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Abstract

Objective—To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses.

Animals—14 healthy adult horses.

Procedure—The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis.

Results—Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease.

Conclusions and Clinical Relevance—Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated. (Am J Vet Res 2005;66:266–270)

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in American Journal of Veterinary Research

Abstract

Objective—To investigate effects of β-aminopropionitrile and a combination of insulin-like growth factor (IGF)-I and β-aminopropionitrile on metabolism of equine tendon fibroblasts.

Sample Population—Flexor tendon explants from 3 horses.

Procedure—Explants received 1 of 4 treatments (control, IGF-I, β-aminopropionitrile, and IGF-I/β-aminopropionitrile) for 10 days, and message expression for collagen types I and III was assessed by use of in situ hybridization. Histologic findings, new protein production, and quantitative determinations of glycosaminoglycan, DNA, and de novo collagen synthesis were made.

Results—Insulin-like growth factor-I stimulated an anabolic response in tendon. Collagen synthesis and glycosaminoglycan and DNA content of explants were all increased. β-Aminopropionitrile significantly suppressed collagen synthesis, which was not ameliorated by concurrent IGF-I treatment. β-Aminopropionitrile caused alterations in cell morphology characterized by large round cells with eccentric nuclei and decreased density of collagen fibers. Protein production and collagen type-III mRNA expression were reduced in these cells.

Conclusion and Clinical Relevance—Treatment with β-aminopropionitrile resulted in decreased production of protein and collagen synthesis, which could be expected to suppress tendon healing. The negative effects of β-aminopropionitrile could not be abrogated by addition of IGF-I to the medium. Treatment resulted in alterations in cell morphology and matrix consistency, which could further delay tendon healing. β-Aminopropionitrile may impair tendon healing at a cellular level by decreasing collagen production or increasing rate of degradation of existing matrix. Because of reduced crosslinking during β- aminopropionitrile treatment, in combination with transiently decreased tensile strength, alterations in collagen content and structure may weaken the healing tendon. (Am J Vet Res 2001;62:1557–1562)

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in American Journal of Veterinary Research

Abstract

Objective—To determine molecular changes in the expression of insulin-like growth factor-I (IGF-I) and transforming growth factor-β1 (TGF-β1) in horses with osteochondrosis, and to characterize expression of matrix aggrecan and collagen types I, II, and X in articular cartilage of affected joints.

Sample Population—Articular cartilage from affected stifle or shoulder joints of 11 horses with naturally acquired osteochondrosis and corresponding joints of 11 clinically normal horses.

Procedure—Harvested specimens were snap frozen in liquid nitrogen, and total RNA was isolated. Specimens were fixed in 4% paraformaldehyde for histologic examinations. Expression of matrix molecules was assessed by analysis of northern blots and in situ hybridization, using equine-specific cDNA probes and riboprobes, respectively. Expression of IGF-I and TGF-β1 was assessed by use of noncompetitive quantitative polymerase chain reaction, in situ hybridization, and immunohistochemical analysis.

Results—Cartilage obtained from osteochondrosis lesions had significantly greater expression of IGF-I, compared with normal cartilage. Expression of TGF- β1 and collagen type I were higher, but not significantly so, in affected tissues. Expression of aggrecan or collagen types II and X did not differ between affected and clinically normal cartilage.

Conclusions and Clinical Relevance—Increased expression of growth factors and collagen type I was found in cartilage from osteochondrosis lesions. However, this probably reflects a healing response to injured tissue rather than a primary alteration. Therefore, methods aimed at altering concentrations of growth factors in cartilage of growing horses would be unlikely to alter the incidence or progress of the disease. (Am J Vet Res 2001;62:1088–1094)

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in American Journal of Veterinary Research

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