Takeda Kugo, Saito Hiroki, Shoji Shintaro, Sekiguchi Hiroyuki, Matsumoto Mitsuyoshi, Ujihira Masanobu, Miyagi Masayuki, Inoue Gen, Takaso Masashi, Uchida Kentaro
Department of Medical Engineering and Technology, Graduate School of Medical Sciences, Kitasato University, 1-15-1 Minami-ku, Kitasato, Sagamihara 252-0375, Kanagawa, Japan.
Department of Orthopaedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku, Kitasato, Sagamihara 252-0374, Kanagawa, Japan.
Bioengineering (Basel). 2024 Oct 18;11(10):1041. doi: 10.3390/bioengineering11101041.
This study aimed to evaluate the osteogenic potential of mesenchymal stromal cell (MSC) spheroids combined with the basic fibroblast growth factor (bFGF) in a mouse femur fracture model. To begin, MSC spheroids were generated, and the expression of key trophic factors (, and ) was assessed using quantitative PCR (qPCR). A binding assay confirmed the interaction between the bFGF and the spheroids' extracellular matrix. The spheroid cultures significantly upregulated , , and expression compared to the monolayers ( < 0.001), and the binding assay demonstrated effective bFGF binding to the MSC spheroids. Following these in vitro assessments, the mice were divided into five groups for the in vivo study: (1) no treatment (control), (2) spheroids alone, (3) bFGF alone, (4) bFGF-loaded spheroids (bFGF-spheroids), and (5) non-viable (frozen) bFGF-loaded spheroids (bFGF-dSpheroids). Bone formation was analyzed by a micro-CT, measuring the bone volume (BV) and bone mineral content (BMC) of the mice four weeks post-fracture. A high dose of the bFGF (10 µg) significantly promoted bone formation regardless of the presence of spheroids, as evidenced by the increases in BV (bFGF, = 0.010; bFGF-spheroids, = 0.006; bFGF-dSpheroids, = 0.032) and BMC (bFGF, = 0.023; bFGF-spheroids, = 0.004; bFGF-dSpheroids, = 0.014), compared to the controls. In contrast, a low dose of the bFGF (1 µg) combined with the MSC spheroids significantly increased BV and BMC compared to the control (BV, = 0.012; BMC, = 0.015), bFGF alone (BV, = 0.012; BMC, = 0.008), and spheroid (BV, < 0.001; BMC, < 0.001) groups. A low dose of the bFGF alone did not significantly promote bone formation ( > 0.05). The non-viable (frozen) spheroids loaded with a low dose of the bFGF resulted in a higher BV and BMC compared to the spheroids alone (BV, = 0.003; BMC, = 0.017), though the effect was less pronounced than in the viable spheroids. These findings demonstrate the synergistic effect of the bFGF and MSC spheroids on bone regeneration. The increased expression of the BMP-2 and VEGF observed in the initial experiments, coupled with the enhanced bone formation in vivo, highlight the therapeutic potential of this combination. Future studies will aim to elucidate the underlying molecular mechanisms and assess the long-term outcomes for bone repair strategies.
本研究旨在评估间充质基质细胞（MSC）球体与碱性成纤维细胞生长因子（bFGF）联合应用于小鼠股骨骨折模型中的成骨潜力。首先，生成MSC球体，并使用定量PCR（qPCR）评估关键营养因子（、和）的表达。结合试验证实了bFGF与球体细胞外基质之间的相互作用。与单层细胞相比，球体培养显著上调了、和的表达（<0.001），结合试验表明bFGF与MSC球体有效结合。在这些体外评估之后，将小鼠分为五组进行体内研究：（1）不治疗（对照组），（2）单独使用球体，（3）单独使用bFGF，（4）负载bFGF的球体（bFGF-球体），以及（5）无活性（冷冻）负载bFGF的球体（bFGF-d球体）。通过微型CT分析骨折后四周小鼠的骨形成情况，测量骨体积（BV）和骨矿物质含量（BMC）。高剂量的bFGF（10μg）无论是否存在球体均显著促进骨形成，与对照组相比，BV（bFGF组，=0.010；bFGF-球体组，=0.006；bFGF-d球体组，=0.032）和BMC（bFGF组，=0.023；bFGF-球体组，=0.004；bFGF-d球体组，=0.014）均增加。相比之下，低剂量的bFGF（1μg）与MSC球体联合应用与对照组（BV，=0.012；BMC，=0.015）、单独使用bFGF组（BV，=0.012；BMC，=0.008）和单独使用球体组（BV，<0.001；BMC，<0.001）相比，显著增加了BV和BMC。单独使用低剂量的bFGF未显著促进骨形成（>0.05）。与单独使用球体相比，负载低剂量bFGF的无活性（冷冻）球体导致更高的BV和BMC（BV，=0.003；BMC，=0.017），尽管效果不如活性球体明显。这些发现证明了bFGF与MSC球体在骨再生方面的协同作用。在初始实验中观察到的BMP-2和VEGF表达增加，以及体内增强的骨形成，突出了这种组合的治疗潜力。未来的研究旨在阐明潜在的分子机制，并评估骨修复策略的长期效果。
