Erythropoietin stimulates patellar tendon healing in rats

Abstract

Backround

Erythropoietin (EPO), regulating erythropoiesis, is used to provide protective and regenerative activity in non-haematopoietic tissues. There is insufficient knowledge about the role of EPO activity in tendon healing. Therefore, we investigated the effect of EPO treatment on healing in rat patellar tendons.

Methods

One hundred and twenty-six, four-month-old male Sprague–Dawley rats were randomly assigned to three experimental groups: 1, no treatment; 2, treatment with isotonic saline (NaCl) and 3, treatment with EPO. Each group was randomly subdivided into two groups for sacrifice at three (1a, 2a, 3a) or six weeks (1b, 2b, 3b). Complete incision of the left patellar tendon from the distal patellar pole was performed. We applied body casts for 20 days after the incised edges of the patellar tendon were brought together with a surgical technique. Both legs were harvested and specimens from each group underwent histological, biomechanical, and protein mRNA expression analyses.

Results

There were statistically significant differences in the ultimate breaking force between the EPO group and others at both weeks three and six (p < 0.05); significant differences in fibroblast proliferation, capillary vessel formation, and local inflammation were found between groups 1a and 3a, and 2a and 3a (p < 0.05). There were statistical differences between 1a, 3a and 2a, 3a for Col III, TGF-β1, and VEGF and between 1b, 3b and 2b, 3b for Col I, Col III, TGF-β1, and VEGF mRNA expressions.

Conclusion

EPO had an additive effect with surgery on the injured tendon healing process in rats compared to the control groups biomechanically, histopathologically and with tissue protein mRNA expression.

Clinical relevance

This is the first experimental study to analyze the relationship between EPO treatment and the patellar tendon repair process by biomechanical, histopathological, and tendon tissue mRNA expression methodologies.

Introduction

Rupture of the patellar tendon is a rare injury that occurs most frequently in patients younger than 40 years of age [1], usually after indirect trauma due to a sudden quadriceps contraction with the knee in slight flexion. It is generally agreed that a healthy patellar tendon will not tear, and that tearing usually involves a tendon weakened by iterative micro trauma [2] or local corticoid injections. Surgery is mandatory, regardless of age and physical activity, and should be undertaken as early as possible after injury [3]. Erythropoietin (EPO) is a glycoprotein hormone produced primarily by the kidneys that regulates the production of red blood cells. The most common indications for EPO treatment are chronic renal failure, anaemia, and anaemia of prematurity [4]. EPO has been shown to exert protective and regenerative activity in a variety of non-haematopoietic tissues [5]. The prevention of programed cell death, reduction of proinflammatory cytokine-induced injury, and provision of tissue protection are some of the functions of EPO [6]. The treatment of patellar tendon rupture by conventional surgical methods alone may not be sufficient to regain initial knee function. Recurrent ruptures of the patellar tendon tend to occur in patients with systemic disease such as diabetes mellitus or chronic renal failure.

Three phases of tendon healing have been identified in animal models [7], [8] and cell cultures [9]: inflammation, fibroplasia, and remodeling. The role of intrinsic and extrinsic healing models has been investigated [10]. Both of these mechanisms are likely critical for proper tendon healing. Both the period of time after tendon injury and local extracellular environmental differences affect the intrinsic and extrinsic tendon healing processes [11], [12].

Tendon rupture is characterized by a disruption of blood vessels and the extracellular matrix (ECM). Platelets interact directly or indirectly with newly exposed collagen, leading to their activation, aggregation, and degranulation [13]. During the early inflammatory phase, biochemical mediators released into the local environment stimulate endothelial cells to express ligands or adhesion molecules. Leukocytes are then recruited into tissues through interactions with the vessel wall, as mediated by selectins and integrins [14]. Proteins such as basic fibroblast growth factor, vascular endothelial growth factor (VEGF), thrombospondin-1, platelet factor-4, intercellular adhesion molecule-2, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor-1, and transforming growth factor (TGF)-β are important molecules in the tendon healing process [15].

In this study, we tested our hypothesis that EPO treatment in combination with patellar tendon surgery would stimulate inflammation and re-vascularization in tendons post-injury and influence cell proliferation, angiogenesis, collagen deposition, and mechanical recovery in a rat model. This study is the first to assess the role of EPO in the tendon healing process with a focus on biomechanical, histopathologic features and tendon tissue protein mRNA expression levels.