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Abstract

Objective—To determine the morphologic and phenotypic effects of transforming growth factor β1 (TGF-β1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes.

Sample Population—Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8- month-old horses.

Procedure—After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-β1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen.

Results—MSC cultures exposed to TGF-β1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-β1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF- β1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-β1 led to dose-related increases in area and intensity of type-II collagen immunoreaction.

Conclusion—Results suggest that TGF-β1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner. (Am J Vet Res 2000;61:1003–1010)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation.

Animals

2 adult horse bone marrow donors without skeletal or hematologic abnormalities.

Procedure

Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the remaining wells on day 12. Medium proteoglycan content was determined for all samples, and proteoglycan monomeric size was determined for pooled samples from days 2-6 and 8-12. Total nucleated cell numbers were determined at culture termination on days 6 and 12. Histologic, histochemical, and collagen immunohistochemical analyses of multiwell chamber slides harvested on day 6 or 12 were performed.

Results

Mesenchymal cells were an abundant cellular constituent of bone marrow aspirates, and separation of hematopoietic elements was achieved by centrifugation and delayed medium exchange. The remaining mesenchymal stem cells progressed from large, spindyloid, fibroblastic-appearing cells to a rounder shaped cell which formed colony plaques; isolated cells remained more spindyloid. Mesenchymal cell transformation toward a chondrocytic phenotype was verified by a shift in expression from collagen type I to type II, and an increase in quantity and molecular size of proteoglycans synthesized over time.

Conclusions

Mesenchymal stem cells obtained from adult horses have the capacity to undergo chondrogenic differentiation in monolayer cultures and may provide a locally recruitable or transplantable autogenous cell source for articular cartilage repair. (Am J Vet Res 1998;59:1182-1187).

Free access
in American Journal of Veterinary Research