Aging is a primary risk factor for osteoarthritis (OA), yet the mechanisms that preserve extracellular matrix integrity in long-lived connective tissues remain poorly defined. The function of articular cartilage depends on maintaining extracellular matrix integrity over a lifetime of mechanical use. Here we identify failure of matrix stabilization as an initiating mechanism of age-associated OA. Cartilage-specific deletion of decorin in mature mice disrupted the superficial collagen II fibrillar network, reduced aggrecan retention, and impaired poroelastic fluid pressurization by 9 months of age, preceding substantial transcriptional changes in resident chondrocytes. With advancing age, these matrix defects culminated in cartilage erosion, fibrotic remodeling, and spontaneous OA by 18 months. Mechanistically, decorin attenuated force-induced collagen II fibril realignment and reinforced the superficial fibrillar network, preserving matrix architecture under sustained physiological loading. These findings establish decorin-mediated extracellular matrix stabilization as a critical determinant of cartilage longevity and support a matrix-first model of age-associated degeneration.

Cartilage extracellular matrix (ECM), a hydrated collagen II-aggrecan composite, undergoes dynamic turnover during both normal homeostasis and disease-associated remodeling. This study elucidates a crucial role for decorin in promoting the retention and stability of nascent aggrecan within this matrix. By applying bio-orthogonal click-labeling, we demonstrate that loss of decorin accelerates the release of nascent aggrecan under both physiological and inflammatory conditions, without affecting its preferential localization to the pericellular matrix. Conversely, supplementation with exogenous decorin mitigates inflammation-induced loss of nascent aggrecan, supporting its potential as a therapeutic target. At the molecular level, decorin exhibits strong binding affinity for aggrecan, and enhances aggrecan-aggrecan and aggrecan-collagen II interactions, reinforcing its direct role in integrating cartilage matrix constituents. Also, by binding to collagen II, decorin stiffens the collagen II fibril network, thereby strengthening the confinement effect that limits the diffusive loss of entrapped aggrecan. Notably, decorin does not alter chondrocyte transcriptomic profiles in vivo, emphasizing its primary role in maintaining matrix integrity through biophysical mechanisms rather than cell signaling. Together, these findings provide a mechanistic foundation for developing decorin-based biomaterials or gene therapies aimed at preserving or regenerating the cartilage matrix for improved outcomes in osteoarthritis. STATEMENT OF SIGNIFICANCE: Development of effective cartilage repair strategies is challenged by the limited understanding of molecular events that regulate the dynamic turnover and degenerative changes of cartilage extracellular matrix. This study shows that decorin, a small proteoglycan, promotes the retention and stability of nascent aggrecan within both normal and degenerative cartilage matrix by augmenting the integration between collagen II and aggrecan molecules and strengthening the collagen II fibril network. In turn, exogenous decorin mitigates the accelerated loss of nascent aggrecan instigated by inflammatory stimulation. Collectively, these findings establish decorin as a therapeutic target for preserving cartilage matrix integrity and improving osteoarthritis intervention.

The pericellular matrix (PCM) is the immediate microniche surrounding cells in various tissues, regulating matrix turnover, cell-matrix interactions, and disease. This study elucidates the structure-mechanical properties and mechanobiology of the PCM in fibrocartilage, using the murine meniscus as the model. The fibrocartilage PCM is comprised of thin, randomly oriented collagen fibrils that entrap proteoglycans, contrasting with the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). Compared to the ECM, the PCM exhibits lower modulus and greater isotropy, but has similar relative viscoelastic properties. In Col5a1+/- menisci, the reduction of collagen V results in thicker, more heterogeneous collagen fibrils, reduced modulus, loss of isotropy and faster viscoelastic relaxation in the PCM. Such altered PCM leads to impaired matrix-to-cell strain transmission, and in turn, disrupts mechanotransduction of meniscal cells, as illustrated by reduced calcium signaling activities and alters expression of matrix genes. In vitro, Col5a1+/- cells produce a weakened PCM with inferior properties and reduced protection of cells against tensile stretch. These findings highlight the PCM as a distinctive microstructure in fibrocartilage mechanobiology, underscoring a pivotal role of collagen V in PCM function. Targeting the PCM or its constituents offers potential for improving meniscus regeneration, osteoarthritis intervention and broader fibrocartilage-related therapies.

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.

The collagenous matrix of tendon provides mechanical integrity, allowing the tissue to withstand large tensile forces. While collagen I-containing fibrils provide the major backbone of the tendon matrix, interactions with other collagen types are critical for matrix formation and maintenance. Of these less abundant collagens, collagen V regulates fibril nucleation and lateral growth. In previous work, mouse models with reduced collagen V production recapitulated the musculoskeletal complications of Classic Ehlers-Danlos Syndrome in tendon development and healing, demonstrating altered structure and inferior mechanical properties. However, the roles of collagen V in homeostasis of mature, healthy tendon remain unknown. Therefore, this study evaluated the role of collagen V in maintaining tendon homeostasis using inducible knockdown of Col5a1in mature mice. After 30 days of reduced collagen V expression, patellar tendons demonstrated changes consistent with impaired matrix remodeling. Extracellular matrix and matrix remodeling genes were differentially expressed, and the distribution of fibril diameters was significantly altered with reduced expression of collagen V. The functional consequence of collagen V knockdown was demonstrated using mechanical testing, which revealed a reduction in failure properties, although sub-failure properties such as stiffness and modulus were not affected. Interestingly, differences between these results and prior studies on the impact of collagen V on development or healing suggest a distinct role for collagen V in maintaining the properties of mature, healthy tendon. In summary, this study demonstrated that collagen V is essential for homeostasis of adult tendons through maintenance of the collagenous matrix.

Understanding matrix molecular activities that regulate the postnatal growth and remodeling of the temporomandibular joint (TMJ) articular disc and condylar cartilage will enable the development of effective regenerative strategies targeting TMJ disorders. This study elucidated the distinct roles of type V collagen (collagen V) in regulating these two units. Studying the TMJ of young adult Col5a1+/- mice, we found that loss of collagen V resulted in substantial changes in the proliferation, clustering and density of progenitors in condylar cartilage, but did not have a major impact on disc cells that are more fibroblast-like. Although loss of collagen V led to thickened collagen fibrils with increased heterogeneity in the disc, there were no significant changes in local micromodulus, except for a reduction at the posterior end of the inferior side. Following the induction of aberrant occlusal loading by the unilateral anterior crossbite (UAC) procedure, both wild-type (WT) and Col5a1+/- condylar cartilage exhibited salient remodeling, and Col5a1+/- condyle developed more pronounced degeneration and tissue hypertrophy at the posterior end than the WT. In contrast, neither UAC nor collagen V deficiency induced marked changes in the morphology or biomechanical properties of the disc. Together, our findings highlight the distinct roles of collagen V in regulating these two units during postnatal growth and remodeling, emphasizing its more crucial role in condylar cartilage due to its impact on the highly mechanosensitive progenitors. These results provide the foundation for using collagen V to improve the regeneration of TMJ and the care of patients with TMJ disorders. Statement of significance Successful regeneration of the temporomandibular joint (TMJ) articular disc and condylar cartilage remains a significant challenge due to the limited understanding of matrix molecular activities that regulate the formation and remodeling of these tissues. This study demonstrates that collagen V plays distinct and critical roles in these processes. In condylar cartilage, collagen V is essential for regulating progenitor cell fate and maintaining matrix integrity. In the disc, collagen V also regulates fibril structure and local micromechanics, but has a limited impact on cell phenotype or its remodeling response. Our findings establish collagen V as a key component in maintaining the integrity of these two units, with a more crucial role in condylar cartilage due to its impact on progenitor cell activities. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Small leucine‐rich proteoglycans, such as decorin and biglycan, play pivotal roles in collagen fibrillogenesis during development, healing, and aging in tendon. Previous work has shown that the absence of decorin and biglycan affects fibril shape and mechanical properties during tendon healing. However, the roles of decorin and biglycan in the healing process of aged tendons are unclear. Therefore the objective of this study was to evaluate the differential roles of decorin and biglycan during healing of patellar tendon injury in aged mice. Aged (300 days old) female Dcn+/+/Bgn+/+ control (WT, n = 52), Dcnflox/flox (I‐Dcn−/−, n = 36), Bgnflox/flox (I‐Bgn−/−, n = 36), and compound Dcnflox/flox/Bgnflox/flox (I‐Dcn−/−/Bgn−/−, n = 36) mice with a tamoxifen‐inducible Cre were utilized. Targeted gene expression, collagen fibril diameter distributions, mechanical properties, and histological assays were employed to assess the effects of knockdown of decorin and/or biglycan at the time of injury. Knockdown resulted in alterations in fibril diameter distribution and scar area, but surprisingly did not lead to many differences in mechanical properties. Biglycan played a larger role in early healing stages, while decorin is more significant in later stages, particularly in scar remodeling. This study highlights some of the differential roles of biglycan and decorin in the regulation of fibril structure and scar area, as well as influencing gene expression during healing in aged mice.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Abstract The aim of this study was to demonstrate the long term safety and the efficacy of the self-gripping Parietex ProGrip™ mesh (Medtronic) used with the laparoscopic approach for inguinal hernia repair. The incidence of chronic pain, post-operative complications, patient satisfaction and hernia recurrence at follow-up after 5 years. Methods Data were collected prospectively using the “hernia-med” register in 1579 primary inguinal hernias. All patients included had undergone surgical repair for inguinal hernia by the laparoscopic transabdominal preperitoneal approach using Parietex ProGrip™ meshes performed in the same hernia referral center in Germany. Pre-, per- and post-operative data were collected, and a follow-up after one and five years was performed prospectively. Complications, pain scored on a 0–10 numeric rating scale (NRS), patient satisfaction and hernia recurrence were assessed. Results The only complications were minor and were post-operative: hematoma/seroma (3 cases), secondary hemorrhage through the trocar's site (5 cases), hematuria, emphysema in the inguinal regions (both sides) and hematoseroma in 3,2%. The 5 year follow-up rate was 76%, respectively 1205 patients. There were only 14 reports of hernia recurrence: 1.2% of the hernias. Most patients (95.9%) were satisfied or very satisfied with their hernia repair with only 1.2% reporting severe pain (NRS score 7–10) and 3.6% reported mild pain. Conclusion This study demonstrates that in experienced hands, inguinal hernia repair surgery performed by laparoscopic transabdominal preperitoneal hernioplasty using Parietex ProGrip™ self-gripping meshes is rapid, efficient and safe with low pain and low hernia recurrence rate. The promising short term results held true also in the long term follow-up.

Collagen XII, a fibril-associated collagen with interrupted triple helices (FACIT), influences fibrillogenesis in numerous tissues. In addition to this extracellular function, collagen XII also directly regulates cellular function. Collagen XII is widely expressed in connective tissues, particularly tendons, ligaments, and the periodontium and periosteum, where it is enriched in the pericellular regions. Mutations in the collagen XII gene cause myopathic Ehlers-Danlos syndrome (mEDS), an early-onset disease characterized by overlapping connective tissue abnormalities and muscle weakness. Patients with mEDS exhibit delayed motor development, muscle weakness, joint laxity, hypermobility, joint contractures, and abnormal wound healing. A mEDS mouse model was generated by deletion of the Col12a1 gene, resulting in skeletal and muscle abnormalities with disorganized tissue structures and altered mechanical properties. Extracellularly, collagen XII interacts with collagen I fibrils and regulates collagen fibril spacing and assembly during fibrillogenesis. Evidence for the binding of collagen XII to other EDS-related molecules (e.g., decorin and tenascin X) suggests that disruption of ECM molecular interactions is one of the causes of connective tissue pathology in mEDS. Collagen XII also has been shown to influence cell behavior, such as cell shape and cell-cell communication, by providing physical connection between adjacent cells during tissue development and regeneration. The focus of this review is on the functions of collagen XII in development, regeneration, and disease.

Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.

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.

Tendon is a vital musculoskeletal tissue that is prone to degeneration. Proper tendon maintenance requires complex interactions between extracellular matrix components that remain poorly understood. Collagen VI and biglycan are two matrix molecules that localize pericellularly within tendon and are critical regulators of tissue properties. While evidence suggests that collagen VI and biglycan interact within the tendon matrix, the relationship between the two molecules and its impact on tendon function remains unknown. We sought to elucidate potential coordinate roles of collagen VI and biglycan within tendon by defining tendon properties in knockout models of collagen VI, biglycan, or both molecules. We first demonstrated co-expression and co-localization of collagen VI and biglycan within the healing tendon, providing further evidence of cooperation between the two molecules during nascent tendon matrix formation. Deficiency in collagen VI and/or biglycan led to significant reductions in collagen fibril size and tendon mechanical properties. However, collagen VI-null tendons displayed larger reductions in fibril size and mechanics than seen in biglycan-null tendons. Interestingly, knockout of both molecules resulted in similar properties to collagen VI knockout alone. These results indicate distinct and non-additive roles for collagen VI and biglycan within tendon. This work provides better understanding of regulatory interactions between two critical tendon matrix molecules.

Collagen XII is a fibril-associated collagen with interrupted triple helices (FACIT). This non-fibrillar collagen is a homotrimer composed of three α1(XII) chains assembled into a collagenous molecule with a C terminal collagenous domain and a large N terminal non-collagenous domain. During tendon development and growth, collagen XII is broadly expressed throughout the extracellular matrix and enriched pericellularly around tenocytes. Tendons in a global Col12a1-/- knockout model demonstrated disrupted fibril and fiber structure and disordered tenocyte organization, highlighting the critical regulatory roles of collagen XII in determining tendon structure and function. However, muscle and bone also are affected in the collagen XII knockout model. Therefore, secondary effects on tendon due to involvement of bone and muscle may occur in the global knockout. The global knockout does not allow the definition of intrinsic mechanisms involving collagen XII in tendon versus extrinsic roles involving muscle and bone. To address this limitation, we created and characterized a conditional Col12a1-null mouse model to permit the spatial and temporal manipulation of Col12a1 expression. Collagen XII knockout was targeted to tendons by breeding conditional Col12a1flox/flox mice with Scleraxis-Cre (Scx-Cre) mice to yield a tendon-specific Col12a1-null mouse line, Col12a1Δten/Δten . Both mRNA and protein expression in Col12a1Δten/Δten mice decreased to near baseline levels in flexor digitorum longus tendons (FDL). Collagen XII immuno-localization revealed an absence of reactivity in the tendon proper, but there was reactivity in the cells of the surrounding peritenon. This supports a targeted knockout in tenocytes while peritenon cells from a non-tendon lineage were not targeted and retained collagen XII expression. The tendon-targeted, Col12a1Δten/Δten mice had significantly reduced forelimb grip strength, altered gait and a significant decrease in biomechanical properties. While the observed decrease in tendon modulus suggests that differences in tendon material properties in the absence of Col12a1 expression underlie the functional deficiencies. Together, these findings suggest an intrinsic role for collagen XII critical for development of a functional tendon.

Decorin and biglycan are two small leucine-rich proteoglycans (SLRPs) that regulate collagen fibrillogenesis and extracellular matrix assembly in tendon. The objective of this study was to determine the individual roles of these molecules in maintaining the structural and mechanical properties of tendon during homeostasis in mature mice. We hypothesized that knockdown of decorin in mature tendons would result in detrimental changes to tendon structure and mechanics while knockdown of biglycan would have a minor effect on these parameters. To achieve this objective, we created tamoxifen-inducible mouse knockdown models targeting decorin or biglycan inactivation. This enables the evaluation of the roles of these SLRPs in mature tendon without the abnormal tendon development caused by conventional knockout models. Contrary to our hypothesis, knockdown of decorin resulted in minor alterations to tendon structure and no changes to mechanics while knockdown of biglycan resulted in broad changes to tendon structure and mechanics. Specifically, knockdown of biglycan resulted in reduced insertion modulus, maximum stress, dynamic modulus, stress relaxation, and increased collagen fiber realignment during loading. Knockdown of decorin and biglycan produced similar changes to tendon microstructure by increasing the collagen fibril diameter relative to wild-type controls. Biglycan knockdown also decreased the cell nuclear aspect ratio, indicating a more spindle-like nuclear shape. Overall, the extensive changes to tendon structure and mechanics after knockdown of biglycan, but not decorin, provides evidence that biglycan plays a major role in the maintenance of tendon structure and mechanics in mature mice during homeostasis.

Decorin and biglycan are two major small leucine-rich proteoglycans (SLRPs) present in the tendon extracellular matrix that facilitate collagen fibrillogenesis, tissue turnover, and cell signal transduction. Previously, we demonstrated that knockout of decorin prevented the decline of tendon mechanical properties that are associated with aging. The objective of this study was to determine the effects of decorin and biglycan knockdown on tendon structure and mechanics in aged tendons using tamoxifen-inducible knockdown models. We hypothesized that the knockdown of decorin and compound knockdown of decorin and biglycan would prevent age-related declines in tendon mechanics and structure compared to biglycan knockdown and wild-type controls, and that these changes would be exacerbated as the tendons progress towards geriatric ages. To achieve this objective, we created tamoxifen-inducible mouse knockdown models to target decorin and biglycan gene inactivation without the abnormal tendon development associated with traditional knockout models. Knockdown of decorin led to increased midsubstance modulus and decreased stress relaxation in aged tendons. However, these changes were not sustained in the geriatric tendons. Knockdown in biglycan led to no changes in mechanics in the aged or geriatric tendons. Contrary to our hypothesis, the compound decorin/biglycan knockdown tendons did not resemble the decorin knockdown tendons, but resulted in increased viscoelastic properties in the aged and geriatric tendons. Structurally, knockdown of SLRPs, except for the 570d I-Dcn-/-/Bgn-/- group, resulted in alterations to the collagen fibril diameter relative to wild-type controls. Overall, this study identified the differential roles of decorin and biglycan throughout tendon aging in the maintenance of tendon structural and mechanical properties and revealed that the compound decorin and biglycan knockdown phenotype did not resemble the single gene decorin or biglycan models and was detrimental to tendon properties throughout aging.

IntroductionLes lésions tendineuses sont une problématique médicale fréquente. L’âge étant un facteur de risque de ces lésions, la compréhension des mécanismes de cicatrisation tendineuse au cours du vieillissement est une étape importante dans le développement de nouvelles stratégies thérapeutiques dans cette population. Les interactions entre les small leucine-rich proteoglycanes (SLRP) de type I, le biglycane (Bgn) et la décorine (Dcn), et les autres molécules de la matrice extracellulaire (MEC) sont essentielles à la régulation de l’assemblage du collagène, ainsi qu’à l’établissement des propriétés mécaniques du tendon. L’objectif de l’étude était de déterminer les rôles spécifiques de la Dcn et du Bgn dans les phases précoce et tardive de la cicatrisation tendineuse en utilisant des souris âgées porteuses d’un knock-out (KO) inductible du gène d’intérêt.Matériels et méthodesDes souris femelles wildtype (WT), Dcnflox/flox (I-Dcn-/-), Bgnflox/flox (I-Bgn-/-), et Dcnflox/flox/Bgnflox/flox (I-Dcn-/-/Bgn-/-) (n=48/groupe) porteuses d’un Cre inductible au tamoxifène (TM) ont subi une lésion patellaire bilatérale (punch de 0,75mm) [1] à l’âge de 300 jours. Le KO de la Dcn, du Bgn ou des 2 a été induit 5 jours après la lésion (phase inflammatoire tardive) ou 21 jours après (début de la phase de remodelage). Aux semaines 3 et 6 (S3 et S6) en cas de KO à j5 ou à S6 en cas de KO à j21, les analyses suivantes ont été effectuées : mesure de la zone cicatricielle (coloration au Bleu Toluidine), mesure du diamètre des fibres de collagène au sein de la lésion (microscopie électronique à transmission), étude de la récupération des propriétés élastiques et viscoélastiques après la lésion (test mécanique avec un pré-conditionnement, 3 phases d’élongation–relaxation et une traction finale jusqu’à la rupture), et étude de l’expression génique des différents collagènes, constituants de la MEC ou facteurs de remodelage (fluidigm dynamic array).RésultatsÀ S3, la cicatrisation était retardée dans le groupe I-Bgn-/- (p=0,02, Mann–Whitney) alors qu’aucune différence n’était constatée à S6 entre les groupes que le KO ait été précoce ou tardif. Les diamètres des fibres de collagène étaient distribués de manière différente entre les génotypes, les tendons des groupes I-Dcn-/- et I-Bgn-/- présentant une plus grande proportion de fibres de petit diamètre (30–40nm) (p<0,0001, Kolmogorov–Smirnov). Malgré la présence d’un tissu cicatriciel plus étendu dans le groupe I-Bgn-/- et un impact des différents KO sur la fibrillogenèse, les propriétés élastiques du tendon (stress maximal et module d’élasticité) n’étaient pas altérées par rapport au groupe WT à S3. Cependant, après induction du KO à j5, une réduction du pourcentage de relaxation et une augmentation du module d’élasticité dynamique à S6 dans le groupe I-Dcn-/- (p=0,001, one-way ANOVA) suggéraient une meilleure récupération des propriétés viscoélastiques. Dans ce même groupe, l’expression de BMP-1 et d’ELASTINE était significativement augmentée à S3 (p=0,04 et p=0,006, one-way ANOVA).ConclusionL’objectif de l’étude était de mieux comprendre le rôle temporel des SLRP de classe I dans la cicatrisation tendineuse. Bien qu’elles partagent le même site de fixation sur le collagène de type I, l’étude a montré un rôle distinct de la Dcn et du Bgn dans la cicatrisation tendineuse ainsi qu’un effet plus marqué de leur absence si l’inactivation était précoce, au cours de la phase inflammatoire.

Tissue turnover requires activation and lineage commitment of tissue-resident stem cells (SCs). These processes are impacted by ageing, but the mechanisms remain unclear. Here, we addressed the mechanisms of ageing in murine hair follicle SCs (HFSCs) and observed a widespread reduction in chromatin accessibility in aged HFSCs, particularly at key self-renewal and differentiation genes, characterized by bivalent promoters occupied by active and repressive chromatin marks. Consistent with this, aged HFSCs showed reduced ability to activate bivalent genes for efficient self-renewal and differentiation. These defects were niche dependent as the transplantation of aged HFSCs into young recipients or synthetic niches restored SC functions. Mechanistically, the aged HFSC niche displayed widespread alterations in extracellular matrix composition and mechanics, resulting in mechanical stress and concomitant transcriptional repression to silence promoters. As a consequence, increasing basement membrane stiffness recapitulated age-related SC changes. These data identify niche mechanics as a central regulator of chromatin state, which, when altered, leads to age-dependent SC exhaustion.

Carlson JA, Sun M, Adams SM, Weiss SN, Birk DE, Soslowsky LJ McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA, University of South Florida, Tampa, FL Disclosures: Carlson JA (N), Sun M (N), Adams SM (N), Weiss SN (N), Birk DE (N), Soslowsky LJ (N) INTRODUCTION: Patients with Classic Ehlers-Danlos syndrome (cEDS), a disorder characterized most commonly by COL5A1 haploinsufficiency, suffer from tissue hyperelasticity, skin hyperextensibility, tendon/ligament fragility and abnormal wound healing. Collagen V (ColV) haploinsufficiency leads to abnormal tissue development and altered collagen assembly, and mechanical loading of the mouse patellar tendon shows a delay in healing and alterations in stiffness and dynamic modulus post-injury. Furthermore, human studies have shown that females have decreased collagen synthesis and altered gene expression during repair, likely influencing the healing potential of cEDS tendons. Therefore, the objective of this study was to determine the effect of ColV deficiency in female mice on wound matrix formation and resultant structure-function relationships when mechanical load is applied post-injury. We hypothesized that ColV deficiency will have effects post-injury, resulting in increased fibril diameter and cellularity, decreased mechanical properties and leading to a delayed healing response when compared to wild-type tendons. METHODS: Adult female wild-type (WT) C57/BL6 and heterozygous Col5a1 mice, a model for cEDS, at 120 days of age (n=84) were used (IACUC approved). Mice were randomly divided into uninjured and injured groups, with injured mice undergoing bilateral patellar tendon injury surgery as described. Injured mice were sacrificed early in the proliferative phase at 1-week (1w), early in the remodeling phase at 3-weeks (3w) or later in remodeling at 6 weeks (6w), and uninjured age-matched mice were also sacrificed. Uninjured and injured patellar tendons of both genotypes were assessed. Gene Expression: Realtime PCR was done as described. Each sample (n= 4) was run in duplicate and data was analyzed using StepOne software v2.0. Transmission Electron Microscopy (TEM): Samples for TEM analysis of fibril structure (n=4) were fixed in situ and processed as described. Mechanics: The patella-patellar tendon-tibia complexes were dissected and prepared for mechanical testing (n=12). Tendons were subjected to a viscoelastic testing protocol containing 3 stress relaxations followed by frequency sweeps, culminating in a ramp-to-failure. Dynamic collagen fiber realignment was quantified using crosspolarization imaging during the ramp-to-failure. Histology: Histological sections of the patellar tendon-bone complex (n=4) were prepared using standard techniques. Cellularity was calculated using a standard grading scale. Statistics: Two-way ANOVAs with post-hoc Bonferroni tests were used to assess the effects of genotype and time on gene expression. Two-way repeated measures ANOVAs with post-hoc Bonferroni tests were used to assess the changes in realignment for increasing strain levels. Kruskal-Wallis non-parametric one-way ANOVA followed by post-hoc Dunn’s test for multiple comparisons were used for histologic data. Significance was set at p≤0.05. RESULTS: Col5a1 expression was significantly increased in WT tendons at 1w and 3w post-injury (PI) compared to uninjured controls. However, no significant changes in Col5a1 expression were seen following injury in Col5a1 tendons (Fig. 1). Genotypic differences in Col5a1 expression were seen at 1w and 3w PI (Fig. 1). Fibrils from the mid-substance of WT and Col5a1 tendons are shown in Figure 2A, with injured tendons having a dominant population of smaller diameter fibrils. Uninjured WT and Col5a1 distributions were comparable (data not shown), however, distinctly different distributions for WT and Col5a1 fibrils PI were seen, with Col5a1 fibrils being larger and more broadly distributed (Fig. 2B). Further, WT and Col5a1 tendons realigned through 5% strain, with Col5a1 tendons continuing to realign through 6% strain (Fig. 3A). Lastly, significant differences in cellularity (Fig. 3B) were seen between uninjured and both 1w and 3w samples in both genotypes, and between 1w and both 3 and 6w samples, with no difference between genotypes at any time-point. Col5a1 tendons had a significant increase in cellularity persisting to 6w PI when compared to uninjured tendons (Fig. 3B). DISCUSSION: ColV plays a key role in fibrillogenesis, matrix remodeling and response to injury, affecting the structure and function of healing tendon. The lack of an increase in Col5a1 expression 1w PI in Col5a1 tendons would affect all stages of later healing and indicates a reduction in regulation of fibrillogenesis throughout healing. WT Col5a1 expression returns to uninjured and Col5a1 levels by 6w PI injury, indicating that the early increase affects fibril diameter, mechanical properties and cellularity throughout healing. Without the initial increase in ColV following injury, fibrillogenesis is less regulated, resulting in a broader distribution of fibril diameter and a shift to larger fibrils of Col5a1 tendons PI, which explains the delayed realignment and is supported by previous work. Following injury, a shift towards smaller fibrils was expected as these are new fibrils from collagen secreting myofibroblasts to fill the injury void. Additionally, increased cellularity in Col5a1 tendons would alter the matrix alignment and architecture, weakening the tissue and affecting mechanical properties. The persistence of increased cellularity in Col5a1 tendons to 6w PI is consistent with viscoelastic and fatigue data, which indicates a delayed healing response in Col5a1 tendons. Qualitative comparisons to male data show Col5a1 fibril distribution was similar to WT and cellularity returned to uninjured levels by 6w PI, unlike the data shown, indicating that sex affects outcomes of reduction in ColV. Future directions may include later healing time points to better understand the extent of the delayed healing. SIGNIFICANCE: This study indicates that the lack of an early increase in Col5a1 expression PI in Col5a1 tendons influences matrix architecture, alignment, and cellularity throughout tendon healing, demonstrating altered and delayed healing compared to WT tendons, REFERENCES: [1] Steinmann et al. Conn Tissue, Heritable Disorders. 2002. [2] Wenstrup et al. J Biol Chem. 2006. [3] Carlson et al. ORS 2020. [4] Carlson et al. ORS 2019. [5] Ainsworth et al. CORR. 1993. [6] Hart et al. CORR. 1998. [7] Beason et al. J. Biomech, 2012. [8] Sun et al. Am J Pathol. 2015. [9] Dunkman et al. Matrix Biol. 2013. [10] Kjaer et al. J. Anat. 2006. [11] Robinson et al. Matrix Biol. 2017. [12] Johnston et al. JOR. 2018. ACKNOWLEDGEMENTS: This study was supported by NIH/NIAMS AR065995 and the Penn Center for Musculoskeletal Disorders (AR069619). Figure 2: (A) TEM Images and (B) Fibril Distribution. Injured tendons shifted to smaller fibrils from uninjured. Col5a1 tendons had a broader distribution with larger fibrils than WT tendons PI.