Introduction and Objective. Achilles tendon defect is difficult problem for orthopedic surgeon, and therefore the development of new treatments is desirable. Platelet-rich fibrin (PRF), dense fibrin scaffold composed of a fibrin matrix containing many growth factors, is recently used as regenerative medicine preparation. However, few data are available on the usefulness of PRF on Achilles tendon healing after injury. The objective of this study is to examine whether PRF promotes the healing of Achilles tendon defect in vivo and evaluated the effects of PRF on tenocytes in vitro. Materials and Methods. PRF were prepared from rats according to international guidelines on the literature. To create rat model for Achilles tendon defect, a 4-mm portion of the right Achilles tendon was completely resected, and PRF was placed into the gap in PRF group before sewing the gap with nylon sutures. To assess the histological healing of Achilles tendon defect, Bonar score was calculated using HE, Alcian-blue, and Picosirius-red staining section. Basso, Beattie, Bresnahan (BBB) score was used for the evaluation of motor functional recovery. Biomechanical properties including failure tensile load, ultimate tensile stress, breaking elongation, and elastic modulus were measured. We examined the effects of PRF on tenocytes isolated from rat Achilles tendon in vitro. The number of viable cells were measured by MTS assay, and immunostaining of ki-67 was used for detection of proliferative cells. Migration of tenocytes was evaluated by wound closure assay. Protein or gene expression level of extracellular matrix protein, such as collagen, were evaluated by immunoblotting, immunofluorescence, or PCR. Phosphorylation level of AKT, FGF receptor, or SMAD3 was determined by western blotting. Inhibitory experiments were performed using MK-2206 (AKT inhibitor), FIIN-2 (FGFR inhibitor), SB-431542 (TGF-B receptor inhibitor), or SIS3 (SMAD3 inhibitor). All p values presented are two-sided and p values < 0.05 were considered statistically significant. Results. In rat Achilles tendon defects, Bonar score was significantly improved in PRF group compared to control group. Collagen deposition at the site of Achilles tendon defect was observed earlier in PRF group. Consistent with the histological findings, BBB score was significantly improved in PRF group. PRF also significantly improved the biomechanical properties of injured Achilles tendon. Furthermore, proliferating tenocytes, labelled by ki-67 were significantly increased in PRF group. These data suggested PRF prompted the healing of Achilles tendon defect. Thus, we further examined the effects of PRF on tenocytes in vitro. PRF significantly increased the number of viable cells, the proliferative cells labelled by ki-67, and migratory ability. Furthermore, PRF significantly increased the protein expression levels of collagen-I, collagen-III, α-SMA, and tenascin-C in tenocytes. Next, we examined the signalling pathway associated with PRF-induced proliferation of tenocytes. PRF increased the phosphorylation level and induced nuclear translocation of AKT, known as key regulator of cell survival. PRF also induced the phosphorylation of FGF receptor. Inhibition of AKT or FGF-receptor completely suppressed the positive effects of PRF on tenocytes. Furthermore, we found that inhibition of FGF receptor partially suppressed the phosphorylation of AKT by PRF. Thus, PRF induced the proliferation of tenocytes via FGFR/AKT axis. We further evaluated the signalling pathway associated with PRF-induced expression of extracellular matrix. PRF increased the phosphorylation levels of SMAD3 and induced nuclear translocation of SMAD3. Furthermore, inhibition of TGF-B receptor or SMAD3 suppressed increased expression level of extracellular matrix by PRF. Thus, PRF increased expression level of extracellular matrix protein via TGF-BR/SMAD3 axis. Conclusions. PRF promotes tendon healing of the Achilles tendon defect and recovery of exercise performance and biomechanical properties. PRF increases the proliferation ability or protein expression level of extracellular matrix protein in tenocytes via FGFR/AKT or TGF-βR/SMAD3 axis, respectively

Achilles tendon mechanical properties depend on a complex hierarchical design, with collagen being the smallest load-bearing unit. At the nanoscale, collagen molecules are organized into fibrils, which at the microscale are assembled into fibers, followed by larger structures such as sub-tendons or fascicles. Degree of in vivo loading affects the collagen content, and organization and consequently the tissue's mechanical response. We aim to unravel how composition, structural organization, and mechanical response are affected by degree of in vivo loading at each length scale. The presentation will outline the results to date about to the use of high-resolution synchrotron-based tissue characterisation methods on several length scales in combination with in situ mechanical tests. We use a rat model, where the tendons are subjected to varying loading in vivo. To characterize the tissue microstructure, phase-contrast enhanced synchrotron micro-tomography is performed. The 3D fiber organization in fully loaded tendons is highly aligned, whereas the fibers in unloaded tendons are significantly more heterogeneously arranged and crimped. To characterize the collagen fibril response, Small Angle X-ray Scattering is performed. Two types of fibril organizations are found; a single population oriented towards the main load direction and two fibril subpopulations with clearly distinct orientations. Scattering during loading showed that the fibrils in unloaded tendons did not strain as much in fully loaded. In situ loading concurrently with high resolution synchrotron experiments show the complex tendon response to in situ load and its relation to in vivo loading and tendon hierarchical structure. Unloading seems to alter the organization of the fibrils and fibers, e.g. increased crimping and more pronounced sub-tendon twists. Acknowledgements: Funding from Knut and Alice Wallenberg Foundation and European Research Council (101002516). Paul Scherrer Institut, Switzerland for beamtime at cSAXS and TOMCAT

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Tendon pathologies represent an unresolved clinical challenge where the patients suffer from pain and impaired mobility. One of the most frequently ruptured tendons is the Achilles tendon and primarily seen in recreational and professional athletes. A study from Sweden reported a significant increase in the incidence of Achilles tendon ruptures of 17% in men and 22% in women due to the demographic changes and the higher sportive activity of older adults (Huttunen TT Am J Sports Med 2014). The re-rupture rate is between 2–10%, and the patients suffer from an impairment over a long time accompanied with incapability to work. The healing process results in the formation of a mechanically insufficient scar tissue. A detailed knowledge on the cellular and molecular processes underlying human Achilles tendon healing is necessary to develop new treatment strategies and judge therapeutic success. The analysis of human Achilles tendon samples at different time points post rupture and the comparison to intact and degenerated tendon tissue provides important information on the healing process

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM

Objectives. After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Methods. Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Results. Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Conclusions. Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses. Bone Joint Res 2017;6:231–244. DOI: 10.1302/2046-3758.64.BJR-2017-0268.R1

Summary Statement. Subject specific FE models of human Achilles tendon were developed and optimum material properties were found. Stress concentration occurred at the midsection but dependent on stiffening and thinning of tendon, indicating that they are two major factors for tendon rupture. Introduction. Achilles tendon injuries are common, occurring about 250,000 per year in the US alone, yet the mechanisms of tendinopathy and rupture remain unknown. Most Achilles tendon ruptures occur at 2 to 6 cm above the insertion to the calcaneus bone. Previous angiographic studies have suggested that there is an avascular area in this region. However, it is not understood why that region receives poor blood supply and prone to rupture. The aim of this study is to investigate influence of geometry and material properties on Achilles tendon rupture with mechanical experiment and corresponding subject-specific finite element (FE) analysis. Patients & Methods. Mechanical experiment was performed on 10 fresh human Achilles tendons. High frequency ultrasound images were used to measure cross sectional areas at the midsection of the tendon. Cyclic testing was performed to measure mechanical properties and failure loads. Subject-specific FE models of these tendons were generated with Free Form Deformation (FFD) technique. FE mechanical simulations that mimic the experimental cyclic loading were performed on these subject specific models. Tendon material properties were described as transversely isotropic hyperelastic and the optimum material parameters for the human Achilles tendon were obtained. Linear portion of the cyclic loading data was used as boundary conditions. Measured strains from the experiment were compared with predicted strains from the FE analysis. This process was repeated until optimum parameters were found. The influence of geometry and material properties on the Achilles tendon rupture was then investigated– first with subject-specific geometry with average material properties and then with subject-specific material properties with average geometry. Results. Our results indicate that a significant variation exist in the geometry and material properties in human Achilles tendons. Stress concentrations occurred at the midsection of the tendon, supporting previous studies that reported tendon rupture at the region. In particular the thinning of midsection in geometry is highly correlated with the collagen uncrimpping rate in material properties where thinner midsection leads to faster uncrimpping of collagen fibres. Variations in geometry led to shifts in the location of stress concentration within the midsection while variations in material property led the change in the magnitude of stress concentration. Discussion/Conclusion. Our results indicate that Achilles tendon rupture is highly dependent on subject-specific geometry and material properties. In particular the mid section is the location of stress concentration but depending on the geometrical shape, multiple stress concentrations occur, making the tendon more prone to rupture while the material properties influenced the magnitude of stress concentration. Our results indicate stiffening and thinning of tendon may lead to higher risk for tendon rupture

Tendon healing is a complex process that often results in compromised healing of the tendon tissue. It has recently been shown that temporal changes in the expression profile and the histological tissue quality of the tendons occur during the early healing process after acute Achilles tendon rupture. Whether these changes are accompanied by an altered healing process, is not yet known and was the aim of the present study. Tendon biopsies were obtained from 24 patients with acute Achilles tendon rupture at the time of surgery (2–9 days after rupture) and examined histologically as well as on RNA level. Histologically, the tendon architecture, the amount of aligned collagen, glycosaminoglycan and fat as well as the cellularity, vascularity and immune cell infiltration were determined. On RNA level the expression of markers for the modeling/remodeling (MMPs and TIMPs), collagens (1, 3, 5), tendon markers (scleraxis, tenomodulin), pro- and anti-inflammatory markers (IL-1beta, IL6, IL10, IL33, TNFa, TGF-beta1, COX2) and immune cell markers (CD3, CD68, CD80, CD206) were analyzed by Real-Time PCR. To determine the clinical outcome, the patients were followed up 12 months after the operation and the following scores were recorded: Subjective score, Tegner score, Visual Analog Scale (VAS) pain, VAS function, Matles Test, Achilles tendon total rupture score (ATRS), Therman 100-points score, Heel rise test. Statistics: Spearman correlation analysis. Correlation analysis shows that early post-rupture surgery is associated with better clinical outcome (ATRS Score: p=0.022). Histologically, a good functional healing outcome shows a positive correlation to the amount of aligned collagen (Heel Rise Test: p = 0.009) and glycosaminoglycans in the tendon (Heel Rise Test: p = 0.026, Matles difference: p = 0.029), as well as a negative correlation to the fat content (Thermann score: p = 0.018, subjective score: p = 0.027, VAS function: p = 0.031). On RNA level, a good healing outcome correlates with increased expression of MMP13, collagen 1, 3, 5 (Heel Rise Test: p = 0.019, p = 0.048, p = 0.030), and TIMP2 (Tegner Score: p = 0.040), TGF-beta1 (Thermann Score: p = 0.032) and CD80 (ATRS: p = 0.025, Thermann score:, p = 0.032). Whereas a limited healing outcome is associated with an increased expression of MMP2 (Heel Rise Test: p = 0.033), MMP3 (Matles Test: p=0.001, Heal Rise test p = 0.017), and IL33 (Tegner Score: p = 0.047). The results of the study show a clear relationship between the tendon biology at the time of the surgery and the clinical and functional healing outcome 12 months after the operation. Especially matrix formation and remodeling play a crucial role, while the examined immunological factors seem to influence the tendon healing to a lesser extent. The modulation of matrix formation could potentially lead to improved treatment options in the future

Achilles tendon rupture may lead to significant functional deficits, which mechanisms are poorly understood. The primary aim was to investigate if the Achilles tendon (AT) was longer, muscles weaker or gait changed on the injured leg 4–5 years after the injury. Secondary aim was to compare functional outcomes with patient reported Achilles Tendon Total Rupture Score (ATRS). We invited all participants from an RCT of conservatively treated AT Rupture (ATR) with or without early weight-bearing (early-WB, non-WB), and 12 moths of follow up. Of the original 56, 37 patients participated, 19 from early-WB (1 re-rupture (RR)), and 18 from non-WB (2 RR). Time from injury to follow up was 4,5 years (4,1 to 5,1). AT length was measured using ultrasound with a validated protocol (Barfod K.W. et al.). Heel raise work was measured on a 10 degree inclining platform. The exercise lasted until the patient could not maintain frequency or height of lift. Number and height of lift was measured using reflective markers in a Vicon system, and total work calculated. Foot pressure mapping (FPM) was measured barefoot, using an EMED platform (novel, Germany). Statistics: T-test for limb to limb comparisons and linear regression for ATRS correlations was applied. Including RR in the sample did not impact the results. We found no differences in any of the variables between the early-WB and non-WB groups. Compared to the uninjured limb, the Achilles tendon was an average of 1,8 (1,2–2,3) cm longer on the injured limb, which produced 40% less work. A smaller calf circumference (p < 0.001), larger dorsiflextion (p = 0.001), and Achilles tendon resting angle (p < 0.001) was found for the injured limb. Difference in mean medial forefoot peak pressure was approaching significance (healthy 484 (SD 165) KPa, injured: 439 (SD 160), p = 0.08). Similarly the difference in pressure / time integral of the medial forefoot was approaching significance (Healthy: 129 (SD 35)KPa, injured: 115 (SD 44)KPa, p = 0.08). Duration of contact time of the heel was extended and heel lift off was delayed in the injured limb (p = 0.02 for both). ATRS could not be linked to Achilles tendon length or total work using linear regression. Conservatively treated Achilles tendon ruptures were approximately 1,8 cm longer. The limb was persistently weaker. A subtle change in heel contact duration and time of heel rise could be detected on the injured limb. ATRS does not appear to correlate directly with AT length or loss of total work

Introduction. Exercise increases tendon collagen synthesis and cross-link formation. Exercise also increases the expression of TGF-β. 1. TGF-β. 1. may contribute to the upregulation of tendon collagen synthesis during exercise, but this relationship has not been established in vivo. The purpose of this study was to evaluate the effects of TGF-β. 1. receptor inhibition on tendon collagen. Materials and Methods. Male Wistar rats were divided into sedentary (SED, n = 9) or exercised (RUN, n=15) groups. Exercised animals completed four days of treadmill exercise (60 minutes/days). The peritendinous space of one Achilles tendon was injected with LY-364947 (ALK5 inhibitor; INHIB) while the opposite leg was injected with a vehicle (SHAM). Injections were given daily after each exercise bout. ERK and Smad 2/3 phosphorylation was evaluated by Western blotting. Collagen I and III gene expression were evaluated via qRT-PCR. Tendon hydroxyproline and hydroxylyslpyridinoline (HP) cross-linking were assayed via HPLC. A longitudinal section of tendon was stained with H&E for evaluation of cell numbers and fibril organization. Results. Phosphorylation of ERK increased by 2.5-fold in both legs given LY-364947 (p<0.05) but was not influenced by exercise (p>0.05). Smad 3 phosphorylation was reduced (p<0.05) in tendons treated with LY-364947. Neither type I nor type III collagen gene expression was affected by TGF-β. 1. receptor inhibition or exercise (p>0.05). Collagen content was not altered by either exercise or LY-364947 (p>0.05). HP cross-linking was 3-fold lower in the RUN-INHIB when compared to the RUN-SHAM tendon (p<0.05). No effect of inhibitor on HP was noted in the sedentary animals. Cell density was greater (p<0.05) in the Achilles tendon of exercised animals (SED: 7.5 cell/100 μm. 2. , RUN: 10.3 cell/100 μm. 2. ) but was not influenced by TGF-β. 1. receptor inhibition (p>0.05). Fiber structure scores were 45% lower (p<0.07) in SED animals treated with inhibitor but normal in RUN animals given inhibitor. Discussion. The changes in ERK and Smad phosphorylation suggest that LY-364947 was effective at altering TGF-β. 1. signaling. Our data suggest that neither acute exercise nor TGF-β. 1. receptor inhibition altered collagen gene expression. In contrast, TGF-β. 1. appears to be important for regulating Achilles tendon cross-link formation during exercise training and inhibition of TGF-β. 1. impacts fiber structure

Achilles tendon (AT) rupture may lead to complaints of heel pain. In forefoot ulcer patients AT lengthening is used to transfer pressure from forefoot to the heel. The primary aim was to investigate if AT was longer or associated with changes in pedobaric measurements, in particular heel pressure, on the injured leg 4–5 years after the injury. Methods. We invited all participants from an RCT (n=56) of conservatively treated AT Rupture (ATR) with or without early weight-bearing (early-WB, non-WB). 37 patients participated, 19 from early-WB (1 re-rupture (RR)), and 18 from non-WB (2 RR). Time from injury to follow up was 4,5 years (4,1 to 5,1). AT length was measured using ultrasound with a validated protocol. Foot pressure mapping (FPM) was measured barefoot, using an EMED platform (novel, Germany), with 5 trials for each foot. Statistics. T-test for limb to limb comparisons and linear regression for correlations was applied. Results. We found no differences in any of the variables between the early-WB and non-WB groups. Compared to the uninjured limb, the Achilles tendon was an average of 1.8 (1.2–2.3) cm longer on the injured limb (p<0.001). When comparing the ratio of the medial (1–2 ray) to lateral (3–5 ray) forefoot mean peak pressure, we found no difference between the injured and healthy limb (p=0.26). Mean heel peak pressure was not different from the injured to the healthy leg (difference was 3,9 (−1,7 – 9,45) p=0,17). Heel lift-off was delayed in the injured limb by 2% (0.4%–4.4%) of the total roll over process (ROP) (p= 0.02). Achilles tendon length could not be linked to either heels lift-off or mean peak pressure of the heel using linear regression (p 0.27 to 0.78). Conclusion. Conservatively treated Achilles tendon ruptures were approximately 1.8 cm longer. A subtle change in the time of heel rise could be detected on the injured limb, but contrary to our expectations AT length did not correlate to time of heel lift or mean heel peak pressure. This is in contrast to the common practice in diabetics, where the Achilles tendon is elongated to relieve pressure from the forefoot – a mechanism we cannot observe from elongation of the tendon after acute rupture, treated conservatively - though this study is underpowered

Introduction. Traditionally Plantaris has been considered of little clinical importance and absent in 8–20% of the population. Recent evidence indicates that it is present in 98–100% of the population and that it may have a contributing role in Achilles tendinopathy due to its close anatomical relationship. The aim of this study was to establish whether Plantaris was present in a sample of cadaveric limbs, to establish its position in relation to the Achilles tendon and to conduct measures of its thickness and width. Materials and Methods. Forty eight cadaveric limbs which had been previously dissected were assessed. Plantaris was looked for in the region of the medial Achilles. If it could not be identified here, Gastrocnemius was reflected back to reveal Plantaris tendon beneath, and was then followed distally. All Plantaris tendon measurements were taken 2- 6 cm from the Achilles insertion using a vernier caliper. Results. Plantaris was present in all of the forty three limbs which were appropriate for assessment. Plantaris was positioned ventromedial to the Achilles tendon in 33 (77%) and medial to the Achilles in 9 (21%) of the limbs. The average width of the Plantaris tendon was 2.8mm (range 1.2–4.9mm) and its average thickness was 0.9mm (range 0.2–1.5mm). Discussion. Plantaris was present in all limbs in keeping with recent studies. This is the first known study, which measures Plantaris tendon in the region of the midportion Achilles. Future studies are planned to compare these measurements with tendinopathic plantaris tendons

Purpose. Functional ultrasound Elastography (FUSE) of Tendo Achilles is an ultrasound technique utilising controlled, measurable movement of the foot to non-invasively evaluate TA elastic and load-deformation properties. The study purpose is to assess Achilles tendons, paratenon and bursa mechanical properties in healthy volunteers and establish an outcome tool for TA treatment. Methods. Forty asymptomatic Achilles tendons of 20 healthy volunteers were recruited (10 men and 10 women, age range 18-55). One patient with Acute Achilles rupture scanned to evaluate the tendon gap. Each volunteer answered the Foot and Ankle Outcome Score (FAOS) and Victorian Institute Sport Assessment score (VISA-A) questionnaires. The Achilles Tendons were divided into three thirds (total 120 Proximal, middle and distal thirds). Three longitudinal images of each third were obtained using portable US scan device (Z.one, Zonare Medical System Inc., USA, 8.5 MHz). Images processing was achieved using a MatLAb software (developed by the research team) in parallel Oxford university computers. Each 1/3rd Achilles tendon under went the following scans: . Free hand US scan. Free hand Compression decompression Elastography scan. Dorsal Flexion elastography. Planter flexion elastography. Zonare real-time Elastography. Elastography scan with the Oxford isometric dynamic foot and Ankle mover (OIDFA). B mode and elasticity images were derived from the raw ultrasound radio frequency data. The anatomical structures mechanical properties were evaluated by a quantitative score of different colours representing stiff tissue (blue) to more soft tissue (green, yellow, red). Results. The Achilles tendons showed mainly a hard structured pattern (82.5%) (99/120 tendon thirds) on sonoelastography; however, mild softening was found in 17.5% (21/120) of the tendons. Therefore, suggesting subclinical changes. The minimal lateral movement of the tendon produced by applying the FOAIDM resulted in well defined elasticity images with tendon in blue colour (stiff) and surrounding soft tissues. The average strain along the tendon was 2% (range 0-6%). The overall correlation (κ) between real-time sonoelastography and ultrasound findings was < 0.3. However, the correlation (κ) between FUSE UEI and US findings was 1.0. Patients with Achilles tendon rupture lateral strain and axial elastography images using FUSE methods revealed a larger gap with spreading of the haematoma along the paratenon. Conclusion. Our findings show that FUSE seems to be a sensitive method for assessment of TA mechanical properties. The B mode and elasticity images must be viewed simultaneously. FUSE method can easily identify the regeneration of ruptured TA. Elasticity and stiffness measurement may offer an invaluable tool to guide TA rupture and tendenopathy treatment and rehabilitation protocols

Summary. Silver nanoparticles improve the tensile property of the repaired Achilles tendon by modulating the synthesis and deposition of collagen. This makes silver nanoparticles a potential drug for tendon healing process with less undesirable side effect. Introduction. Tendon injury is a common injury that usually takes a long time to fully recover and often lead to problems of joint stiffness and re-rupture due to tissue adhesions and scarring on the repaired tendon respectively. Recently, it has been proven that silver nanoparticles (AgNPs) are capable of regenerating skin tissue with minimal scarring and comparable tensile property to normal skin. Hence, it is hypothesised that AgNPs could also improve the healing in tendon injury as both tissues are predominating with fibroblasts. The objective of this study is to look at the in vitro response of primary tenocytes to AgNPs and to investigate the mechanical and histological outcome in vivo. Methods and Materials. Primary tenocytes were harvested from 4 weeks old Sprague Dawley rat. 1.5×10. 4. cells per cm. 2. were seeded in triplicate for BrdU incorporation assay and Sirius red/ fast green staining to study the proliferation and collagen synthesis respectively. In vivo rat Achilles tendon injury model was used to investigate the effect of AgNPs to tendon regeneration. Briefly, the Achilles tendon was transected at 0.5cm from its insertion. The wound was either treated with 1mM AgNPs every 5 days or left untreated as the control. Skin incision was done without transecting the tendon in the sham group. The tendons were harvested on day 42 post operation. Tensile test and immunohistological staining on 7μm cryosections were performed to assess the mechanical property and biological events in healing respectively. SHG imaging was used to determine the collagen fibre orientation and abundance. Results. In vitro BrdU incorporation and Sirius red fast green assay suggested that AgNPs promoted the proliferation and collagen synthesis of tenocytes between 1 to 20μM and 10 to 20μM respectively. Tensile test on in vivo tissue showed that AgNPs-treated samples had significantly better tensile modulus compared to the untreated ones (p<0.05). SHG imaging suggested a better collagen alignment and density in AgNPs-treated samples. Immunohistochemistry demonstrated that AgNPs suppressed tumor necrosis factor (TNF α) whilst promoted fibromodulin (Fmod) and proliferating cell nucleus antigen (PCNA) expression. Discussion. Collagen is the major component that contributes to the tensile strength of a tendon. Its thickness, abundance and alignment directly affect the strength. In this study, it is found that AgNPs stimulate cell proliferation both in vitro and in vivo which is believed to be the reason of the increase in collagen synthesis. Fmod is an important proteoglycan responsible for collagen fibrillogenesis and TNF α is related to ECM degradation which directly affects collagen integrity. Stimulation of Fmod and alleviation of TNF α therefore promote collagen maturity and integrity which attributes to the improvement in the tensile property of the regenerated tissue. Furthermore, inflammation is known to relate to fibrosis and scarring in healing of many types of tissue. It is therefore postulated that the anti-inflammatory effect of AgNPs is one of the major reasons for this phenomenal healing of tendon. To conclude, this study demonstrates a positive effect of AgNPs to the early events of tendon healing which is important for accelerating the whole healing process and shortening of rehabilitation time

Introduction. Tendon cross-sectional area (CSA) and stiffness increase in men during chronic exercise. The increase in tendon CSA and stiffness is not evident in women. In men, exercise increases tendon production of MMPs, IGF-1, and IL-6, which presumably contribute to tendon remodeling during chronic exercise. The purpose of this study was to determine if exercise-induced production of MMPs, IGF-1, and IL-6 are limited in women when compared to men. Materials and Methods. Young men (n=9, 27±1 y) and women (n=8, 26±1 y) performed a single bout of calf press exercise (8 sets of 15 repetitions at 70% of 15-RM). A microdialysis fiber (3000 kDa cut-off) was inserted into the space anterior to the Achilles tendon immediately after exercise and during a control experiment. All proteins were evaluated with ELISA kits. Results. In men IGF-I increased with exercise at 3 (p<0.05) but not 4 hrs. IGF-1 was not elevated at any measured time points in women. IL-6 increased with exercise to a similar extent in men and women at 3 hrs (p<0.05) but values returned to baseline at 4 hrs. MMP-9 increased with exercise at both 2 and 5 hours (p<0.05) in men but not in women. MMP-2 increased with exercise at 2 and 5 hrs to a similar extent in both men and women (p<0.05). In men TIMP-1 increased with exercise at 2 (p<0.05) but not 5 hrs. In women, TIMP-1 levels were elevated post-exercise at both 2 and 5 hrs (p<0.05). Discussion. In men, resistance exercise resulted in a modest and transient increase in tendon production of IGF-1 and IL-6. In women, this is only evident for IL-6. MMP-2 and MMP-9 increased with exercise in men. The exercise-induced increase in MMP-9 was not seen in women. In contrast, MMP-2 increased in women to a comparable magnitude as men. TIMP-1 increased with exercise in men and women but remained elevated out to 5 hrs in women only. The blunted increase in IGF-1 and MMP-9 and a prolonged increase in TIMP-1 may contribute to the lack of tendon adaptations after chronic training in women when compared to men

Introduction

The rabbit common calcanean (Achilles) tendon is a compound apparatus frequently used in studies considering novel interventions to facilitate tendon regeneration. These studies often employ complete surgical transection of the apparatus. Due consideration of the translational relevance to human tendinopathy is often lacking and refinement of this injury model, consistent with the principles of the 3Rs, has not been forthcoming.

Introduction

An additional pathology should be considered for Achillodynia differentials – the intratendinous tear (ITT) – for which we describe symptoms, ultrasound findings and co-presenting pathology.

Introduction

Tendinopathies are among the most common musculoskeletal injuries. Nowadays, part of its diagnosis is established through subjective qualitative evaluation of 2D ultrasound (US). This enables limited diagnostic differentiation or therapeutic optimization and has limited added value to diagnosis in an earlier stage. It is generally accepted that extra diagnostic information can be obtained via strain evaluation. The accurate validation of strain estimation is challenging due to the lack of a ground-truth. Therefore we evaluate the repeatability of displacement and strain estimations in the longitudinal direction, using an easy, fast and interactive application to estimate local strain during dynamic loading of the tendon.

Purpose

Platelet Rich Plasma (PRP) has been shown to have positive effect in tendon regeneration in in-vitro and limited in-vivo animal studies. We aim to study PRP use in acute Achilles tendon rupture (ATR) regeneration in a purposely designed clinical trial.

Introduction

The healing of Achilles tendon rupture is slow and jogging is usually allowed already 6 months after injury. However, the metabolic status of the healing tendon is largely unknown at the time-points when increased loading is allowed. The purpose of this study was to investigate tendon metabolic response and blood flow at 3, 6 and 12 months after Achilles tendon rupture by positron emission tomography (PET) and ultrasound-Power Doppler (UPD).