發(fā)布時(shí)間：2018-07-28 07:00

【摘要】：目的介紹一種可注射明膠原位水凝膠,探討其作為脫鈣骨基質(zhì)(demineralized bone matrix,DBM)粉末輸送載體的可行性。方法首先取明膠制備巰基化明膠,Ellman法檢測(cè)其巰基含量;然后將其與聚乙二醇雙丙烯酸酯交聯(lián)反應(yīng)制備可注射明膠原位水凝膠,采用倒置法檢測(cè)凝膠時(shí)間;最后將可注射明膠原位水凝膠與DBM粉末混合后制備復(fù)合物(以下簡(jiǎn)稱DBM-Gel)。將C2C12細(xì)胞包裹于DBM-Gel內(nèi),采用live/dead染色和Alamar blue法研究材料細(xì)胞毒性。將C2C12細(xì)胞黏附于DBM表面,制備包裹細(xì)胞的DBM-Gel(DBM-Gel組),培養(yǎng)1、3、5、7 d后檢測(cè)細(xì)胞ALP活性;以單純黏附細(xì)胞的DBM作為對(duì)照(DBM組)。采用裸鼠肌肉異位成骨模型進(jìn)行體內(nèi)骨誘導(dǎo)性觀察,于裸鼠腹部肌袋分別植入DBM-Gel(DBM-Gel組)以及DBM、PBS混合液(DBM組),4周后取材進(jìn)行組織學(xué)觀察以及ALP活性檢測(cè)。結(jié)果 Ellman法檢測(cè)制備的巰基化明膠其巰基含量為(0.51±0.03)mmol/g,可注射明膠原位水凝膠凝膠時(shí)間為(6±1)min。將DBM與巰基化明膠、PEGDA溶液混合后,在凝膠時(shí)間內(nèi)可以通過(guò)注射方式到達(dá)植入位點(diǎn)。隨著培養(yǎng)時(shí)間延長(zhǎng),DBM-Gel中細(xì)胞呈鋪展形態(tài),并且部分細(xì)胞之間有連接;培養(yǎng)1、3、7 d細(xì)胞成活率分別為95.4%±1.9%、97.3%±1.3%、96.1%±1.6%;培養(yǎng)1、3、5、7 d細(xì)胞相對(duì)增殖率分別為1.0±0.0、1.1±0.1、1.5±0.1、1.6±0.1。體外誘導(dǎo)培養(yǎng)1、3、5、7 d DBM-Gel組和DBM組細(xì)胞表現(xiàn)相似的ALP活性,組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);隨培養(yǎng)時(shí)間延長(zhǎng),兩組ALP活性均逐漸增加,5、7 d時(shí)ALP活性顯著高于1、3 d(P0.05),1、3 d間比較以及5、7 d間比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。體內(nèi)植入4周后,DBM-Gel組可見骨、軟骨形成,未觀察到骨髓形成;DBM組可見骨髓、骨、軟骨形成。DBM-Gel組、DBM組骨誘導(dǎo)性組織學(xué)評(píng)分分別為4.0、4.5分;ALP活性分別為(119.4±22.7)、(146.7±13.0)μmol/mg protein/min,組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(t= 2.085,P=0.082)。結(jié)論可注射明膠原位水凝膠作為DBM粉末輸送載體可行。
[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 鹵0.03) mmol / g by Ellman, and the in-situ hydrogel time was (6 鹵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% 鹵1.9% 鹵1.31% 鹵1.6.1%, respectively, and the relative proliferation rate of the cells was 1.0 鹵0.01.1 鹵0.11,1.5 鹵0.1 鹵1.6 鹵1.1 鹵0.1 鹵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 鹵22.7), (鹵13.0) 渭 mol/mg protein / min, respectively (119.4 鹵22.7), (鹵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.
【作者單位】： 四川大學(xué)華西醫(yī)院神經(jīng)外科;四川大學(xué)華西醫(yī)院神經(jīng)外科研究室;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(81401528)~~
【分類號(hào)】：R318.08

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