Dept. Oral Cell Biology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Amsterdam, The Netherlands; Dept. Oral and Maxillofacial Surgery, Academic Centre for Dentistry Amsterdam/VU University Medical Center, MOVE Research Institute Amsterdam, Amsterdam, The Netherlands.
Biochimie. 2013 Dec;95(12):2304-13. doi: 10.1016/j.biochi.2013.08.034. Epub 2013 Sep 9.
The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) can be affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more gene products associated with processes such as angiogenesis and bone remodeling. Human ASCs were treated for 15 min with BMP-2 (10 ng/ml) to enhance osteogenic differentiation, or with vehicle. Subsequently, hASCs were seeded on plastic or on biphasic calcium phosphate (BCP) consisting of 60% hydroxyapatite and 40% β-tricalcium phosphate. A PCR array for ~150 trophic factors and differentiation-related genes was performed at day 21 of culture. A limited set of factors was quantified by qPCR at days 0, 4, 14 and 21, and/or ELISA at day 21. Compared to plastic, BCP-cultured hASCs showed ≥2-fold higher expression of ~20 factors, e.g. cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased ≥2-fold on BCP vs. plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2-treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. Substrate affected hASC secretion of Activin A and seemed to affect P1NP release. No clear association between hASC osteogenic differentiation and growth factor expression pattern was observed. Considering our observed lack of association between the degree of differentiation and the expression of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation.
干细胞的分泌组强烈决定组织工程策略的结果。我们研究了人脂肪干细胞（hASCs）的分泌组如何受到基质、BMP-2 处理和分化程度的影响。我们假设，随着分化的进展，hASCs 产生越来越多与血管生成和骨重塑等过程相关的基因产物。将 hASCs 用 BMP-2（10ng/ml）处理 15 分钟以增强成骨分化，或用载体处理。随后，将 hASCs 接种在塑料或由 60%羟基磷灰石和 40%β-磷酸三钙组成的双相磷酸钙（BCP）上。在培养的第 21 天进行了约 150 种营养因子和分化相关基因的 PCR 阵列。在第 0、4、14 和 21 天通过 qPCR 定量了有限数量的因子，并/或在第 21 天通过 ELISA 定量。与塑料相比，BCP 培养的 hASCs 表现出约 20 种因子的表达增加≥2 倍，例如细胞因子如 IL-6、生长因子如 FGF7 和粘附分子如 VCAM1。即使 hASCs 在 BCP 上的分化优于在塑料上，仍有约 50 个基因的表达降低≥2 倍。在 BCP 上接种的 hASCs 经 BMP-2 处理后，约 30 种因子的表达增加，而仅 PGF、PPARG 和 PTN 的表达减少。基质影响 hASC 分泌激活素 A，并似乎影响 P1NP 的释放。hASC 成骨分化与生长因子表达模式之间没有明显的关联。考虑到我们观察到 hASC 分泌的与血管生成和骨重塑相关的因子的表达与分化程度之间缺乏关联，未来的骨再生研究应更侧重于系统地协调干细胞的分泌组，而不仅仅是诱导干细胞的成骨分化。BMP-2 的短孵育可能是一种有前途的治疗方法，可增强成骨分化和环境调节。
