Introduction

The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects.

Methods

We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs.

Results

We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu.

Conclusion

We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.

中文翻译：

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I 型糖尿病通过影响血管生成延迟 3D 构建体的灌注和植入；这可以通过补充肝细胞生长因子来挽救。

介绍

基于细胞的再生疗法的最大瓶颈是缺乏支持移植物的功能性脉管系统。这个问题在糖尿病患者中更加严重，因为糖尿病患者的血管生长受到抑制。为了解决这个问题，我们的目标是确定糖尿病中 3D 工程组织中血管形成不良的原因并扭转其负面影响。

方法

我们使用了由微血管片段组成的 3D 血管化结构，这些微血管片段包含微循环中存在的所有细胞，嵌入 I 型胶原蛋白水凝胶中。构建体在体外培养或皮下植入非糖尿病或 I 型糖尿病（注射链脲佐菌素）小鼠模型中。我们使用 qPCR、ELISA、免疫染色、FAC 和共培养测定来表征糖尿病在工程构建体中的影响。

结果

我们在 3D 血管化结构中证明血管周围细胞分泌肝细胞生长因子 (HGF)，从而驱动微血管发芽。3D 构建体中 HGF 或 HGF 受体信号传导的阻断阻止了血管发芽。此外，体内3D 构建体中的 HGF 表达在糖尿病中下调；而 HGF 受体、VEGF 或 VEGF 受体表达无差异。糖尿病中的低 HGF 表达延迟了移植物和宿主血管的结合，减少了血液灌注并阻止了组织植入。在 3D 结构中补充 HGF，在糖尿病环境中恢复血管发芽。

结论

我们首次表明糖尿病会影响微血管中 HGF 的分泌，从而阻止工程组织的植入。HGF 的外源性补充可恢复 3D 结构中的血管生成生长，显示出在基于细胞的再生疗法中的应用前景。