Cartilage Mineralization Research Articles

GSK3B Overexpression Alleviates Posttraumatic Osteoarthritis in Mice by Promoting DNMT1-Mediated Hypermethylation of NR4A3 Promoter.

Background Glycogen synthase kinase 3β (GSK3B) is reported to be a protective factor for the degradation of chondrocytes by extracellular mechanisms. Nuclear receptor subfamily 4 group A member 3 (NR4A3) is a proinflammatory factor in osteoarthritis. Their regulation mechanism in posttraumatic osteoarthritis (PTOA) is not fully understood. Methods GSK3B expression in the cartilage tissue of PTOA patients was analyzed by western blotting. IL-1β-induced chondrocytes were transfected with pcDNA-GSK3B, and then, the cell viability, apoptosis, expression of the chondrocyte extracellular matrix degradation-related genes MMP13, aggrecan, and type II collagen, and secretion of inflammatory factors TNF-α and IL-6 were detected. Co-IP was used to analyze the interaction between GSK3B and DNMT1. Ch-IP and methylation-specific PCR assays were used for methylation. Also, cells were transfected with pcDNA-GSK3B or together with pcDNA-NR4A3, as well as transfected with si-NR4A3, and then, cell functions were tested. Then, the mice subjected to destabilization of medial meniscus (DMM) surgery were intra-articular injected with 100 μL of the following adeno-related virus vectors (empty vector, Ad-GSK3B, scrambled shRNA, and sh-NR4A3), respectively, and the virus titer was 2 × 108 TU/mL. Cartilage integrity was evaluated by safranin O/fast green staining, HE staining, and Osteoarthritis Research Society International (OARSI) score. Results The expression of GSK3B protein was downregulated in PTOA patients. GSK3B overexpression alleviated IL-1β-induced chondrocyte apoptosis and extracellular matrix degradation, as well as cartilage mineralization in PTOA model mice. NR4A3 overexpression reversed the effect of GSK3B on IL-1β-induced chondrocyte functions. GSK3B could recruit DNMT1 to the NR4A3 promoter region to promote the methylation of NR4A3 and inhibit the expression of NR4A3 protein. Similarly, NR4A3 interference alleviated cartilage degradation under stimulating conditions by inhibiting the activation of the JAK2/STAT3 signaling pathway. Conclusion GSK3B recruits DNMT1 to the NR4A3 promoter region and inhibits the activation of the NR4A3-mediated JAK2/STAT3 signaling pathway, thereby alleviating PTOA.

Nanohydroxyapatite Hydrogel Can Promote the Proliferation and Migration of Chondrocytes and Better Repair Talar Articular Cartilage.

As an important load-bearing part of the body, joints are prone to articular cartilage degradation during exercise, resulting in joint pain, swelling, and deformity, which has an adverse impact on patients' life quality and social medical security. Therefore, this study aims to test an effective biopolymer scaffold in promoting the growth of chondrocytes in talus. Hydrogel (Gel)-nanohydroxyapatite (nHA) was invented as a new type of biopolymer scaffold for osteoarthritis treatment in this research. To detect the effects of Gel-nHA on guidance, cartilage matrix secretion, mineralization, proliferation, and migration of chondrocyte, we cultured chondrocytes to study the biological properties of nHA. It was found that Gel could guide chondrocytes to permeate and migrate, so it could be used as acellular matrix scaffolds for chondrocyte regeneration. In addition, nHA could stimulate chondrocytes to secrete cartilage matrix, such as type II collagen and mucopolysaccharide (GAGs). At the same time, nHA help to induce chondrocyte mineralization and stimulate the secretion of type X collagen, so as to better maintain the integrity of bone cartilage interface. In Gel-nHA, chondrocyte viability could be better maintained, and the proliferation and migration of chondrocytes could be better promoted, so as to better repair the articular cartilage of talus. Therefore, the Gel-nHA scaffold is expected to become an effective method for repairing talus cartilage in the future.

Lef1 ablation alleviates cartilage mineralization following posttraumatic osteoarthritis induction

Cartilage mineralization is a tightly controlled process, imperative for skeletal growth and fracture repair. However, in osteoarthritis (OA), cartilage mineralization may impact the joint range of motion, inflict pain, and increase chances for joint effusion. Here we attempt to understand the link between inflammation and cartilage mineralization by targeting Sirtuin 1 (SIRT1) and lymphoid enhancer binding factor 1 (LEF1), both reported to have contrasting effects on cartilage. We find that inflammatory-dependent cleavage of SIRT1 or its cartilage-specific genetic ablation, directly enhanced LEF1 expression accompanied by a catabolic response. Applying a posttraumatic OA (PTOA) model to cartilage-specific Sirt1 nulls displayed severe OA, which was accompanied by synovitis, meniscal mineralization, and osteophyte formation of the lateral joint compartment. Alternatively, cartilage-specific Lef1 nulls presented reduced lateral mineralization, OA severity, and local pain. Differential gene expression analysis revealed that Lef1 ablation reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like receptor (Tlr) pathways, while enhancing SRY-Box transcription factor 9 (Sox9) and cartilaginous extracellular matrix genes. The results support a link between inflammation and Lef1-dependent cartilage mineralization, mediated by the inactivation of Sirt1. By ablating Lef1 in a PTOA model, the structural and pain-related phenotypes of OA were reduced, in part, by preventing cartilage mineralization of the lateral joint compartment, partially manifested by meniscal tissue mineralization. Overall, these data provide a molecular axis to link between inflammation and cartilage in a PTOA model.

Distribution, structure, and mineralization of calcified cartilage remnants in hard antlers

Antlers are paired deciduous bony cranial appendages of deer that undergo a regular cycle of growth, death and casting, and constitute the most rapidly growing bones in mammals. Antler growth occurs in an appositional mode and involves a modified form of endochondral ossification. In endochondral bones, calcified cartilage is typically a transient tissue that is eventually completely replaced by bone tissue. We studied the distribution and characteristics of calcified cartilage in hard antlers from three deer species (Capreolus capreolus, Cervus elaphus, Dama dama), i.e., in antlers from which the skin (velvet) had been shed. Remnants of calcified cartilage were regularly present as part of the trabecular framework in the late formed, distal antler portions in all three species, whereas this tissue was largely or completely missing in the more proximal antler portions. The presence of calcified cartilage remnants in the distal antler portions is attributed to the limited antler lifespan of only a few months, which is also the reason for the virtual lack of bone remodeling in antlers. The calcified cartilage matrix was more highly mineralized than the antler bone matrix. Mineralized deposits were observed in some chondrocyte lacunae and occasionally also in osteocyte lacunae, a phenomenon that has not previously been reported in antlers. Using synchrotron radiation-induced X-ray fluorescence (SR-XRF) mapping, we further demonstrated increased zinc concentrations in cement lines, along the inner borders of incompletely formed primary osteons, along the walls of partly or completely mineral-occluded chondrocyte and osteocyte lacunae, and in intralacunar mineralized deposits. The present study demonstrates that antlers are a promising model for studying the mineralization of cartilage and bone matrices and the formation of mineralized deposits in chondrocyte and osteocyte lacunae.

Mutation in the CCAL1 locus accounts for bidirectional process of human subchondral bone turnover and cartilage mineralization.

To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humansvia DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.

RNA SEQUENCING REVEALS NOVEL MARKERS AND PATHWAYS OF T3-INDUCED CARTILAGE HYPERTROPHY

Purpose: Hallmark of the osteoarthritis (OA) disease pathophysiological process is the recuperation of OA chondrocytes towards a hypertrophic, growth plate-like morphology and signalling, leading to cartilage degeneration and mineralization. Previously, our group showed by genetics and functional studies that upregulation of thyroid signalling (T3) induces this process and results in an upregulation of known hypertrophic genes such as MMP13, COL1A1, COL10A1 and ALPL. Defining the underlying molecular pathways involved in T3 induced hypertrophy could yield potential markers of hypertrophy and druggable targets.

ANAPHYLATOXINS AMPLIFY CALCIFICATION DURING OSTEOGENIC DIFFERENTIATION OF ARTICULAR CHONDROCYTES

Purpose: Cartilage calcification (CC) is involved in Osteoarthritis (OA) pathogenesis and associated with cartilage degeneration. So far, the initial mechanisms of CC are not clear. It has previously been described that the anaphylatoxin receptors C3aR and especially C5aR are involved in the osteogenic differentiation of human mesenchymal stem cells (MSC). Thus, we suggested that the anaphylatoxins C3a and C5a, released after cartilage trauma and subsequent complement activation, are involved in the initiation of cartilage mineralization.

Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes.

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.

Introduction to the potential of Ferula ovina in dental implant research due to estrogenic bioactive compounds and adhesive properties.

Recent developments in dental implant have heightened the urgent need to natural tissue adhesives estrogenic materials with ability of promoting the proliferation and osteoblastic differentiation in human dental pulp-derived stem cells, to provide better integration of tissue for dentistry. Up to now, far little attention has been paid to adhesives extract of the root of Ferula sp. which contains biomaterial compounds with estrogenic activities. Prior to undertaking the investigation, analysis of the extract of the root of F. ovina revealed a novel terpenoid, and we identified it as Fenoferin. So far, this paper has focused on Fenoferin compared to Ferutinin and root extract to determine if Fenoferin caused changes in craniofacial cartilage, bone (ceratohyal) and tooth mineralization. Following the purpose of study, we used zebrafish as a well-developed model system for studying bone development, so the developing zebrafish larvae were exposed to various concentration of compounds at 2dpf, and the histological analyses were performed at 6dpf. The result of the current study highlights the importance of F. ovina in studies related to dental regenerative medicine.

Fully Ossified Thyroid Cartilage Found among the Skeletal Remains of A 21-Year-Old Slavic Soldier: Interpretation of a Case

The degree of ossification of the thyroid cartilage in anthropological studies is related to other methods of determining the age of a skeleton. The endochondral ossification process begins at the age of approximately 15–20 years and is generally completed in the fifth or sixth decade of life. In the present case, early and complete mineralization of the thyroid cartilage of a skeleton belonging to a 21-year-old young soldier who died in 1946 was observed. Ossified thyroid cartilage at a very young age is rare and may be associated with specific symptoms, such as compression due to trauma, or may also be related to hormonal dysfunction and various diseases that may cause early mineralization. A macroscopic morphological evaluation and radiographic examination of the thyroid cartilage were performed and a decalcification test was applied to a sample taken from the thyroid cartilage to confirm that the structure was indeed mineralized. There is nothing to exclude that this is simply a physiological anatomical variation. Knowledge of this rare anatomical abnormality at a young age would be useful for the diagnosis of various pathological conditions.

Divergent Expression of SPARC, SPARC-L, and SCPP Genes During Jawed Vertebrate Cartilage Mineralization.

While cartilage is an ancient tissue found both in protostomes and deuterostomes, its mineralization evolved more recently, within the vertebrate lineage. SPARC, SPARC-L, and the SCPP members (Secretory Calcium-binding PhosphoProtein genes which evolved from SPARC-L) are major players of dentine and bone mineralization, but their involvement in the emergence of the vertebrate mineralized cartilage remains unclear. We performed in situ hybridization on mineralizing cartilaginous skeletal elements of the frog Xenopus tropicalis (Xt) and the shark Scyliorhinus canicula (Sc) to examine the expression of SPARC (present in both species), SPARC-L (present in Sc only) and the SCPP members (present in Xt only). We show that while mineralizing cartilage expresses SPARC (but not SPARC-L) in Sc, it expresses the SCPP genes (but not SPARC) in Xt, and propose two possible evolutionary scenarios to explain these opposite expression patterns. In spite of these genetic divergences, our data draw the attention on an overlooked and evolutionarily conserved peripheral cartilage subdomain expressing SPARC or the SCPP genes and exhibiting a high propensity to mineralize.

Rickets in Children: An Update.

Rickets refers to a deficient mineralization of the growth plate cartilage, predominantly affecting longer bones. Despite the fact that preventive measures are available, it is still a common disease worldwide; nutritional rickets, due to vitamin D deficiency or dietary calcium inadequate intake, remains the most common form. Medical history, physical examination, radiologic features and biochemical tests are essential for diagnosis. Although recent studies suggest hypophosphatemia as the leading alteration, rickets is classically divided into two categories: calcipenic rickets and phosphopenic rickets. Knowledge of this categorization and of respective clinical and laboratory features is essential for rapid diagnosis and correct management. The aim of this review is to analyze the epidemiological, pathogenetic, clinical, and therapeutic aspects of the different forms of rickets, describing the novelties on this “long-lived” disease.

Mesenchymal stem cell derived extracellular vesicles as treatment for osteoarthritis in a rat high fat diet groove model

Purpose: Mesenchymal stromal/stem cells (MSC) hold a great promise to control the imbalance between anabolic and catabolic processes in an OA joint due to their immunomodulatory and regenerative capacities. Recently, intra-articular injections with human MSC entered the clinical trials as a novel, low invasive and relatively cheap therapy for OA. Although, the results of this novel treatment are promising, it remains unclear how MSC exert their beneficial effects in inflammation and tissue (cartilage) repair. Increasing evidence suggests that the therapeutic efficacy of MSC depends on paracrine signaling. Recently we have shown that MSC-derived extracellular vesicles (MSC-EVs) are an important component of this signaling and have a major contribution to cartilage regeneration and inhibition of TNF-alpha stimulated inflammatory responses in chondrocytes in vitro. Extracellular vesicles (EVs) are secreted by all cell types and range in size from 40 - 100 nm (exosomes) and from 100 -1000 nm (microvesicles). They increasingly appear to play an important role in intercellular communication by transportation of important molecules (peptides, mRNA, miRNA, enzymes). MSC-derived EVs have a great potential to serve as a safer cell free therapy for OA. The purpose of this study was to investigate if MSC-derived EVs can inhibit inflammation and promote cartilage repair to a similar extent as MSCs in a rat high fat diet groove model of OA. Methods: 36 male Wistar-han (Crl:WI(Han) rats were fed a high fat diet for 24 weeks, starting at an age of 12 weeks old. In week 12 of the study the rats received unilateral surgery where local cartilage damage was induced at the right knee joint by making five longitudinal grooves on the femoral condyles. In week 13 the rats were randomized into 3 groups and given an intraarticular injection with either PBS, 2 million human bone marrow derived MSCs or EVs derived from 2 million MSCs. The extracellular vesicles group received 5 of these doses with 5 days interval. Before, during and after the injections pain behavior was monitored by dynamic weight bearing and via measuring the left and right hind paw withdrawal responses to the application of von Frey hairs. uCT scans were made at week 0 and week 24, and used to determine the number of osteophytes and cartilage mineralization in the tibia. Additionally, tibial subchondral plate thickness, trabecular bone thickness, and trabecular bone volume fraction (total volume/trabecular bone volume) were measured using ImageJ 1.47v. At endpoint animals were sacrificed and the knee joints were harvested. Joint tissue is currently under preparation for histological staining to determine the OARSI histopathological score for rats. Data is represented as mean +/- standard deviation (SD) and the difference between the means in the three groups was statistically tested using one way ANOVA. Results: Results from the von Frey tests showed a clear decrease in 50% withdrawal threshold in the right hind paw, which was expected as the right side was the grooved limb. However, no difference between the three treatment groups in either left or right paw was seen. The mean osteophyte number in the PBS group was 4.7 (+/- 0.65) versus 3.6 (+/-2.15) in the MSC group and 4.3 (+/-1.8) in the MSC-EV group (figure 1A). In the MSC and MSC-EV treatment groups the variation in osteophyte number and total osteophyte diameter between the animals was large compared to the PBS group. Cartilage mineralization were observed in 9 of 12 animals in the PBS and MSC group and in 5 of 12 animals in the MSC-EV group. Both the number and total diameter was lowest in the MSC-EV group, but there was no significant difference as compared with control groups (Figure 1B). Other parameters measured on uCT like bone volume fraction, subchondral plate and trabecular bone thickness were similar between the 3 groups. Results from the OARSI histopathological score for rats is currently under preparation. Conclusions: Preliminary results from this in vivo study show no significant differences between PBS, MSC or MSC-EV treatment on uCT data or von Frey test. However, we do observe a large spread in osteophyte score and total diameter in the MSC and MSC-EV treated groups, which could be a sign that individual animals are responding differently to the treatments. Data from the OARSI scoring will help us determine if the response really is variable for MSC and MSC-EV treatment groups. This would be in accordance with observations made in humans, as many clinical trials that test MSC-based therapies report a substantial nonresponder group. In summary, although osteophytes are one of the hallmarks of osteoarthritis, we feel a complete picture of the joint damage including cartilage and synovium is needed to draw conclusions on the effect of the treatments in this study.

Lrp5 mutant and crispant zebrafish faithfully model human osteoporosis, establishing the zebrafish as a platform for CRISPR-based functional screening of osteoporosis candidate genes

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. (C) 2021 American Society for Bone and Mineral Research. (C) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Unravelling the role of osteoprotegerin in osteoarthritis by applying a human isogenic induced pluripotent stem cell model

Purpose: Founded by the hypothesis that the OA disease pathway of the previously identified high impact gain-of-function mutation in TNFRSF11B could be extrapolated to common, age-related, OA disease phenotypes, we set out to functionally study its effect. To this end, we established in vitro hiPS derived spherical chondrogenic and osteogenic pellets and compared TNFRSF11B mutated cells generated from affected family members to CRISPR/Cas9 repaired isogenic control cells. Given that the mutant OPG decoy receptor more efficiently antagonizes osteoclastogenesis, we expect that the mutation affects matrix mineralization hence inflicting the observed chondrocalcinosis phenotype. Methods: Using skin fibroblasts, hiPSCs of an affected family member were created (FOA) and differentiated to induced mesenchymal stromal stem cells (iMSCs). These were differentiated for 6 weeks to deposit neo-cartilaginous and neo-osseous pellets or neo-bone in parallel with a CRISPR-Cas9 repaired hiPSC line (FOA-R) serving as an isogenic control. Presence of glycosaminoglycans was determined by Alcian blue while mineralization was determined by Alizarin red staining. Gene expression analyses were performed to study anabolic markers and hypertrophy (ie, COL2A1, COL1A1, COL10A1) and matrix mineralization (ie, ALPL, MGP). Results: As demonstrated by the intensity of Alcian blue and Alizarin red staining of spherical neo-cartilaginous and neo-osseous pellets, respectively (Figure 1) the bi-directional differentiation via iPSC derived iMSCs appeared successful. Additionally, while only subtle differences in intensity were observed for the Alcian blue staining, the Alizarin red staining intensity in FOA mutated neo-osseous pellets was clearly higher as that of the FOA-repaired pellets, indicating increased matrix mineralization (Figure 1). Gene expression analyses of neo-cartilaginous pellets, showed, however, that in the presence of the FOA TNFRSF11B mutation, MGP levels were considerably decreased (FD=0.14 P=1.6x10-13), indicating cell signaling towards matrix mineralization also in the mutated neo-cartilaginous tissues. In line with this, neo-cartilaginous tissues from FOA mutated cells compared to FOA repaired cells expressed lower levels of COL2A1 (FD=-6.8, P-value=3.9x10-3) concomitant with higher levels of COL1A1 (FD=1.7, P-value=7.4x10-3). Additionally, in the FOA mutated neo-osseous pellets gene expression analysis showed a higher expression of RUNX2 (FD=2.6, P-value= 8.29x10-3) and a trend towards a higher expression of COL1A1 (FD=1.54, P-value= 0.079) and ALPL (FD=1.57, P-value=0.055) relative to FOA repaired neo-osseous pellets.supplements. Conclusions: Employing hiPS-cells from early-onset OA family members (FOA) and CRISPR/Cas9 repaired isogenic controls (FOA-R) in an established spherical 3D in vitro chondrogenic and osteogenic pellet model confirmed that the previously identified gain of function mutation in TNFRSF11B encoding OPG acts via increased matrix mineralization of cartilage and bone. In cartilage, likely, this is modulated via MGP expression as suggested by the significant lower levels in the mutant cells. This may raise the possibility to slow down OA progression in early-onset family members with the use of vitamin K.

Automated analysis of rabbit knee calcified cartilage morphology using micro-computed tomography and deep learning.

Structural dynamics of calcified cartilage (CC) are poorly understood. Conventionally, CC structure is analyzed using histological sections. Micro‐computed tomography (µCT) allows for three‐dimensional (3D) imaging of mineralized tissues; however, the segmentation between bone and mineralized cartilage is challenging. Here, we present state‐of‐the‐art deep learning segmentation for µCT images to assess 3D CC morphology. The sample includes 16 knees from 12 New Zealand White rabbits dissected into osteochondral samples from six anatomical regions: lateral and medial femoral condyles, lateral and medial tibial plateaus, femoral groove, and patella (n = 96). The samples were imaged with µCT and processed for conventional histology. Manually segmented CC from the images was used to train segmentation models with different encoder–decoder architectures. The models with the greatest out‐of‐fold evaluation Dice score were selected. CC thickness was compared across 24 regions, co‐registered between the imaging modalities using Pearson correlation and Bland–Altman analyses. Finally, the anatomical CC thickness variation was assessed via a Linear Mixed Model analysis. The best segmentation models yielded average Dice of 0.891 and 0.807 for histology and µCT segmentation, respectively. The correlation between the co‐registered regions was strong (r = 0.897, bias = 21.9 µm, standard deviation = 21.5 µm). Finally, both methods could separate the CC thickness between the patella, femoral, and tibial regions (p < 0.001). As a conclusion, the proposed µCT analysis allows for ex vivo 3D assessment of CC morphology. We demonstrated the biomedical relevance of the method by quantifying CC thickness in different anatomical regions with a varying mean thickness. CC was thickest in the patella and thinnest in the tibial plateau. Our method is relatively straightforward to implement into standard µCT analysis pipelines, allowing the analysis of CC morphology. In future research, µCT imaging might be preferable to histology, especially when analyzing dynamic changes in cartilage mineralization. It could also provide further understanding of 3D morphological changes that may occur in mineralized cartilage, such as thickening of the subchondral plate in osteoarthritis and other joint diseases.

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage.

Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

The impact of a key nutraceutical complex on chondrocyte cells and matrix Gla protein expression

Osteoarthritis (OA) is the most common type of arthritis among adults, which is associated with cartilage breakdown. Chondrocytes are the only type of cell in articular cartilage that secrete extracellular matrix (ECM). Therefore, to prevent and manage the occurrence of arthritic cartilage, it is important to consider approaches to increase the survival of chondrocyte cells. The production of reactive oxygen species (ROS), the release of pro-inflammatory cytokines (e.g. interleukin-1β (IL-1β)), and cartilage calcification play an imperative role in OA pathophysiology. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) is an enzyme that regulates the production of ROS in chondrocyte cells. ROS and cytokines lead to ECM degradation and chondrocyte apoptosis. Moreover, the matrix γ-carboxyglutamic acid (Gla) protein (MGP) acts as a decalcification agent in chondrocytes, which can inhibit the pathogenic mineralisation of cartilage. The correct balance of nutrients may potentially have a protective effect against OA. In the present study, the effect of essential nutrients on Nox4 activity in chondrocyte cells, chondrocyte viability, and MGP expression was investigated. Chondrocyte cells were pre-treated with the nutrients, and the cells were subsequently assessed for Nox4 activity using the chemiluminescence technique. It was found that the production of ROS significantly decreased after preincubation in the presence of nutrients in comparison to the control samples. Furthermore, it was found that the presence of nutraceuticals improved the viability of chondrocyte cells and the expression of MGP, which acts as a mineralisation inhibitor in the chondrocyte growth plate. The results of this study suggest that the correct balance of key nutrients at the molecular level plays an essential role in supporting chondrocyte cells and promoting cartilage decalcification.

Selection on vocal output affects laryngeal morphology in rats.

Although laryngeal morphology often reflects adaptations for vocalization, the structural consequences of selection for particular aspects of vocal behavior remain poorly understood. In this study, we investigated the effects of increased ultrasonic calling in pups on the adult larynx morphology in selectively bred rat lines. Laryngeal morphology was assessed using multiple techniques: mineralized cartilage volumes were compared in 3D‐models derived from microCT scans, internal structure was compared using clearing and staining procedures combined with microscopy, cellular structure was compared using histology and microscopy, and element composition was assessed with scanning energy dispersive X‐ray spectroscopy. Our results show that adult rats from lines bred to produce ultrasonic calls at higher rates as pups have shorter vocal folds and a more mineralized thyroid cartilage compared to rats bred to produce ultrasonic calls at lower rates. The change in vocal fold length appears to account for differences in low‐frequency calls in these two rat lines. We suggest that the observed increases in mineralization of the thyroid cartilage in the high‐ultrasound lineage provide increased reinforcement of the laryngeal structure during ultrasonic call production. Our findings therefore demonstrate an effect of selection for vocal behavior on laryngeal morphology, with acoustic consequences.

Apoptosis in the Extraosseous Calcification Process.

Extraosseous calcification is a pathologic mineralization process occurring in soft connective tissues (e.g., skin, vessels, tendons, and cartilage). It can take place on a genetic basis or as a consequence of acquired chronic diseases. In this last case, the etiology is multifactorial, including both extra- and intracellular mechanisms, such as the formation of membrane vesicles (e.g., matrix vesicles and apoptotic bodies), mitochondrial alterations, and oxidative stress. This review is an overview of extraosseous calcification mechanisms focusing on the relationships between apoptosis and mineralization in cartilage and vascular tissues, as these are the two tissues mostly affected by a number of age-related diseases having a progressively increased impact in Western Countries.