Subfailure Injury Research Articles

Collagen denaturation is initiated upon tissue yield in both positional and energy-storing tendons

Tendons are collagenous soft tissues that transmit loads between muscles and bones. Depending on their anatomical function, tendons are classified as positional or energy-storing with differing biomechanical and biochemical properties. We recently demonstrated that during monotonic stretch of positional tendons, permanent denatured collagen begins accumulating upon departing the linear region of the stress-strain curve. However, it is unknown if this observation is true during mechanical overload of other types of tendons. Therefore, the purpose of this study was to investigate the onset of collagen denaturation relative to applied strain, and whether it differs between the two tendon types. Rat tail tendon (RTT) fascicles and rat flexor digitorum longus (FDL) tendons represented positional and energy-storing tendons, respectively. The samples were stretched to incremental levels of strain, then stained with fluorescently labeled collagen hybridizing peptides (CHPs); the CHP fluorescence was measured to quantify denatured collagen. Denatured collagen in both positional and energy-storing tendons began to increase at the yield strain, upon leaving the linear region of the stress-strain curve as the sample started to permanently deform. Despite significant differences between the two tendon types, it appears that collagen denaturation is initiated at tissue yield during monotonic stretch, and the fundamental mechanism of failure is the same for the two types of tendons. At tissue failure, positional tendons had double the percentage of denatured collagen compared to energy-storing tendons, with no difference between 0% control groups. These results help to elucidate the etiology of subfailure injury and rupture in functionally distinct tendons.

Single-walled carbon nanohorns modulate tenocyte cellular response and tendon biomechanics.

Subfailure ligament and tendon injury remain a significant burden to global healthcare. Here, we present the use of biocompatible single-walled carbon nanohorns (CNH) as a potential treatment for the repair of sub-failure injury in tendons. First, in vitro exposure of CNH to human tenocytes revealed no change in collagen deposition but a significant decrease in cell metabolic activity after 14 days. Additionally, gene expression studies revealed significant downregulation of collagen Types I and III mRNA at 7 days with some recovery after 14 days of exposure. Biomechanical tests with explanted porcine digitorum tendons showed the ability of CNH suspensions to modulate tendon biomechanics, most notably elastic moduli immediately after treatment. in vivo experiments demonstrated the ability of CNH to persist in the damaged matrix of stretch-injured Sprague Dawley rat Achilles tendon but not significantly modify tendon biomechanics after 7 days of treatment. Although these results demonstrate the early feasibility of utility of CNH as a potential modality for tendon subfailure injury, additional work is needed to further validate and ensure clinical efficacy.

Sub-failure Stretch Injury Response in Rat Achilles Tendon

Sub-failure ligament and tendon injuries are of great concern to athletes and the general population because they account for a significant proportion of all musculoskeletal injuries per year. The goals of this manuscript were to design and evaluate the performance of a reproducible small animal model for tendon sub-failure injury and investigate the tissue response to a singular, controlled sub-failure stretch damage in rat Achilles tendon matrices using biochemical, biomechanical, and image analyses. Utilizing a new tendon stretch injury device, we showed that sub-failure stretch lead to significant changes in the elastic moduli of the toe region of the stress-strain curves of tendons. Scanning electron microscopy was used to examine the ultrastructural damage and disorganization of the normally aligned collagen matrix of the tendon. Additionally, ex vivo biomechanical testing confirmed the persistence of compromised biomechanical properties for up to 7 days post-injury in the healing Achilles tendon. Furthermore, in vivo magnetic resonance imaging and histological analysis showed strong evidence of the injury during repair. Lastly, immunohistochemical staining revealed increased expression of myofibroblast markers elastin and smooth muscle actin, indicative of matrix remodeling facilitated by migrating tissue fibroblasts. These results support the clinical utility of this new in vivo model for the study of sub-failure injury of tendon. Taken together, these findings provide clinically relevant information about the most common type of ligament and tendon injury. Sub-failure sprain and strain injuries to ligaments and tendons are common injuries of the musculoskeletal system. We set out to design a device that could accurately model the response of a sub-failure tendon stretch injury in rat. Using 3D-printed parts, we developed a new injury device and tested the biological and biomechanical response of stretch injury in rat cadavers and live rats for up to 1 week. Our results showed sub-failure stretch of the Achilles tendon led to changes in the biomechanics, most notably the toe region modulus under tension. We saw persistence of compromised biomechanics for up to 1 week, accompanied by a cellular response led by migrating tendon fibroblasts. Future studies will evaluate the long-term response of the stretch injury in rat, in addition to adapting the device for larger animal models.

Response of cartilage and meniscus tissue explants to in vitro compressive overload

To examine the relative susceptibility of cartilage and meniscus tissues to mechanical injury by applying a single, controlled overload and observing cellular, biochemical, and mechanical changes. Cartilage and meniscus tissue explants in radial confinement were subjected to a range of injury by indenting to 40% strain at three different strain rates: 0.5%/s (slow), 5%/s (medium), or 50%/s (fast). Following injury, samples were cultured for either 1 or 9 days. Explants were assayed for cell metabolic activity, water content, and sulfated glycosaminoglycan (sGAG) content. Mechanical properties of explants were determined in torsional shear and unconfined compression. Conditioned medium was assayed for sGAG and lactate dehydrogenase (LDH) release. Peak injury force increased with strain rate but both tissues displayed little to no macroscopic damage. Cell metabolism was lowest in medium and fast groups on day 1. Cell lysis increased with peak injury force and loading rate in both tissues. In contrast, sGAG content and release did not significantly vary with loading rate. Additionally, mechanical properties did not significantly vary with loading rate in either tissue. By use of a custom confinement chamber, large peak forces were obtained without macroscopic destruction of the explants. At the loads achieved in this studied, cell damage was induced without detectable physical or compositional changes. These results indicate that sub-failure injury can induce biologic damage that may not be readily detected and could be an early event in osteoarthritis genesis.

Early response to tendon fatigue damage accumulation in a novel in vivo model

This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.

A119 過負荷による家兎膝蓋腱部分損傷のひずみ速度依存性(A1-5 軟組織のバイオメカニクス3)

A119 過負荷による家兎膝蓋腱部分損傷のひずみ速度依存性(A1-5 軟組織のバイオメカニクス3)

Cervical facet capsular ligament yield defines the threshold for injury and persistent joint-mediated neck pain

The cervical facet joint has been identified as a source of neck pain, and its capsular ligament is a likely candidate for injury during whiplash. Many studies have shown that the mechanical properties of ligaments can be altered by subfailure injury. However, the subfailure mechanical response of the facet capsular ligament has not been well defined, particularly in the context of physiology and pain. Therefore, the goal of this study was to quantify the structural mechanics of the cervical facet capsule and define the threshold for altered structural responses in this ligament during distraction. Tensile failure tests were preformed using isolated C6/C7 rat facet capsular ligaments ( n=8); gross ligament failure, the occurrence of minor ruptures and ligament yield were measured. Gross failure occurred at 2.45±0.60 N and 0.92±0.17 mm. However, the yield point occurred at 1.68±0.56 N and 0.57±0.08 mm, which was significantly less than gross failure ( p<0.001 for both measurements). Maximum principal strain in the capsule at yield was 80±24%. Energy to yield was 14.3±3.4% of the total energy for a complete tear of the ligament. Ligament yield point occurred at a distraction magnitude in which pain symptoms begin to appear in vivo in the rat. These mechanical findings provide insight into the relationship between gross structural failure and painful loading for the facet capsular ligament, which has not been previously defined for such neck injuries. Findings also present a framework for more in-depth methods to define the threshold for persistent pain and could enable extrapolation to the human response.

Injured rabbit ACL treated by radiofrequency. Effects of cyclic loading

Radiofrequency treatment is increasingly used to treat shoulder instability. Patients are asked to restrain their physical activities after this treatment but there is no precise information concerning the necessity for the restrain. There were two groups of ten specimens each. Treatment group specimens were stretched to subfailure injury, treated by a radiofrequency probe, and then cyclically loaded. Control group specimens were stretched to the same subfailure injury, Sham treated, and then cyclically loaded. Between each step of the experiment in both groups there was a relaxation test to examine the ligament viscoelastic properties. At the end, each ligament was stretched to failure and the load-elongation curve obtained. Relaxation forces decreased after the subfailure injury in both groups (average 76% and 81% of intact state, in treatment and control groups, respectively). In the treatment group, the relaxation forces first increased after the radiofrequency treatment (average 99% of intact state), and then decreased after the cyclic loading (average 50% of intact state). The treated ligaments failed at lower loads and smaller deformations than the controls. The radiofrequency treatment restored viscoelastic properties of the injured ligaments, but cyclic loading degraded these. Protection of the treated ligament is advised during the immediate post-operative period.

Tensile cervical facet capsule ligament mechanics: Failure and subfailure responses in the rat

Clinical, epidemiological, and biomechanical studies suggest the involvement of the cervical facet joint in neck pain. Mechanical studies have suggested the facet capsular ligament to be at risk for subfailure tensile injury during whiplash kinematics of the neck. Ligament mechanical properties can be altered by subfailure injury and such loading can induce cellular damage. However, at present, there is no clear understanding of the physiologic context of subfailure facet capsular ligament injury and mechanical implications for whiplash-related pain. Therefore, this study aimed to define a relationship between mechanical properties at failure and a subfailure condition associated with pain for tension in the rat cervical facet capsular ligament. Tensile failure studies of the C6/C7 rat cervical facet capsular ligament were performed using a customized vertebral distraction device. Force and displacement at failure were measured and stiffness and energy to failure were calculated. Vertebral motions and ligament deformations were tracked and maximum principal strains and their directions were calculated. Mean tensile force at failure (2.96±0.69 N) was significantly greater ( p < 0.005 ) than force at subfailure (1.17±0.48 N). Mean ligament stiffness to failure was 0.75±0.27 N/mm. Maximum principal strain at failure (41.3±20.0%) was significantly higher ( p = 0.003 ) than the corresponding subfailure value (23.1±9.3%). This study determined that failure and a subfailure painful condition were significantly different in ligament mechanics and findings provide preliminary insight into the relationship between mechanics and pain physiology for this ligament. Together with existing studies, these findings offer additional considerations for defining mechanical thresholds for painful injuries.

Intrinsic fibroblast-mediated remodeling of damaged collagenous matrices in vivo

Numerous studies have examined wound healing and tissue repair after a complete tissue rupture and reported provisional matrix and scar tissue formation in the injury gap. The initial phases of the repair are largely mediated by the coagulation response and a principally extrinsic inflammatory response followed by type III collagen deposition to form scar tissue that may be later remodeled. In this study, we examine subfailure (Grade II sprain) damage to collagenous matrices in which no gross tissue gap is present and a localized concentration of provisional matrix or scar tissue does not form. This results in extracellular matrix remodeling that relies heavily upon type I collagen, and associated proteoglycans, and less heavily on type III scar tissue collagen. For instance, following subfailure tissue damage, collagen I and III expression was suppressed after 1 day, but by day 7 expression of both genes was significantly increased over controls, with collagen I expression significantly larger than type III expression. Concurrent with increased collagen expression were significantly increased expression of the collagen fibrillogenesis supporting proteoglycans fibromodulin, lumican, decorin, the large aggregating proteoglycan versican, and proteases cathepsin K and L. Interestingly, this remodeling process appears intrinsic with little or no inflammation response as damaged tissues show no changes in macrophage or neutrophils levels following injury and expression of the inflammatory markers, tumor necrosis factor-alpha and tartrate-resistant acid phosphatase were unchanged. Hence, since inflammation plays a large role in wound healing by inducing cell migration and proliferation, and controlling extracellular matrix scar formation, its absence leaves fibroblasts to principally direct tissue remodeling. Therefore, following a Grade II subfailure injury to the collagen matrix, we conclude that tissue remodeling is fibroblast-mediated and occurs without scar tissue formation, but instead with type I collagen fibrillogenesis to repair the tissue. As such, this system provides unique insight into acute tissue damage and offers a potentially powerful model to examine fibroblast behavior.

Cervical Spine Ligament Injury during Simulated Frontal Impact

The supraspinous and interspinous ligaments, ligamentum flavum, and capsular and posterior longitudinal ligament strains were monitored during simulated frontal impact of whole cervical spine specimens with muscle force replication and compared with corresponding physiologic strain limits. To quantify the strains in the cervical spine ligaments during simulated frontal impact and investigate injury mechanisms. Clinical and biomechanical studies have documented injuries to cervical spine ligaments during frontal impact. There are no biomechanical studies investigating subfailure injury mechanisms to these ligaments during simulated frontal impacts of increasing severity. The whole cervical spine with muscle force replication model and a bench-top sled were used to simulate frontal impacts at 4, 6, 8, and 10 g horizontal accelerations of the T1 vertebra. The peak ligament strains during frontal impacts were compared with physiologic strain limits determined during intact flexibility testing. Significant increases (P < 0.05) in the supraspinous and interspinous ligaments and the ligamentum flavum strains beyond physiologic limits were observed throughout the cervical spine, with the highest strains occurring at C3-C4. Significant increases were observed in the capsular ligament strains only during the 10 g impact, whereas the posterior longitudinal ligament strains did not exceed physiologic limits. The supraspinous and interspinous ligaments and the ligamentum flavum may be at risk for injury due to excessive strains during frontal impacts.

Incremental and single trauma produce equivalent subfailure soft tissue injury of the cervical spine

Background. Automotive collision simulations have been performed using either incremental or single trauma. In single trauma, a single impact is performed, while in incremental trauma, a series of impacts of increasing severity are executed. Equivalency of incremental and single trauma for soft tissue injury severity due to the final impact has not been established. Thus, the purpose of the present study was to investigate whether incremental and single trauma produced similar cervical spine subfailure injury severity due to simulated frontal impacts. Methods. Porcine cervical spine specimens (C2–T1) of the incremental trauma group were subjected to five frontal impacts (2, 3.5, 5, 6.5, 8 g), while single trauma specimens were subjected to a single impact (8 g). Flexibility tests were performed on specimens while intact and following each impact. Intact and post 8 g flexibility parameters were compared within incremental and single trauma groups and between groups. Findings. No significant differences ( P<0.05) were found between incremental and single trauma groups when either intact or post 8 g flexibility parameters were compared. Significant increases in flexibility parameters from intact to post 8 g were observed in both groups, indicating soft tissue injury. Interpretation. Incremental and single trauma produced equivalent subfailure cervical spine injury in simulated impacts, for the experimental conditions studied. This study may facilitate greater use of the incremental trauma protocol in future experimental designs.

Radiofrequency probe treatment for subfailure ligament injury: a biomechanical study of rabbit ACL

Objective. Utilizing a rabbit anterior cruciate ligament model of ligamentous subfailure injury, biomechanical properties of injured ligament treated with radiofrequency energy were evaluated. It was hypothesized that an injured ligament treated with radiofrequency probe would demonstrate restoration of biomechanical properties lost through injury. Background. Radiofrequency probe, thermal treatment has been utilized in the clinical setting to address joint instability caused by ligamentous laxity from injury or repetitive microtrauma. The biomechanical effects of radiofrequency probe thermal treatment on injured ligamentous tissues have not been studied in the laboratory. Design. Three groups of specimens: Control, Sham, and Treatment, 10 each, were tested under identical conditions. Methods. Viscoelastic behavior was analyzed using a relaxation test (6% strain, up to 180 s) performed before injury, after injury, and after injury plus sham or injury plus radiofrequency probe treatment. Results. After injury the normalized forces in the relaxation test decreased by approximately 50%. The post-treatment relaxation test revealed significant ( P<0.01) restoration of the average relaxation force in the Treatment group to that of the Control group (0.79, SD 0.11 vs. 0.80, SD 0.10). Both of these groups were significantly different from the Sham group (0.44, SD 0.11). Additionally, stretch-to-failure test showed partial restoration of the toe region of the load–deformation curve by the radiofrequency treatment. Conclusions. The radiofrequency probe treatment is shown to be an effective mechanism for restoring initial ligament tensile stiffness and viscoelastic characteristics lost by the subfailure injury in vitro. Relevance This study employs a proven ligament subfailure injury model and examines the biomechanical consequences of radiofrequency probe thermal treatment, in vitro. These results support the theoretical basis of arthroscopic, electro-thermal capsule shift procedures for subfailure injuries. However, this study does not provide evidence that these improved biomechanical properties are sustained in vivo.

Healing of subfailure ligament injury: comparison between immature and mature ligaments in a rat model

This study evaluated biomechanical properties of healing ligament following subfailure (grade II) injury by comparing young and mature animals in a rat lateral collateral ligament (LCL) model. One randomly selected LCL was stretched in situ using a custom designed device in eighteen young (21 days) and eighteen skeletally mature (8 months) male rats. Animals were euthanized at 0, 7, and 14 days post-surgery, and ligament ultimate stress, strain at failure and laxity were determined ( n=6 pairs per group). At time 0 after introduction of stretch injury, ligament laxity was present in both groups. The mature rats had 54±9% strength of the control while the immature rats had 58±11% of the strength of the control, representing a consistent and significant injury. The immature and mature ligaments showed similar patterns of cellular damage post-injury and had similar modes of mechanical failure. Ligament laxity decreased in each group as healing time increased, however ligament laxity did not completely recover in either group after 2 weeks of healing. After 7 and 14 days of healing, the mature rats, respectively, had only 63±14% and 80±8% strengths of the controls while the immature rats had 94±6% and 94±10%. Hence, mechanical data showed that immature animals recovered their strength after a grade II sprain at a faster rate than mature animals. However, ligament laxity was still present in both groups two weeks after the injury and was not completely removed by growth in the immature group. These findings are clinically relevant since joint laxity after injury is common, and these results may explain the presence of continued instability in a joint injured at a young age. Hence, this study, with a new injury model, showed differences in ligament healing associated with maturity and quantified the clinically observed persistance of ligament laxity.

Subfailure injury affects the relaxation behavior of rabbit ACL

Objective. To study changes in viscoelasticity of a ligament due to an incomplete or a subfailure injury. Design. An in vitro study of anterior cruciate ligament preparations. Background. The viscoelastic properties are an inherent part of the physical characteristics of a ligament. An injury to a ligament alters both its elastic and viscous properties. Although the effects of several parameters on the mechanical properties of a ligament have been studied, there is no information in the literature concerning the effect of an incomplete or subfailure injury on its viscoelastic behavior. Methods. Ten pairs of rabbit femur-anterior cruciate ligament-tibia specimens were used. A standardized relaxation (Relax) test was adopted to quantify the viscoelastic behavior, before and after a subfailure injury. One member of the pair was subjected to three sequential tests: Relax 1; Relax 2; and stretch to failure. The other member of the pair was subjected to other three tests: Relax 3; 80% subfailure injury, i.e. stretch of 80% of failure deformation; and Relax 4. Results. We found that the relaxation test by itself (Relax 1 vs Relax 2), did not affect the viscoelasticity of the ligament. On the other hand, the 80% subfailure injury (Relax 3 vs Relax 4) affected the ligament viscoelastic behavior. The force was decreased by about 50% at time zero (10.46 vs 4.79 N, p = 0.014), and at 180 s (8.14 vs 4.11 N, p = 0.018). Fitting a three-element linear viscoelastic solid model to our data, we found the serial spring stiffness to decrease by about 50% ( p = 0.01), the parallel spring remained unchanged, and there was a tendency for the dashpot coefficient to decrease (by 57%, p = 0.09). Conclusions. The 80% subfailure injury decreased the initial stiffness of the ligament, and tended to decrease its viscoelastic property.

Subfailure injury of the rabbit anterior cruciate ligament.

Ligamentous injuries range in severity from a simple sprain to a complete rupture. Although sprains occur more frequently than complete failures, only a few studies have investigated the phenomena of these subfailure injuries. The purpose of our study was to document the changes in the load-deformation curve until the failure point, after the ligament has been subjected to an 80% subfailure stretch. Thirteen paired fresh rabbit bone-anterior cruciate ligament-bone preparations were used. One of the pairs (control) was stretched until failure; the other (experimental) was first stretched to 80% of the failure deformation of the control and then stretched to failure. Comparisons were made between the load-deformation curves of the experimental and control specimens. The nonlinear load-deformation curves were characterized by eight parameters: failure load (Ffail), failure deformation (Dfail), energy until failure (Efail), deformations measured at 5, 10, 25, and 50% of the failure load (D5, D10, D25, and D50, respectively), and stiffness measured at 50% of the failure force (K50). There were no significant differences in the values for Ffail, Dfail, and Efail between the experimental and control ligaments (p > 0.33). In contrast, the deformation values were all larger for the experimental than the control ligaments (p > 0.01). The deformations D5, D10, D25, and D50 (mean +/- SD) for the control were 0.36 +/- 0.13, 0.49 +/- 0.23, 0.81 +/- 0.35, and 1.23 +/- 0.41 mm. The corresponding deformations for the experimental ligaments were, respectively, 209, 186, 153, and 130% of the control values. K50 was also greater for the experimental ligament (125.0 +/- 41.7 N/mm compared with 108.7 +/- 31.4 N/mm, p < 0.03). These findings indicate that even though the strength of the ligament did not change due to a subfailure injury, the shape of the load-displacement curve, especially at low loads, was significantly altered. Under the dynamic in vivo loading conditions of daily living, this may result in increased joint laxity, additional loads being applied to other joint structures, and, with time, to joint problems.