| Objective:Approximately 5-10% of all bone fractures are complicated by the development of a nonunion, which is difficult to cure in department of orthopedics. In primary 1990, Extracorporeal Shock Wave therapy (ESWT) was used successfully in fundamental and clinical research without combining stem cells, which leading to many positive achievements. In recent years,stem cell transplantation and tissue engineering in repairing bone defects is a hotspots of global study. Bone mesenchymal stem cells(BMSCs) possessing of self-replication, self-renewal, multi-differentiation and the potential to become osteoblast chondrocytes is an ideal seed cell in bone tissue engineering. This study will apply ESWT and autograft of BMSCs and use simple ESWT or BMSCs transplantation to cure rabbits suffered from bone nonuion, observe three different approachs to influence of bone nonuion healing. The research will explore an original treatment method to cure bone nonunion and provide some benefits for clinic.Methods: 39 New Zealand rabbits were confirmed by radiography to have a nonunion in both radius after 10 weeks of rabbits model established. All animals were divided into four groups randomally,which Group A was treated by ESWT combining with autograft of BMSCs,Group B by simple autograft of BMSCs ,Group C by ESWT and Group D by nothing. Under aseptic conditions,BMSCs were collected from the medial femoral of Group A and B's animals. Shock wave was applied in Group A and C while Group A and B received autograft of BMSCs to treat bone nonunion.Before treatment and at 2,6, and 10 weeks after treatment,radiographic follow-up of all rabbits were made. The scalar of bony callus, the gap width of the defect remaining on the radiographs were evaluated on computer and compared in four groups. Tetracycline was injected by intramuscular at 15,7 hours before killing for three days. Osteoblasts and bone trabecular in bone defect were valuated by hematoxylin and eosin staining. The assay of 5-bromo-2-deoxyuridine(BrdU) mark and vascular endothelial cell growth factor (VEGF) immunoreactivity was estimated through immunohistochemistry. New bone forming was evaluated by fluorescence microscope and bone mineralization apposition rate(MAR) was calculated by computer. All datas were expressed as mean±SD;p values of less than 0.05 were considered significant.Results:1BMSCs were harvested purely by density gradient centrifugation of Percoll, and maintained the activity of cells. BMSCs were adhered with round at 24 hours,and shaped with fusiform,clostridial form at 48-72 hours after separating from bone marrow.Primary BMSCs proliferated swiftly and 80～90% of cells could coalite at 10-17 days.The parental BMSCs adhered completely at 24 hours and about 90% of cells could only coalite at 4-5 days with more similar shape than Primary cells.2BMSCs were detected by cell surface molecule(CSM).Cell surface antigen expressed CD44(+) but CD34(-) indicated that we gained uniform surface character of BMSCs.3BMSCs labeled by BrdU occupied with more than 98% of all experimental cells and could be found at 6 weeks after treatment.We can confirm that transplanted BMSCs are involved in the formation of new bone.4At 2,6 weeks,BMSCs gathered and multiplied actively,bone trabecula formed abundantly by HE in Group A better than Group B and C.At 10 weeks, bone union could be found in Group A and part C.But in control group,no obvious bone forming was found all the time.5 The gap changes of bone nonunion was 5.78±0.98mm when animal models were confirmed by radiography. At 2 weeks after treatment, the gap changes of Group A,B,C and D were separately 6.83±0.65, 6.14±0.45, 6.61±0.68 and 5.79±0.68. Group A was statistically significant compared with Group B(P <0.05) and D (P <0.01). Group C was more statistical significant than Group B (P <0.05); At 6 weeks, the control group compared with the experimental group was significant (P <0.01) . The gap changes of Group A was 8.14±0.45,which was better than Group B and C (P <0.05); At 10 weeks, the gap changes of Group A was 9.86±0.26. Group B was 7.67±0.30,which was statistically significant compared with Group A and C(9.13±0.43mm) . The control group compared with the experimental group was significant (P <0.01) .The experimental groups compared with before are statistically significant. The scalar of bony callus in Groups at different time had a similar representation with the gap changes of bone nonunion.6 At 2,6 weeks after treatment,the expressions of VEGF in experimental groups were higher than the control(P <0.01),the amounts of Group A was separately 52.18±5.63,48.27±3.35,higher than Group B,C.At 10 weeks,the expressions of VEGF in Group A and C were similarly stable while Group B was slight. The amounts of VEGF in Group A,B and C was separately 49.40±4.53,30.00±1.14 and 38.69±4.61.The expressions of VEGF in Group A was higher than the other groups.Group C was higher than B7 Golden yellow fluorescence labelling zones were found at 2,6 weeks in Group A,B and C. The zone of bone mineralization was large and bone trabecula was seen clearly. At 10 weeks, golden yellow fluorescence labelling zones in Group A were remained higher than Group C,and Group B was light obviously. Golden yellow fluorescence labelling zones were no found in control group at 2,6 and 10 weeks.MAR in experimental groups was highest at 2 weeks after treatment,was separately 4.53±0.12,3.11±0.08,3.64±0.17um/d.The results of MAR were similar with VEGF.Conclusion: ESWT with autograft of BMSCs in the treatment of nonunion have have an advantage in reducing healing time,elevating the counts of VEGF and the scalar of bony callus,and promoting the heal of nonunion compared with simple ESWT or BMSCs autologous transplantation. |
