GDF-5 deficiency in mice delays Achilles tendon healing.

The aim of this study was to examine the role of one of the growth/differentiation factors, GDF-5, in the process of tendon healing. Specifically, we tested the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, biochemical, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 -/- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Mutant animals took longer to achieve peak cell density, glycosaminoglycan content, and collagen content in the repair tissue, and the time course of changes in collagen fibril size was also delayed. Revascularization was delayed in the mutant mice by 1 week. GDF-5 deficient Achilles tendons also contained significantly more fat within the repair tissue at all time points examined, and was significantly weaker than control tissue at 5 weeks after surgery, but strength differences were no longer detectable by 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair, and are consistent with previously posited roles for GDF-5 in cell recruitment, migration/adhesion, differentiation, proliferation, and angiogenesis.     

The effect of growth/differentiation factor-5 deficiency on femoral composition and mechanical behavior in mice

A subclass of the bone morphogenetic proteins (BMPs), known as growth/differentiation factors (GDFs) 5, 6, and 7, have been shown to affect several skeletal processes, including endochondral ossification, synovial joint formation, and tendon and ligament repair. Mice deficient in GDF-5 have also been shown to exhibit biomechanical abnormalities in tendon that may be associated with altered type I collagen. The purpose of this study was to investigate the effect of GDF-5 deficiency on another type I collagen-rich tissue: cortical bone. Analyses were performed on femora from 8-week-old GDF-5-deficient male brachypodism mice. We hypothesized that GDF-5-deficient bones would exhibit altered geometric, structural, and material properties compared with control littermates. Mutant animals were significantly smaller in body mass than controls (-21%). Geometrically, mutant long bones were significantly shorter (-25%), had a lower polar moment of inertia (-34%), and a lower geometric strength indicator (analogous to the section modulus of a circular section) (-30%). When normalized by body mass, however, geometric differences were no longer significant. Structurally, GDF-5-deficient femora were weaker (-31%) and more compliant (-57%) than controls when tested to failure in torsion. Lower bone structural stiffness in the mutants was not completely explained by the smaller bone geometry, because mutant bones exhibited a significantly lower effective shear modulus (-36%). Although body mass did not fully explain the reduced structural strength in mutant bones, strength differences were adequately explained by bone cross-sectional geometry; maximum effective shear stress was not significantly different between mutants and controls, despite a statistically significant 6% lower ash fraction in mutant femora. No significant difference was detected in collagen content, as indicated by hydroxyproline per dry mass.     

The effects of growth and differentiation factor 5 on bone marrow stromal cell transplants in an in vitro tendon healing model

The effects of growth differentiation factor-5 (GDF-5) and bone marrow stromal cells (BMSCs) on tendon healing were investigated under in?vitro tissue culture conditions. BMSCs and GDF-5 placed in a collagen gel were interpositioned between the cut ends of dog flexor digitorum profundus tendons. The tendons were randomly assigned into four groups: 1) repaired tendon without gel; 2) repaired tendon with BMSC-seeded gel; 3) repaired tendon with GDF-5 gel without cells; and 4) repaired tendon with GDF-5 treated BMSC-seeded gel. At 2 and 4 weeks, the maximal strength of repaired tendons with GDF-5 treated BMSCs-seeded gel was significantly higher than in tendons without gel interposition. However, neither BMSCs nor GDF-5 alone significantly increased the maximal strength of healing tendons at 2 or 4 weeks. These results suggest that the combination of BMSCs and GDF-5 accelerates tendon healing, but either BMSCs or GDF-5 alone are not effective in this model.     

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Healing of the rat Achilles tendon is sensitive to mechanical loading, and the callus strength is reduced by 3/4 after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This implies BMP signaling is crucial during tendon healing and influenced by mechanical loading. We therefore asked if mechanical loading influences the gene expression of the BMP signaling system in intact and healing tendons, and how the BMP signaling system changes during healing. The genes were four BMPs (OP-1/BMP-7, GDF-5/CDMP-1/BMP-14, GDF-6/CDMP-2/BMP-13, and GDF-7/CDMP-3/BMP-12), two receptors (BMPR1b and BMPR2), and the antagonists follistatin and noggin. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. All genes except noggin were expressed at all time points, but followed different patterns during healing. Loading strongly decreased the expression of follistatin, which could lead to increased signaling. The BMP system appears involved in tendon maintenance and healing, and may respond to mechanical loading.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Matrix remodeling in healing rabbit Achilles tendon

Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young's modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.     

Experimental treatment of tendon injury with heparin

The effect of heparin on the progress of healing in the injured tendon was examined after a standard injury had been created in the Achilles tendons of New Zealand White rabbits. Half of the rabbits were then treated by the intratendinous injection of heparin. The diameters of the injured right hind and the uninjured left hind Achilles tendon and overlying skin were measured using vernier calipers before injury and at one month after injury. At this time histological analysis was carried out on all Achilles tendons and tendon dry weight, collagen concentration and DNA concentration were measured. Orientation of collagen fibres was more pronounced in treated than untreated tendons while cellularity and blood vessel density were less pronounced. Analysis by Students t-test demonstrated a statistically significant difference between treated and untreated tendons in terms of leg diameter (p less than 0.005), dry weight (p less than 0.01) and DNA concentration (p less than 0.01).     

Association Between Knee Osteoarthritis and Functional Changes in Ankle Joint and Achilles Tendon

Increasing evidence has shown that biomechanical forces often drive the progression of knee osteoarthritis (OA). Attention should be given to the changes in adjacent joints and their relation to knee OA. The purpose of the present study was to examine the changes in Achilles tendon thickness of individuals with knee OA and to evaluate the correlation between Achilles tendon thickness and knee OA severity in a case-control prospective observational study. A total of 93 participants with no previous ankle injuries were recruited. Of the 93 participants, 63 had knee OA of the medial compartment and 30 served as controls. The subjects underwent a clinical examination that included measurements of weight, height, Achilles tendon thickness, and 1-leg heel rise. The subjects also underwent a computerized gait test and completed the Hebrew version of the Western Ontario and McMaster Osteoarthritis Index and 36-item short-form (SF-36) health survey. Significant difference was found in Achilles tendon thickness between the subjects with knee OA and the healthy controls (17.1 ± 3.4 versus 15.1 ± 3.1; p = .009). Significant differences were also found between the 2 groups in the 1-leg heel rise test, Western Ontario and McMaster Osteoarthritis Index scores, SF-36 scores, and all gait measures. Significant correlations were found between the Achilles tendon thickness and the following measures: weight (r = 0.46), body mass index (r = 0.55), Kellgren and Lawrence OA severity grade (r = 0.25), 1-leg heel rises (r = ?0.50), and SF-36 score (r = ?0.25). Subjects with knee OA presented with a thicker Achilles tendon compared with the healthy controls. Furthermore, a significant correlation between Achilles tendon thickness and knee OA severity was found. A comprehensive assessment of the Achilles tendon and ankle joint should be a part of the knee OA evaluation process.     

Single-row Versus Double-row Repair of the Distal Achilles Tendon: A Biomechanical Comparison

Surgery for recalcitrant insertional Achilles tendinopathy often consists of partial or total release of the insertion site, debridement of the diseased portion of the tendon, calcaneal ostectomy, and reattachment of the Achilles to the calcaneus. Although single-row and double-row techniques exist for repair of the detached Achilles tendon, biomechanical data are lacking to support one technique over the other. Based on data extrapolated from the study of rotator cuff repairs, we hypothesized that a double-row construct would provide superior fixation strength over a single-row repair. Eighteen human cadaveric Achilles tendons (9 matched pairs) with attached calcanei were repaired with single-row or double-row techniques. Specimens were mounted in a servohydraulic materials testing machine, subjected to a preconditioning cycle, and loaded to failure. Failure was defined as suture breakage or pullout, midsubstance tendon rupture, or anchor pullout. Among the failures were 12 suture failures, 5 proximal-row anchor failures, and 1 distal-row anchor failure. No midsubstance tendon ruptures or testing apparatus failures were observed. There were no statistically significant differences in the peak load to failure between the single-row and double-row repairs (p = .46). Similarly, no significant differences were observed with regards to mean energy expenditure to failure (p = .069). The present study demonstrated no biomechanical advantages of the double-row repair over a single-row repair. Despite the lack of a clear biomechanical advantage, there may exist clinical advantages of a double-row repair, such as reduction in knot prominence and restoration of the Achilles footprint.     

Ten weeks of treadmill running decreases stiffness and increases collagen turnover in tendons of old mice

Increased tendon stiffness in response to mechanical loading is well established in young animals. Given that tendons stiffen with aging, we aimed to determine the effect of increased loading on tendons of old animals. We subjected 28‐month‐old mice to 10 weeks of uphill treadmill running; sedentary 8‐ and 28‐month‐old mice served as controls. Following training, plantaris tendon stiffness and modulus were reduced by approximately half, such that the values were not different from those of tendons from adult sedentary animals. The decrease in plantaris tendon stiffness was accompanied by a similar reduction in the levels of advanced glycation end‐product protein adducts in tibialis anterior tendons of trained compared with sedentary old mice. In Achilles tendons, elevated mRNA levels for collagen type 1, matrix‐metalloproteinase‐8, and lysyl oxidase following training suggest that collagen turnover was likely also increased. The dramatic mechanical and structural changes induced by training occurred independent of changes in cell density or tendon morphology. Finally, Achilles tendon calcification was significantly reduced following exercise. These results demonstrate that, in response to exercise, tendons from old animals are capable of replacing damaged and dysfunctional components of extracellular matrix with tissue that is mechanically and structurally comparable to adult tissue. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:346–353, 2016.     

High intratendinous lactate levels in painful chronic Achilles tendinosis. An investigation using microdialysis technique.

In this investigation the microdialysis technique was used to study the concentrations of lactate in Achilles tendons with painful chronic tendinosis and in normal pain-free tendons. In four patients (mean age 40.7 years) with a painful thickening localized at the 2-6 cm level in the Achilles tendon (chronic Achilles tendinosis) and in five controls (mean age 37.2 years) with normal Achilles tendons the local concentrations of lactate were registered under resting conditions. All tendons were examined using ultrasonography. In the tendons with tendinosis the painful thickening corresponded to a widened tendon and structural tendinosis changes. Normal tendons showed no widening and a normal structure. A standard microdialysis catheter was inserted into the Achilles tendon under local anesthesia. Samplings were done every 15 min during a 4 h period. The results showed significantly higher mean concentrations of lactate in tendons with tendinosis compared to normal tendons (2.15 mmol/l vs. 1.14 mmol/l). The lactate concentrations in the tendons with tendinosis were stable, and approximately twofold higher than in the normal tendons during the whole 4 h investigation period. In conclusion, the higher concentrations of lactate in Achilles tendons with painful tendinosis indicate that there are anaerobic conditions in the area with tendinosis. The importance of this finding for the pathogenesis and pain mechanisms in this chronic condition needs to be further investigated.     

Nonsurgical treatment and early return to activity leads to improved Achilles tendon fatigue mechanics and functional outcomes during early healing in an animal model

Achilles tendon ruptures are common and devastating injuries; however, an optimized treatment and rehabilitation protocol has yet to be defined. Therefore, the objective of this study was to investigate the effects of surgical repair and return to activity on joint function and Achilles tendon properties after 3 weeks of healing. Sprague–Dawley rats (N = 100) received unilateral blunt transection of their Achilles tendon. Animals were then randomized into repaired or non‐repaired treatments, and further randomized into groups that returned to activity after 1 week (RTA1) or after 3 weeks (RTA3) of limb casting in plantarflexion. Limb function, passive joint mechanics, and tendon properties (mechanical, organizational using high frequency ultrasound, histological, and compositional) were evaluated. Results showed that both treatment and return to activity collectively affected limb function, passive joint mechanics, and tendon properties. Functionally, RTA1 animals had increased dorsiflexion ROM and weight bearing of the injured limb compared to RTA3 animals 3‐weeks post‐injury. Such functional improvements in RTA1 tendons were evidenced in their mechanical fatigue properties and increased cross sectional area compared to RTA3 tendons. When RTA1 was coupled with nonsurgical treatment, superior fatigue properties were achieved compared to repaired tendons. No differences in cell shape, cellularity, GAG, collagen type I, or TGF‐β staining were identified between groups, but collagen type III was elevated in RTA3 repaired tendons. The larger tissue area and increased fatigue resistance created in RTA1 tendons may prove critical for optimized outcomes in early Achilles tendon healing following complete rupture. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2172–2180, 2016.     

Transiently increased insulin-like growth factor I immunoreactivity in tendons after vibration trauma. An immunohistochemical study on rats

The hind limbs of anaesthetized rats were exposed to vibration trauma (81 Hz; amplitude peak to peak 0.50 mm) for 4 hours during 2 consecutive days. The animals were examined in groups of 4 immediately after the last exposure, and after 1, 2, 3, 5, 7, 10, 14 and 28 days. The Achilles tendons and the tendons of the anterior tibialis muscles were sampled and processed to demonstrate IGF-I immunoreactivity. In the normal Achilles tendon and in the tendon of the anterior tibial muscle, slight IGF-I immunoreactivity was seen in many of the long slender fibroblasts between the collagen bundles. A strong increase in the IGF-I immunoreactivity appeared in the anterior tibialis muscle tendon 3 days after the last vibration exposure. In addition, the tendon fibroblasts became hypertrophic. A similar but less striking increase in IGF-I immunoreactivity appeared in the Achilles tendon. The peak intensity and frequency of stained cells were achieved after 7 days for both tendons. The intensity then levelled off, and was normalized after 28 days. It is concluded that acute exposure to vibrations induces reactive changes in fibroblasts in tendons, which may reflect a change to a more active synthesising state, as a response to the vibration trauma. The transiently altered expression of IGF-I immunoreactivity forms a link in a chain of events regulating the functional activity level of fibroblasts in response to a trauma.     

Complete stress shielding of the Achilles tendon: ultrastructure and level of interleukin-1 and TGF-β

Few studies emphasize the collagen metabolism-related cytokines and ultrastructure of the completely stress-shielded Achilles tendon. In this study, we used a rat model with complete stress shielding of the Achilles tendon to observe the changes in the ultrastructure of the Achilles tendon and concentration of IL-1 and TGF-β 3 weeks after stress shielding. The model group comprised 12 male Sprague-Dawley rats. The stress of the Achilles tendon of the left hind limb was shielded through tendon cerclage combined with sciatic nerve transection, and the right served as a normal control. Three weeks later, the ultrastructure of the Achilles tendon was observed under electron microscopy and IL-1 and TGF-β levels were determined by enzyme-linked immunosorbent assay. Compared with the control side, collagen fibrils of the shielded Achilles tendons were irregularly arranged and loose. The number of small-diameter collagen fibrils increased significantly with the decrease of the average diameter of collagen fibrils. At the same time, IL-1 concentrations increased significantly in the model group as compared to that in the control group, but no significant difference was found in TGF-β levels. These results suggest that IL-1 may play an important role in the change of ultrastructure after stress shielding.     

Pulsed ultrasound treatment accelerates the repair of Achilles tendon rupture in rats.

A rat tenotomy model was used to investigate the effect of combined conservative management and pulsed ultrasound (PUS) on the repair of tenotomized Achilles tendon. Hemitenotomy of right medial Achilles tendon was performed in 48 rats without suture, and patella tenotomy was performed to mimic immobilization and limb disuse of an injured limb. PUS and sham PUS were applied to the healing wound for the treatment group and control group for 5 min, 3 times per week for 2 or 4 weeks, respectively. Tensile tests showed that the ultimate tensile strength (UTS) and stiffness of the repaired tendon in the treatment group at 2 weeks reached 48.92+/-8.39% and 62.48+/-32.46% of the contralateral normal tendon strength, which were significantly higher than those of the control group (UTS, 30.36+/-15.46%; stiffness, 33.90+/-17.59; p<0.05). At 4 weeks, UTS increased to 77.09+/-15.31% and stiffness to 92.48+/-31.12% in the treatment group, significantly higher than those in the control group (UTS, 54.33+/-18.40%, p<0.01; stiffness, 65.02+/-25.48%, p<0.05). Light microscopy revealed more regular, denser, and better aligned collagen fibers in the healing scar of the PUS-treated healing tendons. The findings suggested that PUS were able to accelerate the healing of the ruptured tendons.