University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Taipas, Guimarães, Portugal.
Tissue Eng Part A. 2012 Oct;18(19-20):1979-91. doi: 10.1089/ten.TEA.2012.0083. Epub 2012 Jun 25.
Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.
人关节软骨在滑液关节中承受广泛的机械负荷，静水压力（HP）是主要的作用力量。我们假设通过向在水凝胶中培养的软骨细胞或干细胞施加这种生理刺激，可以增强工程软骨的形成，使用定制设计的 HP 生物反应器。为了验证这一假设，我们通过人鼻软骨细胞（HNCs）或人脂肪干细胞（hASCs）包封在结冷胶（GG）水凝胶中，研究了不同 HP 方案对体外软骨形成的影响。为此，我们通过在 3 周的时间内对 HNC-GG 构建体施加脉动静压或稳定静压负荷，改变低 HP 的频率，并评估它们对软骨组织工程结果的影响。将 10×10(6)个细胞/mL 的 HNCs 包封在 GG 水凝胶（1.5%）中，并在三种方案下在软骨形成培养基中培养 3 周：（1）0.4 MPa 脉动 HP；（2）0.4 MPa 稳定 HP；和（3）静态。随后，我们将脉动方案应用于 hASC-GG 构建体，并通过产生低（0.4 MPa）和生理（5 MPa）HP 水平来改变加载幅度。将 10×10(6)个细胞/mL 的 hASCs 包封在 GG 水凝胶（1.5%）中，并在三种方案下在软骨形成培养基中培养 4 周：（1）0.4 MPa 脉动 HP；（2）5 MPa 脉动 HP；和（3）静态。在 HNC 研究中，脉动 HP 方案可获得最佳的组织发育，而在 hASC 研究中，生理负荷可获得更大的软骨分化和基质沉积，这表现在聚集蛋白聚糖、II 型胶原和 Sox-9 的基因表达；软骨细胞外基质的变色染色；和胶原蛋白的免疫定位。因此，我们提出，HNCs 和 hASCs 都可以检测和响应物理力，从而通过以频率和幅度依赖的方式增强软骨组织的发育，类似于关节加载。
