Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction

Abstract

Purpose

The aim of this study is to examine the capacity of muscle tissue preserved on hamstring tendons forming candy-stripe grafts in order to improve tendon to bone ingrowth and ligamentization. We hypothesized that muscle tissue does possess a stem cell population that could enhance the healing process of the ACL graft when preserved on the tendons.

Methods

Human samples from gracilis and semitendinosus muscles were collected during ACL surgery from ten patients and from these tissue samples human muscle-derived stem cells and tendon-derived stem cells were isolated and propagated. Both stem cell populations were in-vitro differentiated into osteogenic lineage. Alkaline phosphatase activity was determined at days zero and 14 of the osteogenic induction and von Kossa staining to assess mineralization of the cultures. Total RNA was collected from osteoblast cultures and real time quantitative PCR was performed. Western-blot for osteocalcin and collagen type I followed protein isolation. Immunofluorescence double labeling of pericytes in muscle and tendon tissue was performed.

Results

Mesenchymal stem cells from muscle and tendon tissue were isolated and expanded in cell culture. More time was needed to grow the tendon derived culture compared to muscle derived culture. Muscle derived stem cells exhibited more alkaline phosphatase actvity compared to tendon derived stem cells, whereas tendon derived stem cells formed more mineralized nodules after 14 days of osteoinduction. Muscle derived stem cells exhibited higher expression levels of bone sialoprotein, and tendon derived stem cells showed higher expression of dental-matrix-protein 1 and osteocalcin. Immunofluorescent staining against pericytes indicated that they are more abundant in muscle tissue.

Conclusions

These results indicate that muscle tissue is a better source of stem cells than tendon tissue. Achievement of this study is proof that there is vast innate capacity of muscle tissue for enhancement of bone-tendon integration and ligamentization of ACL hamstring grafts and consequently muscle tissue should not be treated as waste after harvesting.

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References

  1. 1.

    Zaffagnini S, Grassi A, Serra M, Marcacci M (2015) Return to sport after ACL reconstruction: how, when and why? A narrative review of current evidence. Joints 3(1):25–30

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Robin BN, Jani SS, Marvil SC, Reid JB, Schillhammer CK, Lubowitz JH (2015) Advantages and disadvantages of transtibial, anteromedial portal, and outside-in femoral tunnel drilling in single-bundle anterior cruciate ligament reconstruction: a systematic review. Arthroscopy 31(7):1412–1417

    Article  PubMed  Google Scholar 

  3. 3.

    Sherman OH, Banffy MB (2004) Anterior cruciate ligament reconstruction: which graft is best? Arthroscopy 20(9):974–980

    Article  PubMed  Google Scholar 

  4. 4.

    Carey JL, Dunn WR, Dahm DL, Zeger SL, Spindler KP (2009) A systematic review of anterior cruciate ligament reconstruction with autograft compared with allograft. J Bone Joint Surg Am 91(9):2242–2250

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Komiyama H, Arai Y, Kajikawa Y, Yoshida A, Morihara T, Terauchi R, Kida Y, Fujiwara H, Kawata M, Kubo T (2012) The fate and role of bone graft-derived cells after autologous tendon and bone transplantation into the bone tunnel. J Orthop Sci 18(6):994–1004

    Article  PubMed  Google Scholar 

  6. 6.

    Scheffler SU, Unterhauser FN, Weiler A (2008) Graft remodeling and ligamentization after cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc 16(9):834–842

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Howell SM (2001) Autogenous graft choices in ACL reconstruction. Curr Opin Orthop 12:149–155

    Article  Google Scholar 

  8. 8.

    Mihelic R, Pecina M, Jelic M, Zoricic S, Kusec V, Simic P, Bobinac D, Lah B, Legovic D, Vukicevic S (2004) Bone morphogenetic protein-7 (osteogenic protein-1) promotes tendon graft integration in anterior cruciate ligament reconstruction in sheep. Am J Sports Med 32(7):1619–1625

    Article  PubMed  Google Scholar 

  9. 9.

    Takigami J, Hashimoto Y, Yamasaki S, Terai S, Nakamura H (2015) Direct bone-to-bone integration between recombinant human bone morphogenetic protein-2-injected tendon graft and tunnel wall in an anterior cruciate ligament reconstruction model. Int Orthop 39(7):1441–1447

    Article  PubMed  Google Scholar 

  10. 10.

    Schwarting T, Schenk D, Frink M, Benolken M, Steindor F, Oswald M, Ruchholtz S, Lechler P (2015) Stimulation with bone morphogenetic protein-2 (BMP-2) enhances bone-tendon integration in vitro. Connect Tissue Res 57(2):99–112

    Article  PubMed  Google Scholar 

  11. 11.

    Bissell L, Tibrewal S, Sahni V, Khan WS (2014) Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther 10(1):19–25

    Article  Google Scholar 

  12. 12.

    Vogrin M, Rupreht M, Dinevski D, Haspl M, Kuhta M, Jevsek M, Knezevic M, Rozman P (2010) Effects of a platelet gel on early graft revascularization after anterior cruciate ligament reconstruction: a prospective, randomized, double-blind, clinical trial. Eur Surg Res 45(2):77–85

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Zhang X, Ma Y, Fu X, Liu Q, Shao Z, Dai L, Pi Y, Hu X, Zhang J, Duan X, Chen W, Chen P, Zhou C, Ao Y (2016) Runx2-modified adipose-derived stem cells promote tendon graft integration in anterior cruciate ligament reconstruction. Sci Rep 6:19073

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Caplan AI (2009) New era of cell-based orthopedic therapies. Tissue Eng Part B Rev 15(2):195–200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Caplan AI (2015) Adult mesenchymal stem cells: when, where, and How. Stem Cells Int 2015:628767

    Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Lavasani M, Lu A, Thompson SD, Robbins PD, Huard J, Niedernhofer LJ (2013) Isolation of muscle-derived stem/progenitor cells based on adhesion characteristics to collagen-coated surfaces. Methods Mol Biol 976:53–65

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Matic I, Antunovic M, Brkic S, Josipovic P, Mihalic KC, Karlak I, Ivkovic A, Marijanovic I (2016) Expression of OCT-4 and SOX-2 in bone marrow-derived human mesenchymal stem cells during osteogenic differentiation. Open Access Maced J Med Sci 4(1):9–16

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G (2005) Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 19(9):1093–1104

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Antunovic M, Kriznik B, Ulukaya E, Yilmaz VT, Mihalic KC, Madunic J, Marijanovic I (2015) Cytotoxic activity of novel palladium-based compounds on leukemia cell lines. Anticancer Drugs 26(2):180–186

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Stevanovic V, Blagojevic Z, Petkovic A, Glisic M, Sopta J, Nikolic V, Milisavljevic M (2013) Semitendinosus tendon regeneration after anterior cruciate ligament reconstruction: can we use it twice? Int Orthop 37(12):2475–2481

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Davies OG, Grover LM, Eisenstein N, Lewis MP, Liu Y (2015) Identifying the cellular mechanisms leading to heterotopic ossification. Calcif Tissue Int 97 (5):432–444

  22. 22.

    Sun L, Hou C, Wu B, Tian M, Zhou X (2013) Effect of muscle preserved on tendon graft on intra-articular healing in anterior cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc 21(8):1862–1868

    Article  PubMed  Google Scholar 

  23. 23.

    Unterhauser FN, Bail HJ, Hoher J, Haas NP, Weiler A (2003) Endoligamentous revascularization of an anterior cruciate ligament graft. Clin Orthop Relat Res 414:276–288

    Article  Google Scholar 

  24. 24.

    Kleiner JB (1986) Origin of replacement cels for the anterior cruciate ligament autograft. J Orthop Res 4(4):466–474

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Caplan AI, Correa D (2011) The MSC: an injury drugstore. Cell Stem Cell 9(1):11–15

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino JP, Lazzari L, Huard J, Peault B (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3(3):301–313

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Shino K (1984) Replacement of the anterior cruciate ligament by an allogenic tendon graft. An experimental study in the dog. J Bone Joint Surg (Br) 66(5):672–681

    CAS  Google Scholar 

  28. 28.

    Kuroda R, Kurosaka M, Yoshiya S, Mizuno K (2000) Localization of growth factors in the reconstructed anterior cruciate ligament: immunohistological study in dogs. Knee Surg Sports Traumatol Arthrosc 8(2):120–126

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Yoshikawa T, Tohyama H, Enomoto H, Matsumoto H, Toyama Y, Yasuda K (2006) Expression of vascular endothelial growth factor and angiogenesis in patellar tendon grafts in the early phase after anterior cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc 14(9):804–810

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Tohyama H, Yoshikawa T, Ju YJ, Yasuda K (2009) Revascularization in the tendon graft following anterior cruciate ligament reconstruction of the knee: its mechanisms and regulation. Chang Gung Med J 32(2):133–139

    PubMed  Google Scholar 

  31. 31.

    Weiler A, Forster C, Hunt P, Falk R, Jung T, Unterhauser FN, Bergmann V, Schmidmaier G, Haas NP (2004) The influence of locally applied platelet-derived growth factor-BB on free tendon graft remodeling after anterior cruciate ligament reconstruction. Am J Sports Med 32(4):881–891

    Article  PubMed  Google Scholar 

  32. 32.

    Hunt P, Rehm O, Weiler A (2006) Soft tissue graft interference fit fixation: observations on graft insertion site healing and tunnel remodeling 2 years after ACL reconstruction in sheep. Knee Surg Sports Traumatol Arthrosc 14(12):1245–1251

    Article  PubMed  Google Scholar 

  33. 33.

    Weiler A, Unterhauser FN, Bail HJ, Huning M, Haas NP (2002) Alpha-smooth muscle actin is expressed by fibroblastic cells of the ovine anterior cruciate ligament and its free tendon graft during remodeling. J Orthop Res 20(2):310–317

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Dustmann M, Schmidt T, Gangey I, Unterhauser FN, Weiler A, Scheffler SU (2008) The extracellular remodeling of free-soft-tissue autografts and allografts for reconstruction of the anterior cruciate ligament: a comparison study in a sheep model. Knee Surg Sports Traumatol Arthrosc 16(4):360–369

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Thomopoulos S, Marquez JP, Weinberger B, Birman V, Genin GM (2006) Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J Biomech 39(10):1842–1851

    Article  PubMed  Google Scholar 

  36. 36.

    Rothrauff BB, Tuan RS (2014) Cellular therapy in bone-tendon interface regeneration. Organogenesis 10(1):13–28

    Article  PubMed  Google Scholar 

  37. 37.

    Kida Y, Morihara T, Matsuda K, Kajikawa Y, Tachiiri H, Iwata Y, Sawamura K, Yoshida A, Oshima Y, Ikeda T, Fujiwara H, Kawata M, Kubo T (2012) Bone marrow-derived cells from the footprint infiltrate into the repaired rotator cuff. J Shoulder Elb Surg 22(2):197–205

    Article  Google Scholar 

  38. 38.

    Kinneberg KR, Galloway MT, Butler DL, Shearn JT (2013) The native cell population does not contribute to central-third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon. J Orthop Res 31(4):638–644

    Article  PubMed  Google Scholar 

  39. 39.

    Bunker DL, Ilie V, Ilie V, Nicklin S (2014) Tendon to bone healing and its implications for surgery. Muscles Ligaments Tendons J 4(3):343–350

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Oguma H, Murakami G, Takahashi-Iwanaga H, Aoki M, Ishii S (2001) Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model. J Orthop Res 19(5):873–880

    CAS  Article  PubMed  Google Scholar 

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Correspondence to Damir Hudetz.

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There is no funding source.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent was obtained from all individual participants included in the study.

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Additional information

An erratum to this article is available at http://dx.doi.org/10.1007/s00264-017-3488-0.

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Ćuti, T., Antunović, M., Marijanović, I. et al. Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction. International Orthopaedics (SICOT) 41, 1189–1198 (2017). https://doi.org/10.1007/s00264-017-3437-y

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Keywords

  • Anterior cruciate ligament
  • Bone-tendon integration
  • Candy-stripe graft
  • Knee
  • Ligamentization
  • Mesenchymal stem cells
  • Pericytes