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  • Author or Editor: Lisa A. Fortier x
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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

Abstract

Objective

To compare chondrocyte proliferation and metabolism in three-dimensional fibrin cultures formed from polymerized autogenous fibrinogen with that of commercially manufactured fractionated fibrinogen.

Animals

Fibrinogen and chondrocytes for in vitro experimentation derived from 2 horses, ages 12 and 14 months, donated for reasons unrelated to skeletal or hematologic abnormalities.

Procedure

Fibrinogen was isolated from whole blood, using plasma cryoprecipitation and centrifugation, and fractionated fibrinogen was purchased. Each was mixed with 10 × 106 chondrocytes/0.5 ml of fibrinogen, and was polymerized by addition of 0.5 ml of calcium-activated thrombin. Thirty 1-ml fibrin-chondrocyte disks were formed from each fibrinogen source and cultured for 0 (n = 6), 7 (n = 12), or 14 (n = 12) days. Chondrocyte metabolism and cell proliferation in each fibrin type were objectively assessed by assays for total proteoglycan content, [35S]proteoglycan accumulation, proteoglycan monomer size, and total DNA. Cell morphology and cartilage-specific cell function was evaluated by routine histologic, alcian blue histochemical, type-II collagen immunohistochemical, and type-II collagen in situ hybridization methods.

Results

Histologic examination indicated better retention of chondrocyte morphology in autogenous composites. Autogenous fibrinogen also stimulated greater chondrocyte proliferation (DNA content increased 1.4-fold on day 14) and supported higher proteoglycan accumulation (increased 1.4-fold on day 14), compared with commercial, fractionated fibrinogen. Abundant intracellular type-II procollagen mRNA was detected in autogenous fibrin cultures by in situ hybridization, and translation was confirmed by extensive pericellular type-II collagen accumulation.

Conclusions

Autogenous fibrinogen has an inherent capacity to maintain chondrocyte phenotypic metabolism that is reduced or absent in commercially prepared fibrinogen. Enhanced, differentiated cell function may be useful for in vivo applications, but represents an added variable that may confound in vitro experiments, and should be considered when designing studies of chondrocyte function. (Am J Vet Res 1998;59:514–520)

Free access
in American Journal of Veterinary Research