Evaluation On The Effects Of Osteogenesis And Angiogenesis After Transplanting Mesenchymal Stem Cells In The Rabbit Model Of Avascular Necronesis Of Femoral Head Induced By Corticosteroid

ObjectiveTo establish the rabbit model of Avascular Necrosis of Femoral Head(ANFH). Mesenchymal stem cells(MSCs)were cultured and labeled in vitro,and then transplanted into the blood-supplying arteries around the femoral head.The expression of CD34 was checked up in the muscles around the femoral head and that of BMP-2 was also checked up in the bone tissues.To evaluate the effects of osteogenesis and angiogenesis after transplanting MSCs in the rabbit model of ANFH induced by corticosteroid.Methods(1)Model-making:Twenty Japanese white rabbits including twelve male ones and eighteen female ones were brought into the study,whose genders weren't limited and mean weight was 2.8±0.9 kg.Ten rabbits were used for preliminary study and twenty ones for formal experiment.Two milliliters of horse serum was injected from auricular veins,and then methylprednisolone(4 mg/kg)was injected from gluteous medius muscles,twice a week.It lasted for six weeks.Three rabbits were dead and seven ones were alive in the preliminary study.Of twenty rabbits used for MSCs transplanting,five rabbits were dead during model-making.Fifteen rabbits left were randomly divided into two groups,of which eight ones were used to transplanting MSCs from right iliac artery(transplanting group),while other ones weren't transplanted MSCs(control group).(2)MSCs culturing:Marrow was obtained from the ilium and the thighbone of the Janpanese rabbit(age of 20 days)under aseptic conditions.PBS was added into the marrow at the propotion of 1:1 and evenly mixed.The mixed marrow was centrifuged at the speed of 1000 rpm for ten minutes.The lipid and impurity in the upper layer were threw away.The marrow left was added into 10 milliliters of Percoll liquid(1.077 g/L) and then centrifuged at the speed of 2500 rpm.Monolayer was extracted from the layer between upper layer and Percoll liquid.These marrow-derived cells were cultured in L-DMEM supplemented wth 10%fetal bovine serum(FBS).The hatching room maintained at 37℃in 5%CO2.Afler primary culture,adhesive cells regenerate when they reach confluency.Meanwhile,cells were also cultured in a 6-well plates in complete medium.Cells were digested by 0.25%trypsin in the culture bottle and blown by a blowtorch.Then they were regenerated by a proportion of 1:2.48 hours before transplanting,BrdU(5 mg/L)was used to label the third generation of MSCs.CD34 and CD44 were respectively detected by immunohistochemistry for the 6-well cells.BrdU in cellular nucleus was also detected by immunofluorescence.Before thransplantation,cells labeled by BrdU were digested by 0.25%trypsin and blown by a blowtorch until cells were all away from the plastic culture bottle.Then they were rinsed by PBS and centrifuged.They were counted and suspensory fluid of 3×10~6 were prepared for transplantation.(3)Transplantation of MSCs:Right femoral artery was isolated in the transplanting rabbits and intubated.The catheter tip was up to the iliac artery level.MSCs labeled with BrdU were slowly injected into the arteries.The right femoral artery was ligated and the rabbit was sent back to the animal center.(4)Sample detection:5 weeks later,the musclular tissues around the right femoral head were obtained.A part of tissues were used for iecd section and fixed in acetone.A mouse anti-BrdU monoclonal primary antibody(1:300)and a rabbit anti-CD34 primary antibody(1:300)were added to the section,while a mouse IgG fluorescein-conjugated Cy3 kit secondary antibody(1:100)and a goat anti-rabbit IgG conjugated with FITC (1:100)were also added for a double immunofluorescent detection.Other tissues were fixed in 10%formaldehyde,then dehydrated in ethanol,embedded in paraffin,and sectioned at a thickness of 5μm.CD34 and BrdU were detected by immunohistochemistry similar to former immunofluorescent detection.Tissues of femoral head were also obtained,fixed in 10%formaldehyde,decalcified in EDTA,dehydrated in ethanol,embedded in paraffin,and also sectioned at a thickness of 5μm.A mouse anti-BrdU monoclonal primary antibody(1:100)and a rabbit anti-BMP-2 primary antibody(1:100)were added to the section,while respective secondary antibody was also added for a double immunohistochemistry detection.HE stain was used to detect osteogenic changes.Results(1)In the preliminary study,three rabbits were dead.Of seven rabbits left,no femoral head was found to cave in.Defuse empty lacunae or shrinked cellular nucleus were found in six rabbits,while only one rabbit was found that the number of empty lacunae was less than 30%.Lipocytes became enlarged in the bone marrow in all the rabbits.Lipocytes also became enlarged in Haverison canal.However,no thrombus or lipoembolus was found in the bone tissues.Well-made model was up to 86%.(2)MSCs extracted from the bone marrow of baby rabbit adhered to the wall of culturing bottle.They grew spindly.The expression of CD44 is positive while CD34 is negative.It showed that what we transplanted were MSCs other than Haematopoietic stem cells(HSC).(3)The nucleus labeled with BrdU showed brown color by immunohistochemistry,meanwhile it detected red light by immunofluorescence.It showed a successful labeling.(4)There were a lot of BrdU-positive cells in muscles around femoral head in transplanting group,of which many MSCs expressed CD34. The iced section also showed similar results by immunofluorecence.(5)BrdU-positive cells were also found in the lacunae of femoral head,in which BMP-2 was expressed in cellular plasm.(6)HE stain showed that cellular nuclei were evidently shrinked and close to empty laculae in control group.Cells evidenly decreased in Haverison canal.Meanwhile,cellular nuclei were much larger in transplanting group than those in contol group.Cells evidently increased in Haverison canal,but they were still less than those in normal rabbits without modeling.Conclusion(1)In the model of ANFH induced by corticosteroid,it is feasible to transplant MSCs from vessels;2 Transplanted marrow-derived stromal cells could reside at muscles around femoral head and osteogenic tissues.MSCs could be transdifferentiated into CD34-positive cells and BMP-2-positive cells.MSCs transplantation could be helpful to the angiogenesis and osteogenesis of osteonecrosis.