Effect of dihydrotestosterone on cultured human tenocytes from intact supraspinatus tendon

  • Vincenzo Denaro
  • Laura Ruzzini
  • Umile Giuseppe Longo
  • Francesco Franceschi
  • Barbara De Paola
  • Achille Cittadini
  • Nicola Maffulli
  • Alessandro Sgambato
Experimental Study

Abstract

The role of hormones in the pathogenesis of tendinopathy is not well recognised, even though the use of anabolic steroids is correlated with a higher incidence of spontaneous tendon ruptures. The aim of this study was to investigate the effects of dihydrotestosterone (DHT) on human tenocyte cultures from the intact supraspinatus tendon of male subjects. Cultured human tenocytes were seeded into culture plates at a density of 5 × 104 cells per well and incubated for 24 h. Then, 10−9 M–10−7 M DHT or Dulbecco’s modified Eagle’s medium (DMEM) only (control) was added to the culture plate wells. Cell morphology assessment and cell proliferation tests were performed 48, 72 and 96 h after DHT treatment. DHT-treated tenocytes showed an increased proliferation rate at DHT concentration higher than 10−8 M. Differences in cell numbers between control and DHT-treated cells were statistically significant (P < 0.05) after 48 and 72 h of treatment with DHT concentrations of 10−8 and 10−7 M. The tenocytes treated with DHT (10−8 and 10−7 M) became more flattened and polygonal compared to control cells that maintained their fibroblast-like appearance during the experiment at each observation time. In conclusion, in vitro, progressive increasing concentration of DHT at doses greater than 10−8 M had direct effects on male human tenocytes, increasing cell number after 48 and 72 h of treatment, and leading to a dedifferentiated phenotype after 48 h of treatment. This effect can be important during tendon-healing and repair, when active proliferation is required. Our results represent preliminary evidence for a possible correlation between testosterone abuse and shoulder tendinopathy.

Keywords

Tenocytes Dihydrotestosterone Anabolic steroids Tendinopathy Shoulder Sports 

Notes

Conflict of interest statement

The authors declare no conflict of interest.

References

  1. 1.
    Ames PR, Longo UG, Denaro V, Maffulli N (2008) Achilles tendon problems: not just an orthopaedic issue. Disabil Rehabil 30:1646–1650CrossRefPubMedGoogle Scholar
  2. 2.
    Andres BM, Murrell GA (2008) Treatment of tendinopathy: what works, what does not, and what is on the horizon. Clin Orthop Relat Res 466:1539–1554CrossRefPubMedGoogle Scholar
  3. 3.
    Arnoczky SP, Lavagnino M, Egerbacher M, Caballero O, Gardner K (2007) Matrix metalloproteinase inhibitors prevent a decrease in the mechanical properties of stress-deprived tendons: an in vitro experimental study. Am J Sports Med 35:763–769CrossRefPubMedGoogle Scholar
  4. 4.
    Arnoczky SP, Lavagnino M, Egerbacher M, Caballero O, Gardner K, Shender MA (2008) Loss of homeostatic strain alters mechanostat “set point” of tendon cells in vitro. Clin Orthop Relat Res 466:1583–1591CrossRefPubMedGoogle Scholar
  5. 5.
    Benz DJ, Haussler MR, Thomas MA, Speelman B, Komm BS (1991) High-affinity androgen binding and androgenic regulation of alpha 1(I)-procollagen and transforming growth factor-beta steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells. Endocrinology 128:2723–2730CrossRefPubMedGoogle Scholar
  6. 6.
    Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, Casaburi R (1996) The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 335:1–7CrossRefPubMedGoogle Scholar
  7. 7.
    De Mos M, van El B, DeGroot J, Jahr H, van Schie HT, van Arkel ER, Tol H, Heijboer R, van Osch GJ, Verhaar JA (2007) Achilles tendinosis: changes in biochemical composition and collagen turnover rate. Am J Sports Med 35:1549–1556CrossRefPubMedGoogle Scholar
  8. 8.
    Egerbacher M, Arnoczky SP, Caballero O, Lavagnino M, Gardner KL (2008) Loss of homeostatic tension induces apoptosis in tendon cells: an in vitro study. Clin Orthop Relat Res 466:1562–1568CrossRefPubMedGoogle Scholar
  9. 9.
    Er F, Gassanov N, Brandt MC, Madershahian N, Hoppe UC (2009) Impact of dihydrotestosterone on L-type calcium channels in human ventricular cardiomyocytes. Endocr Res 34:59–67CrossRefPubMedGoogle Scholar
  10. 10.
    Evans NA, Bowrey DJ, Newman GR (1998) Ultrastructural analysis of ruptured tendon from anabolic steroid users. Injury 29:769–773CrossRefPubMedGoogle Scholar
  11. 11.
    Franceschi F, Longo UG, Ruzzini L, Morini S, Battistoni F, Dicuonzo G, Maffulli N, Denaro V (2008) Circulating substance P levels and shoulder joint contracture after arthroscopic repair of the rotator cuff. Br J Sports Med 42:742–745CrossRefPubMedGoogle Scholar
  12. 12.
    Franceschi F, Longo UG, Ruzzini L, Papalia R, Rizzello G, Denaro V (2007) To detach the long head of the biceps tendon after tenodesis or not: outcome analysis at the 4-year follow-up of two different techniques. Int Orthop 31:537–545CrossRefPubMedGoogle Scholar
  13. 13.
    Franceschi F, Longo UG, Ruzzini L, Rizzello G, Denaro V (2007) Arthroscopic management of calcific tendinitis of the subscapularis tendon. Knee Surg Sports Traumatol Arthrosc 15:1482–1485CrossRefPubMedGoogle Scholar
  14. 14.
    Franceschi F, Longo UG, Ruzzini L, Rizzello G, Maffulli N, Denaro V (2008) No advantages in repairing a type II superior labrum anterior and posterior (SLAP) lesion when associated with rotator cuff repair in patients over age 50: a randomized controlled trial. Am J Sports Med 36:247–253CrossRefPubMedGoogle Scholar
  15. 15.
    Franceschi F, Longo UG, Ruzzini L, Rizzello G, Maffulli N, Denaro V (2007) The Roman Bridge: a “double pulley—suture bridges” technique for rotator cuff repair. BMC Musculoskelet Disord 8:123CrossRefPubMedGoogle Scholar
  16. 16.
    Franceschi F, Longo UG, Ruzzini L, Rizzello G, Maffulli N, Denaro V (2008) Soft tissue tenodesis of the long head of the biceps tendon associated to the Roman Bridge repair. BMC Musculoskelet Disord 9:78CrossRefPubMedGoogle Scholar
  17. 17.
    Franceschi F, Ruzzini L, Longo UG, Martina FM, Zobel BB, Maffulli N, Denaro V (2007) Equivalent clinical results of arthroscopic single-row and double-row suture anchor repair for rotator cuff tears: a randomized controlled trial. Am J Sports Med 35:1254–1260CrossRefPubMedGoogle Scholar
  18. 18.
    Gardner K, Arnoczky SP, Caballero O, Lavagnino M (2008) The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: an in vitro experimental study. Disabil Rehabil 30:1523–1529CrossRefPubMedGoogle Scholar
  19. 19.
    Hartgens F, Kuipers H (2004) Effects of androgenic–anabolic steroids in athletes. Sports Med 34:513–554CrossRefPubMedGoogle Scholar
  20. 20.
    Inhofe PD, Grana WA, Egle D, Min KW, Tomasek J (1995) The effects of anabolic steroids on rat tendon. An ultrastructural, biomechanical, and biochemical analysis. Am J Sports Med 23:227–232CrossRefPubMedGoogle Scholar
  21. 21.
    Jenkins C, Milsted A, Doane K, Meszaros G, Toot J, Ely D (2007) A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production. BMC Cardiovasc Disord 7:13CrossRefPubMedGoogle Scholar
  22. 22.
    Kane TP, Ismail M, Calder JD (2008) Topical glyceryl trinitrate and noninsertional Achilles tendinopathy: a clinical and cellular investigation. Am J Sports Med 36:1160–1163CrossRefPubMedGoogle Scholar
  23. 23.
    Karousou E, Ronga M, Vigetti D, Passi A, Maffulli N (2008) Collagens, proteoglycans, MMP-2, MMP-9 and TIMPs in human achilles tendon rupture. Clin Orthop Relat Res 466:1577–1582CrossRefPubMedGoogle Scholar
  24. 24.
    Knobloch K (2008) The use of a neovascularization score to predict clinical severity in Achilles tendinopathy. Am J Sports Med 36:395–397CrossRefPubMedGoogle Scholar
  25. 25.
    Knobloch K, Grasemann R, Spies M, Vogt PM (2008) Midportion achilles tendon microcirculation after intermittent combined cryotherapy and compression compared with cryotherapy alone: a randomized trial. Am J Sports Med 36:2128–2138CrossRefPubMedGoogle Scholar
  26. 26.
    Knobloch K, Schreibmueller L, Longo UG, Vogt PM (2008) Eccentric exercises for the management of tendinopathy of the main body of the Achilles tendon with or without an AirHeeltrade mark Brace. A randomized controlled trial. B: effects of compliance. Disabil Rehabil 30:1692–1696CrossRefPubMedGoogle Scholar
  27. 27.
    Knobloch K, Schreibmueller L, Longo UG, Vogt PM (2008) Eccentric exercises for the management of tendinopathy of the main body of the Achilles tendon with or without the AirHeeltrade mark Brace. A randomized controlled trial. A: effects on pain and microcirculation. Disabil Rehabil 30:1685–1691CrossRefPubMedGoogle Scholar
  28. 28.
    Lavagnino M, Arnoczky SP, Elvin N, Dodds J (2008) Patellar tendon strain is increased at the site of the jumper’s knee lesion during knee flexion and tendon loading: results and cadaveric testing of a computational model. Am J Sports Med 36:2110–2118CrossRefPubMedGoogle Scholar
  29. 29.
    Longo UG, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli N, Denaro V (2009) Characteristics at Haematoxylin and Eosin staining of ruptures of the long head of the biceps tendon. Br J Sports Med 43:603–607CrossRefPubMedGoogle Scholar
  30. 30.
    Longo UG, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli N, Denaro V (2008) Histopathology of the supraspinatus tendon in rotator cuff tears. Am J Sports Med 36:533–538CrossRefPubMedGoogle Scholar
  31. 31.
    Longo UG, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli N, Forriol F, Denaro V (2007) Light microscopic histology of supraspinatus tendon ruptures. Knee Surg Sports Traumatol Arthrosc 15:1390–1394CrossRefPubMedGoogle Scholar
  32. 32.
    Longo UG, Garau G, Denaro V, Maffulli N (2008) Surgical management of tendinopathy of biceps femoris tendon in athletes. Disabil Rehabil 30:1602–1607CrossRefPubMedGoogle Scholar
  33. 33.
    Longo UG, Olivia F, Denaro V, Maffulli N (2008) Oxygen species and overuse tendinopathy in athletes. Disabil Rehabil 30:1563–1571CrossRefPubMedGoogle Scholar
  34. 34.
    Longo UG, Ramamurthy C, Denaro V, Maffulli N (2008) Minimally invasive stripping for chronic Achilles tendinopathy. Disabil Rehabil 30:1709–1713CrossRefPubMedGoogle Scholar
  35. 35.
    Maffulli N, Ajis A, Longo UG, Denaro V (2007) Chronic rupture of tendo Achillis. Foot Ankle Clin 12:583–596CrossRefPubMedGoogle Scholar
  36. 36.
    Maffulli N, Longo UG (2008) Conservative management for tendinopathy: is there enough scientific evidence? Rheumatology (Oxford) 47:390–391CrossRefGoogle Scholar
  37. 37.
    Maffulli N, Longo UG (2008) How do eccentric exercises work in tendinopathy? Rheumatology (Oxford) 47:1444–1445CrossRefGoogle Scholar
  38. 38.
    Maffulli N, Testa V, Capasso G, Oliva F, Panni AS, Longo UG, King JB (2008) Surgery for chronic Achilles tendinopathy produces worse results in women. Disabil Rehabil 30:1714–1720CrossRefPubMedGoogle Scholar
  39. 39.
    Magra M, Maffulli N (2005) Matrix metalloproteases: a role in overuse tendinopathies. Br J Sports Med 39:789–791CrossRefPubMedGoogle Scholar
  40. 40.
    Magra M, Maffulli N (2005) Molecular events in tendinopathy: a role for metalloproteases. Foot Ankle Clin 10:267–277CrossRefPubMedGoogle Scholar
  41. 41.
    Malliaras P, Richards PJ, Garau G, Maffulli N (2008) Achilles tendon Doppler flow may be associated with mechanical loading among active athletes. Am J Sports Med 36:2210–2215CrossRefPubMedGoogle Scholar
  42. 42.
    Markova MS, Zeskand J, McEntee B, Rothstein J, Jimenez SA, Siracusa LD (2004) A role for the androgen receptor in collagen content of the skin. J Invest Dermatol 123:1052–1056CrossRefPubMedGoogle Scholar
  43. 43.
    Marqueti RC, Parizotto NA, Chriguer RS, Perez SE, Selistre-de-Araujo HS (2006) Androgenic–anabolic steroids associated with mechanical loading inhibit matrix metallopeptidase activity and affect the remodeling of the achilles tendon in rats. Am J Sports Med 34:1274–1280CrossRefPubMedGoogle Scholar
  44. 44.
    Miles JW, Grana WA, Egle D, Min KW, Chitwood J (1992) The effect of anabolic steroids on the biomechanical and histological properties of rat tendon. J Bone Joint Surg Am 74:411–422PubMedGoogle Scholar
  45. 45.
    Millar NL, Wei AQ, Molloy TJ, Bonar F, Murrell GA (2008) Heat shock protein and apoptosis in supraspinatus tendinopathy. Clin Orthop Relat Res 466:1569–1576CrossRefPubMedGoogle Scholar
  46. 46.
    Raivio T, Tapanainen JS, Kunelius P, Jänne OA (2002) Serum androgen bioactivity during 5alpha-dihydrotestosterone treatment in elderly men. J Androl 23:919–921PubMedGoogle Scholar
  47. 47.
    Ray R, Herring CM, Markel TA, Crisostomo PR, Wang M, Weil B, Lahm T, Meldrum DR (2008) Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production. Am J Physiol Regul Integr Comp Physiol 294:R1498–R1503PubMedGoogle Scholar
  48. 48.
    Schwarz R, Colarusso L, Doty P (1976) Maintenance of differentiation in primary cultures of avian tendon cells. Exp Cell Res 102:63–71CrossRefPubMedGoogle Scholar
  49. 49.
    Scott A, Lian O, Bahr R, Hart DA, Duronio V, Khan KM (2008) Increased mast cell numbers in human patellar tendinosis: correlation with symptom duration and vascular hyperplasia. Br J Sports Med 42:753–757CrossRefPubMedGoogle Scholar
  50. 50.
    Sharma P, Maffulli N (2006) Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact 6:181–190PubMedGoogle Scholar
  51. 51.
    Sharma P, Maffulli N (2008) Tendinopathy and tendon injury: the future. Disabil Rehabil 30:1733–1745CrossRefPubMedGoogle Scholar
  52. 52.
    Singh R, Artaza JN, Taylor WE, Gonzalez-Cadavid NF, Bhasin S (2003) Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology 144:5081–5088CrossRefPubMedGoogle Scholar
  53. 53.
    Xu Y, Murrell GA (2008) The basic science of tendinopathy. Clin Orthop Relat Res 466:1528–1538CrossRefPubMedGoogle Scholar
  54. 54.
    Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM (2006) Phenotypic drift in human tenocyte culture. Tissue Eng 12:1843–1849CrossRefPubMedGoogle Scholar
  55. 55.
    Yoo HG, Kim JS, Lee SR, Pyo HK, Moon HI, Lee JH, Kwon OS, Chung JH, Kim KH, Eun HC, Cho KH (2006) Perifollicular fibrosis: pathogenetic role in androgenetic alopecia. Biol Pharm Bull 29:1246–1250CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Vincenzo Denaro
    • 1
  • Laura Ruzzini
    • 1
  • Umile Giuseppe Longo
    • 1
  • Francesco Franceschi
    • 1
  • Barbara De Paola
    • 2
  • Achille Cittadini
    • 2
  • Nicola Maffulli
    • 3
  • Alessandro Sgambato
    • 2
  1. 1.Department of Orthopaedic and Trauma SurgeryCampus Biomedico UniversityRomeItaly
  2. 2.“Giovanni XXIII” Cancer Research Center-Institute of General PathologyCatholic UniversityRomeItaly
  3. 3.Centre for Sports and Exercise MedicineQueen Mary University of London, Mile End HospitalLondonUK

Personalised recommendations