Comparison of equine tendon-, muscle-, and bone marrow–derived cells cultured on tendon matrix

Allison A. Stewart Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Jennifer G. Barrett Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Christopher R. Byron Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Angela C. Yates Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Sushmitha S. Durgam Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Richard B. Evans Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Matthew C. Stewart Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

Objective—To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix.

Sample Population—Cells from 4 young adult horses.

Procedures—Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP).

Results—Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marrow–derived cells (approx 3 to 4 weeks); there were fewer bone marrow–derived cells at confluence than the other 2 cell types. More tendon- and muscle-derived cells were attached to matrices after 7 days than were bone marrow–derived cells. Collagen and proteoglycan synthesis by tendon- and muscle-derived cells was significantly greater than synthesis by bone marrow–derived cells. On a per-cell basis, tendon-derived cells had more collagen synthesis, although this was not significant. Collagen type I mRNA expression was similar among groups. Tendon-derived cells expressed the highest amounts of collagen type III and COMP mRNAs, although the difference for COMP was not significant.

Conclusions and Clinical Relevance—Tendon- and muscle-derived cells yielded greater cell culture numbers in shorter time and, on a per-cell basis, had comparable biosynthetic assays to bone marrow–derived cells. More in vitro experiments with higher numbers may determine whether tendon-derived cells are a useful resource for tendon healing.

Abstract

Objective—To compare viability and biosynthetic capacities of cells isolated from equine tendon, muscle, and bone marrow grown on autogenous tendon matrix.

Sample Population—Cells from 4 young adult horses.

Procedures—Cells were isolated, expanded, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability, proteoglycan synthesis, collagen synthesis, and mRNA expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein (COMP).

Results—Tendon- and muscle-derived cells required less time to reach confluence (approx 2 weeks) than did bone marrow–derived cells (approx 3 to 4 weeks); there were fewer bone marrow–derived cells at confluence than the other 2 cell types. More tendon- and muscle-derived cells were attached to matrices after 7 days than were bone marrow–derived cells. Collagen and proteoglycan synthesis by tendon- and muscle-derived cells was significantly greater than synthesis by bone marrow–derived cells. On a per-cell basis, tendon-derived cells had more collagen synthesis, although this was not significant. Collagen type I mRNA expression was similar among groups. Tendon-derived cells expressed the highest amounts of collagen type III and COMP mRNAs, although the difference for COMP was not significant.

Conclusions and Clinical Relevance—Tendon- and muscle-derived cells yielded greater cell culture numbers in shorter time and, on a per-cell basis, had comparable biosynthetic assays to bone marrow–derived cells. More in vitro experiments with higher numbers may determine whether tendon-derived cells are a useful resource for tendon healing.

  • 1.

    Lam KH, Parkin TD, Riggs CM, et al. Descriptive analysis of retirement of Thoroughbred racehorses due to tendon injuries at the Hong Kong Jockey Club (1992–2004). Equine Vet J 2007;39:143148.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Goodship AE, Birch HL, Wilson AM. The pathobiology and repair of tendon and ligament injury. Vet Clin North Am Equine Pract 1994;10:323349.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Dowling BA, Dart AJ, Hodgson DR, et al. Superficial digital flexor tendonitis in the horse. Equine Vet J 2000;32:369378.

  • 4.

    Alves AG, Nicoletti JM, Thomassian A, et al. Tendon splitting as surgical treatment on experimental equine acute tendinitis. Arch Vet Sci 2002;7:4551.

    • Search Google Scholar
    • Export Citation
  • 5.

    Dahlgren LA, van der Meulen MC, Bertram JE, et al. Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis. J Orthop Res 2002;20:910919.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Chen YJ, Wang CJ, Yang KD, et al. Extracorporeal shock waves promote healing of collagenase-induced Achilles tendinitis and increase TGF-beta1 and IGF-I expression. J Orthop Res 2004;22:854861.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Archambault JM, Wiley JP, Bray RC. Exercise loading of tendons and the development of overuse injuries. A review of current literature. Sports Med 1995;20:7789.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Pufe T, Petersen WJ, Mentlein R, et al. The role of vasculature and angiogenesis for the pathogenesis of degenerative tendons disease. Scand J Med Sci Sports 2005;15:211222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Birch HL, Bailey AJ, Goodship AE. Macroscopic ‘degeneration’ of equine superficial digital flexor tendon is accompanied by a change in extracellular matrix composition. Equine Vet J 1998;30:534539.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Abrahamsson SO. Matrix metabolism and healing in the flexor tendon. Experimental studies on rabbit tendon. Scand J Plast Reconstr Surg Hand Surg Suppl 1991;23:151.

    • Search Google Scholar
    • Export Citation
  • 11.

    Banes AJ, Tsuzaki M, Hu P, et al. PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro. J Biomech 1995;28:15051513.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Dowling BA, Dart AJ, Hodgson DR, et al. Recombinant equine growth hormone does not affect the in vitro biomechanical properties of equine superficial digital flexor tendon. Vet Surg 2002;31:325330.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Butler DL, Juncosa-Melvin N, Boivin GP, et al. Functional tissue engineering for tendon repair: a multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation. J Orthop Res 2008;26:19.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Li HY, Zhou XF. Potential conversion of adult clavicle-derived chondrocytes into neural lineage cells in vitro. J Cell Physiol 2008;214:630644.

  • 15.

    Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 1994;56:283294.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Solchaga LA, Welter JF, Lennon DP, et al. Generation of pluripotent stem cells and their differentiation to the chondrocytic phenotype. Methods Mol Med 2004;100:5368.

    • Search Google Scholar
    • Export Citation
  • 17.

    Fraser JK, Schreiber RE, Zuk PA, et al. Adult stem cell therapy for the heart. Int J Biochem Cell Biol 2004;36:658666.

  • 18.

    Worster AA, Nixon AJ, Brower-Toland BD, et al. Effect of transforming growth factor B1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000;59:10031010.

    • Search Google Scholar
    • Export Citation
  • 19.

    McDuffee LA, Anderson GI. In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors. Vet Surg 2003;32:455463.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Yoshimura H, Muneta T, Nimura A, et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449462.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Izadpanah R, Trygg C, Patel B, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006;99:12851297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Kannus P. Structure of the tendon connective tissue. Scand J Med Sci Sports 2000;10:312320.

  • 23.

    Liu SH, Yang RS, al-Shaikh R, et al. Collagen in tendon, ligament, and bone healing. A current review. Clin Orthop Relat Res 1995;318:265278.

    • Search Google Scholar
    • Export Citation
  • 24.

    Halasz K, Kassner A, Morgelin M, et al. COMP acts as a catalyst in collagen fibrillogenesis. J Biol Chem 2007;282:3116631173.

  • 25.

    Liu X, Wu H, Byrne M, et al. Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development. Proc Natl Acad Sci U S A 1997;94:18521856.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Smith RK, Heinegard D. Cartilage oligomeric matrix protein (COMP) levels in digital sheath synovial fluid and serum with tendon injury. Equine Vet J 2000;32:5258.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Dahlgren LA, Brower-Toland BD, Nixon AJ. Cloning and expression of type III collagen in normal and injured tendons of horses. Am J Vet Res 2005;66:266270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Berglund M, Reno C, Hart DA, et al. Patterns of mRNA expression for matrix molecules and growth factors in flexor tendon injury: differences in the regulation between tendon and tendon sheath. J Hand Surg [Am] 2006;31:12791287.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Dahlgren LA, Mohammed HO, Nixon AJ. Temporal expression of growth factors and matrix molecules in healing tendon lesions. J Orthop Res 2005;23:8492.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Awad HA, Boivin GP, Dressler MR, et al. Repair of patellar tendon injuries using a cell-collagen composite. J Orthop Res 2003;21:420431.

  • 31.

    Guest DJ, Smith MR, Allen WR. Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study. Equine Vet J 2008;40:178181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Young RG, Butler DL, Weber W. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998;16:406413.

  • 33.

    Qu Z, Balkir L, van Deutekom JC, et al. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Biol 1998;142:12571267.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Jankowski RJ, Haluszczak C, Trucco M, et al. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther 2001;12:619628.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Fortier LA, Nixon AJ, Williams J, et al. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res 1998;59:11821187.

    • Search Google Scholar
    • Export Citation
  • 36.

    Mather JP, Robert PE. Introduction to cell and tissue culture. New York: Plenum Press, 1998;6768.

  • 37.

    Diegelmann RF. Analysis of collagen synthesis. Methods Mol Med 2003;78:349358.

  • 38.

    Masuda K, Shirota H, Thonar EJ. Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. Anal Biochem 1994;217:167175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Stewart MC, Saunders KM, Burton-Wurster N, et al. Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J Bone Miner Res 2000;15:166174.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001;25:402408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Ramsey FL, Schafer DW. A closer look at assumptions. In: Ramsey FL, Schafer DW, eds. The statistical sleuth: a course in methods of data analysis. Belmont, Calif: Duxbury Press, 1997;69.

    • Search Google Scholar
    • Export Citation
  • 42.

    Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:429435.

  • 43.

    Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med 2007;13:12191227.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Stewart AA, Byron CR, Pondenis H, et al. Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am J Vet Res 2007;68:941945.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Stewart AA, Byron CR, Pondenis HC, et al. Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells. Am J Vet Res 2008;69:10131021.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46.

    Giannini S, Buda R, Di Caprio F. Effects of freezing on the biomechnical and structural properties of human posterior tibial tendons. Int Orthop 2008;32:145151.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Clavert P, Kempf JF, Bonnomet F, et al. Effects of freezing/thawing on the biomechanical properties of human tendons. Surg Radiol Anat 2001;23:259262.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Salingcarnboriboon R, Yoshitake H, Tsuji K, et al. Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res 2003;287:289300.

    • Crossref
    • Search Google Scholar
    • Export Citation

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