• 1.

    Williams R, Harkins L, Hammond C, et al.Racehorse injuries, clinical problems and fatalities recorded on British racecourses from flat racing and national hunt racing during 1996, 1997 and 1998. Equine Vet J 2001;33:478486.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dyson SJ. Medical management of superficial digital flexor tendonitis: a comparative study of 219 horses (1992–2000). Equine Vet J 2004;36:415419.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ross MW. Superficial digital flexor tendonitis. In: Ross MW, Dyson SJ, eds. Diagnosis and management of lameness in the horse. Philadelphia: Saunders, 2003;628643.

    • Search Google Scholar
    • Export Citation
  • 4.

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

  • 5.

    Gillis C. Rehabilitation of tendon and ligament injuries. 43rd Annu Meet Am Assoc Equine Pract 1997;43:306309.

  • 6.

    Marr CM, Love S, Boyd JS, et al.Factors affecting the clinical outcome of injuries to the superficial digital flexor tendon in National Hunt and point-to-point racehorses. Vet Rec 1993;132:476479.

    • Search Google Scholar
    • Export Citation
  • 7.

    Spurlock SL. Treatment of acute superficial flexor tendon injuries in performance horses with high molecular weight sodium hyaluronate. J Equine Vet Sci 1999;19:338344.

    • Search Google Scholar
    • Export Citation
  • 8.

    Genovese RL. Sonographic response to intralesional therapy with beta-aminopropionitrile fumurate for clinical tendon injuries in horses. 38th Annu Meet Am Assoc Equine Pract 1992;38:265272.

    • Search Google Scholar
    • Export Citation
  • 9.

    Reef VB, Genovese RL, Davis WM. Initial long-term results of horses with superficial digital flexor tendonitis treated with intralesional beta-aminopropionitrile fumurate. 43rd Annu Meet Am Assoc Equine Pract 1997;43:301305.

    • Search Google Scholar
    • Export Citation
  • 10.

    Haupt JL, Donnelly BP, Nixon AJ. Effects of platelet-derived growth factor-BB on the metabolic function and morphologic features of equine tendon in explant culture. Am J Vet Res 2006;67:15951600.

    • Search Google Scholar
    • Export Citation
  • 11.

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

    • Search Google Scholar
    • Export Citation
  • 12.

    Schnabel LV, Mohammed HO, Miller BJ, et al.Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J Orthop Res 2007;25:230240.

    • Search Google Scholar
    • Export Citation
  • 13.

    Henninger RW, Bramlage LR, Bailey M, et al.Effects of tendon splitting on experimentally experimentally-induced acute equine tendinitis. Vet Comp Orthop Traumatol 1992;5:19.

    • Search Google Scholar
    • Export Citation
  • 14.

    Schnabel LV, Lynch ME, van der Meulen MC, et al.Mesenchymal stem cells and insulin-like growth factor–l gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009;10:13921398.

    • Search Google Scholar
    • Export Citation
  • 15.

    Nixon AJ, Sams AE, Ducharme NG. Endoscopically assisted annular ligament release in horses. Vet Surg 1993;22:501507.

  • 16.

    Hogan PM, Bramlage LR. Transection of the accessory ligament of the superficial digital flexor tendon for treatment of tendinits: long term results in 61 standardbred racehorses (1985–1992). Equine Vet J 1995;27:221226.

    • Search Google Scholar
    • Export Citation
  • 17.

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

    • Search Google Scholar
    • Export Citation
  • 18.

    Fortier LA. Stem cells: classifications, controversies, and clinical applications. Vet Surg 2005;34:415423.

  • 19.

    Smith RKW. Mesenchymal stem cell therapy for equine tendinopathy. Disabil Rehabil 2008;30:17521758.

  • 20.

    Young R, Butler D, Weber W, et al.Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. J Orthop Res 1998;16:406413.

    • Search Google Scholar
    • Export Citation
  • 21.

    Awad H, Butler D, Boivin G, et al.Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 1999;5:267277.

  • 22.

    Chong AK, Ang AD, Goh JC, et al.Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit Achilles tendon model. J Bone Joint Surg Am 2007;89:7481.

    • Search Google Scholar
    • Export Citation
  • 23.

    Zachos TA, Bertone AL. Growth factors and their potential therapeutic applications for healing of musculoskeletal and other connective tissues. Am J Vet Res 2005;66:727738.

    • Search Google Scholar
    • Export Citation
  • 24.

    Fortier LA, Smith RK. Regenerative medicine for tendinous and ligamentous injuries of sport horses. Vet Clin North Am Equine Pract 2008;24:191201.

    • Search Google Scholar
    • Export Citation
  • 25.

    Zachos TA, Shields KM, Bertone AL. Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 and -6. J Orthop Res 2006;24:12791291.

    • Search Google Scholar
    • Export Citation
  • 26.

    Chang H, Jiang W, Phillips FM, et al.Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 2003;85-A:15441552.

    • Search Google Scholar
    • Export Citation
  • 27.

    Ishihara A, Shields KM, Litsky AS, et al.Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models. J Orthop Res 2008;26:746771.

    • Search Google Scholar
    • Export Citation
  • 28.

    Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 1998;16:247252.

  • 29.

    Wang Q, Chen Z, Piao Y. Mesenchymal stem cells differentiate into tenocytes by bone morphogenetic protein (BMP) 12 gene transfer. J Biosci Bioeng 2005;100:418422.

    • Search Google Scholar
    • Export Citation
  • 30.

    Lou J, Tu Y, Ludwig FJ, et al.Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells. Clin Orthop Relat Res 1999;369:333339.

    • Search Google Scholar
    • Export Citation
  • 31.

    Seeherman HJ, Archambault JM, Rodeo SA, et al.rhBMP-12 accelerates healing of rotator cuff repairs in a sheep model. J Bone Joint Surg Am 2008;90:22062219.

    • Search Google Scholar
    • Export Citation
  • 32.

    Fu SC, Wong YP, Chan BP. The roles of bone-morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts. Life Sci 2003;72:29652974.

    • Search Google Scholar
    • Export Citation
  • 33.

    Majewski M, Betz O, Ochsner PE, et al.Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model. Gene Ther 2008;15:11391146.

    • Search Google Scholar
    • Export Citation
  • 34.

    Lou J, Tu Y, Burns M, et al.BMP-12 gene transfer augmentation of lacerated tendon repair. J Orthop Res 2001;19:11991202.

  • 35.

    Inada M, Katagiri T, Akiyama S. Bone morphogenetic protein-12 and -13 inhibit terminal differentiation of myoblasts, but do not induce their differentiation into osteoblasts. Biochem Biophys Res Commun 1996;222:317322.

    • Search Google Scholar
    • Export Citation
  • 36.

    Wolfman NM, Hattersly G, Cox K, et al.Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-B gene family. J Clin Invest 1997;100:321330.

    • Search Google Scholar
    • Export Citation
  • 37.

    Mikic B, Bierwert L, Tsou D. Achilles tendon characterization in GDF-7 deficient mice. J Orthop Res 2006;24:831841.

  • 38.

    Wikesjö UM, Sorensen RG, Kinoshita A, et al.Periodontal repair in dogs: effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar bone and periodontal attachment. J Clin Periodontol 2004;31:662670.

    • Search Google Scholar
    • Export Citation
  • 39.

    Hayaskhi K, Frank JD, Dubinsky C, et al.Histologic changes in ruptured canine cranial cruciate ligament. Vet Surg 2003;32:269277.

  • 40.

    Ishihara A, Zachos TA, Bartlett JS, et al.Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery. Am J Vet Res 2006;67:11451155.

    • Search Google Scholar
    • Export Citation
  • 41.

    Vidal MA, Kilroy GE, Johnson JR, et al.Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenesis capacity. Vet Surg 2006;35:601610.

    • Search Google Scholar
    • Export Citation
  • 42.

    Smith RKW, Korda M, Blunn GW, et al.Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet J 2003;35:99102.

    • Search Google Scholar
    • Export Citation
  • 43.

    Shaer BD, Orsini JA. Biopsy techniques. In: Orsini JA, Divers TJ, eds. Manual of equine emergencies: treatment and procedures. 3rd ed. Philadelphia: Saunders, 2008;2728.

    • Search Google Scholar
    • Export Citation
  • 44.

    Bonewald LF, Harris SE, Rosser J, et al.von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif Tissue Int 2003;72:537547.

    • Search Google Scholar
    • Export Citation
  • 45.

    Santangelo KS, Johnson AL, Ruppert AS, et al.Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblastlike synovial cells. Arthritis Res Ther 2007;9:R1R11.

    • Search Google Scholar
    • Export Citation
  • 46.

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

    • Search Google Scholar
    • Export Citation
  • 47.

    Gu W, Bertone AL. Generation and performance of an equinespecific large-scale gene expression microarray. Am J Vet Res 2004;65:16641673.

    • Search Google Scholar
    • Export Citation
  • 48.

    Marcus R, Peritz E, Gabriel K. On closed testing procedures with special reference to ordered analysis of variance. Biometrika 1976;63:655660.

    • Search Google Scholar
    • Export Citation
  • 49.

    Richardson LE, Dudhia J, Clegg PD, et al.Stem cells in veterinary medicine—attempts at regenerating equine tendon after injury. Trends Biotechnol 2007;25:409416.

    • Search Google Scholar
    • Export Citation
  • 50.

    Yoon JH, Halper J. Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 2005;5:2234.

  • 51.

    Yoon JH, Brooks R, Hwan Kim Y, et al.Proteoglycans in chicken gastrocnemius tendons change with exercise. Arch Biochem Biophys 2003;412:279286.

    • Search Google Scholar
    • Export Citation
  • 52.

    Jelinsky SA, Lake SP, Archambault JM, et al.Gene expression in rat supraspinatus tendon recovers from overuse with rest. Clin Orthop Relat Res 2008;466:16121617.

    • Search Google Scholar
    • Export Citation
  • 53.

    Archambault JM, Jelinksy SA, Lake SP, et al.Rat supraspinatus tendon expresses cartilage markers with overuse. J Orthop Res 2007;25:617624.

    • Search Google Scholar
    • Export Citation
  • 54.

    Smith RKW, Birch HL, Goodman S, et al.The influence of ageing and exercise on tendon growth and degeneration—hypothesis for the initiation and prevention of strain-induced tendinopathies. Comp Biochem Physiol A Mol Integr Physiol 2002;133:10391050.

    • Search Google Scholar
    • Export Citation
  • 55.

    Smith RKW, Gerard M, Dowling B, et al.Correlation of cartilage oligomeric protein (COMP) levels in equine tendon with mechanical properties: a proposed role for COMP in determining function-specific mechanical characteristics of locomotor tendons. Equine Vet J Suppl 2002;34:241244.

    • Search Google Scholar
    • Export Citation

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Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow–derived mesenchymal stem cells in vitro

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  • 1 Comparative Orthopedic Molecular Medicine and Applied Research Laboratory, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 2 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 3 Comparative Orthopedic Molecular Medicine and Applied Research Laboratory, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 4 Department of Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 5 Comparative Orthopedic Molecular Medicine and Applied Research Laboratory, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 6 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

Abstract

Objective—To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow–derived mesenchymal stem cells (BMDMSCs).

Animals—9 adult clinically normal horses.

Procedures—BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase–quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic profiling for effects of BMP12 was evaluated by use of an equine gene expression microarray on day 6.

Results—BMDMSCs and SDFTNs had high BMP12 gene expression and remained viable and healthy for at least 6 days. Type l collagen immunocytochemical staining for SDFTNs and tenocyte-like morphology for SDFTNs and BMDMSCs were greatest in BMP12 cells. Cartilage oligomeric matrix protein, as determined via RT-qPCR assay, and chondroitin sulfate, as determined via gene expression microarray analysis, were upregulated relative to control groups in SDFTN-BMP12 cells. The BMDMSCs and SDFTNs became mineralized with BMP2, but not BMP12. Superficial digital flexor tenocytes responded to BMP12 with upregulation of genes relevant to tendon healing and without mineralization as seen with BMP2.

Conclusions and Clinical Relevance—Targeted equine SDFTNs may respond to BMP12 with improved tenocyte morphology and without mineralization, as seen with BMP2. Bone marrow–derived mesenchymal stem cells may be able to serve as a cell delivery method for BMP12.

Abstract

Objective—To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow–derived mesenchymal stem cells (BMDMSCs).

Animals—9 adult clinically normal horses.

Procedures—BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase–quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic profiling for effects of BMP12 was evaluated by use of an equine gene expression microarray on day 6.

Results—BMDMSCs and SDFTNs had high BMP12 gene expression and remained viable and healthy for at least 6 days. Type l collagen immunocytochemical staining for SDFTNs and tenocyte-like morphology for SDFTNs and BMDMSCs were greatest in BMP12 cells. Cartilage oligomeric matrix protein, as determined via RT-qPCR assay, and chondroitin sulfate, as determined via gene expression microarray analysis, were upregulated relative to control groups in SDFTN-BMP12 cells. The BMDMSCs and SDFTNs became mineralized with BMP2, but not BMP12. Superficial digital flexor tenocytes responded to BMP12 with upregulation of genes relevant to tendon healing and without mineralization as seen with BMP2.

Conclusions and Clinical Relevance—Targeted equine SDFTNs may respond to BMP12 with improved tenocyte morphology and without mineralization, as seen with BMP2. Bone marrow–derived mesenchymal stem cells may be able to serve as a cell delivery method for BMP12.

Contributor Notes

This manuscript represents a portion of a thesis submitted by Dr. S. J. Murray to the Department of Veterinary Clinical Sciences, The Ohio State University, as partial fulfillment of the requirements for a Master of Science degree.

Supported by The Comparative Orthopedic Molecular Medicine and Applied Research Laboratory, Department of Veterinary Clinical Sciences, The Ohio State University. Drs. Santangelo and Bertone were supported by NIH grant numbers F32AR053805 and KO8AR4920101A2, respectively, from the National Institute of Arthritis and Muscoloskeletal and Skin Diseases. Dr. Santangelo is presently funded by a GlaxoSmithKline & ACVP/STP coalition Graduate Residency Fellowship.

Presented in abstract form at the 18th Annual Scientific Meeting of the American College of Veterinary Surgeons, San Diego, October 2008.

The authors thank D. Spencer Smith, Marc Hardman, and Amy Stark for technical and statistical assistance.

Address correspondence to Dr. Bertone (bertone.1@osu.edu).