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Home / Equine Research Bank / Tissue Eng Part A / A comparison of three-dimensional culture systems to evaluat...
Tissue engineering. Part A2013; 19(19-20); 2275-2283; doi: 10.1089/ten.TEA.2012.0479

A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells.

Abstract: To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). Results: alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×10(5) cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. Conclusions: equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.
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Publication Date: 2013-07-24 PubMed ID: 23725547PubMed Central: PMC3761431DOI: 10.1089/ten.TEA.2012.0479Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper focuses on comparing different in vitro three-dimensional (3D) culture systems used to model the development of cartilage (chondrogenesis) from horse bone marrow-derived mesenchymal stem cells (MSCs). The study suggests that equine MSCs exhibited chondrogenic transformation in certain 3D culture systems, with the fibrin alginate system potentially being superior for preventing terminal differentiation and hypertrophy.

Methods

  • The researchers collected MSCs from five horses aged between 2-3 years and consolidated these cells in five different three-dimensional (3D) mediums: fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose.
  • The cells in these mediums were maintained in a chondrogenic medium (a type of growth medium ideal for the growth and development of cartilage) that included supplemental TGF-β1 (a protein that regulates cell growth and differentiation) for a period of four weeks.
  • The cell pellets and growth mediums were tested at the 1st, 14th, and 28th day for gene expression of specific markers indicating chondrogenic maturation and hypertrophy, including ACAN, COL2B, COL10, SOX9, and 18S.
  • The cell samples were further evaluated through histology and immunohistochemistry techniques to examine the presence of collagen type II and X.

Results

  • Chondrogenic transformation (conversion to cartilage) was evident in the cells cultured in the alginate, fibrin alginate (FA), and both pellet culture systems. However, no adequate RNA was obtained from the cells cultured in agarose at any time point.
  • On the 14th day, increased expression of specific chondrogenic genes (COL2B, ACAN, and SOX9) was detected in both pellet culture systems.
  • On the last day of observation (Day 28), 5×10(5) cell pellets preserved the heightened expression of COL2B, and there was no discernible difference in the levels of ACAN and SOX9 genes between FA and the two pellet cultures.
  • Notably, the expression of the COL10 gene was considerably lower in the FA cultures by the 28th day, which indicates less occurrence of hypertrophy.
  • Collagen type II was abundantly formed in all culture systems except alginate, while the least amount of collagen type X was found in the FA hydrogels.

Conclusions

  • Equine MSCs responded positively to the three-dimensional culture in the FA blended hydrogel and both pellet culture systems, indicated by chondrogenic induction.
  • The study concludes that for the prevention of terminal differentiation and hypertrophy, FA culture might be superior to pellet culture systems. This implies that the fibrin alginate system may be more advantageous for controlling the differentiation process and maintaining the cells’ undifferentiated state.

Cite This Article

APA
Watts AE, Ackerman-Yost JC, Nixon AJ. (2013). A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells. Tissue Eng Part A, 19(19-20), 2275-2283. https://doi.org/10.1089/ten.TEA.2012.0479

Publication

Tissue engineering. Part A
ISSN: 1937-335X
NlmUniqueID: 101466659
Country: United States
Language: English
Volume: 19
Issue: 19-20
Pages: 2275-2283

Researcher Affiliations

Watts, Ashlee E
  • Comparative Orthopaedics Laboratory, Department of Clinical Sciences, Cornell University, Ithaca, NY 14853, USA.
Ackerman-Yost, Jeremy C
    Nixon, Alan J

      MeSH Terms

      • Animals
      • Bone Marrow Cells / cytology
      • Cell Differentiation / physiology
      • Chondrogenesis / genetics
      • Chondrogenesis / physiology
      • Horses
      • Mesenchymal Stem Cells / cytology

      Grant Funding

      • F32AR057299-01 / NIAMS NIH HHS

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