VEGF Expression in Patellar Tendinopathy: A Preliminary Study

Vascular function and angiogenesis are regulated by vascular endothelial growth factor-A (VEGF). The purpose of this preliminary study was to address the following questions: Is VEGF expression in the patellar tendon more prevalent in patients with patellar tendinopathy than in individuals with normal, pain-free patellar tendons? Which cell populations express VEGF in normal and tendinopathic tendon? Is there a difference in symptom duration between VEGF+ and VEGF? tendons? We collected patellar tendon tissue from 22 patients undergoing open débridement of the patellar tendon and from 10 patients undergoing intramedullary nailing of the tibia. VEGF expression was assessed immunohistochemically. Relevant inflammatory and repair cell types were immunolabeled. VEGF expression was absent from control tendons, but was present in a subset of patients with histopathological evidence of angiofibroblastic tendinosis. VEGF was expressed in the intimal layer of tendon vessels, but was absent in other cell types. Patients demonstrating VEGF expression in the patellar tendon had a shorter symptom duration (12 ± 7.8 months) than patients with no detectable VEGF (32.8 ± 23.5 months). VEGF may contribute to the vascular hyperplasia that is a cardinal feature of symptomatic tendinosis, particularly in cases with more recent onset.     

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy‐like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early‐stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1888–1896, 2015.     

Temporal effect of in vivo tendon fatigue loading on the apoptotic response explained in the context of number of fatigue loading cycles and initial damage parameters

Accumulation of damage is a leading factor in the development of tendinopathy. Apoptosis has been implicated in tendinopathy, but the biological mechanisms responsible for initiation and progression of these injuries are poorly understood. We assessed the relationship between initial induced damage and apoptotic activity 3 and 7 days after fatigue loading. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles, and (ii) will be higher at 7 than at 3 days after loading. Left patellar tendons were fatigue loaded for either 100 or 7,200 cycles. Diagnostic tests were applied before and after fatigue loading to determine the effect of fatigue loading on hysteresis, elongation, and loading and unloading stiffness (damage parameters). Cleaved Caspase‐3 staining was used to identify and calculate the percent apoptosis in the patellar tendon. While no difference in apoptotic activity occurred between the 100 and 7,200 cycle groups, greater apoptotic activity was associated with greater induced damage. Apoptotic activity was higher at 7 than 3 days after loading. We expect that the decreasing number of healthy cells that can repair the induced damage in the tendon predispose it to further injury. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1097–1103, 2014.     

Production of PGE2 increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non‐tenocytes

Whether tendon inflammation is involved in the development of tendinopathy or degenerative changes of the tendon remains a matter of debate. We explored this question by performing animal and cell culture experiments to determine the production and effects of PGE₂, a major inflammatory mediator in tendons. Mouse tendons were subjected to repetitive mechanical loading via treadmill running, and the effect of PGE₂ on proliferation and differentiation of tendon stem cells (TSCs) was assessed in vitro. Compared to levels in cage control mice, PGE₂ levels in mouse patellar and Achilles tendons were markedly increased in response to a bout of rigorous treadmill running. PGE₂ treatment of TSCs in culture decreased cell proliferation and induced both adipogenesis and osteogenesis of TSCs, as evidenced by accumulation of lipid droplets and calcium deposits, respectively. Effects of PGE₂ on both TSC proliferation and differentiation were apparently PGE₂–dose‐dependent. These findings suggest that high levels of PGE₂, which are present in tendons subjected to repetitive mechanical loading conditions in vivo as shown in this study, may result in degenerative changes of the tendon by decreasing proliferation of TSCs in tendons and also inducing differentiation of TSCs into adipocytes and osteocytes. The consequences of this PGE₂ effect on TSCs is the reduction of the pool of tenocytes for repair of tendons injured by mechanical loading, and production of fatty and calcified tissues within the tendon, often seen at the later stages of tendinopathy. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:198–203, 2010     

Eccentric training improves tendon biomechanical properties: A rat model

The treatment of choice for tendinopathies is eccentric reeducation. Although the clinical results appear favorable, the biomechanical changes to the tissue are not yet clear. Even if the mechanotransduction theory is commonly accepted, the physiology of tendons is not clearly understood. We aimed to better define the biomechanical and histological changes that affect healthy tendon after eccentric and concentric training. This study compared the effects of two methods of training (eccentric [E] training and concentric [C] training) with untrained (U) rats. The animals were trained over a period of 5 weeks. The tricipital, patellar, and Achilles tendons were removed, measured and a tensile test until failure was performed. A histological analysis (hematoxylin and eosin and Masson's trichrome stains) was also realized. There was a significant increase in the rupture force of the patellar and tricipital tendons between the U and E groups. The tricipital tendons in the control group presented a significantly smaller cross‐sectional area than the E‐ and C‐trained groups, but none was constated between E and C groups. No significant difference was observed for the mechanical stress between the three groups for all three tendons. Histological studies demonstrated the development of a greater number of blood vessels and a larger quantity of collagen in the E group. The mechanical properties of tendons in rats improve after specific training, especially following eccentric training. Our results partly explained how mechanical loading, especially in eccentric mode, could improve the healing of tendon. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:119–124, 2012     

Dose–response effect of an intra‐tendon application of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) in a rat Achilles tendinopathy model

The purpose of this study was to assess whether intra‐tendon delivery of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) would improve Achilles tendon repair in a rat collagenase‐induced tendinopathy model. Seven days following collagenase induction of tendinopathy, one of four intra‐tendinous treatments was administered: (i) Vehicle control (sodium acetate buffer), (ii) 1.02 µg rhPDGF‐BB, (iii) 10.2 µg rhPDGF‐BB, or (iv) 102 µg rhPDGF‐BB. Treated tendons were assessed for histopathological (e.g., proliferation, tendon thickness, collagen fiber density/orientation) and biomechanical (e.g., maximum load‐to‐failure and stiffness) outcomes. By 7 days post‐treatment, there was a significant increase in cell proliferation with the 10.2 and 102 µg rhPDGF‐BB‐treated groups (p = 0.049 and 0.015, respectively) and in thickness at the tendon midsubstance in the 10.2 µg of rhPDGF‐BB group (p = 0.005), compared to controls. All groups had equivalent outcomes by Day 21. There was a dose‐dependent effect on the maximum load‐to‐failure, with no significant difference in the 1.02 and 102 µg rhPDGF‐BB doses but the 10.2 µg rhPDGF‐BB group had a significant increase in load‐to‐failure at 7 (p = 0.003) and 21 days (p = 0.019) compared to controls. The rhPDGF‐BB treatment resulted in a dose‐dependent, transient increase in cell proliferation and sustained improvement in biomechanical properties in a rat Achilles tendinopathy model, demonstrating the potential of rhPDGF‐BB treatment in a tendinopathy application. Consequently, in this model, data suggest that rhPDGF‐BB treatment is an effective therapy and thus, may be an option for clinical applications to treat tendinopathy. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 413–420, 2013     

Molecular response of the patellar tendon to fatigue loading explained in the context of the initial induced damage and number of fatigue loading cycles

Accumulation of sub-rupture fatigue damage has been implicated in the development of tendinopathy. We previously developed an in vivo model of damage accumulation using the rat patellar tendon. Our model allows us to control the input loading parameters to induce fatigue damage in the tendon. Despite this precise control, the resulting induced damage could vary among animals because of differences in size or strength among their patellar tendons. In this study, we used number of applied cycles and initial (day-0) parameters that are indicative of induced damage to assess the molecular response 7 days after fatigue loading. We hypothesized that day-0 hysteresis, elongation, and stiffness of the loading and unloading load-displacement curves would be predictive of the 7-day molecular response. Results showed correlations between the 7-day molecular response and both day-0 elongation and unloading stiffness. Additionally, loading resulted in upregulation of several extracellular matrix genes that suggest adaptation; however, several of these genes (Col-I, -XII, MMP 2, and TIMP 3) shut down after a high level of damage was induced. We showed that evaluating the 7-day molecular profile in light of day-0 elongation provides important insight that is lost from comparing number of fatigue loading cycles only. Our data showed that loading generally results in an adaptive response. However, the tendon's ability to effectively respond deteriorates as greater damage is induced.     

Growth factor and protease expression during different phases of healing after rabbit deep flexor tendon repair

The purpose of the study was to contribute to the mapping of molecular events during flexor tendon healing, in particular the growth factors insulin‐like growth factor‐1 (IGF‐1), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), matrix metalloproteinases (MMP‐3 and MMP‐13) and their inhibitors (tissue inhibitors of metalloproteinases, TIMP‐1 and TIMP‐3, and the protease cathepsin K. In a rabbit model of flexor tendon injury, the mRNA expression for the growth factors, MMPs and TIMPs were measured in tendon and tendon sheath tissue at several time points (3, 6, 21, and 42 days) representing different phases of the healing process. We found that MMP‐13 remained increased during the study period, whereas MMP‐3 returned to normal levels within the first week after injury. TIMP‐3 was down‐regulated in the tendon sheaths. Cathepsin K was up‐regulated in tendons and sheaths after injury. NGF was present in both tendons and sheaths, but unaltered. IGF‐1 exhibited a late increase in the tendons, while VEGF was down‐regulated at the later time points. In conclusion, we have demonstrated the presence of NGF in flexor tendons. MMP‐13 expression appears to play a more protracted role in flexor tendon healing than MMP‐3. The relatively low levels of endogenous IGF‐1 and VEGF mRNA following injury support their potential beneficial role as exogenous modulators to optimize tendon healing and strength without increasing adhesion formation. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:886–892     

Effect of shock-wave therapy on patellar tendinopathy in a rabbit model.

This study investigated the effect of shock-wave therapy (SWT) on collagenase induced tendinopathy in the rabbit patellar tendon. Eighteen rabbits were treated by ultrasonography-guided injection of 0.025 ml collagenase into the patellar tendon in both knees. After tendinopathy was confirmed at 3 weeks post-treatment by the histological examination, SWT was initiated to the right patellar tendon involving 1500 cycles at 0.29 mJ/mm2 in two separated weekly courses from 4 weeks post-treatment. The rabbits were randomly divided into two groups, which were sacrificed at the 4th and 16th week after SWT, respectively. The histological examination, the mechanical and biochemical tests then were performed. The ultimate tensile load in the SWT tendon increased 7.03% at 4 week and 10.34% at 16 week after treatment as compared to the sham group. Hydroxyproline concentrations increased in the SWT tendons over both the 4 and 16 weeks after treatment. Moreover, the pyridinoline concentration increased at the 4th week but decreased at 16th week as compared to the sham group. The histological examination demonstrated increased blast-like tenocyte at the 4th week, while more mature tenocyte with neovasculization at the 16th week. The result obtained here validates the effectiveness of the SWT in the established tendinopathy. SWT may increase collagen synthesis and collagen crosslink formation during early healing process.     

Injury response of geriatric mouse patellar tendons

Injury adversely impacts the structure and mechanical properties of a tendon, thus causing pain and disability. Previously, we demonstrated that patellar tendons in mature (P150) and aged (P300) mice do not recover original functionality, even 6 weeks after injury, and that uninjured geriatric tendons (P570) are functionally inferior to uninjured mature tendons. In this study, we hypothesized that the repair response in injured geriatric mice would be further compromised, thus undermining patellar tendon function post‐injury. Patellar tendons from wild‐type mice were injured at 540 days. At 3 and 6 weeks post‐surgery, structural, mechanical, and biochemical analyses were performed and compared to uninjured controls. Mechanical properties of geriatric tendons failed to improve after injury. When compared to mature and aged tendons post‐injury, it was determined that at no age was there a suitable repair response. In previous studies, we were able to associate the absence of SLRPs with phenotypic changes both early and late in repair. Here we found that SLRPs were significantly decreased after injury, thus offering a possible explanation for why geriatric tendons were unable to mount an adequate repair response. Thus, we conclude that regardless of age after maturity, tendon healing ultimately results in a substandard outcome. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1256–1263, 2016.     

Effect of NKISK on tendon lengthening: An in vivo model for various clinically applicable dosing regimens

One proposed mechanism of tendon lengthening is the “sliding fibril” hypothesis, in which tendons lengthen through the sliding of discontinuous fibrils after release of decorin‐fibronectin interfibrillar bonds. The pentapeptide NKISK has been reported to inhibit the binding of decorin, a proteoglycan on the surface of collagen fibrils, to fibronectin, a tissue adhesion molecule, which are thought to play a role in interfibrillar binding. Prior investigations have demonstrated that NKISK produces in vivo tendon lengthening. This study investigates the effect of potential clinically applicable NKISK injection regimens in an in vivo model. One hundred and thirteen male Sprague‐Dawley rats were divided into 15 different treatment groups, each receiving percutaneous patellar tendon injections of NKISK, QKTSK (a “nonsense” pentapeptide), or PBS of varying volumes, concentrations, and injection schedules. Following sacrifice, the patellar tendon lengths were measured in all groups, and biomechanical testing was performed with comparisons made to the contralateral, untreated control limbs. Tendon lengthening was significantly greater (p ≤ 0.05) in nearly all NKISK‐treated tendons as compared to PBS‐ and QKTSK‐treated tendons and was dose‐dependent. Measured lengthening was less in rats whose sacrifice was delayed following the final injection of NKISK, which likely indicates recontraction of lengthened tendons, but they remained significantly longer than the controls. Biomechanical testing did not reveal significant differences in ultimate load, modulus, stiffness, or displacement. This study demonstrates that NKISK given in clinically plausible dosing regimens produces dose‐dependent tendon lengthening in an in vivo setting with minimal effects on the mechanical properties of the treated tendons. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:971–976, 2008     

The native cell population does not contribute to central‐third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon

Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon‐to‐bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central‐third patellar tendon (PT) defect site. To do this, we contrasted the histochemical and biomechanical properties of de‐cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post‐surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12, or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 638–644, 2013     

Effect of overuse‐induced tendinopathy on tendon healing in a rat supraspinatus repair model

Supraspinatus tears often result in the setting of chronic tendinopathy. However, the typical repair model utilizes an acute injury. In recognition of that distinction, our laboratory developed an overuse animal model; however it is unclear whether induced overuse is necessary in the repair model. We studied the repair properties of overuse‐induced tendons compared to normal tendons. We hypothesized that histological and mechanical properties would not be altered between the overuse‐induced and normal tendons 1 and 4 weeks after repair. Thirty‐one adult male Sprague‐Dawley rats were subjected to either overuse or cage activity for 4 weeks prior to bilateral supraspinatus tendon repair surgery. Rats were sacrificed at 1 and 4 weeks post‐surgery and evaluated for histology and mechanics. Results at 1 week showed no clear histologic changes, but increased inflammatory protein expression in overuse tendons. At 4 weeks, percent relaxation was slightly increased in the overuse group. No other alterations in mechanics or histology were observed. Our results suggest that the effects of the surgical injury overshadow the changes evoked by overuse. Because clinically relevant mechanical parameters were not altered in the overuse group, we conclude that when examining tendons 4 weeks after repair in the classic rat supraspinatus model, inducing overuse prior to surgery is likely to be unnecessary. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:161–166, 2016.     

Cycle‐dependent matrix remodeling gene expression response in fatigue‐loaded rat patellar tendons

Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low‐cycle (100) and high‐cycle (7,200) sub‐failure‐fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real‐time RT‐PCR at 1‐ and 7‐day post‐loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high‐cycle group showed upregulation of TIMP‐1, ‐2, Col3a1, and Col5a1, and downregulation TIMP‐4 at both time points, upregulation of MMP‐2 at 7‐day post‐loading and downregulation of MMP‐13 and ‐14 at 1‐day post‐loading, suggesting overall repair/remodeling. In contrast, the low‐cycle loaded group showed upregulation of MMP‐2, ‐3, ‐13, and Col12a1 at both time points, upregulation of TIMP‐1, ‐2, ‐3, Col3a1, and integrin β1 and downregulation of integrin α11 at 1‐day post‐loading and upregulation of Col1a1 at 7‐day post‐loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high‐ and low‐cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response. Published by Wiley Periodicals, Inc. J Orthop Res 28:1380–1386, 2010     

Patellar Tendon Ruptures

Rupture of the patellar tendon is a relatively infrequent, yet disabling, injury, which is most commonly seen in patients less than 40 years of age. It tends to occur during athletic activities when a violent contraction of the quadriceps muscle group is resisted by the flexed knee. Rupture usually represents the final stage of a degenerative tendinopathy resulting from repetitive microtrauma to the patellar tendon. This injury may also occur during less strenuous activity in patients whose tendons are weakened by systemic illness or the administration of local or systemic corticosteroid medications. The diagnosis is made on the basis of the presence of a painful, palpable defect in the substance of the tendon; an inability to completely extend the knee against gravity; and the existence of patella alta confirmed by lateral radiographs. Ultrasonography and magnetic resonance imaging are useful in identifying a neglected rupture, as well as when the diagnosis is in question or an intra-articular injury is suspected. The prognosis after a patellar tendon rupture depends in large part on the interval between injury and repair. Surgery soon after the injury is recommended for optimal results. This is best accomplished by accurate reapproximation of the ruptured tendon ends, repair of the torn extensor retinacula, and placement of a reinforcing cerclage suture. An aggressive rehabilitation program, emphasizing early range-of-motion exercises, protected weight bearing, and quadriceps strengthening, will enhance the results of surgery. Patients who undergo delayed repair are at risk for a compromised result secondary to loss of full knee flexion and decreased quadriceps strength, although a functional extensor mechanism is likely to be reestablished.