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International Orthopaedics

, Volume 40, Issue 7, pp 1523–1530 | Cite as

Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ

  • Weili Fu
  • Qi Li
  • Xin Tang
  • Gang Chen
  • Chenghao Zhang
  • Jian LiEmail author
Original Paper

Abstract

Purpose

It has been reported that the anterior cruciate ligament (ACL) has certain self-healing ability after acute injury or with primary suture repair. Many studies have confirmed that a remnant preservation technique with ACL reconstruction contributes to biological augmentation for ACL healing. However, it remains unclear whether mesenchymal stem cells (MSC) reside in ACL remnants in situ. The aim of this study was to investigate the methods of culture and identification of MSC derived from the remnants of ACL rupture patients and to analyse these MSC’s properties.

Methods

The cells of ACL remnants from the ACL rupture patients were isolated by the methods of enzymatic digestion and cultured in vitro to the third passage under the microscope to observe their morphology and growth status. The third passage of isolated cells was analysed for the identification of immunophenotype, osteogenic, adipogenic and chondrogenic differentiation.

Results

On the third to fifth days of in vitro culture, a few cells of long fusiform shape appeared and were adherent to the plastic walls. On the sixth to ninth days, cells clustered and colonies were observed. The third passage cells showed uniform cell morphology and good proliferation, with appearance of the typical surface markers of MSC, CD29, CD44, CD90 and CD105. The surface markers of CD34 and CD45 of haematopoietic stem cells were not expressed. Under appropriate conditions of in vitro culture, isolated cells could be differentiated into osteoblasts that deposit mineralised matrix and express early osteogenic markers, adipocytes that accumulate lipid droplets in cytoplasm and chondrocytes that secrete chondrogenic-specific matrix aggrecan and collagen II. Real-time polymerase chain reaction (PCR) analysis demonstrated that the specific mRNA expression of osteogenesis, adipogenesis and chondrogenesis increased significantly compared with the control groups at day zero.

Conclusions

Stem cells derived in situ from the human ACL stump were successfully isolated and characterised. Those isolated cells were identified as MSC according to their adherent ability, morphology, surface markers and multilineage differentiation potential. MSC derived from ACL remnants could be a potential source of seeding cells for ligament regeneration.

Keywords

Anterior cruciate ligament remnants Mesenchymal stem cells Tissue-specific stem cells Multipotent differentiation Ligament regeneration 

Notes

Acknowledgments

This study was funded by National Natural Science Foundation of China (No. 81301560), China Postdoctoral Science Foundation (No. 2012 M521698).

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Stevanović V, Blagojević Z, Petković A, Glišić M, Sopta J, Nikolić V, Milisavljević M (2013) Semitendinosus tendon regeneration after anterior cruciate ligament reconstruction: can we use it twice? Int Orthop 37:2475–2481CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sun L, Wu B, Tian M, Liu B, Luo Y (2013) Comparison of graft healing in anterior cruciate ligament reconstruction with and without a preserved remnant in rabbits. Knee 20:537–544CrossRefPubMedGoogle Scholar
  3. 3.
    Tashman S, Kopf S, Fu FH (2008) The kinematic basis of ACL reconstruction. Oper Tech Sports Med 16:116–118CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Oiestad BE, Holm I, Aune AK, Gunderson R, Myklebust G, Engebretsen L, Fosdahl MA, Risberg M (2010) Knee function and prevalence of knee osteoarthritis after anterior cruciate ligament reconstruction: a prospective study with 10 to 15 years of follow-up. Am J Sports Med 38:2201–2210CrossRefPubMedGoogle Scholar
  5. 5.
    Chen CH (2009) Graft healing in anterior cruciate ligament reconstruction. Sports Med Arthrosc Rehabil Ther Technol 1:21CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Schwarting T, Benölken M, Ruchholtz S, Frink M, Lechler P (2015) Bone morphogenetic protein-7 enhances bone-tendon integration in a murine in vitro co-culture model. Int Orthop 39:799–805CrossRefPubMedGoogle Scholar
  7. 7.
    Struewer J, Roessler PP, Schuettler KF, Ruppert V, Stein T, Timmesfeld N, Paletta JR, Efe T (2014) Influence of cyclical mechanical loading on osteogenic markers in an osteoblast-fibroblast co-culture in vitro: tendon-to-bone interface in anterior cruciate ligament reconstruction. Int Orthop 38:1083–1089CrossRefPubMedGoogle Scholar
  8. 8.
    Leong NL, Petrigliano FA, McAllister DR (2014) Current tissue engineering strategies in anterior cruciate ligament reconstruction. J Biomed Mater Res A 102:1614–1624CrossRefPubMedGoogle Scholar
  9. 9.
    Yang G, Rothrauff BB, Tuan RS (2013) Tendon and ligament regeneration and repair: clinical relevance and developmental paradigm. Birth Defects Res C Embryo Today 99:203–222CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Park SY, Oh H, Park SW, Lee JH, Lee SH, Yoon KH (2012) Clinical outcomes of remnant-preserving augmentation versus double-bundle reconstruction in the anterior cruciate ligament reconstruction. Arthroscopy 28:1833–1841CrossRefPubMedGoogle Scholar
  11. 11.
    Kazusa H, Nakamae A, Ochi M (2013) Augmentation technique for anterior cruciate ligament injury. Clin Sports Med 32:127–140CrossRefPubMedGoogle Scholar
  12. 12.
    Wu B, Zhao Z, Li S, Sun L (2013) Preservation of remnant attachment improves graft healing in a rabbit model of anterior cruciate ligament reconstruction. Arthroscopy 29:1362–1371CrossRefPubMedGoogle Scholar
  13. 13.
    Eguchi A, Adachi N, Nakamae A, Usman MA, Deie M, Ochi M (2014) Proprioceptive function after isolated single-bundle posterior cruciate ligament reconstruction with remnant preservation for chronic posterior cruciate ligament injuries. Orthop Traumatol Surg Res 100:303–308CrossRefPubMedGoogle Scholar
  14. 14.
    Lee DC, Shon OJ, Kwack BH, Lee SJ (2013) Proprioception and clinical results of anterolateral single-bundle posterior cruciate ligament reconstruction with remnant preservation. Knee Surg Relat Res 25:126–132CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kim SJ, Kim SH, Chun YM, Hwang BY, Choi DH, Yoon JY (2012) Clinical comparison of conventional and remnant-preserving transtibial single-bundle posterior cruciate ligament reconstruction combined with posterolateral corner reconstruction. Am J Sports Med 40:640–649CrossRefPubMedGoogle Scholar
  16. 16.
    Lui PP (2013) Identity of tendon stem cells--how much do we know? J Cell Mol Med 17:55–64CrossRefPubMedGoogle Scholar
  17. 17.
    Lui PP, Chan KM (2011) Tendon-derived stem cells (TDSCs): from basic science to potential roles in tendon pathology and tissue engineering applications. Stem Cell Rev 7:883–897CrossRefPubMedGoogle Scholar
  18. 18.
    Fu WL, Zhou CY, Yu JK (2014) A new source of mesenchymal stem cells for articular cartilage repair: MSCs derived from mobilized peripheral blood share similar biological characteristics in vitro and chondrogenesis in vivo as MSCs from bone marrow in a rabbit model. Am J Sports Med 42:592–601CrossRefPubMedGoogle Scholar
  19. 19.
    Fu WL, Zhang JY, Fu X, Duan XN, Leung KK, Jia ZQ, Wang WP, Zhou CY, Yu JK (2012) Comparative study of the biological characteristics of mesenchymal stem cells from bone marrow and peripheral blood of rats. Tissue Eng Part A 18:1793–1803CrossRefPubMedGoogle Scholar
  20. 20.
    Segawa Y, Muneta T, Makino H, Nimura A, Mochizuki T, Ju YJ, Ezura Y, Umezawa A, Sekiya I (2009) Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res 27:435–441CrossRefPubMedGoogle Scholar
  21. 21.
    Asari T, Furukawa K, Tanaka S, Kudo H, Mizukami H, Ono A, Numasawa T, Kumagai G, Motomura S, Yagihashi S, Toh S (2012) Mesenchymal stem cell isolation and characterization from human spinal ligaments. Biochem Biophys Res Commun 417:1193–1199CrossRefPubMedGoogle Scholar
  22. 22.
    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, Péault B (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3:301–313CrossRefPubMedGoogle Scholar
  23. 23.
    da Silva Meirelles L, Caplan AI, Nardi NB (2008) In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26:2287–2299CrossRefPubMedGoogle Scholar
  24. 24.
    Caplan AI (2008) All MSCs are pericytes? Cell Stem Cell 3:229–230CrossRefPubMedGoogle Scholar
  25. 25.
    Mifune Y, Matsumoto T, Ota S, Nishimori M, Usas A, Kopf S, Kuroda R, Kurosaka M, Fu FH, Huard J (2012) Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. Cell Transplant 21:1651–1665CrossRefPubMedGoogle Scholar
  26. 26.
    Nohmi S, Yamamoto Y, Mizukami H, Ishibashi Y, Tsuda E, Maniwa K, Yagihashi S, Motomura S, Toh S, Furukawa K (2012) Post injury changes in the properties of mesenchymal stem cells derived from human anterior cruciate ligaments. Int Orthop 36:1515–1522CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kwapisz A, Chojnacki M, Domzalski M, Grzegorzewski A, Synder M (2014) Do gene expression changes in articular cartilage proteases of the synovial membrane correlate with expression changes of the same genes in systemic blood cells? Int Orthop 38:649–654CrossRefPubMedGoogle Scholar
  28. 28.
    Morito T, Muneta T, Hara K, Ju YJ, Mochizuki T, Makino H, Umezawa A, Sekiya I (2008) Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford) 47:1137–1143CrossRefGoogle Scholar
  29. 29.
    Zhang J, Pan T, Im HJ, Fu FH, Wang JH (2011) Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Med 9:68CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhang J, Wang JH (2010) Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord 11:10CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bi Y, Ehirchiou D, Kilts TM, Inkson CA, Embree MC, Sonoyama W, Li L, Leet AI, Seo BM, Zhang L, Shi S, Young MF (2007) Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med 13:1219–1227CrossRefPubMedGoogle Scholar
  32. 32.
    Pauly S, Klatte F, Strobel C, Schmidmaier G, Greiner S, Scheibel M, Wildemann B (2010) Characterization of tendon cell cultures of the human rotator cuff. Eur Cell Mater 20:84–97PubMedGoogle Scholar
  33. 33.
    Van Eijk F, Saris DB, Riesle J, Willems WJ, Van Blitterswijk CA, Verbout AJ, Dhert WJ (2004) Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source. Tissue Eng 10:893–903CrossRefPubMedGoogle Scholar
  34. 34.
    Cheng MT, Yang HW, Chen TH, Lee OK (2009) Isolation and characterization of multipotent stem cells from human cruciate ligaments. Cell Prolif 42:448–460CrossRefPubMedGoogle Scholar
  35. 35.
    Cheng MT, Liu CL, Chen TH, Lee OK (2010) Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering. Tissue Eng Part A 16:2237–2253CrossRefPubMedGoogle Scholar
  36. 36.
    Randelli P, Conforti E, Piccoli M, Ragone V, Creo P, Cirillo F, Masuzzo P, Tringali C, Cabitza P, Tettamanti G, Gagliano N, Anastasia L (2013) Isolation and characterization of 2 new human rotator cuff and long head of biceps tendon cells possessing stem cell-like self-renewal and multipotential differentiation capacity. Am J Sports Med 41:1653–1664CrossRefPubMedGoogle Scholar

Copyright information

© SICOT aisbl 2015

Authors and Affiliations

  • Weili Fu
    • 1
  • Qi Li
    • 1
  • Xin Tang
    • 1
  • Gang Chen
    • 1
  • Chenghao Zhang
    • 1
  • Jian Li
    • 1
    Email author
  1. 1.Department of Orthopaedics, West China HospitalSichuan UniversityChengduChina

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