Tendon injuries - part 9 ppt

tionnaire-based instrument to measure the severity of Achilles tendinopathy, the VISA-A [84]. There is a need for a quantitative index of pain and function in patients with Achilles tendinopathy. The VISA-A questionnaire appears a valid, reliable and easy-to-administer measure of the severity of Achilles tendinopathy, and seems suitable for both clinical rating and quantitative research. Conclusions Surgery for chronic overuse tendon conditions, even when successful, does not reconstitute a normal tendon. Mostly, the result is functionally satisfactory despite mor- phological differences and biomechanical weakness com- pared to a normal tendon. The therapeutic use of growth factors by gene transfer, it seems, may produce a tendon which is biologically, biomechanically, biochemically, and physiologically more “normal.” References 1. Józsa L, Kannus P. (1997) Human Tendon: Anatomy, Phys- iology and Pathology. Champaign, IL: Human Kinetics. 2. Maffulli N, Benazzo F. (2000) Basic sciences of tendons. Sports Med Arthroscopy Rev. 8:1–5. 3. Birk DE, Zycband EI, Woodruff S, Winkelmann DA, Trelstad RL. (1997) Collagen fibrillogenesis in situ: fibril segments become long fibrils as the developing tendon matures. Developmental Dynamics. 208:291–298. 4. Puddu G, Ippolito E, Postacchini F. (1976) A classification of Achilles tendon disease. Am J Sports Med. 4:145–150. 5. Selvanetti A, Cipolla M, Puddu, G. (1997) Overuse tendon injuries: basic science and classification. Operative Tech- niques Sports Med. 5:110–117. 6. Khan KM, Maffulli N. (1998) Tendinopathy: an Achilles’ heel for athletes and clinicians. Clin J Sport Med. 8:151–154. 7. Maffulli N, Khan KM, Puddu G. (1998) Overuse tendon conditions: time to change a confusing terminology. Arthroscopy. 14:840–843. 8. Maffulli N, Binfield PM, King JB. (1998) Tendon problems in athletic individuals. J Bone Joint Surg. (Am) 80-A:142– 143. 9. Astrom M, Rausing A. (1995) Chronic Achilles tendinopa- thy. a survey of surgical and histopathologic findings. Clin Orthop Rel Res. 316:151–164. 10. Leadbetter WB. (1992) Cell-matrix response in tendon injury. Clin Sports Med. 11:533–578. 11. Movin T, Gad A, Reinholt FP, et al. (1997) Tendon pathol- ogy in long-standing achillodynia. biopsy findings in 40 patients. Acta Orthop Scand. 68:170–175. 12. Khan KM, Cook JL, Bonar F, et al. (1999) Histopathology of common tendinopathies. update and implications for clinical management. Sports Med. 27:393–408. 13. Kvist M. (1991) Achilles tendon injuries in athletes. Ann Chirurg Gynaecol. 80:188–201. 274 N. Maffulli et al. 14. Józsa L, Balint J, Kannus P, et al. (1993) Mechanoreceptors in human myotendinous junction. Muscle Nerve. 16: 453– 457. 15. Khan K, Jill L, Cook PT. (2000) Overuse tendon injuries: where does the pain come from. Sports Med Arthroscopy Rev. 8:17–31. 16. Khan KM, Cook JL, Maffulli N, et al. (2000) Where is the pain coming from in tendinopathy? it may be biochemical, not only structural, in origin. Br J Sports Med. 34:81–83. 17. Alfredson H,Thorsen K, Lorentzon R.(1999) In situ micro- dialysis in tendon tissue: high levels of glutamate, but not prostaglandin E2 in chronic Achilles tendon pain. Knee Surg Sports Traumatol Arthroscopy. 7:378–381. 18. Baker BE. (1984) Current concepts in the diagnosis and treatment of musculotendinous injuries. Med Sci Sports Exerc. 16:323–327. 19. Blazina ME, Kerlan RK, Jobe FW, Carter VS. (1973) Jumper’s knee. Orthop Clin North Am. 4:665–672. 20. Leadbetter WB, Mooar OA, Lane GJ, Lee SJ. (1992) The surgical treatment of tendinitis. Clinical rationale and bio- logical basis. Clin Sports Med. 11:679–712. 21. Martens M, Wouters P, Burssens A, Mulier JC. (1982) Patellar tendinitis: pathology and results of treatment. Acta Orthop Scand. 53:445–450. 22. Phillips BB. (1992) Traumatic disorders of tendon. In: Cren- shaw AH, ed. Campbell’s Operative Orthopaedics. St. Louis: Mosby-Year Book; 1921–1922. 23. Orava S, Osterback L, Hurme M. (1986) Surgical treatment of patellar tendon pain in athletes. Br J Sports Med. 20: 167–169. 24. Paavola M, Orava S, Leppilahti J, et al. (2000) Chronic Achilles tendon overuse injury: complications after surgical treatment. an analysis of 432 consecutive patients. Am J Sports Med. 28(1):77–82. 25. Paavola M, Kannus P, Paakkala T, et al. (2000) Long-term prognosis of patients with Achilles tendinopathy. Am J Sports Med. 28(5):634–642. 26. Maffulli N, Binfield PM, Moore D, King JB. (1999) Surgical decompression of chronic central core lesions of the Achilles tendon. Am J Sports Med. 27:747–752, 27. Gigante A, Specchia N, Rapali S, Ventura A, de Palma L. (1996) Fibrillogenesis in tendon healing: an experimental study. Bollettino della Societa’ Italiana di Biologia Speri- mentale. 72(7–8):203–210. 28. Wang ED. (1998) Tendon repair. J Hand Ther. 11:105–110. 29. Reddy GK, Stehno-Bittel L, Enwemeka CS. (1999) Matrix remodeling in healing rabbit Achilles tendon. Wound Repair Regeneration. 7:518–527. 30. Maffulli N, Ewen SW, Waterston SW, Reaper J, Barrass V. (2000) Tenocytes from ruptured and tendinopathic achilles tendons produce greater quantities of type III collagen than tenocytes from normal Achilles tendons. an in vitro model of human tendon healing. Am J Sports Med. 28(4):499–505. 31. Józsa L, Reffy A, Kannus P, Demel S, Elek E. (1990) Patho- logical alterations in human tendons. Arch Orthop Trauma Surg. 110(1):15–21. 32. Grotendorst GR. (1988) Growth factors as regulators of wound repair. Int J Tissue React. 10(6):337–344. 33. Duffy FJ Jr, Seiler JG, Gelberman RH, Hergrueter CA. (1995) Growth factors and canine flexor tendon healing: initial studies in uninjured and repair models. J Hand Surg. (Am) 20(4):645–649. 34. Pierce GF, Tarpley JE, Tseng J, Bready J, Chang D, Kenney WC, Rudolph R, Robson MC, Vande Berg J, Reid P. (1995) Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds. J Clin Investig. 96(3):1336–1350. 35. Chan BP, Chan KM, Maffulli N, Webb S, Lee KK. (1997) Effect of basic fibroblast growth factor. an in vitro study of tendon healing. Clin Orthop and RelRes. (342):239–247. 36. Gabra N, Khayat A, Calabresi P, Khayat A. (1994) Detec- tion of elevated basic fibroblast growth factor during early hours of in vitro angiogenesis using a fast ELISA immunoassay. Biochem Biophys Res Commun. 205:1423– 1430. 37. Enzura Y, Rosen V, Nifuji A. (1996) Induction of hypertro- phy in healing patellar tendon by implantation of human recombinant BMP 12. J Bone Mineral Res. 11:401. 38. Ignotz RA, Massague J. (1986 Mar) Transforming growth factor-beta stimulates the expression of fibronectin and col- lagen and their incorporation into the extracellular matrix. J Biol Chem. 261(9):1337–1345. 39. Kvist H, Kvist M. (1980) The operative treatment of chronic calcaneal paratenonitis. J Bone Joint Surg. (Br) 62:353–357. 40. Leppilahti J, Orava S, Karpakka J, et al. (1991) Overuse injuries of the Achilles tendon. Ann Chirurg Gynaecol. 80:202–207. 41. Rolf C, Movin T. (1997) Etiology, histopathology, and outcome of surgery in achillodynia. Foot Ankle Int. 18: 565–569. 42. Nelen G, Martens M, Burssens A. (1989) Surgical treatment of chronic Achilles tendinitis. Am J Sports Med. 17:754–759. 43. Clancy WG, Heiden EA. (1997) Achilles tendinitis treat- ment. In: The Athletes. Foot and Ankle Clinics Contempo- rary Approaches to the Achilles Tendon, 1083–1095. 44. Leach RE, Schepsis AA,Takai H. (1992) Long-term results of surgical management of Achilles tendinitis in runners. Clin Orthop Rel Res. 282:208–212. 45. Schepsis AA, Leach RE. (1987) Surgical management of Achilles tendinitis. Am J Sports Med. 15:308–315. 46. Testa V, Maffulli N, Capasso G, Bifulco, G. (1996) Percuta- neous longitudinal tenotomy in chronic Achilles tendonitis. Bull Hosp Joint Dis. 54:241–244. 47. Testa V, Maffulli N, Capasso G, Benazzo F. (2002) Manage- ment of Achilles tendinopathy by ultrasound guided per- cutaneous longitudinal tenotomy. Med Sci Sports Exerc. 34:573–580. 48. Astrom M. (1997) On the nature and etiology of chronic Achilles tendinopathy. Lund University, Sweden. 49. Benazzo F, Maffulli N. (2000) An operative approach to Achilles tendinopathy. Sports Med Arthroscopy Rev. 8:96– 101. 50. Clancy WGJ, Neidhart D, Brand RL. (1976) Achilles ten- donitis in runners: a report of five cases. Am J Sports Med. 4:46–57. 51. Friedrich T, Schmidt W, Jungmichel D, et al. (2001) Histopathology in rabbit Achilles tendon after operative tenolysis (longitudinal fiber incisions). Scand J Med Sci Sports. 11(1):4–8. 52. Ljungqvist R. (1967) Subcutaneous partial rupture of the Achilles tendon. Acta Orthop Scand. (Suppl). 53. Maffulli N, Testa V, Capasso G. (1993) Use of a tourniquet in the internal fixation of fractures of the distal part of the fibula. a prospective, randomized trial. J Bone Joint Surg. (Am) 75(5):700–703. 54. Binfield PM, Maffulli N. (1997) Surgical management of common tendinopathies of the lower limb. Sports Exerc Inj. 3:116–122. 55. Maffulli N, Testa V, Capasso G, Bifulco G, Binfield PM. (1997) Results of percutaneous longitudinal tenotomy for Achilles tendinopathy in middle- and long-distance runners. Am J Sports Med. 25:835–840. 56. Williams JG. (1986) Achilles tendon lesions in sport. Sports Med. 3:114–135. 57. Wilcox DK, Bohay DR, Anderson JG. (2000) Treatment of chronic Achilles tendon disorders with flexor hallucis longus tendon transfer/augmentation. Foot Ankle Int. 21(12):1004–1010. 58. Tallon C, Coleman BD, Khan KM, et al. (2001) Outcome of surgery for chronic Achilles tendinopathy: a critical review. Am J Sports Med. 29:315–320. 59. Tallon C, Maffulli N, Ewen SWB. (2001) Ruptured Achilles tendons are significantly more degenerated than tendino- pathic tendons. Med Sci Sports Exerc. 33:1983–1990. 60. Kaikkonen A, Kannus P, Jarvinen M. (1994) A performance test protocol and scoring scale for the evaluation of ankle injuries. Am J Sports Med. 22(4):462–469. 61. Kitaoka HB, Patzer GL. (1997) Analysis of clinical grading scales for the foot and ankle. Foot Ankle Int. 18(7):443–446. 62. Pintore E, Barra V, Pintore R, Maffulli N. (2001) Peroneus brevis tendon transfer in neglected tears of the Achilles tendon. J Trauma. 50:71–78. 63. Den Hartog BD. (2003) Flexor hallucis longus transfer for chronic Achilles tendonosis. Foot Ankle Int. 24(3):233–237. 64. Maffulli N, Pintore E, Petricciuolo F. (1991) Arthroscopic wounds: to suture or not to suture. Acta Orthop Belg. 57: 154–156. 65. Maffulli N, Dymond NP, Regine R. (1990) Surgical repair of ruptured Achilles tendon in sportsmen and sedentary patients: a longitudinal ultrasound assessment. Int J Sports Med. 11:78–84. 66. Williams JGP, Sperryn PN, Boardman S, Street M, Mellett S. (1976) Post-operative management of chronic Achilles tendon pain in sportsmen. Physiotherapy. 62:256–259. 67. Bittar ES. (2002) Arthroscopic management of massive rotator cuff tears. Arthroscopy. 18(9)(Suppl 2):104–106. 68. Maquirriain J, Sammartino M, Ghisi JP, Mazzuco J. (2003) Tibialis anterior tenosynovitis: Avoiding extensor retinacu- lum damage during endoscopic debridement. Arthroscopy. 19(2):9E. 69. Maquirriain J. (1998) Endoscopic release of Achilles peritenon. Arthroscopy. 14(2):182–185. 70. Maquirriain J,Ayerza M, Costa-Paz M, Muscolo DL. (2002) Endoscopic surgery in chronic Achilles tendinopathies: a preliminary report. Arthroscopy. 18(3):298–303. 71. Romeo AA, Larson RV. (1999) Arthroscopic treatment of infrapatellar tendonitis. Arthroscopy. 15(3):341–345. 72. van Dijk CN, Kort N. (1998) Tendoscopy of the peroneal tendons. Arthroscopy. 14(5):471–478. 25. Surgery for Chronic Overuse Tendon Problems in Athletes 275 73. van Dijk CN, Kort N, Scholten PE. (1997) Tendoscopy of the posterior tibial tendon. Arthroscopy. 13(6):692– 698. 74. Grifka J, Boenke S, Kramer J. (1995) Endoscopic therapy in epicondylitis radialis humeri. Arthroscopy. 11(6):743– 748. 75. Gartsman GM, Hammerman SM. (2000) Arthroscopic biceps tenodesis: operative technique. Arthroscopy. 16(5): 550–552. 76. Coleman BD, Khan KM, Kiss ZS, Bartlett J, Young DA, Wark JD. (2000) Open and arthroscopic patellar tenotomy for chronic patellar tendinopathy. a retrospective outcome study. Am J Sports Med. 28(2):183–190. 77. Benazzo F, Stennardo G, Mosconi M, et al. (2001) Muscle transplant in the rabbit’s Achilles tendon. Med Sci Sports Exerc. 33(5):696–701. 78. Galloway MT, Jokl P, Drayton OW. (1992) Achilles tendon overuse injuries. Clin Sports Med. 11:771–782. 79. Yodlowski ML, Scheller AD Jr, Minos L. (2002) Surgical treatment of Achilles tendinitis by decompression of the 276 N. Maffulli et al. retrocalcaneal bursa and the superior calcaneal tuberosity. Am J Sports Med. 30:318–321. 80. Alfredson H, Pietila T, Jonsson P, et al. (1998) Heavy-load eccentric calf muscle training for the treatment of chronic Achilles tendinosis. Am J Sports Med. 26:360–366. 81. Kolodziej P, Glisson RR, Nunley JA. (1999) Risk of avul- sion of the Achilles tendon after partial excision for treat- ment of insertional tendonitis and Haglund’s deformity: a biomechanical study. Foot Ankle Int. 20(7):433–437. 82. Subotnick S, Sisney P. (1986) Treatment of Achilles tendinopathy in the athlete. J Am Podiatr Med Assoc. 76: 552–557. 83. Visentini PJ, Khan KM, Cook JL, et al. (1998) The VISA score: an index of the severity of jumper’s knee (patellar tendinosis). J Sci Med Sports. 1:22–28. 84. Robinson JM, Cook JL, Purdan C, Visentini PJ, Ross J, Maffulli N, Taunton JE, Khan KM. (2001) The VISA-A questionnaire: a valid and reliable index of the clinical severity of Achilles tendinopathy. Br J Sports Med. 35(5):335–341. Part IV New Developments Tendon injuries arising from overuse are a difficult clini- cal problem. Lack of information about their etiology makes the pursuit of effective treatments almost a random process. Some components of the mechanical environment seem to contribute to the manifestation of tendinopathies.To devise proper treatments,we must first understand how loading affects tendons at the cellular and molecular levels.This chapter will review some of the current basic science methods used to understand tendon biology and mechanics, both in injury and non-injury sit- uations. The first 2 parts will focus on studies done to understand tendinopathies in animal models (in vivo), while the third and fourth parts will describe some of the research methodologies (ex vivo and in vitro) that have been used to understand the mechanisms that control a tendon’s response to mechanical loads. Animal Models of Tendinopathies Induced by Overuse This section describes animal models in which tendons have been subjected to repeated mechanical loads, usu- ally via muscle stimulation. In some cases, these loading protocols resulted in an injury reaction, but in other cases they did not. One of the first models of acute overuse injury to a tendon was presented by Rais [1].An inflammation of the Achilles paratenon with crepitation was induced in the Achilles tendon of young rabbits.With a kicking machine, 150 flexion/extension movements of the ankle were done per minute with simultaneous muscle contraction of the plantar flexors. Edema and an inflammatory cell infiltrate were present after 6 hours of exercise. The changes were dependent on the duration of exposure. Two weeks after the single loading session, there was new collagen for- mation, along with hypercellularity and hypervascularity in the paratenon. Backman and coworkers [2] used a similar protocol to the one used by Rais [1], but in a chronic manner. Load- ing sessions of 2 hours in duration were done 3 times per week, for 5.5 weeks. All animals had irregular thickening over their Achilles tendon and palpable nodules. In the paratenon, there was an increased number of fibrob- lasts and capillaries, as well as edema and an infiltration of inflammatory cells, mostly lymphocytes. Backman and coworkers [2] also described degenerative processes that included changes in the staining affinity of the tendon, fibrils of varying thickness, and nuclei of varying size. Although the inflammatory changes in the paratenon were consistent, degenerative changes in the tendon were of varying severity. In a related study, it was also reported that blood flow to the tendon and paratenon of the exper- imental leg was twice that of the contralateral leg after the 5.5 weeks of loading [3]. Archambault and coworkers [4] reproduced this rabbit model but used skeletally mature animals, a longer pro- tocol and a lower movement frequency (75 contractions per minute vs. 150 contractions per minute used by Backman and Rais in their respective studies). After 11 weeks of loading the rabbit Achilles tendon, no changes were observed in the paratenon or the tendon that were suggestive of an injury response. One major difference in the models is that Archambault and coworkers [4] used a lower movement frequency. It has been suggested that rapid movement of a tendon in its sheath may result in injury [5]. A higher movement frequency results in more gliding events between the sheath (or paratenon) and the tendon, possibly increasing the likelihood of injury. Smutz and coworkers [6] evaluated a low-force, high- frequency protocol on the wrist tendons of adult rhesus monkeys. For 6 hours per day, 5 days per week, and for 3 weeks, the wrist flexor and extensor muscles were elec- trically stimulated. This repetitive motion protocol had no detectable effect on the wrist tendons, and no signs of pathology or inflammation were noted in the carpal tunnel. This report and the one by Archambault and 279 26 Research Methodology and Animal Modeling in Tendinopathy Joanne M. Archambault and Albert J. Banes coworkers [4] suggest that tendons are not easily injured in adult animals using a controlled loading protocol. Note that tendon injuries were induced in immature animals in the studies of Backman and Rais. Recently, Soslowsky and coworkers [7] were successful in inducing degenerative changes in the rat supraspina- tus tendon. Rats were trained to run downhill on a tread- mill for 1 hour per day, 5 days per week, for 4, 8, or 16 weeks. The experimental tendons had a statistically lower maximum load to failure, and a lower maximum stress capacity than tendons from unexercised (control) animals, but had a greater cross-sectional area. There was increased cellularity, collagen disorganization, and changes in cell shape in the experimental tendons when compared with the control tendons (Figure 26-1). This model of tendinopathy in the rat shoulder most closely approximates a reproducible tendinopathy. With this model, it may be possible to probe how the tendon cells respond to overuse, infer causative mechanisms and derive scientifically-based therapeutic interventions. There have been 2 reports of the induction of overuse tendinopathy in chickens [8] and of partial failure at the bone-tendon junction of the humeral epicondyle of rabbits induced by muscle stimulation [9]. However, details of these models are lacking. Tendinopathies have been reported to occur sponta- neously in dogs [10] and horses [11,12]. Very good work has been done in the veterinary field on the diagnosis and histopathology of injuries to the equine superficial digital flexor tendon (SDFT). Ultrasound imaging, thermogra- phy and ground reaction force patterns have been cor- related to injury development, often in a prospective manner [13–15]. In addition, a good relationship between ultrasonographic findings and histological changes in injured horse tendons was demonstrated by Marr and 280 J.M. Archambault and A.J. Banes coworkers [16]. Ultrasound technology allows for the monitoring of tendon changes during an overuse proto- col without sacrificing the animal at a time point earlier than necessary to demonstrate changes. In this same paper [16], the only real staging of tendinopathies that is currently available has been described. Injuries of less than 2 weeks duration showed hemorrhage, edema, fibrolysis, fibrin deposition, and inflammatory tissue. Injuries of one to 5 months duration showed fibroplasia and granulation tissue. Injuries of over a one-year duration showed various degrees of fibrosis, increased number of cells, irregular arrangement of collagen, widespread scar formation and hemosiderin deposition. Fibrosis of the paratenon was also common at this stage. Marr and coworkers [16] suggested a mechanical etiology to the observed pathology: the initial disruption of the tendon induces dissolution of the tendon matrix and fiber lysis caused by the release of col- lagenases and proteases from damaged cells and from inflammatory cells attracted to the site of injury. What follows is a typical wound healing response, with new matrix deposition and remodeling. Inflammation was observed in the very early stages of the pathology, but not at the later stages. There has been ongoing controversy about the role of inflammation in overuse tendinopathies [17]. In tendon specimens from end-stage tendinopathy, few signs of clas- sical inflammation have been observed [18,19]. This has led researchers to conclude that inflammation is not part of tendon degeneration. This is an erroneous conclusion since these observations only indicate that there is no inflammation with cellular infiltrates at the end-stage of pathology. Moreover, since tendinopathies have not been staged, the pathology may have been initiated months or years prior to detection. There may have been inflamma- Figure 26-1. Histological section of representative control rat supraspinatus tendon (left photo) and tendon from a rat with 8 weeks of downhill treadmill running (right photo). Tendons from the overuse running group had increases in cellularity and collagen disorganization when compared to tendons from control animals. (From [7], with permission.) tion at some point in the initial stages of tendon degen- eration, which would have resolved by the time a tendon rupture occurred or the tendon was biopsied during surgery. Cytokine induction of matrix metalloproteinases and other proteases might be a mechanism by which matrix degeneration occurs. Animal Models of Tendinopathies Induced by Chemical Means Collagenase has been injected into tendons in an effort to model the pathological changes observed in human tissues.The validity of this approach has been questioned because it does not directly simulate the overuse process. In addition, other models have been developed to study rheumatoid tenosynovitis [20] and antibiotic-induced tendinopathy. Williams and coworkers [21,22] injected collagenase into the superficial digital flexor tendon of horses to induce tendinopathy and investigated the sequence of events associated with the healing of this injury. The severity of the pathology was related to the amount of collagenase injected. After a transient inflammatory response, a classical wound healing process occurred with formation of granulation tissue and increased staining for Type III collagen and fibronectin. Reorganization of the matrix continued until 14 months, the last time point studied, but normal structure had not been reestablished. The authors pointed out that the morphological char- acteristics and time course of repair following the col- lagenase injection were similar to those of a natural, exercise-induced trauma. Collagenase has also been injected into the supraspina- tus tendon of the rat [23]. This procedure produced a marked disruption of the collagen matrix and fibroplasia. At 12 weeks following the injection, these changes were still present, but to a lesser extent than the earlier time points, suggestive of a resolution process. Stone and coworkers [24] injected collagenase into the patellar tendon of rabbits, and compared it to the injection of a cytokine cocktail. The exact composition of the cytokine preparation was not described in their publication, but it included Interleukin-1a, Transforming Growth Factor-b, basic Fibroblast Growth Factor and other cytokines that would have been produced by rabbit synovial fibroblasts exposed to phorbol 12-myristate acetate, a strong tumour promoting agent, in culture.At 16 weeks post-collagenase injection, small myxoid foci were detected with disorga- nized collagen. The cytokine injection did not cause the collagen matrix disorganization observed in the collage- nase-injected tendons, however there was an increase in cellularity. Since the cytokine preparation included agents that could both stimulate (i.e. IL-1a) and inhibit collagenase expression (i.e. TGFb) in fibroblasts, the experimental effect were weaker than the effects associ- ated with the collagenase-only injections (Figure 26-2). Collagenase injections produce tendinopathy that is remarkably similar to the histologic appearance of end- 26. Research Methodology and Animal Modeling in Tendinopathy 281 Figure 26-2. Histological section of rabbit patellar tendon 4 weeks after injection of saline (A), cytokine preparation (B) or collagenase (C). The cytokine-injected tendons demonstrated a slight increase in cellularity, while collagenases-injected tendons were hypercellular, hypervascular and had disorganized matrix. (From [24], with permission.) stage pathological specimens in humans. However, there is no evidence that collagenase is involved in human tendinopathies. Collagenase might be released into the tendon during the inflammation that some have sug- gested accompanies microtrauma [25] or tendon cells may produce collagenase in response to excessive loading. Preliminary results suggest that the rabbit Achilles tendon does not produce collagenase in response to one bout of overuse of up to 6 hours duration, but does produce collagenase when placed in organ culture [26]. If active collagenase was found in surgical specimens or ruptured tendons, this would give credence to the col- lagenase injection approach. However, evidence that collagenase is produced over time in an animal model of tendon overuse would be stronger support for its role in tendinopathies. Achilles tendinopathy and ruptures have been associ- ated with fluoroquinolone antibiotics [27]. Ruptured tendons had irregular collagen fiber arrangement, hyper- cellularity, and increased glycosaminoglycan staining— pathological features that are very similar to the degenerative changes described previously in athletic tendinopathies [28]. The mechanisms involved in this induction of tendinopathy are not known, however this class of antibiotics appears to have a toxic effect on tendon cells [29]. After 3 days of oral antibiotic adminis- tration to rats, cells in the Achilles tendon showed degen- erative alterations, and there was a loss of cell-matrix interactions. These results are mentioned to provide an appreciation of the powerful effects that biochemical agents can have on tendons—tendon ruptures occured within 2 weeks of the start of antibiotic treatment in 50% of the cases [30]. Tendon degeneration in antibiotics- related cases occurs much more quickly than in overuse- related cases, indicating that healthy tendon cells are the key to maintaining normal tendon structure. It would be short-sighted to think that tendinopathies are only a function of overuse and loading. If this were the case, every person who engages in sports or work that involves repetitive movement might sustain a tendinopathy. How- ever, as this does not occur, there might be a genetic pre- disposition to these injuries. Research Methodology (ex vivo Systems) Although investigations with in vivo animal models might be the best way to simulate and stage the tendinopathy process that occurs in humans, the in vivo situation is very complex.The animal’s age, gender, phys- iology, genetics, behavior, nutrition, etc. may all have an effect on the outcome, or be confounding variables in the 282 J.M. Archambault and A.J. Banes process. Research done with tendons in culture allows for a single experimental variable to be manipulated. For example, ex vivo experiments can be used to study the effects of repetitive loading on tendons, without the systematic biochemical and hormonal responses to exer- cise that occur simultaneously in the animal. Slack and coworkers [31] first showed that tendons could be maintained in organ culture. Using embryonic chick tendons, they showed that protein and DNA syn- thesis increased in tendons that were experimentally loaded in culture for 48 to 72 hours, versus tendons that were cultured on steel grids. Hannafin and coworkers [32] reported that cyclic tension in culture for 2 hours per day could maintain the mechanical properties of the tendon. If tendons were cultured without tension, the tensile modulus decreased to 68% of control tendon (noncul- tured) in a 4-week period.This is an important finding for ex vivo research methodologies, since it indicates that tension is necessary for the tendon properties to remain as close as possible to those of the in vivo system, where the tendon is always under some degree of tension. Also, collagenase is expressed and produced very quickly in tendons in culture when they are not under tension (J. Archambault, S. Arnoczky, unpublished work). This tendon-derived collagenase could be responsible for degrading tendon matrix, and lead to the reduced mechanical properties observed in cultured, unloaded tendons. Mechanical loading devices are important research tools that allow for multiple tendons to be loaded simul- taneously in culture. Such a device has been used at the University of North Carolina to cyclically load tendons in culture and evaluate the corresponding biological responses (Figure 26-3). Banes and coworkers [33] reported that 8 hours of load per day at 0.65% elonga- tion for 3 days stimulated DNA and collagen synthesis in whole avian flexor tendons. The load effect could be blocked by gap junction inhibitors, indicating the impor- tance of intercellular communication in tendons. In addi- tion, cyclically loaded tendons at 0.5Hz for 5 days at about 20% of the tendon’s ultimate tensile stress resulted in significantly greater strain accumulation in the loaded compared to the unloaded tendons [34]. The mechanical stimulus also reduced the number of apoptotic cells observed in the unloaded cultured tendons. An important application of tendon loading in culture is the evaluation of the biological responses to fatigue loading protocols. As a tendon fatigues, either with static or cyclic loading, there is a significant decline in its mechanical properties [35]. Overuse tendinopathies may have a component of material fatigue, as the tendon is loaded throughout its lifetime. A tendon rupture could be considered the equivalent of a fatigue rupture in an engineering material. The amount of damage done by a loading protocol is a function of the number of loading cycles and the amount of stress. Tendons differ from engineering materials in that they have the ability to repair damage. One of the causes for overuse injuries in tendons may be a breakdown in the balance between fatigue damage and routine repair [36]. If either the damage resulting from the loading regime is excessive or the ability of the cells to repair damage is compromised, the damage may accumulate and result in a tendinopa- thy. An ex vivo tendon loading system could be used to evaluate the biological response of tendons to various amounts of fatigue damage. A threshold of loading may exist above which a tendon cannot fabricate enough matrix to keep pace with the rate of damage, leading to a decline in the tendon’s mechanical properties. Such information may be important to understanding if repet- itive loading results in a disengagement of anabolic and catabolic states in tendon cells. Organ cultures have been used recently to evaluate the effect of hypoxia on tendons [37]. Flexor digitorum profundus tendons of rabbits were incubated under various degrees of oxygen tension. Although cell proliferation, synthesis of non-collagenous proteins, and synthesis of proteoglycans were not affected, hypoxia inhibited collagen synthesis. This result suggests that hypoxia may reduce collagen synthesis, or the repair rate of tendons, a potentially deleterious effect if it were combined with damage from cyclic loading. Research Methodology (in vitro Systems) In vitro experiments allow researchers to apply well- defined mechanical stimulation to isolated tendon cells. Cells can be subjected to fluid-induced shear stress, com- pression, or stretching. In the latter case, cells attached to a substrate are deformed when the substrate is deformed. These systems are best suited for evaluating the molecu- lar and cellular responses of tendon cells to mechanical stimulation, the mechanisms of transduction of these mechanical signals and the effect of various drugs. They may provide some clues as to which biochemical factors could then be evaluated in more complex systems, such as the ex vivo and in vivo models described previously. Banes and coworkers [38] first showed that tenocytes responded to mechanical loading in vitro by altering expression of actin and tubulin. Almekinders and coworkers [39] reported that human tenocytes responded to repetitive stretching by producing PGE 2 . The produc- tion of PGE 2 was greater at higher frequencies and could be blocked by the NSAID indomethacin. Wang and coworkers had similar findings when they stretched human tenocytes residing on a microgrooved surface [40]. They also demonstrated that PGE 2 production depended on the stretching magnitude: with 4% strain, PGE 2 production was similar to non-stretched cells, but, at 8% and 12% strain, PGE 2 was significantly increased. Since PGE 2 is thought to be important in inflammation and pain, general and specific COX inhibitors have been used as a treatment for tendinopathies. It is difficult to justify this type of treatment since PGE 2 has not been found in painful human Achilles tendons [41]. It is also possible that tenocytes release PGE 2 in response to mechanical stimulation as do bone cells [42], and that blocking this release might be detrimental to normal functioning of the tendon. Stretching of tenocytes has also been shown to induce the expression of novel genes [43], and activate the asso- ciation of proteins that are part of focal adhesions [44]. Other in vitro work with tenocytes has shown that cytokines and growth factors can interact with mechani- cal stimulation to produce effects that do not occur when the mechanical load is applied alone. Banes and cowork- ers [45] showed that mechanical load by itself does not stimulate cell division, but that it does when growth factors such as IGF-I and PDGF-BB are present. Archambault and coworkers [46] showed that stretching tenocytes in the presence of IL-1b leads to a greater release of MMP-3 than when either stretching or IL-1b were applied in isolation. Such combination effects may be important to the in vivo system: mechanical loading by itself may not cause a cellular response, but an altered 26. Research Methodology and Animal Modeling in Tendinopathy 283 Figure 26-3. Photograph of a chicken flexor digitorum profun- dus tendon clamped into a loading frame of a multi-station tendon loading device. (From [33], with permission.) biochemical environment may be enough to produce a tendon injury. Carpenter and coworkers [47] reported that damage to the supraspinatus tendon in rats was greater when the overuse protocol was combined with an intrinsic (collagenase injection) or extrinsic (external compression on tendon) injury. Recent data demonstrated that cyclic straining of tenocytes led to an immediate upregulation of a stress- activated protein kinase, pJNK, that is an upstream regulator of apoptosis. This up-regulation was strain but not frequency dependent, with a much stronger up- regulation observed at 6% than 3% strain [48]. pJNK upregulation returned to normal levels after 2 hours of stretching, but persisted if the tendon cells were simulta- neously exposed to environmental stresses such as hyperthermia and hyperosmolarity [49]. This is another example of combined stimuli producing a greater cellu- lar response than mechanical loading alone. Conclusions Although there is interest in understanding the causes of tendinopathies, research into this topic is still in its infancy. Hopefully this chapter has provided insights into the current knowledge of experimental aspects of tendon overuse injury and damage. Table 26-1 summa- rizes the important features of the animal models of tendinopathies discussed in this chapter. In vivo animal 284 J.M. Archambault and A.J. Banes models are the gold standard for understanding tendinopathies, but research done with ex vivo and in vitro systems have made an important contribution to our understanding. References 1. Rais O. (1961) Heparin treatment of peritenomyosis (peri- tendinitis) crepitans acuta: a clinical and experimental study including the morphological changes in peritenon and muscle. Acta Chir Scandinav. 268(Suppl):1–88. 2. Backman C, Boquist L, Friden J, Lorentzon R,Toolanen G. (1990) Chronic Achilles paratenonitis with tendinosis: an experimental model in the rabbit. J Orthop Res. 8:541–547. 3. Backman C, Friden J, Widmark A. (1991) Blood flow in chronic Achilles tendinosis. Radioactive microsphere study in rabbits. Acta Orthop Scand. 62:386–387. 4. Archambault JM, Hart DA, Herzog W. (2001) Response of rabbit Achilles tendon to chronic repetitive loading. Connect Tissue Res. 42:13–23. 5. Moore A, Wells R, Ranney D. (1991) Quantifying exposure in occupational manual tasks with cumulative trauma dis- order potential. Ergonomics. 34:1433–1453. 6. Smutz WP, Miller SC, Eaton CJ, Bloswick DS, France EP. (1994) Investigation of low-force high-frequency activities on the development of carpal-tunnel syndrome. Clin Biomech. 9:15–20. 7. Soslowsky LJ, Thomopoulos S, Tun S, Flanagan CL, Keefer CC, Mastaw J, Carpenter JE. (2000) Neer Award 1999. Overuse activity injures the supraspinatus tendon in an Table 26-1. Summary of animal models of tendinopathy Reference Animal Target tendon Means Result Rais 1961 Rabbit Achilles Acute muscle stimulation Paratenon inflammation Backman et al. 1990 Rabbit Achilles Chronic muscle stimulation Paratenon inflammation and tendon degeneration Archambault et al. 2001a Rabbit Achilles Chronic muscle stimulation No changes Smutz et al. 1994 Monkey Wrist flexors Chronic muscle stimulation No changes Soslowsky et al. 2000 Rat Supraspinatus Chronic treadmill running Tendon degeneration and decline Carpenter et al. 1998 of mechanical properties Lai et al. 1995 Chicken Gastrocnemius Ablation overload Less collagen material Han et al. 1995 Rabbit Lateral common extensor Acute muscle stimulation Partial failure at lateral epicondyle Sakata et al. 1988 Rabbit Tibialis anterior Antigen injection Inflammation of synovial sheath Williams et al. 1984a, 1984b Horse Superficial digital flexor Collagenase injection Acute inflammation, disruption of matrix, more type III collagen & fibronectin Soslowsky et al. 1996 Rat Supraspinatus Collagenase injection Disruption of matrix, hypercellularity Stone et al. 1999 Rabbit Patellar Collagenase injection Cytokine Disruption of matrix, injection hypercellularity Shakibaei et al. 2001 Rat Achilles Oral fluoroquinolone Degenerative alterations, loss of antibiotics cell-matrix interactions [...]... Tissues Organs 165:30– 39 Moore RH, Dowling DA ( 198 2) Effects of enkephalins on perfusion pressure in isolated hindlimb preparations Life Sci 31:15 59 1566 Nakamura-Craig M, Smith TW ( 198 9) Substance P and peripheral inflammatory hyperalgesia Pain 38 :91 98 Neely FG ( 199 8) Biomechanical risk factors for exerciserelated lower limb injuries Sports Med 26: 395 –413 Nozaki-Taguchi N, Yamamoto T ( 199 8) Involvement of... Figure 3 0-1 Principle of viral gene transfer 30 Gene Therapy in Tendon Ailments 3 09 Table 3 0-2 In vivo therapeutic gene therapy in tendons Abbreviations: HVJhemagglutinating virus of Japan; PDGF-B-platelet derived growth factor B; HGF/ SF-hepatocyte growth factor/scatter factor; BMP-2-bone morphogenetic protein 2; ODN-oligodeoxynucleotide Author/Year Species Nakamura, 199 8 [21] Natsuume, 199 8 [22]... 54:1 797 –1802 Marr CM ( 199 2) Microwave thermography: a non-invasive technique for investigation of injury of the superficial digital flexor tendon in the horse Equine Vet J 24:2 69 273 Marr CM, McMillan I, Boyd JS, Wright NG, Murray M ( 199 3) Ultrasonographic and histopathological findings in equine superficial digital flexor tendon injury Equine Vet J 25:23– 29 Maffulli N, Khan KM, Puddu G ( 199 8) Overuse tendon. .. 328:425–427 297 67 Strand FL, et al ( 199 1) Neuropeptide hormones as neurotrophic factors Physiol Rev 71:1017–1046 68 Szolcsanyi J, Helyes Z, Oroszi G, Nemeth J, Pinter E ( 199 8) Release of somatostatin and its role in the mediation of the anti-inflammatory effect induced by antidromic stimulation of sensory fibres of rat sciatic nerve Br J Pharmacol 123: 93 6 94 2 69 Taiwo YO, Levine JD ( 199 1) Kappa- and delta-opioids... 14: 498 7– 499 7 73 Wenk HN, Honda CN ( 199 9) Immunohistochemical localization of delta opioid receptors in peripheral tissues J Comp Neurol 408:567–5 79 74 Wiesenfeld-Hallin Z, et al ( 198 4) Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat Neurosci Lett 52: 199 –204 75 Willson NJ, et al ( 197 6)... surface The nerves of tendons are composed of myelinated, fast-transmitting Aa- and Ab-fibers and unmyelinated, slow-transmitting Ag-, Ad-, B- and C-fibers The nerve endings of myelinated fibers (Aa, Ab) are type I-III specialized mechanoreceptors, whose main function is to mediate physical energy (pressure, tension) into afferent nerve signals The unmyelinated nerve endings of Ag-, Ad-, and C-fibers are type... (TNF-a) can stimulate metalloproteinase expression in tendon cells (MMP-1,2,3 and 13) [17,18, 19] Inflammatory cytokines such as IL-1b and TNF-a elaborated by lymphocytes and macrophages can stimulate tendon cells to produce interstitial collagenase (MMP-1), gelatinase (MMP-2), stromelysin (MMP-3) as well as MMP-13 which can activate other MMPs to degrade collagens and aggrecans [18] Moreover, IL-1b... Doctrow SR ( 199 3) Regulation of growthplate chondrocytes by insuline-like growth-factor and basic fibroblast growth factor J Bone Joint Surg 75A: 177–1 89 7 Tsuzaki M, Xiao H, Brigman B, Yamamoto J, Lawrence WT, Van Wyk J, Banes AJ (2000) IGF-I is expressed by avian flexor tendon cells J Orthop Res 8:546–556 8 Abrahamsson S-O, Lohmander S ( 199 6) Differential effects of insulin-like growth factor-I on matrix... Robbins PD, Evans CH ( 199 9) The role of gene therapy fact or fiction? Clin Sports Med 18:223–2 39 28 The Use of Growth Factors in the Management of Tendinopathies 29 Archambault JM, Wiley JP, Bray RC ( 199 5) Exercise loading of tendons and the development of overuse injuries a review of current literature Sports Med 20:77– 89 30 Backman C, Boquist L, Friden J, Lorentzon R, Toolanen G ( 199 0) Chronic Achilles... Stricker BH ( 199 9) Achilles tendinitis associated with fluoroquinolones Br J Clin Pharmacol 48:433–437 28 Movin T, Gad A, Guntner P, Foldhazy Z, Rolf C ( 199 7) Pathology of the Achilles tendon in association with ciprofloxacin treatment Foot Ankle Int 18: 297 – 299 29 Shakibaei M, Stahlmann R (2001) Ultrastructure of Achilles tendon from rats after treatment with fleroxacin Arch Toxicol 75 :97 –102 30 McGarvey . Speri- mentale. 72(7–8):203–210. 28. Wang ED. ( 199 8) Tendon repair. J Hand Ther. 11:105–110. 29. Reddy GK, Stehno-Bittel L, Enwemeka CS. ( 199 9) Matrix remodeling in healing rabbit Achilles tendon. . 316:151–164. 10. Leadbetter WB. ( 199 2) Cell-matrix response in tendon injury. Clin Sports Med. 11:533–578. 11. Movin T, Gad A, Reinholt FP, et al. ( 199 7) Tendon pathol- ogy in long-standing achillodynia FP,Rolf C. ( 199 7) Tendon pathol- ogy in long-standing achillodynia. biopsy findings in 40 patients. Acta Orthop Scand. 68:170–175. 19. Khan KM, Cook JL,Bonar F, Harcourt P,Astrom M. ( 199 9) Histopathology