采用改良多层 PCL-nHA-nZnO 静电纺丝支架在灌注式生物反应器中研究成骨和血管生成。

Investigation of osteogenesis and angiogenesis in perfusion bioreactors using improved multi-layer PCL-nHA-nZnO electrospun scaffolds.

机构信息

Biomedical Engineering Department, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

出版信息

Biotechnol Lett. 2023 Sep;45(9):1223-1243. doi: 10.1007/s10529-023-03411-w. Epub 2023 Jul 13.

DOI:10.1007/s10529-023-03411-w

PMID:37439932

Abstract

PURPOSE

Bone tissue engineering aims to create a three-dimensional, matured, angiogenic scaffold with a suitable thickness that resembles a natural bone matrix. On the other hand, electrospun fibers, which researchers have considered due to their good biomimetic properties, are considered 2D structures. Due to the highly interwoven network and small pore size, achieving the desired thickness for bone lesions has always been challenging. In bone tissue engineering, bioreactors are crucial for achieving initial tissue maturity and introducing certain signals as flow parameters for differentiation.

METHODS

In the present study, Human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured in a perfusion bioreactor on treated (improved pore size by gelatin sacrification and subsequent ultrasonication) 5-layer polycaprolactone-nano hydroxyapatite-nano zinc oxide (T-PHZ) scaffolds to investigate osteogenesis and angiogenesis simultaneously. The flow parameters and stresses on the cells were studied using two patterns of parallel and vertical scaffolds relative to the flow of the culture medium. In dynamic vertical flow (DVF), the culture medium flows perpendicular to the scaffolds, and in dynamic parallel flow (DPF), the culture medium flows parallel to the scaffolds. In all evaluations, static samples (S) served as the control group.

RESULTS

Live/dead, and MTT assays demonstrated the biocompatibility of the 5-layer scaffolds and the suitability of the bioreactor's functional conditions. ALP activity, EDAX analysis, and calcium content measurements exhibited greater osteogenesis for T-PHZ scaffolds in DVF conditions. Calcium content increased by a factor of 2.2, 1.8, and 1.6 during days 7 to 14 of culture under DVF, DPF and S conditions, respectively. After 21 days of co-culturing, an immunohistochemistry (IHC) test was performed to investigate angiogenesis and osteogenesis. Five antibodies were investigated in DVF, CD31, VEGFA, and VEGFR2 for angiogenesis, osteocalcin, and RUNX2 for osteogenesis. Compressive stress applied in DVF mode has increased osteogenic activity compared to DPF.

CONCLUSION

The results indicated the development of ideal systems for osteogenesis and angiogenesis on the treated multilayer electrospun scaffolds in the perfusion bioreactor.

摘要

目的

骨组织工程旨在创建具有合适厚度的三维成熟、血管生成支架，其类似于天然骨基质。另一方面，由于具有良好的仿生特性，研究人员已经考虑使用静电纺丝纤维，但它们被认为是 2D 结构。由于高度交织的网络和较小的孔径，为骨病变获得所需的厚度一直具有挑战性。在骨组织工程中，生物反应器对于实现组织初始成熟和引入某些信号（作为分化的流动参数）至关重要。

方法

在本研究中，人骨髓间充质干细胞（hBMSCs）和人脐静脉内皮细胞（HUVECs）在灌注生物反应器中于经处理的（通过明胶牺牲和随后的超声处理改善孔径）5 层聚己内酯-纳米羟基磷灰石-纳米氧化锌（T-PHZ）支架上共培养，以同时研究成骨和血管生成。使用平行和垂直支架相对于培养基流动的两种模式研究细胞的流动参数和应力。在动态垂直流动（DVF）中，培养基垂直于支架流动，而在动态平行流动（DPF）中，培养基平行于支架流动。在所有评估中，静态样本（S）作为对照组。

结果

活/死和 MTT 测定表明 5 层支架具有生物相容性，并且生物反应器的功能条件合适。碱性磷酸酶（ALP）活性、能谱分析（EDAX）和钙含量测量表明，在 DVF 条件下，T-PHZ 支架的成骨作用更好。在 DVF、DPF 和 S 条件下，培养第 7 天至第 14 天，钙含量分别增加了 2.2、1.8 和 1.6 倍。在共培养 21 天后，进行免疫组织化学（IHC）测试以研究血管生成和成骨作用。在 DVF 中研究了 5 种抗体，CD31、VEGFA 和 VEGFR2 用于血管生成，骨钙素和 RUNX2 用于成骨作用。与 DPF 相比，在 DVF 模式下施加的压缩应力增加了成骨活性。