Biomarkers affected by impact velocity and maximum strain of cartilage during injury.

Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated with traumatic injury resulting in post-traumatic osteoarthritis (PTOA). Our objective was to develop a single impact model with cartilage "injury level" defined in terms of controlled combinations of strain rate to a maximum strain (both independent of cartilage load resistance) to study their sensitivity to articular cartilage cell viability and potential PTOA biomarkers. A servo-hydraulic test machine was used to measure canine humeral head cartilage explant thickness under repeatable pressure, then subject it (except sham and controls) to a single impact having controlled constant velocity V=1 or 100mm/s (strain rate 1.82 or 182/s) to maximum strain ε=10%, 30%, or 50%. Thereafter, explants were cultured in media for twelve days, with media changed at day 1, 2, 3, 6, 9, 12. Explant thickness was measured at day 0 (pre-injury), 6 and 12 (post-injury). Cell viability, and tissue collagen and glycosaminoglycan (GAG) were analyzed immediately post-injury and day 12. Culture media were tested for biomarkers: GAG, collagen II, chondroitin sulfate-846, nitric oxide, and prostaglandin E2 (PGE2). Detrimental effects on cell viability, and release of GAG and PGE2 to the media were primarily strain-dependent, (PGE2 being more prolonged and sensitive at lower strains). The cartilage injury model appears to be useful (possibly superior) for investigating the relationship between impact severity of injury and the onset of PTOA, specifically for discovery of biomarkers to evaluate the risk of developing clinical PTOA, and to compare effective treatments for arthritis prevention.

Age-dependent regulation of tendon crimp structure, cell length and gap width with strain.

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0-1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell-cell communication after overstretching.

Characteristics of myogenic response and ankle torque recovery after lengthening contraction-induced rat gastrocnemius injury.

BACKGROUND: Although muscle dysfunction caused by unfamiliar lengthening contraction is one of most important issues in sports medicine, there is little known about the molecular events on regeneration process. The purpose of this study was to investigate the temporal and spatial expression patterns of myogenin, myoD, pax7, and myostatin after acute lengthening contraction (LC)-induced injury in the rat hindlimb. METHODS: We employed our originally developed device with LC in rat gastrocnemius muscle (n = 24). Male Wistar rats were anesthetized with isoflurane (aspiration rate, 450 ml/min, concentration, 2.0%). The triceps surae muscle of the right hindlimb was then electrically stimulated with forced isokinetic dorsi-flexion (180°/sec and from 0 to 45°). Tissue contents of myoD, myogenin, pax7, myostatin were measured by western blotting and localizations of myoD and pax7 was measured by immunohistochemistry. After measuring isometric tetanic torque, a single bout of LC was performed in vivo. RESULTS: The torque was significantly decreased on days 2 and 5 as compared to the pre-treatment value, and recovered by day 7. The content of myoD and pax7 showed significant increases on day 2. Myogenin showed an increase from day 2 to 5. Myostatin on days 5 and 7 were significantly increased. Immunohistochemical analysis showed that myoD-positive/pax7-positive cells increased on day 2, suggesting that activated satellite cells play a role in the destruction and the early recovery phases. CONCLUSION: We, thus, conclude that myogenic events associate with torque recovery after LC-induced injury.

A mathematical model of the process of ligament repair: effect of cold therapy and mechanical stress.

This article proposes a mathematical model that predicts the wound healing process of the ligament after a sprain, grade II. The model describes the swelling, expression of the platelet-derived growth factor (PDGF), formation and migration of fibroblasts into the injury area and the expression of collagen fibers. Additionally, the model can predict the effect of ice treatment in reducing inflammation and the action of mechanical stress in the process of remodeling of collagen fibers. The results obtained from computer simulation show a high concordance with the clinical data previously reported by other authors.

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy.

We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues.

Protective effects of therapeutic cold and heat against the oxidative damage induced by a muscle strain injury in rats.

The mechanisms of action of physical agents commonly used to treat skeletal muscle lesions are not well understood. In this study, we examined whether the modulation of oxidative stress is involved in the beneficial effects of cold and heat on gastrocnemius muscle strain injury. Adult male Wistar rats were submitted to a strain injury and treated with therapeutic agents in an isolated or combined form. Strain damage caused an increase in muscle and blood oxidative damage. We suggest that this oxidative damage might be related to the impairment of the muscle cell structure, since we observed a significant positive correlation between increased plasma creatine kinase activity and both oxidized dichlorofluoresceine and lipid peroxidation levels in muscle and blood. The intensity of the inflammatory response appears also to be an important factor in the genesis of oxidative damage immediately following a muscle strain injury. Therapeutic cold seems to be more effective in preventing the damage induced by a strain injury, possibly due to its capacity to control the impairment of muscle cell structure and to modulate the intensity of the inflammatory response that follows a muscle strain injury.

Identification of ankle sprain motion from common sporting activities by dorsal foot kinematics data.

This study presented a method to identify ankle sprain motion from common sporting activities by dorsal foot kinematics data. Six male subjects performed 300 simulated supination sprain trials and 300 non-sprain trials in a laboratory. Eight motion sensors were attached to the right dorsal foot to collect three-dimensional linear acceleration and angular velocity kinematics data, which were used to train up a support vector machine (SVM) model for the identification purpose. Results suggested that the best identification method required only one motion sensor located at the medial calcaneus, and the method was verified on another group of six subjects performing 300 simulated supination sprain trials and 300 non-sprain trials. The accuracy of this method was 91.3%, and the method could help developing a mobile motion sensor system for ankle sprain detection.

A micromechanical model to predict damage and failure in biological tissues. Application to the ligament-to-bone attachment in the human knee joint.

Computational models are developed in injury biomechanics to assess lesions in biological tissues based on mechanical measurements. The linear mechanics of fracture theory (LMFT) is a common approach to establish injuries based on thresholds (such as force or strain thresholds) which are straightforward to implement and computationally efficient. However, LMFT does not apply to non-linear heterogeneous materials and does not have the ability to predict failure onset. This paper proposes the cohesive zone model theory (CZMT) as an alternative. CZMT focuses on the development of behaviour laws for crack initiation and propagation at an interface that apply within a fibrous material or at the interface between materials. With the view of evaluating CZMT for biological tissues, the model developed by Raous et al. [1999. A consistent model coupling adhesion, friction and unilateral contact. Comput. Methods Appl. Mech. Eng., 177, 383-399] was applied to the ligament-to-bone interface in the human knee joint. This model accounts for adhesion, friction and damage at the interface and provides a smooth transition from total adhesion to complete failure through the intensity of adhesion variable. A 2D finite element model was developed to mimic previous experiments, and the model parameters were determined using a dichotomy method. The model showed good results by its ability to predict damage. The extension to a 3D geometry, with an inverse problem approach, is, however, required to better estimate the model parameters values. Although it is computationally costly, CZMT supplements the improvements achieved in microimaging techniques to support the development of micro/macro approaches in biomechanical modelling.

Optimization of flexor tendon tissue engineering with a cyclic strain bioreactor.

PURPOSE: Mechanical manipulation of cultured tendon cells can enhance cell proliferation and matrix production. This study aims to determine the bioreactor strain patterns (amplitude, frequency, and on/off ratio) that favor cellular proliferation, promote collagen production, and maintain morphology in candidate cell lines cultured for flexor tendon tissue engineering, including multipotent stromal cells. METHODS: We studied epitenon tenocytes (Es), sheath fibroblasts (Ss), bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived stem cells (ASCs). We examined the effects of 3 patterns of cyclic uniaxial strain on cell proliferation, collagen I production, and cell morphology. RESULTS: Adipoderived stem cells (33% adhesion) and Ss (29%) adhered more strongly to bioreactor membranes than did Es (15%) and BMSCs (7%), p=.04. Continuous cyclic strain (CCS, 8%, 1 Hz) inhibited cell proliferation (p=.01) and increased per-cell collagen production (p=.04) in all cell types. Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/5 hours off) increased proliferation in ASCs (p=.06) and Ss (p=.04). Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/2 hours off) increased total collagen production by 25% in ASCs (p=.004) and 20% in Ss (p=.05). Cyclic strain resulted in cell alignment perpendicular to the strain axis, cytoskeletal alignment, and nuclear elongation. These morphological characteristics are similar to those of tenocytes. CONCLUSIONS: These results demonstrate that intermittent cyclic strain can increase cell proliferation, promote collagen I production, and maintain tenocyte morphology in vitro. Use of a cell bioreactor might accelerate the in vitro stage of tendon tissue engineering.

Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin.

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.

The free diffusion of macromolecules in tissue-engineered skeletal muscle subjected to large compression strains.

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in compressed muscle tissue overlying a bony prominence and progresses to more superficial tissues until penetrating the skin. Individual subjects with impaired motor and/or sensory capacities are at high risk of developing DTI. Impaired diffusion of critical metabolites in compressed muscle tissue may contribute to DTI, and impaired diffusion of tissue damage biomarkers may further impose a problem in developing early detection blood tests. We hypothesize that compression of muscle tissue between a bony prominence and a supporting surface locally influences the diffusion capacity of muscle. The objective of this study was therefore, to determine the effects of large compression strains on free diffusion in a tissue-engineered skeletal muscle model. Diffusion was measured with a range of fluorescently labeled dextran molecules (10, 20, 150kDa) whose sizes were representative of both hormones and damage biomarkers. We used fluorescence recovery after photobleaching (FRAP) to compare diffusion coefficients (D) of the different dextrans between the uncompressed and compressed (48-60% strain) states. In a separate experiment, we simulated the effects of local partial muscle ischemia in vivo, by reducing the temperature of compressed specimens from 37 to 34 degrees C. Compared to the D in the uncompressed model system, values in the compressed state were significantly reduced by 47+/-22% (p<0.02). A 3 degrees C temperature decrease further reduced D in the compressed specimens by 10+/-6% (p<0.05). In vivo, the effects of large strains and ischemia are likely to be summative, and hence, the present findings suggest an important role of impaired diffusion in the etiology of DTI, and should also be considered when developing biochemical screening methods for early detection of DTI.

Transforming growth factor-beta following skeletal muscle strain injury in rats.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine implicated in inflammatory processes, wound healing, and fibrosis. In muscle diseases (i.e., dystrophy and inflammatory myopathy) and in animal models of muscle injury (i.e., produced by cardiotoxin, laceration, and eccentric contractions), increased TGF-beta was associated with muscle fibrosis and healing. Although TGF-beta transcript abundance was increased following injury, many studies presume that TGF-beta protein was also active as evident by increases in collagen transcript abundance. The purpose was to determine whether TGF-beta protein is present and active 48 h following injury. Using female rats, muscle strains were produced by stretching (50 stretches) the plantar flexor muscles. Forty-eight hours following injury, the medial gastrocnemius was removed and compartmentalized into five equal segments. Damaged myofibers with intracellular concanavalin A staining were counted. The percentage of damaged myofibers was significantly greater in the distal-most segment. TGF-beta was assessed by using immunohistochemistry, RT-PCR, and immunoblot analysis. Immunohistochemistry revealed the presence of TGF-beta1 in areas of myofiber injury, whereas TGF-beta2 was not detected. Increases in TGF-beta1 and TGF-beta2 transcript abundance following strain injury were documented by RT-PCR analysis. Increases in TGF-beta1 and TGF-beta2 precursor abundance were observed following strain injury by using immunoblot analysis but there was no change in active TGF-beta abundance. Although there was no correlation between the amount of cellular injury and TGF-beta transcript and protein abundance, elevated levels of TGF-beta1 and TGF-beta2 precursor proteins were present in strain-injured skeletal muscles 48 h after injury.

Effects of estrogen on gastrocnemius muscle strain injury and regeneration in female rats.

AIM: To study the effects of estrogen on muscle damage and regeneration after acute passive gastrocnemius muscle strain injury in female Sprague-Dawley rats. METHODS: Rats were divided into 5 groups: ovariectomized, strained and treated with low-dosage estradiol (20 microg/d) (E(low)), treated with high-dosage estradiol (200 microg/d) (E(high)), treated with oil placebo (Oil), strained with no ovariectomy (Strain), and sham operated with no strain and no ovariectomy (Con). Muscle damage index [plasma creatine kinase (CK)], antioxidant indexes [glutathione (GSH), Vitamin E (Vit E), total antioxidant capability (TAC)], and muscle regeneration index (desmin) were investigated at 7 d. RESULTS: The plasma CK activity increased but GSH, Vit E, and TAC levels decreased after muscle strain injury (Strain vs Con P<0.05). Plasma CK activity was the greatest while GSH, Vit E, and TAC were the lowest in the Oil group among the five groups (P<0.01). Plasma CK in the E(high) and Strain groups was lower than that in the E(low) group. Plasma GSH, Vit E, and TAC were higher in the E(high) and Strain groups compared with the E(low) group (P<0.05). The expression of desmin in the E(high) and Strain groups was higher than that in the E(low) group (P<0.01) while that in the Oil group was the lowest in all the five groups (P<0.01). CONCLUSION: Endogenous estrogen in normal female rats or exogenous estrogen in ovariectomized rats could improve antioxidant capability in vivo, so that reduced muscle damage and accelerated muscle regeneration post gastronemius muscle strain injury.

On the fracture of human dentin: is it stress- or strain-controlled?

Despite substantial clinical interest in the fracture resistance of human dentin, there is little mechanistic information in archival literature that can be usefully used to model such fracture. In fact, although the fracture event in dentin, akin to other mineralized tissues like bone, is widely believed to be locally strain-controlled, there has never been any scientific proof to support this belief. The present study seeks to address this issue through the use of a novel set of in vitro experiments in Hanks' balanced salt solution involving a double-notched bend test geometry, which is designed to discern whether the critical failure events involved in the onset of fracture are locally stress- or strain-controlled. Such experiments are further used to characterize the notion of "plasticity" in dentin and the interaction of cracks with the salient microstructural features. It is observed that fracture in dentin is indeed locally strain-controlled and that the presence of dentinal tubules does not substantially affect this process of crack initiation and growth. The results presented are believed to be critical steps in the development of a micromechanical model for the fracture of human dentin that takes into consideration the influence of both the microstructure and the local failure mode.

Mechanisms of muscle injury gleaned from animal models.

Eccentric contractions of skeletal muscles produce injury and, ultimately, muscle strengthening. Current data suggest that the earliest events associated with injury are mechanical in nature and may be based primarily on the sarcomere strain experienced by the muscle. In this review, recent experimental data, primarily from rabbit dorsiflexor muscles, are used to provide general information regarding the factors that cause injury and means for preventing injury. Mechanical experiments reveal that excessive sarcomere strain is the primary cause of injury. We hypothesize that excessive strain permits extracellular or intracellular membrane disruption that may permit hydrolysis of structural proteins, leading to the myofibrillar disruption that is commonly observed. Inflammation that occurs after injury further degrades the tissue, but prevention of the inflammation leads to a long-term loss in muscle function. Simple preventative treatments such as increasing muscle oxidative capacity (getting into shape) or cyclic stress-relaxation of tissue (stretching out) have no measurable effect on the magnitude of muscle injury that occurs. Ultimately, an improved understanding of the damage mechanism may improve our ability to provide rehabilitative and strengthening prescriptions that have a rational scientific basis.

Fibrosis and intercellular collagen connections from four weeks of muscle strains.

The effect of repeated cycles of muscle strain was studied in the soleus muscle of female rats. Muscle strains were repeated 3X/week for 1 month using two different strain protocols. Striking changes, including marked variability in fiber size, evidence of degradation and regeneration, and an expanded extracellular matrix were pronounced in the fast-stretched muscles but not in the slow-stretched muscles. However, the slow-stretched muscles did contain struts of connective tissue joining adjacent myofibers. Therefore, repeated muscle strains at high strain rates produced morphological changes similar to many myopathies, including fibrosis, whereas adaptation occurred in response to the same number of strains at slow strain rates. Such diverse tissue responses have relevance to the understanding of the mechanisms of skeletal muscle dysfunction in cumulative trauma disorders and in the design of preventive actions and treatments.

Initial events in exercise-induced muscular injury.

Immediately following unaccustomed exercise, particularly that with eccentric contractions, there is evidence of injury to skeletal muscle fibers: a) disruption of the normal myofilament structures in some sarcomeres, observable with both light and electron microscope and b) loss of intramuscular proteins (e.g., creatine kinase enzymes) into the plasma, indicating damage to sarcolemma. This pathology is probably responsible for the temporary reductions in muscle force and delayed-onset soreness that can occur following eccentric exercise. The mechanisms underlying this injury are not known, although loss of intracellular Ca2+ homeostasis could play a primary role. In other experimental muscle injury models, elevated [Ca2+]i appears to cause release of muscle enzymes through activation of phospholipase A2, which in turn could induce injury to sarcolemma through production of leukotrienes and prostaglandins, through free O2 radical formation (in the subsequent lipoxygenase and cyclooxygenase reactions), and/or through release of detergent lysophospholipids. On the other hand, the mechanism responsible for the rapid damage to myofibrils caused by increased [Ca2+]i is unknown. Regardless of the cause(s), the initial and early events in the injury process are autogenetic; i.e., they are indigenous to the muscle cells and occur before phagocytic cells enter the injury site.