The Actin Cytoskeleton as a Regulator of Proteoglycan 4.

OBJECTIVE: The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro. There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies. MATERIALS AND METHODS: A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed. RESULTS: PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro. Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling. CONCLUSION: Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

3D characterization of the triple-bundle Achilles tendon from in vivo high-field MRI.

The Achilles tendon consists of three subtendons that transmit force from the triceps surae muscles to the calcaneus. Individual differences have been identified in Achilles subtendon morphology and twist in cadavers, which may have implications for triceps surae mechanics and function. High-field magnetic resonance imaging (MRI) can be used to identify boundaries within multi-bundle tissues, which could then enable studies of subtendon structure-function relationships in humans. The objective of this study was to use high-field MRI (7T) to image and reconstruct Achilles subtendons arising from the triceps surae muscles. We imaged the dominant lower leg of a cohort of healthy human subjects (n = 10) using a tuned musculoskeletal sequence (double echo steady state sequence, 0.4 mm isotropic voxels). We then characterized the cross-sectional area and orientation of each subtendon between the MTJ and calcaneal insertion. Image collection and segmentation was repeated to assess repeatability. Subtendon morphometry varied across subjects, with average subtendon areas of 23.5 ± 8.9 mm2 for the medial gastrocnemius, 25.4 ± 8.9 mm2 for the lateral gastrocnemius, and 13.7 ± 5.9 mm2 for the soleus subtendons. Repeatable subject-specific variations in size and position of each subtendon were identified over two visits, expanding on prior knowledge that high variability exists in Achilles subtendon morphology across subjects.

A Single-Sensor Approach for Noninvasively Tracking Phase Velocity in Tendons during Dynamic Movement.

Shear wave tensiometry is a noninvasive method for directly measuring wave speed as a proxy for force in tendons during dynamic activities. Traditionally, tensiometry has used broadband excitation pulses and measured the wave travel time between two sensors. In this work, we demonstrate a new method for tracking phase velocity using shaped excitations and measurements from a single sensor. We observed modulation of phase velocity in the Achilles tendon that was generally consistent with wave speed measures obtained via broadband excitation. We also noted a frequency dependence of phase velocity, which is expected for dispersive soft tissues. The implementation of this method could enhance the use of noninvasive wave speed measures to characterize tendon forces. Further, the approach allows for the design of smaller shear wave tensiometers usable for a broader range of tendons and applications.

A Kalman Filter Approach for Estimating Tendon Wave Speed from Skin-Mounted Accelerometers.

Shear wave tensiometry is a noninvasive approach for assessing in vivo tendon forces based on the speed of a propagating shear wave. Wave speed is measured by impulsively exciting a shear wave in a tendon and then assessing the wave travel time between skin-mounted accelerometers. Signal distortion with wave travel can cause errors in the estimated wave travel time. In this study, we investigated the use of a Kalman filter to fuse spatial and temporal accelerometer measurements of wave propagation. Spatial measurements consist of estimated wave travel times between accelerometers. Temporal measurements are the change in wave arrival at a fixed accelerometer between successive impulsive taps. The Kalman filter substantially improved the accuracy of estimated wave speeds when applied to simulated tensiometer data. The variability of estimated wave speed was reduced by ~55% in the presence of random sensor noise. It was found that increasing the number of accelerometers from two to three further reduced wave speed errors by 45%. The use of redundant accelerometers (>2) also improved the robustness of wave speed measures in the presence of uncertainty in accelerometer location. We conclude that the use of a Kalman filter and redundant accelerometers can enhance the fidelity of using shear wave tensiometers to track tendon wave speed and loading during movement.

Canine ACL rupture: a spontaneous large animal model of human ACL rupture.

BACKGROUND: Anterior cruciate ligament (ACL) rupture in humans is a common condition associated with knee pain, joint instability, and secondary osteoarthritis (OA). Surgical treatment with an intraarticular graft provides reasonable outcomes at mid and long-term follow-up. Non-modifiable and modifiable factors influence risk of ACL rupture. The etiology, mechanobiology, causal biomechanics, and causal molecular pathways are not fully understood. The dog model has shared features of ACL rupture that make it a valuable spontaneous preclinical animal model. In this article, we review shared and contrasting features of ACL rupture in the two species and present information supporting spontaneous canine ACL rupture as a potentially useful preclinical model of human ACL rupture with a very large subject population. RESULTS: ACL rupture is more common in dogs than in humans and is diagnosed and treated using similar approaches to that of human patients. Development of OA occurs in both species, but progression is more rapid in the dog, and is often present at diagnosis. Use of client-owned dogs for ACL research could reveal impactful molecular pathways, underlying causal genetic variants, biomechanical effects of specific treatments, and opportunities to discover new treatment and prevention targets. Knowledge of the genetic contribution to ACL rupture is more advanced in dogs than in humans. In dogs, ACL rupture has a polygenetic architecture with moderate heritability. Heritability of human ACL rupture has not been estimated. CONCLUSION: This article highlights areas of One Health research that are particularly relevant to future studies using the spontaneous canine ACL rupture model that could fill gaps in current knowledge.

Age- and Sex-Specific Joint Biomechanics in Response to Partial and Complete Anterior Cruciate Ligament Injury in the Porcine Model.

CONTEXT: Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients. OBJECTIVE: To evaluate the effects of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model. DESIGN: Descriptive laboratory study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: We studied 60 male and female Yorkshire crossbreed pigs aged 1.5, 3, 4.5, 6, and 18 months (n = 6 pigs per age per sex). MAIN OUTCOME MEASURE(S): Joint laxity was measured in intact, anteromedial bundle-transected, and ACL-transected joints under applied anterior-posterior drawer and varus-valgus torque using a robotic testing system. Loading of the soft tissues in the stifle joint was measured under each condition. RESULTS: Anterior-posterior joint laxity increased by 13% to 50% (P < .05) after anteromedial bundle transection and 75% to 178% (P < .05) after ACL transection. Destabilization after anteromedial bundle transection increased with age (P < .05) and was greater in late female than late male adolescents (P < .05). In anteromedial bundle-transected joints, the posterolateral bundle resisted the anterior load. In ACL-transected joints, the medial collateral ligament (MCL) contribution was largest, followed by the medial meniscus. The MCL contribution was larger and the medial meniscus contribution was smaller in male versus female specimens. CONCLUSIONS: Partial ACL transection resulted in moderate increases in joint laxity, with the remaining bundle performing the primary ACL function. Destabilization due to partial ACL transection (anteromedial bundle) was largest in late adolescent joints, indicating that operative treatment should be considered in active, late-adolescent patients with this injury. Increased forces on the MCL and medial meniscus after ACL transection suggested that rehabilitation protocols may need to focus on protecting these tissues.

Sex-specific biomechanics and morphology of the anterior cruciate ligament during skeletal growth in a porcine model.

Pediatric anterior cruciate ligament (ACL) injuries are on the rise, and females experience higher ACL injury risk than males during adolescence. Studies in skeletally immature patients indicate differences in ACL size and joint laxity between males and females after the onset of adolescence. However, functional data regarding the ACL and its anteromedial and posterolateral bundles in the pediatric population remain rare. Therefore, this study uses a porcine model to investigate the sex-specific morphology and biomechanics of the ACL and its bundles throughout skeletal growth. Hind limbs from male and female Yorkshire pigs aged early youth to late adolescence were imaged using magnetic resonance imaging to measure the size and orientation of the ACL and its bundles, then biomechanically tested under anterior-posterior drawer using a robotic testing system. Joint laxity decreased (p < 0.001) while joint stiffness increased (p < 0.001) throughout skeletal growth in both sexes. The ACL was the primary stabilizer against anterior tibial loading, while the functional role of the anteromedial bundle increased with age (p < 0.001), with an earlier increase in males. ACL and posterolateral bundle cross-sectional area and ACL and anteromedial bundle length were larger in males than females during adolescence (p < 0.01 for all), while ACL and bundle sagittal angle remained similar between sexes. Additionally, in situ ACL stiffness versus cross-sectional area regressions were significant across skeletal growth (r2 = 0.75, p < 0.001 in males and r2 = 0.64, p < 0.001 in females), but not within age groups. This study has implications for age and sex-specific surgical intervention strategies and suggests the need for human studies.

Age- and sex-specific differences in ACL and ACL bundle size during adolescent growth.

Anterior cruciate ligament (ACL) injuries are increasingly common in adolescents, and injuries in this age-group are associated with many unique challenges. Recent large animal studies suggest that the size and function of the major bundles of the ACL change differently throughout skeletal growth. To better aid clinical treatment of pediatric partial ACL tears and better predict outcomes from age-specific treatments, there is a need to measure changes in ACL bundle size in humans during growth. As such, the objective of this study was to compare changes in the length and cross-sectional area (CSA) of the ACL and its primary bundles in adolescent human subjects. Magnetic resonance imaging (MRI) scans were analyzed to determine the visibility and integrity of the ACL and its anteromedial and posterolateral bundles. MRI scans were considered from a retrospective database of subjects ranging from 10 to 18 years of age. The ACL and its anteromedial and posterolateral bundles were segmented and reconstructed into 3D models, and length and CSA were calculated. Total ACL length and CSA were greater in males compared with females, with a statistically significant interaction between age and sex for CSA. Sex had a significant effect on the CSA of both bundles. These sex-dependent differences emerge with moderate to large effect sizes (range: d = 0.50 to d = 1.23) beginning around 13 years of age. Along with ACL bundle structure-function relationships previously established in preclinical animal models, these findings may point toward biomechanical changes in the adolescent human ACL.

Joint laxity varies in response to partial and complete anterior cruciate ligament injuries throughout skeletal growth.

Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model. To do so, we applied anterior-posterior tibial loads and varus-valgus moments to stifle joints of female pigs ranging from early juvenile to late adolescent ages and assessed both kinematics and in-situ loads carried in the bundles of the ACL and other soft tissues including the collateral ligaments and the menisci. Partial ACL injury led to increased anterior tibial translation only in late adolescence and small increases in varus-valgus rotation at all ages. Complete ACL injury led to substantial increases in translation and rotation at all ages. At all ages, the medial collateral ligament and the medial meniscus combined to resist the majority of applied anterior tibial load following complete ACL transection. Across all ages and flexion angles, the contribution of the MCL ranged from 45 to 90% of the anterior load and the contribution of the medial meniscus ranged from 14 to 35% of the anterior load. These findings add to our current understanding of age-specific functional properties of both healthy and injured knees during skeletal growth.

Tissue-specific changes in size and shape of the ligaments and tendons of the porcine knee during post-natal growth.

Prior studies have analyzed growth of musculoskeletal tissues between species or across body segments; however, little research has assessed the differences in similar tissues within a single joint. Here we studied changes in the length and cross-sectional area of four ligaments and tendons, (anterior cruciate ligament, patellar tendon, medial collateral ligament, lateral collateral ligament) in the tibiofemoral joint of female Yorkshire pigs through high-field magnetic resonance imaging throughout growth. Tissue lengths increased by 4- to 5-fold from birth to late adolescence across the tissues while tissue cross-sectional area increased by 10-20-fold. The anterior cruciate ligament and lateral collateral ligament showed allometric growth favoring change in length over change in cross-sectional area while the patellar tendon and medial collateral ligament grow in an isometric manner. Additionally, changes in the length and cross-sectional area of the anterior cruciate ligament did not increase as much as in the other ligaments and tendon of interest. Overall, these findings suggest that musculoskeletal soft tissue morphometry can vary within tissues of similar structure and within a single joint during post-natal growth.

In Situ Joint Stiffness Increases During Skeletal Growth but Decreases Following Partial and Complete Anterior Cruciate Ligament Injury.

Partial and complete anterior cruciate ligament (ACL) injuries occur in both pediatric and adult populations and can result in loss of joint stability and function. The sigmoidal shape of knee joint function (load-translation curve) under applied loads includes a low-load region (described by slack length) followed by a high-load region (described by stiffness). However, the impact of age and injury on these parameters is not fully understood. The current objective was to measure the effects of age and injury on the shape of joint function in a porcine model. In response to an applied anterior-posterior tibial load, in situ slack did not change (p > 0.05), despite sevenfold increases in joint size with increasing age. Joint stiffness increased from an average of 10 N/mm in early youth to 47 N/mm in late adolescence (p < 0.05). In situ ACL stiffness increased similarly, and changes in in situ joint stiffness and ACL stiffness were highly correlated across ages. With complete ACL injury, in situ slack length increased by twofold to fourfold and in situ stiffness decreased threefold to fourfold across ages (p < 0.05). Partial ACL injury resulted in less dramatic, but statistically significant, increases in joint slack and significant decreases in in situ joint stiffness in the adolescent age groups (p < 0.05). This work furthers our understanding of the interaction between joint biomechanics and ACL function throughout growth and the impact of ACL injury in the skeletally immature joint.

Biomechanical Function and Size of the Anteromedial and Posterolateral Bundles of the ACL Change Differently with Skeletal Growth in the Pig Model.

BACKGROUND: ACL injuries are becoming increasingly common in children and adolescents, but little is known regarding age-specific ACL function in these patients. To improve our understanding of changes in musculoskeletal tissues during growth and given the limited availability of pediatric human cadaveric specimens, tissue structure and function can be assessed in large animal models, such as the pig. QUESTIONS/PURPOSES: Using cadaveric porcine specimens ranging throughout skeletal growth, we aimed to assess age-dependent changes in (1) joint kinematics under applied AP loads and varus-valgus moments, (2) biomechanical function of the ACL under the same loads, (3) the relative biomechanical function of the anteromedial and posterolateral bundles of the ACL; and (4) size and orientation of the anteromedial and posterolateral bundles. METHODS: Stifle joints (analogous to the human knee) were collected from female Yorkshire crossbreed pigs at five ages ranging from early youth to late adolescence (1.5, 3, 4.5, 6, and 18 months; n = 6 pigs per age group, 30 total), and MRIs were performed. A robotic testing system was used to determine joint kinematics (AP tibial translation and varus-valgus rotation) and in situ forces in the ACL and its bundles in response to applied anterior tibial loads and varus-valgus moments. To see if morphological changes to the ACL compared with biomechanical changes, ACL and bundle cross-sectional area, length, and orientation were calculated from MR images. RESULTS: Joint kinematics decreased with increasing age. Normalized AP tibial translation decreased by 44% from 1.5 months (0.34 ± 0.08) to 18 months (0.19 ± 0.02) at 60° of flexion (p < 0.001) and varus-valgus rotation decreased from 25° ± 2° at 1.5 months to 6° ± 2° at 18 months (p < 0.001). The ACL provided the majority of the resistance to anterior tibial loading at all age groups (75% to 111% of the applied anterior force; p = 0.630 between ages). Anteromedial and posterolateral bundle function in response to anterior loading and varus torque were similar in pigs of young ages. During adolescence (4.5 to 18 months), the in situ force carried by the anteromedial bundle increased relative to that carried by the posterolateral bundle, shifting from 59% ± 22% at 4.5 months to 92% ± 12% at 18 months (data for 60° of flexion, p < 0.001 between 4.5 and 18 months). The cross-sectional area of the anteromedial bundle increased by 30 mm throughout growth from 1.5 months (5 ± 2 mm) through 18 months (35 ± 8 mm; p < 0.001 between 1.5 and 18 months), while the cross-sectional area of the posterolateral bundle increased by 12 mm from 1.5 months (7 ± 2 mm) to 4.5 months (19 ± 5 mm; p = 0.004 between 1.5 and 4.5 months), with no further growth (17 ± 7 mm at 18 months; p = 0.999 between 4.5 and 18 months). However, changes in length and orientation were similar between the bundles. CONCLUSION: We showed that the stifle joint (knee equivalent) in the pig has greater translational and rotational laxity in early youth (1.5 to 3 months) compared with adolescence (4.5 to 18 months), that the ACL functions as a primary stabilizer throughout growth, and that the relative biomechanical function and size of the anteromedial and posterolateral bundles change differently with growth. CLINICAL RELEVANCE: Given the large effects observed here, the age- and bundle-specific function, size, and orientation of the ACL may need to be considered regarding surgical timing, graft selection, and graft placement. In addition, the findings of this study will be used to motivate pre-clinical studies on the impact of partial and complete ACL injuries during skeletal growth.

Size and Shape of the Human Anterior Cruciate Ligament and the Impact of Sex and Skeletal Growth: A Systematic Review.

BACKGROUND: High rates of anterior cruciate ligament (ACL) injury and surgical reconstruction in both skeletally immature and mature populations have led to many studies investigating the size and shape of the healthy ligament. The purposes of the present study were to compile existing quantitative measurements of the geometry of the ACL, its bundles, and its insertion sites and to describe effects of common covariates such as sex and age. METHODS: A search of the Web of Science was conducted for studies published from January 1, 1900, to April 11, 2018, describing length, cross-sectional area, volume, orientation, and insertion sites of the ACL. Two reviewers independently screened and reviewed the articles to collect quantitative data for each parameter. RESULTS: Quantitative data were collected from 92 articles in this systematic review. In studies of adults, reports of average ACL length, cross-sectional area, and volume ranged from 26 to 38 mm, 30 to 53 mm, and 854 to 1,858 mm, respectively. Reported values were commonly found to vary according to sex and skeletal maturity as well as measurement technique. CONCLUSIONS: Although the geometry of the ACL has been described widely in the literature, quantitative measurements can depend on sex, age, and measurement modality, contributing to variability between studies. As such, care must be taken to account for these factors. The present study condenses measurements describing the geometry of the ACL, its individual bundles, and its insertion sites, accounting for common covariates when possible, to provide a resource to the clinical and scientific communities. CLINICAL RELEVANCE: Quantitative measures of ACL geometry are informative for developing clinical treatments such as ACL reconstruction. Age and sex can impact these parameters.

Rise of the Pigs: Utilization of the Porcine Model to Study Musculoskeletal Biomechanics and Tissue Engineering During Skeletal Growth.

Large animal models play an essential role in the study of tissue engineering and regenerative medicine (TERM), as well as biomechanics. The porcine model has been increasingly used to study the musculoskeletal system, including specific joints, such as the knee and temporomandibular joints, and tissues, such as bone, cartilage, and ligaments. In particular, pigs have been utilized to evaluate the role of skeletal growth on the biomechanics and engineered replacements of these joints and tissues. In this review, we explore the publication history of the use of pig models in biomechanics and TERM discuss interspecies comparative studies, highlight studies on the effect of skeletal growth and other biological considerations in the porcine model, and present challenges and emerging opportunities for using this model to study functional TERM.

Orientation changes in the cruciate ligaments of the knee during skeletal growth: A porcine model.

Musculoskeletal injuries in pediatric patients are on the rise, including significant increases in anterior cruciate ligament (ACL) injuries. Previous studies have found major anatomical changes during skeletal growth in the soft tissues of the knee. Specifically, the ACL and the posterior cruciate ligament (PCL) change in their relative orientation to the tibial plateau throughout growth. In order to develop age-specific treatments for ACL injuries, the purpose of this study was to characterize orientation changes in the cruciate ligaments of the Yorkshire pig, a common pre-clinical model, during skeletal growth in order to verify the applicability of this model for pediatric musculoskeletal studies. Hind limbs were isolated from female Yorkshire pigs ranging in age from newborn to late adolescence and were then imaged using high field strength magnetic resonance imaging. Orientation changes were quantified from the magnetic resonance images using image segmentation software. Statistically significant increases were found in the coronal and sagittal angles of the ACL relative to the tibial plateau during pre-adolescent growth. Additional changes were observed in the PCL angle, Blumensaat angle, intercondylar roof angle, and the aspect ratio of the intercondylar notch. Only the sagittal angle of the ACL relative to the tibial plateau experienced statistically significant changes through late adolescence. The age-dependent properties of the ACL and PCL in the female pig mirrored results found in female human patients, suggesting that the porcine model may provide a pre-clinical platform to study the cruciate ligaments during skeletal growth. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2725-2732, 2017.

Engineering anisotropic biphasic Janus-type polymer nanofiber scaffold networks via centrifugal jet spinning.

Biphasic materials, comprised of an ordered arrangement of two different material phases within a material, have the potential for a wide variety of applications including filtration, protective clothing and tissue engineering. This study reports for the first time, a process for engineering biphasic Janus-type polymeric nanofiber (BJPNF) networks via the centrifugal jet spinning technique. BJPNF alignment and fiber diameter was dependent on fabrication rotational speed as well as solution composition. The biphasic character of these BJPNFs, which was controlled via the rotational speed of fabrication, was confirmed at the individual nanofiber scale using energy dispersive X-ray spectroscopy, and at the bulk, macro-scale using attenuated total reflectance-Fourier transform infrared spectroscopy. Biphasic character was also demonstrated at the functional level via differing affinities on either side of the BJPNF for cell attachment. Our work thus presents a method for fabricating BJPNF scaffold networks where there might be a need for different properties on either side of a material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2455-2464, 2017.

Microfluidic multiplexed partitioning enables flexible and effective utilization of magnetic sensor arrays.

We demonstrate microfluidic partitioning of a giant magnetoresistive sensor array into individually addressable compartments that enhances its effective use. Using different samples and reagents in each compartment enables measuring of cross-reactive species and wide dynamic ranges on a single chip. This compartmentalization technique motivates the employment of high density sensor arrays for highly parallelized measurements in lab-on-a-chip devices.