| Purpose:To isolate and culture stromal vascular fraction cells(SVF cells) from adipose tissue of rabbits, and to testify SVF cells are adipose derived mesenchymal stem cells(ADSCs) by examining mesenchymal stem cell surface antigens and potential of multidirectional differentiation. To prepare porcine small intestinal submucosa, and examine its histocompatibility and cell compatibility. To observe chondrogenic effect of chondrogenic medium on ADSCs-SIS composite in vitro. To observe reparative effect of ADSCs-SIS composites induced by chondrogenic medium in vitro on treatment of growth plate defects in rabbits.Method:Part 1 Dissected the subcutaneous fat from groin of rabbit, digested with type I Collagenase to obtain stromal vascular fraction cells (SVF cells), the surface antigens including CD29, CD44, CD45 of 3rd passsge SVF cells were analyzed by flow cytometry, multi-directional differentiation potential of SVF cells were examined by adipogenic,osteogenic,and chondrogenic differentiation, alkaline Phosphatase staining, alizarin red staining, Von Kossa staining were used to identify osteogenic differentiation, oil red staining were used to identify adipogenic differentiation, type II collagen mRNA RT-PCR, type II collagen immunohistological staining, toluidine blue staining and safranin O staining were used to identify chondrogenic differentiation.Part 2 Small intestinal submucosa were processed by enzyme digestion-hypertonic saline decellularization, lyophilized at -52℃in high vaccum, and sterilized by gamma radiation, paraffin slices were used to observe the effect of decellularization, the surface structure of SIS were observed by scan electron microscope(SEM).SIS were implant into sacrospinous muscle pocket of rabbits, the specimens were examined by paraffin slice to observe degradation and histocompatibility of SIS, ADSCs of rabbits were isolated and cultured in vitro, 3rd passage of ADSCs were seeded onto one side or both sides of SIS, after one week of cocultivation, ADSCs-SIS composite were observed by paraffin slice and SEM.Part 3 3rd passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro, 7 days and 14 days after being induced, real-time fluorescent quantitative RT-PCR was used to analyze mRNA level of typeⅡcollagen, GAPDH was used as internal control gene, 2-△△CT method was used to calculate. ADSCs-SIS composites which were not induced were used as control, typeⅡcollagen protein was examined by histoimmunologic staining, toluidine blue staining and safranin O staining were used to observe extracellular matrix. SEM was used to observe cellular morphology after 14 days of chondrogenic differentiation.Part 4 3rd passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro for 14 days before being implanted. 36 New Zealand rabbits of 6-8 weeks old were divided into 3 groups randomly, 50% of medial growth plate in proximal right tibia was excised, group A was blank control group, the defects of growth plates were not filled, the defects of growth plates in group B were filled with SIS without cells, the defects of growth plates in group C were filled with ADSCs-SIS composites induced by chondrogenic medium in vitro for 14 days. 4 rabbits of each group were sacrificed at 4, 8 and 16 weeks, leg bones of both sides were taken off, fixed with 10% formalin solution, X ray photographs were taken to measure tibial length and proximal tibial articular surface angle. Calculated the differences of proximal articular surface angle and length of left and right tibias in each rabbit. Compared proximal tibial articular surface angle and length of left and right tibia in each group of different time interval, and compared difference of proximal articular surface angle and length of left and right tibias among 3 groups of different time interval. Paraffin slices stained with HE were used to observe reparative effect of ADSCs-SIS composites on growth plate cartilage.Spss 13.0 statitical package was used to deal with all data. Calculate a p-Value for paired T test and one-factor analysis of variance.Result:Part 1 Primary SVF cells were multi-angular or short spindle shaped, 3rd passage SVF cells were long spindle shaped. Cell surface antigen of 3rd passage SVF cells were CD44+, CD29+, CD45-. Oil red staining was positive in adipogenic differentiation group, ALP staining, alizarin red staining and Von Kossa staining were positive in osteogenic differentiation group. RT-PCR of typeⅡcollagen mRNA showed that SVFs cells also expressed typeⅡcollagen mRNA, but the product bands were brighter in cells after 7 and 14 days of chondrogenic differentiation. After 14 days of chondrogenic differentiation, histoimmunologic staining of typeⅡcollagen was positive, toluidine blue staining showed that metachromatic staining was observed, and safranin O staining showed that cytoplasms were stained red.Part 2 SIS was white and semi opaque membrane, paraffin slices showed that no cell was observed in SIS, loose weave structure of serosal surface and compact structure of mucosal surface were observed by SEM, after one week of cocultivation, plenty of cells could be observed on the upside and few cells were observed on the downside of SIS when ADSCs were seeded only onto upside of SIS, plenty of cells could be observed on both sides of SIS when ADSCs were seeded onto both sides of SIS. Cells adhering to the fibers of SIS could be observed in paraffin slice after ADSCs were implanted onto SIS. After SIS were implanted into the muscle pocket of rabbits, infection and immunologic reaction were not observed, paraffin slice showed that only few of SIS fibers were not absorbed at 2 weeks after implantation, and completely abosorbed at 4 weeks after implantation.Part 3 Standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 7 days and 14 days were larger than that in uninduced ADSCs-SIS composites(P < 0.05), standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 14 days was larger than that in ADSCs-SIS composites which were induced for 7 days(P<0.05) . Histoimmunologic staining of typeⅡcollagen protein was positive in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium, but was negative in uninduced ADSCs-SIS composites. Extracellular matrix was metachromatic stained by toluidine blue, and red stained by safranin O in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium. Plenty of ADSCs adhered to both sides of SIS after 14 days of chondrogenic differentiation were observed by SEM.Part 4 After operation, incisions were not infected, and no immunologic rejection was observed, feeding and activity of animals were normal. The direct-viewing of specimens showed that 4 weeks after operation, obvious angular deformity of tibias was observed in group A, and slight angular deformity of tibias was observed in group B, no angular deformity of tibias was observed in group C, and no length discrepancy was observed in all 3 groups; 8 weeks after operation, obvious angular deformity and length discrepancy of tibias were observed in group A, angular deformity and length discrepancy of tibias were observed in group B, no length discrepancy or angular deformity was observed in group C; 16 weeks after operation, obvious angular deformity and obvious length discrepancy of tibias were observed in group A and group B, no angular deformity or length discrepancy of tibias was observed in group C. Comparison of X ray photograph measurement in each group showed that, in group A, 4, 8, 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle(P< 0.05) and length(P<0.05); in group B, 4 weeks after operation, there was significant difference between right and left tibias in length(P<0.05) , there was no significant difference between right and left tibias in the proximal articular surface angle(P> 0.05), 8 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle or length(P>0.05), 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle and length(P<0.05) ; in group C, 4, 8, 16 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle(P>0.05) or length(P>0.05). Comparison of X ray photograph measurement among 3 groups showed that 4 weeks after operation, the proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) ,there was no significant difference between group B and group C in proximal tibial articular surface angle difference(P> 0.05), and there was no significant difference among all 3 groups in tibial length difference(P>0.05); 8 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P <0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , there was no significant difference between group B and group C in tibial length difference(P>0.05) or proximal tibial articular surface angle difference(P>0.05); 16 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , proximal tibial articular surface angle difference of group B was larger than that of group C(P<0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05), tibial length difference of group B was larger than that of group C(P<0.05) . Paraffin slices stained with HE showed that 4 weeks after operation, bone bridges could be seen in growth plate cartilages and no reparative cartilage could be seen in growth plates of group A, bone bridges could be seen in growth plates and few reparative cartilages could be seen in growth plates of group B, and no bone bridges could be seen and reparative cartilages could be seen in growth plates of group C; 8 weeks after operation, bone bridges could still be seen in group A, bone bridges were not absorbed and growth plates were still discontinuous in group B, the reparative cartilages in growth plates rearranged in group C; 16 weeks after operation, bone bridges could be seen in growth plates of group A and group B, and cartilages rearranged like column in the medial half of growth plates in group C. Conclusion:Part 1 Stromal vascular fraction cells have identical surface antigens of mesenchymal stem cells and potential of multidirectional differentiation. So SVF cells were identified as adipose derived mesenchymal stem cellsPart 2 Small intestinal submucosa has good histocompatibility and good cell compatibility. It can be used as scaffold for ADSCs.Part 3 ADSCs could differentiated into chondrogenic cells after being induced by chondrogenic medium in SIS scaffold.Part 4 ADSCs-SIS composites induced by chondrogenic medium in vitro could prevent angular deformity and length discrepancy of tibias effectively after being implanted into medial growth plate defect of proximal tibia, and could repair cartilage of growth plates. This experimental study can give some suggestion to clinical treatment of growth plate injury by using the ADSCs-SIS composites which are induced by chondrogenic medium in vitro.
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