Mao Beini, Zhang Zhong, Lai Sike, Zhang Kaibo, Li Jian, Fu Weili
Department of Orthopedics, Orthopedic Research Institute, West China Hospital, Sichuan University, Chengdu, China.
Department of Orthopaedics, No.3 People's Hospital of Chengdu, Chengdu, China.
Front Bioeng Biotechnol. 2022 Apr 27;10:855103. doi: 10.3389/fbioe.2022.855103. eCollection 2022.
Tissue engineering is a promising treatment strategy for meniscal regeneration after meniscal injury. However, existing scaffold materials and seed cells still have many disadvantages. The objective of the present study is to explore the feasibility of peripheral blood-derived mesenchymal stem cells (PBMSCs) augmented with demineralized cortical bone matrix (DCBM) pretreated with TGF-β3 as a tissue-engineered meniscus graft and the repair effect. PBMSCs were collected from rabbit peripheral blood and subjected to three-lineage differentiation and flow cytometry identification. DCBM was prepared by decalcification, decellularization, and cross-linking rabbit cortical bone. Various characteristics such as biomechanical properties, histological characteristics, microstructure and DNA content were characterized. The cytotoxicity and the effects of DCBM on the adhesion and migration of PBMSCs were evaluated separately. The meniscus-forming ability of PBMSCs/DCBM complex induced by TGF-β3 was also evaluated at the molecular and genetic levels, respectively. Eventually, the present study evaluated the repair effect and cartilage protection effect of PBMSCs/DCBM as a meniscal graft in a rabbit model of medial meniscal reconstruction in 3 and 6 months. The results showed PBMSCs positively express CD29 and CD44, negatively express CD34 and CD45, and have three-lineage differentiation ability, thus can be used as tissue engineering meniscus seed cells. After the sample procedure, the cell and DNA contents of DCBM decreased, the tensile modulus did not decrease significantly, and the DCBM had a pore structure and no obvious cytotoxicity. PBMSCs could adhere and grow on the scaffold. Under induction of TGF-β3, PBMSCs/DCBM composites expressed glycosaminoglycan (GAG), and the related gene expression also increased. The results of the experiments that the PBMSCs/DCBM group had a better repair effect than the DCBM group and the control group at both 12 and 24 weeks, and the protective effect on cartilage was also better. Therefore, the application of DCBM augmented with PBMSCs for meniscus injury treatment is a preferred option for tissue-engineered meniscus.
组织工程是半月板损伤后半月板再生的一种很有前景的治疗策略。然而，现有的支架材料和种子细胞仍存在许多缺点。本研究的目的是探讨用经转化生长因子-β3（TGF-β3）预处理的脱矿皮质骨基质（DCBM）增强的外周血间充质干细胞（PBMSCs）作为组织工程半月板移植物的可行性及修复效果。从兔外周血中采集PBMSCs，并进行三系分化及流式细胞术鉴定。通过对兔皮质骨进行脱钙、脱细胞和交联制备DCBM。对其生物力学性能、组织学特征、微观结构和DNA含量等各种特性进行表征。分别评估DCBM的细胞毒性及其对PBMSCs黏附与迁移的影响。还分别在分子和基因水平评估了TGF-β3诱导的PBMSCs/DCBM复合物的半月板形成能力。最终，本研究在兔内侧半月板重建模型中评估了PBMSCs/DCBM作为半月板移植物在3个月和6个月时的修复效果及软骨保护效果。结果显示，PBMSCs阳性表达CD29和CD44，阴性表达CD34和CD45，且具有三系分化能力，因此可用作组织工程半月板种子细胞。经过样本处理后，DCBM的细胞和DNA含量降低，拉伸模量无明显下降，且DCBM具有孔隙结构且无明显细胞毒性。PBMSCs可在支架上黏附生长。在TGF-β3诱导下，PBMSCs/DCBM复合材料表达糖胺聚糖（GAG），相关基因表达也增加。实验结果表明，在12周和24周时，PBMSCs/DCBM组的修复效果均优于DCBM组和对照组，对软骨的保护作用也更好。因此，应用PBMSCs增强的DCBM治疗半月板损伤是组织工程半月板的首选方案。
