Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo
Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo
复制标题
DOI：
10.1089/scd.2010.0082
复制
发表时间：
2011-03-01
影响因子：
4
通讯作者：
Longaker, Michael T.
中科院分区：
文献类型：
--
作者：
James, Aaron W.;Levi, Benjamin;Longaker, Michael T.
人脂肪源性基质细胞（hASCs）是一种多能性基质细胞，具有成骨分化的能力。然而，目前对hASC成骨的认识与其在骨骼组织再生中的潜在未来用途之间存在许多障碍。例如，冷冻储存对hASC成骨分化的影响尚未详细研究。为了检查冷冻储存的效果，从脂肪抽吸物收获hASC，并在标准培养条件下维持或在标准条件（90%胎牛血清，10%二甲基亚砜）下冷冻2周。接下来，进行细胞形态学（表面电子显微镜[EM]）、细胞活力和生长（台盼蓝;溴脱氧尿苷掺入）、成骨分化（碱性磷酸酶、茜素红和定量实时（RT）-聚合酶链反应）和成脂分化（油红O染色和定量RT-聚合酶链反应）的体外参数。最后，在无胸腺小鼠中使用临界尺寸的颅骨缺损评估体内骨形成，利用羟基磷灰石（HA）-聚（乳酸-共-乙醇酸）支架进行ASC递送。通过连续显微计算机断层扫描和组织学评估愈合情况。在检查的所有标记物中，新鲜来源的ASC与冻融ASC显着不同。表面电镜显示细胞形态有明显差异。体外冻融过程明显抑制了细胞增殖、成骨和成脂分化（*P < 0.01）。在体内，在用新鲜hASC移植的颅骨缺损中观察到接近完全愈合。这是与移植有冻融hASC的组相比的，冻融hASC显示出很少的愈合（*P < 0.01）。最后，观察到重组胰岛素样生长因子1或重组骨形态发生蛋白4增加或拯救冷冻hASCs的体外成骨分化（*P < 0.01）。冷冻储存ASC显著影响其体外和体内生物学。在体外和体内，ASC在冻融后成功经历成骨分化的能力基本上是沉默的。然而，重组蛋白的使用可用于减轻冻融过程的有害影响。
Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze-thaw process.