, and dark blue stained particles were detected in the limbus corneae, which indicated that hAM-dMSCs had survived at this site. While, there were no hAM-dMSCs in the near central and central cornea.9. Western-blot detection of rabbit corneal alkali burn of cornea for TNF-a and IL-1β expression after different treatment methods:Western-blot results showed that TNF-a and IL-1β in corneal alkali burns in treatment of the various stages of the process are expressed. Besides, with the extension of treatment time, the expression intensity was decreased obviously.10. Concentrations of TNF-a and IL-1β in the aqueous humor of the control and study groups are detected using ELISA. The average TNF-a concentration in the study groups decreased with time, and the difference at different times was statistically significant. In the control group, the average TNF-a concentration also decreased with time. At corresponding times, the TNF-a concentration in the study group was significantly lower than in the control group.The average IL-1βconcentration in each group decreased with time, and the content in the study was significantly lower than in the control group. The difference between study groups and control one at day1and day3, association group and control group at day7and day14was statistically significant.11. The concentration of LF at each observation time point was significantly decreased in the tears of control subjects compared with experimental ones and blank ones (P<0.01or P<0.05); the concentration of LF in the experimental group has no distinction when compared with the control group (P>0.05). There is a positive relationship between LF in tears and results from Schirmer’s I (P<0.01).Conclusion1. Enzymatic digestion and cell adherent culture method was used to isolate cells from amniotic membrane, and then amniotic membrane-derived mesenchymal stem cells were successfully identified through cell morphology, and flow cytometry. It was confirmed that the amniotic membrane-derived mesenchymal stem cells possess the ability of MSCs and thus provide seed cells for the treatment of wound repair.2. It is safe and feasible for the treatment of ocular trauma disease using hAM-dMSCs transplantation through localized subconjunctival injection, which conducive to the creation of a new treatment method for ocular trauma disease, that is, widely stem cell source, no immune rejection, and minimally invasive.3. Through the experiment of SPIONs-labeling methods, it became definite that less than or equal to14μg/ml is the safe concentration of SPIONs to label amniotic membrane-derived mesenchymal stem cells, which did not influence the cell biological characteristic after it was labeled by SPIONs. Amniotic membrane-derived mesenchymal stem cells labeled by14μg/ml SPIONs can provide practical basis for tracking of the treatment of related diseases by cell transplantation.4. HAM-dMSCs after early being subconjunctivally transplantation into severe alkali burned rabbit cornea could promote cornea repair through restrain cornea angiogenesis. Migration and differentiation of hAM-dMSCs depend on the local microenvironment. The action mechanism of hAM-dMSCs mainly through the regulation of cytokines and improving the located microenvironment of residuary cells, and promoting residual cell growth to play its repairment, not differentiate into the corresponding target cells.5. As a kind of treatment of corneal alkali burn, hAM-dMSCs combined with amniotic membrane transplantation can improve the speed of corneal epithelial healing, reduce corneal turbidity and neovascularization, and be conducive to the secretion of tear proteins. Addition to urgent and effective surgical methods for the treatment of corneal alkali burn, supplement of tear proteins possess biologically active can not only accelerate wound healing, but improve the quality of visual and life after patients cured.