Louis J. Soslowsky

Collagen V is a key matrix protein involved in fibril nucleation and lateral fibril growth during extracellular matrix assembly. Genetic mouse models have been used to investigate the role of collagen V in tendon, which showed deficient mechanical properties and aberrant fibril structure in the absence of collagen V. However, the lasting effects of collagen V deficiency later into adulthood remain unknown, as well as the role of collagen V in maintaining a mature matrix. This study therefore investigated the long-term effects of collagen V reduction on tendon as well as its role in mature tendon matrix in adulthood. Tendon-targeted conditional Col5a1 knockout, which excises Col5a1 alleles early in development, had long-term impact on tendon structure and function in 300-day old mice. Gene expression was altered with differential expression of primarily matrix and matrix remodeling genes. Regional changes in cellular shape and density were consistent with typical behavior in tendinopathy. Fibril diameters were increased due to dysregulated lateral growth. Deficits in mechanical properties indicate a weaker tendon matrix after knockout, although deformation patterns of collagen fibrils were not affected. In contrast, inducing collagen V knockdown in a mature tendon matrix at 120-days old did not cause substantial changes in any of the above-mentioned properties in 300-day old mice. In conclusion, these findings highlight the important function of collagen V in matrix assembly that has lasting effects into later ages, even though collagen V has little role in homeostatic maintenance of a mature tendon matrix.

Tendons are soft musculoskeletal tissues that transfer tensile loads from muscle to bone and consist of a highly organized extracellular matrix. Highly and repetitively loaded tendons such as the Achilles tendon are more susceptible to injury, and injuries are prevalent in the older population. Glycosaminoglycans (GAGs) are long polysaccharide chains that decrease in number with age in tendon and other musculoskeletal tissues. Yet, little is known of the role of GAGs in the tensile mechanics of the ageing Achilles tendon. The objective of this study was to investigate the mechanical role of GAGs in the ageing Achilles tendon. We enzymatically digested GAGs from the Achilles tendons of young, middle-aged and old C57BL/6 mice, removing a total of 64% of GAGs from the tendon insertion and midsubstance. GAG removal did not affect viscoelastic or structural properties across age in the Achilles tendon, paired with no changes to fibril realignment. However, removal of GAGs altered regional material properties at the Achilles tendon insertion in young mice in the absence of any material property changes to the Achilles tendon midsubstance. Finally, we found no changes to regional properties at the Achilles tendon insertion or midsubstance in middle-aged or old mice. In summary, GAG content influences regional mechanical properties at the calcaneal insertion of the Achilles tendon in young mice. KEY POINTS: The Achilles tendon is among the most commonly injured tendons, and aged tendon injuries are becoming an increasingly present societal burden. Glycosaminoglycans decrease across age in the Achilles tendon, yet their effect on structural properties, viscoelasticity and fibre realignment is minimal. In young Achilles tendons, glycosaminoglycans influence the elastic modulus near the calcaneal insertion, which was 60% greater following chondroitinase treatment. These data elucidate the mechanical role of glycosaminoglycans in the healthy Achilles tendon across age.

Rotator cuff disorders are common in the aging population, often leading to pain, disability, and reduced quality of life. Age-associated changes can occur disproportionately in regions of high mechanical stress, such as the insertion site of the supraspinatus tendon, leading to increased injury rates and tendinopathies. We previously demonstrated site-specific differences in structure, function, and gene expression between the insertion and midsubstance regions of supraspinatus tendons in mature, Day 150 mice. However, a comprehensive study examining the role of aging on site-specific supraspinatus tendon multiscale structure, function, and gene expression is needed. Therefore, our objective was to elucidate the role of aging on supraspinatus tendon multiscale structure, function, and gene expression. Whole-tissue and regional mechanics, as well as regional fibril response to load, cellularity and cell shape, collagen fibril morphology, and gene expression were evaluated in supraspinatus tendons of Day 300 and Day 570 mice. Aging resulted in decreased stiffness, dynamic modulus, and decreased midsubstance modulus. Collagen fibril morphology analysis revealed a more noticeable shift toward smaller fibril diameters with aging in the midsubstance region. Histological evaluation demonstrated reduced cellularity and a transition to more elongated cell morphology in aged tendons. Gene expression profiling highlighted upregulation of pro-inflammatory and catabolic markers and downregulation of major collagens in Day 570 tendons relative to Day 300 tendons. Understanding the effects of aging on tendon structure, function and gene expression provides critical insights into the mechanisms underlying aging-related injury and tendinopathy.

We demonstrated that 16 wk of high-speed treadmill running did not induce structural, functional, or mechanical changes consistent with Achilles tendinopathy in the rat. These findings underscore the importance of exploring alternative approaches to generating reliable and clinically relevant animal models of Achilles tendinopathy.

The hierarchical structure of tendon dictates its ability to effectively transmit loads from muscle to bone. Tendon- and site-specific differences in mechanical loading result in the establishment and remodeling of structure, as well as associated changes in composition throughout development and healing. Previous work has demonstrated region-specific differences in the response of collagen fibrils to mechanical loading within the insertion region and midsubstance regions of mouse supraspinatus tendons using atomic force microscopy. However, multiscale structure, function, and gene expression differences between the insertion and midsubstance of the supraspinatus tendon have not yet been linked together in a comprehensive study. Therefore, the purpose of this study was to elucidate site-specific hierarchical structure, function, and gene expression differences in mouse supraspinatus tendons. Supraspinatus tendons from day 150 wild-type C57BL/6 mice were harvested for regional mechanics, histology, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (qPCR). Mechanical testing revealed that the midsubstance region demonstrated a greater modulus and increased collagen fiber realignment compared to the insertion region. Histological scoring demonstrated greater cellularity and more rounded cells in the insertion region. TEM analysis showed differences in collagen fibril diameter distributions between the two regions, with a shift towards smaller diameters observed at the insertion region. Gene expression analysis identified several genes that were differentially expressed between regions, with principal component analysis revealing distinct clustering based on region. These findings provide insight into the regional heterogeneity of the supraspinatus tendon and underscore the importance of considering these differences in the context of tendon injury and repair, contributing to a better understanding of tendon structure-function and guiding future studies aimed at elucidating the mechanisms underlying tendon pathology.

Exercise influences clinical Achilles tendon health in humans, but animal models of exercise-related Achilles tendon changes are lacking. Moreover, previous investigations of the effects of treadmill running exercise on rat Achilles tendon demonstrate variable outcomes. Our objective was to assess the functional, structural, cellular, and biomechanical impacts of treadmill running exercise on rat Achilles tendon with sensitive in and ex vivo approaches. Three running levels were assessed over the course of 8 weeks: control (cage activity), moderate-speed (treadmill running at 10 m/min, no incline), and high-speed (treadmill running at 20 m/min, 10° incline). We hypothesized that moderate-speed treadmill running would beneficially impact tendon biomechanics through increased tenocyte cellularity, metabolism, and anabolism whereas high-speed treadmill running would cause a tendinopathic phenotype with compromised tendon biomechanics due to pathologic tenocyte differentiation, metabolism, and catabolism. Contrary to our hypothesis, treadmill running exercise at these speeds had a nominal effect on the rat Achilles tendon. Treadmill running modestly influenced tenocyte metabolism and nuclear aspect ratio as well as viscoelastic tendon properties but did not cause a tendinopathic phenotype. These findings highlight the need for improved models of exercise- and loading-related tendon changes that can be leveraged to develop strategies for tendinopathy prevention and treatment.

Orthopaedic researchers need new strategies for engaging underrepresented minority (URM) students. Our field has demonstrated noticeable gaps in racial, ethnic, and gender diversity, which inhibit our ability to innovate and combat the severe socioeconomic burden of musculoskeletal disorders. Towards this goal, we designed, implemented, and evaluated Learning on a Limb (LoaL), an orthopaedic research outreach module to teach URM high school students about orthopaedic research. During the 4-h module, students completed hands-on activities to learn how biomechanical testing, microcomputed tomography, cell culture, and histology are used in orthopaedic research. Over 3 years, we recruited 32 high school students from the Greater Philadelphia Area to participate in LoaL. Most participants identified as racial/ethnic or gender minorities in orthopaedic research. Using pre/post-tests, we found that students experienced significant learning gains of 51 percentage points from completing LoaL. In addition to teaching students about orthopaedic research, post-survey data demonstrated that participating in LoaL strongly influenced students' interest in orthopaedic research and scientific confidence. Several students acted on this interest by completing summer research experiences in the McKay Orthopaedic Research Laboratory at the University of Pennsylvania. LoaL instructors also benefited by having the opportunity to "pay it forward" to the next generation of students and build community within their department. Empowering institutions to host modules like LoaL would synergistically inspire URM high school students and strengthen community within orthopaedic departments to ultimately enhance orthopaedic research innovations.

BACKGROUND: Collagen XI is a fibril-forming collagen typically associated with type II collagen tissues but is also expressed in type I collagen-rich tendons, especially during development. We previously showed that tendon-targeted (Scx-Cre) Col11a1 knockout mice have smaller tendons in adulthood with aberrant fibril structure and impaired mechanical properties. However, the manifestation of this phenotype is not clearly understood. Therefore, our objective is to define the spatiotemporal roles of collagen XI in tendon structure-function during postnatal development. Given the high expression of collagen XI during embryonic development, we hypothesized that collagen XI knockout leads to the deposition of weakened extracellular matrix during early postnatal timepoints, disrupting the establishment of tendon structure and function. METHODS: Patellar and Achilles tendons from postnatal (P) days 0, 10, 20, and 30 tendon-targeted scleraxis-Cre heterozygous and homozygous Col11a1 knockout mice were evaluated for morphology, nuclear organization, fibril morphology, mechanical properties, and gene expression. RESULTS: At P0, there were no differences in tendon length or fibril diameter of either tendon. By P10, striking structural and functional differences emerged, with collagen XI deficiency resulting in increased tendon length, a heterogeneous and larger diameter population of fibrils, and inferior mechanical properties in both patellar and Achilles tendons. Differences increased in magnitude through P30, supporting our hypothesis that impaired structure-function during postnatal development may drive tendon lengthening and reduced mechanical properties. CONCLUSIONS: Though collagen XI is a quantitatively minor component of the tendon extracellular matrix, these results highlight the critical role of collagen XI in the acquisition of tendon structure-function.

Collagen XI is ubiquitous in tissues such as joint cartilage, cancellous bone, muscles, and tendons and is an important contributor during a crucial part in fibrillogenesis. The COL11A1 gene encodes one of three alpha chains of collagen XI. The present study elucidates the role of collagen XI in the establishment of mechanical properties of tendons and ligaments. We investigated the mechanical response of three tendons and one ligament tissues from wild type and a targeted mouse model null for collagen XI: Achilles tendon (ACH), the flexor digitorum longus tendon (FDL), the supraspinatus tendon (SST), and the anterior cruciate ligament (ACL). Area was substantially lower in Col11a1ΔTen/ΔTen ACH, FDL, and SST. Maximum load and maximum stress were significantly lower in Col11a1ΔTen/ΔTen ACH and FDL. Stiffness was lower in Col11a1ΔTen/ΔTen ACH, FDL, and SST. Modulus was reduced in Col11a1ΔTen/ΔTen FDL and SST (both insertion site and midsubstance). Collagen fiber distributions were more aligned under load in both wild type group and Col11a1ΔTen/ΔTen groups. Results also revealed that the effect of collagen XI knockout on collagen fiber realignment is tendon-dependent and location-dependent (insertion versus midsubstance). In summary, this study clearly shows that the regulatory role of collagen XI on tendon and ligament is tissue specific and that joint hypermobility in type II Stickler's Syndrome may in part be due to suboptimal mechanical response of the soft tissues surrounding joints.

Orthopaedic researchers need new strategies for engaging diverse students. Our field has demonstrated noticeable gaps in racial, ethnic, and gender diversity, which inhibit our ability to innovate and combat the severe socioeconomic burden of musculoskeletal disorders. Towards this goal, we designed, implemented, and evaluated Learning on a Limb, an orthopaedic research outreach module to teach diverse high school students about orthopaedic research. During the 4-hr module, students completed hands-on activities to learn how biomechanical testing, microcomputed tomography, cell culture, and histology are used in orthopaedic research. Over three years, we recruited 32 high school students from the Greater Philadelphia Area to participate in Learning on a Limb. Most participants identified as racial/ethnic or gender minorities in orthopaedic research. Using pre/post-tests, we found that students experienced significant learning gains of 51 percentage points from completing Learning on a Limb. In addition to teaching students about orthopaedic research, post-survey data demonstrated that participating in Learning on a Limb strongly influenced students' interest in orthopaedic research. Several students acted on this interest by completing summer research experiences in the McKay Orthopaedic Research Laboratory at the University of Pennsylvania. Learning on a Limb instructors also benefited by having the opportunity to "pay it forward" to the next generation of students and build community within their department. Empowering institutions to host modules like Learning on a Limb would synergistically inspire diverse high school students and strengthen community within orthopaedic departments to ultimately enhance orthopaedic research innovations.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both in vitro and in vivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in in vivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of in situ tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

Tendon injury and healing involve significant changes to tissue biology and composition. Current techniques often require animal sacrifice or tissue destruction, limiting assessment of dynamic changes in tendons, including treatment response, disease development, rupture risk, and healing progression. Changes in tendon composition, such as altered collagen content, can significantly impact tendon mechanics and function. Analyses of compositional changes typically require ex vivo techniques with animal sacrifice or destruction of the tissue. In vivo evaluation of tendons is critical for longitudinal assessment. We hypothesize that photoacoustic ultrasound detects differences in collagen concentration throughout healing. We utilized photoacoustic ultrasound, a hybrid imaging modality that combines ultrasound and laser-induced photoacoustic signals to create detailed and high-resolution images of tendons, to identify its endogenous collagen composition. We correlated the photoacoustic signal to picrosirius red staining. The results show that the photoacoustic ultrasound-estimated collagen content in tendons correlates well with picrosirius red staining. This study demonstrates that photoacoustic ultrasound can assess injury-induced compositional changes within tendons and is the first study to image these targets in rat Achilles tendon in vivo.

Abstract Collagen V (Col5) is a quantitatively minor component of collagen fibrils comprising tendon, however, plays a crucial role in regulation of development and dynamic healing processes. Clinically, patients with COL5a1 haploinsufficiency, known as classic Ehlers‐Danlos Syndrome ( c EDS), present with hyperextensible skin, joint instability and laxity, with females more likely to be affected. Previous studies in Col5‐deficient mice indicated that reduced Col5a1 expression leads to a reduction in stiffness, fibril deposition, and altered fibril structure. Additionally, Col5‐deficient male tendons demonstrated altered healing compared to wild‐type tendons, however female mice have not yet been studied utilizing this model. Along with clinical differences between sexes in c EDS patient populations, differences in hormone physiology may be a factor influencing tendon health. Therefore, the objective of this study was to utilize a Col5a1 +/ − female mouse model, to determine the effect of Col5 on tendon cell morphology, cell density, tissue composition, and mechanical properties throughout healing. We hypothesized that reduction in Col5 expression would result in an abnormal wound matrix post‐injury, resulting in reduced mechanical properties compared to normal tendons. Following patellar tendon surgery, mice were euthanized at 1, 3, and 6‐week post‐injury. Col5‐deficient tendons demonstrated altered and decreased healing compared to WT tendons. The lack of resolution in cellularity by 6‐week post‐injury in Col5‐deficient tendons influenced the decreased mechanical properties. Stiffness did not increase post‐injury in Col5‐deficient mice, and collagen fiber realignment was delayed during mechanical loading. Therefore, increased Col5a1 expression post‐injury is necessary to re‐establish matrix engagement and cellularity throughout tendon healing.

This study adapts a novel application of microdialysis to rat tendon models, offering a minimally invasive avenue for longitudinal tendon assessment. Successfully detecting changes in tendon healing after acute injury, it showcases strong correlations between extracellular fluid and dialysate concentrations. The results highlight the potential of microdialysis as a direct measure of tendon metabolism, biology, and inflammation, bypassing the need for animal euthanasia and tissue destruction.

The Journal of Orthopaedic Research, a publication of the Orthopaedic Research Society (ORS), is the forum for the rapid publication of high quality reports of new information on the full spectrum of orthopaedic research, including life sciences, engineering, translational, and clinical studies.

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.

Abstract Biglycan, a small leucine‐rich proteoglycan (SLRP), is involved in collagen fibrillogenesis and also acts as a signaling molecule. Although decorin has been considered as the primary SLRP in developing and maintaining tendon structure and mechanics, more recent work using inducible knockdown models suggests that biglycan is involved in tendon homeostasis. The purpose of the study was to determine the role of biglycan in tendon homeostasis to maintain mechanical and structural integrity in aged mice. Aged (485 days old) female Bgn +/+ control (wild type [WT], n = 16) and 16 bitransgenic conditional Bgn flox/flox mice (I‐ Bgn −/− , n = 16) with a tamoxifen‐inducible Cre (driven by ROSA) were utilized. After biglycan knockdown, the transgenic model demonstrated effective knockdown of the target gene without any compensation from other SLRPs or type I collagen. Patellar tendon cellularity was not modified after biglycan knockdown. However, biglycan knockdown had an impact on collagen fibrillogenesis with a higher percentage of small diameter fibrils (25–45 nm) and a lower percentage of medium size fibrils (150–165 nm) in I‐ Bgn −/− tendons. Biglycan knockdown also induced a reduction in the midsubstance modulus and maximum stress compared to WT. Stress relaxation was reduced at 4% strain in I‐ Bgn −/− tendons but no changes were observed in dynamic modulus and tan delta. As in mature tendons (120 days old), this study showed significant effects of biglycan knockdown on mechanical and structural properties of aged tendons only 30 days after knockdown. These data suggest that biglycan has a major role in maintaining homeostasis in aged tendon.