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

发布时间：2018-07-28 07:00

【摘要】：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(P0.05);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(P0.05),1、3 d间比较以及5、7 d间比较,差异均无统计学意义(P0.05)。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。
[Abstract]:Objective to introduce an injectable gelatin in situ hydrogel and to investigate its feasibility as a powder carrier for decalcified bone matrix (demineralized bone matrix. Methods the sulfhydryl group of gelatin was prepared by Ellman method, then the injection gelatin in situ hydrogel was prepared by crosslinking the gelatin with polyethylene glycol bisacrylates, and the gel time was measured by inversion method. Finally, the injectable gelatin in situ hydrogel was mixed with DBM powder to prepare the composite (hereinafter referred to as DBM-Gel). C2C12 cells were encapsulated in DBM-Gel. Cytotoxicity of the material was studied by live/dead staining and Alamar blue method. C2C12 cells were attached to the surface of DBM to prepare DBM-Gel coated cells (DBM-Gel group). The ALP activity of the cells was detected after 5 days of culture and the DBM of the cells was used as control group (DBM group). Bone induction was observed in nude mice model of myoectopic osteogenesis. DBM-Gel (DBM-Gel group) and DBM group were implanted into abdominal muscle bag in nude mice for 4 weeks. Histological observation and ALP activity were performed. Results the sulfhydryl group content of the prepared gelatin was (0.51 卤0.03) mmol / g by Ellman, and the in-situ hydrogel time was (6 卤1) min. After mixing DBM with sulfhydrylated gelatin (PEGDA) solution, the implantation site could be reached by injection within the gel time. With the prolongation of culture time, the cells in DBM-Gel showed spreading morphology and some cells were connected with each other, the survival rate of the cells was 95.4% 卤1.9% 卤1.31% 卤1.6.1%, respectively, and the relative proliferation rate of the cells was 1.0 卤0.01.1 卤0.11,1.5 卤0.1 卤1.6 卤1.1 卤0.1 卤1.6. The activity of ALP in DBM-Gel group and DBM group was similar to that in DBM group on 7 days after induction in vitro (P0.05), but there was no significant difference between the two groups (P0.05), but with the increase of culture time, there was no significant difference between the two groups (P0.05). The activity of ALP in the two groups was significantly higher than that in 1D (P0.05) and 5d (P0.05), and there was no significant difference in the activity of ALP between the two groups at the 7th day (P0.05). Bone and cartilage were found in DBM-Gel group 4 weeks after implantation in vivo. Bone marrow and bone were not observed in DBM group. The osteoinducible histological score of the DBM-Gel group was (119.4 卤22.7), (卤13.0) 渭 mol/mg protein / min, respectively (119.4 卤22.7), (卤13.0) 渭 mol/mg / min, and there was no significant difference between the two groups (t = 2.085). Conclusion it is feasible to inject gelatin in situ hydrogel as DBM powder carrier.
【作者单位】：四川大学华西医院神经外科;四川大学华西医院神经外科研究室;
【基金】：国家自然科学基金资助项目(81401528)~~
【分类号】：R318.08

【相似文献】