Summary Objective Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly ...systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. Design Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. Results 3D cultures specifically expressed chondrogenic mRNAs collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. Conclusions For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.
Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, ...such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration-response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.
Purpose
A systematic review, to study treatment effects for osteoporotic vertebral fractures (OVFs) in the elderly including all available evidence from controlled trials on percutaneous cement ...augmentation.
Methods
Primary studies, published up to December, 2019, were searched in PubMed and the Cochrane Library. Selected were all prospective controlled studies including patients > 65 years of age and reporting on at least one main outcome. Main outcomes were pain, disability and quality of life (QOL) 1 day post-intervention and at 6 months postoperatively. Excluded were meta-analyses or reviews, retrospective or non-controlled studies, case studies, patients’ groups with neoplastic and/or traumatic fractures and/or neurologically compromised patients.
Results
Eighteen studies comprising 2165 patients (
n
= 1117 percutaneous cement augmentation,
n
= 800 conservative treatment (CT),
n
= 248 placebo) with a mean follow-up of up to 12 months were included. Pooled results showed significant pain relief in favor of percutaneous cement augmentation compared to CT, direct postoperative and at 6 months follow-up. At 6 months, a significant difference was observed for functional disability scores in favor of percutaneous cement augmentation. When comparing percutaneous cement augmentation to placebo, no significant differences were observed.
Conclusion
This review incorporates all current available evidence (RCTs and non-RCTs) on the efficacy of percutaneous cement augmentation in the treatment of OVFs in the elderly. Despite methodological heterogeneity of the included studies, this review shows overall significant sustained pain relief and superior functional effect in the short- and long term for percutaneous cement augmentation compared to conservative treatment.
Graphic abstract
These slides can be retrieved under Electronic Supplementary Material.
Cost-effective preventive interventions are necessary for tackling the increasing number of hip fractures, which are frequently occuring as a serious consequence of osteoporosis. Several ...interventions have been available for preventing and treating osteoporosis. The aim of this study was to systematically review and critically appraise studies that assessed cost-effectiveness of hip protectors for the prevention of hip fractures and to investigate the effects of age, gender and residence situation on cost-effectiveness. A systematic review was conducted in order to identify economic evaluation studies examining the hip protector solely or compared to no treatment according to the Preferred Reported Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Synthesis of results was performed to observe trends between the studies. Methodological quality of the studies was assessed by the use of the Quality of Health Economic Studies (QHES) instrument. A total of 15 economic evaluation studies were included for analysis. The methodological quality was high in most studies (13/15). The hip protector was solely evaluated in three studies and within 12 other studies compared with no intervention. All studies that investigated the cost-effectiveness in long-term care facilities revealed that hip protector use is a cost-effective strategy for the prevention of hip fractures in elderly. Cost-effectiveness was also observed in two studies that provided hip protectors in a geriatric hospital ward. Four studies included both community-dwelling residents and residents living in a long-term care facility in their study. These studies showed more variability regarding cost-effectiveness. One study did not report information regarding the residence situation of their cohort, but also observed cost-effectiveness. In conclusion, this review suggests that hip protectors are a cost-effective approach in the prevention of hip fractures in populations with high risk of hip fractures especially in long-term care facilities and a geriatric ward in a hospital.
Summary Objective Bone morphogenic protein (BMP)-2 and BMP-7 are clinically approved and their recombinant proteins are used for bone tissue regenerative purposes and widely evaluated for cartilage ...regeneration. Previous comparison of the in vitro chondrogenic characteristics of BMP-2 vs BMP-7 did not address hypertrophic differentiation and characterizing their chondrogenic properties with a focus in on chondrocyte hypertrophy was topic of investigation in this study. Design Equimolar concentrations of BMP-2 or BMP-7 were added to chondrogenic differentiating ATDC5, human bone marrow stem cells or rabbit periosteal explants. Expression of Col2a1, Sox9, Acan, Col10a1, Runx2, ALP, Mmp13, Mef2c and Bapx1/Nkx3.2 was determined by reverse transcription-quantitative PCR (RT-qPCR) and immunoblotting. Glycosaminoglycan content, cell proliferation capacity and ALP activity were analysed by colourimetric analyses. Expression of Bapx1/Nkx3.2 and Sox9 was targeted by transfection of target specific siRNA duplexes. Results BMP-2 dose-dependently increased chondrocyte hypertrophy during chondrogenic differentiation of progenitor cells, whereas BMP-7 acted hypertrophy-suppressive and chondro-promotive. Both BMPs did not influence cell proliferation, but they did increase total glycosaminoglycan content. In a candidate approach Bapx1/Nkx3.2 was found to be involved in the BMP-7 mediated suppression of chondrocyte hypertrophy in ATDC5 cells. Conclusions BMP-2 and BMP-7 display opposing actions on the chondrogenic outcome of differentiating progenitor cells: BMP-2 acts a specific inducer of chondrocyte hypertrophy, while BMP-7 appears to increase or maintain chondrogenic potential and prevent chondrocyte hypertrophy. Our results pave the way for an application-dependent differential use of BMP-2 or BMP-7.
Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is ...unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis.
Sequencing-based rRNA 2′-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC–MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation.
We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2′-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2′-O-me levels. The 2′-O-me status of 5.8S-U14 (one of identified differential 2′-O-me sites; decreased by 7.7%, 95% CI 0.9–14.5%) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI 33–64%). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI 2.2–4.1) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI 1.3–2.0).
Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.
Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human ...articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients.
Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation.
Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation.
Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.
Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from ...the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification.
A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT + MMT) osteoarthritis model.
A cyclic peptide spanning AA 1–30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM–pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1–30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1–30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1–30 peptide treated animals.
The cyclic-inverso AHSG 1–30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis.
Since the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage ...osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes.
Protein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway–phenotype relationships were evaluated using pharmacological inhibitors.
Compared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI3K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI3K/AKT routes, and enabling of chondrocyte proliferation.
This study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI3K/AKT as crucial OA-associated intracellular signaling routes.
For many proteins from osteoarthritic synovial fluid, their intra-articular tissue of origin remains unknown. In this study we performed comparative proteomics to identify osteoarthritis-specific and ...joint tissue-dependent secreted proteins that may serve as candidates for osteoarthritis biomarker development on a tissue-specific basis.
Protein secretomes of cartilage, synovium, Hoffa's fat pad and meniscus from knee osteoarthritis patients were determined using liquid chromatography tandem mass spectrometry, followed by label-free quantification. Validation of tissue-dependent protein species was conducted by ELISA on independent samples. Differential proteomes of osteoarthritic and non-osteoarthritic knee synovial fluids were obtained via similar proteomics approach, followed by ELISA validation.
Proteomics revealed 64 proteins highly secreted from cartilage, 94 from synovium, 37 from Hoffa's fat pad and 21 from meniscus. Proteomic analyses of osteoarthritic vs non-osteoarthritic knee synovial fluid revealed 70 proteins with a relatively higher abundance and 264 proteins with a relatively lower abundance in osteoarthritic synovial fluid. Of the 70 higher abundance proteins, 23 were amongst the most highly expressed in the secretomes of a specific intra-articular tissue measured. Tissue-dependent release was validated for SLPI, C8, CLU, FN1, RARRES2, MATN3, MMP3 and TNC. Abundance in synovial fluid of tissue-dependent proteins was validated for IGF2, AHSG, FN1, CFB, KNG and C8.
We identified proteins with a tissue-dependent release from intra-articular human knee OA tissues. A number of these proteins also had an osteoarthritis-specific abundance in knee synovial fluid. These proteins may serve as novel candidates for osteoarthritis biomarker development on a tissue-specific basis.
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