Fülber Joice, Agreste Fernanda R, Seidel Sarah R T, Sotelo Eric D P, Barbosa Ângela P, Michelacci Yara M, Baccarin Raquel Y A
Departamento de Clínica Médica, Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo 05506-270, Brazil.
Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil.
World J Stem Cells. 2021 Jun 26;13(6):645-658. doi: 10.4252/wjsc.v13.i6.645.
Mesenchymal stem cells (MSCs) represent a promising therapy for the treatment of equine joint diseases, studied due to their possible immunomodulatory characteristics and regenerative capacity. However, the source of most suitable MSCs for producing cartilage for regenerative processes in conjunction with biomaterials for an enhanced function is yet to be established.
To compare the chondrogenicity of MSCs derived from synovial fluid, bone marrow, and adipose tissue of horses, using the aggrecan synthesis.
MSCs from ten horses were cultured, phenotypic characterization was done with antibodies CD90, CD44 and CD34 and were differentiated into chondrocytes. The 3D cell culture system in which biocompatible nanoparticles consisting of gold, iron oxide, and poly-L-lysine were added to the cells, and they were forced by magnets to form one microspheroid. The microspheroids were exposed to a commercial culture medium for 4 d, 7 d, 14 d, and 21 d. Proteoglycan extraction was performed, and aggrecan was quantified by enzyme-linked immunosorbent assay. Keratan sulfate and aggrecan in the microspheroids were identified and localized by immunofluorescence.
All cultured cells showed fibroblast-like appearance, the ability to adhere to the plastic surface, and were positive for CD44 and CD90, thus confirming the characteristics and morphology of MSCs. The soluble protein concentrations were higher in the microspheroids derived from adipose tissue. The aggrecan concentration and the ratio of aggrecan to soluble proteins were higher in microspheroids derived from synovial fluid than in those derived from bone marrow, thereby showing chondrogenic superiority. Microspheroids from all sources expressed aggrecan and keratan sulfate when observed using confocal immunofluorescence microscopy. All sources of MSCs can synthesize aggrecan, however, MSCs from synovial fluid and adipose tissue have demonstrated better biocompatibility in a 3D environment, thus suggesting chondrogenic superiority.
All sources of MSCs produce hyaline cartilage; however, the use of synovial liquid or adipose tissue should be recommended when it is intended for use with biomaterials or scaffolds.
间充质干细胞（MSCs）是治疗马关节疾病的一种有前景的疗法，因其可能具有的免疫调节特性和再生能力而受到研究。然而，与生物材料结合用于再生过程以增强功能的最合适的用于产生软骨的间充质干细胞来源尚未确定。
通过聚集蛋白聚糖合成来比较源自马的滑液、骨髓和脂肪组织的间充质干细胞的软骨形成能力。
培养来自十匹马的间充质干细胞，用抗体CD90、CD44和CD34进行表型鉴定，并将其分化为软骨细胞。在三维细胞培养系统中，将由金、氧化铁和聚-L-赖氨酸组成的生物相容性纳米颗粒添加到细胞中，并用磁铁迫使它们形成一个微球。将微球暴露于商业培养基中4天、7天、14天和21天。进行蛋白聚糖提取，并通过酶联免疫吸附测定法定量聚集蛋白聚糖。通过免疫荧光鉴定并定位微球中的硫酸角质素和聚集蛋白聚糖。
所有培养的细胞均呈现成纤维细胞样外观，具有粘附于塑料表面的能力，并且CD44和CD90呈阳性，从而证实了间充质干细胞的特征和形态。源自脂肪组织的微球中可溶性蛋白浓度更高。源自滑液的微球中聚集蛋白聚糖浓度以及聚集蛋白聚糖与可溶性蛋白的比率高于源自骨髓的微球，从而显示出软骨形成优势。当使用共聚焦免疫荧光显微镜观察时，所有来源的微球均表达聚集蛋白聚糖和硫酸角质素。所有来源的间充质干细胞均可合成聚集蛋白聚糖，然而，滑液和脂肪组织来源的间充质干细胞在三维环境中表现出更好的生物相容性，因此表明具有软骨形成优势。
所有来源的间充质干细胞均可产生透明软骨；然而，当打算与生物材料或支架一起使用时，建议使用滑液或脂肪组织。
