可注射明胶原位水凝胶作为脱钙骨基质粉末输送载体可行性的初步研究

Feasibility of an injectable and in situ gelling gelatin hydrogel for demineralized bone matrix powder delivery

目的介绍一种可注射明胶原位水凝胶,探讨其作为脱钙骨基质(demineralized bone matrix,DBM)粉末输送载体的可行性。方法首先取明胶制备巯基化明胶,Ellman法检测其巯基含量;然后将其与聚乙二醇双丙烯酸酯交联反应制备可注射明胶原位水凝胶,采用倒置法检测凝胶时间;最后将可注射明胶原位水凝胶与DBM粉末混合后制备复合物(以下简称DBM-Gel)。将C2C12细胞包裹于DBM-Gel内,采用live/dead染色和Alamar blue法研究材料细胞毒性。将C2C12细胞黏附于DBM表面,制备包裹细胞的DBM-Gel(DBM-Gel组),培养1、3、5、7 d后检测细胞ALP活性;以单纯黏附细胞的DBM作为对照(DBM组)。采用裸鼠肌肉异位成骨模型进行体内骨诱导性观察,于裸鼠腹部肌袋分别植入DBM-Gel(DBM-Gel组)以及DBM、PBS混合液(DBM组),4周后取材进行组织学观察以及ALP活性检测。结果 Ellman法检测制备的巯基化明胶其巯基含量为(0.51±0.03)mmol/g,可注射明胶原位水凝胶凝胶时间为(6±1)min。将DBM与巯基化明胶、PEGDA溶液混合后,在凝胶时间内可以通过注射方式到达植入位点。随着培养时间延长,DBM-Gel中细胞呈铺展形态,并且部分细胞之间有连接;培养1、3、7 d细胞成活率分别为95.4%±1.9%、97.3%±1.3%、96.1%±1.6%;培养1、3、5、7 d细胞相对增殖率分别为1.0±0.0、1.1±0.1、1.5±0.1、1.6±0.1。体外诱导培养1、3、5、7 d DBM-Gel组和DBM组细胞表现相似的ALP活性,组间比较差异无统计学意义(P>0.05);随培养时间延长,两组ALP活性均逐渐增加,5、7 d时ALP活性显著高于1、3 d(P<0.05),1、3 d间比较以及5、7 d间比较,差异均无统计学意义(P>0.05)。体内植入4周后,DBM-Gel组可见骨、软骨形成,未观察到骨髓形成;DBM组可见骨髓、骨、软骨形成。DBM-Gel组、DBM组骨诱导性组织学评分分别为4.0、4.5分;ALP活性分别为(119.4±22.7)、(146.7±13.0)μmol/mg protein/min,组间比较差异无统计学意义(t=–2.085,P=0.082)。结论可注射明胶原位水凝胶作为DBM粉末输送载体可行。

Objective To introduce an injectable and in situ gelling gelatin hydrogel, and to explore the possibility as a carrier for demineralized bone matrix （DBM） powder delivery. Methods First, thiolated gelatin was prepared and the thiol content was determined by Ellman method, and then the injectable and in situ gelling gelatin hydrogel （Gel） was formed by crosslinking of the thiolated gelatin and poly （ethylene oxide） diacrylate and the gelation time was determined by inverted method. Finally, the DBM-Gel composite was prepared by mixing Gel and DBM powder. The cytotoxicity was tested by live/dead staining and Alamar blue assay of the encapsulated cells in the DBM-Gel. For in vitro cell induction, C2C12 cells were firstly incubated onto the surface of the DBM and then the composite was prepared. The experiment included two groups： DBM-Gel and DBM. The alkaline phosphatase （ALP） activity was determined at 1, 3, 5,and 7 days after culture. In vivo osteoinductivity was evaluated using ectopic bone formation model of nude rats. Histological observation and the ALP activity was measured in DBM-Gel and DBM groups at 4 weeks after implantation. Results The thiol content in the thiolated gelatin was （0.51±0.03） mmol/g determined by Ellman method. The gelation time of the hydrogel was （6±1） minutes. DBM powder can be mixed with the hydrogel and injected into the implantation site within the gelation time. The cells in the DBM-Gel exhibited spreading morphology and connected each other in part with increasing culture time. The viability of the cells was 95.4%±1.9%, 97.3%±1.3%, and 96.1%±1.6% at 1, 3, and 7 days after culture, respectively. The relative proliferation was 1.0±0.0, 1.1±0.1, 1.5±0.1, and 1.6±0.1 at 1, 3, 5, and 7 days after culture respectively. In vitro induction showed that the ALP activity of the DBM-Gel group was similar to that of the DBM group, showing no significant difference （P〉0.05）. With increasing culture time, the ALP activities in both groups increased gradually and the activity at 5 and 7 days was significantly higher than that at 1 and 3 days （P〈0.05）, while there was no significant difference between at 1 and 3 days, and between 5 and 7 days （P〉0.05）. At 4 weeks after implantation in vivo, new bone and cartilage were observed, but no bone marrow formation in DBM-Gel group; in DBM group, new bone, new cartilage, and bone marrow formation were observed. The histological osteoinduction scores of DBM-Gel and DBM groups were 4.0 and 4.5, respectively. The ALP activities of DBM-Gel and DBM groups were respectively （119.4±22.7） and （146.7±13.0）μmol/mg protein/min, showing no significant difference （t=-2.085, P=0.082）. Conclusion The injectable and in situ gelling gelatin hydrogel for delivery of DBM is feasible.