Background

Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis.

Methods

The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 μm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior.

Results

Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 μm pores and vessels bridging across 250 μm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally.

Conclusion

The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths.

中文翻译：

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支架结构和基质应变以时间依赖性方式调节间充质细胞和微血管的生长和发育

 背景

由于依赖于良好形成的血管网络，体积组织工程结构的发展受到限制。支架孔径和基质的机械特性决定细胞附着、增殖和连续的组织形态发生。我们假设支架孔隙结构也控制形态发生过程中的基质-血管相互作用。

 方法

体外评估了接种在 450、340 和 250 μm 孔的羟基磷灰石支架上的间充质干细胞 (MSC) 与接种在 20 mg/mL 纤维蛋白水凝胶内的微血管碎片 (MVF) 之间的相互作用，微血管碎片 (MVF) 被浇注到细胞接种的支架中超过 21 天，并与不含支架但含有 MSC 和 MVF 的纤维蛋白水凝胶进行比较。对所有组进行了 mRNA 测序，并开发了计算力学模型来验证结构对预测血管化的影响，该血管化是由支架表面与孔内部相比更硬的基质行为驱动的。

 结果

脱钙支架的凝集素染色显示第 14 天时血管持续生长、分支和网络形成。纤维蛋白凝胶对展开的毛细血管网络的形成没有抵抗力，在 450 μm 的孔内有更大的血管环，并且血管桥接在 250 μm 的孔之间。观察到缺氧和连续的血管生成信号刺激了支架中的血管生长。纤维蛋白凝胶显示 VEGF 表达呈线性倍数增加，而 BMP2 没有变化。在支架内，相对于纤维蛋白，第 7 天到第 14 天之间 VEGF 增加了数倍，第 3 天到第 7 天之间 BMP2 早期增加了数倍。有证据表明支架组中存在基于 yap/taz 的河马信号传导和机械转导。通过计算模型确定的血管生长模型与实验观察到的趋势相匹配。

 结论

支架系统和机械传导传感基质力学之间缺氧信号传导的不同性质是成骨细胞和微血管生长差异的主要原因。计算模型暗示支架结构决定了孔内水凝胶的分支形态和应变，从而决定了血管长度。