High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-β1 (TGF-β1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-β1 (pTGF-β1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-β1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

当培养基中添加生长因子（GF）等蛋白质时，高密度间充质干细胞（MSC）聚集体可以在体外通过软骨内骨化引导形成骨样组织，首先引导软骨模板的形成，然后进行肥大因子培养。最近的报告通过受控时空递送软骨转化生长因子-β1 (TGF-β1) 和软骨形成和成骨骨形态发生蛋白-2 (BMP-2) 从嵌入人 MSC 聚集体中的微粒来概括这些结果，以避免扩散限制和需要重复外源性补充，进行漫长且昂贵的体外培养。然而，由于 GF 的稳定性有限，因此局部基因递送是蛋白质使用的一种有前途的替代方案。在这里，能够局部递送编码TGF-β1（pTGF-β1）和BMP-2（pBMP-2）的Lipofectamine-质粒DNA（pDNA）纳米复合物的矿物包被的羟基磷灰石微粒（MCM）单独或组合地掺入，来自三个健康猪供体的 MSC 聚集体诱导这些转基因的持续产生。由于文献中记录的 MSC 供体与供体之间分化能力存在差异，因此本工作对三个供体群体进行了研究。供体 1 聚集体中 pBMP-2 的递送促进了第 2 周的软骨形成，随后在第 4 周增强了成骨表型。供体 2 和 3 聚集体在第 2 周没有促进强劲的糖胺聚糖 (GAG) 产生，但到了第 4 周，供体 2与对照相比，与 pTGF-β1/pBMP-2 的聚集体和与卸载的 MCM 和 pBMP-2 的供体 3 聚集体增强了成骨作用。 这些结果证明了通过细胞团内受控的非病毒基因传递促进干细胞聚集体中成骨的能力。这些发现还表明，在临床应用之前需要筛选供体间充质干细胞对基因转移的再生潜力。总而言之，这项工作展示了一种有前途的基因治疗方法，可以控制仿生 3D 凝结中的干细胞命运，从而治疗骨缺陷。