Lasers in Medical Science

, Volume 28, Issue 5, pp 1281–1288 | Cite as

LLLT improves tendon healing through increase of MMP activity and collagen synthesis

  • Flávia Da Ré Guerra
  • Cristiano Pedrozo Vieira
  • Marcos Santos Almeida
  • Letícia Prado Oliveira
  • Andrea Aparecida de Aro
  • Edson Rosa Pimentel
Original Article


The Achilles tendon has a high incidence of rupture, and the healing process leads to a disorganized extracellular matrix (ECM) with a high rate of injury recurrence. To evaluate the effects of different conditions of low-level laser (LLL) application on partially tenotomized tendons, adult male rats were divided into the following groups: G1, intact; G2, injured; G3, injured + LLL therapy (LLLT; 4 J/cm2 continuous); G4, injured + LLLT (4 J/cm2, 20 Hz); G5, injured; G6, injured + LLLT (4 J/cm2 continuous); and G7, injured + LLLT (4 J/cm2, 20 Hz until the 7th day and 2 kHz from 8 to 14 days). G2, G3, and G4 were euthanized 8 days after injury, and G5, G6, and G7 were euthanized on the 15th day. The quantification of hydroxyproline (HOPro) and non-collagenous protein (NCP), zymography for matrix metalloproteinase (MMP)-2 and MMP-9, and Western blotting (WB) for collagen types I and III were performed. HOPro levels showed a significant decrease in all groups (except G7) when compared with G1. The NCP level increased in all transected groups. WB for collagen type I showed an increase in G4 and G7. For collagen type III, G4 presented a higher value than G2. Zymography for MMP-2 indicated high values in G4 and G7. MMP-9 increased in both treatment groups euthanized at 8 days, especially in G4. Our results indicate that the pulsed LLLT improved the remodeling of the ECM during the healing process in tendons through activation of MMP-2 and stimulation of collagen synthesis.


Achilles tendon Rupture LLLT Collagen Matrix metalloproteinase 9 Matrix metalloproteinase 2 


  1. 1.
    O’Brien M (1997) Structure and metabolism of tendons. Scand J Med Sci Sport 7:55–61CrossRefGoogle Scholar
  2. 2.
    Ahtikoski AM, Koskinen SO, Virtanen P, Kovanen V, Risteli J, Takala TE (2003) Synthesis and degradation of type IV collagen in rat skeletal muscle during immobilization in shortened and lengthened positions. Acta Physiol Scand 177(4):473–481PubMedCrossRefGoogle Scholar
  3. 3.
    Benevides GP, Pimentel ER, Toyama MH et al (2004) Biochemical and biomechanical analysis of tendons of caged and penned chickens. Connect Tissue Res 45:206–215PubMedCrossRefGoogle Scholar
  4. 4.
    Yoon JH, Halper J (2005) Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 5(1):22–34PubMedGoogle Scholar
  5. 5.
    James R, Kesturu G, Balian G, Chhabra AB (2008) Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options. JHS 33:102–112Google Scholar
  6. 6.
    Kannus P, Natri A (1997) Etiology and pathophysiology of tendon ruptures in sports. Scand J Med Sci Sports 7(2):107–112PubMedCrossRefGoogle Scholar
  7. 7.
    Järvinen T et al (2005) Achilles tendon disorders: etiology and epidemiology. Foot Ankle Clin 10(2):255–266PubMedCrossRefGoogle Scholar
  8. 8.
    Enwemeka CS, Reddy GK (2000) The biological effects of laser therapy and other modalities on connective tissue repair processes. Laser Therapy 12:22–30CrossRefGoogle Scholar
  9. 9.
    Lesic A, Bumbasirevic M (2004) Disorders of the Achilles tendon. Current Orthopaedics 18:63–75CrossRefGoogle Scholar
  10. 10.
    Sharma P, Maffuli N (2005) Basic biology of tendon injury and healing. Surgeon 3:309–316PubMedCrossRefGoogle Scholar
  11. 11.
    Chan HKF, Fung DT, Ng GY (2007) Effects of low-voltage microamperage stimulation on tendon healing in rats. J Orthop Sports Phys Ther 37(7):399–403PubMedCrossRefGoogle Scholar
  12. 12.
    Oliveira FS, Pinfild CE, Parizoto NA et al (2009) Effect of low level laser therapy (830 nm) with different therapy regimes on the process of tissue repair in partial lesion calcaneous tendon. Lasers Surg Med 41:271–276PubMedCrossRefGoogle Scholar
  13. 13.
    Chen CH, Tsai LL, Wang YH et al (2009) Low-level laser irradiation promotes cell proliferation and mRNA expression of type I collagen and decorin in porcine Achilles tendon fibroblasts in vitro. J of Orthopaedic Research 27:646–650CrossRefGoogle Scholar
  14. 14.
    Wren TA, Yerby SA, Beaupré GS, Carter DR (2001) Mechanical properties of the human Achilles tendon. Clin Biomech (Bristol, Avon) 16(3):245–251CrossRefGoogle Scholar
  15. 15.
    Demir H, Menku P, Kirnap M (2004) Comparison of the effects of laser, ultrasound, and combined laser + ultrasound treatments in experimental tendon healing. Lasers Surg Med 35:84–89PubMedCrossRefGoogle Scholar
  16. 16.
    Aro AA, Vidal BC, Tomiosso TC, Gomes L, Martiello-Rosa SMG, Pimentel ER (2008) Structural and biomechanical analysis of the effect of immobilization followed by stretching on the calcaneal tendon of rats. Connect Tissue Res 49:443–454PubMedCrossRefGoogle Scholar
  17. 17.
    Enwemeka CS (1991) Connective tissue plasticity: ultrastructural, biomechanical, and morphometric effects of physical factors on intact and regenerating tendons. J Orthop Sports Phys Ther 14:198–212PubMedCrossRefGoogle Scholar
  18. 18.
    Elwakil TF (2007) An in-vivo experimental evaluation of He–Ne laser photostimulation in healing Achilles tendons. Lasers Med Sci 22:53–59PubMedCrossRefGoogle Scholar
  19. 19.
    Pinfild CE, Liebano RE, Hochman BS, Ferreira LM (2005) Helium–neon laser in viability of random skin flap in rats. Lasers Surg Med 37:89–91CrossRefGoogle Scholar
  20. 20.
    Ng GYF, Fung DTC, Leung MCP et al (2004) Ultrastructural comparison of medial collateral ligament repair after single or multiple applications of GaAlAs laser in rats. Lasers Surg Med 35:317–323PubMedCrossRefGoogle Scholar
  21. 21.
    de Aro AA, Pimentel ER (2010) Retainer of rats for laser beam and biocompounds application. MU9000622-4, 16 April 2010Google Scholar
  22. 22.
    Heinegard D, Sommarin Y (1987) Isolation and characterization of proteoglycans. Methods Enzymol 144:319–372PubMedCrossRefGoogle Scholar
  23. 23.
    Bradford MM (1976) A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  24. 24.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76(9):4350–4354PubMedCrossRefGoogle Scholar
  25. 25.
    Marquetti RC, Parizotto NA, Chriguer RS, Perez SEA, 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(8):1274–1280CrossRefGoogle Scholar
  26. 26.
    Stegeman H, Stalder K (1967) Determination of hydroxyproline. Clin Chim Acta 18:267–273CrossRefGoogle Scholar
  27. 27.
    Baranauskas V, Vidal BC, Parizoto NA (1998) Observation of geometric structure of collagen molecules by atomic force microscopy. Appl Biochem Biotechnol 69(2):91–97PubMedCrossRefGoogle Scholar
  28. 28.
    Murphy PG, Loltz BJ, Frank CB, Hart DA (1994) Influence of exogenous growth factors on the synthesis and secretion of collagen types I and III by explants or normal and healing rabbit ligaments. Biochem Cell Biol 72:403–409PubMedCrossRefGoogle Scholar
  29. 29.
    Oshiro W, Lou J, Xing X, Tu Y, Manske PR (2003) Flexor tendon healing in the rat: a histologic and gene expression study. J Hand Surg (Am) 28:814–823CrossRefGoogle Scholar
  30. 30.
    Snoek-Van BP, Von Den Hoff JW (2005) Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 38:73–83CrossRefGoogle Scholar
  31. 31.
    Gavish L, Perez L, Gertz SD (2006) Low-level laser irradiation modulates matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells. Lasers Surg Med 38(8):779–786PubMedCrossRefGoogle Scholar
  32. 32.
    Bosman F, Stamenkovic I (2003) Functional structure and composition of the extracellular matrix. J of Pathology 200:423–428CrossRefGoogle Scholar
  33. 33.
    Clutterbuck AL, Harris P, Allaway D et al (2008) Matrix metalloproteinases in inflammatory pathologies of the horse. Vet J 183(1):27–38PubMedCrossRefGoogle Scholar
  34. 34.
    Abrahamsson SO (1991) Matrix metabolism and healing in the flexor tendon. Experimental studies on rabbit tendon. Scand J Plast Reconstr Hand Surg Suppl 23:1–51Google Scholar
  35. 35.
    Yamaguchi M, Hayashi M, Fujita S, Yoshida T, Utsunomiya T, Yamamoto H (2010) Low-energy laser irradiation facilitates the velocity of tooth movement and the expressions of matrix metalloproteinase-9, cathepsin K, and alpha(v) beta(3) integrin in rats. Eur J Orthod 32:131–139PubMedCrossRefGoogle Scholar
  36. 36.
    Gillard JA, Reed MW, Buttle D, Cross SS, Brown NJ (2004) Matrix metalloproteinase activity and immunohistochemical profile of matrix metalloproteinase-2 and −9 and tissue inhibitor of metalloproteinase-1 during human dermal wound healing. Wound Repair Regen 12:295–304PubMedCrossRefGoogle Scholar
  37. 37.
    Karousou E, Vigetti D, Maffulli N (2008) Collagens, proteoglycans, MMP-2, MMP-9 and TIMPs in human Achilles tendon rupture. Clin Orthop Relat Res 466:1577–1582PubMedCrossRefGoogle Scholar
  38. 38.
    Pugliese LS, Medrado AP, Reis SR, Andrade ZA (2003) The influence of low-level laser therapy on biomodulation of collagen and elastic fibers. Pesqui Odontol Bras 17(4):307–313PubMedCrossRefGoogle Scholar
  39. 39.
    Amiel D, Akeson W, Harwood FL et al (1983) Stress deprivation effect on metabolic turnover of medial collateral ligament collagen: a comparison between nine- and 12-week immobilization. Clin Orthop 172:265–270PubMedGoogle Scholar
  40. 40.
    Aro AA, Vidal BC, Biancalana A, Tolentino FT, Gomes L, Mattiello SM, Pimentel ER (2012) Analysis of the deep digital flexor tendon in rats submitted to stretching after immobilization. Connect Tissue Res 53(1):29–38PubMedCrossRefGoogle Scholar
  41. 41.
    Vieira CP, de Aro AA, de Almeida MS, de Mello GC, Antunes E, Rosa Pimentel E (2012) Effects of acute inflammation induced in the rat paw on the deep digital flexor tendon. Connect Tissue Res 53(2):160–168PubMedCrossRefGoogle Scholar
  42. 42.
    Oakes BW (2003) Tissue healing and repairs: tendons and ligaments. In: Frontera WR (ed) Rehabilitation of sports injuries: scientific basis. Blackwell, Boston, pp 56–98Google Scholar
  43. 43.
    Sharma P, Maffulli N (2006) Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact 6(2):181–190PubMedGoogle Scholar
  44. 44.
    Salate ACB, Barbosa G, Gaspar P et al (2005) Effect of In-Ga-Al-P diode laser irradiation on angiogenesis in partial ruptures of Achilles tendon in rats. Photmed Laser Surg 23(5):470–475CrossRefGoogle Scholar

Copyright information

© Springer-Verlag London 2012

Authors and Affiliations

  • Flávia Da Ré Guerra
    • 1
  • Cristiano Pedrozo Vieira
    • 1
  • Marcos Santos Almeida
    • 1
  • Letícia Prado Oliveira
    • 1
  • Andrea Aparecida de Aro
    • 1
  • Edson Rosa Pimentel
    • 1
  1. 1.Department of Anatomy, Cell Biology and Physiology and Biophysics, Institute of BiologyUniversity of Campinas—UNICAMPCampinasBrazil

Personalised recommendations