复合透明质酸钠的硫酸钙可注射材料促进骨再生的实验研究

Effect of injectable composites of calcium sulfate and hyaluronate in enhancing osteogenesis

目的制备复合透明质酸钠的硫酸钙可注射材料,观察其促进骨再生作用,为骨缺损修复提供一种可注射材料。方法将硫酸钙分别与透明质酸钠溶液、交联透明质酸钠溶液、PBS溶液,按照2∶1(W/V)比例混合,制备3种复合材料(记作CA+HA、CA+HAC以及CA)。通过将3种复合材料浸泡于PBS溶液中观察其形变并进行X线衍射分析,观察材料稳定性。参照ISO10993-5制备3种复合材料浸提液,用于培养小鼠前成骨细胞(MC3T3-E1),并采用细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)方法检测材料的生物相容性和不同浓度浸提液促进细胞增殖的能力,以单纯培养基培养细胞作为对照组。采用成骨分化培养基制作促进细胞增殖的最佳浓度浸提液,用于培养MC3T3-E1细胞,ELISA法检测成骨分化相关蛋白ALP、Ⅰ型胶原(collagen typeⅠ,COL-Ⅰ)及骨钙蛋白(osteocalcin,OCN)浓度。取新西兰大白兔制作股骨髁骨缺损模型,分别植入CA+HA、CA+HAC以及CA材料,于6、12周取标本行Micro-CT扫描并计算骨组织占组织体积百分比(bone volume/tissue volume,BV/TV),然后标本切片行HE染色,观察缺损区新骨形成情况。结果复合材料稳定性检测示,CA+HA及CA+HAC具有良好可注射性,在PBS溶液中不易溃散,优于CA。生物相容性实验显示,培养6、12、24 h,CA组吸光度(A)值均低于对照组(P<0.05);CA+HA组、CA+HAC组A值与对照组比较,差异无统计学意义(P>0.05)。细胞增殖实验显示,25%、50%浓度浸提液培养5 d时CA组、CA+HA组和CA+HAC组A值均显著高于对照组(P<0.05);75%、100%浓度浸提液培养后,仅CA+HA组A值高于对照组(P<0.05)。选择50%浓度浸提液进行成骨分化实验。ELISA检测示,培养14、21 d时CA+HA组和CA+HAC组ALP、COL-Ⅰ、OCN浓度均显著高于对照组、CA组(P<0.05)。Micro-CT检查示,6、12周时CA+HA组、CA+HAC组间BV/TV差异无统计学意义(P>0.05),但均显著高于CA组(P<0.05);HE染色示,6周时CA组缺损处无成形骨组织,CA+HA组和CA+HAC组可见少量骨组织;12周时,CA组可见到少量细条索状骨组织形成,CA+HA组和CA+HAC组均见较多条索状骨组织,明显优于CA组,但两组间无显著差异。结论硫酸钙与透明质酸钠溶液及其交联产品按照2∶1(W/V)比例混合制备的复合材料具有稳定性和可注射性,体外能促进小鼠MC3T3-E1细胞增殖和分化,植入新西兰大白兔体内后具有良好成骨能力,有望作为一种可注射材料用于骨缺损微创治疗。

Objective To fabricate an injectable composite bone substitute with hyaluronic acid （HA） and calcium sulfate and to evaluate the biocompatibility and effect of the composite on cell proliferation, osteogenic differentiation in vitro and osteogenic capability in vivo. Methods Calcium sulfate powder was mixed with HA solution, cross-linked HA solution, and phosphate buffer solution （PBS） in a ratio of 2 ： 1 （W/V） to get composites of CA＋HA, CA＋HAC, and CA. The standard extracts from above 3 materials were prepared according to ISO10993-5, and were used to culture mouse MC3T3-E1 cells. The composite biocompatibility and cell proliferation in different concentrations of extract were tested with cell counting kit-8 （CCK-8）. The cells were cultured with standard medium as a control. The optimal concentration was selected for osteogenic differentiation test, and ELISA Kit was used to determine the alkaline phosphatase （ALP）, collagen type I （COL-I）, and osteocalcin （OCN）. The femoral condylar bone defect was made on New Zealand white rabbits and repaired with CA＋HA, CA＋HAC, and CA. Micro-CT was done to evaluate new bone formation with bone volume/tissue volume （BV/TV） ratio at 6 and 12 weeks. HE staining was used to observe bone formation. Results CA＋HA and CA＋HAC were better in injectability and stability in PBS than CA. The biocompatibility test showed that absorbance （A） value of CA group was significantly lower than that of control group （P〈0.05） at 6, 12, and 24 hours after culture, but no significant difference was found in A values between CA＋HA group or CA＋HAC group and control group （P〉0.05）. The proliferation test showed 25% and 50% extract of all 3 materials had significantly higher A value than control group （P〈0.05）. For 75% and 100% extract, only CA＋HA group had significantly higher A value than control group （P〈0.05）. And 50% extract was selected for osteogenic differentiation test. At 14 and 21 days, ALP, COL-I and OCN concentrations of CA＋HA group and CA＋HAC group were significantly higher than those of CA group and control group （P〈0.05）. Micro-CT results showed higher BV/TV in CA＋HA group and CA＋HAC group than CA group at 6 and 12 weeks （P〈0.05）, but no significant difference was found between CA＋HA group and CA＋HAC group （P〉0.05）. HE staining revealed that a little bone tissue was seen in CA＋HA group and CA＋HAC group, but there was no bone formation in CA group at 6 weeks; more streak bone tissue in CA＋HA group and CA＋HAC group than CA group at 12 weeks. Conclusion Composites prepared with calcium sulfate and HA or with cross-linked HA are stable, injectable, and biocompatible. The materials have excellent effect on proliferation and differentiation of mouse MC3T3-E1 cells. They also show good osteogenic capability in vivo. So it is a potential bone substitutes for bone defective diseases.