Nokhbatolfoghahaei Hanieh, Paknejad Zahrasadat, Bohlouli Mahboubeh, Rezai Rad Maryam, Aminishakib Pouyan, Derakhshan Samira, Mohammadi Amirabad Leila, Nadjmi Nasser, Khojasteh Arash
Dental Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Tehran 1983969413, Iran.
Student Research Committee, Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran.
ACS Omega. 2020 Dec 2;5(49):31943-31956. doi: 10.1021/acsomega.0c04861. eCollection 2020 Dec 15.
Extracellular matrix (ECM)-contained grafts can be achieved by decellularization of native bones or synthetic scaffolds. Limitations associated with harvesting the native bone has raised interest in preparing in vitro ECM bioscaffold for bone tissue engineering. Here, we intend to develop an ECM-contained construct via decellularizing an engineered gelatin-coated β-tricalcium phosphate (gTCP) scaffold. In order to find an optimal protocol for decellularization of cell-loaded gTCP scaffolds, they were seeded with buccal fat pad-derived stem cells. Then, four decellularization protocols including sodium dodecyl sulfate, trypsin, Triton X-100, and combined solution methods were compared for the amounts of residual cells and remnant collagen and alteration of scaffold structure. Then, the efficacy of the selected protocol in removing cells from gTCP scaffolds incubated in a rotating and perfusion bioreactor for 24 days was evaluated and compared with static condition using histological analysis. Finally, decellularized scaffolds, reloaded with cells, and their cytotoxicity and osteoinductive capability were evaluated. Complete removal of cells from gTCP scaffolds was achieved from all protocols. However, treatment with Triton X-100 showed significantly higher amount of remnant ECM. Bioreactor-incubated scaffolds possessed greater magnitude of ECM proteins including collagen and glycosaminoglycans. Reseeding the decellularized scaffolds also represented higher osteoinductivity of bioreactor-based scaffolds. Application of Triton X-100 as decellularization protocol and usage of bioreactors are suggested as a suitable technique for designing ECM-contained grafts for bone tissue engineering.
含有细胞外基质（ECM）的移植物可以通过对天然骨或合成支架进行脱细胞处理来实现。与获取天然骨相关的局限性引发了人们对制备用于骨组织工程的体外ECM生物支架的兴趣。在此，我们打算通过对工程化明胶包被的β-磷酸三钙（gTCP）支架进行脱细胞处理来开发一种含有ECM的构建体。为了找到细胞负载的gTCP支架脱细胞的最佳方案，将颊脂垫来源的干细胞接种到这些支架上。然后，比较了包括十二烷基硫酸钠、胰蛋白酶、Triton X-100和联合溶液法在内的四种脱细胞方案在残留细胞量、残留胶原蛋白量以及支架结构改变方面的情况。接着，使用组织学分析评估并比较了所选方案在旋转和灌注生物反应器中孵育24天的gTCP支架上去除细胞的效果与静态条件下的效果。最后，对重新接种细胞的脱细胞支架及其细胞毒性和骨诱导能力进行了评估。所有方案都实现了从gTCP支架上完全去除细胞。然而，用Triton X-100处理显示残留ECM的量明显更高。生物反应器孵育的支架含有更多的ECM蛋白，包括胶原蛋白和糖胺聚糖。重新接种脱细胞支架也表明基于生物反应器的支架具有更高的骨诱导性。建议将Triton X-100用作脱细胞方案并使用生物反应器，作为设计用于骨组织工程的含有ECM的移植物的合适技术。
