Archives of Orthopaedic and Trauma Surgery

, Volume 127, Issue 9, pp 815–821 | Cite as

Tissue engineering of tendons and ligaments by human bone marrow stromal cells in a liquid fibrin matrix in immunodeficient rats: Results of a histologic study

  • Stefan Hankemeier
  • Martijn van Griensven
  • Marco Ezechieli
  • Tanja Barkhausen
  • Matthew Austin
  • Michael Jagodzinski
  • Rupert Meller
  • Ulrich Bosch
  • Christian Krettek
  • Johannes Zeichen
Arthroscopy and Sports Medicine



The original complex structure and mechanical properties are not fully restored after ligament and tendon injuries. Due to their high proliferation rate and differentiation potential, Bone Marrow Stromal Cells (BMSC) are considered to be an ideal cell source for tissue engineering to optimize the healing process. Ideal matrices for tissue engineering of ligaments and tendons should allow for homogenous cell seeding and offer sufficient stability.

Material and methods

A mixture of human BMSC and liquid fibrin glue was injected into a standardized full-thickness window defect of the patellar tendon of immunodeficient rats (BMSC group). The histology of the tissue was analysed 10 and 20 days postoperatively and compared to four control groups. These groups consisted of a cohort with a mixture of human fibroblasts and fibrin glue, fibrin glue without cells, a defect group without treatment, and a group with uninjured patellar tendon tissue.


Tendon defects in the BMSC group revealed dense collagen fibres and spindle-shaped cells, which were mainly orientated along the loading axis. Histologic sections of the control groups, especially of untreated defects and of defects filled with fibrin glue only, showed irregular patterns of cell distribution, irregular formed cell nucleoli and less tissue maturation. Compared to healthy tendon tissue, higher numbers of cells and less intense matrix staining was observed in the BMSC group. No ectopic bone or cartilage formation was observed in any specimen.


Injection of human BMSC in a fibrin glue matrix appears to lead to more mature tissue formation with more regular patterns of cell distribution. Advantages of this “in-vivo” tissue engineering approach are a homogenous cell-matrix mixture in a well-known and approved biological matrix, and simple, minimally-invasive application by injection.


BMSC Tissue engineering Tendon Ligament Fibrin glue Histology Healing 



The authors would like to thank the “German Speaking Arthroscopy Association” (AGA) for financial support.


  1. 1.
    Altman GH, Horan RL, Martin I, Farhadi J, Stark PR, Volloch V, Richmond JC, Vunjak-Novakovic G, Kaplan DL (2002) Cell differentiation by mechanical stress. FASEB J 16:270–272PubMedGoogle Scholar
  2. 2.
    Awad HA, Boivin GP, Dressler MR, Smith FN, Young RG, Butler DL (2003) Repair of patellar tendon injuries using a cell-collagen composite. J Orthop Res 21:420–431PubMedCrossRefGoogle Scholar
  3. 3.
    Badylak SF, Tullius R, Kokini K, Shelbourne KD, Klootwyk T, Voytik SL, Kraine MR, Simmons C (1995) The use of xenogeneic small intestinal submucosa as a biomaterial for achilles tendon repair in a dog model. J Biomed Mater Res 29:977–985PubMedCrossRefGoogle Scholar
  4. 4.
    Battaglia TC, Clark RT, Chhabra A, Gaschen V, Hunziker EB, Mikic B (2003) Ultrastructural determinants of murine achilles tendon strength during healing. Connect Tissue Res 44:218–224PubMedCrossRefGoogle Scholar
  5. 5.
    Becker JC, Domschke W, Pohle T (2004) Biological in vitro effects of fibrin glue: fibroblast proliferation, expression and binding of growth factors. Scand J Gastroenterol 39:927–932PubMedCrossRefGoogle Scholar
  6. 6.
    Brandt KD, Myers SL, Burr D, Albrecht M (1991) Osteoarthritic changes in canine articular cartilage, subchondral bone, and synovium fifty-four months after transection of the anterior cruciate ligament. Arthritis Rheum 34:1560–1570PubMedCrossRefGoogle Scholar
  7. 7.
    Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889–895PubMedCrossRefGoogle Scholar
  8. 8.
    Bruns J, Kampen J, Kahrs J, Plitz W (2000) Achilles tendon rupture: experimental results on spontaneous repair in a sheep model. Knee Surg Sports Traumatol Arthrosc 8:364–369PubMedCrossRefGoogle Scholar
  9. 9.
    Butler DL, Juncosa N, Dressler MR (2004) Functional efficacy of tendon repair processes. Annu Rev Biomed Eng 6:303–329PubMedCrossRefGoogle Scholar
  10. 10.
    Butler DL, Kay MD, Stouffer DC (1986) Comparison of material properties in fascicle-bone units from human patellar tendon and knee ligaments. J Biomech 19:425–432PubMedCrossRefGoogle Scholar
  11. 11.
    Caplan AI (1994) The mesengenic process. Clin Plast Surg 21:429–435PubMedGoogle Scholar
  12. 12.
    Cartmell JS, Dunn MG (2004) Development of cell-seeded patellar tendon allografts for anterior cruciate ligament reconstruction. Tissue Eng 10:1065–1075PubMedGoogle Scholar
  13. 13.
    Christman KL, Fok HH, Sievers RE, Fang Q, Lee RJ (2004) Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction. Tissue Eng 10:403–409PubMedCrossRefGoogle Scholar
  14. 14.
    Dahlgren LA, Mohammed HO, Nixon AJ (2005) Temporal expression of growth factors and matrix molecules in healing tendon lesions. J Orthop Res 23:84–92PubMedCrossRefGoogle Scholar
  15. 15.
    Dressler MR, Butler DL, Boivin GP (2005) Effects of age on the repair ability of mesenchymal stem cells in rabbit tendon. J Orthop Res 23:287–293PubMedCrossRefGoogle Scholar
  16. 16.
    Frank C, Hart DA, Shrive NG (1999) Molecular biology and biomechanics of normal and healing ligaments-a review. Osteoarthr Cartil 7:130–140PubMedCrossRefGoogle Scholar
  17. 17.
    Garvin J, Qi J, Maloney M, Banes AJ (2003) Novel system for engineering bioartificial tendons and application of mechanical load. Tissue Eng 9:967–979PubMedCrossRefGoogle Scholar
  18. 18.
    Gentlemen E, Lay AN, Dickerson DA, Nauman EA, Livesay GA, Dee KC (2003) Mechanical characterization of collagen fibres and scaffolds for tissue engineering. Biomaterials 24:3805–3813CrossRefGoogle Scholar
  19. 19.
    Hankemeier S, Grassel S, Plenz G, Spiegel HU, Bruckner P, Probst A (2001) Alteration of fracture stability influences chondrogenesis, osteogenesis and immigration of macrophages. J Orthop Res 19:531–538PubMedCrossRefGoogle Scholar
  20. 20.
    Hankemeier S, Keus M, Zeichen J, Jagodzinski M, Barkhausen T, Bosch U, Krettek C, van Griensven M (2005) Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng 11:41–49PubMedCrossRefGoogle Scholar
  21. 21.
    Horch RE, Bannasch H, Kopp J, Andree C, Stark GB (1998) Single-cell suspensions of cultured human keratinocytes in fibrin-glue reconstitute the epidermis. Cell Transplant 7:309–317PubMedCrossRefGoogle Scholar
  22. 22.
    Korbling M, Estrov Z (2003) Adult stem cells for tissue repair—a new therapeutic concept? N Engl J Med 349:570–582PubMedCrossRefGoogle Scholar
  23. 23.
    Lee OK, Blunn GW (2002) Use of fibrin as a carrier for mesenchymal stem cell delivery. Abstract book 48th meeting Orthopaedic Research Society, DallasGoogle Scholar
  24. 24.
    Lin TW, Cardenas L, Soslowsky LJ (2004) Biomechanics of tendon injury and repair. J Biomech 37:865–877PubMedCrossRefGoogle Scholar
  25. 25.
    Lusardi DA, Cain JE (1994) The effect of fibrin sealant on the strength of tendon repair of full thickness tendon lacerations in the rabbit achilles tendon. J Foot Ankle Surg 33:443–447PubMedGoogle Scholar
  26. 26.
    Muraglia A, Cancedda R, Quarto R (2000) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 113:1161–1166PubMedGoogle Scholar
  27. 27.
    Murrell GA, Lilly EG, Collins A, Seaber AV, Goldner RD, Best TM (1993) Achilles tendon injuries: a comparison of surgical repair versus no repair in a rat model. Foot Ankle 14:400–406PubMedGoogle Scholar
  28. 28.
    Musgrave DS, Fu FH, Huard J (2002) Gene therapy and tissue engineering in orthopedic surgery. J Am Acad Orthop Surg 10:6–15PubMedGoogle Scholar
  29. 29.
    Orlic D, Kaystura J, Chimenti S, Jakoniuk I, Anderson SM, Ki B, Pickel J, McKay R, Quiani F, Nadal-Ginard B, Bodine DM, Leri A, Anversa P (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410:640–641CrossRefGoogle Scholar
  30. 30.
    Ouyang HW, Goh JC, Lee EH (2004) Viability of allogeneic bone marrow stromal cells following local delivery into patella tendon in rabbit model. Cell Transplant 13:649–657PubMedGoogle Scholar
  31. 31.
    Pevny T, Hunter RE (2001) Current concepts in anterior cruciate ligament reconstruction. In: Chow JCY (ed) Advanced arthroscopy. Springer, New York, pp S381–S392Google Scholar
  32. 32.
    Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147PubMedCrossRefGoogle Scholar
  33. 33.
    Proctor CS, Jackson DW, Simon TM (1997) Characterization of the repair tissue after removal of the central one-third of the patellar tendon. An experimental study in a goat model. J Bone Joint Surg Am 79:997–1006PubMedGoogle Scholar
  34. 34.
    Provenzano PP, Hurschler C, Vanderby R Jr (2001) Microstructural morphology in the transition region between scar and intact residual segments of a healing rat medial collateral ligament. Connect Tissue Res 42:123–133PubMedGoogle Scholar
  35. 35.
    Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344:385–386PubMedCrossRefGoogle Scholar
  36. 36.
    Rokito AS, Cuomo F, Gallagher MA, Zuckerman JD (1999) Long-term functional outcome of repair of large and massive chronic tears of the rotator cuff. J Bone Joint Surg Am 81:991–997PubMedCrossRefGoogle Scholar
  37. 37.
    Saito T, Dennis JE, Lenon DP, Young RG, Caplan A (1995) Myogenic expression of mesenchymal stem cells within myotubes of mdx mice in vitro and in vivo. Tissue Eng 1:327–343CrossRefPubMedGoogle Scholar
  38. 38.
    Schlag G, Redl H (1998) Fibrin sealant in orthopedic surgery. Clin Orthop 227:269–285Google Scholar
  39. 39.
    Schultheiss D, Gabouev AI, Cebotari S, Tudorache I, Walles T, Schlote N, Wefer J, Kaufmann PM, Haverich A, Jonas U, Stief CG, Mertsching H (2005) Biological vascularized matrix for bladder tissue engineering: matrix preparation, reseeding technique and short-term implantation in a porcine model. J Urol 173:276–280PubMedCrossRefGoogle Scholar
  40. 40.
    Takahashi Y, Tabata Y (2003) Homogenous seeding of mesenchymal stem cells into nonwoven fabric for tissue engineering. Tissue Eng 9:931–938PubMedCrossRefGoogle Scholar
  41. 41.
    Van Griensven M, Zeichen J, Tschernig T, Seekamp A, Pape HC (2002) A modified method to culture human osteoblasts from bone tissue specimens using fibrin glue. Exp Toxicol Pathol 54:25–29PubMedCrossRefGoogle Scholar
  42. 42.
    Woo SL, Danto MI, Ohland KJ, Lee TQ, Newton PO (1990) The use of a laser micrometer system to determine the cross-sectional shape and area of ligaments: a comparative study with two existing methods. J Biomech Eng 112:426–431PubMedGoogle Scholar
  43. 43.
    Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ (1998) Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. J Orthop Res 16:406–413PubMedCrossRefGoogle Scholar
  44. 44.
    Zeichen J, van Griensven M, Bosch U (2000) The proliferative response of isolated human tendon fibroblasts to cyclic biaxial mechanical strain. Am J Sports Med 28:888–892PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Stefan Hankemeier
    • 1
  • Martijn van Griensven
    • 2
  • Marco Ezechieli
    • 1
  • Tanja Barkhausen
    • 1
  • Matthew Austin
    • 3
  • Michael Jagodzinski
    • 1
  • Rupert Meller
    • 1
  • Ulrich Bosch
    • 4
  • Christian Krettek
    • 1
  • Johannes Zeichen
    • 1
  1. 1.Trauma DepartmentHanover Medical School (MHH)HanoverGermany
  2. 2.Ludwig Boltzmann InstituteResearch Centre for TraumatologyViennaAustria
  3. 3.Rothman InstituteOrthopaedic SurgeryPhiladelphiaUSA
  4. 4.Orthopaedic DepartmentInternational Neuroscience InstituteHanoverGermany

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