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Compared to articular cartilage, the biomechanical properties of costal cartilage have not yet been extensively explored. The research presented addresses this problem by studying for the first time the anisotropic elastic behavior of human costal cartilage. Samples were taken from 12 male and female cadavers and unconfined compression and indentation tests were performed in mediolateral and dorsoventral direction to determine Young’s Moduli EC for compression and Ei5%, Ei10% and Eimax at 5%, 10% and maximum strain for indentation. Furthermore, the crack direction of the unconfined compression samples was determined and histological samples of the cartilage tissue were examined with the picrosirius-polarization staining method. The tests revealed mean Young’s Moduli of EC = 32.9 ± 17.9 MPa (N = 10), Ei5% = 11.1 ± 5.6 MPa (N = 12), Ei10% = 13.3 ± 6.3 MPa (N = 12) and Eimax = 14.6 ± 6.6 MPa (N = 12). We found that the Young’s Moduli in the indentation test are clearly anisotropic with significant higher results in the mediolateral direction (all P = 0.002). In addition, a dependence of the crack direction of the compressed specimens on the load orientation was observed. Those findings were supported by the orientation of the structure of the collagen fibers determined in the histological examination. Also, a significant age-related elastic behavior of human costal cartilage could be shown with the unconfined compression test (P = 0.009) and the indentation test (P = 0.004), but no sex effect could be detected. Those results are helpful in the field of autologous grafts for rhinoplastic surgery and for the refinement of material parameters in Finite Element models e.g., for accident analyses with traumatic impact on the thorax.

This work deals with the examination of tool marks in human cartilage. We compared the effectiveness of several cleaning methods on cut marks in porcine cartilage. The method cleaning by multiple casts achieved the significantly highest scores (P = 0.02). Furthermore, we examined the grain-like elevations (dots) located on casts of cut cartilage. The results of this study suggest that the casting material forms these dots when penetrating cartilage cavities, which are areas where the strong collagen fibres leave space for the chondrocytes. We performed fixation experiments to avoid this, without success. In addition, 31 casting materials were compared regarding contrast under light-microscope and 3D tool marks scanner. Under the light-microscope, brown materials achieved significantly higher values than grey (P = 0.02) or black (P = 0.00) whereas under the 3D scanner, black materials reached higher contrast values than grey (P = 0.04) or brown (P = 0.047). To compare the accuracy and reproducibility of 6 test materials for cartilage, we used 10 knives to create cut marks that were subsequently scanned. During the alignment of the individual signals of each mark, the cross-correlation coefficients (Xmax) and lags (LXmax) were calculated. The signals of the marks in agarose were aligned with significantly fewer lags and achieved significantly higher cross-correlation coefficients compared to all tested materials (both P = 0.00). Moreover, we determined the cross-correlation coefficients (XC) for known-matches (KM) per material. Agarose achieved significantly higher values than AccuTrans®, Clear Ballistics™, and gelatine (all P = 0.00). The results of this work provide valuable insights for the forensic investigation of marks in human costal cartilage.

Summary Objective The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. Methods A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. Results The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. Conclusions The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFβ, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.

Summary Objective The vascular invasion of cartilage is an essential process in the endochondral ossification of long bones. In contrast, vascularization of articular cartilage constitutes a pathological mechanism in the development of osteoarthritis. Polymorphisms of Col9a1 have been described as risk factors for hip osteoarthritis (OA) and the loss of collagen IX is known to lead to premature OA of the hip joint in mice but the underlying mechanism is so far unknown. Design To understand the contribution of collagen IX to OA development in the hip joint, we analyzed the early development of murine Col9a1−/− femoral heads between newborn stage and 16 weeks of age. Results We found significantly accelerated ossification of the femoral heads in the absence of collagen IX as well as premature vascular and osteoclast invasion, even though hypertrophic differentiation was delayed. The loss of collagen IX led to anatomically altered femoral heads lacking the epiphyseal tubercle. Interestingly, this region was found to contain highest levels of the antiangiogenic protein thrombospondin-1 (TSP-1). Hence, TSP-1 levels were strongly reduced in the Col9a1−/− femoral heads. In addition, antiangiogenic matrilin-1 was found to be decreased, while proangiogenic active MMP-9 levels were increased in the collagen IX deficient mice compared to wildtype controls. Conclusion We conclude that collagen IX protects against premature vascularization and cartilage to bone transition in femoral heads by increasing the levels of antiangiogenic TSP-1 and matrilin-1 and decreasing the levels of proangiogenic active MMP-9.

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived upon C-terminal cleavage of fibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the mechanisms controlling the bioavailability of asprosin in tissues. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. Our analyses show that asprosin already multimerizes intracellularly, but that stable multimerization via covalent bonds is facilitated by transglutaminase activity. Further, asprosin fiber formation requires an intact fibrillin-1 fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in an oligomerized form which may regulate its cellular bioavailability in tissues.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1(−/−) femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Collagen XII, belonging to the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The trimeric molecule consists of three N-terminal noncollagenous NC3 domains joined by disulfide bonds followed by a short interrupted collagen triple helix at the C-terminus. Collagen XII is expressed widely in the musculoskeletal system and mutations in the COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome, which is associated with skeletal abnormalities and muscle weakness. Our study defines the role of collagen XII in patella development using the Col12a1-/- mouse model. Deficiency in Col12a1 expression causes malformed facies patellaris femoris grooves at an early stage, which leads to patella subluxation and growth retardation. Due to the patella subluxation, more muscle fibers with centralized nuclei occur in the quadriceps than in the gastrocnemius muscles indicating a local effect. To further understand the role of collagen XII in the skeletal tissues single cell RNAseq (scRNA-seq) was performed. Comparison of the gene expression in the tenocyte cell sub-population of wild type and Col12a1-/- mice showed that several matrix genes are altered. Finally, we reinvestigated collagen XII deficient patients and observed a patella instability.

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal–epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1−/− mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1−/− mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Knee osteoarthritis (OA) involves several structures and molecules in the joint, which interact in a pathophysiological process. One of these molecules is the cartilage oligomeric matrix protein (COMP). Elevated COMP levels in the synovial fluid as well as in the serum have been described in OA patients. However, this has not been described in the infrapatellar fat pad (IPFP) tissue before. In this prospective trial, we collected 14 IPFPs from patients with high-grade OA (mean age 63.8 ± 17.6 years) who underwent total knee replacement (OA group) and from 11 healthy patients (mean age 33.7 ± 14.8 years) who underwent anterior cruciate ligament reconstruction (control group). The presence of macrophages (CD68 and CD206) and proinflammatory cytokines (interleukin 1β [IL-1β] and IL-6) was analyzed. Histological and immunohistological examinations as well as immunoblotting analysis for COMP, leptin, and matrix-metalloproteinase-3 were performed. The IPFPs of both the OA and control group consisted of adipose tissue and fibrous tissue, and the fibrous tissue showed higher score values than the adipose tissue for COMP staining (intensity as well as stained area) in both groups. Although COMP could be detected in most samples, leptin expression was found only in single specimens. COMP could be detected mostly in the fibrous tissue portion of the IPFP. We speculate that it is involved in a remodeling process taking place in the IPFP during OA. Presence of leptin was irregular in immunohistology, and the control group showed higher scores in case of presence. Interestingly, immunoblotting could detect leptin in all analyzed samples. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:747-758, 2020.

Children with mitochondrial diseases often present with slow growth and short stature, but the underlying mechanism remains unclear. In this study, Holzer et al. provide in vivo evidence that mitochondrial respiratory chain dysfunction induces cartilage degeneration coincident with altered metabolism, impaired extracellular matrix formation, and cell death at the cartilage–bone junction., In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage–bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca2+-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged, however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca2+-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.

The purpose of the study was to investigate the effect of an increase in mechanical knee joint loading during running on the serum COMP level. On two different test days, 20 healthy men ran with knee orthoses for 30 min on a treadmill (v = 2.2 m/s). On day 1, the orthoses were passive, whereas on day 2 they were pneumatically driven (active) and thus increased the external knee flexion moments (+30.9 Nm) during stance phase. Lower-limb mechanics and serum COMP levels (baseline; 0, 0.5, 1, 2 h post running) were analyzed. COMP levels increased immediately after running with passive (+35%; pre: 7.5 U/l, 95%CI: 6.4, 8.7, post: 9.8 U/l, 95%CI: 8.8, 10.8, p < 0.001) and active orthoses (+45%; pre: 7.6 U/l; 95%CI: 6.4, 8.8, post: 10.3 U/l, 95%CI: 9.2, 11.5, p < 0.001), but they did not differ between interventions. While running with active orthoses, greater ankle dorsiflexion angles, knee flexion angles, and moments occurred (p < 0.05). Comparing both interventions, the Δ COMP pre-post, meaning the difference (Δ) between running with active and passive orthoses in pre to post COMP level change (=level after (post) running minus level before (pre) running), correlated negatively with Δ COMP baseline (difference between the baseline COMP level before running with active and passive orthoses, r = -0.616; p = 0.004), and with a positive tendence with the Δ maximum knee flexion (r = 0.388; p = 0.091). Therefore, changes in COMP concentration after physical activity seem to be highly influenced by the COMP baseline level. In addition, correlation analysis indicates that modifications in knee joint kinematics have a greater effect on cartilage metabolism than an increase in joint moments. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1937-1946, 2018.

In order to maintain their native properties, cryopreserved tendons are usually used in biomechanical research and in transplantation of allogenic tendon grafts. The use of different study protocols leads to controversy in literature and thus complicates the evaluation of the current literature. The aim of this study consisted in examining the influence of different freezing and thawing temperatures on the mechanical properties of tendons. 60 porcine tendons were frozen at either -80°C or -20°C for 7days and thawed at room or body temperature for 240 or 30min, respectively. A subgroup of ten tendons was quick-frozen with liquid nitrogen (-196°C) for 2s before cryopreservation. Biomechanical testing was performed with a material testing machine and included creep, cyclic and load-to-failure tests. The results showed that freezing leads to a reduced creep strain after constant loading and to an increased secant modulus. Freezing temperature of -80°C increased the secant modulus and decreased the strain at maximum stress, whereas thawing at room temperature reduced the maximum stress, the strain at initial tendon failure and the Young's Modulus. Quick-freezing led to increased creep strain after constant loading, increased strain at initial failure in the load-to-failure test, and decreased strain at maximum stress. When cryopreserving, tendons for scientific or medical reasons, freezing temperature of -20°C and thawing temperature of 37.5°C are recommended to maintain the native properties of tendons. A treatment with liquid nitrogen in the sterilization process of tendon allografts is inadvisable because it alters the tendon properties negatively.

Human skeletal muscle atrophies in response to reduced mechanical loading. The study aimed to define the muscle‐specific anatomical cross‐sectional area (ACSA) location that is most sensitive to immobilization and to investigate the effectiveness of resistive vibration exercise, alone, or combined with protein supplementation, against muscle atrophy. Eleven individuals (35.2 ± 8.1 years, 22.6 ± 1.7 kg/m2) were analysed in trial 1, and eight subjects (37.1 ± 6.7 years, 23.0 ± 1.8 kg/m2) in trial 2. Subject received control (CON), vibration training (RVE) (25 minutes à 2 sessions/wk), or RVE + nutrition (NEX) interventions (whey protein + potassium/bicarbonate). Magnetic resonance images (MRI) of both thighs were acquired before, during, and 6 days after 21‐days of bed rest to determine ACSAs in regions of interest (ROI) between 10% and 90% of the proximal end of the M. rectus femoris tendon and the end of the femoral neck. Muscle atrophy was highest at the location of their greatest ACSA. ACSA of all muscles at 70% of ROI was reduced after bed rest during CON (range −4.7% to −14.8%) and remained reduced after 6 days of recovery for the majority of muscles (range −3.4% to −8.3%) except for M. vastus lateralis, M. vastus medialis, M. sartorius, and knee flexors. Applied countermeasures had no effect. In conclusion, thigh muscle atrophy can be monitored using ACSA and RVE or NEX was not sufficient to prevent muscle atrophy during 21‐days bed rest.

Biomarkers of cartilage metabolism are sensitive to changes in the biological and mechanical environment and can indicate early changes in cartilage homeostasis. The purpose of this study was to determine if a daily locomotion replacement program can serve as a countermeasure for changes in cartilage biomarker serum concentration caused by immobilization. Ten healthy male subjects (mean ± 1 standard deviation; age: 29.4 ± 5.9 years; body mass: 77.7 ± 4.1 kg) participated in the crossover 5-day bed rest study with three interventions: control (CON), standing (STA), and locomotion replacement training (LRT). Serum samples were taken before, during, and after bed rest. Biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays. Cartilage oligomeric matrix protein (COMP) levels after 24 hours of bed rest decreased independently of the intervention (-16.8% to -9.8%) and continued to decrease until 72 hours of bed rest (minimum, -23.2% to -20.6%). LRT and STA did not affect COMP during bed rests (P = .056) but there was a strong tendency for a slower decrease with LRT (-9.4%) and STA (-11.7%) compared with CON (-16.8%). MMP-3 levels decreased within the first 24 hours of bed rest (CON: -22.3%; STA: -14.7%; LRT: -17%) without intervention effect. Both COMP and MMP-3 levels recovered to baseline levels during the 6-day recovery period. MMP-1, MMP-9, and TNF-α levels were not affected by immobilization or intervention. COMP and MMP-3 are mechano-sensitive cartilage biomarkers affected by immobilization, and simple interventions such as standing upright or LRT during bed rest cannot prevent these changes. Clinical significance: simple locomotion interventions cannot prevent cartilage biomarker change during bed rest.

The aim of the study was to examine the effect of mechanical knee joint loading on the fragmentation pattern of serum cartilage oligomeric matrix protein (COMP). Ten healthy men ran with knee orthoses that were passive or active (+30.9 N·m external flexion moments) on a treadmill (30 minute; v = 2.2 m/s). Lower-limb mechanics, serum COMP levels, and fragmentation patterns (baseline; 0, 0.5, 1, 2 hours postrunning) were analyzed. Running with active orthoses enhanced knee flexion moments, ankle dorsiflexion, and knee flexion angles (P

Gelenkknorpel ist essenziell fur die uneingeschrankte Funktion des muskuloskelettalen Systems. Da es sich um ein bradytrophes Gewebe handelt, ist mechanische Belastung fur die Knorpelgesundheit von hoher Bedeutung. Der Einfluss von reduzierter mechanischer Belastung, wie zum Beispiel durch Immobilisation oder Mikrogravitation, auf den hyalinen Knorpel ist jedoch noch weitgehend unbekannt. Die niedrige Stoffwechselaktivitat von Knorpelgewebe macht lange Studiendauern erforderlich, um Effekte von mechanischen Interventionen messen zu konnen. Aufgrund der besonderen Gewebeeigenschaften ist die Regenerationskapazitat von Knorpel sehr gering, sobald degenerative Prozesse eingesetzt haben. Daher ist die Durchfuhrung von Immobilisationsexperimenten in gesunden Personen schwierig. Einige wenige Studien wurden in der Vergangenheit in Patientengruppen durchgefuhrt, die aufgrund von Verletzungen immobilisiert waren. Die Ergebnisse weisen darauf hin, dass Immobilisation zu degenerativen Prozessen im Gelenkknorpel fuhren kann. Forschung im Weltraumkontext bietet die einzigartige Moglichkeit die Anpassung des muskuloskelettalen Systems an Immobilisation durch Bettruhe oder Mikrogravitation in gesunden Probanden zu untersuchen. Bisher stehen wenige Daten zur Knorpelgesundheit unter diesen Bedingungen zur Verfugung, aber die bisherigen Ergebnisse zeigen, dass es wichtig ist, das Risiko einer Knorpeldegeneration wahrend langerer Raumfahrtmissionen genauer zu untersuchen. Ergebnisse aus diesem Forschungsfeld wurden das Verstandnis der Rolle von mechanischer Belastung fur die Knorpelgesundheit masgeblich erweitern und wichtige Informationen fur Patienten liefern, die aufgrund von Verletzungen und Erkrankungen einer langen Immobilisation unterliegen.

SMPD3 deficiency in the neutral sphingomyelinase (Smpd3-/-) mouse results in a novel form of juvenile dwarfism, suggesting smpd3 is a polygenetic determinant of body height. SMPD3 controls homeostasis of the sphingomyelin cycle in the Golgi compartment, essential for membrane remodeling, initiating multiform vesicle formation and transport in the Golgi secretory pathway. Using the unbiased Smpd3-/- genetic model, this study shows that the perturbed Golgi secretory pathway of chondrocytes of the epiphyseal growth zone leads to dysproteostasis, skeletal growth inhibition, malformation, and chondrodysplasia, but showed unimpaired mineralization in primary and secondary enchondral ossification centers. This has been elaborated by biochemical analyses and immunohistochemistry of long bones of Smpd3-/- mice. A more precise definition of the microarchitecture and three-dimensional structure of the bone was shown by peripheral quantitative computed tomography, high-resolution microcomputed tomography, and less precisely by dual-energy X-ray absorptiometry for osteodensitometry. Ablation of the Smpd3 locus as part of a 980-kb deletion on chromosome 8 in the fro/fro mutant, generated by chemical mutagenesis, is held responsible for skeletal hypomineralization, osteoporosis, and multiple fractures of long bones, which are hallmarks of human osteogenesis imperfecta. The phenotype of the genetically unbiased Smpd3-/- mouse, described here, precludes the proposed role of Smpd3 as a candidate gene of human osteogenesis imperfecta, but suggests SMPD3 deficiency as the pathogenetic basis of a novel form of chondrodysplasia.

Over 200 million people suffer from osteoporosis worldwide, one third of which will develop osteoporotic bone fractures. Unfortunately, no effective cure exists. Mutations in plastin 3 (PLS3), an F-actin binding and bundling protein, cause X-linked primary osteoporosis in men and predisposition to osteoporosis in postmenopausal women. Moreover, the strongest association so far for osteoporosis in elderly women after menopause was connected to a rare SNP in PLS3, indicating a possible role of PLS3 in complex osteoporosis as well. Interestingly, 5% of the general population are overexpressing PLS3, with yet unknown consequences. Here, we studied ubiquitous Pls3 knockout and PLS3 overexpression in mice and demonstrate that both conditions influence bone remodeling and structure: while Pls3 knockout mice exhibit osteoporosis, PLS3 overexpressing mice show thickening of cortical bone and increased bone strength. We show that unbalanced PLS3 levels affect osteoclast development and function, by misregulating the NF kappa B pathway. We found upregulation of RELA (NF kappa B subunit p65) in PLS3 overexpressing mice-known to stimulate osteoclastogenesis-but strikingly reduced osteoclast resorption. We identify NF kappa B repressing factor (NKRF) as a novel PLS3 interactor, which increasingly translocates to the nucleus when PLS3 is overexpressed. We show that NKRF binds to the NF kappa B downstream target and master regulator of osteoclastogenesis nuclear factor of activated T cells 1 (Nfatc1), thereby reducing its transcription and suppressing osteoclast function. We found the opposite in Pls3 knockout osteoclasts, where decreased nuclear NKRF augmented Nfatc1 transcription, causing osteoporosis. Regulation of osteoclastogenesis and bone remodeling via the PLS3-NKRF-NF kappa B-NFATC1 axis unveils a novel possibility to counteract osteoporosis.

cope The aim of our study was to investigate dose-dependent effects of isoflavone (ISO) exposure during adolescence on the androgen sensitivity of various physiological end points in male Wistar rats. Methods and results During embryogenesis and adolescence, rats were exposed to an ISO-depleted diet (IDD) or one of two diets enriched with different concentrations of a soy-based ISO extract causing plasma concentrations observed averagely (ISO-rich diet [IRD]low) and maximally (IRDhigh) in Asian men. Most of the rats were orchiectomized at postnatal day (PND) 81 and were treated with testosterone propionate (TP) or vehicle from PND 89 to 99. In intact rats (PND 99) body weight, food intake, and fat mass were not influenced by ISO, but serum triglycerides and hepatic fatty acid synthase expression were decreased. Trabecular bone mineral density (BMD) was reduced in IRDlow, but not in IRDhigh rats. Orchiectomy (ORX) induced loss of BMD, which was antagonized by IRDhigh. ISO increased androgen sensitivity of seminal vesicle and levator ani. Besides, ISO plasma levels were reduced by ORX compared to intact and TP-treated rats. Conclusion In summary, the results of this study indicate that exposure to ISO during adolescence affects bone homeostasis, lipid metabolism, and modulates androgen sensitivity in young adult male rats.

SummaryActivation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-β induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.

Previous wound healing studies have failed to define a role for either α1β1 or α2β1 integrin in fibroblast-mediated wound contraction, suggesting the involvement of another collagen receptor in this process. Our previous work demonstrated that the integrin subunit α11 is highly induced during wound healing both at the mRNA and protein level, prompting us to investigate and dissect the role of the integrin α11β1 during this process. Therefore, we used mice with a global ablation of either α2 or α11 or both integrin subunits and investigated the repair of excisional wounds. Analyses of wounds demonstrated that α11β1 deficiency results in reduced granulation tissue formation and impaired wound contraction, independently of the presence of α2β1. Our combined in vivo and in vitro data further demonstrate that dermal fibroblasts lacking α11β1 are unable to efficiently convert to myofibroblasts, resulting in scar tissue with compromised tensile strength. Moreover, we suggest that the reduced stability of the scar is a consequence of poor collagen remodeling in α11-/- wounds associated with defective transforming growth factor-β–dependent JNK signaling. publishedVersion

The trabecular extracellular matrix (ECM) forms a three-dimensional scaffold to stabilize the bone marrow, provide substrates for cell-matrix interactions and retain, present or release signals to modulate hematopoietic stem and progenitor cell development. However, the impact of trabecular ECM components on hematopoiesis has been poorly studied. Using collagen IX alpha1 - deficient (Col9a1(−/−)) mice, we revealed that a lack of collagen IX alpha1 results in a disorganized trabecular network enriched in fibronectin, and in a reduction in myeloid cells, which was accompanied by a decrease in colony-stimulating factor 1 receptor expression on monocytes from the bone marrow. In contrast, B-cell numbers in the bone marrow and T-cell numbers in the thymus remained unchanged. Alterations in the bone marrow microenvironment may not only reduce myeloid cell numbers, but also have long-term implications for myeloid cell function. Mice were infected with Listeria moncytogenes to analyze the function of myeloid cells. In this case, an inadequate macrophage-dependent clearance of bacterial infections was observed in Col9a1(−/−) mice in vivo. This was mainly caused by an impaired interferon-gamma/tumor necrosis factor-alpha-mediated activation of macrophages. The loss of collagen IX alpha1 therefore destabilizes the trabecular bone network, impairs myeloid cell differentiation, and affects the innate immune response against Listeria.

The purpose of the study was to investigate the effect of an increase in mechanical knee joint loading during running on the serum COMP level. On two different test days, 20 healthy men ran with knee orthoses for 30 min on a treadmill (v = 2.2 m/s). On day 1, the orthoses were passive, whereas on day 2 they were pneumatically driven (active) and thus increased the external knee flexion moments (+30.9 Nm) during stance phase. Lower-limb mechanics and serum COMP levels (baseline; 0, 0.5, 1, 2 h post running) were analyzed. COMP levels increased immediately after running with passive (+35%; pre: 7.5 U/l, 95%CI: 6.4, 8.7, post: 9.8 U/l, 95%CI: 8.8, 10.8, p 0.001) and active orthoses (+45%; pre: 7.6 U/l; 95%CI: 6.4, 8.8, post: 10.3 U/l, 95%CI: 9.2, 11.5, p 0.001), but they did not differ between interventions. While running with active orthoses, greater ankle dorsiflexion angles, knee flexion angles, and moments occurred (p 0.05). Comparing both interventions, the Δ COMP pre-post, meaning the difference (Δ) between running with active and passive orthoses in pre to post COMP level change (=level after (post) running minus level before (pre) running), correlated negatively with Δ COMP baseline (difference between the baseline COMP level before running with active and passive orthoses, r = -0.616; p = 0.004), and with a positive tendence with the Δ maximum knee flexion (r = 0.388; p = 0.091). Therefore, changes in COMP concentration after physical activity seem to be highly influenced by the COMP baseline level. In addition, correlation analysis indicates that modifications in knee joint kinematics have a greater effect on cartilage metabolism than an increase in joint moments. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1937-1946, 2018.

Perifibrillar adapter proteins, interconnecting collagen fibrils, and linking the collagen network with the aggrecan matrix seem to play a crucial role in the pathogenesis of osteoarthritis (OA). Therefore, we examined immunohistochemically the extracellular distribution of collagen II and the main perifibrillar adapter proteins—collagen IX, decorin, cartilage oligomeric matrix protein (COMP), and matrilin-3—in human samples of healthy (n=4) and OA (n=42) knee joint cartilage. Histopathology assessment was performed using an OA score. Staining patterns were evaluated in relation to the disease stage. The perifibrillar adapter proteins were uniformly distributed in the upper zones of healthy cartilage. In moderate OA (n=8; score 14.3 ± 4.7), all proteins analyzed were locally absent in the fibrillated area or the superficial and upper mid zone. In advanced OA (n=20; score 18.9 ± 5.3), they were uniformly distributed in these zones and accumulated pericellularly. Perifibrillar adapter proteins are important for the stabilization of the collagen network in the upper zones of healthy cartilage. Their degradation might be a critical event in early OA. In advanced OA, there are indications for an increased synthesis in an attempt to regenerate the lost tissue and to protect the remaining cartilage from further destruction.

The purpose of this study was to investigate the combinatory effects of an isoflavone (ISO)-rich diet and exercise on fat mass and lipid metabolism in ovariectomized (OVX) rats. Therefore the female Wistar rats were sedentary, performed an intense treadmill uphill running, received ISOs, or a combination of ISOs and running after ovariectomy. The exercise reduced visceral fat mass, adipocyte size and serum leptin in Sham animals and antagonized the increases of these parameters induced by OVX. ISOs reduced OVX induced increase of serum leptin. The combination of training and ISOs was most effective in reducing serum triglyceride levels. In OVX rats the training stimulated the expression of genes associated with fatty acid synthesis (SREBP-1c and FAS) in adipose tissue, soleus muscle, liver and genes associated with fatty acid oxidation (PPARδ and PGC-1α) in adipose tissue. ISOs stimulated the expression of SREBP-1c and FAS in soleus muscle and PGC-1α in adipose tissue, whereas suppressed hepatic SREBP-1c and FAS expression. Strong additive effects of ISOs combined with the training were observed for PPARδ and PGC-1α expressions in soleus muscle. In conclusion our results demonstrate that both the training and ISOs affect fat mass and fatty acid metabolism in OVX rats. The training seems to have a higher impact than ISO exposure in regulating gene expression in adipose tissue. However, the strongest effects for several of the addressed parameters could be observed in the combination group especially in the soleus muscle. Therefore a combination of training and an ISO-rich diet may have beneficial effects on fatty acid metabolism and could be a concept for the prevention of obesity in postmenopausal females.

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4&minus, /&minus, mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4&minus, mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4&minus, mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4&minus, mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

In osteoarthritis animal models the rat knee is one of the most frequently investigated joint. However, it is unknown whether topographical variations in articular cartilage and subchondral bone of the normal rat knee exist and how they are linked or influenced by growth and maturation. Detailed knowledge is needed in order to allow interpretation and facilitate comparability of published osteoarthritis studies. For the first time, the present study maps topographical variations in cartilage thickness, cartilage compressive properties and subchondral bone microarchitecture between the medial and lateral tibial compartment of normal growing rat knees (7 vs. 13 weeks). Thickness and compressive properties (aggregate modulus) of cartilage were determined and the subchondral bone was analyzed by micro-computed tomography. We found that articular cartilage thickness is initially homogenous in both compartments, but then differentiates during growth and maturation resulting in greater cartilage thickness in the medial compartment in the 13-week-old animals. Cartilage compressive properties did not vary between the two sites independently of age. In both age-groups, subchondral plate thickness as well as trabecular bone volume ratio and trabecular thickness were greater in the medial compartment. While a high porosity of subchondral bone plate with a high topographical variation (medial/lateral) could be observed in the 7-week-old animals, the porosity was reduced and was accompanied by a reversion in topographical variation when reaching maturity. Our findings highlight that there is a considerable topographical variation in articular cartilage and subchondral bone within the normal rat knee in relation to the developmental status.

Botulinum toxin A (BTX)-induced muscle paralysis results in pronounced bone degradation with substantial bone loss. We hypothesized that whole-body vibration (WBV) and insulin-like growth factor-I (IGF-I) treatment can counteract paralysis-induced bone degradation following BTX injections by activation of the protein kinase B (Akt) signaling pathway. Female C57BL/6 mice (n = 60, 16 weeks) were assigned into six groups (n = 10 each): SHAM, BTX, BTX+WBV, BTX+IGF-I, BTX+WBV+IGF-I, and a baseline group, which was killed at the beginning of the study. Mice received a BTX (1.0 U/0.1 mL) or saline (SHAM) injection in the right hind limb. The BTX+IGF-I and BTX+WBV+IGF-I groups obtained daily subcutaneous injections of human IGF-I (1 μg/day). The BTX+WBV and BTX+WBV+IGF-I groups underwent WBV (25 Hz, 2.1 g, 0.83 mm) for 30 min/day, 5 days/week for 4 weeks. Femora were scanned by pQCT, and mechanical properties were determined. On tibial sections TRAP staining, static histomorphometry, and immunohistochemical staining against Akt, phospho-Akt, IGF-IR (IGF-I receptor), and phospho-IGF-IR were conducted. BTX injection decreased trabecular and cortical bone mineral density. The WBV and WBV+IGF-I groups showed no difference in trabecular bone mineral density compared to the SHAM group. The phospho-IGF-IR and phospho-Akt stainings were not differentially altered in the injected hind limbs between groups. We found that high-frequency, low-magnitude WBV can counteract paralysis-induced bone loss following BTX injections, while we could not detect any effect of treatment with IGF-I.