Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects

BackgroundHuman adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect.
Methodology/Principal FindingsHuman ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo.
Conclusions/SignificanceHuman ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

研究背景：人类脂肪来源基质细胞（human adipose-derived stromal cells, hASCs）是一类多能基质细胞，已被证实具备向成骨谱系分化的能力。这提示其可用于修复颅面或附肢骨骼的缺损。本研究旨在证实未分化的hASCs在修复难以愈合的小鼠骨骼缺损中的应用价值。
研究方法与主要结果：人类脂肪来源基质细胞取自女性脂肪抽吸物。研究人员于成年雄性裸鼠顶骨处构建临界尺寸（4 mm）的颅骨缺损模型。缺损分为三组：空白对照组（仅保留缺损）、仅接受羟基磷灰石涂层聚乳酸-羟基乙酸共聚物（poly(lactic-co-glycolic acid), PLGA）支架组，以及搭载人类脂肪来源基质细胞的PLGA支架组。于损伤后分层时间点开展MicroCT扫描，后续进行组织学检测、原位杂交及组织形态计量分析。结果显示，搭载hASCs的颅骨缺损模型实现了近乎完全的骨愈合；相较之下两组对照组仅出现极少量骨愈合（*P<0.01）。通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）及绿色荧光蛋白（green fluorescent protein, GFP）标记细胞的组织学共表达实验证实，移植后的hASCs可向成骨谱系分化。性染色体分析及荧光素酶（Luciferase）标记hASCs的定量实验显示，hASCs可在缺损部位存活两周。最后，重组骨形态发生蛋白2（recombinant bone morphogenetic protein 2, rBMP-2）可在体外增强hASCs的成骨分化能力，并在体内促进骨愈合。
结论与意义：hASCs无需预先诱导分化即可修复小鼠临界尺寸颅骨缺损。重组分化因子（如BMP-2）可用于辅助hASCs介导的骨修复。值得注意的是，hASCs会逐渐从颅骨缺损部位消失。本研究证实了hASCs应用于人类骨骼缺损治疗的转化潜力。