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BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyt

Cartilage defects and osteoarthritis are health problems which are major burdens on health care systems globally, especially in aging populations. Cartilage is a vulnerable tissue, which generally faces a progressive degenerative process when injured. This makes it the 11th most common cause of global disability. Conservative methods are used to treat the initial phases of the illness, while orthopedic management is the method used for more progressed phases. These include, for instance, arthroscopic shaving, microfracturing and mosaicplasty, and joint replacement as the final treatment. Cell-based implantation methods have also been developed. Despite reports of successful treatments, they often suffer from the non-optimal nature of chondrocyte phenotype in the repair tissue. Thus, improved strategies to control the phenotype of the regenerating cells are needed. Avascular tissue cartilage relies on diffusion for nutrients acquisition and the removal of metabolic waste products. A low oxygen content is also present in cartilage, and the chondrocytes are, in fact, well adapted to it. Therefore, this raises an idea that the regulation of oxygen tension could be a strategy to control the chondrocyte phenotype expression, important in cartilage tissue for regenerative purposes. This narrative review discusses the aspects related to oxygen tension in the metabolism and regulation of articular and growth plate chondrocytes and progenitor cell phenotypes, and the role of some microenvironmental factors as regulators of chondrocytes.

Cartilage defects and osteoarthritis are health problems which are major burdens on health care systems globally, especially in aging populations. Cartilage is a vulnerable tissue, which generally faces a progressive degenerative process when injured. This makes it the 11th most common cause of global disability. Conservative methods are used to treat the initial phases of the illness, while orthopedic management is the method used for more progressed phases. These include, for instance, arthroscopic shaving, microfracturing and mosaicplasty, and joint replacement as the final treatment. Cell-based implantation methods have also been developed. Despite reports of successful treatments, they often suffer from the non-optimal nature of chondrocyte phenotype in the repair tissue. Thus, improved strategies to control the phenotype of the regenerating cells are needed. Avascular tissue cartilage relies on diffusion for nutrients acquisition and the removal of metabolic waste products. A low oxygen content is also present in cartilage, and the chondrocytes are, in fact, well adapted to it. Therefore, this raises an idea that the regulation of oxygen tension could be a strategy to control the chondrocyte phenotype expression, important in cartilage tissue for regenerative purposes. This narrative review discusses the aspects related to oxygen tension in the metabolism and regulation of articular and growth plate chondrocytes and progenitor cell phenotypes, and the role of some microenvironmental factors as regulators of chondrocytes.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet–based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

Introduction: Osteoarthritis (OA) affects over 500 million people worldwide. OA patients are symptomatically treated, and current therapies exhibit marginal efficacy and frequently carry safety-risks associated with chronic use. No disease-modifying therapies have been approved to date leaving surgical joint replacement as a last resort. To enable effective patient care and successful drug development there is an urgent need to uncover the pathobiological drivers of OA and how these translate into disease endotypes. Endotypes provide a more precise and mechanistic definition of disease subgroups than observable phenotypes, and a panel of tissue- and pathology-specific biochemical markers may uncover treatable endotypes of OA. Areas covered: We have searched PubMed for full-text articles written in English to provide an in-depth narrative review of a panel of validated biochemical markers utilized for endotyping of OA and their association to key OA pathologies. Expert opinion: As utilized in IMI-APPROACH and validated in OAI-FNIH, a panel of biochemical markers may uncover disease subgroups and facilitate the enrichment of treatable molecular endotypes for recruitment in therapeutic clinical trials. Understanding the link between biochemical markers and patient-reported outcomes and treatable endotypes that may respond to given therapies will pave the way for new drug development in OA.

Introduction: Osteoarthritis (OA) affects over 500 million people worldwide. OA patients are symptomatically treated, and current therapies exhibit marginal efficacy and frequently carry safety-risks associated with chronic use. No disease-modifying therapies have been approved to date leaving surgical joint replacement as a last resort. To enable effective patient care and successful drug development there is an urgent need to uncover the pathobiological drivers of OA and how these translate into disease endotypes. Endotypes provide a more precise and mechanistic definition of disease subgroups than observable phenotypes, and a panel of tissue- and pathology-specific biochemical markers may uncover treatable endotypes of OA. Areas covered: We have searched PubMed for full-text articles written in English to provide an in-depth narrative review of a panel of validated biochemical markers utilized for endotyping of OA and their association to key OA pathologies. Expert opinion: As utilized in IMI-APPROACH and validated in OAI-FNIH, a panel of biochemical markers may uncover disease subgroups and facilitate the enrichment of treatable molecular endotypes for recruitment in therapeutic clinical trials. Understanding the link between biochemical markers and patient-reported outcomes and treatable endotypes that may respond to given therapies will pave the way for new drug development in OA.

Cartilage defects and osteoarthritis are health problems which are major burdens on health care systems globally, especially in aging populations. Cartilage is a vulnerable tissue, which generally faces a progressive degenerative process when injured. This makes it the 11th most common cause of global disability. Conservative methods are used to treat the initial phases of the illness, while orthopedic management is the method used for more progressed phases. These include, for instance, arthroscopic shaving, microfracturing and mosaicplasty, and joint replacement as the final treatment. Cell-based implantation methods have also been developed. Despite reports of successful treatments, they often suffer from the non-optimal nature of chondrocyte phenotype in the repair tissue. Thus, improved strategies to control the phenotype of the regenerating cells are needed. Avascular tissue cartilage relies on diffusion for nutrients acquisition and the removal of metabolic waste products. A low oxygen content is also present in cartilage, and the chondrocytes are, in fact, well adapted to it. Therefore, this raises an idea that the regulation of oxygen tension could be a strategy to control the chondrocyte phenotype expression, important in cartilage tissue for regenerative purposes. This narrative review discusses the aspects related to oxygen tension in the metabolism and regulation of articular and growth plate chondrocytes and progenitor cell phenotypes, and the role of some microenvironmental factors as regulators of chondrocytes.

Purpose To assess the ability of MRI-DTI to evaluate growth plate morphology and activity compared with that of histomorphometry and micro-CT in rabbits. Methods The hind limbs of female rabbits aged 16, 20, and 24 wk (n = 4 per age group) were studied using a 9.4T MRI scanner with a multi-gradient echo 3D sequence and DTI in 14 directions (b-value = 984 s/mm(2)). After MRI, the right and left hind limb were processed for histological analysis and micro-CT, respectively. The Wilcoxon signed-rank test was used to evaluate the height and volume of the growth plate. Intraclass correlation and Pearson correlation coefficient were used to evaluate the association between DTI metrics and age. Results The growth plate height and volume were similar for all modalities at each time point and age. Age was correlated with all tractography and DTI metrics in both the femur and tibia. A correlation was also observed between all the metrics at both sites. Tract number and volume declined with age; however, tract length did not show any changes. The fractional anisotropy color map showed lateral diffusion centrally in the growth plate and perpendicular diffusion in the hypertrophic zone, as verified by histology and micro-CT. Conclusion MRI-DTI may be useful for evaluating the growth plates., open access

Purpose To assess the ability of MRI-DTI to evaluate growth plate morphology and activity compared with that of histomorphometry and micro-CT in rabbits. Methods The hind limbs of female rabbits aged 16, 20, and 24 wk (n = 4 per age group) were studied using a 9.4T MRI scanner with a multi-gradient echo 3D sequence and DTI in 14 directions (b-value = 984 s/mm(2)). After MRI, the right and left hind limb were processed for histological analysis and micro-CT, respectively. The Wilcoxon signed-rank test was used to evaluate the height and volume of the growth plate. Intraclass correlation and Pearson correlation coefficient were used to evaluate the association between DTI metrics and age. Results The growth plate height and volume were similar for all modalities at each time point and age. Age was correlated with all tractography and DTI metrics in both the femur and tibia. A correlation was also observed between all the metrics at both sites. Tract number and volume declined with age; however, tract length did not show any changes. The fractional anisotropy color map showed lateral diffusion centrally in the growth plate and perpendicular diffusion in the hypertrophic zone, as verified by histology and micro-CT. Conclusion MRI-DTI may be useful for evaluating the growth plates., open access

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

BackgroundTo assess the compound effects of BMI and sustained depressive symptoms on changes in knee structure, cartilage composition, and knee pain over 4 years using statistical interaction analyses.MethodsOne thousand eight hundred forty-four individuals from the Osteoarthritis Initiative Database were analyzed at baseline and 4-year follow-up. Individuals were categorized according to their BMI and presence of depressive symptoms (based on the Center for Epidemiological Studies Depression Scale (threshold≥16)) at baseline and 4-year follow-up. 3 T MRI was used to quantify knee cartilage T2 over 4 years, while radiographs were used to assess joint space narrowing (JSN). Mixed effects models examined the effect of BMI-depressive symptoms interactions on outcomes of cartilage T2, JSN, and knee pain over 4-years.ResultsThe BMI-depressive symptoms interaction was significantly associated with knee pain (p < 0.001) changes over 4 years, but not with changes in cartilage T2 (p = 0.27). In women, the BMI-depressive symptoms interaction was significantly associated with JSN (p = 0.01). In a group-based analysis, participants with obesity and depression had significantly greater 4-year changes in knee pain (coeff.(obesity + depression vs. no_obesity + no_depression) = 4.09, 95%CI = 3.60-4.58, p < 0.001), JSN (coeff. = 0.60, 95%CI = 0.44-0.77, p < 0.001), and cartilage T2 (coeff. = 1.09, 95%CI = 0.68-1.49, p < 0.001) than participants without depression and normal BMI.ConclusionsThe compound effects of obesity and depression have greater impact on knee pain and JSN progression compared to what would be expected based on their individual effects.

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhanc

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhanc

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

Objective: To provide orientation-independent MR parameters potentially sensitive to articular cartilage degeneration by measuring isotropic and anisotropic components of T₂ relaxation, as well as 3D fiber orientation angle and anisotropy via multi-orientation MR scans. Approach: Seven bovine osteochondral plugs were scanned with a high angular resolution of thirty-seven orientations spanning 180° at 9.4 T. The obtained data was fitted to the magic angle model of anisotropic T₂ relaxation to produce pixel-wise maps of the parameters of interest. Quantitative Polarized Light Microscopy (qPLM) was used as a reference method for the anisotropy and fiber orientation. Main results: The number of scanned orientations was found to be sufficient for estimating both fiber orientation and anisotropy maps. The relaxation anisotropy maps demonstrated a high correspondence with qPLM reference measurements of the collagen anisotropy of the samples. The scans also enabled calculating orientation-independent T₂ maps. Little spatial variation was observed in the isotropic component of T₂ while the anisotropic component was much faster in the deep radial zone of cartilage. The estimated fiber orientation spanned the expected 0°–90° in samples that had a sufficiently thick superficial layer. The orientation-independent magnetic resonance imaging (MRI) measures can potentially reflect the true properties of articular cartilage more precisely and robustly. Significance: The methods presented in this study will likely improve the specificity of cartilage qMRI by allowing the assessment of the physical properties such as orientation and anisotropy of collagen fibers in articular cartilage.

BackgroundHip minimum joint space width (mJSW) provides a proxy for cartilage thickness. This study aimed to conduct a genome-wide association study (GWAS) of mJSW to (i) identify new genetic determinants of mJSW and (ii) identify which mJSW loci convey hip osteoarthritis (HOA) risk and would therefore be of therapeutic interest.MethodsGWAS meta-analysis of hip mJSW derived from plain X-rays and DXA was performed, stratified by sex and adjusted for age and ancestry principal components. Mendelian randomisation (MR) and cluster analyses were used to examine causal effect of mJSW on HOA.Findings50,745 individuals were included in the meta-analysis. 42 SNPs, which mapped to 39 loci, were identified. Mendelian randomisation (MR) revealed little evidence of a causal effect of mJSW on HOA (ORIVW 0.98 [95% CI 0.82-1.18]). However, MR-Clust analysis suggested the null MR estimates reflected the net effect of two distinct causal mechanisms cancelling each other out, one of which was protective, whereas the other increased HOA susceptibility. For the latter mechanism, all loci were positively associated with height, suggesting mechanisms leading to greater height and mJSW increase the risk of HOA in later life.InterpretationsOne group of mJSW loci reduce HOA risk via increased mJSW, suggesting possible utility as targets for chondroprotective therapies. The second group of mJSW loci increased HOA risk, despite increasing mJSW, but were also positively related to height, suggesting they contribute to HOA risk via a growth-related mechanism.FundingPrimarily funded by the Medical Research Council and Wellcome Trust.

Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene (DDR2), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.

BackgroundThe use of coring instrumentation for osteochondral allograft (OCA) transplantation of the femoral trochlea is challenging due to the complex topography of this anatomical area.PurposeWe sought to determine the effect of flat guides versus guides contoured to the surface of the trochlea on graft step-off in trochlear OCAs performed on a foam bone model. We also determined the effect of surgeon experience level and graft size on step-off.Study designControlled laboratory study.MethodsAllograft harvesters were produced in 3 different sequential sizes with either a flat or a contoured undersurface. The guides matched one another in all aspects of shape and size except for the undersurface contour. The contoured undersurface generally matched the surface of the bone model trochlea but was not customized to that surface. A total of 72 foam femora were obtained. Identical trochlear stellate lesions of 3 different sizes (small, medium, and large) were created using 3-dimensional printed surface templates. A total of 6 surgeons (3 attending and 3 resident surgeons) performed OCAs of each trochlear lesion. Each surgeon performed 1 graft for each size and each guide type (n = 6 per surgeon). A specialized digital caliper was prepared that allowed the measurement of graft step-off to within 0.01 mm at a distance of 5 mm between the 2 sensors. The measurements were performed at 8 positions around the clockface of the grafts.ResultsGrafts performed with the contoured guides had a mean step-off of 0.43 ± 0.37 mm. Grafts performed with the flat guides had a mean step-off of 0.74 ± 0.78 mm (P < .0001). Experience level did not have a significant effect on graft step-off (P = .81). There was no identifiable pattern indicating higher step-off at any one position on the clockface.ConclusionIn this study, contoured grafts had significantly lower step-offs compared with flat guides. Experience level, clockface position, and graft size did not affect step-off.

SignificanceCartilage tissue engineering is a promising strategy for effective curative therapies for treatment of osteoarthritis. However, tissue engineers depend predominantly on time-consuming, expensive, and destructive techniques as quality control to monitor the maturation of engineered cartilage. This practice can be impractical for large-scale biomanufacturing and prevents spatial and temporal monitoring of tissue growth, which is critical for the fabrication of clinically relevant-sized cartilage constructs. Nondestructive multimodal imaging techniques combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) hold great potential to address this challenge.AimThe feasibility of using multimodal FLIm-OCT for nondestructive, spatial, and temporal monitoring of self-assembled cartilage tissue maturation in a preclinical mouse model is investigated.ApproachSelf-assembled cartilage constructs were developed for 4 weeks in vitro followed by 4 weeks of in vivo maturation in nude mice. Sterile and nondestructive in situ multispectral FLIm and OCT imaging were carried out at multiple time points ( t=2 , 4, and 8 weeks) during tissue development. FLIm and 3D volumetric OCT images were reconstructed and used for the analysis of tissue biochemical homogeneity, morphology, and structural integrity. A biochemical homogeneity index was computed to characterize nonhomogeneous tissue growth at different time points. OCT images were validated against histology.ResultsFLIm detects heterogenous extracellular matrix (ECM) growth of tissue-engineered cartilage. The outer edge of the tissue construct exhibited longer fluorescence lifetime in 375 to 410 and 450 to 485 nm spectral channels, indicating increase in collagen content. Significant ( p<0.05 ) decrease of construct homogeneity index was observed between t=2 weeks and t=4 weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture

Femoroacetabular impingement (FAI) has a strong clinical association with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain poorly understood. The purpose of this study is to use whole-genome RNA sequencing to identify and subsequently validate differentially expressed genes (DEGs) in femoral head articular cartilage samples from patients with FAI and hip OA secondary to FAI. Thirty-seven patients were included in the study with whole-genome RNA sequencing performed on 10 gender-matched patients in the FAI and OA cohorts and the remaining specimens were used for validation analyses. We identified a total of 3531 DEGs between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways. Quantitative reverse transcription-polymerase chain reaction validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage compared to FAI samples with severe cartilage damage or OA cartilage. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. We independently validated the results of the sequencing analysis and found increased expression of anabolic markers in patients with FAI and minimal histologic cartilage damage, suggesting that anabolic signaling may be increased in early FAI with a transition to catabolic and inflammatory gene expression as FAI progresses towards more severe hip OA. Clinical significance:Cam-type FAI has a strong clinical association with hip OA; however, the cellular pathophysiology of disease progression remains poorly understood. Several previous studies have demonstrated increased expression of inflammatory markers in FAI cartil

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by pathogenic variants of cartilage oligomeric matrix protein (COMP). Clinical symptoms of PSACH are characterized by growth disturbances after the first year of life. These disturbances lead to severe short stature with short limbs, brachydactyly, scoliosis, joint laxity, joint pain since childhood, and a normal face. Epimetaphyseal dysplasia, shortened long bones, and short metacarpals and phalanges are common findings on radiological examination. Additionally, anterior tonguing of the vertebral bodies in the lateral view is an important finding in childhood because it is specific to PSACH and normalizes with age. Here, we report five Japanese patients with PSACH, with one recurrent (p.Cys351Tyr) and four novel heterozygous pathogenic COMP variants (p.Asp437Tyr, p.Asp446Gly, p.Asp507Tyr, and p.Asp518Val). These five pathogenic variants were located in the calcium-binding type 3 (T3) repeats. In four of the novel variants, the affected amino acid was aspartic acid, which is abundant in each of the eight T3 repeats. We describe the radiological findings of these five patients. We also retrospectively analyzed the sequential changes in the vertebral body and epimetaphysis of the long bones from the neonatal to infantile periods in a patient with PSACH and congenital heart disease.

In this work a mathematical model for the interaction of two key signalling molecules in rat tibia ossification is presented and discussed. The molecules under consideration are Indian hedgehog (Ihh) and parathyroid hormone-related peptide (PTHrP). These are known to be major agents in the dynamics of the so-called growth plate, where transition from pristine cartilage to advancing bone takes place. Our model consists in a steady-state linear approximation to a reaction-diffusion system where only diffusion and absorption mechanisms are retained. Estimates on some system parameters are given, on the basis of the knowledge of a few measurable quantities. This allows for explicitly solving our model, whereupon a discussion on robustness and regulatory properties thereof is provided. In particular, we show that the size of the Proliferative Zone in the growth plate is rather insensitive to variations in the flux coefficients for Ihh and PTHrP at their boundaries. Besides, we also show that the model is also insensitive to large changes in the (comparatively small) critical value of the PTHrP concentration which marks the transition form Proliferative to Hyperthropic Regions within the Growth Plate. These results hold irrespective of the particular diffusivities selected for Ihh and PTHrP., MICINN, Depto. de Análisis Matemático y Matemática Aplicada, Fac. de Ciencias Matemáticas, TRUE, pub

OBJECTIVE: The medial femoral condyle of the knee exhibits some of the highest incidences of chondral degeneration. However, a dearth of healthy human tissues has rendered it difficult to ascertain whether cartilage in this compartment possesses properties that predispose it to injuries. Assessment of young, healthy tissue would be most representative of the tissues intrinsic properties. DESIGN: This work examined the topographical differences in tribological, tensile, and compressive properties of young (n = 5, 26.2 ± 5.6 years old), healthy, human medial femoral condyles, obtained from viable allograft specimens. Corresponding to clinical incidences of pathology, it was hypothesized that the lowest mechanical properties would be found in the posterior region of the medial condyle, and that tissue composition would correspond to the established structure-function relationships of cartilage. RESULTS: Youngs modulus, ultimate tensile strength, aggregate modulus, and shear modulus in the posterior region were 1.0-, 2.8-, 1.1-, and 1.0-fold less than the values in the anterior region, respectively. Surprisingly, although glycosaminoglycan content is thought to correlate with compressive properties, in this study, the aggregate and shear moduli correlated more robustly to the amount of pyridinoline crosslinks per collagen. Also, the coefficient of friction was anisotropic and ranged 0.22-0.26 throughout the condyle. CONCLUSION: This work showed that the posteromedial condyle displays lower tensile and compressive properties, which correlate to collagen crosslinks and may play a role in this regions predisposition to injuries. Furthermore, new structure-function relationships may need to be developed to account for the role of collagen crosslinks in compressive properties.

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

Background: Early detection of post-traumatic cartilage damage in the ankle joint in magnetic resonance images can be difficult due to disturbances to structures usually appearing over time. Purpose: To study the articular cartilage of unilateral Weber type-B/SER-type ankle fractures shortly post-trauma using T2 relaxation time. Material and Methods: Fifty one fractured ankles were gathered from consecutively screened patients, compiled initially for RCT studies, and treated at Oulu University Hospital and classified as stable (n = 28) and unstable fractures (n = 23) based on external-rotation stress test: medial clear space of ≥5 mm was interpreted as unstable. A control group of healthy young individuals (n = 19) was also gathered. All ankles were imaged on average 9 (range: 1 to 25) days after injury on a 3.0T MRI unit for T2 relaxation time assessment, and the cartilage was divided into sub-regions for comparison. Results: Control group displayed significantly higher T2 values in tibial cartilage compared to stable (six out of nine regions, p-values = .003–.043) and unstable (six out of nine regions, p-values = .001–.037) ankle fractures. No differences were detected in talar cartilage. Also, no differences were observed between stable and unstable fractures in tibial or talar cartilage. Conclusions: Lower T2 relaxation times of tibial cartilage in fractured ankles suggest intact extra cellular matrix (ECM) of the cartilage. Severity of the ankle fracture, measured by ankle stability, does not seem to increase ECM degradation immediately after trauma.

[Abstract] It is well known that patients with attention deficit hyperactivity disorder treated with stimulants, such as methylphenidate hydrochloride (MPH), have reduced height and weight. Even though MPH has an anorexigenic effect, an additional impact of this drug on the growth plate cannot be discarded. In this study, we aimed to determine the cellular effect of MPH on an in vitro growth plate model. We tested the effects of MPH on the viability and proliferation of a prechondrogenic cell line via an MTT assay. In vitro differentiation of this cell line was performed, and cell differentiation was evaluated through the expression of cartilage- and bone-related genes as measured via RT-PCR. MPH did not alter the viability or proliferation of prechondrogenic cells. However, it reduced the expression of cartilage extracellular matrix-related genes (type II collagen and aggrecan) and increased the expression of genes involved in growth plate calcification (Runx2, type I collagen, and osteocalcin) at different phases of their differentiation process. Our results evidence that MPH upregulates genes associated with growth plate hypertrophic differentiation. This may induce premature closure of the growth plate, which would contribute to the growth retardation that has been described to be induced by this drug.

The biological and biomechanical functions of cartilage, bone and osteochondral tissue are naturally orchestrated by a complex crosstalk between zonally dependent cells and extracellular matrix components. In fact, this crosstalk involves biomechanical signals and the release of biochemical cues that direct cell fate and regulate tissue morphogenesis and remodelling in vivo. Three-dimensional bioprinting introduced a paradigm shift in tissue engineering and regenerative medicine, since it allows to mimic native tissue anisotropy introducing compositional and architectural gradients. Moreover, the growing synergy between bioprinting and drug delivery may enable to replicate cell/extracellular matrix reciprocity and dynamics by the careful control of the spatial and temporal patterning of bioactive cues. Although significant advances have been made in this direction, unmet challenges and open research questions persist. These include, among others, the optimization of scaffold zonality and architectural features; the preservation of the bioactivity of loaded active molecules, as well as their spatio-temporal release; the in vitro scaffold maturation prior to implantation; the pros and cons of each animal model and the graft-defect mismatch; and the in vivo non-invasive monitoring of new tissue formation. This work critically reviews these aspects and reveals the state of the art of using three-dimensional bioprinting, and its synergy with drug delivery technologies, to pattern the distribution of cells and/or active molecules in cartilage, bone and osteochondral engineered tissues. Most notably, this work focuses on approaches, technologies and biomaterials that are currently under in vivo investigations, as these give important insights on scaffold performance at the implantation site and its interaction/integration with surrounding tissues

Compositional changes in the chemical makeup of hydrogels offer a powerful strategy for fine tuning of mechanical properties, enabling specific targeting for different applications. The chemical versatility exhibited by the tunable system introduced here can be leveraged to address a broad range of characteristics across the field of tissue engineering-from blood vessels to cartilage, for example-which demands materials with very different mechanical profiles. Furthermore, we rely exclusively on dynamic, non-covalent cross-linking to provide opportunities for 3D printing and injectability. This work describes a highly tunable system based on hydrogen bonding and ionic interactions. Single network hydrogels were made by exploiting various acrylic monomers including N-acryloyl glycinamide (NAGA) and acrylic acid (AAc). Additionally, hybrid hydrogels were explored by combining these acrylic networks with an ionically cross-linked alginate network. By combining orthogonal cross-linking strategies and altering the ratio between different components in these hybrid gels, a broad range of mechanical properties is demonstrated. The characteristics were extensively investigated using tensile testing, compression testing, and rheological measurements. The final scaffolds were also shown to be non-cytotoxic in preliminary cell viability studies for human dermal fibroblasts.

Objective: Quantitative Magnetic Resonance Imaging (MRI) holds great promise for the early detection of cartilage deterioration. Here, a Magnetic Resonance Fingerprinting (MRF) framework is proposed for comprehensive and rapid quantification of T 1, T 2 ∗, and T RAFF2 with whole-knee coverage. Methods: A MRF framework was developed to achieve quantification of Relaxation Along a Fictitious Field in the 2nd rotating frame of reference (T RAFF2) along with T 1 and T 2 ∗. The proposed sequence acquires 65 measurements of 25 high-resolution slices, interleaved with 7 inversion pulses and 40 RAFF2 trains, for whole-knee quantification in a total acquisition time of 3:25 min. Comparison with reference T 1, T 2 ∗, and T RAFF2 methods was performed in phantom and in seven healthy subjects at 3 T. Repeatability (test-retest) with and without repositioning was also assessed. Results: Phantom measurements resulted in good agreement between MRF and the reference with mean biases of -54, 2, and 5 ms for T 1, T 2 ∗, and T RAFF2, respectively. Complete characterization of the whole-knee cartilage was achieved for all subjects, and, for the femoral and tibial compartments, a good agreement between MRF and reference measurements was obtained. Across all subjects, the proposed MRF method yielded acceptable repeatability without repositioning (R 2≥ 0.94) and with repositioning (R 2≥ 0.57) for T 1, T 2 ∗, and T RAFF2. Significance: The short scan time combined with the whole-knee coverage makes the proposed MRF framework a promising candidate for the early assessment of cartilage degeneration with quantitative MRI, but further research may be warranted to improve repeatability after repositioning and assess clinical value in patients., ImPhys/Weingärtner group, ImPhys/Medical Imaging, ImPhys/Computational Imaging

The imbalance between life expectancy and quality of life is increasing due to the raising prevalence of chronic diseases. Musculoskeletal disorders and chronic wounds affect a growing percentage of people and demand more efficient tools for regenerative medicine. Scaffolds that can better mimic the natural physical stimuli that tissues receive under healthy conditions and during healing may significantly aid the regeneration process. Shape, mechanical properties, pore size and interconnectivity have already been demonstrated to be relevant scaffold features that can determine cell adhesion and differentiation. Much less attention has been paid to scaffolds that can deliver more dynamic physical stimuli, such as electrical signals. Recent developments in the precise measurement of electrical fields in vivo have revealed their key role in cell movement (galvanotaxis), growth, activation of secondary cascades, and differentiation to different lineages in a variety of tissues, not just neural. Piezoelectric scaffolds can mimic the natural bioelectric potentials and gradients in an autonomous way by generating the electric stimuli themselves when subjected to mechanical loads or, if the patient or the tissue lacks mobility, ultrasound irradiation. This review provides an analysis on endogenous bioelectrical signals, recent developments on piezoelectric scaffolds for bone, cartilage, tendon and nerve regeneration, and their main outcomes in vivo. Wound healing with piezoelectric dressings is addressed in the last section with relevant examples of performance in animal models. Results evidence that a fine adjustment of material composition and processing (electrospinning, corona poling, 3D printing, annealing) provides scaffolds that act as true emitters of electrical stimuli that activate endogenous signaling pathways for more efficient and long-term tissue repair

Current regenerative cartilage therapies are associated with several drawbacks such as dedifferentiation of chondrocytes during expansion and the formation of fibrocartilage. Optimized chondrocyte expansion and tissue formation could lead to better clinical results of these therapies. In this study, a novel chondrocyte suspension expansion protocol that includes the addition of porcine notochordal cell-derived matrix was used to self-assemble human chondrocytes from osteoarthritic (OA) and nondegenerate (ND) origin into cartilage organoids containing collagen type II and proteoglycans. Proliferation rate and viability were similar for OA and ND chondrocytes and organoids formed had a similar histologic appearance and gene expression profile. Organoids were then encapsulated in viscoelastic alginate hydrogels to form larger tissues. Chondrocytes on the outer bounds of the organoids produced a proteoglycan-rich matrix to bridge the space between organoids. In hydrogels containing ND organoids some collagen type I was observed between the organoids. Surrounding the bulk of organoids in the center of the gels, in both OA and ND gels a continuous tissue containing cells, proteoglycans and collagen type II had been produced. No difference was observed in sulphated glycosaminoglycan and hydroxyproline content between gels containing organoids from OA or ND origin after 28 days. It was concluded that OA chondrocytes, which can be harvested from leftover surgery tissue, perform similar to ND chondrocytes in terms of human cartilage organoid formation and matrix production in alginate gels. This opens possibilities for their potential to serve as a platform for cartilage regeneration but also as an in vitro model to study pathways, pathology, or drug development.

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhanc

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p ​= ​0.390) and led to a higher migration score ex vivo (p ​< ​0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p ​= ​0.031) and ex vivo (p ​< ​0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell mig, Biomaterials & Tissue Biomechanics

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhanc

Repair of osteochondral defects and regeneration of cartilage is a major challenge. In this work, the mesenchymal stem cells (MSCs)-laden hydrogel was designed using silk fibroin (SF) and gelatin methacrylate (GelMA), to encapsulate platelet-rich plasma (PRP). Initially, GelMA was synthesized, and SF was prepared using silkworm cocoon, then MSCs-laden SF/GelMA (SG) hydrogel was fabricated. The physicochemical properties of the hydrogels were evaluated using Fourier-transform infrared spectroscopy, scanning electron microscope, and rheometry. After hydrogel preparation, the viability of MSCs in the hydrogels was investigated via CCK-8 analysis and fluorescent images. The MSCs-laden SG hydrogel containing PRP was subsequently injected into the cartilage defect area in Sprague Dawley rats. Hematoxylin and eosin (H&E), Masson staining, and Mankin scores evaluation confirmed the new cartilage formation in 8 weeks. The results presented in the study, therefore, showed that the prepared MSCs-laden SG hydrogel loaded with PRP has the potential for cartilage reconstruction, which is crucial to the treatment of knee osteoarthritis., SCOPUS: ar.j, info:eu-repo/semantics/published

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from

The identification of skeletal elements, the analysis of their developmental sequence, and the time of their appearance during larval development are essential to broaden the knowledge of each fish species and to recognize skeletal abnormalities that may affect further fish performance. Therefore, this study aimed to provide a general description of the development of the entire skeleton highlighting its variability in Cichlasoma dimerus. Larvae of C. dimersus were stained with alcian blue and alizarin red from hatching to 25 days posthatching. Skeletogenesis began with the endoskeletal disk and some cartilage structures from the caudal fin and the splachnocranium, while the first bony structure observed was the cleithrum. When larvae reached the free-swimming and exogenous feeding stage, mostly bones from the jaws, the branchial arches, and the opercle series evidenced some degree of ossification, suggesting that the ossification sequence of C. dimerus adjusts to physiological demands such as feeding and ventilation. The caudal region was the most variable regarding meristic counts and evidenced higher incidence of bone deformities. In conclusion, this work provides an overview of C. dimerus skeletogenesis and lays the groundwork for further studies on diverse topics, like developmental plasticity, rearing conditions, or phylogenetic relationships.

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.