Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo.

Statement of significance

Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.

中文翻译：

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间充质干细胞来源的细胞外基质通过体外和体内封装的软骨细胞增强软骨的表型和软骨形成。

间充质干细胞衍生的细胞外基质（MSC-ECM）是一种天然生物材料，具有强大的生物活性和良好的生物相容性，并已被研究用作组织工程的支架。在这项研究中，我们测试了使用脱细胞的人骨髓来源的MSC-ECM（hBMSC-ECM）作为体外软骨细胞扩增的培养基质的适用性，以及用于基于软骨细胞的软骨修复的支架。收获由在组织培养塑料（TCP）上培养的hBMSC沉积的hBMSC-ECM，然后进行脱细胞过程以去除hBMSC。与在TCP上生长的软骨细胞相比，接种在hBMSC-ECM上的软骨细胞显示出显着增加的增殖速率，并且保持了比TCP组更好的软骨细胞表型。在扩展到相同的细胞数量并置于高密度微团培养物中后，来自ECM组的软骨细胞显示出比TCP组更好的软骨分化特性。为了测试软骨的形成能力，将经过软骨细胞浸渍的hBMSC-ECM的复合材料进行简短的胰蛋白酶处理，以实现细胞介导的收缩，并折叠形成3D软骨细胞浸润的hBMSC-ECM（Cell / ECM构建体）。在文化上体外在软骨形成培养基中培养21天，在Cell / ECM构建体中观察到了牢固的软骨形成。通过皮下植入SCID小鼠体内测试了类似制备的Cell / ECM构建体。植入后14天，在植入的Cell / ECM构建体中观察到明显的软骨形成，与由软骨细胞片组成的对照组相比，sGAG沉积更高。综上所述，这些发现表明hBMSC-ECM是软骨细胞扩增的优良培养底物和潜在可用于体内软骨再生的生物活性基质。

重要声明

当前针对局灶性软骨缺损的基于细胞的治疗面临挑战，包括软骨细胞去分化，异种支架的需求以及软骨形成欠佳。我们在这里提出一种新技术，利用成人干细胞衍生的细胞外基质，作为人类成人软骨细胞的培养基质和/或封装支架，修复软骨缺损。在干细胞来源的基质中培养的软骨细胞在体外培养扩增后显示出更高的增殖能力，更好的软骨细胞表型和更高的再分化能力。最重要的是，由折叠在干细胞基质中的软骨细胞形成的三维构建体在体外和体内均表现出优异的软骨形成。这些发现证明了干细胞来源的细胞外基质适合作为软骨细胞扩增的培养底物以及软骨再生的候选生物活性基质。