Engineering the healing of the rabbit medial collateral ligament

  • S. L. -Y. Woo
  • D. W. Smith
  • K. A. Hildebrand
  • J. A. Zeminski
  • L. A. Johnson
Cellular Engineering

Abstract

A biological approach to improve healing of the medical collateral ligament (MCL) was investigated by exploring the use of therapeutic growth factors based on in vitro and in vivo experiments. The in vitro cell culture studies involved screening a variety of growth factors to select those that exhibit the most positive effects on cell proliferation and extracellular matrix synthesis. The selected growth factors were applied in vivo to a rabbit model where the MCL was ruptured. Biomechanical and histological evaluations are performed to determine whether the selected growth factors can enhance the properties of the healed MCL, whether these improvements are dose dependent, and whether combinations of growth factors can enhance MCL healing to a greater extent than individual growth factors. In vitro studies showed that epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) have the greatest effect on ligament fibroblast proliferation, whereas transforming growth factor-β1 (TGF1) superiorly promotes extracellular matrix synthesis. These growth factors were then applied in vivo at different dosages, in isolation and in combination, and the ligaments were evaluated six weeks post-operatively. Tensile testing of the femur-MCL-tibia complexes (FMTCs) revealed that the specimens treated with a high dose of PDGF-BB have ultimate load, ultimate elongation and energy absorbed to failure values that are significantly greater than those from the other groups. The high dose of PDGF-BB was more effective than the low dose, indicating a dose dependency. The addition ofTGF1 to PDGF-BB did not lead to any further increases in the structural properties of the FMTC. These encouraging results suggest that PDGF-BB may be a potential growth factor to enhance the quality of the healing ligament.

Keywords

Growth factors Healing Cell proliferation Matrix synthesis Tensile testing MCL 

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References

  1. Batten, M. L., Hansen, J. C. andDahners, L. E. (1996): ‘Influence of dosage and timing of application of platelet-derived growth factor on early healing of the rat medial collateral ligament’,J. Orthop. Res.,14, pp. 736–741CrossRefGoogle Scholar
  2. Bauer, E. A., Cooper, T. W., Huang, J. S., Altman, J. andDeuel, T. F. (1985): ‘Stimulation ofin vitro human skin collagenase expression by platelet-derived growth factor’,Proc. Natl. Acad. Sci. USA,82, pp. 4132–4136CrossRefGoogle Scholar
  3. Centrella, M., McCarthy, T. L. andCanalis, E. (1991): ‘Current concepts review: Transforming growth factor-beta and remodeling of bone’,J. Bone Joint. Surg. [Am.],73, pp. 1418–1428Google Scholar
  4. Conti, N. A. andDahners, L. E. (1993): ‘The effect of exogeneous growth factors on the healing of ligaments’,Trans. Orthop. Res. Soc.,18, p. 60Google Scholar
  5. Dahners, L. andBurroughs, P. (1990): ‘The effect of enforced exercise on the healing of ligament injuries’,Am. J. Sports Med.,18, (4), pp. 376–378Google Scholar
  6. Desrosiers, E. A., Yahia, L. andRivard, C.-H. (1996): ‘Proliferative and matrix synthesis response of canine anterior cruciate ligament fibroblasts submitted to combined growth factors’,J. Orthop. Res.,14, pp. 200–208CrossRefGoogle Scholar
  7. FCrank, E. Woo, S. L.-Y., Amiel, D., Harwood, F., Gomez, M. andAkeson, W. (1983): ‘Medial collateral ligament healing: A multidisciplinary assessment in rabbits’,Am. J. Sports Med.,11, pp. 379–389Google Scholar
  8. Hom, D. B. (1995): ‘Growth factors in would healing’,Otolaryngol. Clin. North Am.,28, (5), pp. 933–953Google Scholar
  9. Indelicato, P. A. (1995): ‘Isolated medial collateral ligament injuries in the knee’,J. Am. Acad. Orthop. Surg.,3, pp. 9–14Google Scholar
  10. Inoue, M., McGurk-Burleson, E., Hollis, J. M. andWoo, S. L.-Y. (1987): ‘Treatment of the medial collateral ligament injury I: The importance of anterior cruciate ligament on the varus-valgus knee laxity’,Am. J. Sports Med.,15, pp. 15–21MATHGoogle Scholar
  11. Lee, T. Q. andWoo, S. L.-Y. (1988): ‘A new method for determining cross-sectional shape and area of soft tissues’,J. Biomech. Eng.,110, pp. 110–114CrossRefGoogle Scholar
  12. Letson, A. K. andDahners, L. E. (1994): ‘The effect of combinations of growth factors on ligament healing’,Clin. Orthop.,308, pp. 207–212Google Scholar
  13. Lynch, S. E., Nixon, J. C., Colvin, R. B. andAntoniades, H. N. (1987): ‘Role of platelet-derived growth factor in wound healing: Synergistic effects with other growth factors’,Proc. Natl. Acad. Sci. USA,84, pp. 7696–7700CrossRefGoogle Scholar
  14. Marui, T., Niyibizi, C., Georgescu, H. I., Cao, M., Kavalkovich, K. W., Levine, R. E. andWoo, S. L.-Y. (1997): ‘The effect of growth factors on matrix synthesis by ligament fibroblasts’,J. Orthop. Res.,15, pp. 18–23CrossRefGoogle Scholar
  15. Matsuda, N., Lin, W.-L., Kumar, N. M., Cho, M. I. andGenco, R. J. (1992): ‘Mitogenic chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro’,J. Periodontol.,63, p 6Google Scholar
  16. Miyasaka, K. C., Daniel, D. M., Stone, M. L. andHirshman, P. (1991): ‘The incidence of knee ligament injuries in the general population’,Am. J. Knee Surg.,4, pp. 3–8Google Scholar
  17. Nagineni, C. N., Amiel, D., Green, M. H., Berchuck, M. andAkeson, W. H. (1992): ‘Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: An invitro cell culture study’,J. Orthop. Res.,10, pp. 465–475CrossRefGoogle Scholar
  18. Peterkofsky, B. andDiegelmann, R. (1971): ‘Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins’,Biochem.10, pp. 988–994CrossRefGoogle Scholar
  19. Pierce, G.F., Mustoe, T. A., Senior, R. M., Reed, J., Griffin, G. L., Thompson, A. andDeuel, T. F. (1988): ‘In vivo incisional would healing augmented by platelet-derived growth factor and recombinant c-sis gene homodimeric proteins’,J. Exp. Med.,167, pp. 974–987CrossRefGoogle Scholar
  20. Pierce, G. F., Mustoe, T. A., Lingelbach, J., Masakowski, V. R., Griffin, G. L., Senior, R. M. andDeuel, T. F. (1989): ‘Platelet-derived growth factor and transforming growth factor-β enhance tissue repair activities by unique mechanisms’,J. Cell Biol.,109, 429–440CrossRefGoogle Scholar
  21. Pledger, W. J., Stiles, C. D., Antoniades, H. N. andScher, C. D. (1977): ‘Induction of DNA synthesis in BALB/c3T3 cells byt serum components: Reevaluation of the commitment process’,Proc. Natl. Acad. Sci. USA,74, (10) pp. 4481–4485CrossRefGoogle Scholar
  22. Pledger, W. J., Stiles, C. D., Antoniades, H. N. andScher, C. D. (1978): ‘An ordered sequence of events is required before BALB/c-3T3 cells become committed to DNA synthesis’,Proc. Natl. Acad. Sci. USA,75, (6), pp. 2839–2843CrossRefGoogle Scholar
  23. Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H. andFauci, A. S. (1986): ‘Transforming growth factor type β: Rapid induction of fibroblasts and angiogenesisin vivo and stimulation of collagen formationin vitro’,Proc. Natl. Acad. Sci. USA,83, pp. 4167–4171CrossRefGoogle Scholar
  24. Roberts, A. B., Thompson, N. L. andHeine, U. (1988): ‘Transforming growth factor-beta: possible roles in carcinogenesis’,Br. J. Cancer,57, pp. 594–600Google Scholar
  25. Schering, Jr., S. C., Schmidt, C. D., Georgescu, H. I., Kwoh, C. K., Evans, C. H. andWoo, S. L.-Y. (1997): ‘Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits’,Connect. Tissue Res.,36, pp. 1–8CrossRefGoogle Scholar
  26. Schmidt, C. C., Georgescu, H. I., Kwoh, C. K., Blomstrom, G. L., Engle, C. P., Larkin, L. A., Evans, C. H. andWoo, S. L.-Y.: (1995): ‘Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments’,J. Orthop. Res.,13, pp. 184–190CrossRefGoogle Scholar
  27. Singh, J. P., Adams, L. D. andBonin, P. D. (1988): ‘Mode of fibroblast growth enhancement by Human Interleukin-1’,J. Cell Biol.,106, pp. 813–819CrossRefGoogle Scholar
  28. Spindler, K. P., Dawson, J. M. Stahlman, G. C. andDavidson, J. M. (1996): ‘Collagen synthesis and biomechanical response toTGF1 in the healing rabbit MCL’,Trans. Orthop. Res. Soc.,21, p. 793Google Scholar
  29. Starksen, N. F., Harsh IV, G. R. andGibbs, V. C. (1987): ‘Regulated expression of the platelet-derived growth factor A chain gene in microvascular endothelial cells’,J. Biol. Chem.,262, pp. 14381–14384Google Scholar
  30. Stiles, C. D., Capone, G. T., Scher, C. D., Antoniades, H. N., Vanwyk, J. J. andPledger, W. J. (1979). ‘Dual control of cell growth by somatomedins and platelet-deriver growth factor’,Proc. Natl. Acad. Sci. USA,76 (3), pp. 1279–1283CrossRefGoogle Scholar
  31. Tzeng, D. Y., Deuel, T. F., Huang, J. S., Senior, R. M., Boxer, L. A. andBaehner, R. L. (1984): ‘Platelet-derived growth factor promotes polymorphonuclear leukocyte activation’,Blood,64, pp. 1123–1128Google Scholar
  32. Tzeng, D. Y., Deuel, T. F., Huang, J. S. andBaehner, R. L. (1985): ‘Platelet-derived growth factor promotes human peripheral monocyte activation’,Blood,66, pp. 179–183Google Scholar
  33. Weiss, J. A., Woo, S. L.-Y., Ohland, K. J., Horibe S. andNewton, P. O. (1991): ‘Evaluation of a new injury model to study medial collateral ligament healing: Primary repair vs. nonoperative treatment’,J. Orthop. Res.,9, pp. 516–528CrossRefGoogle Scholar
  34. Weiss, J. A., Beck, C. L., Levine, R. E. andGreenwald, R. M. (1995): ‘Effects of platelet-derived growth factor on early medial collateral ligament healing’,Trans. Orthop. Res. Soc.,20, p. 159.Google Scholar
  35. Wilder, R. L., Lafyatis, R. andRemmers, E. F. (1980): ‘Platelet-derived growth factor and transforming growth factor beta in wound healing and repair’,inLeadbetter, W. B., Buckwalter, J. A. andGordon, S. L. (Eds.): ‘Sports-induced inflammation’ (Am. Acad. Orthopaedic Surgeons, Park Ridge, Illinois) pp. 301–313Google Scholar
  36. Woo, S. L.-Y., Gomez, M. A., Seguchi, Y., Endo, C. M. andAkeson, W. H. (1983): ‘Measurement of mechanical properties of ligament substance from a bone-ligament-bone preparation’,J. Orthorp. Res.,1, pp. 22–29CrossRefGoogle Scholar
  37. Woo, S. L.-Y., Gomez, M. A., Inoue, M. andAkeson, W. H. (1987): ‘New experimental procedures to evaluate the biomechanical properties of healing canine medial collateral ligaments’,Orthop. Res.,5, pp. 425–432CrossRefGoogle Scholar
  38. Woo, S. L.-Y., Danto, M. I., Ohland, K. J., Lee, T. Q. andNewton, P. O. (1990a): ‘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, pp. 426–431Google Scholar
  39. Woo, S. L.-Y., Young, E. P., Ohland, K. J., Marcin, J. P., Horibe, S. andLin, H. C. (1990b) The effects of transection of the anterior cruciate ligament on healing of the medial collateral ligament: A biomechanical study of the knee in dogs’,J. Bone Joint. Surg. Am.,72, pp. 382–392Google Scholar

Copyright information

© IFMBE 1998

Authors and Affiliations

  • S. L. -Y. Woo
    • 1
  • D. W. Smith
    • 1
  • K. A. Hildebrand
    • 1
  • J. A. Zeminski
    • 1
  • L. A. Johnson
    • 1
  1. 1.Musculoskeletal Research Center, Department of Orthopaedic Surgery, E1641 Biomedical Science TowerUniversity of PittsburghPittsburghUSA

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