The Biomechanical, Biochemical, and Morphological Properties of 19 Human Cadaveric Lower Limb Tendons and Ligaments: An Open-Access Data Set.

BACKGROUND: Methodological heterogeneity hinders data comparisons across isolated studies of tendon and ligament properties, limiting clinical understanding and affecting the development and evaluation of replacement materials. PURPOSE: To create an open-access data set on the morphological, biomechanical, and biochemical properties of clinically important tendons and ligaments of the lower limb, using consistent methodologies, to enable direct tendon/ligament comparisons. STUDY DESIGN: Descriptive laboratory study. METHODS: Nineteen distinct lower limb tendons and ligaments were retrieved from 8 fresh-frozen human cadavers (5 male, 3 female; aged 49-65 years) including Achilles, tibialis posterior, tibialis anterior, fibularis (peroneus) longus, fibularis (peroneus) brevis, flexor hallucis longus, extensor hallucis longus, plantaris, flexor digitorum longus, quadriceps, patellar, semitendinosus, and gracilis tendons; anterior cruciate, posterior cruciate, medial collateral, and lateral collateral ligaments; and 10 mm-wide grafts from the contralateral quadriceps and patellar tendons. Outcomes included morphology (tissue length, ultrasound-quantified cross-sectional area [CSAUS], and major and minor axes), biomechanics (failure load, ultimate tensile strength [UTS], failure strain, and elastic modulus), and biochemistry (sulfated glycosaminoglycan [sGAG] and hydroxyproline contents). Tissue differences were analyzed using mixed-model regression. RESULTS: There was a range of similarities and differences between tendons and ligaments across outcomes. A key finding relating to potential graft tissue suitability was the comparable failure loads, UTS, CSAUS, sGAG, and hydroxyproline present between hamstring tendons (a standard graft source) and 5 tendons not typically used for grafting: fibularis (peroneus) longus and brevis, flexor and extensor hallucis longus, and flexor digitorum longus tendons. CONCLUSION: This study of lower limb tendons and ligaments has enabled direct comparison of morphological, biomechanical, and biochemical human tissue properties-key factors in the selection of suitable graft tissues. This analysis has identified 6 potential new donor tissues with properties comparable to currently used grafts. CLINICAL RELEVANCE: This extensive data set reduces the need to utilize data from incompatible sources, which may aid surgical decisions (eg, evidence to expand the range of tendons considered suitable for use as grafts) and may provide congruent design inputs for new biomaterials and computational models. The complete data set has been provided to facilitate further investigations, with the capacity to expand the resource to include additional outcomes and tissues.

Tendon biomechanical properties are altered by storage duration but not freeze-thaw temperatures or cycles.

Tendon allograft and xenograft processing often involves one or more steps of freezing and thawing. As failure strength is an important graft consideration, this study aimed to evaluate effects on failure properties when varying freeze-thaw conditions. Kangaroo tendons, a potential xenograft source, were used to evaluate changes in ultimate tensile strength (UTS), failure strain and elastic modulus after exposure to different freezer-storage temperatures (-20°C vs. -80°C), storage durations (1, 3, 6, 9, or 12 months), number of freeze-thaw cycles (1, 2, 3, 4, 5, or 10), or freeze-thaw temperature ranges (including freezing in liquid nitrogen to thawing at 37°C). Tendons stored for 6 or more months had significantly increased UTS and elastic modulus compared with 1 or 3 months of storage. This increase occurred irrespective of the freezing temperature (-20°C vs. -80°C) or the number of freeze-thaw cycles (1 vs. 10). In contrast, UTS, failure strain and the elastic modulus were no different between storage temperatures, number of freeze-thaw cycles and multiple freeze-thaw cycles across a range of freeze and thaw temperatures. Common freeze-thaw protocols did not negatively affect failure properties, providing flexibility for graft testing, storage, transportation and decellularisation procedures. However, the change in properties with the overall storage duration has implications for assessing the consistent performance of grafts stored for short versus extended periods of time (<6 months vs. >6 months), and the interpretation of data obtained from tissues of varying or unknown storage durations.

Streamlining quantitative joint-wide medial femoro-tibial histopathological scoring of mouse post-traumatic knee osteoarthritis models.

OBJECTIVES: Histological scoring remains the gold-standard for quantifying post-traumatic osteoarthritis (ptOA) in animal models, allowing concurrent evaluation of numerous joint tissues. Available systems require scoring multiple sections/joint making analysis laborious and expensive. We investigated if a single section allowed equivalent quantitation of pathology in different joint tissues and disease stages, in three ptOA models. METHOD: Male 10-12-week-old C57BL/6 mice underwent surgical medial-meniscal-destabilization, anterior-cruciate-ligament (ACL) transection, non-invasive-ACL-rupture, or served as sham-surgical, non-invasive-ACL-strain, or naïve/non-operated controls. Mice (n = 12/group) were harvested 1-, 4-, 8-, and 16-week post-intervention. Serial sagittal toluidine-blue/fast-green stained sections of the medial-femoro-tibial joint (n = 7/joint, 84 µm apart) underwent blinded scoring of 40 histology-outcomes. We evaluated agreement between single-slide versus entire slide-set maximum or median scores (weighted-kappa), and sensitivity/specificity of single-slide versus median/maximum to detect OA pathology. RESULTS: A single optimal mid-sagittal section showed excellent agreement with median (weighted-kappa 0.960) and maximum (weighted-kappa 0.926) scores. Agreement for individual histology-outcomes was high with only 19/240 median and 15/240 maximum scores having a weighted-kappa ≤0.4, the majority of these (16/19 and 11/15) in control groups. Statistically-significant histology-outcome differences between ptOA models and their controls detected with the entire slide-set were reliably reproduced using a single slide (sensitivity >93.15%, specificity >93.10%). The majority of false-negatives with single-slide scoring were meniscal and subchondral bone histology-outcomes (89%) and occurred in weeks 1-4 post-injury (84%). CONCLUSION: A single mid-sagittal slide reduced the time needed to score diverse histopathological changes by 87% without compromising the sensitivity or specificity of the analysis, across a variety of ptOA models and time-points.

Challenging the Perceptions of Human Tendon Allografts: Influence of Donor Age, Sex, Height, and Tendon on Biomechanical Properties.

BACKGROUND: The use of allograft tendons has increased for primary and revision anterior cruciate ligament reconstruction, but allograft supply is currently limited to a narrow range of tendons and donors up to the age of 65 years. Expanding the range of donors and tendons could help offset an increasing clinical demand. PURPOSE: To investigate the effects of donor age, sex, height, and specific tendon on the mechanical properties of a range of human lower leg tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine tendons were retrieved from 39 fresh-frozen human cadaveric lower legs (35 donors [13 female, 22 male]; age, 49-99 years; height, 57-85 inches [145-216 cm]) including: Achilles tendon, tibialis posterior and anterior, fibularis longus and brevis, flexor and extensor hallucis longus, plantaris, and flexor digitorum longus. Tendons underwent tensile loading to failure measuring cross-sectional area (CSA), maximum load, strain at failure, ultimate tensile strength, and elastic modulus. Results from 332 tendons were analyzed using mixed-effects linear regression, accounting for donor age, sex, height, and weight. RESULTS: Mechanical properties were significantly different among tendons and were substantially greater than the effects of donor characteristics. Significant effects of donor sex, age, and height were limited to specific tendons: Achilles tendon, tibialis posterior, and tibialis anterior. All other tendons were unaffected. The Achilles tendon was most influenced by donor variables: greater CSA in men (ß = 15.45 mm2; Sidák adjusted P < .0001), decreased maximum load with each year of increased age (ß = -17.20 N per year; adjusted P = .0253), and increased CSA (ß = 1.92 mm2 per inch; adjusted P < .0001) and maximum load (ß = 86.40 N per inch; adjusted P < .0001) with each inch of increased height. CONCLUSION: Mechanical properties vary significantly across different human tendons. The effects of donor age, sex, and height are relatively small, are limited to specific tendons, and affect different tendons uniquely. The findings indicate that age negatively affected only the Achilles tendon (maximum load) and challenge the exclusion of donors aged >65 years across all tendon grafts. CLINICAL RELEVANCE: The findings support including a broader range of tendons for use as allografts for anterior cruciate ligament reconstruction and reviewing the current exclusion criterion of donors aged >65 years.

Animal models of osteoarthritis : the benefits and costs of reducing variability.

Cite this article: Bone Joint Res 2022;11(8):514-517.

Long-term Effect of a Single Subcritical Knee Injury: Increasing the Risk of Anterior Cruciate Ligament Rupture and Osteoarthritis.

BACKGROUND: Rupture of the anterior cruciate ligament (ACL) is a well-known risk factor for the development of posttraumatic osteoarthritis (PTOA), but patients with the "same injury" can have vastly different trajectories for the onset and progression of disease. Minor subcritical injuries preceding the critical injury event may drive this disparity through preexisting tissue pathologies and sensory changes. PURPOSE: To investigate the role of subcritical injury on ACL rupture risk and PTOA through the evaluation of pain behaviors, joint mechanics, and tissue structural change in a mouse model of knee injury. STUDY DESIGN: Controlled laboratory study. METHODS: Ten-week-old male C57BL/6J mice were allocated to naïve control and subcritical knee injury groups. Injury was induced by a single mechanical compression to the right hindlimb, and mice were evaluated using joint histopathology, anteroposterior joint biomechanics, pain behaviors (mechanical allodynia and hindlimb weightbearing), and isolated ACL tensile testing to failure at 1, 2, 4, or 8 weeks after injury. RESULTS: Subcritical knee injury produced focal osteochondral lesions in the patellofemoral and lateral tibiofemoral compartments with no resolution for the duration of the study (8 weeks). These lesions were characterized by focal loss of proteoglycan staining, cartilage structural change, chondrocyte pathology, microcracks, and osteocyte cell loss. Injury also resulted in the rapid onset of allodynia (at 1 week), which persisted over time and reduced ACL failure load (P = .006; mean ± SD, 7.91 ± 2.01 N vs 9.37 ± 1.01 N in naïve controls at 8 weeks after injury), accompanied by evidence of ACL remodeling at the femoral enthesis. CONCLUSION: The present study in mice establishes a direct effect of a single subcritical knee injury on the development of specific joint tissue pathologies (osteochondral lesions and progressive weakening of the ACL) and allodynic sensitization. These findings demonstrate a predisposition for secondary critical injuries (eg, ACL rupture) and an increased risk of PTOA onset and progression (structurally and symptomatically). CLINICAL RELEVANCE: Subcritical knee injuries are a common occurrence and, based on this study, can cause persistent sensory and structural change. These findings have important implications for the understanding of risk factors of ACL injury and subsequent PTOA, particularly with regard to prevention and management strategies following an often underreported event.

Sex- and injury-based differences in knee biomechanics in mouse models of post-traumatic osteoarthritis.

Sex and joint injury are risk factors implicated in the onset and progression of osteoarthritis (OA). In mouse models of post-traumatic OA (ptOA), the pathogenesis of disease is notably impacted by sex (often worse in males) and injury model (e.g. meniscal versus ligament injury). Increasing ptOA progression and severity is often associated with greater relative instability of the joint but few studies have directly quantified changes in joint mechanics after injury and compared outcomes across multiple models in both male and female mice. Passive anterior-posterior knee biomechanics were evaluated in 10-week-old, male and female C57BL/6J mice. PtOA injury models included destabilisation of the medial meniscus (DMM), anterior cruciate ligament transection (ACLT) or mechanical rupture (ACLR), and combined DMM and ACLT (DMM + ACLT). Sham operated and non-operated controls (NOC) were included for baseline comparisons. The test apparatus loaded hindlimbs at 60° flexion between ± 1 N at 0.5 mm/s (build specifications available for download: https://doi.org/10.17632/z754455x3c.1). Measures of joint laxity (range of motion, neutral zone) and stiffness were calculated. Joint laxity was comparable between male and female mice while joint stiffness was greater in females (P ≤ 0.002, correcting for body-mass and injury-model). Anterior-posterior joint mechanics were minimally altered by DMM but significantly affected by loss of the ACL (P < 0.001), with equivalent changes between ACL-injury models despite different injury mechanisms and adjacent meniscal damage. These findings suggest that despite the important role of joint injury; sex- and model-specific differences in ptOA progression and severity are not primarily driven by altered anterior-posterior knee biomechanics.

Using mouse models to investigate the pathophysiology, treatment, and prevention of post-traumatic osteoarthritis.

Post-traumatic osteoarthritis (PTOA) is defined by its development after joint injury. Factors contributing to the risk of PTOA occurring, the rate of progression, and degree of associated disability in any individual, remain incompletely understood. What constitutes an "OA-inducing injury" is not defined. In line with advances in the traumatic brain injury field, we propose the scope of PTOA-inducing injuries be expanded to include not only those causing immediate structural damage and instability (Type I), but also those without initial instability/damage from moderate (Type II) or minor (Type III) loading severity. A review of the literature revealed this full spectrum of potential PTOA subtypes can be modeled in mice, with 27 Type I, 6 Type II, and 4 Type III models identified. Despite limitations due to cartilage anatomy, joint size, and bio-fluid availability, mice offer advantages as preclinical models to study PTOA, particularly genetically modified strains. Histopathology was the most common disease outcome, cartilage more frequently studied than bone or synovium, and meniscus and ligaments rarely evaluated. Other methods used to examine PTOA included gene expression, protein analysis, and imaging. Despite the major issues reported by patients being pain and biomechanical dysfunction, these were the least commonly measured outcomes in mouse models. Informative correlations of simultaneously measured disease outcomes in individual animals, was rarely done in any mouse PTOA model. This review has identified knowledge gaps that need to be addressed to increase understanding and improve prevention and management of PTOA. Preclinical mouse models play a critical role in these endeavors. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:424-439, 2017.

Joint loads resulting in ACL rupture: Effects of age, sex, and body mass on injury load and mode of failure in a mouse model.

Anterior cruciate ligament (ACL) tears are a common knee injury with a known but poorly understood association with secondary joint injuries and post-traumatic osteoarthritis (OA). Female sex and age are known risk factors for ACL injury but these variables are rarely explored in mouse models of injury. This study aimed to further characterize a non-surgical ACL injury model to determine its clinical relevance across a wider range of mouse specifications. Cadaveric and anesthetized C57BL/6 mice (9-52 weeks of age) underwent joint loading to investigate the effects of age, sex, and body mass on ACL injury mechanisms. The ACL injury load (whole joint load required to rupture the ACL) was measured from force-displacement data, and mode of failure was assessed using micro-dissection and histology. ACL injury load was found to increase with body mass and age (p < 0.001) but age was not significant when controlling for mass. Sex had no effect. In contrast, the mode of ACL failure varied with both age and sex groups. Avulsion fractures (complete or mixed with mid-substance tears) were common in all age groups but the proportion of mixed and mid-substance failures increased with age. Females were more likely than males to have a major avulsion relative to a mid-substance tear (p < 0.01). This data compliments studies in human cadaveric knees, and provides a basis for determining the severity of joint injury relative to a major ACL tear in mice, and for selecting joint loading conditions in future experiments using this model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1754-1763, 2017.