Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts
Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts
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DOI：
10.1089/scd.2010.0250
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发表时间：
2011-02-01
影响因子：
4
通讯作者：
Longaker, Michael T.
中科院分区：
文献类型：
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作者：
Levi, Benjamin;James, Aaron W.;Longaker, Michael T.
人脂肪源性基质细胞（hASCs）具有经证实的使骨骼缺损骨化的能力。hASC刺激骨修复的机制尚未完全了解。在这项研究中，我们研究了hASCs刺激小鼠颅骨缺损的自体修复的潜力。免疫荧光、成骨染色和表面电子显微镜用于证明hASCs的成骨分化。使用骨传导支架将hASC移植到无胸腺小鼠的4 mm颅骨缺损中。分析包括微计算机断层扫描，组织学，原位杂交，定量实时聚合酶链反应。接下来，通过条件培养基和共培养测定评估hASC和小鼠颅骨成骨细胞（mOBs）之间的体外相互作用。培养基补充有Hedgehog信号传导调节剂，包括重组N-末端Sonic hedgehog、smoothened激动剂和环巴胺。最后，将环巴胺在体内递送至hASC移植的缺陷。与对照组（无处理或仅支架）相比，在hASC移植的缺损中观察到显著的颅骨愈合（*P < 0.05）。hASC显示刺激宿主小鼠成骨的证据，包括（1）通过原位杂交增加缺损边缘的骨标记物表达，和（2）通过种特异性定量实时聚合酶链反应增加宿主成骨基因表达。使用条件培养基或共培养测定，hASCs刺激mOB成骨分化，伴随着Hedgehog信号转导激活。N-末端Sonic hedgehog或smoothened激动剂复制，而环巴胺逆转，条件培养基对mOBs的促成骨作用。最后，环巴胺注射在体内抑制骨形成。hASC愈合临界大小的小鼠颅骨缺损，这至少部分是通过刺激宿主缺损的自体愈合。我们的研究表明，hASC衍生的Hedgehog信号可能在骨骼修复中发挥旁分泌作用。
Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P < 0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.