Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Locally Directed Recombinant Adeno- Associated Virus-Mediated IGF-1 Gene Therapy Enhances Osteochondral Repair and Counteracts Early Osteoarthritis In Vivo.

BACKGROUND: Restoration of osteochondral defects is critical, because osteoarthritis (OA) can arise. HYPOTHESIS: Overexpression of insulin-like growth factor 1 (IGF-1) via recombinant adeno-associated viral (rAAV) vectors (rAAV-IGF-1) would improve osteochondral repair and reduce parameters of early perifocal OA in sheep after 6 months in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral defects were created in the femoral trochlea of adult sheep and treated with rAAV-IGF-1 or rAAV-lacZ (control) (24 defects in 6 knees per group). After 6 months in vivo, osteochondral repair and perifocal OA were assessed by well-established macroscopic, histological, and immunohistochemical scoring systems as well as biochemical and micro-computed tomography evaluations. RESULTS: Application of rAAV-IGF-1 led to prolonged (6 months) IGF-1 overexpression without adverse effects, maintaining a significantly superior overall cartilage repair, together with significantly improved defect filling, extracellular matrix staining, cellular morphology, and surface architecture compared with rAAV-lacZ. Expression of type II collagen significantly increased and that of type I collagen significantly decreased. Subchondral bone repair and tidemark formation were significantly improved, and subchondral bone plate thickness and subarticular spongiosa mineral density returned to normal. The OA parameters of perifocal structure, cell cloning, and matrix staining were significantly better preserved upon rAAV-IGF-1 compared with rAAV-lacZ. Novel mechanistic associations between parameters of osteochondral repair and OA were identified. CONCLUSION: Local rAAV-mediated IGF-1 overexpression enhanced osteochondral repair and ameliorated parameters of perifocal early OA. CLINICAL RELEVANCE: IGF-1 gene therapy may be beneficial in repair of focal osteochondral defects and prevention of perifocal OA.

Subchondral bone remodeling patterns in larger animal models of meniscal injuries inducing knee osteoarthritis - a systematic review.

PURPOSE: Elucidating subchondral bone remodeling in preclinical models of traumatic meniscus injury may address clinically relevant questions about determinants of knee osteoarthritis (OA). METHODS: Studies on subchondral bone remodeling in larger animal models applying meniscal injuries as standardizing entity were systematically analyzed. Of the identified 5367 papers reporting total or partial meniscectomy, meniscal transection or destabilization, 0.4% (in guinea pigs, rabbits, dogs, minipigs, sheep) remained eligible. RESULTS: Only early or mid-term time points were available. Larger joint sizes allow reporting higher topographical details. The most frequently reported parameters were BV/TV (61%), BMD (41%), osteophytes (41%) and subchondral bone plate thickness (39%). Subchondral bone plate microstructure is not comprehensively, subarticular spongiosa microstructure is well characterized. The subarticular spongiosa is altered shortly before the subchondral bone plate. These early changes involve degradation of subarticular trabecular elements, reduction of their number, loss of bone volume and reduced mineralization. Soon thereafter, the previously normal subchondral bone plate becomes thicker. Its porosity first increases, then decreases. CONCLUSION: The specific human topographical pattern of a thinner subchondral bone plate in the region below both menisci is present solely in the larger species (partly in rabbits), but absent in rodents, an important fact to consider when designing animal studies examining subchondral consequences of meniscus damage. Large animal models are capable of providing high topographical detail, suggesting that they may represent suitable study systems reflecting the clinical complexities. For advanced OA, significant gaps of knowledge exist. Future investigations assessing the subchondral bone in a standardized fashion are warranted.

A Photopolymerizable Biocompatible Hyaluronic Acid Hydrogel Promotes Early Articular Cartilage Repair in a Minipig Model In Vivo.

Articular cartilage defects represent an unsolved clinical challenge. Photopolymerizable hydrogels are attractive candidates supporting repair. This study investigates the short-term safety and efficacy of two novel hyaluronic acid (HA)-triethylene glycol (TEG)-coumarin hydrogels photocrosslinked in situ in a clinically relevant large animal model. It is hypothesized that HA-hydrogel-augmented microfracture (MFX) is superior to MFX in enhancing early cartilage repair, and that the molar degree of substitution and concentration of HA affects repair. Chondral full-thickness defects in the knees of adult minipigs are treated with either 1) debridement (No MFX), 2) debridement and MFX, 3) debridement, MFX, and HA hydrogel (30% molar derivatization, 30 mg mL-1 HA; F3) (MFX+F3), and 4) debridement, MFX, and HA hydrogel (40% molar derivatization, 20 mg mL-1 HA; F4) (MFX+F4). After 8 weeks postoperatively, MFX+F3 significantly improves total macroscopic and histological scores compared with all other groups without negative effects, besides significantly enhancing the individual repair parameters "defect architecture," "repair tissue surface" (compared with No MFX, MFX), and "subchondral bone" (compared with MFX). These data indicate that photopolymerizable HA hydrogels enable a favorable metastable microenvironment promoting early chondrogenesis in vivo. This work also uncovers a mechanism for effective HA-augmented cartilage repair by combining lower molar derivatization with higher concentrations.

Quantifying the Human Subchondral Trabecular Bone Microstructure in Osteoarthritis with Clinical CT.

Osteoarthritis (OA) is characterized by critical alterations of the subchondral bone microstructure, besides the well-known cartilaginous changes. Clinical computed tomography (CT) detection of quantitative 3D microstructural subchondral bone parameters is applied to monitor changes of subchondral bone structure in different stages of human OA and is compared with micro-CT, the gold standard. Determination by clinical CT (287 µm resolution) of key microstructural parameters in tibial plateaus with mild-to-moderate and severe OA reveals strong correlations to micro-CT (35 µm), high inter- and intraobserver reliability, and small relative differences. In vivo, normal, mild-to-moderate, and severe OA are compared with clinical CT (331 µm). All approaches detect characteristic expanded trabecular structure in severe OA and fundamental microstructural correlations with clinical OA stage. Multivariate analyses at various in vivo and ex vivo imaging resolutions always reliably separate mild-to-moderate from severe OA (except mild-to-moderate OA from normal), revealing a striking similarity between 287 µm clinical and 35 µm micro-CT. Thus, accurate structural measurements using clinical CT with a resolution near the trabecular dimensions are possible. Clinical CT offers an opportunity to quantitatively monitor subchondral bone microstructure in clinical and experimental settings as an advanced tool of investigating OA and other diseases affecting bone architecture.

Impaired Intracellular Ca2+ Dynamics, M-Band and Sarcomere Fragility in Skeletal Muscles of Obscurin KO Mice.

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

Axial alignment is a critical regulator of knee osteoarthritis.

Although osteoarthritis (OA), a leading cause of disability, has been associated with joint malalignment, scientific translational evidence for this link is lacking. In a clinical case study, we provide evidence of osteochondral recovery upon unloading symptomatic isolated medial tibiofemoral knee OA associated with varus malalignment. By mapping response correlations at high resolution, we identify spatially complex degenerative changes in cartilage after overloading in a clinically relevant ovine model. We further report that unloading diminishes OA cartilage degeneration and alterations of critical parameters of the subchondral bone plate in a similar topographic fashion. Last, therapeutic unloading shifted the articular cartilage and subchondral bone phenotype to normal and restored several physiological correlations disturbed in neutral and varus OA, suggesting a protective effect on the integrity of the entire osteochondral unit. Collectively, these findings identify modifiable trajectories with considerable translational potential to reduce the burden of human OA.

Comparative anatomy and morphology of the knee in translational models for articular cartilage disorders. Part I: Large animals.

BACKGROUND: The human knee is a complex joint, and affected by a variety of articular cartilage disorders. Large animal models are critical to model the complex disease mechanisms affecting a functional joint. Species-dependent differences highly affect the results of a pre-clinical study and need to be considered, necessitating specific knowledge not only of macroscopic and microscopic anatomical and pathological aspects, but also characteristics of their individual gait and joint movements. METHODS: Literature search in Pubmed. RESULTS AND DISCUSSION: This narrative review summarizes the most relevant anatomical structural and functional characteristics of the knee (stifle) joints of the major translational large animal species, comprising dogs, (mini)pigs, sheep, goats, and horses in comparison with humans. Specific characteristics of each species, including kinematical gait parameters are provided. Considering these multifactorial dimensions will allow to select the appropriate model for answering the research questions in a clinically relevant fashion.

Septins, a cytoskeletal protein family, with emerging role in striated muscle.

Appropriate organization of cytoskeletal components are required for normal distribution and intracellular localization of different ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Proteins of the contractile system are in direct or indirect connection with the extrasarcomeric cytoskeleton. A number of other molecules which have essential role in regulating stretch-, voltage-, and chemical signal transduction from the surface into the cytoplasm or other intracellular compartments are already well characterized. Sarcomere, the basic contractile unit, is comprised of a precisely organized system of thin (actin), and thick (myosin) filaments. Intermediate filaments connect the sarcomeres and other organelles (mitochondria and nucleus), and are responsible for the cellular integrity. Interacting proteins have a very diverse function in coupling of the intracellular assembly components and regulating the normal physiological function. Despite the more and more intense investigations of a new cytoskeletal protein family, the septins, only limited information is available regarding their expression and role in striated, especially in skeletal muscles. In this review we collected basic and specified knowledge regarding this protein group and emphasize the importance of this emerging field in skeletal muscle biology.

Comparative anatomy and morphology of the knee in translational models for articular cartilage disorders. Part II: Small animals.

BACKGROUND: Small animal models are critical to model the complex disease mechanisms affecting a functional joint leading to articular cartilage disorders. They are advantageous for several reasons and significantly contributed to the understanding of the mechanisms of cartilage diseases among which osteoarthritis. METHODS: Literature search in Pubmed. RESULTS AND DISCUSSION: This narrative review summarizes the most relevant anatomical structural and functional characteristics of the knee (stifle) joints of the major small animal species, including mice, rats, guinea pigs, and rabbits compared with humans. Specific characteristics of each species, including kinematical gait parameters are provided and compared with the human situation. When placed in a proper context respecting their challenges and limitations, small animal models are important and appropriate models for articular cartilage disorders.

Analysis of spatial osteochondral heterogeneity in advanced knee osteoarthritis exposes influence of joint alignment.

Osteoarthritis (OA) is considerably affected by joint alignment. Here, we investigate the patterns of spatial osteochondral heterogeneity in patients with advanced varus knee OA together with clinical data. We report strong correlations of osteochondral parameters within individual topographical patterns, highlighting their fundamental and location-dependent interactions in OA. We further identify site-specific effects of varus malalignment on the lesser loaded compartment and, conversely, an unresponsive overloaded compartment. Last, we trace compensatory mechanisms to the overloaded subarticular spongiosa in patients with additional high body weight. We therefore propose to consider and to determine axial alignment in clinical trials when selecting the location to assess structural changes in OA. Together, these findings broaden the scientific basis of therapeutic load redistribution and weight loss in varus knee OA.

Improved Calcium Homeostasis and Force by Selenium Treatment and Training in Aged Mouse Skeletal Muscle.

During aging reduction in muscle mass (sarcopenia) and decrease in physical activity lead to partial loss of muscle force and increased fatigability. Deficiency in the essential trace element selenium might augment these symptoms as it can cause muscle pain, fatigue, and proximal weakness. Average voluntary daily running, maximal twitch and tetanic force, and calcium release from the sarcoplasmic reticulum (SR) decreased while reactive oxygen species (ROS) production associated with tetanic contractions increased in aged - 22-month-old - as compared to young - 4-month-old - mice. These changes were accompanied by a decline in the ryanodine receptor type 1 (RyR1) and Selenoprotein N content and the increased amount of a degraded RyR1. Both lifelong training and selenium supplementation, but not the presence of an increased muscle mass at young age, were able to compensate for the reduction in muscle force and SR calcium release with age. Selenium supplementation was also able to significantly enhance the Selenoprotein N levels in aged mice. Our results describe, for the first time, the beneficial effects of selenium supplementation on calcium release from the SR and muscle force in old age while point out that increased muscle mass does not improve physical performance with aging.

N-methyl-D-aspartate (NMDA) receptor expression and function is required for early chondrogenesis.

BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

Topographic modeling of early human osteoarthritis in sheep.

Articular cartilage damage occurring during early osteoarthritis (OA) is a key event marking the development of the disease. Here, we modeled early human OA by gathering detailed spatiotemporal data from surgically induced knee OA development in sheep. We identified a specific topographical pattern of osteochondral changes instructed by a defined meniscal injury, showing that both cartilage and subchondral bone degeneration are initiated from the region adjacent to the damage. Alterations of the subarticular spongiosa arising locally and progressing globally disturbed the correlations of cartilage with subchondral bone seen at homeostasis and were indicative of disease progression. We validated our quantitative findings against human OA, showing a similar pattern of early OA correlating with regions of meniscal loss and an analogous late critical disturbance within the entire osteochondral unit. This translational model system can be used to elucidate mechanisms of OA development and provides a roadmap for investigating regenerative therapies.

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.

Asymptomatic focal calcium pyrophosphate crystal deposition within partially failed repair tissue after matrix-assisted autologous chondrocyte implantation.

Possible failures of autologous chondrocyte implantation (ACI), a cell-based technique for articular cartilage repair, are not always clinically apparent and the underlying mechanisms largely remain unknown. This case report presents the first scenario in the literature highlighting an association of a medium-term partial failure of an advanced ACI procedure (matrix-assisted ACI) in the knee with focal asymptomatic calcium pyrophosphate deposition disease, a common inflammatory pyrophosphate arthropathy. The specific presence of CPPDs, resulting from increased biomechanical stresses in the repair tissue-cartilage and repair tissue-subchondral bone integration sites, together with the absence of cartilage regeneration was identified and possibly contributed to the partial failure.Level of evidence V.

Author Correction: CB1 receptor activation induces intracellular Ca2+ mobilization and 2-arachidonoylglycerol release in rodent spinal cord astrocytes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

CB1 receptor activation induces intracellular Ca2+ mobilization and 2-arachidonoylglycerol release in rodent spinal cord astrocytes.

Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca2+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid 2-arachidonoylglycerol, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and 2-arachidonoylglycerol production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the 2-arachidonoylglycerol synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca2+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca2+ transients lead to the production of 2-arachidonoylglycerol in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.

Reliable landmarks for precise topographical analyses of pathological structural changes of the ovine tibial plateau in 2-D and 3-D subspaces.

Selecting identical topographical locations to analyse pathological structural changes of the osteochondral unit in translational models remains difficult. The specific aim of the study was to provide objectively defined reference points on the ovine tibial plateau based on 2-D sections of micro-CT images useful for reproducible sample harvesting and as standardized landmarks for landmark-based 3-D image registration. We propose 5 reference points, 11 reference lines and 12 subregions that are detectable macroscopically and on 2-D micro-CT sections. Their value was confirmed applying landmark-based rigid and affine 3-D registration methods. Intra- and interobserver comparison showed high reliabilities, and constant positions (standard errors < 1%). Spatial patterns of the thicknesses of the articular cartilage and subchondral bone plate were revealed by measurements in 96 individual points of the tibial plateau. As a case study, pathological phenomena 6 months following OA induction in vivo such as osteophytes and areas of OA development were mapped to the individual subregions. These new reference points and subregions are directly identifiable on tibial plateau specimens or macroscopic images, enabling a precise topographical location of pathological structural changes of the osteochondral unit in both 2-D and 3-D subspaces in a region-appropriate fashion relevant for translational investigations.