脱细胞猪软骨微分散组织特异性3D基质的生物相容性和功能特性

E. A. Nemets, A. E. Lazhko, A. Grigoriev, Y. Basok, A. Kirillova, V. Sevastianov
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引用次数: 1

摘要

与在组织工程构建中用作组织特异性基质的软组织脱细胞相比,软骨组织的脱细胞需要几种处理技术,这可能会对天然细胞外基质(ECM)的生物相容性和功能特性产生负面影响。目的:研究猪软骨微分散组织特异性3D基质的生物相容性和功能特性,并通过化学、物理和酶技术进行脱细胞处理。材料和方法。为了进行脱细胞,通过低温研磨获得的微分散软骨颗粒(MCPs)在洗涤剂溶液(十二烷基硫酸钠和Triton X-100)中孵卵,然后用10%乙醇和dna酶i的超临界二氧化碳(scCO2)处理。采用Ames试验(鼠伤寒沙门菌反向突变试验)测定脱细胞微分散软骨颗粒(dMCPs)的遗传毒性。在性成熟的远交种大鼠体内研究了dMCPs的局部和全身毒性作用以及吸收。将脱细胞的MCP标本(10 mg)植入大腿肌肉组织。人脂肪来源的间充质干细胞/基质细胞(hAdMSCs)在dMCPs上培养时,通过荧光钙黄蛋白AM染色的活体显微镜分析其活力。使用PrestoBlue™细胞活力试剂评估细胞代谢活性。经实验证明,经scCO2处理后,将猪dMCPs植入大鼠肌肉,不表现出局部和全身毒性作用,也不表现出遗传毒性和对动物生殖系统的负面影响。经过6个月的体内实验,大部分(87%)移植的脱细胞软骨被吸收。结果表明,所得基质能够支持hAdMSCs的粘附和增殖。结论。猪dMCP标本在局部和一般毒性作用、遗传毒性和生殖毒性方面适用于生物相容性医疗产品,并且可以用作创建细胞和组织工程软骨构建的基质。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Biocompatible and functional properties of a microdispersed tissue-specific 3D matrix from decellularized porcine cartilage
In contrast to decellularization of soft tissues for use as tissue-specific matrices in the creation of tissue-engineered constructs, decellularization of cartilage tissue requires several processing techniques, which can negatively affect the biocompatibility and functional properties of the native extracellular matrix (ECM).Objective: to study the biocompatible and functional properties of microdispersed tissue-specific 3D matrix from a porcine cartilage that is decellularized by sequential use of chemical, physical and enzymatic techniques.Materials and methods. For decellularization, microdispersed cartilage particles (MCPs), obtained by cryomilling, were incubated in detergent solutions (sodium dodecyl sulfate and Triton X-100), then treated with supercritical carbon dioxide (scCO2) with 10% ethanol and DNase I. The Ames test (Salmonella typhimurium reverse mutation assay) was used to determine the genotoxicity of decellularized microdispersed cartilage particles (dMCPs). Local and general toxic effects, as well as resorption of dMCPs were studied in vivo on sexually mature outbred rats. Decellularized MCP specimens (10 mg) were implanted into the thigh muscle tissue. Viability of human adipose-derived mesenchymal stem/stromal cells (hAdMSCs), when cultured on dMCPs, was analyzed by in vivo microscopy, stained with fluorescent Calcein AM dye. Cell metabolic activity was assessed using PrestoBlue™ Cell Viability Reagent.Results. It has been proven that porcine dMCPs implanted in rat muscle after treatment with scCO2 do not exhibit local and general toxic effects, and do not show genotoxicity and negative effects on the reproductive system of animals. After 6 months of in vivo experiment, most (87%) of the implanted decellularized cartilage was resorbed. It was shown that the resulting matrices are able to support adhesion and proliferation of hAdMSCs. Conclusion. Porcine dMCP specimens are suitable for biocompatible medical products in terms of local and general toxic effects, genotoxicity and reproductive toxicity, and can be used as a matrix for creating cell- and tissue-engineered cartilage constructs.
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