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Tendons play fundamental roles in the execution of human movement and therefore understanding tendon function, health and disease is important for everyday living and sports performance. The acute mechanical behavioural and physiological responses to short-term loading of tendons, as well as more chronic morphological and mechanical adaptations to longer term loading, differ between sexes. This has led some researchers to speculate that there may be a sex-specific injury risk in tendons. However, the link between anatomical, physiological and biomechanical sex-specific differences in tendons and their contributory role in the development of tendon disease injuries has not been critically evaluated. This review outlines the evidence surrounding the sex-specific physiological and biomechanical responses and adaptations to loading and discusses how this evidence compares to clinical evidence on tendon injuries and rehabilitation in the Achilles and patellar tendons in humans. Using the evidence available in both sports science and medicine, this may provide a more holistic understanding to improve our ability to enhance human tendon health and performance in both sexes., (© 2024. The Author(s).)

Background: Achilles tendon is vital in maintaining the stability and function of ankle joint. It is quite difficult to achieve the structural and functional repair of Achilles tendon in tissue engineering., Methods: A tissue-engineered tendon micro-tissue was prepared using rat tail tendon extracellular matrix (TECM) combined with rat adipose stem cells (ADSCs) to repair Achilles tendon injuries. The TECM was prepared by repeated freezing and thawing. The in vitro characteristics of TECM and its effect on ADSCs proliferation were detected. This tissue-engineered tendon micro-tissue for Achilles tendon repair in vivo was evaluated based on general characteristics, gait analysis, ultrasound findings, histological analysis, and biomechanical testing., Results: The results showed that the TECM scaffold had good biocompatibility for ADSCs. At 2 weeks post-surgery, collagen types I and III and tenomodulin expression were higher, and vascular endothelial growth factor expression was lower in the micro-tissue group than other groups. At 4 and 8 weeks post-surgery, the results of histological analysis and ultrasound findings showed that the repaired tendon tissue was smooth and lustrous, and was arranged regularly and evenly in the micro-tissue group. Gait analysis confirmed that better motor function recovery was noted in micro-tissue group than other groups. In addition, the mechanical properties of the repaired tendon tissue in micro-tissue group were better than other groups., Conclusion: Tissue-engineered tendon micro-tissue fabricated by TECM and ADSCs has good biocompatibility and can promote structural and functional repair of tendon in vivo. This composite biomaterial has broad application prospects in tissue engineering., (© 2024. The Author(s).)

Tendon xanthoma and altered mechanical properties have been demonstrated in people with familial hypercholesterolaemia. However, it is unclear whether mild, untreated hypercholesterolaemia alters musculotendinous mechanical properties and muscle architecture. We conducted a case-control study of adults aged 50 years and over, without lower limb injury or history of statin medication. Based on fasting low-density lipoprotein (LDL) cholesterol levels, 6 participants had borderline high LDL (>3.33 mmol/L) and 6 had optimal LDL cholesterol (<2.56 mmol/L). Using shear wave elastography, shear wave velocity (SWV) of the Achilles tendon and gastrocnemius medialis muscle (a proxy for stiffness), along with muscle fascicle length and pennation angle were measured under four passive tensile loads (0, 0.5, 1.0, 1.5 kg) applied via a pulley system. Differences between groups were found for tendon SWV but not muscle SWV, fascicle length or pennation angle. Participants with hypercholesterolaemia showed greater SWV (mean difference, 95 % CI: 2.4 m/s, 0.9 to 4.0, P = 0.024) compared to the control group across all loads. These findings suggest that adults with mild hypercholesterolaemia have increased tendon stiffness under low passive loads, while muscle was not affected. Future research is needed to confirm findings in a larger cohort and explore the impact of hypercholesterolaemia on tendon fatigue injury and tendinopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tendons are collagen-based connective tissues where the composition, structure and mechanics respond and adapt to the local mechanical environment. Adaptation to prolonged inactivity can result in stiffer tendons that are more prone to injury. However, the complex relation between reduced loading, structure, and mechanical performance is still not fully understood. This study combines mechanical testing with high-resolution synchrotron X-ray imaging, scattering techniques and histology to elucidate how reduced loading affects the structural properties and mechanical response of rat Achilles tendons on multiple length scales. The results show that reduced in vivo loading leads to more crimped and less organized fibers and this structural inhomogeneity could be the reason for the altered mechanical response. Unloading also seems to change the fibril response, possibly by altering the strain partitioning between hierarchical levels, and to reduce cell density. This study elucidates the relation between in vivo loading, the Achilles tendon nano-, meso‑structure and mechanical response. The results provide fundamental insights into the mechanoregulatory mechanisms guiding the intricate biomechanics, tissue structural organization, and performance of complex collagen-based tissues. STATEMENT OF SIGNIFICANCE: Achilles tendon properties allow a dynamic interaction between muscles and tendon and influence force transmission during locomotion. Lack of physiological loading can have dramatic effects on tendon structure and mechanical properties. We have combined the use of cutting-edge high-resolution synchrotron techniques with mechanical testing to show how reduced loading affects the tendon on multiple hierarchical levels (from nanoscale up to whole organ) clarifying the relation between structural changes and mechanical performance. Our findings set the first step to address a significant healthcare challenge, such as the design of tailored rehabilitations that take into consideration structural changes after tendon immobilization., Competing Interests: Declaration of Competing Interest The authors have no financial/commercial Conflict of Interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Structural alterations of the triceps surae and Achilles tendon (AT) can promote plantarflexion weakness one-year following an AT repair, influencing the activation strategies of the Gastrocnemius Medialis (GM) muscle. However, this is yet to be demonstrated. We aimed to determine whether patients with plantar flexion weakness one-year after AT repair show altered GM spatial activation. In this cross-sectional and case-control study, ten middle-aged men (age 34 ± 7 years old, and 12.9 ± 1.1 months post-surgery) with a high AT total rupture score who attended conventional physiotherapy for six months after surgery, and ten healthy control men (age 28 ± 9 years old), performed maximal and submaximal (40, 60 and 90%) voluntary isometric plantarflexion contractions on a dynamometer. The peak plantar flexor torque was determined by isokinetic dynamometry and the GM neuromuscular activation was measured with a linear surface-electromyography (EMG) array. Overall EMG activation (averaged channels) increased when the muscle contraction levels increased for both groups. EMG spatial analysis in AT repaired group showed an increased activation located distally at 85-99%, 75-97%, and 79-97% of the electrode array length for 40%, 60%, and 90% of the maximal voluntary isometric contractions, respectively. In conclusion, patients with persistent plantar flexion weakness after AT rupture showed higher distal overactivation in GM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tendon injuries are extremely common, resulting in mechanically weaker tendons that could lead to tendon rupture. Dry needling (DN) is widely used to manage pain and function after injury. However, DN is invasive and high inter-practitioner variability has led to mixed success rates. Focused ultrasound (fUS) is a non-invasive medical technology that directs ultrasound energy into a well-defined focal volume. fUS can induce thermal ablation or mechanical fractionation, with bioeffect type controlled through ultrasound parameters. Tendons must withstand high physiological loads, thus treatments maintaining tendon mechanical properties while promoting healing are needed. Our objective was to evaluate mechanical effects of DN and 3 fUS parameter sets, chosen to prioritize mechanical fractionation, on Achilles and supraspinatus tendons. Ex vivo rat Achilles and supraspinatus tendons (50 each) were divided into sham, DN, fUS-1, fUS-2, and fUS-3 (n = 10/group). Following treatment, tendons were mechanically tested. Elastic modulus of supraspinatus tendons treated with DN (126.64 ± 28.1 MPa) was lower than sham (153.02 ± 29.3 MPa; p = 0.0280). Stiffness and percent relaxation of tendons treated with DN (Achilles: 114.40 ± 31.6 N/mm; 49.10 ± 6.1%; supraspinatus: 109.53 ± 30.8 N/mm; 50.17 ± 7.6%) were lower (all p < 0.0334) than sham (Achilles: 141.34 ± 20.9 N/mm; 60.30 ± 7.7%; supraspinatus: 135.14 ± 30.2 N/mm; 60.85 ± 15.4%). Modulus of Achilles and supraspinatus tendons treated with fUS-1 (159.88 ± 25.7 MPa; 150.12 ± 22.0 MPa, respectively) were similar to sham (156.35 ± 23.0 MPa; 153.02 ± 29.3 MPa, respectively). These results suggest that fUS preserves mechanical properties better than DN, with fUS-1 performing better than fUS-2 and fUS-3. fUS should be studied further to fully understand its mechanical and healing effects to help evaluate fUS as an alternative, non-invasive treatment for tendon injuries., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mechano-regulation during tendon healing, i.e. the relationship between mechanical stimuli and cellular response, has received more attention recently. However, the basic mechanobiological mechanisms governing tendon healing after a rupture are still not well-understood. Literature has reported spatial and temporal variations in the healing of ruptured tendon tissue. In this study, we explored a computational modeling approach to describe tendon healing. In particular, a novel 3D mechano-regulatory framework was developed to investigate spatio-temporal evolution of collagen content and orientation, and temporal evolution of tendon stiffness during early tendon healing. Based on an extensive literature search, two possible relationships were proposed to connect levels of mechanical stimuli to collagen production. Since literature remains unclear on strain-dependent collagen production at high levels of strain, the two investigated production laws explored the presence or absence of collagen production upon non-physiologically high levels of strain (>15%). Implementation in a finite element framework, pointed to large spatial variations in strain magnitudes within the callus tissue, which resulted in predictions of distinct spatial distributions of collagen over time. The simulations showed that the magnitude of strain was highest in the tendon core along the central axis, and decreased towards the outer periphery. Consequently, decreased levels of collagen production for high levels of tensile strain were shown to accurately predict the experimentally observed delayed collagen production in the tendon core. In addition, our healing framework predicted evolution of collagen orientation towards alignment with the tendon axis and the overall predicted tendon stiffness agreed well with experimental data. In this study, we explored the capability of a numerical model to describe spatial and temporal variations in tendon healing and we identified that understanding mechano-regulated collagen production can play a key role in explaining heterogeneities observed during tendon healing., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To compare the biomechanical and histological properties of Achilles tendons repaired at different time points during the acute injury period., Methods: Thirty-six skeletally mature Sprague-Dawley rats underwent bilateral mid-substance Achilles tenotomy. The Achilles tendons were repaired either in the first 24 h (group 1), 24-48 h (group 2), 48-72 h (group 3), or > 72 h (mean: 120 ± 5.2 h) (group 4) after tenotomy. Six weeks after repair, nine tendons per group were assessed biomechanically and histologically. The Stoll histological scoring system was used for histological examination. The groups were compared with each other and native tendons (control group). The correlations between biomechanical and histological results were analysed., Results: There were no significant differences between groups 1, 2 and 3 regarding the mean load to failure; it was significantly lower in group 4. Healed tendons in groups 1, 2 and 3 had significantly greater stiffness than native tendons and group 4 tendons. All healed tendons had a larger cross-sectional area than native tendons. There was no significant difference in tendon length between the groups. There was no significant difference in Young's modulus between the groups; Young's modulus was lower in all the groups than in the control group. Group 1 had significantly higher extracellular matrix organization, cell alignment, cell distribution and nucleus morphology scores and total scores than group 4. Group 1 had significantly higher extracellular matrix organization, cell distribution, vascularization and inflammation scores and total scores than group 3. A significant positive correlation was detected between the maximum load to failure and total histological score., Conclusion: Repair of acute Achilles tendon rupture within 48 h, and especially in the first 24 h, provides better biomechanical and histological outcomes. In the clinical practice, the data could be used to decrease re-rupture rates, to achieve more anatomical tendon healing and to implement more effective post-operative rehabilitation programme.

The pathologic conditions of the Achilles tendon are best understood in the context of its unique anatomy and functional demands. Some of these unique considerations include its high physiologic load demands, microscopic tissue composition, muscular origin spanning the knee joint, intimate insertional relationship with the plantar fascia, sensory innervation, and vascular supply with watershed areas. Risks of both acute rupture and chronic tendinopathy are affected by the tendon's anatomy and its functional demands. The tendon's functional anatomy changes with advancing age, notably in its collagen composition and vascular supply., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Conservative (non-operative) treatment of Achilles tendon ruptures is a common alternative to operative treatment. Following rupture, ankle immobilization in plantarflexion is thought to aid healing by restoring tendon end-to-end apposition. However, early activity may improve limb function, challenging the role of immobilization position on tendon healing, as it may affect loading across the injury site. This study investigated the effects of ankle immobilization angle in a rat model of Achilles tendon rupture. We hypothesized that manipulating the ankle from full plantarflexion into a more dorsiflexed position during the immobilization period would result in superior hindlimb function and tendon properties, but that prolonged casting in dorsiflexion would result in inferior outcomes. After Achilles tendon transection, animals were randomized into eight immobilization groups ranging from full plantarflexion (160°) to mid-point (90°) to full dorsiflexion (20°), with or without angle manipulation. Tendon properties and ankle function were influenced by ankle immobilization position and time. Tendon lengthening occurred after 1 week at 20° compared to more plantarflexed angles, and was associated with loss of propulsion force. Dorsiflexing the ankle during immobilization from 160° to 90° produced a stiffer, more aligned tendon, but did not lead to functional changes compared to immobilization at 160°. Although more dorsiflexed immobilization can enhance tissue properties and function of healing Achilles tendon following rupture, full dorsiflexion creates significant tendon elongation regardless of application time. This study suggests that the use of moderate plantarflexion and earlier return to activity can provide improved clinical outcomes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP -tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)-positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx -deficient mice had migration of Sca-1-positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx -null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro , and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx -null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body., (© 2018 Sakabe et al.)

Conservative treatment (non-operative) of Achilles tendon ruptures is suggested to produce equivalent capacity for return to function; however, long term results and the role of return to activity (RTA) for this treatment paradigm remain unclear. Therefore, the objective of this study was to evaluate the long term response of conservatively treated Achilles tendons in rodents with varied RTA. Sprague Dawley rats (n=32) received unilateral blunt transection of the Achilles tendon followed by randomization into groups that returned to activity after 1-week (RTA1) or 3-weeks (RTA3) of limb casting in plantarflexion, before being euthanized at 16-weeks post-injury. Uninjured age-matched control animals were used as a control group (n=10). Limb function, passive joint mechanics, tendon properties (mechanical, histological), and muscle properties (histological, immunohistochemical) were evaluated. Results showed that although hindlimb ground reaction forces and range of motion returned to baseline levels by 16-weeks post-injury regardless of RTA, ankle joint stiffness remained altered. RTA1 and RTA3 groups both exhibited no differences in fatigue properties; however, the secant modulus, hysteresis, and laxity were inferior compared to uninjured age-matched control tendons. Despite these changes, tendons 16-weeks post-injury achieved secant stiffness levels of uninjured tendons. RTA1 and RTA3 groups had no differences in histological properties, but had higher cell numbers compared to control tendons. No changes in gastrocnemius fiber size or type in the superficial or deep regions were detected, except for type 2x fiber fraction. Together, this work highlights RTA-dependent deficits in limb function and tissue-level properties in long-term Achilles tendon and muscle healing., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Loading influences tendon healing, and so does inflammation. We hypothesized that the two are connected. 48 rats underwent Achilles tendon transection. Half of the rats received Botox injections into calf muscles to reduce mechanical loading. Cells from the regenerating tissue were analyzed by flow cytometry. In the loaded group, the regenerating tissue contained 83% leukocytes (CD45(+)) day 1, and 23% day 10. The M1/M2 macrophage ratio (CCR7/CD206) peaked at day 3, while T helper (CD3(+)CD4(+)) and Treg cells (CD25(+) Foxp3(+)) increased over time. With Botox, markers associated with down-regulation of inflammation were more common day 5 (CD163, CD206, CD25, Foxp3), and M1 or M2 macrophages and Treg cells were virtually absent day 10, while still present with full loading. The primary variable, CCR7/CD206 ratio day 5, was higher with full loading (p = 0.001) and the Treg cell fraction was lower (p < 0.001). Free cage activity loading is known to increase size and strength of the tendon in this model compared to Botox. Loading now appeared to delay the switch to an M2 type of inflammation with more Treg cells. It seems a prolonged M1 phase due to loading might make the tendon regenerate bigger.

Background: The aim of this study was to examine the potential biomechanical and histological benefits of systemic erythropoietin administration during the healing of Achilles tendon injury in a rat experimental model., Methods: Eighty Sprague-Dawley female rats were included in this study. Animals were randomly assigned into two groups with 40 animals in each: erythropoietin group and control group. Then each group was further divided into four subgroups corresponding to four time points with 10 animals in each. A full-thickness cut was made on the Achilles tendon of each animal and then the tendon was sutured with modified Kessler method. Erythropoietin groups received intraperitoneal erythropoietin (500 IU/kg/day) every day at same time throughout the study period, and the control groups received saline in a similar manner. Animals were sacrificed at four time points, and tensile test was performed on each tendon sample to assess maximum load for each sample. In addition, histopathological examination and scoring was done., Results: Both groups had improvement on tensile test (maximum load) over time. However, groups did not differ with regard to maximum load in any of the time points. Similarly, groups did not differ with regard to any of the histopathological scores over time., Conclusions: The findings of this study do not support the benefit of systemic erythropoietin administration in Achilles tendon healing process. Further evidence from larger experimental studies is required to justify any such potential benefit.

Background: Tendon injury is one of the most frequent injuries in sports activities. TENS is a physical agent used in the treatment of pain but its influence on the tendon's healing process is unclear., Objective: To evaluate the influence of TENS on the healing of partial rupture of the Achilles tendon in rats., Method: Sixty Wistar rats were submitted to a partial rupture of the Achilles tendon by direct trauma and randomized into six groups (TENS or Sham stimulation) and the time of evaluation (7, 14, and 21 days post-injury). Burst TENS was applied for 30 minutes, 6 days, 100 Hz frequency, 2 Hz burst frequency, 200 µs pulse duration, and 300 ms pulse train duration. Microscopic analyses were performed to quantify the blood vessels and mast cells, birefringence to quantify collagen fiber alignment, and immunohistochemistry to quantify types I and III collagen fibers., Results: A significant interaction was observed for collagen type I (p=0.020) where the TENS group presented lower percentage in 14 days after the lesion (p=0.33). The main group effect showed that the TENS group presented worse collagen fiber alignment (p=0.001) and lower percentage of collagen III (p=0.001) and the main time effect (p=0.001) showed decreased percentage of collagen III at 7 days (p=0.001) and 14 days (p=0.001) after lesion when compared to 21 days., Conclusions: Burst TENS inhibited collagen I and III production and impaired its alignment during healing of partial rupture of the Achilles tendon in rats.

Background: Surgical repair around the bone-tendon insertion (BTI) may involve bone-to-bone (BB), bone-to-tendon (BT), or tendon-to-tendon (TT) reattachment with varying healing outcome., Hypothesis: The repair of Achilles tendon-calcaneus (ATC) by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time., Study Design: Controlled laboratory study., Methods: Forty-seven adolescent male Chinese goats were divided into BB, BT, and TT groups. Osteotomy of the calcaneus, reattachment of Achilles tendon to the calcaneus after removal of the insertion, and tenotomy of the Achilles tendon were performed to simulate BB, BT, and TT repair, respectively. The ATC healing complexes were harvested at 6, 12, or 24 weeks postoperatively. Mechanical and morphological properties of the healing ATC complexes were assessed by tensile testing and qualitative histology, respectively. The contralateral intact ATC complex was used as the control., Results: Failure load of BT was 33.4% lower than that of TT (P = .0243) at week 12. Ultimate strength of BT was 50.2% and 45.3% lower than that of TT at weeks 12 (P = .0002) and 24 (P = .0001), respectively. Tissue morphological characteristics of the BB and TT groups showed faster remodeling. The BT group showed limited regeneration of fibrocartilage zone and excessive formation of fibrous tissue at the healing interface., Conclusion: BTI repair between homogeneous tissues (BB and TT healing) showed better healing quality with respect to mechanical and histological assessments than did healing between heterogeneous tissues (BT healing)., Clinical Relevance: Anatomic reconstruction of ATC complex injury may be a primary concern when selecting the proper surgical approach. However, it is recommended to select fracture fixation (BB) or tendon repair (TT) instead of bone-tendon reattachment (BT) if possible to ensure better outcome at the healing interface., (© 2015 The Author(s).)

Background: Tendon lacerations are most commonly managed with surgical repair. Postoperative complications such as adhesions and ruptures often occur with immobilization. Early postoperative mobilization is therefore advised to minimize complications and time required to return to daily life. The aim of this study was to evaluate whether botulinum neurotoxin type-A (BoNT-A) can be used to enhance healing and prevent rupture in mobilized animals with Achilles tenotomy., Methods: Twenty-seven rabbits were divided into 3 groups, namely, I, II, and III, after surgical 1-sided Achilles tenotomy and end-to-end repair. The control group for biomechanical comparisons consisted of randomly selected contralateral (unoperated) healthy Achilles tendons. Group I received BoNT-A (4 U/kg) injection into the calf muscles. One week later, electromyographical confirmation was performed to establish the effects of injection. Surgery was then performed. Animals in the second group (n = 9, group II) were immobilized with a cast postoperatively. The third group (n = 9, group III) was mobilized immediately with no cast or BoNT-A. Tendons were harvested and gap formation or ruptures as well as strength of the repaired tendon were assessed 6 weeks after surgery., Results: Achilles tendons healed in all animals injected with BoNT-A, whereas all were ruptured in group III. All Achilles tendons of animals in groups I and II healed. However, group I repaired tendons were biomechanically equivalent to healthy tendons, whereas group II repaired tendons demonstrated significantly decreased tensile strength (P = 0.009)., Conclusions: The present study suggests that local injection of BoNT-A can be used for treatment of tendon rupture and may replace the use of cast for immobilization. However, further studies are needed to determine whether BoNT-A injection can have a beneficial effect on the healing of tendon repairs in humans.

Background and Objective: Tissue engineering is an option in reconstructing large tendon defects and managing their healing and regeneration. We designed and produced a novel xenogeneic-based bovine platelet, embedded it within a tissue-engineered collagen implant (CI) and applied it in an experimentally induced large tendon defect model in rabbits to test whether bovine platelets could stimulate tendon healing and regeneration in vivo., Methods: One hundred twenty rabbits were randomly divided into two experimental and pilot groups. In all the animals, the left Achilles tendon was surgically excised and the tendon edges were aligned by Kessler suture. Each group was then divided into three groups of control (no implant), treated with CI and treated with collagen-platelet implant. The pilot groups were euthanized at 10, 15, 30 and 40 days post-injury (DPI), and their gross and histologic characteristics were evaluated to study host-graft interaction mechanism. To study the tendon healing and its outcome, the experimental animals were tested during the experiment using hematologic, ultrasonographic and various methods of clinical examinations and then euthanized at 60 DPI and their tendons were evaluated by gross pathologic, histopathologic, scanning electron microscopic, biophysical and biochemical methods., Results: Bovine platelets embedded within a CI increased inflammation at short term while it increased the rate of implant absorption and matrix replacement compared with the controls and CI alone. Treatment also significantly increased diameter, density, amount, alignment and differentiation of the collagen fibrils and fibers and approximated the water uptake and delivery behavior of the healing tendons to normal contralaterals (p < 0.05). Treatment also improved echogenicity and homogenicity of the tendons and reduced peritendinous adhesion, muscle fibrosis and atrophy, and therefore, it improved the clinical scores and physical activity related to the injured limb when compared with the controls (p < 0.05)., Conclusion: The bovine platelet gel embedded within the tissue-engineered CI was effective in healing, modeling and remodeling of the Achilles tendon in rabbit. This strategy may be a valuable option in the clinical setting.

Achilles tendon injuries affect both athletes and the general population, and their incidence is rising. In particular, the Achilles tendon is subject to dynamic loading at or near failure loads during activity, and fatigue induced damage is likely a contributing factor to ultimate tendon failure. Unfortunately, little is known about how injured Achilles tendons respond mechanically and structurally to fatigue loading during healing. Knowledge of these properties remains critical to best evaluate tendon damage induction and the ability of the tendon to maintain mechanical properties with repeated loading. Thus, this study investigated the mechanical and structural changes in healing mouse Achilles tendons during fatigue loading. Twenty four mice received bilateral full thickness, partial width excisional injuries to their Achilles tendons (IACUC approved) and twelve tendons from six uninjured mice were used as controls. Tendons were fatigue loaded to assess mechanical and structural properties simultaneously after 0, 1, 3, and 6 weeks of healing using an integrated polarized light system. Results showed that the number of cycles to failure decreased dramatically (37-fold, p<0.005) due to injury, but increased throughout healing, ultimately recovering after 6 weeks. The tangent stiffness, hysteresis, and dynamic modulus did not improve with healing (p<0.005). Linear regression analysis was used to determine relationships between mechanical and structural properties. Of tendon structural properties, the apparent birefringence was able to best predict dynamic modulus (R(2)=0.88-0.92) throughout healing and fatigue life. This study reinforces the concept that fatigue loading is a sensitive metric to assess tendon healing and demonstrates potential structural metrics to predict mechanical properties., (© 2013 Published by Elsevier Ltd.)

Background: Many unsolved problems in plastic and hand surgery are related to poor healing of acute and chronic tendon injuries. The authors hypothesized that tendon healing could be augmented by the addition of a tendon-derived, extracellular matrix hydrogel that would guide tissue regeneration., Methods: Both Achilles tendons of 36 Wistar rats were given full-thickness injuries approximately 5 mm long and 0.5 mm wide from the tendon insertion at the calcaneus to the midsubstance. The hydrogel was injected into the injury site of one leg and compared with control saline in the other. The ultimate failure load, ultimate tensile stress, and stiffness were evaluated at 2, 4, and 8 weeks. Tendon cross-sections underwent histologic analysis (hematoxylin and eosin and picrosirius red) after the animals were killed. Statistical analysis of biomechanical data was performed using a paired t test., Results: There was no significant difference in strength between gel and saline injections in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.42), or stiffness (p = 0.76) at 2 weeks. However, there was a significant difference in ultimate failure load (74.8 ± 11.6 N versus 58.4 ± 14.2 N; p = 0.02) at 4 weeks. The difference in ultimate tensile stress (p = 0.63) and stiffness (p = 0.08) remained insignificant. By 8 weeks, there was no significant difference in strength in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.39), or stiffness (p = 0.75)., Conclusions: Treatment with the tendon hydrogel significantly increases the ultimate failure load of tendons at the critical 4-week time point, and is a promising method for augmentation of tendon healing.

Background: Acute Achilles tendon rupture is a frequent and potentially disabling injury. Over the past decade a change in treatment of acute Achilles tendon rupture away from operative towards non-operative treatment has taken place. However, the optimal non-operative treatment protocol remains to be clarified, particularly the role of weight-bearing during early rehabilitation. Also, there is a need for a clinically applicable and accurate measurement to detect patients in risk of developing Achilles tendon elongation., Purpose: The aim of this PhD thesis was to evaluate non-operative treatment of acute Achilles tendon rupture., Methods: In study I, a cross-sectional survey was performed investigating the chosen treatment protocols across Scandinavia. In study II, the effect of immediate weight-bearing on patient reported and functional outcomes was investigated in a randomized controlled trial (RCT). In study III, the effect of immediate weight-bearing on the biomechanical properties of the plantar flexor muscle-tendon complex was investigated in an RCT. In study IV, validity, reliability and agreement of a novel ultrasound measurement of Achilles tendon length and elongation was tested., Results: Study I found surgery to be the preferred treatment in 83% of departments in Denmark, 92% in Norway, 65% in Sweden, and 30% in Finland (p < 0.001). Study II and III showed no statistically significant effects of controlled early weight-bearing at one year follow-up except from a better health-related quality of life in the weight-bearing group (p=0.009). Compared to the unaffected limb, the affected limb had decreased stiffness (77%, p < 0.001) and strength (93%, p = 0.009) of the plantar flexor muscle-tendon complex. Study IV showed excellent intra-rater reliability (ICC 0.96, SEM 3.7 mm and MDC 10.3 mm), inter-rater reliability (ICC 0.97, SEM 3.3 mm and MDC 9.3 mm) and validity (measurement error 2%)., Conclusion: Treatment algorithms across Scandinavia showed considerable variation, though operative treatment and controlled early weight-bearing was the preferred treatment in Denmark, Norway and Sweden. Immediate weight-bearing was found to be safe and recommendable in non-operative treatment of acute Achilles tendon rupture. The novel ultrasound measurement showed excellent reliability and acceptable validity and agreement.

Background: Restoration of biomechanical strength following surgical reconstruction of tendon or ligament insertion tears is challenging because these injuries typically heal as fibrous scars. The authors hypothesize that injuries at the tendon-bone interface would benefit from reconstruction with decellularized composite tendon-bone grafts., Methods: Tendon-bone grafts were harvested from Sprague-Dawley rats. Grafts subjected to decellularization were compared histologically and biomechanically with untreated grafts ex vivo and in a new in vivo model. Wistar rats underwent Sprague-Dawley allograft reconstruction using a pair-matched design. The rats were killed at 2 or 4 weeks. B-cell and macrophage infiltration was determined using immunohistochemistry, and explants were tested biomechanically., Results: Decellularization resulted in a decrease in cells from 164 ± 61 (untreated graft) to 13 ± 7 cells per high-power field cells (p < 0.005) and a corresponding significant decrease in DNA content, and preserved scaffold architecture of the tendon-bone interface. Biomechanical comparison revealed no difference in failure load (p = 0.32), ultimate tensile stress (p = 0.76), or stiffness (p = 0.22) between decellularized grafts and untreated controls. Following in vivo reconstruction with tendon-bone interface grafts, decellularized grafts were stronger than untreated grafts at 2 weeks (p = 0.047) and at 4 weeks (p < 0.005). A persistent increase in B-cell and macrophage infiltration was observed in both the capsule surrounding the tendon-bone interface and the tendon substance in untreated controls., Conclusion: Decellularized tendon-bone grafts display better biomechanical properties at early healing time points and a decreased immune response compared with untreated grafts in vivo.

Background: Weightbearing in a fixed brace after acute Achilles tendon ruptures does not necessarily lead to mechanical tension in the tendon. Early motion has a positive effect on the clinical outcome, but it is not clear if this is because of effects on tendon strength or unspecific effects. The aim of this study was to examine if tensional loading leads to an improvement in the mechanical properties of the healing Achilles tendon., Hypothesis: The elastic modulus of the tendon callus is increased by early tensional loading., Study Design: Randomized controlled trial; Level of evidence, 2., Methods: Thirty-five patients with an acute Achilles tendon rupture were recruited consecutively. They underwent surgery with a single suture and received metal markers in the distal and proximal parts of the tendon. After surgery, the patients were randomized to either cast immobilization for 7 weeks or tensional loading. The latter group wore a cast for 2 weeks and then a removable foam walker boot for 5 weeks. They were instructed to remove the boot twice daily and push a special training pedal to produce a predetermined, gradually increasing tensional load on the healing tendon. At 7, 19, and 52 weeks postoperatively, the patients were investigated with roentgen stereophotogrammetric analysis under different loading conditions and computed tomography. The collected data allowed calculation of the modulus of elasticity. At 52 weeks, the clinical outcome was also examined using the Achilles tendon Total Rupture Score (ATRS) and the heel-raise index., Results: The elastic modulus at 19 and 52 weeks was higher in the tensional loading group. There was no significant difference in the ATRS or heel-raise index at 52 weeks. As in previous studies, there was a significant correlation between the modulus at 7 weeks and the heel-raise index at 52 weeks. There were no signs of tendon elongation., Conclusion: Early tensional loading improves the mechanical properties of the healing Achilles tendon.

Heterotopic mineralization may result in tendon weakness, but effects on other biomechanical responses have not been reported. We used a needle injury, which accelerates spontaneous mineralization of murine Achilles tendons, to test two hypotheses: that injured tendons would demonstrate altered biomechanical responses; and that unilateral injury would accelerate mineralization bilaterally. Mice underwent left hind (LH) injury (I; n = 11) and were euthanized after 20 weeks along with non-injured controls (C; n = 9). All hind limbs were examined by micro computed tomography followed by biomechanical testing (I = 7 and C = 6). No differences were found in the biomechanical responses of injured tendons compared with controls. However, the right hind (RH) tendons contralateral to the LH injury exhibited greater static creep strain and total creep strain compared with those LH tendons (p ≤ 0.045) and RH tendons from controls (p ≤ 0.043). RH limb lesions of injured mice were three times larger compared with controls (p = 0.030). Therefore, despite extensive mineralization, changes to the responses we measured were limited or absent 20 weeks postinjury. These results also suggest that bilateral occurrence should be considered where tendon mineralization is identified clinically. This experimental system may be useful to study the mechanisms of bilateral new bone formation in tendinopathy and other conditions., (Copyright © 2013 Orthopaedic Research Society.)

Injured tendons have limited repair ability after full-thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. The multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham-operated controls). Animals were euthanized 1 or 6 months after surgery, and tendons were subjected to histological and Synchrotron Radiation-based Computed Microtomography (SRµCT) examination and 3D structure analysis. Histological and SRµCT findings showed satisfactory graft integration with native tendon. Histological examination also showed ongoing angiogenesis. Adverse side-effects (inflammation, rejection, calcification) were not observed. The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation., (Copyright © 2013 Orthopaedic Research Society.)

Purpose: Ideally, a classification should have some prognostic value, and should therefore include precise information upon extent and location of the Achilles tendon disorders. We propose a new imaging and anatomical system to classify Achilles tendon disorders at imaging using US and MRI., Approach: We consider the non-insertional region as the tendon mid-portion, and distinguish the insertional component into a pre-insertion site, located about two centimetres above the calcaneum, and a calcaneal insertion, where the tendon is attached to the bone. On sagittal scans, we introduced a new classification which considers two main portions: "musculotendinous" and "insertional". In the context of the muscolotendinous portion, it is possible to find muscle fibres proximally, and the free tendon distally. This latter is made up of proximal, middle and distal portions. We also propose a 5 grade Doppler classification system to quantify blood flow, in which Grades I and II are respectively characterised by the presence of one and two vessels within the tendon; in Grades III, IV and V, the neovascularisation respectively involves less than 50 %, from 50 to 90 %, and more than 90 % of the tendon tissue. These proposed systems will require validation and possible modification to be applied to different tendons.

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., (Copyright © 2012 Orthopaedic Research Society.)

Purpose: Achilles tendon ruptures are devastating and recover slowly and incompletely. There is a great demand for biomolecular therapies to improve recovery, yet little is understood about growth factors in a healing tendon. Here, the role of growth factors during tendon healing in a rat model and their reaction to single and multiple growth factor treatment are explored., Methods: Rat tendons were transected surgically and resutured. The expression of bFGF, BMP-12, VEGF and TGF-β1 was assessed by immunohistochemical analysis one to 8 weeks after surgery. Paracrine effects of TGF-β1 or BMP-12 added by adenoviral transfer, as well as the effect of autologous conditioned serum (ACS) on growth factor expression, were evaluated., Results: bFGF, BMP-12 and VEGF expression was highest 1 week after transection. bFGF and BMP-12 declined during the remaining period whereas VEGF expression persisted. TGF-β1 expression dramatically increased after 8 weeks. ACS treatment increased bFGF (P = 0.007) and BMP-12 (P = 0.004) expression significantly after 8 weeks. Also overall expression of bFGF, BMP-12 and TGF-β1 regardless of time point was significantly greater than controls with ACS treatment (P < 0.05). Both BMP-12 and TGF-β1 treatments had no significant effect. No effect was observed in VEGF with any treatment., Conclusion: bFGF, BMP-12, VEGF and TGF-β1 are differentially expressed during tendon healing. Additional BMP-12 or TGF-β1 has no significant influence, whereas ACS generally increases expression of all factors except VEGF. Staged application of multiple growth factors may be the most promising biomolecular treatment.

The purpose of this study was to determine the long-term impact of surgical repair and subsequent 6-week immobilization of an Achilles tendon rupture on muscle strength, muscle strength endurance and muscle activity. 63 patients participated in this study on average 10.8 ± 3.4 years after surgically repaired Achilles tendon rupture and short-term immobilization. Clinical function was assessed and muscle strength, strength endurance and muscle activity were measured using a dynamometer and electromyography. Ankle ROM, heel height during heel-raise tests and calf circumference were smaller on the injured than on the contralateral side. Ankle torques during the concentric dorsiflexion tasks at 60 °/sec and 180 °/sec and ankle torques during the eccentric plantarflexion task and during the concentric plantarflexion task at 60 °/sec for the injured leg were significantly lower than those for the contralateral leg. The total work during a plantarflexion exercise at 180 °/sec was 14.9% lower in the injured compared to the contralateral leg (p < 0.001). Muscle activity for the gastrocnemius muscle during dorsiflexion tasks was significantly higher in the injured than in the contralateral limb. Limited ankle joint ROM and increased muscle activity in the injured leg suggest compensatory mechanisms to account for differences in muscle morphology and physiology caused by the injury., (© Georg Thieme Verlag KG Stuttgart · New York.)

During neonatal development, tendons undergo a well orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm for elucidation of mechanistic insight required to develop treatment modalities to improve adult tissue healing. The objective of this study was to compare and contrast normal development with injury during early and late developmental healing. Using backwards multiple linear regressions, quantitative and objective information was obtained into the structure-function relationships in tendon. Specifically, proteoglycans were shown to be significant predictors of modulus during early developmental healing but not during late developmental healing or normal development. Multiple independent parameters predicted percent relaxation during normal development, however, only biglycan and fibril diameter parameters predicted percent relaxation during early developmental healing. Lastly, multiple differential predictors were observed between early development and early developmental healing; however, no differential predictors were observed between late development and late developmental healing. This study presents a model through which objective analysis of how compositional and structural parameters that affect the development of mechanical parameters can be quantitatively measured. In addition, information from this study can be used to develop new treatment and therapies through which improved adult tendon healing can be obtained., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During neonatal development, tendons undergo a well-orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm through which improved adult tissue healing may occur. By examining injury at distinctly different stages of development, vital information can be obtained into the structure-function relationships in tendon. The mouse is an intriguing developmental model due to the availability of assays and genetically altered animals. However, it has not previously been used for mechanical analysis of healing tendon due to the small size and fragile nature of neonatal tendons. The objective of this study was to evaluate the differential healing response in tendon at two distinct stages of development through mechanical, compositional, and structural properties. To accomplish this, a new in vivo surgical model and mechanical analysis method for the neonatal mouse Achilles tendons were developed. We demonstrated that injury during early development has an accelerated healing response when compared to injury during late development. This accelerated healing model can be used in future mechanistic studies to elucidate the method for improved adult tendon healing., (Copyright © 2011 Orthopaedic Research Society.)

Non-operative treatment of Achilles tendon ruptures is associated with an increased risk of rerupture. We hypothesized that this is due to inferior mechanical properties during an early phase of healing, and performed a randomized trial, using a new method to measure the mechanical properties. Tantalum markers were inserted in the tendon stumps, and tendon strain at different loadings was measured by stereo-radiography (Roentgen stereophotogrammetric analysis) at 3, 7 and 19 weeks and 18 months after injury. Thirty patients were randomized to operative or non-operative treatment. The primary out-come variable was an estimate for the modulus of elasticity at 7 weeks. Strain per force, cross-sectional area and tendon elongation were also measured. The functional outcome variable was the heel-raise index after 18 months. There was no difference in the mean modulus of elasticity or other mechanical or functional variables between operative and non-operative treatments at any time-point, but strain per force at 7 and 19 weeks had a significantly larger variation in the non-operative group. This group, therefore, might contain more outliers with poor healing. The modulus of elasticity at 7 weeks correlated with the heel-raise index after 18 months in both treatment groups (r(2) =0.75; P=0.0001). This correlation is an intriguing finding., (© 2010 John Wiley & Sons A/S.)

The process by which collagen fibrils are aligned following tendon injury remains unknown. Therefore, we analyzed the process of tendon regeneration by transmission electron microscopy, using a film model method. In mice, the Achilles tendon of medial head was transected. On day 3, after only the proximal end of the transected tendon was placed on film and kept in vivo, a translucent substance containing granules, called tendon gel, was secreted. On day 5, the granules assembled in a loose (L) layer, and coalesced tightly in a dense (D) layer, forming an L-D-L layered pattern. On day 10, granules showed high electron density in H layers, which developed into D-H-D layers on day 13. The distal end was placed on film to face the proximal end. On day 10, the tendon gel showed a D-H-D layer pattern. On day 11, mechanical stress from muscular constriction changed the tendon gel to aligned collagen fibrils (6 ± 2 nm in diameter). Thereafter, the diameter of the fibrils increased. Tendon gel harvested on day 5 or day 10 was pulled manually or by hanging weights (about 0.6 MPa). Aligned collagen fibrils (32 ± 7 nm in diameter) were created by traction using tendon gel harvested on day 10., (Copyright © 2011 Orthopaedic Research Society.)

Achilles tendon ruptures are treated with an initial period of immobilization, which obstructs the healing process partly by a reduction of blood circulation. Intermittent pneumatic compression (IPC) has been proposed to enhance tendon repair by stimulation of blood flow. We hypothesized that daily IPC treatment can counteract the deficits caused by 2 weeks of immobilization post tendon rupture. Forty-eight Sprague-Dawley SD) rats, all subjected to blunt Achilles tendon transection, were divided in three equal groups. Group A was allowed free cage activity, whereas groups B-C were immobilized at the operated hindleg. Group C received daily IPC treatment. Two weeks postrupture the rats were euthanatized and the tendons analyzed with tensile testing and histological assessments of collagen organization and collagen III-LI occurrence. Immobilization significantly reduced maximum force, energy uptake, stiffness, tendon length, transverse area, stress, organized collagen diameter and collagen III-LI occurrence by respectively 80, 75, 77, 22, 47, 65, 49, and 83% compared to free mobilization. IPC treatment improved maximum force 65%, energy 168%, organized collagen diameter 50%, tendon length 25%, and collagen III-LI occurrence 150% compared to immobilization only. The results confirm that immobilization impairs healing after tendon rupture and furthermore demonstrate that IPC-treatment can enhance proliferative tendon repair by counteracting biomechanical and morphological deficits caused by immobilization., ((c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen.

Collagen content and cross-linking are believed to be major determinants of tendon structural integrity and function. The current study aimed to investigate the effects of transforming growth factor (TGF)-beta1 on the collagen content and cross-linking of Achilles tendons, and on the histological and biomechanical changes occurring during Achilles tendon healing in rabbits. Bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the TGF-beta1 gene were surgically implanted into experimentally injured Achilles tendons. Collagen proteins were identified by immunohistochemical staining and fiber bundle accumulation was revealed by Sirius red staining. Achilles tendons treated with TGF-beta1-transfected BMSCs showed higher concentrations of collagen I protein, more rapid matrix remodeling, and larger fiber bundles. Thus TGF-beta1 can promote mechanical strength in healing Achilles tendons by regulating collagen synthesis, cross-link formation, and matrix remodeling.

Background: The abstract goes here and covers two columns. Accelerated rehabilitation has been advocated after Achilles tendon repair, but it produces significant forces at the repair site. The abstract goes here and covers two columns., Hypothesis: Stresses applied to the repaired Achilles tendon simulating postoperative forces may exceed the strength of some repairs., Study Design: Controlled laboratory study., Methods: Fifteen Achilles tendons were incised 4 cm proximal to the calcaneal insertion, then were repaired using either a percutaneous, 4-strand Krackow, or an epitendinous augmented 4-strand Krackow technique. Tendons were cyclically loaded to 1000 cycles each at 100, 190, and 369 N. The number of cycles to initial gapping, 5-mm gapping, and total failure were compared using Mann-Whitney U tests with adjustments for multiple comparisons., Results: Gap resistance was significantly greater for augmented Krackow repairs (2208 cycles to initial gapping) versus nonaugmented repairs (502 cycles, P = .024) and for nonaugmented Krackow repairs versus percutaneous repairs (5 cycles, P = .024). All percutaneous repairs failed during the 100-N cycling (102 +/- 135 cycles). All nonaugmented Krackow repairs failed during the 190-N cycles (total cycles to failure: 1268 +/- 345). All augmented Krackow repairs were intact (no gapping) after the 190-N cycles. Four failed during 369-N cycling (total cycles to failure, 2017 +/- 11), and 1 remained intact for 3000 cycles., Conclusion: Epitendinous cross-stitch weave augmentation of Achilles tendon repairs significantly increased repair strength and gap resistance., Clinical Relevance: Epitendinous cross-stitch weave augmentation of Achilles tendon repairs may better allow for early stretching and ambulation after Achilles tendon repair.

Objective and Design: In the presented study we compared the effect of stable peptide BPC 157 and methylprednisolone on early functional recovery after Achilles tendon to bone transection in a rat model before collagen healing started., Material and Methods: Surgical transection of the right Achilles tendon to bone area was performed in seventy two Wistar Albino male rats. Healing Achilles tendon edges were harvested at days 1-4 following the transection. Using Achilles functional index (AFI), myeloperoxidase activity, histological inflammatory cell influx and vascular index early functional recovery was evaluated., Treatment: Agents (stable peptide BPC 157 10 microg methylprednisolone 5 mg, normal saline 5 ml) were given alone (/kg b.w., intraperitoneally, once daily, first 30 min after surgery, last 24 h before analysis). Control group received normal saline 5 ml/kg., Results: BPC 157 improved functional recovery (AFI values increased at all time points, p <0.05) by anti-inflammatory (decreased myeloperoxidase (MPO) activity and histological inflammatory cell influx, p <0.05) and increased new blood vessel formation (increased vascular index, p <0.05). Methyprednisolone decreased MPO activity and histological inflammatory cell influx, (p <0.05) but also decreased new blood vessel formation and did not affect early functional recovery., Conclusions: Stable peptide BPC 157 with combined anti-inflammatory action and induction of early new blood vessel formation facilitates early functional recovery in Achilles tendon to bone healing.

Purpose: The purpose of this study was to determine age-related differences in the human plantar flexor muscles and tendon., Methods: Four age groups--a 20-yr group (20-27 yr, N = 19), 30-yr group (31-38 yr, N = 15), 50-yr group (46-57 yr, N = 10) and 70-yr group (62-77 yr, N = 15)--volunteered to take part in the present study. Muscle thickness, strength, and activation level (using twitch-interpolation technique) of plantar flexor muscles were measured. Elongation of the Achilles tendon was determined using ultrasonography while subjects performed ramp isometric plantar flexion up to the voluntary maximum., Results: No significant difference in relative muscle thickness (to limb length) was observed among the four age groups. Muscle strength and activation level of the 20-yr group were significantly higher than those of the 50- and 70-yr groups (activation levels were not measured in the 70-yr group), and maximal strain (elongation/initial tendon length) of the Achilles tendon decreased with aging. Although there were no differences in muscle strength and activation levels between the 20- and 30-yr groups, maximal strain of the Achilles tendon of the 30-yr group was already lower than that of the 20-yr group (P = 0.062)., Conclusion: These results suggest that the processes of age-related changes in the muscle and tendon are different. Furthermore, the differences in age-related changes of muscle and tendon might play a role in the frequency of Achilles tendon ruptures among men in their 30s.

Background: Platelet concentrate application with added thrombin improves Achilles tendon repair in the rat. Upon tissue injury, platelets are activated by thrombin, which has many biological properties in common with growth factors. We wanted to differentiate the effect of platelets from that of thrombin., Methods: The Achilles tendon was transected in 50 rats. Platelet gel was prepared from the blood of 10 other rats. The rats were given either platelet gel with active or neutralized thrombin implanted into the defect during the operation, or a local injection 6h postoperatively with 50 microL of either platelet concentrate, thrombin or saline. The rats were killed after 14 days and the tendons were mechanically tested., Results: Compared to saline, platelet gel caused a 42% increase in force at failure, a 90% increase in energy, and a 61% increase in ultimate stress. Platelet gel with neutralized thrombin caused a 22% increase in force at failure, and energy and stress were less elevated. Injected platelet concentrate caused a 24% increase in force at failure, and thrombin caused a 10% increase. These effects and the differences between treatments were statistically significant., Interpretation: Platelets and thrombin had independent and additive stimulatory effects on tendon repair. The clinical relevance is so far unknown.

Background: Mechanical stimulation improves the repair of ruptured tendons. Injection of a platelet concentrate (platelet-rich plasma, PRP) can also improve repair in several animal models. In a rat Achilles tendon transection model, 1 postoperative injection resulted in increased strength after 4 weeks. Considering the short half-lives of factors released by platelets, this very late effect calls for an explanation., Methods: We studied the effects of platelets on Achilles tendon regenerates in rats 3, 5 and 14 days after transection. The tendons were either unloaded by Botulinum toxin A (Botox) injections into the calf muscles, or mechanically stimulated in activity cages. No Botox injections and ordinary cages, respectively, served as controls. Repair was evaluated by tensile testing., Results: At 14 days, unloading (with Botox) abolished any effect of the platelets and reduced the mechanical properties of the repair tissue to less than half of normal. Thus, some mechanical stimulation is a prerequisite for the effect of platelets at 14 days. Without Botox, both activity and platelets increased repair independently of each other. However, at 3 and 5 days, platelets improved the mechanical properties in Botox-treated rats., Interpretation: Platelets influence only the early phases of regeneration, but this allows mechanical stimulation to start driving neo-tendon development at an earlier time point, which kept it constantly ahead of the controls.

Stable gastric pentadecapeptide BPC 157 (BPC 157, as an antiulcer agent in clinical trials for inflammatory bowel disease; PLD-116, PL 14736, Pliva, no toxicity reported) alone (without carrier) ameliorates healing of tendon and bone, respectively, as well as other tissues. Thereby, we focus on Achilles tendon-to-bone healing: tendon to bone could not be healed spontaneously, but it was recovered by this peptide. After the rat's Achilles tendon was sharply transected from calcaneal bone, agents [BPC 157 (10 microg, 10 ng, 10 pg), 6alpha-methylprednisolone (1 mg), 0.9% NaCl (5 mL)] were given alone or in combination [/kg body weight (b.w.) intraperitoneally, once time daily, first 30-min after surgery, last 24 h before analysis]. Tested at days 1, 4, 7, 10, 14, and 21 after Achilles detachment, BPC 157 improves healing functionally [Achilles functional index (AFI) values substantially increased], biomechanically (load to failure, stiffness, and Young elasticity modulus significantly increased), macro/microscopically, immunohistochemistry (better organization of collagen fibers, and advanced vascular appearance, more collagen type I). 6alpha-Methylprednisolone consistently aggravates the healing, while BPC 157 substantially reduces 6alpha-methylprednisolone healing aggravation. Thus, direct tendon-to-bone healing using stabile nontoxic peptide BPC 157 without a carrier might successfully exchange the present reconstructive surgical methods., (Copyright 2006 Orthopaedic Research Society.)

The prevalence of Achilles tendon (AT) injury is high in various sports, and AT rupture patients have been reported to have a 200-fold risk of sustaining a contralateral rupture. Tendon adaptation to different exercise modes is not fully understood. The present study investigated the structural properties of the AT in male elite athletes that subject their AT to different exercise modes as well as in Achilles rupture patients. Magnetic resonance imaging of the foot and leg, anthropometric measurements, and maximal isometric plantar flexion force were obtained in 6 male AT rupture patients and 25 male elite athletes (kayak/control group n = 9, volleyball n = 8 and endurance running n = 8). AT cross-sectional area (CSA) was normalized to body mass. Runners had a larger normalized AT CSA along the entire length of the tendon compared with the control group (P < 0.05). The volleyball subjects had a larger normalized CSA compared with the control group (P < 0.05) in the area of thinnest tendon CSA. No structural differences of the AT were found in the rupture subjects compared with the control group. Rupture subjects did not subject their AT to greater force or stress during a maximal voluntary isometric plantar flexion than the other groups. The CSA of the triceps surae musculature was the strongest predictor of AT CSA (r(s) = 0.569, P < 0.001). This study is the first to show larger CSA in tendons that are subjected to intermittent high loads. AT rupture patients did not display differences in structural or loading properties of the tendons.

Objectives: Versican is the principal large proteoglycan expressed in mid-tendon, but its role in tendon pathology is unknown. Our objective was to define the expression of versican isoform splice variant messenger ribonucleic acid (mRNA) in normal Achilles tendons, in chronic painful tendinopathy and in ruptured tendons., Methods: Total RNA isolated from frozen tendon samples (normal n = 14; chronic painful tendinopathy n = 10; ruptured n = 8) was assayed by relative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for total versican, versican variants V0, V1, V2, V3 and type I collagen alpha1 mRNA, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Differences between sample groups were tested by Wilcoxon statistics., Results: Painful and ruptured tendons showed a significant decrease (median 2-fold) in the expression of versican mRNA, in contrast to an increased expression (median 8-fold) of type I collagen alpha1 mRNA in painful tendons. Versican splice variants V0 and V1 mRNA were readily detected in normal samples, V3 levels were substantially lower, and V2 levels were more variable. Each of V1, V2 and V3 mRNA showed significant decreases in expression in painful and ruptured tendons, but V0 was not significantly changed., Conclusions: Changes in versican expression relative to that of collagen, and alterations in the balance of versican splice variants, may contribute to changes in matrix structure and function in tendinopathies.

Adenovirus-mediated gene transfer is a potential method for enhancing tendon healing. We investigated the transfection of Ad5CMVntLacZ, an adenovirus containing the reporter gene LacZ, in primary cultured human rotator cuff tendon cells and in a rat Achilles tendon healing model in vivo. Ad5CMVempty, the adenoviral vector containing no inserted gene, was used as a control for adenoviral transfection alone. Activity of beta-galactosidase,the protein expressed by LacZ gene, was measured using a beta-galactosidase assay and detected visually by X-gal staining. Cultured cells were successfully transfected without impairing cell viability. Maximal beta-galactosidase activity was detected when cells were transfected at the dose of 1000 PFU/cell. The duration of LacZ expression was six days with a peak value at 24 h post-transfection. A transfection rate of 100% was obtained at 5000 PFU/cell. Successful in vivo transfection by Ad5CMVntLacZ was obtained in healing rat Achilles tendon as confirmed by X-gal staining. 0.4% of tendon cells were transfected when Ad5CMVntLacZ was injected into the tendon at a dose of 10(6) PFU. The rate rose to 2% with 10(8) PFU and 3% with 10(9) PFU. The duration of LacZ expression in vivo was 17 days. Transfection efficiency was enhanced threefold and localization improved when a gelatin sponge was used to deliver the adenovirus. The results demonstrate that adenovirus can be used to deliver a gene of interest to cultured human rotator cuff tendon cells and healing tendon, with gelatin sponge implantation enhancing adenoviral transfection efficiency in vivo.

Flexor tendon injuries are a common problem requiring sutured repair followed by early postoperative mobilisation. Results are better if active rather than passive flexion is allowed, however active movement increases the incidence of dehiscence. This has prompted examination of thermal bonding as a strategy for strengthening tendon repairs. Three experiments are described, using method and experimental design similar to those previously described. The strength of thermal bonds created between sections of bovine Achilles tendon at a range of temperatures and times was measured. Bonds were created by heating compressed sections in a water bath. Bond strength was measured on a tensiometer as load (N) and stress (N/mm2 and N m kg-1) then calculated using measured cross-sectional area, weight and length. Results were in broad agreement with those previously described, showing peak bond strength in samples heated to 62 degrees C for 8 min (median 17.11 N/cm2, SD 2.13). The effects of cooling to 0 degree C for 10 min immediately after bonding was to increase median bond strength when compared with non-cooled samples bonded under similar conditions (non-cooled, 9.11 N/cm2, SD 3.11; cooled, 11.96 N/cm2, SD 2.93). Statistical analysis using a quadratic regression model showed a close correlation between experimental data and curves modelled to best fit the data. Peak values for thermal bonds created using the methods described approached and in some cases exceeded that reported for the epitendinous element of modified Kessler tendon repairs in human digital flexor tendons. If such bonds could be created in vivo, thermal bonding may have a role in augmenting the strength of tendon repairs. The methods described are impractical for a non-laboratory environment. A laser would seem the most attractive alternative source of heat energy.

Bone repair is controlled by signalling proteins as well as mechanical stimuli. Bone morphogenetic proteins (BMPs), implanted or injected into a fracture site, can enhance bone healing, and so can mechanical stimulation. Possibly, mechanical signals can alter BMP expression, but furthermore, tissue differentiation initiated by an implanted BMP could be guided into the required direction by mechanical signals. Mechanical stimulation also has profound effects on tendon repair. We studied the effects of 3 different BMPs in a rat tendon repair model. One of these, osteogenic protein 1 (OP-1) induced bone formation in the tendon at the expense of mechanical strength and thus overrode any mechanical control system. On the other hand, cartilage derived morphogenetic protein 1 and 2 (CDMP-1 and -2) both made the tendon callus bigger and stronger at an early time point.

We studied the effects of insulin-like growth factor I on Achilles tendon healing in a rat model. Rats were randomized into groups of six each: sham surgery, transection alone, and transection plus growth factor. Postoperatively, rats treated with growth factor had a significantly smaller maximum functional deficit and a decreased time to functional recovery than rats in the untreated groups. Biomechanical testing revealed no significant differences in the measured parameters between the treated and the untreated groups after transection. To study the mechanism of action, six additional animals received an Achilles tendon injection of the inflammatory agent carrageenan alone and six received carrageenan plus growth factor. Rats treated with growth factor did not show the inflammation-induced functional deficit experienced by the control rats. Spectrometric myeloperoxidase assays on the remaining eight rats after Achilles tendon transection demonstrated no significant difference between the untreated and the growth factor-treated groups, indicating a mechanism other than neutrophil recruitment by which the growth factor limits inflammation. Histologic studies were performed on carrageenan-injected rats at postinjection day 2 and on surgically treated rats at postoperative day 15. No gross histologic differences were seen between untreated and growth factor-treated groups. This study demonstrated that via a possible antiinflammatory mechanism, insulin-like growth factor I reduces maximum functional deficit and accelerates recovery after Achilles tendon injury.