Injectable Composites Of Calcium Sulfate And Hyaluronate Enhances Osteogenesis

Background:Bone defect is common in skeletal diseases such as trauma,infection,tumor and bone necrosis.Autografting is the golden standard for bone grafting materials,however the bone quantity is limited and related complications such as pain and nerve injury is not rare in harvesting of bone graft.Allografting is an alternative with copius supply but combined with the risk of transmission of infective agents or immunogenicity.Bone tissue engineering is fast developing with material field progress.The material available in clinic is still limited and calcium sulphate and calcium phosphate is among the few available choice.Injectable materials have advantage in treating deep and irregular bone defect,and can act as carrier of antibiotics and other agents.Objective:To fabricate an injectable material based on available product,with the addition of biopolymer to improve the osteogenetic capacity of the old material.The injectable bone substitute has the potential for treatment of bone defect,especially easily handable in minimally invasive surgery.Method:Physically mix calcium sulphate powder with hyaluronic acid solution(ARTZ 0.1%w/v,Mw 900kDa)in a 2g:1ml ratio to get a putty composite(CA+HA).Replace ARTZ with its cross-linked product(Gel-One 0.1%w/v)in the same ratio to get another composite(CA+HAC).Mixation of CaS04 with PBS solution in 2g:1ml to yields control group(CA).Make standard extracts using above 3 materials and the extract are diluted to get 25%,50%and 75%extract.In vitro test was done with mouse MC3T3-E1 cells.Biocompatability test done using CCK-8 test with standard extract and OD value was compared.Proliferative test done using CCK-8 test with each diluted exact and the concentration with best result was selected for osteogenic differentiation test with ELISA Kit.Iv vivo test was performed with Newzealand rabbit with femoral condylar bone defect model.X-ray,Micro-CT and tissue histological evaluation with H-E stain was performed at 6 and 12 weeks to evaluate bone formation.Results:Compared to CA material,the 2 composites(CA+HA,CA+HAC)are more injectable and stable in PBS solution.In biocompatibility test,the 2 composite materials show similar OD value with culture medium(p>0.05).In proliferative test,25%and 50%diluted extract of all 3 materials shows higher OD value at 6,12,24hr(p<0.05)than standard medium while using 75%and 100%extract,only CA+HA group is better than control group(p<0.05),the other 2 groups show similar OD value with standard medium(p>0.05).Differentiation test show that on day 21,ALP,COL-1 and OCN of CA+HA and CA+HAC groups are higher than CA and standard medium group(p<0.05).Micro-CT test shows higher BV/TV in 2 composites than CA group.Histology with H-E stain revealed more bone formation in 2 composite groups than the CA group at 6 and 12 weeks.Conclusion:Composites made with calcium sulfate and hyaluronate or with cross-linked hyaluronate are stable,injectable and biocompatible.The materials have excellent proliferative and differentiative effect on mouse MC3T3-E1 cells.They also show good bone formation capability in Newzealand rabbit bone defect model.The materials are potentiale bone substitutes for treating bone defective diseases.