Cell and Tissue Research

, Volume 355, Issue 2, pp 327–335 | Cite as

Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold

  • Laurie A. McDuffee
  • Blanca P. Esparza Gonzalez
  • Rodolfo Nino-Fong
  • Enrique Aburto
Regular Article


Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6–12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2–5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.


Mesenchymal stem cells Bone marrow Osteogenic differentiation Fibrin glue Equine Nude mice 



The authors thank Dr. Glenda Wright for support and highly valuable comments on the manuscript and Dr. Jonathan Spears (Atlantic Veterinary College, UPEI) for assistance with the laboratory animals.


  1. Auer JA, Stick JA (2012) Equine surgery, 4th edn. Elsevier/Saunders, St. LouisGoogle Scholar
  2. Aughey E, Frye FL, Johnston H, Ebrary I (2001) Comparative veterinary histology with clinical correlates. Manson/Veterinary Press, LondonCrossRefGoogle Scholar
  3. Bacha WJ, Bacha LM (2011) Color atlas of veterinary histology, 3rd edn. Wiley-Blackwell, ChichesterGoogle Scholar
  4. Banks WJ (1986) Applied veterinary histology, 2nd edn. Williams & Wilkins, BaltimoreGoogle Scholar
  5. Barry S (2010) Non-steroidal anti-inflammatory drugs inhibit bone healing: a review. Vet Comp Orthop Traumatol 23:385–392PubMedCrossRefGoogle Scholar
  6. Bueno DF, Kerkis I, Costa AM, Martins MT, Kobayashi GS, Zucconi E, Fanganiello RD, Salles FT, Almeida AB, do Amaral CE, Alonso N, Passos-Bueno MR (2009) New source of muscle-derived stem cells with potential for alveolar bone reconstruction in cleft lip and/or palate patients. Tissue Eng Part A 15:427–435PubMedCrossRefGoogle Scholar
  7. Carpenter RS, Goodrich LR, Frisbie DD, Kisiday JD, Carbone B, McIlwraith CW, Centeno CJ, Hidaka C (2010) Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone. J Orthop Res 28:1330–1337PubMedCentralPubMedCrossRefGoogle Scholar
  8. Cauvin ER, Munroe GA (1998) Septic osteitis of the distal phalanx: findings and surgical treatment in 18 cases. Equine Vet J 30:512–519PubMedCrossRefGoogle Scholar
  9. Crovace A, Lacitignola L, Rossi G, Francioso E (2010) Histological and immunohistochemical evaluation of autologous cultured bone marrow mesenchymal stem cells and bone marrow mononucleated cells in collagenase-induced tendinitis of equine superficial digital flexor tendon. Vet Med Int 2010:250978PubMedCentralPubMedCrossRefGoogle Scholar
  10. Dresdale A, Rose EA, Jeevanandam V, Reemtsma K, Bowman FO, Malm JR (1985) Preparation of fibrin glue from single-donor fresh-frozen plasma. Surgery 97:750–755PubMedGoogle Scholar
  11. Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P (2007) Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng 13:1299–1312PubMedCrossRefGoogle Scholar
  12. Ferris D, Frisbie D, Kisiday J, McIlwraith CW (2012) In vivo healing of meniscal lacerations using bone marrow-derived mesenchymal stem cells and fibrin glue. Stem Cells Int 2012:691605PubMedCentralPubMedCrossRefGoogle Scholar
  13. Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RK (2012) Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine Vet J 44:25–32PubMedCrossRefGoogle Scholar
  14. Hale BW, Goodrich LR, Frisbie DD, McIlwraith CW, Kisiday JD (2012) Effect of scaffold dilution on migration of mesenchymal stem cells from fibrin hydrogels. Am J Vet Res 73:313–318PubMedCrossRefGoogle Scholar
  15. Hance SR, Bramlage LR, Schneider RK, Embertson RM (1992) Retrospective study of 38 cases of femur fractures in horses less than one year of age. Equine Vet J 24:357–363PubMedCrossRefGoogle Scholar
  16. Henrotin Y (2011) Muscle: a source of progenitor cells for bone fracture healing. BMC Med 9:136PubMedCentralPubMedCrossRefGoogle Scholar
  17. Ishihara A, Zekas LJ, Litsky AS, Weisbrode SE, Bertone AL (2010a) Dermal fibroblast-mediated BMP2 therapy to accelerate bone healing in an equine osteotomy model. J Orthop Res 28:403–411PubMedGoogle Scholar
  18. Ishihara A, Zekas LJ, Weisbrode SE, Bertone AL (2010b) Comparative efficacy of dermal fibroblast-mediated and direct adenoviral bone morphogenetic protein-2 gene therapy for bone regeneration in an equine rib model. Gene Ther 17:733–744PubMedCrossRefGoogle Scholar
  19. Ishimura M, Ohgushi H, Habata T, Tamai S, Fujisawa Y (1997) Arthroscopic meniscal repair using fibrin glue. Part I: Experimental study. Arthroscopy 13:551–557PubMedCrossRefGoogle Scholar
  20. Ito K, Yamada Y, Naiki T, Ueda M (2006) Simultaneous implant placement and bone regeneration around dental implants using tissue-engineered bone with fibrin glue, mesenchymal stem cells and platelet-rich plasma. Clin Oral Implants Res 17:579–586PubMedCrossRefGoogle Scholar
  21. Kalbermatten DF, Kingham PJ, Mahay D, Mantovani C, Pettersson J, Raffoul W, Balcin H, Pierer G, Terenghi G (2008) Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit. J Plast Reconstr Aesthet Surg 61:669–675PubMedCrossRefGoogle Scholar
  22. Kim KS, Lee JH, Ahn HH, Lee JY, Khang G, Lee B, Lee HB, Kim MS (2008) The osteogenic differentiation of rat muscle-derived stem cells in vivo within in situ-forming chitosan scaffolds. Biomaterials 29:4420–4428PubMedCrossRefGoogle Scholar
  23. Kisiday JD, Hale BW, Almodovar JL, Lee CM, Kipper MJ, Wayne MC, Frisbie DD (2011) Expansion of mesenchymal stem cells on fibrinogen-rich protein surfaces derived from blood plasma. J Tissue Eng Regen Med 5:600–611PubMedCrossRefGoogle Scholar
  24. Lacitignola L, Crovace A, Rossi G, Francioso E (2008) Cell therapy for tendinitis, experimental and clinical report. Vet Res Commun 32 (Suppl 1):S33–S38PubMedCrossRefGoogle Scholar
  25. Liu R, Schindeler A, Little DG (2010) The potential role of muscle in bone repair. J Musculoskelet Neuronal Interact 10:71–76PubMedGoogle Scholar
  26. McDuffee LA (2012) Comparison of isolation and expansion techniques for equine osteogenic progenitor cells from periosteal tissue. Can J Vet Res 76:91–98PubMedCentralPubMedGoogle Scholar
  27. Murphy JM, Fink DJ, Hunziker EB, Barry FP (2003) Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 48:3464–3474PubMedCrossRefGoogle Scholar
  28. Nino-Fong R, McDuffee LA, Esparza Gonzalez BP, Kumar MR, Merschrod SEF, Poduska KM (2013) Scaffold effects on osteogenic differentiation of equine mesenchymal stem cells: an in vitro comparative study. Macromol Biosci 13:348–355PubMedCrossRefGoogle Scholar
  29. Penny J, Harris P, Shakesheff KM, Mobasheri A (2012) The biology of equine mesenchymal stem cells: phenotypic characterization, cell surface markers and multilineage differentiation. Front Biosci 17:892–908CrossRefGoogle Scholar
  30. Radtke CL, Nino-Fong R, Esparza Gonzalez BP, Stryhn H, McDuffee LA (2013) Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells. Am J Vet Res 74:790–800PubMedCrossRefGoogle Scholar
  31. Ryu JH, Kim IK, Cho SW, Cho MC, Hwang KK, Piao H, Piao S, Lim SH, Hong YS, Choi CY, Yoo KJ, Kim BS (2005) Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium. Biomaterials 26:319–326PubMedCrossRefGoogle Scholar
  32. Schnabel LV, Lynch ME, van der Meulen MC, Yeager AE, Kornatowski MA, Nixon AJ (2009) Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 27:1392–1398PubMedCrossRefGoogle Scholar
  33. Smith RK, Korda M, Blunn GW, Goodship AE (2003) 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 35:99–102PubMedCrossRefGoogle Scholar
  34. Song IH, Caplan AI, Dennis JE (2009) In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo. J Orthop Res 27:916–921PubMedCrossRefGoogle Scholar
  35. Stewart AA, Byron CR, Pondenis H, Stewart MC (2007) Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am J Vet Res 68:941–945PubMedCrossRefGoogle Scholar
  36. Sun B, Ma W, Su F, Wang Y, Liu J, Wang D, Liu H (2011) The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/beta-glycerophosphate hydrogel: in vitro and in vivo. J Mater Sci Mater Med 22:2111–2118PubMedCrossRefGoogle Scholar
  37. Toupadakis CA, Wong A, Genetos DC, Cheung WK, Borjesson DL, Ferraro GL, Galuppo LD, Leach JK, Owens SD, Yellowley CE (2010) Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue. Am J Vet Res 71:1237–1245PubMedCrossRefGoogle Scholar
  38. Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM, Gimble JM (2007) Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet Surg 36:613–622PubMedCrossRefGoogle Scholar
  39. Wright V, Peng H, Usas A, Young B, Gearhart B, Cummins J, Huard J (2002) BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther 6:169–178PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Laurie A. McDuffee
    • 1
  • Blanca P. Esparza Gonzalez
    • 1
  • Rodolfo Nino-Fong
    • 1
  • Enrique Aburto
    • 2
  1. 1.Comparative Orthopaedic Research Laboratory, Department of Health Management, Atlantic Veterinary CollegeUniversity of Prince Edward IslandCharlottetownCanada
  2. 2.Department of Pathology and MicrobiologyUniversity of Prince Edward IslandCharlottetownCanada

Personalised recommendations