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Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells

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  • 1 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 2 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 3 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 4 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

Abstract

Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.

Sample Population—MSCs obtained from 5 young horses.

Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.

Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.

Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.

Abstract

Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.

Sample Population—MSCs obtained from 5 young horses.

Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.

Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.

Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.

Contributor Notes

Supported by the Veterinary Orthopedic Society through the Hohn-Johnson Research Award.

Address correspondence to Dr. Stewart.