Experimental Study On The Ectopia Osteogenesised By Small Intestinal Submucosa Combined With Bone Marrow Stem Cells In Vivo

Objective:To explore the appropriate mode of fabricating tissue engineered bone, the ectopia osteogenesis of the tissue engineered bone periosteum which construced by BMSCs combined with SIS in vivo was observed.Methods:One 2-month-old New Zealand white rabbit BMSCs was cultured.Parts of the 2nd BMSCs were induced for osteoblast.Spread to 3rd generation induced cells to carry on ALP dyeing and CD molecular identification.The 3rd induced and no-induced BMSCs was cultured with SIS,the marking is M2 and M1.The simple SIS was a control. After 3d and 5d,three membranes were observed by scanning electron microscope. Twenty-four rabbits(2 months old)were randomly divided into three groups,each with eight.10 days After cultured,M2,M1 and SIS were implanted into muscular pouch of thigh of rabbits respectively,as experimental group 1,experimental group 2 and control group(n=8).The effectiveness of bone formation was assessed separately by X-ray,gross observation,histology,ALP quantitative and immunohistochemistry of osteocalcin and collagen-Ⅰafter implantation for 4,8,12 weeks.Results:The cultured cell proliferation was active.Spreading to the third generation, cell membrane surface molecular identification showed more than 90 per cent purity of BMSCs.5 days after compound cultured,the SEM observation shows that BMSCs grow and attach very good on SIS.After operation,all animals had normal daily activities; without incision red swelling;no one died.After four and eight weeks surgery,X-ray, histology and immunohistochemistry began to show new bone formation in group M1 and M2,and group SIS was no new bone formation;at each time point,M2 Group's assessment was higher than that of group M1and SIS(p＜0.05).After 12 weeks,the bone formation of M1 and M2 groups are on the rise,more obvious characteristics.Masson stain showed the mean bone formation in group M2,M1,SIS was(25.08±0.79),14.21±0.68)and(10.62±0.56),and the mean ALP quantitation of samples was(10.26±0.38), 6.28±0.17)and(1.55±0.21).The group M2 was higher than group M1 or SIS(p＜0.05), the group M1 was higher than group SIS(p＜0.05).As time extension,the mean bone formation in group M2 or M1 was increased(p＜0.05),and that in group SIS did not change significantly(p＞0.05).Conclusions:The tissue engineered bone periosteum which constructed by SIS combined with BMSCs have abilities of ectopia osteogenesis in vivo.Moreover,bone formation of after induction was more visible,which indicates that it might be an excellent scaffold material in the bone tissue engineering.Rabbit muscle bag model is a better model of ectopia osteogenesis.