ICON 2023: International Scientific Tendinopathy Symposium Consensus - the core outcome set for Achilles tendinopathy (COS-AT) using a systematic review and a Delphi study of professional participants and patients.

To develop a core outcome set for Achilles tendinopathy (COS-AT) for use in clinical trials we performed a five-step process including (1) a systematic review of available outcome measurement instruments, (2) an online survey on truth and feasibility of the available measurement instruments, (3) an assessment of the methodological quality of the selected outcome measurement instruments, (4) an online survey on the outcome measurement instruments as COS and (5) a consensus in-person meeting. Both surveys were completed by healthcare professionals and patients. The Outcome Measures in Rheumatology guidelines with a 70% threshold for consensus were followed. We identified 233 different outcome measurement instruments from 307 included studies; 177 were mapped within the International Scientific Tendinopathy Symposium Consensus core domains. 31 participants (12 patients) completed the first online survey (response rate 94%). 22/177 (12%) outcome measurement instruments were deemed truthful and feasible and their measurement properties were evaluated. 29 participants (12 patients) completed the second online survey (response rate 88%) and three outcome measurement instruments were endorsed: the Victorian Institute of Sports Assessment-Achilles questionnaire, the single-leg heel rise test and evaluating pain after activity using a Visual Analogue Scale (VAS, 0-10). 12 participants (1 patient) attended the final consensus meeting, and 1 additional outcome measurement instrument was endorsed: evaluating pain during activity/loading using a VAS (0-10). It is recommended that the identified COS-AT will be used in future clinical trials evaluating the effectiveness of an intervention. This will facilitate comparing outcomes of intervention strategies, data pooling and further progression of knowledge about AT. As COS-AT is implemented, further evidence on measurement properties of included measures and new outcome measurement instruments should lead to its review and refinement.

Dexamethasone treatment influences tendon healing through altered resolution and a direct effect on tendon cells.

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.

Elemental and Structural Characterization of Heterotopic Ossification during Achilles Tendon Healing Provides New Insights on the Formation Process.

Heterotopic ossification (HO) in tendons can lead to increased pain and poor tendon function. Although it is believed to share some characteristics with bone, the structural and elemental compositions of HO deposits have not been fully elucidated. This study utilizes a multimodal and multiscale approach for structural and elemental characterization of HO deposits in healing rat Achilles tendons at 3, 6, 12, 16, and 20 weeks post transection. The microscale tomography and scanning electron microscopy results indicate increased mineral density and Ca/P ratio in the maturing HO deposits (12 and 20 weeks), when compared to the early time points (3 weeks). Visually, the mature HO deposits present microstructures similar to calcaneal bone. Through synchrotron-based X-ray scattering and fluorescence, the hydroxyapatite (HA) crystallites are shorter along the c-axis and become larger in the ab-plane with increasing healing time, while the HA crystal thickness remains within the reference values for bone. At the mineralization boundary, the overlap between high levels of calcium and prominent crystallite formation was outlined by the presence of zinc and iron. In the mature HO deposits, the calcium content was highest, and zinc was more present internally, which could be indicative of HO deposit remodeling. This study emphasizes the structural and elemental similarities between the calcaneal bone and HO deposits.

The Delayed Presentation of Achilles Tendon Ruptures Is Associated With Marked Alterations in the Gene Expression of COL1A1, MMPs, TIMPs, and IL-6.

BACKGROUND: Both acute and chronic Achilles tendon ruptures are affected by alterations in the extracellular matrix during the healing process of the tendon. Yet, these alterations in gene expression patterns are not well characterized. PURPOSE: To characterize temporal and spatial differences in gene expression patterns after an Achilles tendon rupture and to evaluate if cells from chronic Achilles tendon ruptures have the same ability to form new tendon tissue (tendon constructs) as healthy tendon cells. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 35 patients with surgically treated Achilles tendon ruptures were included in the study and divided into 3 groups: acute (<4 weeks), short-term chronic (1-6 months), and long-term chronic (>6 months). Biopsy specimens were collected during surgical repair and were used to analyze the gene expression within the different groups and to compare mRNA levels in the proximal and distal tendon ends. A complementary in vitro experiment was performed to evaluate if cells from chronic Achilles tendon ruptures can form tendon constructs. RESULTS: The mRNA levels for COL1A1 and COL3A1 were significantly higher in the short-term chronic group compared with the acute group (P < .05). Both MMP-1 and MMP-13 had the highest mRNA levels in the acute group (P < .01) compared with the long-term chronic group, while MMP-2 had the highest mRNA level in the short-term chronic group. Significant differences between the proximal and distal tendon ends were only detected for the monocyte and macrophage marker CD163 (P < .05), which was more expressed proximally. Cells extracted from chronic Achilles tendon ruptures displayed a similar ability and effectiveness to form tendon constructs as healthy tendon cells. CONCLUSION: A high collagenase gene activity after an Achilles tendon rupture indicated possible rapid matrix degradation in the acute phase. Chronic ruptures appeared to initiate the healing process even before treatment, indicated by the higher expression of collagen in the short-term chronic group. Cells from chronic Achilles tendon ruptures also displayed an ability to form new tendon tissue in vitro. CLINICAL RELEVANCE: The study shows a rapid increase in collagenase gene expression, which could lead to matrix degradation that continues for months after an Achilles tendon rupture.

Micro- and nanostructure specific X-ray tomography reveals less matrix formation and altered collagen organization following reduced loading during Achilles tendon healing.

Recovery of the collagen structure following Achilles tendon rupture is poor, resulting in a high risk for re-ruptures. The loading environment during healing affects the mechanical properties of the tendon, but the relation between loading regime and healing outcome remains unclear. This is partially due to our limited understanding regarding the effects of loading on the micro- and nanostructure of the healing tissue. We addressed this through a combination of synchrotron phase-contrast X-ray microtomography and small-angle X-ray scattering tensor tomography (SASTT) to visualize the 3D organization of microscale fibers and nanoscale fibrils, respectively. The effect of in vivo loading on these structures was characterized in early healing of rat Achilles tendons by comparing full activity with immobilization. Unloading resulted in structural changes that can explain the reported impaired mechanical performance. In particular, unloading led to slower tissue regeneration and maturation, with less and more disorganized collagen, as well as an increased presence of adipose tissue. This study provides the first application of SASTT on soft musculoskeletal tissues and clearly demonstrates its potential to investigate a variety of other collagenous tissues. STATEMENT OF SIGNIFICANCE: Currently our understanding of the mechanobiological effects on the recovery of the structural hierarchical organization of injured Achilles tendons is limited. We provide insight into how loading affects the healing process by using a cutting-edge approach to for the first time characterize the 3D micro- and nanostructure of the regenerating collagen. We uncovered that, during early healing, unloading results in a delayed and more disorganized regeneration of both fibers (microscale) and fibrils (nanoscale), as well as increased presence of adipose tissue. The results set the ground for the development of further specialized protocols for tendon recovery.

Secretome from myoblasts statically loaded at low intensity promotes tenocyte proliferation via the IGF-1 receptor pathway.

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle-derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA-seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin-like growth factor-1 (IGF-1). Confirmation of IGF-1 expression and secretion was carried out using qPCR and enzyme-linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF-1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome-induced tenocyte proliferation. In conclusion, IGF-1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise-induced healing.

Multimodal and multiscale characterization reveals how tendon structure and mechanical response are altered by reduced loading.

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-, meso­structure 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.

Muscle fascicle and sarcomere adaptation in response to Achilles tendon elongation in an animal model.

Permanent loss of muscle function seen after an Achilles tendon rupture may partly be explained by tendon elongation and accompanying shortening of the muscle. Muscle fascicle length shortens, serial sarcomere number is reduced, and the sarcomere length is unchanged after Achilles tendon transection (ATT), and these changes are mitigated with suturing. The method involved in this study was a controlled laboratory study. Two groups of rats underwent ATT on one side with a contralateral control (CTRL): A) ATT with 3 mm removal of the Achilles tendon and no suturing (substantial tendon elongation), and B) ATT with suture repair (minimal tendon elongation). The operated limb was immobilized for 2 wk to reduce load. Four weeks after surgery the rats were euthanized, and hindlimbs were analyzed for tendon length, gastrocnemius medialis (GM) muscle mass, length, fascicle length, sarcomere number and length. No differences were observed between the groups, and in both groups the Achilles tendon length was longer (15.2%, P < 0.001), GM muscle mass was smaller (17.5%, P < 0.001), and muscle length was shorter (8.2%, P < 0.001) on the ATT compared with CTRL side. GM fascicle length was shorter (11.2%, P < 0.001), and sarcomere number was lower (13.8%, P < 0.001) on the ATT side in all regions. Sarcomere length was greater in the proximal (5.8%, P < 0.001) and mid (4.2%, P = 0.003), but not distal region on the ATT side. In this animal model, regardless of suturing, ATT resulted in tendon elongation, loss of muscle mass and length, and reduced serial sarcomere number, which resulted in an "overshoot" lengthening of the sarcomeres.NEW & NOTEWORTHY Following acute Achilles tendon rupture, patients are often left with functional deficits. The specific reason remains largely unknown. The shortened muscle leads to reduced fascicle length, in turn leading to adaptation by reduced serial sarcomere numbers. Surprisingly, this adaptation appears to "overshoot" and lead to increased sarcomere length. The present animal model advances understanding of how muscle sarcomeres, which are difficult to measure in humans, are affected when undue elongation takes place after tendon rupture.

Spatiotemporal and microstructural characterization of heterotopic ossification in healing rat Achilles tendons.

Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.

Heterotopic mineral deposits in intact rat Achilles tendons are characterized by a unique fiber-like structure.

Heterotopic mineralization entails pathological mineral formation inside soft tissues. In human tendons mineralization is often associated with tendinopathies, tendon weakness and pain. In Achilles tendons, mineralization is considered to occur through heterotopic ossification (HO) primarily in response to tendon pathologies. However, refined details regarding HO deposition and microstructure are unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomography. Furthermore, we test the potential of studying local tissue injury by needling intact Achilles tendons and the relation between tissue microdamage and HO. The results show that HO occurs in all intact Achilles tendons at 16 weeks of age. HO deposits are characterized by an elongated ellipsoidal shape and by a fiber-like internal structure which suggests that some collagen fibers have mineralized. The data indicates that deposition along fibers initiates in the pericellular area, and propagates into the intercellular area. Within HO deposits cells are larger and more rounded compared to tenocytes between unmineralized fibers, which are fewer and elongated. The results also indicate that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption may also occur. Additionally, phase contrast synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level in response to needling. The needle injury protocol could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition.

Nanoscale characterization of collagen structural responses to in situ loading in rat Achilles tendons.

The specific viscoelastic mechanical properties of Achilles tendons are highly dependent on the structural characteristics of collagen at and between all hierarchical levels. Research has been conducted on the deformation mechanisms of positional tendons and single fibrils, but knowledge about the coupling between the whole tendon and nanoscale deformation mechanisms of more commonly injured energy-storing tendons, such as Achilles tendons, remains sparse. By exploiting the highly periodic arrangement of tendons at the nanoscale, in situ loading of rat Achilles tendons during small-angle X-ray scattering acquisition was used to investigate the collagen structural response during load to rupture, cyclic loading and stress relaxation. The fibril strain was substantially lower than the applied tissue strain. The fibrils strained linearly in the elastic region of the tissue, but also exhibited viscoelastic properties, such as an increased stretchability and recovery during cyclic loading and fibril strain relaxation during tissue stress relaxation. We demonstrate that the changes in the width of the collagen reflections could be attributed to strain heterogeneity and not changes in size of the coherently diffracting domains. Fibril strain heterogeneity increased with applied loads and after the toe region, fibrils also became increasingly disordered. Additionally, a thorough evaluation of radiation damage was performed. In conclusion, this study clearly displays the simultaneous structural response and adaption of the collagen fibrils to the applied tissue loads and provide novel information about the transition of loads between length scales in the Achilles tendon.

Early Tensile Loading in Nonsurgically Treated Achilles Tendon Ruptures Leads to a Larger Tendon Callus and a Lower Elastic Modulus: A Randomized Controlled Trial.

BACKGROUND: Early tensile loading improves material properties of healing Achilles tendon ruptures in animal models and in surgically treated human ruptures. However, the effect of such rehabilitation in patients who are nonsurgically treated remains unknown. HYPOTHESIS: In nonsurgically treated Achilles tendon ruptures, early tensile loading would lead to higher elastic modulus 19 weeks after the injury compared with controls. STUDY DESIGN: Randomized controlled trial; Level of evidence, 2. METHODS: Between October 2015 and November 2018, a total of 40 nonsurgically treated patients with acute Achilles tendon rupture were randomized to an early tensile loading (loaded group) or control group. Tantalum bead markers were inserted percutaneously into the tendon stumps 2 weeks after the injury to allow high-precision measurements of callus deformation under mechanical testing. The loaded group used a training pedal twice daily to produce a gradual increase in tensile load during the following 5 weeks. Both groups were allowed full weightbearing in an ankle orthosis and unloaded range of motion exercises. Patients were followed clinically and via roentgen stereophotogrammetric analysis and computed tomography at 7, 19, and 52 weeks after the injury. RESULTS: The mean ± standard deviation elastic modulus at 19 weeks was 95.6 ± 38.2 MPa in the loaded group and 108 ± 45.2 MPa in controls (P = .37). The elastic modulus increased in both groups, although it was lower in the loaded group at all time points. Tendon cross-sectional area increased from 7 weeks to 19 weeks, from 231 ± 99.5 to 388 ± 142 mm2 in the loaded group and from 188 ± 65.4 to 335 ± 87.2 mm2 in controls (P < .001 for the effect of time). Cross-sectional area for the loaded group versus controls at 52 weeks was 302 ± 62.4 mm2 versus 252 ± 49.2 mm2, respectively (P = .03). Gap elongation was 7.35 ± 13.9 mm in the loaded group versus 2.86 ± 5.52 mm in controls (P = .27). CONCLUSION: Early tensile loading in nonsurgically treated Achilles tendon ruptures did not lead to higher elastic modulus in the healing tendon but altered the structural properties of the tendon via an increased tendon thickness. REGISTRATION: NCT0280575 (ClinicalTrials.gov identifier).

Dexamethasone Enhances Achilles Tendon Healing in an Animal Injury Model, and the Effects Are Dependent on Dose, Administration Time, and Mechanical Loading Stimulation.

BACKGROUND: Corticosteroid treatments such as dexamethasone are commonly used to treat tendinopathy but with mixed outcomes. Although this treatment can cause tendon rupture, it can also stimulate the tendon to heal. However, the mechanisms behind corticosteroid treatment during tendon healing are yet to be understood. PURPOSE: To comprehend when and how dexamethasone treatment can ameliorate injured tendons by using a rat model of Achilles tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: An overall 320 rats were used for a sequence of 6 experiments. We investigated whether the drug effect was time-, dose-, and load-dependent. Additionally, morphological data and drug administration routes were examined. Healing tendons were tested mechanically or used for histological examination 12 days after transection. Blood was collected for flow cytometry analysis in 1 experiment. RESULTS: We found that the circadian rhythm and drug injection timing influenced the treatment outcome. Dexamethasone treatment at the right time point (days 7-11) and dose (0.1 mg/kg) significantly improved the material properties of the healing tendon, while the adverse effects were reduced. Local dexamethasone treatment did not lead to increased peak stress, but it triggered systemic granulocytosis and lymphopenia. Mechanical loading (full or moderate) is essential for the positive effects of dexamethasone, as complete unloading leads to the absence of improvements. CONCLUSION: We conclude that dexamethasone treatment to improve Achilles tendon healing is dose- and time-dependent, and positive effects are perceived even in a partly unloaded condition. CLINICAL RELEVANCE: These findings are promising from a clinical perspective, as the positive effect of this drug was seen even when given at lower doses and in a moderate loading condition, which better mimics the load level in patients with tendon ruptures.

A quality optimization approach to image Achilles tendon microstructure by phase-contrast enhanced synchrotron micro-tomography.

Achilles tendons are mechanosensitive, and their complex hierarchical structure is in part the result of the mechanical stimulation conveyed by the muscles. To fully understand how their microstructure responds to mechanical loading a non-invasive approach for 3D high resolution imaging suitable for soft tissue is required. Here we propose a protocol that can capture the complex 3D organization of the Achilles tendon microstructure, using phase-contrast enhanced synchrotron micro-tomography (SR-PhC-µCT). We investigate the effects that sample preparation and imaging conditions have on the resulting image quality, by considering four types of sample preparations and two imaging setups (sub-micrometric and micrometric final pixel sizes). The image quality is assessed using four quantitative parameters. The results show that for studying tendon collagen fibers, conventional invasive sample preparations such as fixation and embedding are not necessary or advantageous. Instead, fresh frozen samples result in high-quality images that capture the complex 3D organization of tendon fibers in conditions as close as possible to natural. The comprehensive nature of this innovative study by SR-PhC-µCT breaks ground for future studies of soft complex biological tissue in 3D with high resolution in close to natural conditions, which could be further used for in situ characterization of how soft tissue responds to mechanical stimuli on a microscopic level.

Effect of storage and preconditioning of healing rat Achilles tendon on structural and mechanical properties.

Tendon tissue storage and preconditioning are often used in biomechanical experiments and whether this generates alterations in tissue properties is essential to know. The effect of storage and preconditioning on dense connective tissues, like tendons, is fairly understood. However, healing tendons are unlike and contain a loose connective tissue. Therefore, we investigated if storage of healing tendons in the fridge or freezer changed the mechanical properties compared to fresh tendons, using a pull-to-failure or a creep test. Tissue morphology and cell viability were also evaluated. Additionally, two preconditioning levels were tested. Rats underwent Achilles tendon transection and were euthanized 12 days postoperatively. Statistical analyzes were done with one-way ANOVA or Student's t-test. Tissue force and stress were unaltered by storage and preconditioning compared to fresh samples, while high preconditioning increased the stiffness and modulus (p ≤ 0.007). Furthermore, both storage conditions did not modify the viscoelastic properties of the healing tendon, but altered transverse area, gap length, and water content. Cell viability was reduced after freezing. In conclusion, preconditioning on healing tissues can introduce mechanical data bias when having extensive tissue strength diversity. Storage can be used before biomechanical testing if structural properties are measured on the day of testing.

Glutamate triggers the expression of functional ionotropic and metabotropic glutamate receptors in mast cells.

Mast cells are emerging as players in the communication between peripheral nerve endings and cells of the immune system. However, it is not clear the mechanism by which mast cells communicate with peripheral nerves. We previously found that mast cells located within healing tendons can express glutamate receptors, raising the possibility that mast cells may be sensitive to glutamate signaling. To evaluate this hypothesis, we stimulated primary mast cells with glutamate and showed that glutamate induced the profound upregulation of a panel of glutamate receptors of both the ionotropic type (NMDAR1, NMDAR2A, and NMDAR2B) and the metabotropic type (mGluR2 and mGluR7) at both the mRNA and protein levels. The binding of glutamate to glutamate receptors on the mast cell surface was confirmed. Further, glutamate had extensive effects on gene expression in the mast cells, including the upregulation of pro-inflammatory components such as IL-6 and CCL2. Glutamate also induced the upregulation of transcription factors, including Egr2, Egr3 and, in particular, FosB. The extensive induction of FosB was confirmed by immunofluorescence assessment. Glutamate receptor antagonists abrogated the responses of the mast cells to glutamate, supporting the supposition of a functional glutamate-glutamate receptor axis in mast cells. Finally, we provide in vivo evidence supporting a functional glutamate-glutamate receptor axis in the mast cells of injured tendons. Together, these findings establish glutamate as an effector of mast cell function, thereby introducing a novel principle for how cells in the immune system can communicate with nerve cells.

Diminishing effects of mechanical loading over time during rat Achilles tendon healing.

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.

Correction to: Glutamate triggers the expression of functional ionotropic and metabotropic glutamate receptors in mast cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Elastin levels are higher in healing tendons than in intact tendons and influence tissue compliance.

Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28 days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14 days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20× 10 N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28 days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. PLAIN LANGUAGE SUMMARY: Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. When a tendon is injured and ruptured the tissue heals through scar formation. This scar tissue is different from a normal intact tendon and it is important to understand how the tendons heal. Little is known about the presence and function of elastin during healing of tendon injuries. We have shown, in animal experiments, that healing tendons have higher amounts of elastin compared to intact tendons. The elastin is also spread throughout the tissue. When we reduced the levels of this protein, we discovered altered mechanical properties of the tendon. The healing tendon can normally extend quite a lot, but after elastin removal this extensibility was less obvious. The ability of the healing tissue to extend is probably important to protect the tendon from re-injuries during the first months after rupture. We therefore propose that the tendons heal with a large amount of elastin to prevent re-ruptures during early locomotion.

Response to mechanical loading in rat Achilles tendon healing is influenced by the microbiome.

We have previously shown that changes in the microbiome influence how the healing tendon responds to different treatments. The aim of this study was to investigate if changes in the microbiome influence the response to mechanical loading during tendon healing. 90 Sprague-Dawley rats were used. Specific Opportunist and Pathogen Free (SOPF) rats were co-housed with Specific Pathogen Free (SPF) rats, carrying Staphylococcus aureus and other opportunistic microbes. After 6 weeks of co-housing, the SOPF rats were contaminated which was confirmed by Staphylococcus aureus growth. Clean SOPF rats were used as controls. The rats were randomized to full loading or partial unloading by Botox injections in their calf muscles followed by complete Achilles tendon transection. Eight days later, the healing tendons were tested mechanically. The results were analysed by a 2-way ANOVA with interaction between loading and contamination on peak force as the primary outcome and there was an interaction for both peak force (p = 0.049) and stiffness (p = 0.033). Furthermore, partial unloading had a profound effect on most outcome variables. In conclusion, the response to mechanical loading during tendon healing is influenced by changes in the microbiome. Studies aiming for clinical relevance should therefore consider the microbiome of laboratory animals.