| Object: Investigate the adhesive, proliferative and differentiative biological behavior of bone mesenchymal stem cells (BMSCs) into absorbable gelatin sponge, to conclude the biocompatibility of absorbable gelatin sponge and the possibility of gelatin sponge as BMSCs scaffold in intraocular trasplantation .Method: Sacrifice the neonate rat , collecte BMSCs suspension from the thighbone and tibia ,culture and amplicate BMSCs in vitro by adherence cultivation. Purify BMSCs by passage. Choose the well-grown 4th passage and co-culture them with absorbable gelatin sponge which was modified by Poly-L-Lysine(PLYS). After 7 days'cultivation, BMSCs were induced by conditioned medium of neonate rat retinal cells for 24 hours. Observe adhesive and proliferative biological activity and the morphological changes of induced BMSCs on absorbable gelatin sponge with light microscope and scanning electron microscope(SEM). Purify and identify the 4th passage by flow cytometer(FCM). Use MMT colorimetry to determine proliferative vitality . Detect the expression of neuron specific enolase(NSE)of induced cells by immunohistochemical.Result1. morphological observation and identification of BMSCs Observation by light microscope : cells began to adhere 24 hours after inoculation , presenting large polygon. after 48 hours , the number of cellsgradually increased and some of them stretched out pseudopod,forming comma or short stick shape . Meanwhile they began to proliferate , forming cell groups which had new round cells located in the center ,while peripheral cells were relatively larger with rhombus or long-spindle shape .They also displayed monolayer and were crosslinked.Detect the 4th passage by FCM : CD71 positve , positive rate 92.73%; CD 90 positve , positive rate 94.33%; CD45 negative, negative rate 95%.2. morphological observation and proliferative vitality determination of BMSCs co-cultured with absorbable gelatin sponge. Observation by light microscope : the 4th passage adhered agglomerately on the surface of gelatin sponge 24 hours after inoculation, and might fall off easily when shaked .After 72 hours, the cell agglomerate became bigger ,and adhered deep into gelatin sponge, which cannot be ramshackle when shaked .after 7 days , the cell agglomerate enveloped the whole gelatin sponge.Observation by SEM: The absorbable gelatin sponge with multiporous three-dimensional structure, which had different sizes of pores , of which the diameters ranged from 150-400um, had 98% open pore rate. The cells adhered on the surface of gelatin sponge with round shape 24 hours after inoculation , having granulated ecphyma on their surface.72 hours later , the cells adhered deep inside the gelatin sponge, stretching out many pseudopod linked with the gelatin surface.7days later, the cells became long-spindle, full of microvilli on the surface, and grew along the framework . The cells were crosslinked andsecreted large quantity of extra-cell matrix surrounding the gelatin sponge.MTT colorimetry determines cells proliferative vitality : there was no significant difference of proliferative vitality between inoculated and uninoculated BMSCs.3. morphological observation and immunohistochemical identification of neural induced BMSCs co-cultured with absorbable gelatin sponge.Observation by SEM : after induction , the cells contracted into cone or ball shape, the ecphymas became thinner and longer ,and the ends of some long ecphymas formed cone-like intumescences which were interconnected.Result of immunohistochemistry : NSE coloration was positive for partial cells after induction ,with their cytoplasm dyed deep brown.Discussion: this experiment used the whole marrow adherent culture to isolate, culture and purify BMSCs. Morphological observation of adhesive cells with light microscope displayed that cells began to adhere 24 hours after inoculation , presenting large polygon. after 48 hours , the number of cells gradually increased and some of them stretched out pseudopod forming comma or short stick shape . Meanwhile they began to proliferate , forming cell groups which had new round cells located in the center ,while peripheral cells were relatively larger with rhombus or long-spindle shape .They also displayed monolayer and were crosslinked. The shape of adhesive cells was in line with the morphological features of BMSCs , thus initially testifying that the adhesive cells were BMSCs . The recognition of BMSCs surface antigens and the application of FCM provide fairly exact method for purification andidentification of BMSCs . At present , the researches show that BMSCs express various surface antigens, such as SH2, SH3, Stro-1, CD29, CD44, CD71, CD90, CD106, CD120 etc ,without expressing hematopoietic cell surface antigens , like hematopoietic stem cell marker antigen CD34, white blood cell marker antigen CD45, lymphocyte surface antigen CD11a and monocyte , macrophage surface antigen CD14 etc. Accordingly , at the present stage , Using FCM to identify whether correlated antigens are expressed , so as to exclude the hematopoietic cells and prove that the obtained BMSCs are pure . This experiment chose 4th passage and used FCM to identify the expression of CD71, CD90, CD45 .The result showed that CD71positve, positive rate 92.73%; CD 90 positve, positive rate 94.33%; CD45 negative, negative rate 95%, which demonstrated that the 4th passage was mainly BMSCs, and the morphological observation also supported this conclusion.The absorbable gelatin sponge used in the experiment had better biological safety and degradation . SEM observed that the absorbable gelatin sponge with multiporous three-dimensional structure, which had different sizes of pores , of which the diameters range from 150-400um, had 98% open pore rate. The observation by SEM revealed that the absorbable gelatin sponge had preferable three-dimensional structure . To study the biocompatibility of absorbable gelatin sponge, we adopted combined cell culturing method in vitro and evaluated the biocompatibility by directly observing the adhesive, proliferative and differentiative biological behavior ofBMSCs co-cultured with absorbable gelatin sponge . The observation by SEM showed: the cells adhered on the surface of gelatin sponge with round shape 24 hours after inoculation , having granulated ecphyma on its surface. 72 hours later , the cells adhered deep inside the gelatin sponge, stretching out many pseudopod linked with the gelatin surface. 7 days later, the cells became long-spindle, full of microvilli on the surface, and grew along the framework . The cells were crosslinked and secreted large quantity of extra-cell matrix surrounding the gelatin spon. In summary, these results show that BMSCs can adhere and proliferate on the absorbable gelatin sponge modified by PLYS. MTT colorimetry determined cells proliferative vitality : there was no significant difference of proliferative vitality between inoculated and uninoculated BMSCs (P >0.05). 24 hours after the induction by retinal cells condition medium , the cells were observed by SEM. after induction , the cells contracted into cone or ball shape, the ecphymas became thinner and longer ,and the ends of some long ecphymas formed cone-like intumescences which were interconnected . Result of immunohistoch- emistry : NSE coloration was positive for partial cells after induction ,with their cytoplasm dyed deep brown .The result signified that BMSCs could differentiate into retinal neuron on absorbable gelatin sponge.Conclusion: BMSCs are able to adhere , proliferate and differentiate on the absorbable gelatin sponge .So the conclusion is that the absorbable gelatin sponge has nicer biocompatibility and could be used as effective BMSCs scaffold in intraocular trasplantation . However , the feasibility ofintraocular trasplantation using BMSCs co-cultured with absorbable gelatin sponge has to be verified through in vivo experiment . Besides , how to massively amplify BMSCs under stable culture condition? How to make the differentiative state finer ? these are questions remained to be settled ,so as to offer nicer sources of materials for ocular treatment.
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