Date Published: February 13, 2018
Publisher: Public Library of Science
Author(s): Cesar de Cesar Netto, Alexandre Leme Godoy-Santos, Pedro Augusto Pontin, Renato Jose Mendonça Natalino, Cesar Augusto Martins Pereira, Francisco Diego de Oliveira Lima, Lucas Furtado da Fonseca, Jackson Rucker Staggers, Leonardo Muntada Cavinatto, Lew Charles Schon, Olavo Pires de Camargo, Túlio Diniz Fernandes, Chunfeng Zhao.
Our goal was to develop a novel technique for inducing Achilles tendinopathy in animal models which more accurately represents the progressive histological and biomechanical characteristic of chronic Achilles tendinopathy in humans. In this animal research study, forty-five rabbits were randomly assigned to three groups and given bilateral Achilles injections. Low dose (LD group) (n = 18) underwent a novel technique with three low-dose (0.1mg) injections of collagenase that were separated by two weeks, the high dose group (HD) (n = 18) underwent traditional single high-dose (0.3mg) injections, and the third group were controls (n = 9). Six rabbits were sacrificed from each experimental group (LD and HD) at 10, 12 and 16 weeks. Control animals were sacrificed after 16 weeks. Histological and biomechanical properties were then compared in all three groups. At 10 weeks, Bonar score and tendon cross sectional area was highest in HD group, with impaired biomechanical properties compared to LD group. At 12 weeks, Bonar score was higher in LD group, with similar biomechanical findings when compared to HD group. After 16 weeks, Bonar score was significantly increased for both LD group (11,8±2,28) and HD group (5,6±2,51), when compared to controls (2±0,76). LD group showed more pronounced histological and biomechanical findings, including cross sectional area of the tendon, Young’s modulus, yield stress and ultimate tensile strength. In conclusion, Achilles tendinopathy in animal models that were induced by serial injections of low-dose collagenase showed more pronounced histological and biomechanical findings after 16 weeks than traditional techniques, mimicking better the progressive and chronic characteristic of the tendinopathy in humans.
Achilles tendinopathy is one of the most common overuse injuries of the foot and ankle and often results in chronic pain and functional impairment.[2,3] The disease is commonly diagnosed in physically active individuals, affecting 9% of recreational runners and up to 5% of professional athletes. It also affects 5.6% of the sedentary population and its chronic presentation is associated with advanced age.
Institutional review board approval was obtained prior to this controlled experimental study. This animal study was approved by the University of Sao Paulo Medical School Ethics Committee [Comissão de Ética no Uso de Animais (CEUA) do Comitê de Ética em Pesquisa da Faculdade de Medicina da Universidade de São Paulo (CEP-FMUSP)], under the protocol number 148/12.
There were a total of seven losses (15.5%), including: two animals in LD group (11.1%), four in HD group (22.2%) and one in the C group (11.1%). Two deaths were due to anesthetic complications, two were sacrificed secondary to deep postoperative infection, one was sacrificed secondary to a fracture of the left hind paw inside the cage, one was secondary to an urinary infection, and there was one sudden death of unknown cause.
Tendinopathy is a common disease and can be debilitating. Despite the significant prevalence, its pathophysiology is not fully elucidated. Animal models represent useful tools in the study of the physiopathology of the tendinopathy since human samples usually only come from late stage disease.[13,21,37–39] Bacterial collagenase type I, injected in a single large dose (0.3–0.5mg) is the most used technique in the literature for induction of tendinopathy in various animal models. However, to date, there were no studies evaluating the role of multiple consecutive injections of collagenase. We have shown in this study that serial application of a lower dose collagenase, when compared to a single high-dose injection, can induce a more progressive disease, with persistent findings after 16 weeks, replicating better in animals the histological and biomechanical findings proposed in the continuum model of human tendinopathy.
In conclusion, the biomechanical and histological findings in our study demonstrate that the serial application of lower doses (0.1mg) of collagenase type 1A in the Achilles tendon of rabbits results in a more progressive tendinopathic change when compared to the single higher dose (0.3mg) injection and to controls. The single collagenase injection model of induced tendinopathy, the most commonly used in the recent literature, might not be adequate enough to demonstrate the progressive development of the disease. We believe the proposed model of tendinopathy induced by serial injections of low-dose collagenase may represent a better option for the study of Achilles tendinopathy, with findings that mimic better the progressive nature of the chronic disease in humans. It is our hope that the results of this study can foster development of additional animal models of chronic Achilles tendinopathy, aiming more accurate elucidation of the pathophysiology of the disease and the search for better therapeutic possibilities.