Sutherland Amanda J, Beck Emily C, Dennis S Connor, Converse Gabriel L, Hopkins Richard A, Berkland Cory J, Detamore Michael S
University of Kansas Medical Center, Kansas City, Kansas, United States of America.
Bioengineering Graduate Program, University of Kansas, Lawrence, Kansas, United States of America.
PLoS One. 2015 May 12;10(5):e0121966. doi: 10.1371/journal.pone.0121966. eCollection 2015.
Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.
基于细胞外基质(ECM)的材料在再生医学中具有吸引力,因为它们有潜力在不添加生物因子的情况下帮助干细胞募集、浸润和分化。在肌肉骨骼组织工程中,脱矿骨基质被广泛使用,但最近软骨基质作为一种潜在的软骨诱导材料受到了关注。因此,本研究的目的是建立一种用于关节软骨的化学脱细胞方法,以量化细胞的去除情况,并分析脱细胞过程中各个阶段软骨的生化成分,该过程包括一个物理灭活步骤。为了研究细胞对软骨基质的反应,将大鼠骨髓间充质干细胞(rBMSCs)培养在仅含细胞的细胞团(对照)、软骨分化培养基(TGF-β)、化学脱细胞软骨颗粒(DCC)或物理灭活软骨颗粒(DVC)中。化学脱细胞过程去除了基质中绝大多数的DNA和约一半的糖胺聚糖(GAG),但对羟脯氨酸的含量没有显著影响。最值得注意的是,在rBMSCs的软骨诱导方面,DCC组明显优于TGF-β组,其II型胶原基因表达高出一个数量级或更多。虽然DVC没有表现出与DCC相同程度的软骨形成反应,但DVC对I型胶原、X型胶原和Runx2有更大程度的下调。本研究引入了一种新的软骨灭活和脱细胞方案,并具有软骨诱导性的证据。这种生物活性以及提供再生软骨的“原材料”构建块,可能表明DCC在依赖募集内源性细胞进行软骨再生的生物材料中具有广阔前景。