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Mechanical loading affects tendon healing and recovery. However, our understanding about how physical loading affects recovery of viscoelastic functions, collagen production and tissue organisation is limited. The objective of this study was to investigate how different magnitudes of loading affects biomechanical and collagen properties of healing Achilles tendons over time. Achilles tendon from female Sprague Dawley rats were cut transversely and divided into two groups; normal loading (control) and reduced loading by Botox (unloading). The rats were sacrificed at 1, 2- and 4-weeks post-injury and mechanical testing (creep test and load to failure), small angle x-ray scattering (SAXS) and histological analysis were performed. The effect of unloading was primarily seen at the early time points, with inferior mechanical and collagen properties (SAXS), and reduced histological maturation of the tissue in unloaded compared to loaded tendons. However, by 4 weeks no differences remained. SAXS and histology revealed heterogeneous tissue maturation with more mature tissue at the peripheral region compared to the center of the callus. Thus, mechanical loading advances Achilles tendon biomechanical and collagen properties earlier compared to unloaded tendons, and the spatial variation in tissue maturation and collagen organization across the callus suggests important regional (mechano-) biological activities that require more investigation., Competing Interests: The authors have declared that no competing interests exist.

Background: The immune system reflects the microbiome (microbiota). Modulation of the immune system during early tendon remodeling by dexamethasone treatment can improve rat Achilles tendon healing. The authors tested whether changes in the microbiota could influence the effect of dexamethasone treatment., Hypothesis: A change in microbiome would influence the response to dexamethasone on regenerate remodeling, specifically tendon material properties (peak stress)., Study Design: Controlled laboratory study., Methods: Specific opportunist and pathogen-free female rats were housed separately (n = 41) or together with specific pathogen-free rats carrying opportunistic microbes such as Staphylococcus aureus (n = 41). After 6 weeks, all co-housed rats appeared healthy but now carried S aureus. Changes in the gut bacterial flora were tested by API and RapID biochemical tests. All rats (clean and contaminated) underwent Achilles tendon transection under aseptic conditions. Flow cytometry was performed 8 days postoperatively on tendon tissue. Sixty rats received subcutaneous dexamethasone or saline injections on days 5 through 9 after transection. The tendons were tested mechanically on day 12. The predetermined primary outcome was the interaction between contamination and dexamethasone regarding peak stress, tested by 2-way analysis of variance., Results: Dexamethasone increased peak stress in all groups but more in contaminated rats (105%) than in clean rats (53%) (interaction, P = .018). A similar interaction was found for an estimate of elastic modulus ( P = .021). Furthermore, dexamethasone treatment reduced transverse area but had small effects on peak force and stiffness. In rats treated with saline only, contamination reduced peak stress by 16% ( P = .04) and elastic modulus by 35% ( P = .004). Contamination led to changes in the gut bacterial flora and higher levels of T cells (CD3 + CD4 + ) in the healing tendon ( P < .05)., Conclusion: Changes in the microbiome influence tendon healing and enhance the positive effects of dexamethasone treatment during the early remodeling phase of tendon healing., Clinical Relevance: The positive effect of dexamethasone on early tendon remodeling in rats is strikingly strong. If similar effects could be shown in humans, immune modulation by a few days of systemic corticosteroids, or more specific compounds, could open new approaches to rehabilitation after tendon injury.

Background: Mechanical loading stimulates Achilles tendon healing. However, various degrees of loading appear to have different effects on the mechanical properties of the healing tendon, and strong loading might create microdamage in the tissue. This suggests that different mechanisms might be activated depending on the magnitude of loading. The aim of this study was to investigate these mechanisms further., Methods: Female rats had their right Achilles tendon cut transversely and divided into three groups: 1) unloading (calf muscle paralysis by Botox injections, combined with joint fixation by a steel-orthosis), 2) mild loading (Botox only), 3) strong loading (free cage activity). Gene expression was analyzed by PCR, 5 days post-injury, and mechanical testing 8 days post-injury. The occurrence of microdamage was analyzed 3, 5, or 14 days post-injury, by measuring leakage of injected fluorescence-labelled albumin in the healing tendon tissue., Results: Peak force, peak stress, and elastic modulus of the healing tendons gradually improved with increased loading as well as the expression of extracellular matrix genes. In contrast, only strong loading increased transverse area and affected inflammation genes. Strong loading led to higher fluorescence (as a sign of microdamage) compared to mild loading at 3 and 5 days post-injury, but not at 14 days., Discussion: Our results show that strong loading improves both the quality and quantity of the healing tendon, while mild loading only improves the quality. Strong loading also induces microdamage and alters the inflammatory response. This suggests that mild loading exert its effect via mechanotransduction mechanisms, while strong loading exert its effect both via mechanotransduction and the creation of microdamage., Conclusion: In conclusion, mild loading is enough to increase the quality of the healing tendon without inducing microdamage and alter the inflammation in the tissue. This supports the general conception that early mobilization of a ruptured tendon in patients is advantageous., Competing Interests: The authors have declared that no competing interests exist.

The role of inflammation and the mechanism of tendon healing after rupture has historically been a matter of controversy. The purpose of the present study is to investigate the role of mast cells and their relation to the NMDA receptor-1 (a glutamate receptor) during healing after Achilles tendon rupture. Eight female Sprague Dawley rats had their right Achilles tendon transected. Three weeks after rupture, histological quantification of mast cell numbers and their state of degranulation was assessed by histochemistry. Co-localization of mast cell tryptase (a mast cell marker) and NMDA receptor-1 was determined by immunofluorescence. The intact left Achilles tendon was used as control. An increased number of mast cells and a higher proportion of degranulated mast cells were found in the healing Achilles tendon compared to the intact. In addition, increased co-localization of mast cell tryptase and NMDA receptor-1 was seen in the areas of myotendinous junction, mid-tendon proper and bone tendon junction of the healing versus the intact tendon. These findings introduce a possible role for mast cells in the healing phase after Achilles tendon rupture.

Tendon function and homeostasis rely on external loading. This study investigates the biological mechanisms behind tendon biomechanical function and how the mechanical performance is affected by reduced daily loading. The Achilles tendons of 16 weeks old female Sprague Dawley rats (n = 40) were unloaded for 5 weeks by inducing muscle paralysis with botulinum toxin injections in the right gastrocnemius and soleus muscles. The contralateral side was used as control. After harvest, the tendons underwent biomechanical testing to assess viscoelasticity (n = 30 rats) and small angle X-ray scattering to determine the structural properties of the collagen fibrils (n = 10 rats). Fourier transform infrared spectroscopy and histological staining (n = 10 rats) were performed to investigate the collagen and proteoglycan content. The results show that the stiffness increased in unloaded tendons, together with an increased collagen content. Creep and axial alignment of the collagen fibers were reduced. Stress-relaxation increased whereas hysteresis was reduced in response to unloading with botox treatment. Our findings indicate that altered matrix deposition relies on mechanical loading to reorganize the newly formed tissue, without which the viscoelastic behavior is impaired. The results demonstrate that reduced daily loading deprives tendons of their viscoelastic properties, which could increase the risk of injury.

Mechanical loading stimulates tendon healing both when applied in the inflammatory phase and in the early remodeling phase of the process, although not necessarily via the same mechanisms. We investigated the gene response to mechanical loading in these two phases of tendon healing. The right Achilles tendon in rats was transected, and the hindlimbs were unloaded by tail suspension. The rats were exposed to 5 min of treadmill running 3 or 14 days after tendon transection. Thereafter, they were resuspended for 15 min or 3 h until euthanasia. The controls were suspended continuously. Gene analysis was first performed by microarray analysis followed by quantitative RT-PCR on selected genes, focusing on inflammation. Fifteen minutes after loading, the most important genes seemed to be the transcription factors EGR1 and C-FOS, regardless of healing phase. These transcription factors might promote tendon cell proliferation and differentiation, stimulate collagen production, and regulate inflammation. Three hours after loading on day 3 , inflammation was strongly affected. Seven inflammation-related genes were upregulated according to PCR: CCL20, CCL7, IL-6, NFIL3, PTX3, SOCS1, and TLR2. These genes can be connected to macrophages, T cells, and recruitment of leukocytes. According to Ingenuity Pathway Analysis, the recruitment of leukocytes was increased by loading on day 3 , which also was confirmed by histology. This inflammation-related gene response was not seen on day 14 Our results suggest that the immediate gene response after mechanical loading is similar in the early and late phases of healing but the late gene response is different. NEW & NOTEWORTHY This study investigates the direct effect of mechanical loading on gene expression during different healing phases in tendon healing. One isolated episode of mechanical loading was studied in otherwise unloaded healing tendons. This enabled us to study a time sequence, i.e., which genes were the first ones to be regulated after the loading episode., (Copyright © 2017 the American Physiological Society.)

Inflammation initiates tendon healing and then normally resolves more or less completely. Unresolved inflammation might disturb the remodeling process. We hypothesized that suppression of inflammation during the early remodeling phase by systemic dexamethasone treatment can improve healing. 36 rats underwent Achilles tendon transection and were randomized to dexamethasone or saline on days 0-4 after surgery (early inflammatory phase), and euthanasia day 7. Another 54 rats received injections days 5-9 (early remodeling phase) and were euthanized day 12 for mechanical, histological and flow cytometric evaluation. Dexamethasone treatment days 0-4 reduced the cross-sectional area, peak force and stiffness by day 7 to less than half (p < 0.001 for all), while material properties (peak stress and elastic modulus) were not significantly affected. In contrast, dexamethasone treatment days 5-9 increased peak force by 39% (p = 0.002) and stiffness by 58% (p < 0.001). The cross-sectional area was reduced by 42% (p < 0.001). Peak stress and elastic modulus were more than doubled (p < 0.001 for both). Semi-quantitative histology at day 12 showed that late dexamethasone treatment improved collagen alignment, and flow cytometry revealed reduced numbers of CD8a + cytotoxic T cells in the tendon callus. These results suggest that downregulation of lingering inflammation during the early remodeling phase can improve healing.

Background and purpose - In 3 papers in Acta Orthopaedica 10 years ago, we described that platelet-rich plasma (PRP) improves tendon healing in a rat Achilles transection model. Later, we found that microtrauma has similar effects, probably acting via inflammation. This raised the suspicion that the effect ascribed to growth factors within PRP could instead be due to unspecific influences on inflammation. While testing this hypothesis, we noted that the effect seemed to be related to the microbiota. Material and methods - We tried to reproduce our old findings with local injection of PRP 6 h after tendon transection, followed by mechanical testing after 11 days. This failed. After fruitless variations in PRP production protocols, leukocyte concentration, and physical activity, we finally tried rats carrying potentially pathogenic bacteria. In all, 242 rats were used. Results - In 4 consecutive experiments on pathogen-free rats, no effect of PRP on healing was found. In contrast, apparently healthy rats carrying Staphylococcus aureus showed increased strength of the healing tendon after PRP treatment. These rats had lower [corrected] levels of cytotoxic T-cells in their spleens. Interpretation - The failure to reproduce older experiments in clean rats was striking, and the difference in response between these and Staphylococcus-carrying rats suggests that the PRP effect is dependent on the immune status. PRP functions may be more complex than just the release of growth factors. Extrapolation from our previous findings with PRP to the situation in humans therefore becomes even more uncertain.

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.

Early tendon healing can be stimulated by mechanical loading and inhibited by cyclooxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs). Therefore, we investigated if impairment of tendon healing by a COX-2 inhibitor (parecoxib) is related to loading. Because loading might infer microdamage, which also stimulates healing, we also investigated if this effect is inhibited by parecoxib. The Achilles tendon was transected in 114 rats. Three degrees of loading were used: full loading, partial unloading, and unloading (no unloading, Botox injections in the plantar flexor muscles, or Botox in combination with tail suspension). For each loading condition, the rats received either parecoxib or saline. In a second experiment, rats were unloaded with Botox, and the tendon was subjected to microdamage by needling combined with either saline or parecoxib. Mechanical testing day 7 showed that there was a significant interaction between loading and parecoxib for peak force at failure (P < 0.01). However, logarithmic values showed no significant interaction, meaning that we could not exclude that the inhibitory effect of parecoxib was proportionate to the degree of loading. Microbleeding was common in the healing tissue, suggesting that loading caused microdamage. Needling increased peak force at failure (P < 0.01), and this effect of microdamage was almost abolished by parecoxib (P < 0.01). Taken together, this suggests that COX-2 inhibition impairs the positive effects of mechanical loading during tendon healing, mainly by reducing the response to microdamage., (Copyright © 2015 the American Physiological Society.)

Background: During early tendon healing, the cells within the regenerating tissue are, to a large part, inflammatory leukocytes (CD45+). In a rat Achilles tendon healing model, the inflammation resolves between 5 and 10 days. In the same model, Cox inhibitors (NSAIDs) impair healing when given during the first 5 days, but have a positive effect if given later. We tested the hypothesis that a Cox inhibitor would exert these effects by influencing inflammation, and thereby the composition of the inflammatory cell subpopulations. Methods: Achilles tendon transection was performed in 44 animals. Animals were randomized to either parecoxib or saline injections. Healing was evaluated by mechanical testing day 7 after surgery and by flow cytometry day 3 and 10. Results: Cross-sectional area, peak force and stiffness were reduced by parecoxib 31, 33, and 25% respectively (p=0.005, p=0.002, and p=0.005). By flow cytometry, there was a strong effect of time (p<0.001) on virtually all inflammatory cell subpopulations (CD45, CD11b, CD68, CCR7, CD163, CD206, CD3, CD4), but no significant effect of parecoxib at any time point. Conclusion: The results suggest that the negative effects of Cox inhibitors on tendon healing might be exerted mainly via mechanisms not directly related to inflammatory cells. [ABSTRACT FROM AUTHOR]