Study Of VEGF Modified Adipose-derived Stem Cells In Repairing The Bone Defect With Diabetic Osteoporosis

IntroductionBone tissue engineering has become the main current treatment in bone defects.This method is also fit to repair bone defect with diabetic osteoprosis, however,bone metabolic unbanlance and cell factors dysfunction make the bone defect repairement much more challengable under the diabetic condition.The in-vitro constructed engineering bone is the combination of cells and vector; therefore,there is no source of nutrition to this engineering bone.After being implanted,the blood and cell liquid are the major nutrition source for the engineering bone.Necorosis and scelorosis of the capillaries,hige blood glucose level and accumulation of AGEs in the bone matrix often cause poor blood surply to the implant and delay the vasculization process aroud the implant.The seed cells proliferation,transformation and secretion are also affected by these bad factors,eventually the seed cells death will become manifested and the implant will be out of effects.The inter-growth between engineering bone and self bone is nearly impossible under this situation.According to the above reasons,the early vasculization aroud the implant maybe is promising in sloving the problem of inter-growth between the engineering bone and self bone.In present study,we wil implant VEGF gene modified ADSCs with absorbable gelatin sponge into the diabetic rats models with femeral bone defects.Through the detection of the bone morpholage,histology and CD31/CD43,We will verify the fact that the interface barrier problem can be sloved by vasculization aroud the implant under diabetic conditions and prove that VEGF modified ADSCs can be an effective method in repairing the bone defet under diabetic situation.Material and Methods1.Adult ADSCs were isolated from adult rat fat tissues in vitro.Cell proliferation was observed by drafting cell growth curve.Labeling protein of stem cells was detected by immol/Lunofluorescence technique.2.Recombinant adenoviral vector with VEGF were constructed using the second generation adenoviral vector constructed system,pAdEasy-1.Human VEGF gene was inserted pAdshutle vector by enzyme cut and ligation.Positive clonies were screened and proliferation.Recombinant plasmids were isolated and cotransformed into E.coli BJ5183,and then kanamycin resistant recombinant plasmids were transfected into 293 cells for production of recombinant adenoviruses expressing VEGF.AV-VEGF was transfected into COS-7 cells and western blots carried out to confirm the expression of VEGF.3.Selection of concentration of high blood glucose and AGEs through MTT method.Based on the suppression rate calculation,we calculated the results of suppression rate of ADSCs in 1d,2d,3d,4d,5d,6d after cluture.From the suppression results,We choosed the concentration of AGEs and glcouse which not only has the biological effects on the ADSCs but also will not cause severe damage to the ADSCs.4.The effects of high glucose and AGEs on ADSCs osteogensis.In order to see what effects would high glucose and AGEs on the ADSCs osteogensis,we used Real-timeQ-PCR technique to detect the CNI,ALP,and OCN mRNA relative content secreted by ADSCs on 4d,7d,14d and 21d after osteogensis induce.The number and diameter of the calcification node was also counted.5.The effects of VEGF on ADSCs osteogensis under high glucose and AGEs condition.The high glucose together with AGEs had the strongest suppression power on the ADSCs osteogensis.In order to testify the function of VEGF on the ADSCs osteogensis under this condition,we used Real-timeQ-PCR technique to detect the CNI,ALP,and OCN mRNA relative content secreted by ADSCs.The number and diameter of the calcification node was also counted.6.The models of diabetic rat were constructed by STZ induced method.The blood glucose level,physical changes were recorded.The weight change between normal and diabetic group on 0w,4w,6w,8w,10w and 12w were statistically analyzed.On the third month after diabetic model constructed,2 diabetic rats were randomly choosed and killed by over-dose anesthesia.The femeral bones were isolated and decalcified.HE stain was followed to show the bone density change and to verfy the diabetic model type.7.The bone defect was made on the diabetic rats femoral and then the rats were divided into different groups.The blood level of Ca²⁺,P,and ALP were recorded.The area of defect bone implanted with VEGF modified ADSCs was observed under microscope,histological methods and CD₃₁,CD₃₄stain.The microvessel density and electric scan microscop were alson applied in the part of reseach.Results1.ADSCs were obtained from adult rat fat tissues and cultured with the DMEM medium containedβ-glycerophosphate sodium and ascorbic acid. Individual cells grew adherent to the wall of the bottle at post-24h.There are very few cells grew adherent to the wall of bottle.At post-72h,assumed fiber-like cells, mainly in long streak shape and few in polygonal shape were seen.These cells showed a clone like growth.These results indicated that in 3 generation time the capability of ADSCs proliferation there are no significant difference(P＜0.05) compared with 6^thgeneration.It was decreased after 9^thgeneration.The amplified times of different generation is 1.61 day for 3^rdgeneration,1.73 day for 6^th generation and 2.25 day for 9^thgeneration by calculated cell growth curve.ADScs of the 3^rdgeneration were detected by immol/Lunofluorescence staining for antigen of stem cells.The results showed that these cells were positive for CD44, CD29,CD166,CD99 and CD105,but negative for CD34,CD31,CD45 and HLA-DR.2.ADSCs wih Ad-VEGF transferred were cultured and detected the VEGF concentration by ELISA assay in different time to confirm AV-VEGF transfected efficiency to ADSCs.MTT assay were used for ADSCs proliferation,conceptive radiommol/Lunoassays(RIA)were used for Osteocalcin,enzyme activity detection for ALP.The formation process of mineralization nodes were observed with microscope and calcified nodules stain was made,and Collagen I were analyzed by immol/Lunofluorescence technique.Finally,RT-PCR assay were used to detecte cbfal and osterix mRNA level in osteoblatic differentiation. Recombinant adenovirus with VEGF were produced by pADEasy-1 system had a high transferred efficiency to COS-7 cells and expressed the VEGF protein by Western Blot confirmation.ADSCs were transfected with 1×10¹⁰VP/ml of AV-VEGF and expressed VEGF positive cells was up to 86%.The concentration of VEGF in culture medium increased in post-transfected 24h than control and reach to peak at post-transfected 72h.However it still was detected VEGF level at 21 day after adenovirus transfected.3.High blood glucose and AGEs surpressed the ADSCs osteogensis ability as these two factors decreased the relative expression of CNI,ALP and OCN mRNA content(the mean decrease expression content was 20-30%of control group).In this suppression process,the suppression ability of AGEs was stronger than that of high glucose and the combination group(high blood glucose plus AGEs)had the the strongest suppression ability.High glucose and AGEs could both upgrade the ALP expression content.The mean upgrade rate is 2.7-3.3 times ast the control group.The calcification node was decreased under high glucose and AGEs conditions.4.VEGF gene could upgrade the mRNA expression content of CNI,ALP and OCN under high glucose and AGEs conditions.The upgrade rate of VEGF on 21^thday to CNI and OCN was 2.23 and 1.87 times as control group respectively. There was no significant statistic difference in ALP expression from VEGF, however the occurrence of mRNA expression was early than the other group. VEGF could enhance the osteogensis ability of ADSCs,as a result of this,the clacification nodes were increased as time went on. 5.We successfully constructed the diabetic rat models.The blood glucose was all above 16.7mmol/Lol/L.the rats all had diabetic syndroms and the weight was significantly decreased.HE stain proved that there were osteoprosis in the diabetic rat models.the recovery rate,motality rate,construction rate was 10.25%, 15.38%,74.4%respectively.6.The blood Ca²⁺level was lower than the control group in VEGF transinfected group on 1^stand 2^ndweek.The blood P level of VEGF group was higher than that in the control group.ALP level was also significantly increased in the VEGF group.The local vasculization effects aroud the VEGF modified ADSCs implant was significant.On 2^ndand 4^thweek,the CD31 and CD34 stain aroud the VEGF modified ADSCs implant were positive and were negative in the other groups.ConclusionRecombinant adenovirus with VEGF were produced by pADEasy-1 system had a high transferred efficiency to ADSCs cells and expressed the VEGF protein; Through the down-grade the mRNA expression of CNI,ALP and OCN,the high glucose and AGEs suppressed the osteogensis of ADSCs(the mean decrease expression content was 20-30%of control group).In this suppression process the AGEs suppression ability was stronger than that of high glucose and weaker than that of combination group(AGEs plus high glucose);The VEGF gene could enhance the osteogensis ability of ADSCs.The bone density and number of blood vessels were significantly increased aroud the VEGF modified ADSCs implant. There was noted intergrowth zone between VEGF modified ADSCs implant and self bone.VEGF modified ADSCs could be an effective way in repairing the bone defect with diabetic osteoprosis.