Many skeletal tissue regenerative strategies centre around the multifunctional properties of bone marrow derived stromal cells (BMSC) or mesenchymal stem/stromal cells (MSC)/bone marrow derived skeletal stem cells (SSC). Specific identification of these particular stem cells has been inconclusive. However, enriching these heterogeneous bone marrow cell populations with characterised skeletal progenitor markers has been a contributing factor in successful skeletal bone regeneration and repair strategies. In the current studies we have isolated, characterised and enriched ovine bone marrow mesenchymal stromal cells (oBMSCs) using a specific antibody, Stro-4, examined their multipotential differentiation capacity and, in translational studies combined Stro-4+ oBMSCs with a bovine extracellular matrix (bECM) hydrogel and a biocompatible melt electro-written medical-grade polycaprolactone scaffold, and tested their bone regenerative capacity in a small in vivo, highly vascularised, chick chorioallantoic membrane (CAM) model and a preclinical, critical-sized ovine segmental tibial defect model. Proliferation rates and CFU-F formation were similar between unselected and Stro-4+ oBMSCs. Col1A1, Col2A1, mSOX-9, PPARG gene expression were upregulated in respective osteogenic, chondrogenic and adipogenic culture conditions compared to basal conditions with no significant difference between Stro-4+ and unselected oBMSCs. In contrast, proteoglycan expression, alkaline phosphatase activity and adipogenesis were significantly upregulated in the Stro-4+ cells. Furthermore, with extended cultures, the oBMSCs had a predisposition to maintain a strong chondrogenic phenotype. In the CAM model Stro-4+ oBMSCs/bECM hydrogel was able to induce bone formation at a femur fracture site compared to bECM hydrogel and control blank defect alone. Translational studies in a critical-sized ovine tibial defect showed autograft samples contained significantly more bone, (4250.63 mm3, SD = 1485.57) than blank (1045.29 mm3, SD = 219.68) ECM-hydrogel (1152.58 mm3, SD = 191.95) and Stro-4+/ECM-hydrogel (1127.95 mm3, SD = 166.44) groups. Stro-4+ oBMSCs demonstrated a potential to aid bone repair in vitro and in a small in vivo bone defect model using select scaffolds. However, critically, translation to a large related preclinical model demonstrated the complexities of bringing small scale reported stem-cell material therapies to a clinically relevant model and thus facilitate progression to the clinic.

中文翻译：

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使用牛细胞外基质水凝胶和新型生物相容性熔体电写医疗级聚己内酯支架对 Stro-4+ 富集骨髓间充质基质细胞的再生能力进行表征和评估。

许多骨骼组织再生策略以骨髓来源的基质细胞（BMSC）或间充质干/基质细胞（MSC）/骨髓来源的骨骼干细胞（SSC）的多功能特性为中心。这些特定干细胞的具体鉴定尚无定论。然而，用特征性骨骼祖细胞标记丰富这些异质骨髓细胞群一直是成功的骨骼骨再生和修复策略的一个促成因素。在目前的研究中，我们使用特异性抗体 Stro-4 分离、表征和富集了绵羊骨髓间充质基质细胞 (oBMSC)，检查了它们的多向分化能力，并在转化研究中将 Stro-4+ oBMSC 与牛细胞外基质相结合（bECM）水凝胶和生物相容性熔体电写医疗级聚己内酯支架，并在小型体内、高度血管化的雏鸡绒毛尿囊膜（CAM）模型和临床前、临界尺寸的绵羊节段性胫骨缺损中测试了它们的骨再生能力模型。未选择的 oBMSC 和 Stro-4+ oBMSC 之间的增殖率和 CFU-F 形成相似。与基础条件相比，Col1A1、Col2A1、mSOX-9、PPARG 基因表达在各自的成骨、软骨形成和脂肪形成培养条件下均上调，且 Stro-4+ 和未选择的 oBMSC 之间没有显着差异。相反，Stro-4+细胞中的蛋白多糖表达、碱性磷酸酶活性和脂肪生成显着上调。此外，通过扩展培养，oBMSC 具有维持强软骨形成表型的倾向。在 CAM 模型中，与单独的 bECM 水凝胶和对照空白缺损相比，Stro-4+ oBMSCs/bECM 水凝胶能够诱导股骨骨折部位的骨形成。对临界大小的绵羊胫骨缺损的转化研究表明，自体移植物样品含有明显更多的骨（4250.63 mm3，SD = 1485.57），比空白（1045.29 mm3，SD = 219.68）ECM-水凝胶（1152.58 mm3，SD = 191.95）和Stro- 4+/ECM-水凝胶（1127.95 mm3，SD = 166.44）组。Stro-4+ oBMSC 在体外和使用精选支架的小型体内骨缺损模型中显示出帮助骨修复的潜力。然而，至关重要的是，转化为大型相关临床前模型证明了将小规模报道的干细胞材料疗法引入临床相关模型并从而促进临床进展的复杂性。