Skip to main content
SpringerLink
Log in

In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers

  • Experimental Study
  • Published:
Knee Surgery, Sports Traumatology, Arthroscopy Aims and scope

Abstract

Purpose

Results of in vitro cell models are commonly used to promote new therapies (e.g., platelet-rich plasma), and clinicians have to be aware of the specific limitations of such models. To gain a sufficient and effective cell load, many current in vitro models use cells multiplied through various passages. This is especially important in tendon-like cell (TLC) models, since native tendon tissue is not available unlimited and contains limited amount of tenocytes. The purpose was to determine the occurrence of phenotypic changes following extended monolayer culture of TLCs, according to cell-passage number.

Methods

Tendon samples were obtained from 15 healthy patients undergoing biceps tenodesis. Tendons were digested and cultured (monolayer) for six passages. Tendon-specific markers (collagens I and III, decorin, tenascin-C, and tenomodulin) and their histology were analyzed using gene expression and protein content assays. Native cells, the cells cultured and cells passaged one to six times were analyzed at each passage.

Results

Gene expression of types I and III collagen of cultured TLCs significantly decreased after two passages. Gene expression of decorin, tenascin-C, and tenomodulin exhibited a trend of decreased gene expression with increased passage. Protein levels of types I and III collagen and decorin decreased after four passages.

Conclusions

The significant findings let conclude that tenocyte-like cells obtained from human LHB can be maintained in monolayer culture at low passages, before the signs of phenotypic drift are present. But researchers must be aware of rapid phenotypic drift at higher passage numbers. Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if TLCs passaged multiple times are used. The clinical relevance of this data is that understanding of in vitro TLC models, and their limitations may finally help the clinician to judge the potential of experimental data of new biologic treatment options.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Almarza AJ, Augustine SM, Woo SL (2008) Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts. Ann Biomed Eng 36(12):1927–1933

    Article  PubMed  Google Scholar 

  2. Bernard-Beaubois K, Hecquet C, Houcine O, Hayem G, Adolphe M (1997) Culture and characterization of juvenile rabbit tenocytes. Cell Biol Toxicol 13(2):103–113

    Article  PubMed  CAS  Google Scholar 

  3. de Wreede R, Ralphs JR (2009) Deposition of collagenous matrices by tendon fibroblasts in vitro: a comparison of fibroblast behavior in pellet cultures and a novel three-dimensional Long-term Scaffoldless culture system. Tissue Eng Part A 15(9):2707–2715

    Article  PubMed  Google Scholar 

  4. Denaro V, Ruzzini L, Longo UG, Franceschi F, De Paola B, Cittadini A, Maffulli N, Sgambato A (2010) Effect of dihydrotestosterone on cultured human tenocytes from intact supraspinatus tendon. Knee Surg Sports Traumatol Arthrosc 18(7):971–976

    Article  PubMed  Google Scholar 

  5. Doroski DM, Brink KS, Temenoff JS (2007) Techniques for biological characterization of tissue-engineered tendon and ligament. Biomaterials 28(2):187–202

    Article  PubMed  CAS  Google Scholar 

  6. Fu SC, Cheuk YC, Chan KM, Hung LK, Wong MW (2008) Is cultured tendon fibroblast a good model to study tendon healing? J Orthop Res 26(3):374–383

    Article  PubMed  CAS  Google Scholar 

  7. Joseph M, Maresh CM, McCarthy MB, Kraemer WJ, Ledgard F, Arciero CL, Anderson JM, Nindl BC, Mazzocca AD (2009) Histological and molecular analysis of the biceps tendon long head post-tenotomy. J Orthop Res 27(10):1379–1385

    Article  PubMed  Google Scholar 

  8. Kovacevic D, Rodeo SA (2008) Biological augmentation of rotator cuff tendon repair. Clin Orthop Relat Res 466(3):622–633

    Article  PubMed  Google Scholar 

  9. Kuc IM, Scott PG (1997) Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues. Connect Tissue Res 36(4):287–296

    Article  PubMed  CAS  Google Scholar 

  10. Maffulli N, Yao L, Bestwick C, Bestwick L, Aspden R (2008) Phenotypic drift in human tenocyte culture. J Bone Joint Surg Br 90-B (SUPP_III):494-a

    Google Scholar 

  11. Mazzocca AD, McCarthy MB, Arciero C, Jhaveri A, Obopilwe E, Rincon L, Wyman J, Gronowicz GA, Arciero RA (2007) Tendon and bone responses to a collagen-coated suture material. J Shoulder Elbow Surg 16(5 Suppl):S222–S230

    Article  PubMed  Google Scholar 

  12. Moffat KL, Kwei AS, Spalazzi JP, Doty SB, Levine WN, Lu HH (2009) Novel nanofiber-based scaffold for rotator cuff repair and augmentation. Tissue Eng Part A 15(1):115–126

    Article  PubMed  CAS  Google Scholar 

  13. 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 20:84–97

    PubMed  CAS  Google Scholar 

  14. Pringle GA, Dodd CM (1990) Immunoelectron microscopic localization of the core protein of decorin near the d and e bands of tendon collagen fibrils by use of monoclonal antibodies. J Histochem Cytochem 38(10):1405–1411

    Article  PubMed  CAS  Google Scholar 

  15. Rodeo SA (2007) Biologic augmentation of rotator cuff tendon repair. J Shoulder Elbow Surg 16(5, Supplement 1):S191–S197

    Article  PubMed  Google Scholar 

  16. Schulze-Tanzil G, Mobasheri A, Clegg PD, Sendzik J, John T, Shakibaei M (2004) Cultivation of human tenocytes in high-density culture. Histochem Cell Biol 122(3):219–228

    Article  PubMed  CAS  Google Scholar 

  17. Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ (2001) Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 128(19):3855–3866

    PubMed  CAS  Google Scholar 

  18. Stoll C, John T, Endres M, Rosen C, Kaps C, Kohl B, Sittinger M, Ertel W, Schulze-Tanzil G (2010) Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering. J Orthop Res 28(9):1170–1177

    Article  PubMed  CAS  Google Scholar 

  19. Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD (2009) Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems. BMC Musculoskelet Disord 10:27

    Article  PubMed  Google Scholar 

  20. Teicher BA, Maehara Y, Kakeji Y, Ara G, Keyes SR, Wong J, Herbst R (1997) Reversal of in vivo drug resistance by the transforming growth factor-beta inhibitor decorin. Int J Cancer 71(1):49–58

    Article  PubMed  CAS  Google Scholar 

  21. Tenni R, Viola M, Welser F, Sini P, Giudici C, Rossi A, Tira ME (2002) Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen. Eur J Biochem 269(5):1428–1437

    Article  PubMed  CAS  Google Scholar 

  22. Theisen C, Fuchs-Winkelmann S, Knappstein K, Efe T, Schmitt J, Paletta JR, Schofer MD (2010) Influence of nanofibers on growth and gene expression of human tendon derived fibroblast. Biomed Eng Online 9:9

    Article  PubMed  Google Scholar 

  23. Wong MW, Tang YY, Lee SK, Fu BS, Chan BP, Chan CK (2003) Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor. J Bone Joint Surg Am 85-A(10):1914–1920

    PubMed  Google Scholar 

  24. Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM (2006) Phenotypic drift in human tenocyte culture. Tissue Eng 12(7):1843–1849

    Article  PubMed  CAS  Google Scholar 

  25. Yoon JH, Brooks RL Jr, Zhao JZ, Isaacs D, Halper J (2004) The effects of enrofloxacin on decorin and glycosaminoglycans in avian tendon cell cultures. Arch Toxicol 78(10):599–608

    Article  PubMed  CAS  Google Scholar 

  26. Zhang L, Tran N, Chen HQ, Kahn CJ, Marchal S, Groubatch F, Wang X (2008) Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching. Cell Tissue Res 332(1):101–109

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The University of Connecticut Health Center/New England Musculoskeletal Institute has received direct funding, and material support for this study was provided by Arthrex Inc. (Naples, Fl). The company had no influence on study design, data collection or interpretation of the results. Augustus D Mazzocca receives research support and is a consultant for Arthrex, Inc.; all other authors, their immediate family, and any research foundation with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT, 06030, USA

    Augustus D. Mazzocca, David Chowaniec, Mary Beth McCarthy, Knut Beitzel, Mark P. Cote, William McKinnon & Robert Arciero

Authors
  1. Augustus D. Mazzocca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Chowaniec
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Beth McCarthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Knut Beitzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark P. Cote
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. William McKinnon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert Arciero
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Augustus D. Mazzocca.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mazzocca, A.D., Chowaniec, D., McCarthy, M.B. et al. In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers. Knee Surg Sports Traumatol Arthrosc 20, 1666–1672 (2012). https://doi.org/10.1007/s00167-011-1711-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00167-011-1711-x

Keywords

Search

Navigation