The Experimental Study Of The Pravastatin's Effect On The Transplantation Of BMSCs And FS For Curing Early-stage SANFH Of Ribbits

Objective:To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits through twice injection of E.coli endotoxin(LPS) and three times injection of methylprednisolone(MPS), to explore the mechanism of the avascular necrosis of femoral head. To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded,we verify the tissular and cellular biocompatibility of the engineering tissue;to observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF,BMP-2 for BMSCs/FS complex; to observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression and we also explore the mechanism of the pravastatin for the SANFH.Methods:Chapter 1To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits:80 New Zealand rabbits (both male and female available) were obtained,and then randomly divided into 4 groups.Group A(control group):rabbits were intramuscular injected with normal saline at the same time point as the same as group D;Group B:injection of horse sera and three times of methylprednisolone;Group C:injection of single E.coli endotoxin and three times of methylprednisolone;Group D:injection of twice E.coli endotoxin and three times of methylprednisolone. We observe the animal's general condition.and we also detect the level of cholesterol(CHOL),triglycerides(TG),blood rheology,coagulation state,thrombomodulin.All the data was analysed by SPSS 17.0.We used the T test to analyse the data between two sets and used the q test to analyse the data among multiple sets.We identified the statistical significant difference with P<0.01.We obtained the bilateral general femoral head sample after 6,8,10,12 weeks and we check the general pathological section through light microscopy and carry out immunohistochemical staining of vascular endothelial growth factor(VEGF) and microthrobus(MSB). we performed the X-ray,MRI to elucidate the medical imaging changes of the SANFH after 8,10,12 weeks.Chapter 2To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded:Isolating the BMSCs from the femoral marrow cavity of the New Zealand rabbits,purifying the BMSCs through the repeated medium changes and passaging by the adherence,and the we identified the BMSCs through CD34,CD44 test through FCM. We divided the P3 BMSCs into 4 groups.Group A:P3 BMSCs+osteoinduction medium,Group B:P3BMSCs+Brdu+osteoinduction medium,Group C: P3 BMSCs+fibrin glue+ osteoinduction medium,Group D:P3 BMSCs+fibrin glue+ Brdu+osteoinduction medium. On the 1,2,4,7,14,21 day after osteoinduction,we performed the cell kinetics tests,light microscopy observation trypan blue staining,calcium-cobalt staining and Von-Kossa staining and examined the amount of ALP and Ca quantitatively.We evaluated the data by SPSS 17.0.Chapter 3To observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF,BMP-2 for BMSCs/FS complex:P3 BMSCs+FS+osteogenic medium:group A(control),group B(100 umol/L pravatatin).Investigate the cell growth kinetics,observe the cell morphology under inverted microscope at day 1,4,7,14; check the ALP quantity at day 14;check the osteogenic ability using Von-Kossa staining at day 21;extract the mRNA,compound cDNA using reverse transcription,to hybrid the FITC-cDNA with genomic microarray chip,to discriminate the differentially expressed genes.To check the VEGF and BMP-2 expression.To analyse the data with SPSS 17.0.Chapter 4To observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression:60SANFH-rabbits were randomly divided into four groups(15each group).Group A:control;Group B:core decompression only;Group C:Brdu marked BMSCs only;Group D:pravastatin+Brdu marked BMSCs. We test the level of blood fat at 4,8,12, 16 weeks after operation.At 8,12,16 weeks after operation,rabbits were sacrificed,the femoral heads were observed through naked eye,light microscopy,electron microscope.We also examined expression of VEGF and Cbfa1 through real-time PCR at 8,12,16 weeks after operation.We examined the Brdu marked BMSCs in the femoral head at 8,12,16 week after operation and performed the MRI. To analyse the data with SPSS 17.0.Results:Chapter 1At 4 week after the final injection,the rabbits'weight of group A increased and the rabbits' weight of group B,C,D decreased.The rabbits of group B,C,D showed listlessness,less activity,reduced diet and less response.4animals died at 10 days after final injection in group B,5 animals died at 10 days after final injection in group C,7 animals died at 10days after final injection in group D.Compared with group A,the level of CHOL,TG,the whole blood viscosity low shear rate,viscosity of plasma,erythrocyte aggregation index significantly increased(P<0.05),which group D increased more significantly.The MRI showed no change before injection and 2 week after injection.The signal intensity of MRI in group C and D decreased slightly and the signal intensity of MRI in group A and B at four week after injection.The MRI detected small necrotic area of femoral head in group C and D and the signal intensity of MRI in group B decreased slightly at 8 week after injection.The necrotic area of femoral head enlarged at 12 week after injection in group D.The rate of empty lacuna is 10.4%±4.4% in group B,19.7%±4.7 in group C,33.2%±5.6% in group D and the area of bone trabecula is 64.4%±8.4% in group B,53.2%±5.7% in group C, 41.3%±5.1% in group D.Compared with group B and C,the group D showed the statistical difference(P<0.01).Compared with group A,B,C on medical imaging and pathology, the necrotic area of femoral head,fat cells in intramedullary,empty lacuna in femoral head increased significantly in group D and the bone trabecula became thin and more fractured.Chapter 2Little cells can attach on the culturing bottle 48 hours later,presenting like fibroblast;most cells present long fusiform 4d later.The P3 BMSCs expressed CD44 through FCM. The Brdu marked BMSCs embedded in the fibrin glue can survive. The BMSCs presented like the fibroblast 3 days later;the brim of fibrin glue began to degrade 6 days later and the BMSCs fell onto the plat; the most of the fibrin glue degraded and more BMSCs fell onto the plate and the BMSCs presented the normal morphous 14 days later;all the fibrin glue degraded 21 days later and the fibrin displayed no bad effect on the morphous of BMSCs. More than 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy,more 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy at 12,24,48,72 hour after cultivation.there is no statistical differences in each group(P>0.05).The OD of MTT,quantity of ALP and Ca showed no statistical differences in each group(P>0.05).Chapter 3The Brdu marked BMSCs embedded in the the FS in group B can survive;the cells present typical fusiform 4d later;the margin of the fibrin glue began to degrade and the cells fell onto the plate 7d later; the BMSCs proliferate good and most FS degradated;more and more BMSCs fell onto the plate and the morphology of adherent cells presented normal 14d later. The quantity of ALP in group B are more than that in group A(P<0.05) and the pravastatin can upregulate the VEGF and BMP2 through ELISA(P<0.05). Among 2723 genes,there are 326 differentially expressed genes including ALP1,BMP-2,OCN,DLX5, Cbfa1MMP13 which are closely correlated with osteogenic differentiation through the microarray chip.Chapter 4The level of CHOL,TG,HDL showed no statistical differences in each group(P>0.05) at 1 week after operation. The level of CHOL,TG in group D decreased and had statistical difference compared with other groups.there is no no statistical difference among group A,B,C at 4 week after operation. The level of CHOL,TG in group D decreased significantly and other groups decreased slightly at 8 week after operation and is no no statistical difference between group B and C(P>0.05). At 12 week after operation,the level of CHOL,TG in group D decreased more significantly and had statistical difference compared with other groups(P<0.01). The level of HDL in group A,B,C decreased,but increased in group D at 4 week after operation.At 8,12 week after operation,there were no statistical differences between group B and C(P>0.05),but there were statistical differences in group D compared with other groups.By pathological examination,the necrotic area of femoral head,fat cells in intramedullary,the rate of empty lacuna decreased but the density of bone trabecular increased in group D compared with other groups.The expression of VEGF,Cbfa1 showed no differences in each group through real-time PCR at 4 week after operation. At 8 week after operation,the expression of VEGF,Cbfa1 upregulated in all groups but group D upregulated more significantly. At 16 week after operation,the group showed the statistical differences compared with other groups(P<0.05).The necrotic area of femoral head enlarged in group A,no changing in group B,decreased in group C,decreasing significantly in group D at 4 week after operation. The necrotic area of femoral head enlarged slightly in group A,decreasing slightly in group B and C,disappearing almostly in group D.Conclusion:Chapter 11. Under the identical experimental condition,the appearance of SANFH were the earliest and most obvious in the group which receive the twice injection of E.coli endotoxin and thrice injection of glucocorticoid and the empty lacuna increased day by day.lt can provide good animal model of SANFH for the clinical research.2. The micrangium endothelial cells in the necrotic femoral head were impaired after the rabbits SANFH model made.The arteriolae in the femoral head were impaired and thrombosis;also resulted in the dyslipidemia; and also resulted in dysfunction of blood coagulation and hypercoagulative state.This may underlie the pathological mechanism of early stage SANFH.Chapter 21. The biological fibrin glue are satisfactory compatible,plastical and biological degradable,it has trhee-dimensional structure.lt can be used as injectable scaffold for cell cultivation and transplant.2. The use of Brdu for marking is easy,swift,safe,sensitive,and it can reflect the proliferation and position of BMSCs.3. The engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded present excellent biocompatibility Chapter 31.The pravastatin can induce the BMSCs to differentiate to osteoblast and the mechanism maybe correlate to upregulate the osteogenic genes expressions.2.The pravatatin can induce the BMSCs/FS to secrete the VEGF and BMP2.Chapter 41. The BMSCs combined with FS can be transplanted into the femoral head through decompression,and the BMSCs can differentiated into osteoblast and osteocyte and it can help repair and regenerate the bone tissue of the SANFH.2. Pravastatin can improve the lipid metabolism in vivo and ameliorate the toxic effect of the adipocyte on the osteocyte.It can upregulate the VEGF expression in the femoral head and improve the blood supply in the femoral head.It can also upregulate the Cbfal expression in the femoral head and help form new bone.3. The combination of the systemic application of pravastatin and local application of BMSCs for curing SANFH are feasible and can be hoped to apply clinically in the therapy for early-stage SANFH.