Effect And Biological Properties Of DOAA In The Treatment Of Large-scale Osteoarticular Defects

Objective:Establish New Zealand white rabbits’models of proximal radius articulatio defect,prepare radius demineralized osteoarticular allograft（DOAA）,and explore its repair to New Zealand white rabbit radius osteoarticular defects and the biological properties of demineralized osteoarticular allograft And the effect on human bone marrow mesenchymal stem cells.Methods:Thirty New Zealand white rabbits were divided into an exclusion group,an autogenous bone graft group,and DOAA group to create large proximal radius osteoarticular defect models of New Zealand white rabbits.The postoperative signs and complications of experimental animals were observed at 4,8,12,and 16 weeks,and the repair of osteoarticular defects in experimental animals was evaluated by Lane-Sandhu X-ray score.The obtained proximal radius joints and limb bones were demineralized for 12h,24h,and 36h,and the proximal radius articulatio were subjected to infrared spectroscopy and X-ray diffraction analysis.The limbs long bones were cultured with saline and L-DMEM respectively The base is used as an extraction medium to prepare extracts A（demineralized 12h）,B（demineralized 24h）,C（demineralized 36h）and extract a（demineralized 12h）,b（demineralized 24h）,c（d emineralized 36h）.For local acute toxicity,physiological saline was used as the negative control group,25%alcohol was used as the positive control group,and the extracts A,B,and C were injected into the skin of 3 New Zealand white rabbits to evaluate the local effects of the demineralized bone extract on experimental animals.With physiological saline as the negative control group,PEI as the positive control group,and extracts A,B,and C,after adding the red blood cell suspension,the optical density value was measured at 560 nm with a microplate reader to calculate the hemolysis index and evaluate the same The hemolytic reaction of a kind of allogeneic demineralized bone extract to human blood.CCK-8 was used to detect the activity of demineralized 12h,24h,36h extracts on h BMSCs;and the scratch test was used to detect the effects of different demineralized bone extracts on h BMSCs.Results:At the 4th and 8th week,the healing of radius defects in each group was statistically significant（P<0.01）;at the 12th and 16th week,the healing of bone defects in the exclusion group,autogenous bone transplantation group and DOAA group were statistically significant（P<0.01）,but the healing of autogenous bone transplantation group and DOAA group was not statistically significant（P>0.05）.The results of flow cytometry showed that the expression rates of CD73,CD90,and CD105 were 99.96%,93.02%,and 97.69%respectively,and the expression rates of CD34,CD45,and HLA-DR were 0.1%,0.17%,and 0.04%respectively,which met the criteria of h BMSCs.ATR-FTIR analysis showed that the absorption peaks at1243cm^-1,1546cm^-1,and 1653cm^-1were N-H vibration absorption peak of collagen amide III,N-H vibration absorption peak of collagen amide II and C=O vibration absorption peak of collagen amide I.The absorption peaks at 1027cm^-1,and 3006cm^-1were PO₄^3-and OH^-of hydroxyapatite.The results of X-ray diffraction analysis showed that the X-ray diffraction peaks of different DOAA were diffuse wide peaks,indicating poor crystallinity.After 12h,24h,and 36h decalcification,the diffraction peaks of hydroxyapatite gradually became smaller and wider in the range of 32°to35°.The local acute toxicity of demineralized bone extract on experimental animals showed that the PII of injection A,B,C,and normal saline group was 0,which was very mild stimulation.The local primary stimulation index of 25%alcohol injectionsite was 6.3,which was a severe stimulation response.Hemolysis reaction showed that the hemolysis index of A,B and C were 2.29%,2.78%,and 2.40%,which were all less than 5%,meeting the requirements of national standards.CCK-8test showed that there was no statistical significance between the negative control group and extract group A（P>0.05）after the 5th day（i.e.logarithmic growth phase and plateau phase）,and there was no statistical significance between extract group B and extract group C（P>0.05）,but there was a significant difference between extract group B and extract group C and negative control group and extract group A（P<0.01）.Scratch test showed that there was no significant difference between the negative control group and extract group A（P>0.05）,and there was no significant difference between extract group B and extract group C（P>0.05）,but there was a significant difference between extract group B and extract group C and the negative control group and extract group A（P<0.05）.Conclusions:The demineralized osteoarticular allograft made by this method had a similar curative effect with autogenous bone transplantation in the treatment of large segmental osteoarticular defects of proximal radius in New Zealand white rabbits.It is a kind of scaffold material that mainly contains collagen,a small amount of calcium and phosphorus inorganic salt,and has the same function as autologous bone in recruiting h BMSCs to the injured site to repair the injured tissue;The results showed that DOAA with 24h and 36h decalcification time had better biological properties and could improve the activity and recruitment ability of h BMSCs.