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Diese Arbeit beschäftigt sich mit der Austestung verschiedener Antikörper zum Nachweis der Proteine Kollagen Typ I, II und X. Es wird bezüglich Kollagene viel auf RNA-Ebene geforscht. Leider ist in der Literatur nur sehr wenig auf Protein-Ebene zu finden. Kollagen ist das im menschlichen Körper am häufigsten vorkommende Protein. Vor allem ist es in Sehnen, Knorpel, Knochen und Haut zu finden. Zelladhäsion, Zellmigration, Angiogenese und Gewebereparatur zählen zu den verschiedenen Aufgaben der Kollagene. Kollagen Typ I ist Teil von Bändern, Sehnen, Haut, Blutgefäßen und Basalmembranen, denen es auch seine Festigkeit und Dehnbarkeit verleiht. Kollagen Typ II ist das wichtigste Kollagen der Epiphysenfuge, auch zu finden im Corpus vitreum, in den Bandscheiben und im Innenohr. Während der endochondralen Ossifikation dient Kollagen Typ X als bedeutender Marker für hypertrophe Chondrozytendifferenzierung. Defekte oder Mutationen in Kollagenen sind mit verschiedenen Krankheiten assoziiert. Nach Differenzierung der murinen chondrogenen Zelllinie ATDC5 wurde Protein aus den Zellen gewonnen. Die Austestung der Antikörper erfolgte mittels Western-Blot-Verfahren und dem Gerät „Jess“ der Firma Bio-Techne, welches die Automatisierung der Proteinanalyse ermöglicht. „Jess“ steht der Universitätsklinik für Kinder- und Jugendheilkunde seit Februar 2022 zur Verfügung und wird während dieser Arbeit ausgetestet. Mit dem Western-Blot-Verfahren gelang der Nachweis von Kollagen Typ I mit drei von fünf Antikörpern. Für Kollagen Typ II wurden drei Antikörper ausgetestet, wobei nur ein Antikörper ein spezifisches Signal im Western Blot zeigte. Der Nachweis von Kollagen Typ X gelang lediglich mit einem Antikörper, wobei zwei ausgetestet wurden. Bei der Durchführung der Proteinanalyse mit dem Gerät „Jess“ war nur ein Antikörper in der Lage Kollagen Typ I zu detektieren. Kollagen Typ X konnte mit beiden ausgewählten Antikörpern detektiert werden. Es gelang nicht Kollagen Typ II mit „Jess“ nachzuweisen. Auf Basis der Zelllinie ATDC5 funktionierten die meisten der ausgewählten Antikörper nicht. Die Antikörper gingen viele unspezifische Bindungen in der Probe ein. This work deals with the testing of various antibodies to detect the proteins collagen type I, II, and X. There is a lot of research being done on collagens at the RNA level, but unfortunately, very little can be found on the protein level in the literature. Collagen is the most common protein in the human body and is mainly found in tendons, cartilage, bones, and skin. Cell adhesion, cell migration, angiogenesis, and tissue repair are among the various functions of collagens. Collagen type I is part of ligaments, tendons, skin, blood vessels, and basement membranes, which also give it its strength and elasticity. Collagen type II is the most important collagen in the epiphyseal plate, and is also found in the vitreous body, intervertebral discs, and inner ear. During endochondral ossification, collagen type X serves as a significant marker for hypertrophic chondrocyte differentiation. Defects or mutations in collagens are associated with various diseases. After differentiation of the murine chondrogenic cell line ATDC5, protein was obtained from the cells. The antibodies were tested using the Western blot method and the "Jess" device from Bio-Techne, which allows for the automation of protein analysis. "Jess" has been available to the University Hospital for Pediatrics and Adolescent Medicine since February 2022 and is being tested during this work. With the Western blot method, collagen type I was detected using three out of five antibodies. For collagen type II, three antibodies were tested, with only one antibody showing a specific signal in the Western blot. Collagen type X was detected with one antibody, although two were tested. When performing protein analysis with the "Jess" device, only one antibody was able to detect collagen type I. Collagen type X was detected with both selected antibodies. However, collagen type II could not be detected with "Jess". Based on the ATDC5 cell line, most of the selected antibodies did not work. The antibodies formed many non-specific bindings in the sample.

As a widely used steroid hormone medicine, glucocorticoids have the potential to cause steroid-induced osteonecrosis of the femoral head (SONFH) due to mass or long-term use. The non-coding RNA hypothesis posits that they may contribute to the destruction and dysfunction of cartilages as a possible etiology of SONFH. MiR-30b-5p was identified as a regulatory factor in cartilage degeneration caused by methylprednisolone (MPS) exposure in our study through cell transfection. The luciferase reporter assay confirmed that miR-30b-5p was downregulated and runt-related transcription factor 2 (Runx2) was mediated by miR-30b-5p. The nobly increased expression of matrix metallopeptidase 13 (MMP13) and type X collagen (Col10a1) as Runx2 downstream genes contributed to the hypertrophic differentiation of chondrocytes, and the efficiently upregulated level of matrix metallopeptidase 9 (MMP9) may trigger chondrocyte apoptosis with MPS treatments. The cell transfection experiment revealed that miR-30b-5p inhibited chondrocyte hypertrophy and suppressed MPS-induced apoptosis. As a result, our findings showed that miR-30b-5p modulated Runx2, MMP9, MMP13, and Col10a1 expression, thereby mediating chondrocyte hypertrophic differentiation and apoptosis during the SONFH process. These findings revealed the mechanistic relationship between non-coding RNA and SONFH, providing a comprehensive understanding of SONFH and other bone diseases.

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Background Developmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MicroRNAs (miRNA) are important regulators of cartilage development. In a previous study, high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim here was to investigate the effects of miR-1-3p in the ARC. Methods The diagnosis of acetabular dysplasia was confirmed with X-ray examination, while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model. FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes. The miR-1-3p target genes were predicted by a bioinformatics database. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X was evaluated by qPCR analysis. The protein expression of SOX9, RUNX2 and collagen type X was detected by western blot analysis. The levels of SOX9, RUNX2, and collagen type X in the ARC were further assessed via immunohistochemistry analysis. Finally, Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes. Results We observed low expression of miR-1-3p in the ARC of animals with DDH. SOX9 is a miR-1-3p target gene. Using miR-1-3p silencing technology in vitro, we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control. We also confirmed that with miR-1-3p silencing, SOX9 expression was upregulated, whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression. Finally, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry, RT-PCR, and western blots. Conclusion The relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.

Objective: We here aimed to characterize changes of Matrix Gla Protein (MGP) expression in relation to its recently identified OA risk allele rs1800801-T in OA cartilage, subchondral bone and human ex vivo osteochondral explants subjected to OA related stimuli. Given that MGP function depends on vitamin K bioavailability, we studied the effect of frequently prescribed vitamin K antagonist warfarin. Methods: Differential (allelic) mRNA expression of MGP was analyzed using RNA-sequencing data of human OA cartilage and subchondral bone. Human osteochondral explants were used to study exposures to interleukin one beta (IL-1b; inflammation), triiodothyronine (T3; Hypertrophy), warfarin, or 65% mechanical stress (65%MS) as function of rs1800801 genotypes. Results: We confirmed that the MGP risk allele rs1800801-T was associated with lower expression and that MGP was significantly upregulated in lesioned as compared to preserved OA tissues, mainly in risk allele carriers, in both cartilage and subchondral bone. Moreover, MGP expression was downregulated in response to OA like triggers in cartilage and subchondral bone and this effect might be reduced in carriers of the rs1800801-T risk allele. Finally, warfarin treatment in cartilage increased COL10A1 and reduced SOX9 and MMP3 expression and in subchondral bone reduced COL1A1 and POSTN expression. Discussion & conclusions: Our data highlights that the genetic risk allele lowers MGP expression and upon OA relevant triggers may hamper adequate dynamic changes in MGP expression, mainly in carti-lage. The determined direct negative effect of warfarin on human explant cultures functionally un-derscores the previously found association between vitamin K deficiency and OA. (c) 2021 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-β1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

COL10A1 is a secreted, short-chain collagen found in several types of cancer. Studies have shown that COL10A1 aberrant expression is considered an oncogenic factor. However, its underlying mechanisms and regulation of gastric cancer remain undefined.The data on the expression of COL10A1, clinicopathological characteristics, and outcome of patients with GC were obtained from The Cancer Genome Atlas. The ALGGEN-PROMO database defined the related transcription factors. Quantitative real-time reverse transcription-polymerase chain reaction and western blotting analysis were used to identify the differential expression levels of COL10A1 and related transcription factors.We found that high COL10A1 expression is an independent risk factor for gastric cancer. Upregulation of LEF1 and Wnt2 was also observed in gastric cancer, suggesting a potential correlation between LEF1/COL10A1 regulation in the Wnt2 signaling pathway.High COL10A1 expression may contribute to poor outcomes via upregulation of LEF1 and Wnt2 in gastric cancer.

Summary Objective The present study is to characterize the role of long intergenic non-coding RNA, regulator of reprogramming (linc-ROR) in bone marrow mesenchymal stem cell (BMSCs) chondrogenesis, cartilage formation and OA development. Methods Linc-ROR expression pattern in articular cartilage tissue sample from OA patients were studied by real-time PCR. Linc-ROR lentivirus mediated BMSCs were constructed. In vitro micromass cultured BMSCs chondrogenesis or in vivo MeHA hydrogel encapsulated BMSCs cartilage formation activity were studied. Linc-ROR associating miRNAs which repressed SOX9 expression were characterized by luciferase assay, real-time PCR and Western blot. Linc-ROR was co-transfected with miRNAs into BMSCs to study its rescue effect on SOX9 expression and chondrogenesis activity. Results Linc-ROR was down-regulated in articular cartilage tissue from OA patients and was positively correlated with the expression level of SOX9 (R2 = 0.43). Linc-ROR expression was upregulated during BMSCs chondrogenesis. Linc-ROR ectopic expression significantly promoted in vitro BMSCs chondrogenesis and in vivo cartilage formation activities as revealed by safranin O, alcian blue and COL II staining. The mRNA expression level of chondrogenesis markers including COL II, SOX9 and ACAN were increased, and the hypertrophy markers MMP13 and COL X were decreased upon linc-ROR overexpression in BMSCs. Linc-ROR functioned as a miRNA sponge for miR-138 and miR-145. Both miR-138 and miR-145 suppressed BMSCs chondrogenesis activity and SOX9 expression, while co-expression of linc-ROR displayed a rescuing effect. Conclusions Taken together, linc-ROR modulated BMSCs chondrogenesis differentiation and cartilage formation by acting as a competing endogenous RNA for miR-138 and miR-145 and activating SOX9 expression. Linc-ROR could be considered as a new diagnostic and therapeutic target for OA treatment.

Summary Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3., Highlights • A new induction method enables hypertrophic chondrocyte differentiation from iPSCs • Chondrodysplasia mutants show intracellular MATN3 or COL10 accumulation • Some, but not all, mutants have ER stress with an unfolded protein response • This induction system is applicable to transcriptomic analysis and drug development, Chondrodysplasias are highly heterogeneous genetic cartilage diseases. Here, Toguchida and colleagues establish a new induction system to differentiate hypertrophic chondrocytes from iPSCs and analyze these disorders in vitro. They found that different chondrodysplasia mutants, despite mutations being in the same gene, showed varying phenotypes and transcriptomic changes. This system provides an initial platform for further investigation and drug development.

Context Height velocity (HV) is difficult to assess because growth is very slow. The current practice of calculating it from measurements taken at several-month intervals is insufficient for managing children with growth disorders. We identified a bone growth by-product (collagen X biomarker, CXM) in blood that in preliminary analysis in healthy children correlated strongly with conventionally determined HV and displayed a pattern resembling published norms for HV vs age. Objective The goal was to confirm our initial observations supporting the utility of CXM as an HV biomarker in a larger number of individuals and establish working reference ranges for future studies. Design, Settings, and Participants CXM was assessed in archived blood samples from 302 healthy children and 10 healthy adults yielding 961 CXM measurements. A total of 432 measurements were plotted by age, and sex-specific reference ranges were calculated. Serial values from 116 participants were plotted against observed HV. Matched plasma, serum, and dried blood spot readings were compared. Results A correlation of blood CXM with conventional HV was confirmed. Scatter plots of CXM vs age showed a similar pattern to current HV norms, and CXM levels demarcated the pubertal growth spurt both in girls and boys. CXM levels differed little in matched serum, plasma, and dried blood spot samples. Conclusions Blood CXM offers a potential means to estimate HV in real time. Our results establish sex-specific, working reference ranges for assessing skeletal growth, especially over time. CXM stability in stored samples makes it well suited for retrospective studies.

As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.

Acute kidney injury (AKI) is an independent risk factor for chronic kidney disease (CKD). However, the role and mechanism of microRNA (miRNA, miR) in AKI-CKD transition are elusive. In this study, a murine model of renal ischemia/reperfusion was established to investigate the repairing effect and mechanism of miR-101a-3p on renal injury. The pathological damage of renal tissue was observed by hematoxylin and eosin and Masson staining. The levels of miR-101, profibrotic cytokines, and epithelial–mesenchymal transition (EMT) markers were analyzed using Western blotting, real-time polymerase chain reaction, and/or immunofluorescence. MiR-101 overexpression caused the downregulation of α-smooth muscle actin, collagen-1, and vimentin, as well as upregulation of E-cadherin, thereby alleviating the degree of renal tissue damage. MiR-101 overexpression mitigated hypoxic HK-2 cell damage. Collagen, type X, alpha 1 and transforming growth factor β receptor 1 levels were downregulated in hypoxic cells transfected with miR-101 mimic. Our study indicates that miR-101 is an anti-EMT miRNA, which provides a novel therapeutic strategy for AKI-CKD transition.

OBJECTIVE: Inflammation and innate immune responses may contribute to development and progression of Osteoarthritis (OA). Chondrocytes are the sole cell type of the articular cartilage and produce extracellular-matrix molecules. How inflammatory mediators reach chondrocytes is incompletely understood. Previous studies have shown that chondrocytes express mRNA encoding complement proteins such as C1q, suggesting local protein production, which has not been demonstrated conclusively. The aim of this study is to explore C1q production at the protein level by chondrocytes. DESIGN: We analysed protein expression of C1q in freshly isolated and cultured human articular chondrocytes using Western blot, ELISA and flow cytometry. We examined changes in mRNA expression of collagen, MMP-1 and various complement genes upon stimulation with pro-inflammatory cytokines or C1q. mRNA expression of C1 genes was determined in articular mouse chondrocytes. RESULTS: Primary human articular chondrocytes express genes encoding C1q, C1QA, C1QB, C1QC, and secrete C1q to the extracellular medium. Stimulation of chondrocytes with pro-inflammatory cytokines upregulated C1QA, C1QB, C1QC mRNA expression, although this was not confirmed at the protein level. Extracellular C1q bound to the chondrocyte surface dose dependently. In a pilot study, binding of C1q to chondrocytes resulted in changes in the expression of collagens with a decrease in collagen type 2 and an increase in type 10. Mouse articular chondrocytes also expressed C1QA, C1QB, C1QC, C1R and C1S at the mRNA level. CONCLUSIONS: C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression. ispartof: OSTEOARTHRITIS AND CARTILAGE vol:28 issue:5 pages:675-684 ispartof: location:England status: published

The treatment for intervertebral disc degeneration (IDD) has drawn great attention and recent studies have revealed that the p38 MAPK pathway is a potential therapeutic target for delaying the degeneration of intervertebral discs. In this study, we analyzed a nature-derived protein tyrosine kinase inhibitor, Genistein, and its function in delaying IDD in rats both in vitro and in vivo via the p38 MAPK pathway. Nucleus pulposus cells treated with Genistein showed better function compared with untreated cells. Further study revealed that Genistein could play a protective role in IDD by inhibiting phosphorylation of p38, consequently inhibiting the p38 pathway-mediated inflammatory response. The rat IDD model also demonstrated that Genistein could effectively delay the degeneration of intervertebral disc tissue. The current study reveals new biological functions of Genistein, further demonstrates the effects of the p38 MAPK pathway on intervertebral disc degeneration, and deepens our understanding of the treatment and prevention of IDD.

Background. Collagen type X alpha 1 (COL10A1) is a structural component of the extracellular matrix that is aberrantly expressed in a variety of cancer tissues. However, its role in pancreatic cancer progression is not well understood. Methods. The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Gene Expression Profiling Interaction Analysis (GEPIA) data were employed to explore the expression of COL10A1 in normal and tumor tissues and its prognostic value in pancreatic adenocarcinoma. The clinical data of pancreatic cancer in TCGA were used to explore the relationship between COL10A1 and clinical features. Genes coexpressed with COL10A1 were explored using multiple databases and analyzed for functional enrichment. In addition, the lncRNA/miRNA/COL10A1 axis that may be involved in COL10A1 regulation in pancreatic cancer was explored by constructing a competitive endogenous RNA (ceRNA) regulatory axis. Finally, COL10A1 was analyzed for correlation with immune cell infiltration and various immune checkpoint molecules in pancreatic cancer. Results. It was found that the expression of COL10A1 was significantly increased in pancreatic cancer tissues. High expression of COL10A1 was related to the clinicopathological characteristics and the worse prognosis of pancreatic cancer patients. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer. Besides, in pancreatic adenocarcinoma, the expression level of COL10A1 showed a significant positive correlation with tumor immune cell infiltration, biomarkers of immune cells, and expression of immune checkpoint molecules. Conclusion. COL10A1 is an early diagnostic marker, and its high expression correlates with immune infiltration in pancreatic cancer. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased (p 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

Background The collagen alpha‐1(X) chain gene (COL10A1) is a known causative gene for Schmid metaphyseal chondrodysplasia (SMCD). This study clinically examined a Chinese family (n = 42) for SMCD and inheritance pattern. Fifteen individuals were diagnosed with SMCD based on characteristic skeletal phenotypes with autosomal dominant inheritance mode. Methods Four clinically diagnosed patients and three healthy relatives were selected for subsequent genetic tests. Trio‐whole exome sequencing (Trio‐WES) followed by Sanger sequencing and familial co‐segregation analysis were performed to identify SMCD‐associated variants. Results COL10A1 (NM_000493.4):c.1952 G>T(p.Trp651Leu) variant was detected only in the four patients and not in the three healthy relatives. The variant was evaluated as “likely pathogenic” according to the American College of Medical Genetics and Genomics variation classification guidelines with evidence of PM2, PM5, PP1, and PP3. To test the presence of the target variant in proband's fetal offspring, we developed a noninvasive prenatal testing method by extracting cell‐free fetal DNA in maternal plasma followed by high‐depth sequencing. The variant was also detected in the fetus and later confirmed by amniocentesis. Conclusion We identified a new disease‐causing variant in COL10A1. Cell‐free fetal DNA in maternal peripheral blood can be used as the rapid and noninvasive prenatal diagnostic method to detect the pathogenic/or likely pathogenic variant., Diagnosing SMCD is challenging and is misdiagnosed in many cases. Prenatal testing is done only if there is a family history of the disease. Moreover, the present diagnostic tests are invasive and potentially harmful to the developing fetus. Therefore, a noninvasive accurate testing method is particularly significant. Moreover, this testing method can be useful in detecting a variety of inherited variations. Hence, I believe this study may be relevant in the clinical scenario and definitely contributes to the existing literature in this field.

Summary A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation., Graphical Abstract, Highlights • Combination of a GSK3 inhibitor and an RAR agonist induces chondrocytes from hPSCs • The present protocol shows excellent differentiation efficiency • The potential to form hyaline cartilaginous tissues in vivo is demonstrated • RA and Wnt/β-catenin signals collaboratively regulate key genes for chondrogenesis, Saito and colleagues show that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, a GSK3 inhibitor and an RAR agonist, within 5–9 days. Genome-wide analysis of RAR and β-catenin association with DNA for sequential samples at each protocol stage demonstrates that RA and Wnt/β-catenin signaling is collaboratively involved in direct regulation of chondrocyte differentiation.

Summary Objective We aimed to assess whether an increase in chondrocyte size might be a feature of the articular cartilage (AC) hypertrophic-like phenotype both in experimental and in human osteoarthritis (OA). The anatomical location of these enlarged cells in the cartilage layers was also evaluated. Methods Experimental OA was carried out in female rabbits alone or in combination with osteoporosis (OPOA). The rabbits were subjected to destabilization knee surgery to develop OA. Osteoporosis was induced with ovariectomy and methylprednisolone administration. Human OA samples obtained from knee replacement surgery were also studied. Cartilage lesions and chondrocyte size were assessed in AC sections. Immunostaining of type-X collagen and metalloproteinase-13 were used as markers of the AC hypertrophic transformation. Both the cell size and the gene expression of type-X collagen were further analyzed in primary murine chondrocyte cultures. Results Compared to healthy AC, chondrocyte size was increased both in experimental and in human OA, in correlation with the severity of cartilage damage. No differences in chondrocyte size were found between deeper or more superficial regions of AC. In cell cultures, accretion of hypertrophic markers and cell enlargement were found to occur synchronized. Conclusions We observed an enhancement in the mean size of chondrocytes at the OA cartilage, which showed correlation with cartilage damage, both in human and in experimental OA. The enlarged chondrocytes were homogeneously distributed throughout the AC. Our results suggest that chondrocyte size could be a reliable measure of disease progression, of potential use in the histopathological assessment of OA cartilage.

Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. During this process, bi-directional communication among tumor cells and cancer associated fibroblasts (CAF) regulate extracellular matrix (ECM) deposition and remodeling. As a result of this dynamic process, soluble ECM proteins can be released into the bloodstream and may represent novel circulating biomarkers useful for cancer diagnosis. The aim of the present study was to measure the levels of three circulating ECM related proteins (COL11A1, COL10A1 and SPARC) in plasma samples of lung cancer patients and in healthy heavy-smokers controls and test whether such measurements have diagnostic or prognostic value.Gene expression profiling of lung fibroblasts isolated from paired normal and cancer tissue of NSCLC patients was performed by gene expression microarrays. The prioritization of the candidates for the study of circulating proteins in plasma was based on the most differentially expressed genes in cancer associated fibroblasts. Soluble ECM proteins were assessed by western blot in the conditioned medium of lung fibroblasts and by ELISA assays in plasma samples.Plasma samples from lung cancer patients and healthy heavy-smokers controls were tested for levels of COL11A1 and COL10A1 (n = 57 each) and SPARC (n = 90 each). Higher plasma levels of COL10A1 were detected in patients (p ≤ 0.001), a difference that was driven specifically by females (p 0.001). No difference in COL11A1 levels between patients and controls was found. SPARC levels were also higher in plasma patients than controls (p 0.001) with good performance in discriminating the two groups (AUC = 0.744). No significant association was observed between plasma proteins levels and clinicopathological features or survival.Soluble factors related to proficient tumor-stroma cross-talk are detectable in plasma of primary lung cancer patients and may represent a valuable complementary diagnostic tool to discriminate lung cancer patients from healthy heavy-smokers individuals as shown for the SPARC protein.

Summary Objective Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. Methods Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. Results Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. Conclusion Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Endochondral ossification (EO) is the process by which the long bones of the body form and has proven to be a promising method in tissue engineering for achieving cell-mediated bone formation. The present review centred on state-of-the-art research pertaining to mesenchymal stem cells (MSCs)-mediated endochondral bone formation, focusing on the role of donor cells, extracellular matrix and host immune cells during tissue-engineered bone formation. Possible research avenues to improve graft outcome and bone output were highlighted, as well as emerging research that, when applied to tissue-engineered bone grafts, offers new promise for improving the likelihood of such grafts transition from bench to bedside.

This study aims to investigate the effect of enriched plasma cells on the production of inflammatory cytokines and development of osteoarthritis (OA) in mice with B-cell-specific conditional deletion of the tuberous sclerosis 1 gene (TSC1). OA was induced by destabilization of the medial meniscus (DMM) in mice with TSC1 disruption in B cells (CD19-TSC1) and in littermate control mice (CON). The effects of DMM and incidence of OA were evaluated histologically, mRNA levels of inflammatory cytokines were detected by polymerase chain reaction, and serum cytokine levels were detected by enzyme-linked immunosorbent assay. Deletion of TSC1 caused constitutive activation of mechanistic target of rapamycin complex 1 mTORC1 in B cells. CON mice subjected to DMM exhibited a severe OA phenotype with increased inflammatory cytokines in B cells, serum, and the synovial membrane. Importantly, inflammatory cytokine production was also increased in B cells from the spleen of CD19-TSC1 conditional KO mice, but the OA phenotype was significantly elevated in conditional KO mice after DMM surgery compared with CON mice, as indicated by more severe articular cartilage destruction, increased protein expression of matrix metalloproteinase-13 and mRNA of type X collagen in the articular cartilage, decreased mRNA expression of type II collagen in the articular cartilage, and increased inflammatory cytokines in serum and the synovial membrane. The results demonstrate that inflammatory cytokine synthesis by B cells was enriched in CD19-TSC1 conditional KO mice, and this enhanced synthesis of inflammatory cytokines accelerated the incidence of OA.

Background Schmid‐type metaphyseal chondrodysplasia (SMCD) is a rare autosomal dominant skeletal dysplasia caused by heterozygous mutations in COL10A1, the gene which encodes collagen type X alpha 1 chain. However, its genotype–phenotype relationship has not been fully determined. Subjects and Methods The proband is a 2‐year‐old boy, born of non‐consanguineous Chinese parents. We conducted a systematic analysis of the clinical and radiological characteristics and a follow‐up study of the proband. Whole‐exome sequencing was applied for the genetic analysis, together with bioinformatic analysis of predicted consequences of the identified variant. A homotrimer model was built to visualize the affected region and predict possible outcomes of this variant. Furthermore, a literature review and genotype–phenotype analysis were performed by online searching all cases with SMCD. Results A novel heterozygous variant (NM_000493.4: c.1863_1866delAATG, NP_000484.2: p.(Met622 Thrfs*54)) was identified in COL10A1 gene in the affected child. And it was predicted to be pathogenic by in silico analysis. Protein modeling revealed that the variant was located in the NC1 domain, which was predicted to produce truncated collagen and impair the trimerization of collagen type X alpha 1 chain and combination with molecules in the matrix. Moreover, genotype–phenotype correlation analysis demonstrated that patients with truncating variants or variants in NC1 domain often presented earlier onset and severer symptoms compared with those with non‐truncating or variants in non‐NC1 domains. Conclusion The NC1 domain of COL10A1 was proved to be the hotspot region underlying SMCD, patients with variants in NC1 domain were more likely to present severer manifestations at an earlier age., This study characterized a novel COL10A1 variant (c.1863_1866delAATG, p.M622 Tfs*54) associated with Schmid type metaphyseal chondrodysplasia in a two‐year‐old Chinese patient. The novel variant was predicted to impair the trimerization of collagen X (α1) and combination with molecules in the matrix by in silico analysis. The NC1 domain of COL10A1 was a key region underlying SMCD, patients with NC1 mutations tend to present severer manifestations at earlier age.

Background Osteoarthritis (OA) is a disease of the whole joint, with articular cartilage breakdown as a major characteristic. Inflammatory mediators, proteases, and oxidants produced by chondrocytes are known to be responsible for driving cartilage degradation. Nevertheless, the early pathogenic events are still unclear. To investigate this, we employed an antibody that is specific to oxidative post-translationally modified collagen type II (anti-oxPTM-CII) to detect early cartilage pathogenic changes in two rat models of OA. Methods The animals underwent surgery for destabilization of the medial meniscus (DMM) and were sacrificed after 3, 5, 7, 14, and 28 days. Alternatively, anterior cruciate ligament transection with partial meniscectomy (ACLT+pMx) was performed and animals were sacrificed after 1, 3, 5, 7, and 14 days. Joints were stained with toluidine blue and saffron du Gatinais for histological scoring, anti-oxPTM-CII, and anti-collagen type X antibodies (anti-CX). Results We observed positive oxPTM-CII staining as early as 1 or 3 days after ACLT+pMx or DMM surgeries, respectively, before overt cartilage lesions were visible. oxPTM-CII was located mostly in the deep zone of the medial tibial cartilage, in the pericellular and territorial matrix of hypertrophic chondrocytes, and co-localized with CX staining. Staining was weak or absent for the lateral compartment or the contralateral knees except at later time points. Conclusion The results demonstrate that oxidant production and chondrocyte hypertrophy occur very early in the onset of OA, possibly initiating the pathogenic events of OA. We propose to use anti-oxPTM-CII as an early biomarker for OA ahead of radiographic changes.

Collagen type X alpha 1 (COL10A1) is a member of the collagen family and the main matrix component. However, COL10A1 expression and prognosis relationship remains unclear in gastric cancer (GC). Through the analysis of database of Oncomine, the Cancer Genome Atlas (TCGA) as well as the Gene Expression Omnibus (GEO), in contrast to the tissue of normal gastric, COL10A1 in gastric cancer, had been upregulated. The high expression of COL10A1 was obviously related to T stage (P = 0.025) and lymph node metastasis (P = 0.025). It has been illustrated by the analysis of logistic regression that COL10A1’s heightened expression in gastric cancer had been essentially linked with pathological stage, tumor differentiation, and T classification. The Kaplan–Meier curve in the Kaplan-Meier plotter database (P = 0.0371) and GSE84437 (P = 0.002) indicate that patients with high COL10A1 expression possess poor prognosis, specifically GC patients with lymph node metastasis have it. TCGA’s Multivariate analysis (P = 0.025) and GSE84437 dataset (P = 0.034) show that high expression COL10A1 is a key independent predictor of poor overall survival. Searching KEGG pathway enrichment by GSEA, the results suggested that 29 pathways were enriched. qRT-PCR technique was used for verification of the COL10A1’s high expression in gastric cancer in contrast to the normal gastric tissues. In conclusion, COL10A1 is of great importance in predicting the survival rate of GC patients., Graphical abstract

BACKGROUND Breast cancer, a common malignant tumor, has been considered as the leading cause of cancer-related death in women. Collagen type X alpha 1 (COL10A1) is overexpressed in breast cancer. The current study was designed to determine the functional involvement and regulatory mechanism of COL10A1 on the growth and metastasis of breast cancer. MATERIAL AND METHODS COL10A1 and Prolyl 4-hydroxylase beta polypeptide (P4HB) expressions in normal tissues and tumor tissues of breast cancer patients were obtained from the GEPIA dataset. COL10A1 and P4HB levels in breast cancer cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Furthermore, the interaction between COL10A1 and P4HB was confirmed by co-immunoprecipitation (Co-IP) assay. Cell Counting Kit-8 (CCK-8) and colony formation assay were applied to evaluate cell proliferation and clone-forming abilities of breast cancer cells. In addition, wound healing assay and transwell assay were performed to measure cell migration and invasion capabilities, respectively, in breast cancer. RESULTS The GEPIA dataset presented overexpressed COL10A1 and P4HB in tumor tissues of breast cancer patients. COL10A1 and P4HB expression levels were greatly upregulated in breast cancer cell lines. In addition, COL10A1 could directly interact with P4HB. Functionally, overexpressed COL10A1 boosted the proliferation and metastasis of breast cancer cells and silenced COL10A1 impeded the progression of breast cancer. More importantly, knockdown of P4HB weakened the promoting effects of overexpressed COL10A1 on cell proliferation, migration, and invasion in breast cancer. CONCLUSIONS COL10A1 promotes the malignant progression of breast cancer by upregulating P4HB expression, indicating that COL10A1 functions as an oncogene in breast cancer.

Background Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets. Methods The miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients. Results Within the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found. Conclusions Although miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

Thyroid hormone has been implicated in the normal growth and development of articular cartilage; however, its effect on a disease state, such as hypothyroidism, is unknown. The purpose of this investigation was to compare normal articular cartilage from proximal femurs of immature miniature swine to proximal femurs from hypothyroid-induced immature miniature swine.Two 11-week-old male Sinclair miniature swine were made hypothyroid by administration of 6-propyl-2-thiouracil (PTU) in their drinking water; two control animals did not receive PTU. At 25 weeks of age, the animals were euthanized and their proximal femurs were fixed and decalcified. Samples were sectioned and analyzed by histology to define extracellular matrix (ECM) structure, immunohistochemistry (IHC) to identify types II and X collagen, and histomorphometry to assess articular cartilage mean total and localized height and cell density. Statistics included nested mixed-effects ANOVA with p ≤ 0.05 considered statistically significant.Compared to controls, hypothyroid articular cartilage demonstrated statistically significant quantitative differences in mean tissue height, mean cell density and type II collagen localized zone height. Qualitative differences in ECM proteoglycans and overall collagen types were also found. Type X collagen was not detected in either hypothyroid or control articular cartilage specimens.Significant changes in articular cartilage structure in hypothyroid compared to control immature miniature swine suggest that thyroid hormone is critical in the growth and development of articular cartilage.Understanding articular cartilage development in immature animal models may provide insight into healing or repair of degenerative human articular cartilage.

Histone deacetylase 4 (HDAC4) plays a vital role in chondrocyte hypertrophy and bone formation. To investigate the function of HDAC4 in postnatal skeletal development, the present study developed lineage-specific HDAC4-knockout mice [collagen type 2α1 (Col2α1)-Cre, HDAC4(d/d) mice] by crossing transgenic mice expressing Cre recombinase. Thus, a specific ablation of HDAC4 was performed in Col2α1-expressing mice cells. The knee joints of HDAC4(fl/fl) and Col2α1-Cre, HDAC4(d/d) mice were analyzed at postnatal day (P)2-P21 using an in vivo bromodeoxyuridine (BrdU) assay, and Safranin O, Von Kossa and whole-body staining were used to evaluate the developmental growth plate, hypertrophic differentiation, mineralization and skeletal mineralization patterns. The trabecular bone was analyzed using microcomputed tomography. The expressions of BrdU, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-13, runt-related transcription factor (Runx)-2, osteoprotegerin (OPG), CD34, type X collagen (ColX), osteocalcin and Wnt5a were determined using immunohistochemistry, in situ hybridization (ISH) and reverse transcription-quantitative (RT-q)PCR. The results demonstrated that HDAC4-null mice (HDAC4(d/d) mice) were severely runted; these mice had a shortened hypertrophic zone (histopathological evaluation), accelerated vascular invasion and articular mineralization (Von Kossa staining), elevated expressions of MMP-13, Runx2, OPG and CD34 (RT-qPCR and immunohistochemistry), downregulated expression of the proliferative marker BrdU and PCNA (immunohistochemistry), increased expression of ColX and decreased expression of Wnt5a (ISH). In conclusion, chondrocyte-derived HDAC4 was responsible for regulating chondrocyte proliferation and differentiation as well as endochondral bone formation.

OBJECTIVE:As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors. DESIGN:Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX) 9, transforming growth factor beta (TGFB) 1 or bone morphogenetic protein (BMP) 2 cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy. RESULTS:Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenes SOX9, TGFB1 and BMP2 as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9). CONCLUSIONS:Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factors SOX9, TGFB1 and BMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.

BACKGROUND: Alimentary tract cancers (ATCs) are the most malignant cancers in the world. Numerous studies have revealed the tumorigenesis, diagnosis and treatment of ATCs, but many mechanisms remain to be explored. METHODS: To identify the key genes of ATCs, microarray datasets of oesophageal cancer, gastric cancer and colorectal cancer were obtained from the Gene Expression Omnibus (GEO) database. In total, 207 differentially expressed genes (DEGs) were screened. KEGG and GO function enrichment analyses were conducted, and a protein-protein interaction (PPI) network was generated and gene modules analysis was performed using STRING and Cytoscape. RESULTS: Five hub genes were screened, and the associated biological processes indicated that these genes were mainly enriched in cellular processes, protein binding and metabolic processes. Clinical survival analysis showed that COL10A1 and KIF14 may be significantly associated with the tumorigenesis or pathology grade of ATCs. In addition, relative human ATC cell lines along with blood samples and tumour tissues of ATC patients were obtained. The data proved that high expression of COL10A1 and KIF14 was associated with tumorigenesis and could be detected in blood. CONCLUSION: In conclusion, the identification of hub genes in the present study helped us to elucidate the molecular mechanisms of tumorigenesis and identify potential diagnostic indicators and targeted treatment for ATCs.

Osteoarthritis (OA) impacts the quality of life in middle-aged and elderly people by inducing immobility. The severe inflammation in chondrocytes is reported to be related to the development and process of OA. The present study aims to investigate the protective effects of Apremilast on injured chondrocytes induced by interleukin-1α (IL-1α) and the underlying mechanism. 10 ng/mL IL-1α was used to induce the in vitro injured chondrocytes. QRT-PCR was used to evaluate the expression level of Sry-type high-mobility-group box 9 (SOX-9), collagen type II alpha-1 gene (COL2A1), Aggrecan (ACAN) and collagen type X alpha 1 chain (COL10A1). SiRNA technology was utilized to knock down the expression of SOX-9 in the chondrocytes. The expression of SOX-9 was determined by Western Blot assay and/or immunofluorescence assay. Western Blot was used to evaluate the expression level of phosphorylated cyclic AMP response element binding (CREB). SOX9, Col2a1 and Acan were significantly up-regulated and Col10a1 was significantly down-regulated in the chondrocytes by Apremilast in a dose-dependent manner. IL-1α induced the injured chondrocytes by decreasing the expression of SOX9, Col2a1, Acan and increasing the expression of Col10a1, which were greatly reversed by Apremilast. By silencing SOX-9, the effects of Apremilast on SOX9 and marker genes were abolished. Phosphorylated CREB was up-regulated by Apremilast in a time-dependent manner. The up-regulated SOX-9 by Apremilast was reversed by the protein kinase A (PKA)/CREB pathway inhibitor H89. Apremilast may protect chondrocytes from inflammation by up-regulating SOX9.

Background The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. Methods Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. Results The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. Conclusions CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.

Human disorders of the post-squalene cholesterol biosynthesis pathway frequently result in skeletal abnormalities, yet our understanding of the mechanisms involved is limited. In a forward-genetic approach, we have found that a late-onset skeletal mutant, named kolibernu7, is the result of a cis-acting regulatory mutation leading to loss of methylsterol monooxygenase 1 (msmo1) expression within pre-hypertrophic chondrocytes. Generated msmo1nu81 knockdown mutation resulted in lethality at larval stage. We demonstrated that this is a result of both cholesterol deprivation and sterol intermediate accumulation by creating a mutation eliminating activity of Lanosterol synthase (Lss). Our results indicate that double lssnu60;msmo1nu81 and single lssnu60 mutants survive significantly longer than msmo1nu81 homozygotes. Liver-specific restoration of either Msmo1 or Lss in corresponding mutant backgrounds suppresses larval lethality. Rescued mutants develop dramatic skeletal abnormalities, with a loss of Msmo1 activity resulting in a more-severe patterning defect of a near-complete loss of hypertrophic chondrocytes marked by col10a1a expression. Our analysis suggests that hypertrophic chondrocytes depend on endogenous cholesterol synthesis, and blocking C4 demethylation exacerbates the cholesterol deficiency phenotype. Our findings offer new insight into the genetic control of bone development and provide new zebrafish models for human disorders of the cholesterol biosynthesis pathway., Summary: Characterization of three new zebrafish mutants highlights the role of cholesterol biosynthesis genes in skeletogenesis and establishes zebrafish models for disorders of the post-squalene cholesterol biosynthesis pathway, including chondrodysplasia punctata.

Functional repair of diseased or injured tissues remains a significant challenge for regenerative medicine. Extracellular matrix (ECM) composition during tissue assembly (e.g., development) is dramatically different from that of the homeostatic adult and may aid the design of engineered therapeutics that will promote regrowth and functionality of damaged tissues. Implementing a top-down approach, we evaluated perlecan (HSPG2), a pericellular ECM protein critical for proper cartilage development. When HSPG2 is knocked down, the non-lethal phenotype mimics the musculoskeletal defects observed in human Schwartz-Jampel syndrome. We previously demonstrated that HSPG2 knockdown significantly decreased the stiffness of the interstitial matrix and chondrocytes in developing cartilage (Xu et al., 2016b). However, it is not clear what changes occur in ECM structure and organization to cause the observed decrease in stiffness when HSPG2 is knocked down. Therefore, we performed proteomic analysis using mass spectrometry to test the hypothesis that ECM components that contribute to the developing structural integrity of cartilage will be in lower abundance in Hspg2C1532Y-Neo mutants (Neo/Neo) than in wildtype littermates (+/+). Relative increases in the abundance of ECM and associated proteins highlighted the expected developmental changes in ECM composition between embryonic day (E)16.5 and postnatal day (P)3 timepoints. The relative abundance of multiple proteins was significantly higher in Neo/Neo vs. +/+ P3 mice, contrasting our original hypothesis. Further investigation confirmed that the total collagen content increased with HSPG2 knockdown. However, similar collagen fibril diameter and ECM volume fractions between P3 Neo/Neo and +/+ littermates suggested HSPG2 knockdown did not affect matrix protein organization and assembly. Sulfated glycosaminoglycan (GAG) abundance showed no difference between groups, but safranin O staining indicated atypical GAG deposition. This, and increased hyaluronic acid binding protein expression, suggested increased hyaluronic acid deposition leads to decreased mechanical stiffness in Neo/Neo cartilage. Chondrocytes in perlecan-deficient cartilage may upregulate the synthesis of key ECM to compensate for decreased matrix stiffness; however, without HSPG2, GAGs accumulate and the matrix assembles into a structure with less mechanical integrity. Overall, the study of perlecan-deficient mice will provide insight into the influence of HSPG2 on chondrogenesis, matrix secretion, and functional cartilage assembly to enhance our design of engineering scaffolds that mimic cartilage to promote restoration of tissue function.

Psoriasis (Ps) and Psoriatic Arthritis (PsA) are characterized by a multifactorial etiology, involving genetic and environmental factors. The present study aimed to investigate polymorphisms (SNPs) within genes involved in extracellular matrix and cell homeostasis and microRNA genes as susceptibility biomarkers for Ps and PsA. Bioinformatic analysis on public RNA-seq data allowed for selection of rs12488457 (A/C, COL6A5), rs13081855 (G/T, COL8A1), rs3812111 (A/T, COL10A1) and rs2910164 (C/G, MIR146A) as candidate biomarkers. These polymorphisms were analyzed by Real-Time PCR in a cohort of 1417 Italian patients (393 Ps, 424 PsA, 600 controls). Statistical and bioinformatic tools were utilized for assessing the genetic association and predicting the effects of the selected SNPs. rs12488457, rs13081855 and rs2910164 were significantly associated with both Ps (p = 1.39 &times, 10&minus, 8, p = 4.52 &times, 4, p = 0.04, respectively) and PsA (p = 5.12 &times, 5, p = 1.19 &times, 6, p = 0.01, respectively). rs3812111, instead, was associated only with PsA (p = 0.005). Bioinformatic analysis revealed common and differential biological pathways involved in Ps and PsA. COL6A5 and COL8A1 take part in the proliferation and angiogenic pathways which are altered in Ps/PsA and contribute to inflammation together with MIR146A. On the other hand, the exclusive association of COL10A1 with PsA highlighted the specific involvement of bone metabolism in PsA.