Chronic changes in muscle architecture and aponeurosis structure following calf muscle strain injuries.

BACKGROUND: Muscle strain injuries in the human calf muscles are frequent sports injuries with high recurrence. Potential structural and functional changes in the medial head of the musculus gastrocnemius (GM) and the associated aponeurosis are not well documented. PURPOSE: To test whether a GM muscle strain injury affects muscle fascicle length, pennation angle, and the morphology of the deep aponeurosis at rest and during muscle contraction long time after the injury. Additionally, electromyography (EMG) of the GM and the soleus muscle during a unilateral heel rise was measured in the injured and uninjured calf. METHODS: GM fascicle length, pennation angle, and aponeurosis thickness was analyzed on dynamic ultrasonography (US) recordings in 10 participants with a chronic calf strain. In addition, US images taken across the distal portion and mid-belly of the GM were analyzed at three different ankle positions. EMG recordings were obtained during a unilateral heel rise. RESULTS: The pennation angle of the injured distal GM was significantly larger compared to the uninjured GM in the contracted, but not the relaxed state. Pennation angle increased more in the injured compared to the uninjured GM during contraction. Fascicle length was shorter in the most distal portion of the injured GM. Fascicles at the distal portion of the injured GM showed a pronounced curvilinear shape as the muscle contracted and the aponeurosis was enlarged in the injured compared to the uninjured GM. The ratio between GM and soleus EMG activity showed a significantly higher relative soleus activity in the injured compared to the healthy calf. CONCLUSION: The greater change in pennation angle and curvilinear fascicle shape during contraction suggest that a long-term consequence after a muscle strain injury is that some muscle fibers at the distal GM are not actively engaged. The significantly enlarged aponeurosis indicates a substantial and long-lasting connective tissue involvement following strain injuries.

Autophagy guards tendon homeostasis.

Tendons are vital collagen-dense specialized connective tissues transducing the force from skeletal muscle to the bone, thus enabling movement of the human body. Tendon cells adjust matrix turnover in response to physiological tissue loading and pathological overloading (tendinopathy). Nevertheless, the regulation of tendon matrix quality control is still poorly understood and the pathogenesis of tendinopathy is presently unsolved. Autophagy, the major mechanism of degradation and recycling of cellular components, plays a fundamental role in the homeostasis of several tissues. Here, we investigate the contribution of autophagy to human tendons' physiology, and we provide in vivo evidence that it is an active process in human tendon tissue. We show that selective autophagy of the endoplasmic reticulum (ER-phagy), regulates the secretion of type I procollagen (PC1), the major component of tendon extracellular matrix. Pharmacological activation of autophagy by inhibition of mTOR pathway alters the ultrastructural morphology of three-dimensional tissue-engineered tendons, shifting collagen fibrils size distribution. Moreover, autophagy induction negatively affects the biomechanical properties of the tissue-engineered tendons, causing a reduction in mechanical strength under tensile force. Overall, our results provide the first evidence that autophagy regulates tendon homeostasis by controlling PC1 quality control, thus potentially playing a role in the development of injured tendons.

Chronic Sequelae After Muscle Strain Injuries: Influence of Heavy Resistance Training on Functional and Structural Characteristics in a Randomized Controlled Trial.

BACKGROUND: Muscle strain injury leads to a high risk of recurrent injury in sports and can cause long-term symptoms such as weakness and pain. Scar tissue formation after strain injuries has been described, yet what ultrastructural changes might occur in the chronic phase of this injury have not. It is also unknown if persistent symptoms and morphological abnormalities of the tissue can be mitigated by strength training. PURPOSE: To investigate if heavy resistance training improves symptoms and structural abnormalities after strain injuries. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A total of 30 participants with long-term weakness and/or pain after a strain injury of the thigh or calf muscles were randomized to eccentric heavy resistance training of the injured region or control exercises of the back and abdominal muscle. Isokinetic (hamstring) or isometric (calf) muscle strength was determined, muscle cross-sectional area measured, and pain and function evaluated. Scar tissue ultrastructure was determined from biopsy specimens taken from the injured area before and after the training intervention. RESULTS: Heavy resistance training over 3 months improved pain and function, normalized muscle strength deficits, and increased muscle cross-sectional area in the previously injured region. No systematic effect of training was found upon pathologic infiltration of fat and blood vessels into the previously injured area. Control exercises had no effect on strength, cross-sectional area, or scar tissue but a positive effect on patient-related outcome measures, such as pain and functional scores. CONCLUSION: Short-term strength training can improve sequelae symptoms and optimize muscle function even many years after a strain injury, but it does not seem to influence the overall structural abnormalities of the area with scar tissue. REGISTRATION: NCT02152098 (ClinicalTrials.gov identifier).

Influence of the integrin alpha-1 subunit and its relationship with high-fat diet upon extracellular matrix synthesis in skeletal muscle and tendon.

Integrins are important for mechanosensation in tissue and play, together with nutrition, a role in regulating extracellular matrix (ECM) in skeletal muscle and tendon. Integrin receptors are dimers that consist of an α and ß subunit and bridge extracellular and intracellular signals. The present study investigates whether the deletion of the integrin receptor α1 subunit influences collagen and other matrix proteins in the musculotendinous tissue and whether it causes any compensatory changes in other integrin subunits in C57BL/6J mice. In addition, we study whether a high-fat diet (HFD) influences these responses in muscle or tendon. Mice on a HFD had a higher number of non-enzymatic cross-links in skeletal muscle ECM and increased gene expression of collagen and other extracellular matrix proteins. In contrast to gene expression, total collagen protein content was decreased by HFD in the muscle with no change in tendon. Integrin α1 subunit knockout resulted in a decrease of collagen type I and III, TGF-ß1 and IGF-1 gene expression in muscle of HFD mice but did not affect total collagen protein compared with wild-type (WT) littermates in either muscle or tendon. There was no compensatory increase in the genes that express other integrin subunits. In conclusion, HFD induced a significant increase in expression of ECM genes in muscle. On the protein level, HFD resulted in a lower collagen content in muscle. Tendons were unaffected by the diet. Deletion of the integrin α1 subunit did not affect collagen protein or gene expression in muscle or tendon.

Muscle-strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans.

Traumatic strain injury in skeletal muscle is often associated with fluid accumulation at the site of rupture, but the role of this injury exudate (EX) in cellular responses and healing is unknown. We aimed to characterize the EX sampled from human hamstring or calf muscles following a strain injury (n = 12). The cytokine and growth-factor profile, gene expression, and transcriptome analysis of EX-derived cells were compared with blood taken simultaneously from the same individuals. Cellular responses to the EX were tested in 3-dimensional (3D) culture based on primary human fibroblasts and myoblasts isolated from hamstring muscles. The EX contained a highly proinflammatory profile with a substantial expression of angiogenic factors. The proinflammatory profile was present in samples taken early postinjury and in samples aspirated several weeks postinjury, suggesting persistent inflammation. Cells derived from the EX demonstrated an increased expression of fibrogenic, adipogenic, and angiogenesis-related genes in comparison with blood cells. The injury EX stimulated fibroblast proliferation 2-fold compared with plasma, whereas such an effect was not seen for myoblasts. Finally, in 3D cell culture, the EX induced an up-regulation of connective tissue-related genes. In summary, EX formation following a muscle-strain injury stimulates fibroblast proliferation and the synthesis of connective tissue in fibroblasts. This suggests that the EX promotes an acute tissue-healing response but potentially also contributes to the formation of fibrotic tissue in the later phases of tissue repair.-Bayer, M. L., Bang, L., Hoegberget-Kalisz, M., Svensson, R. B., Olesen, J. L., Karlsson, M. M., Schjerling, P., Hellsten, Y., Hoier, B., Magnusson, S. P., Kjaer, M. Muscle-strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans.

[Treatment of acute muscle injuries].

This review summarises the treatment of acute muscle injuries. Muscle injuries are frequent traumatic injuries caused by either excessive strain on the muscle tendon unit (strain injury) or a forceful blow to the muscle (contusion). An early start of rehabilitation after acute strain injuries is a key to shortening the time to return to sport. The application of ice, compression and elevation is well tolerated by patients, but there is no evidence that these methods enhance tissue repair. Complications after strain injuries include risk of recurrence, muscle atrophy and development of scar tissue. Ectopic bone formation may, however rarely, occur after severe contusion injuries.

Role of tissue perfusion, muscle strength recovery, and pain in rehabilitation after acute muscle strain injury: A randomized controlled trial comparing early and delayed rehabilitation.

Muscle strain injuries disrupt the muscle-tendon unit, early rehabilitation is associated with a faster return to sports (RTS), but the time course of tissue healing remains sparsely described. The purpose was to examine tissue regeneration and the effectiveness of early versus delayed rehabilitation onset on functional and structural recovery after strain injuries. A total of 50 recreational athletes with a severe acute strain injury in their thigh or calf muscles were randomized to early or delayed rehabilitation onset. Magnetic resonance imaging (MRI) was obtained initially, 3 and 6 months postinjury, and dynamic contrast-enhanced MRI (DCE-MRI) estimated tissue inflammation initially and after 6 months. Muscle strength was determined 5 weeks, 3 months, and 6 months postinjury, and a questionnaire determined soreness, pain, and confidence. DCE-MRI microvascular perfusion was higher in the injured compared to an uninjured muscle acutely (P < 0.01) and after 6 months (P < 0.01), for both groups (P > 0.05) and unrelated to RTS (P > 0.05). Total volume of the injured muscle decreased from the acute to the 3-month scan, and to the 6-month scan (P < 0.01) in both groups. Muscle strength was similar in both groups at any time. There was a nonsignificant trend (P ≤ 0.1) toward less pain and higher confidence with early rehabilitation. One reinjury was recorded. In conclusion, our data showed prolonged tissue repair with the initial response linked to muscle atrophy but did not explain why early rehabilitation onset accelerated recovery considering that structural and functional recovery was similar with early and delayed rehabilitation.

Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue.

OBJECTIVE: Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We investigated the influence of insulin-like growth factor I (IGF-I) on tendon construct formation in 3D cell culture. DESIGN: Tendon constructs were grown in 0.5 or 10% FBS with or without IGF-I (250 mg/ml) supplementation. Collagen content (fluorometric), mRNA levels (PCR) and fibril diameter (transmission electron microscopy) were determined at 7, 10, 14, 21 and 28 days. RESULTS: IGF-I revealed a stimulating effect on fibril diameter (up to day 21), mRNA for collagen (to day 28), tenomodulin (to day 28) and scleraxis (at days 10 and 14), and on overall collagen content. 10% FBS diminished the development of fibril diameter (day 14), collagen content (at days 21 and 28) and mRNA expression for collagen, tenomodulin and scleraxis. CONCLUSION: IGF-I supplementation promotes early onset of tensile load induced collagen formation and tendon structural arrangement, whereas the FBS concentration routinely used in cultures diminishes collagen expression, collagen content and fibril formation.

Gremlin-2 is a BMP antagonist that is regulated by the circadian clock.

Tendons are prominent members of the family of fibrous connective tissues (FCTs), which collectively are the most abundant tissues in vertebrates and have crucial roles in transmitting mechanical force and linking organs. Tendon diseases are among the most common arthropathy disorders; thus knowledge of tendon gene regulation is essential for a complete understanding of FCT biology. Here we show autonomous circadian rhythms in mouse tendon and primary human tenocytes, controlled by an intrinsic molecular circadian clock. Time-series microarrays identified the first circadian transcriptome of murine tendon, revealing that 4.6% of the transcripts (745 genes) are expressed in a circadian manner. One of these genes was Grem2, which oscillated in antiphase to BMP signaling. Moreover, recombinant human Gremlin-2 blocked BMP2-induced phosphorylation of Smad1/5 and osteogenic differentiation of human tenocytes in vitro. We observed dampened Grem2 expression, deregulated BMP signaling, and spontaneously calcifying tendons in young CLOCKΔ19 arrhythmic mice and aged wild-type mice. Thus, disruption of circadian control, through mutations or aging, of Grem2/BMP signaling becomes a new focus for the study of calcific tendinopathy, which affects 1-in-5 people over the age of 50 years.

Release of tensile strain on engineered human tendon tissue disturbs cell adhesions, changes matrix architecture, and induces an inflammatory phenotype.

Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-ß1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.

What is the impact of inflammation on the critical interplay between mechanical signaling and biochemical changes in tendon matrix?

Mechanical loading can influence tendon collagen homeostasis in animal models, while the dynamics of the human adult tendon core tissue are more debatable. Currently available data indicate that human tendon adaptation to loading may happen primarily in the outer tendon region. A role of inflammation in this peritendinous adaptation is supported by a rise in inflammatory mediators in the peritendinous area after physiological mechanical loading in humans. This plays a role in the exercise-induced rise in tendon blood flow and peritendinous collagen synthesis. Although inflammatory activity can activate proteolytic pathways in tendon, mechanical loading can protect against matrix degradation. Acute tendon injury displays an early inflammatory response that seems to be lowered when mechanical loading is applied during regeneration of tendon. Chronically overloaded tendons (tendinopathy) do neither at rest nor after acute exercise display any enhanced inflammatory activity, and thus the basis for using anti-inflammatory medication to treat tendon overuse seems limited.

In vitro tendon tissue development from human fibroblasts demonstrates collagen fibril diameter growth associated with a rise in mechanical strength.

BACKGROUND: Collagen-rich tendons and ligaments are important for joint stability and force transmission, but the capacity to form new tendon is poorly understood. In the present study, we investigated mechanical strength, fibril size, and structure during development of tendon-like tissue from adult human tenocytes (termed tendon constructs) in vitro over 5 weeks in 3D tissue culture. RESULTS: The constructs displayed large elongated tendon cells aligned along the tendon axis together with collagen fibrils that increased in diameter by 50% from day 14 to 35, which approaches that observed in adult human tendon in vivo. The increase in diameter was accompanied by a 5-fold increase in mechanical strength (0.9±0.1 MPa to 4.9±0.6 MPa) and Young's modulus (5.8±0.9 MPa to 32.3±4.2 MPa), while the maximal strain at failure (16%) remained constant throughout the 5-week culture period. CONCLUSIONS: The present study demonstrates that 3D tendon constructs can be formed by isolated human tendon fibroblasts, and when these constructs are subjected to static self-generated tension, the fibrils will grow in size and strength approaching that of adult human tendon in vivo.

No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts.

The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-ß1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells.

Effects of aging on muscle mechanical function and muscle fiber morphology during short-term immobilization and subsequent retraining.

Very little attention has been given to the combined effects of aging and disuse as separate factors causing deterioration in muscle mechanical function. Thus the purpose of this study was to investigate the effects of 2 wk of immobilization followed by 4 wk of retraining on knee extensor muscle mechanical function (e.g., maximal strength and rapid force capacity) and muscle fiber morphology in 9 old (OM: 67.3 ± 1.3 yr) and 11 young healthy men (YM: 24.4 ± 0.5 yr) with comparable levels of physical activity. Following immobilization, OM demonstrated markedly larger decreases in rapid force capacity (i.e., rate of force development, impulse) than YM (∼ 20-37 vs. ∼ 13-16%; P < 0.05). In contrast, muscle fiber area decreased in YM for type I, IIA, and IIx fibers (∼ 15-30%; P < 0.05), whereas only type IIa area decreased in OM (13.2%; P < 0.05). Subsequent retraining fully restored muscle mechanical function and muscle fiber area in YM, whereas OM showed an attenuated recovery in muscle fiber area and rapid force capacity (tendency). Changes in maximal isometric and dynamic muscle strength were similar between OM and YM. In conclusion, the present data reveal that OM may be more susceptible to the deleterious effects of short-term muscle disuse on muscle fiber size and rapid force capacity than YM. Furthermore, OM seems to require longer time to recover and regain rapid muscle force capacity, which may lead to a larger risk of falling in aged individuals after periods of short-term disuse.

Increased cellular proliferation in rat skeletal muscle and tendon in response to exercise: use of FLT and PET/CT.

PURPOSE: The purpose of this study is to investigate exercise-induced cellular proliferation in rat skeletal muscle/tendon with the use of 3'-[F-18]fluoro-3'deoxythymidine (FLT) and to quantitatively study concomitant changes in the proliferation-associated factor, Ki67. PROCEDURES: Wistar rats (n = 13) performed 3 days of treadmill running. Cellular proliferation was investigated 3 days before and 48 h after the running exercise with the use of FLT and positron emission tomography/computed tomography (PET/CT). Results were compared to a sedentary control group (n = 10). Image-derived standardized uptake values were calculated for Achilles tendons and calf muscles and compared to gene expression and immunohistochemical evaluations of Ki67. RESULTS: Treadmill running induced increased uptake of FLT uptake in calf muscles (30%; p < 0.001) and in Achilles tendon (21%, p < 0.001). The image-derived results were supported by a correlation in calf muscle to Ki67 (protein and mRNA level), while this coherence was not found in tendon. CONCLUSION: FLT-PET seems to be a promising tool for imaging of exercise-induced cellular proliferation in musculo-tendinous tissue.

The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension.

Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.