Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jun 26, 2021; 13(6): 645-658
Published online Jun 26, 2021. doi: 10.4252/wjsc.v13.i6.645
Chondrogenic potential of mesenchymal stem cells from horses using a magnetic 3D cell culture system
Joice Fülber, Fernanda R Agreste, Sarah R T Seidel, Eric D P Sotelo, Ângela P Barbosa, Yara M Michelacci, Raquel Y A Baccarin
Joice Fülber, Fernanda R Agreste, Sarah R T Seidel, Eric D P Sotelo, Ângela P Barbosa, Raquel Y A Baccarin, Departamento de Clínica Médica, Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo 05506-270, Brazil
Yara M Michelacci, Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil
Author contributions: Fülber J, Michelacci YM, and Baccarin RYA designed the study and wrote the manuscript; Baccarin RYA was responsible for obtaining funds; Fülber J, Agreste FR, Seidel SRT, Sotelo EDP, Barbosa AP collected tissue samples; Fülber J, Michelacci YM, and Baccarin RYA conducted the experimental analysis; Fülber J performed cell culture, chondrogenic differentiation of mesenchymal stem cells and all laboratory tests; all authors read and approved the final manuscript.
Supported by Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, No. 001.
Institutional review board statement: The study was reviewed and approved by Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of São Paulo (FMVZ/USP).
Institutional animal care and use committee statement: The present work was approved by the Committee for Ethics in Research of the University of São Paulo (CEUA/USP, 1143080617), and it was carried out in accordance with USP guidelines, ARRIVE guidelines, and the EC Directive 2010/63/EU for animal experiments (http://ec.europa.eu/environment/chemicals/Lab_animals/Legislation_en.htm).
Conflict-of-interest statement: Authors of this manuscript have no conflicts of interest to disclose.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Joice Fülber, PhD, Postdoc, Departamento de Clínica Médica, Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87 Cidade Universitária, São Paulo 05506-270, Brazil. jfulber@usp.br
Received: February 5, 2021
Peer-review started: February 5, 2021
First decision: March 17, 2021
Revised: March 29, 2021
Accepted: June 4, 2021
Article in press: June 4, 2021
Published online: June 26, 2021
Processing time: 140 Days and 15.2 Hours
Abstract
BACKGROUND

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.

AIM

To compare the chondrogenicity of MSCs derived from synovial fluid, bone marrow, and adipose tissue of horses, using the aggrecan synthesis.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

Keywords: Cellular therapy; Chondrocytes; Aggrecan; Keratan sulfate

Core Tip: As a method no yet studied in equine chondrocytes, chondrogenic differentiation was performed in three-dimensional plate culture, using technology with biocompatible nanoparticles consisting of gold, iron oxide and poly-L-lysine, forming microspheroids. It has been shown that this technique is advantageous, as it allows for aggregation and a lower number of cells, ensuring high cell density, providing an adequate microenvironment for the differentiation and phenotypic expression of chondrocytes.