Membrane culture and reduced oxygen tension enhances cartilage matrix formation from equine cord blood mesenchymal stromal cells in vitro.
Abstract: Ongoing research is aimed at increasing cartilage tissue yield and quality from multipotent mesenchymal stromal cells (MSC) for the purpose of treating cartilage damage in horses. Low oxygen culture has been shown to enhance chondrogenesis, and novel membrane culture has been proposed to increase tissue yield and homogeneity. The objective of this study was to evaluate and compare the effect of reduced oxygen and membrane culture during in vitro chondrogenesis of equine cord blood (CB) MSC. Methods: CB-MSC (n = 5 foals) were expanded at 21% oxygen prior to 3-week differentiation in membrane or pellet culture at 5% and 21% oxygen. Assessment included histological examination (H&E, toluidine Blue, immunohistochemistry (IHC) for collagen type I and II), protein quantification by hydroxyproline assay and dimethylmethylene assay, and mRNA analysis for collagen IA1, collagen IIA1, collagen XA1, HIF1α and Sox9. Results: Among treatment groups, 5% membrane culture produced neocartilage most closely resembling hyaline cartilage. Membrane culture resulted in increased wet mass, homogenous matrix morphology and an increase in total collagen content, while 5% oxygen culture resulted in higher GAG and type II collagen content. No significant differences were observed for mRNA analysis. Conclusions: Membrane culture at 5% oxygen produces a comparatively larger amount of higher quality neocartilage. Matrix homogeneity is attributed to a uniform diffusion gradient and reduced surface tension. Membrane culture holds promise for scale-up for therapeutic purposes, for cellular preconditioning prior to cytotherapeutic applications, and for modeling system for gas-dependent chondrogenic differentiation studies.
Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.
Publication Date: 2014-01-11 PubMed ID: 24418676DOI: 10.1016/j.joca.2013.12.021Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research studied the effects of membrane culture and reduced oxygen tension on the formation of cartilage tissue from horse cord blood stem cells in a lab setting. The findings suggest that the combination of these techniques produces a larger amount of high-quality cartilage tissue, which holds potential for therapies involving cartilage repair in horses.
Research Methodology
- The scientists obtained mesenchymal stromal cells (MSC), a type of adult stem cell, from the cord blood of five foals. This cell type has the ability to differentiate into various tissue types, including cartilage.
- These cells were expanded—meaning their numbers were increased—under conditions of 21% oxygen, which is the typical concentration of oxygen in air.
- After expansion, the cells were made to differentiate into cartilage either in a pellet or a novel membrane culture, and under conditions of 5% or 21% oxygen, for three weeks.
- The characteristics of the resulting cartilage were examined through techniques such as histology (examination of tissue under a microscope), immunohistochemistry (detection of proteins in cells), protein quantification, and analysis of specific messenger RNAs (products of gene expression).
Research Findings
- The researchers found that using membrane culture at 5% oxygen yielded cartilage most similar to hyaline cartilage, the most abundant type of cartilage in the body.
- This method also resulted in an increased wet mass, implying a larger amount of cartilage tissue, and a more homogeneous structure of the produced cartilage matrix, a supportive framework within the tissue.
- Additionally, the approach led to an increase in total collagen content, the main protein that makes up cartilage. Likewise, using a lower oxygen concentration resulted in higher levels of GAG, and type II collagen, key components of cartilage.
- However, no significant differences were seen in the production of mRNA for various genes involved in the production and structure of collagen.
Significance and Applications
- The use of membrane culture at a reduced oxygen tension seems to provide a viable approach to producing a larger amount of high-quality cartilage in the lab. This could potentially be used for transplantation into horses to repair damaged cartilage.
- Applying this method can lead to more homogeneous distribution of the constituents of the cartilage, attributed to a uniform diffusion of substances and reduced surface tension in membrane culture.
- Finally, the research also suggests that membrane culture has potential for scaling up production for therapeutic applications, for conditioning cells prior to applying other cell therapies, and for modeling gas-dependent differentiation of stem cells into cartilage for further studies.
Cite This Article
APA
Co C, Vickaryous MK, Koch TG.
(2014).
Membrane culture and reduced oxygen tension enhances cartilage matrix formation from equine cord blood mesenchymal stromal cells in vitro.
Osteoarthritis Cartilage, 22(3), 472-480.
https://doi.org/10.1016/j.joca.2013.12.021 Publication
Researcher Affiliations
- Department of Biomedical Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada. Electronic address: cco@uoguelph.ca.
- Department of Biomedical Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada. Electronic address: mvickary@uoguelph.ca.
- Department of Biomedical Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada; Department of Clinical Medicine, Orthopaedic Research Laboratory, Aarhus University, Aarhus, Denmark. Electronic address: tkoch@uoguelph.ca.
MeSH Terms
- Animals
- Biomarkers / metabolism
- Cartilage / growth & development
- Cell Culture Techniques / methods
- Chondrocytes / drug effects
- Chondrocytes / physiology
- Chondrogenesis / drug effects
- Chondrogenesis / physiology
- Fetal Blood / cytology
- Horses
- In Vitro Techniques
- Mesenchymal Stem Cells / cytology
- Oxygen / metabolism
Citations
This article has been cited 16 times.- López-Jiménez C, Chiu LLY, Waldman SD, Guilak F, Koch TG. TRPV4 activation enhances compressive properties and glycosaminoglycan deposition of equine neocartilage sheets. Osteoarthr Cartil Open 2022 Jun;4(2):100263.
- Dai W, Wu T, Leng X, Yan W, Hu X, Ao Y. Advances in biomechanical and biochemical engineering methods to stimulate meniscus tissue. Am J Transl Res 2021;13(8):8540-8560.
- Salcedo-Jiménez R, Koenig JB, Lee OJ, Gibson TWG, Madan P, Koch TG. Extracorporeal Shock Wave Therapy Enhances the In Vitro Metabolic Activity and Differentiation of Equine Umbilical Cord Blood Mesenchymal Stromal Cells. Front Vet Sci 2020;7:554306.
- Loibl M, Wuertz-Kozak K, Vadala G, Lang S, Fairbank J, Urban JP. Controversies in regenerative medicine: Should intervertebral disc degeneration be treated with mesenchymal stem cells?. JOR Spine 2019 Mar;2(1):e1043.
- Roberts EL, Dang T, Lepage SIM, Alizadeh AH, Walsh T, Koch TG, Kallos MS. Improved expansion of equine cord blood derived mesenchymal stromal cells by using microcarriers in stirred suspension bioreactors. J Biol Eng 2019;13:25.
- Barboni B, Russo V, Berardinelli P, Mauro A, Valbonetti L, Sanyal H, Canciello A, Greco L, Muttini A, Gatta V, Stuppia L, Mattioli M. Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance. Cell Transplant 2018 Jan;27(1):93-116.
- Desancé M, Contentin R, Bertoni L, Gomez-Leduc T, Branly T, Jacquet S, Betsch JM, Batho A, Legendre F, Audigié F, Galéra P, Demoor M. Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy. Int J Mol Sci 2018 Feb 10;19(2).
- Gómez-Leduc T, Desancé M, Hervieu M, Legendre F, Ollitrault D, de Vienne C, Herlicoviez M, Galéra P, Demoor M. Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges. Int J Mol Sci 2017 Sep 8;18(9).
- Ng J, Wei Y, Zhou B, Burapachaisri A, Guo E, Vunjak-Novakovic G. Extracellular matrix components and culture regimen selectively regulate cartilage formation by self-assembling human mesenchymal stem cells in vitro and in vivo. Stem Cell Res Ther 2016 Dec 9;7(1):183.
- Russell KA, Chow NH, Dukoff D, Gibson TW, LaMarre J, Betts DH, Koch TG. Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells. PLoS One 2016;11(12):e0167442.
- Gómez-Leduc T, Hervieu M, Legendre F, Bouyoucef M, Gruchy N, Poulain L, de Vienne C, Herlicoviez M, Demoor M, Galéra P. Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering. Sci Rep 2016 Sep 8;6:32786.
- Williams LB, Co C, Koenig JB, Tse C, Lindsay E, Koch TG. Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media. Front Vet Sci 2016;3:56.
- Williams LB, Russell KA, Koenig JB, Koch TG. Aspiration, but not injection, decreases cultured equine mesenchymal stromal cell viability. BMC Vet Res 2016 Mar 7;12:45.
- Russell KA, Gibson TW, Chong A, Co C, Koch TG. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells. PLoS One 2015;10(9):e0136621.
- Peng L, Shu X, Lang C, Yu X. Effects of hypoxia on proliferation of human cord blood-derived mesenchymal stem cells. Cytotechnology 2016 Aug;68(4):1615-22.
- Lach M, Trzeciak T, Richter M, Pawlicz J, Suchorska WM. Directed differentiation of induced pluripotent stem cells into chondrogenic lineages for articular cartilage treatment. J Tissue Eng 2014;5:2041731414552701.
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