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通过双交联策略的 3D 生物打印生成富含脂肪干细胞的透明质酸支架用于软骨生成。

Generating adipose stem cell-laden hyaluronic acid-based scaffolds using 3D bioprinting via the double crosslinked strategy for chondrogenesis.

机构信息

Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan; Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.

Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; Ph.D Program in Life Sciences, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.

出版信息

Mater Sci Eng C Mater Biol Appl. 2021 May;124:112072. doi: 10.1016/j.msec.2021.112072. Epub 2021 Mar 26.

Abstract

Bioprinting of most cell-laden hydrogel scaffolds with the required structural integrity, mechanical modulus, cell adhesion, cell compatibility, and chondrogenic differentiation are still significant issues that affect the application of bioinks in cartilage tissue engineering. This study focuses on constructing printable bioinks by combining adipose-derived stem cells (ADSCs), hyaluronic acid (HA)-based hydrogels and analyzing their ability to induce chondrogenesis using 3D bioprinting technology. First, biotinylated hyaluronic acid was synthesized via an adipic acid dihydrazide (ADH) linker with amide bond formation to form HA-biotin (HAB). Both HAB and the as-received streptavidin were mixed to form a partially cross-linked HA-biotin-streptavidin (HBS) hydrogel through noncovalent bonding. After that, the partially cross-linked HBS hydrogel was mixed with sodium alginate and subsequently printed to form the HBSA hydrogel 3D scaffolds using a bioprinter. Finally, the 3D scaffolds of the HBSA (HBS + alginate) hydrogel were submerged into CaCl solution to achieve a stable 3D HBSAC (HBSA + Ca) hydrogel scaffold through ion transfer crosslinking. The physical-chemical characteristics of the hybrid bioink compositions have been evaluated to determine the desired 3D bioprinting structure. Cytotoxicity and chondrogenic differentiation were also assessed to confirm that the double cross-linked HBSAC hydrogel scaffold was useful for chondrogenic formation. The results showed that partially crosslinking the biotinylated HA-based hydrogel with streptavidin has a significant effect on printability and structural integrity. Morphological analysis of a suitable 3D printed HBSAC hydrogel scaffold showed visible pores with the desired shape and geometry. We have concluded that the HBSAC hydrogel possesses a favorable biocompatibility profile. The HBSAC hydrogel can also secrete significantly higher amounts of chondrogenic marker genes at day 5 and sulfated glycosaminoglycans (sGAGs) from days 7 to 14 compared to the HA hydrogel, as determined via quantitative real-time PCR assay and Alcian blue staining and the DMMB assay.

摘要

用具有所需结构完整性、机械模量、细胞黏附性、细胞相容性和软骨分化能力的大多数细胞负载水凝胶支架进行生物打印仍然是影响生物墨水在软骨组织工程中应用的重要问题。本研究通过将脂肪来源干细胞(ADSCs)、基于透明质酸(HA)的水凝胶结合在一起,并使用 3D 生物打印技术分析其诱导软骨生成的能力,来构建可打印的生物墨水。首先,通过己二酰肼(ADH)连接物与酰胺键形成将生物素化透明质酸合成,形成 HA-生物素(HAB)。将 HAB 和接收的链霉亲和素混合,通过非共价键形成部分交联的 HA-生物素-链霉亲和素(HBS)水凝胶。之后,将部分交联的 HBS 水凝胶与海藻酸钠混合,随后使用生物打印机打印形成 HBSA 水凝胶 3D 支架。最后,将 HBSA(HBS+海藻酸钠)水凝胶的 3D 支架浸入 CaCl2溶液中,通过离子转移交联形成稳定的 3D HBSAC(HBS+Ca)水凝胶支架。已经评估了混合生物墨水成分的物理化学特性,以确定所需的 3D 生物打印结构。还评估了细胞毒性和软骨分化,以确认双交联 HBSAC 水凝胶支架有利于软骨形成。结果表明,部分交联基于生物素化 HA 的水凝胶与链霉亲和素对可打印性和结构完整性有显著影响。适当的 3D 打印 HBSAC 水凝胶支架的形态分析显示出具有所需形状和几何形状的可见孔。我们得出结论,HBSAC 水凝胶具有良好的生物相容性。与 HA 水凝胶相比,HBSAC 水凝胶在第 5 天还可以显著分泌更多的软骨形成标记基因,并且在第 7 天至第 14 天分泌更多的硫酸化糖胺聚糖(sGAGs),通过定量实时 PCR 测定、阿利新蓝染色和 DMMB 测定来确定。

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