Bone morphogenetic protein-2 gene-transfected human amniotic fluid-derived stem cells combined with β-tricalcium phosphate stents promote bone regeneration

Abstract

Objective To study the in vitro osteoblastic differentiation of human amniotic fluidderived stem cells (hAFSCs) transfected with bone morphogenetic protein-2 (BMP-2) gene,and to evaluate the in vivo bone formation ability of genetically modified hAFSCs when loaded onto porous β-tricalcium phosphate (β-TCP) stents.Methods hAFSCs were primarily cultured from mid-stream amniotic fluid collected after artificial rupture of amniotic sac.CD117/c-kit positive hAFSCs were obtained by using immunoselection with magnetic beads,and were then subjected to adenovirus-mediated transfection of BMP2 and enhanced green fluorescent protein (EGFP) (tranfection group),or EGFP alone (control group) or no transfection (non-transfection group).Forty-eight hours later,the hAFSCs were examined for EGFP expression and cell growth under fluorescent microscopy,and for rate of transfection by flow cytometry.At 7,14,21 and 28 days after transfection,ELISA was used to measure the level of BMP-2 in the cell culture of each group,so as to confirm the expression of target proteins.Osteogenesis ability was assessed with alkaline phosphatase (ALP) staining on day 14 and alizarin red staining on day 28.The activity of ALP was quantitatively assessed in all groups after 3,7,and 14 days of osteogenic induction.A total of 20 8-week-old nude mice were randomized into four groups (n=5 each) to receive femoral implantation of different complex of hAFSCs and β-TCP stents:Adv-hBMP-2-hAFSC-β-TCP,Adv-EGFP-hAFSC-β-TCP,hAFSC-β-TCP or β-TCP.Ectopic bone formation was evaluated and compared among these groups at week 8.Results The hAFSCs grew well.At 48 h after Adv-hBMP-2 or Adv-EGFP transfection,fluorescent microscopy showed that the hAFSCs appeared normally wall-adhering,shining with strong green fluorescence,with a positive rate of transfection being (89.00±4.25)％,and no cell death.The level of BMP-2 in the supernatant of hAFSCs culture was (3.405±0.229)μg/L on day 7,(4.575±0.179)μg/L on day 14,(3.910±0.175)μg/L on day 21,and (2.694±0.205)μg/L on day 28,respectively.The protein level of BMP-2 peaked on day 14,remained detectable on day 28,and was higher in the transfection group than those in the control group and nontransfection group at all these time points (all P＜0.05).On day 14 of osteoblastic induction,ALP staining showed that the patches of cross-linking hAFSCs in the transfection group,with larger area and number of ALP positive cells as compared with the other two groups; on day 28,alizarin red staining showed multicenter cell aggregates with massive formation of red calcium nodules in the transfection group.The number and size of these nodules were more prominent as compared with the other two groups.After osteoblastic induction,the three groups had increased expression of ALP compared with baseline.The level of ALP expression gradually increased in the transfection group,and was higher than those in the control group and non-transfection group at all time points (all P＜0.05).The level of ALP expression in the control group and non-transfection group was increased on days 3,7,and 14,and did not differ between the two groups at all time points.At 8 weeks after in vivo implantation,Adv-hBMP-2-hAFSC-β-TCP was demonstrated to result in more rapid degradation of the biomaterial,formation of more new bones and no fibrosis,as compared with the other three groups.In contrast,Adv-EGFP-hAFSC-β-TCP and hAFSC-β-TCP resulted in little formation of new bones with slower biomaterial degradation,and β-TCP resulted in little formation of new bones with no biomaterial degradation.Conclusion hAFSCs modified by the BMP-2 gene may be beneficial to the enhancement of bone regeneration. Key words: Bone morphogenetic proteins;  Amniotic fluid stem cells;  Genes;  Transfection; β-tricalcium phosphate stents ;  Bone regeneration