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Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_I | Pages 77 - 78
1 Jan 2003
Pullig O Weseloh G Klatt A Wagener R Swoboda B
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Introduction

Matrilin-3 is a member of the recently described matrilin family of extracellular matrix proteins containing von Willebrand factor A-like domains. The matrilin-3 subunit can form homotetramers as well as hetero-oligomers together with subunits of matrilin-1 (cartilage matrix protein). It has a restricted tissue distribution and is strongly expressed in growing skeletal tissues. Detailed information on expression and distribution of extracellular matrix proteins is important to understand cartilage function in health and in disease like osteoarthritis.

Methods

Matrilin-3 expression was analysed on decalcified normal cartilage/bone sections (N = 5) and decalcified cartilage/ bone sections with minor (N= 10), moderate (N = 10), and severe osteoarthritic lesions (N = 10). Osteoarthritic changes were classified histomorphologically, using the grading system of Mankin. Matrilin-3 expression was investigated by immunohistochemistry, in situ hybridization, Western blot analysis, and quantitative PCR. For immunohistochemistry, a polyclonal antibody against matrilin-3 was used. For Western blot analysis, cartilage extracts were obtained from normal and osteoarthritic samples, partially purified, and separated in SDS poly-acrylamide gelelectrophoreses. After blotting onto nitro-cellulose, matrilin-3 was visualized by incubation with the polyclonal anti-matrilin-3 antibody and chemiluminescence detection. Matrilin-3 -mRNA expression was determined by in situ hybridization using a digoxigenin-labeled anti-sense probe.

Results

Our results indicate that matrilin-3 is a mandatory component of mature articular cartilage with its expression being restricted to chondrocytes from the tangential zone and the upper middle cartilage zone. Osteoarthritic cartilage samples with only moderate morphological osteoarthritic destructions have elevated levels of matrilin-3 mRNA. In parallel, we found an increased deposition of matrilin-3 protein in the cartilage matrix. Matrilin-3 staining was diffusely distributed in the cartilage matrix, with no cellular staining being detectable. In cartilage samples with minor osteoarthritic lesions, matrilin-3 deposition was restricted to the middle zone and to the upper deep zone. A strong correlation was found between enhanced matrilin-3 gene and protein expression and the extent of tissue damage. Sections with severe osteoarthritic destruction showed the highest amount of matrilin-3 mRNA, strong signals in in situ hybridization, and prominent protein deposition in the middle and deep cartilage zone.

Conclusion

We conclude that matrilin-3 is an integral component of human articular cartilage matrix and that the enhanced expression of matrilin-3 in osteoarthritis may be a cellular response to the modified microenvironment in the disease.


Bone & Joint Research
Vol. 12, Issue 2 | Pages 147 - 154
20 Feb 2023
Jia Y Qi X Ma M Cheng S Cheng B Liang C Guo X Zhang F

Aims. Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD. Methods. We conducted an integrative analysis of BMD genome-wide association study (GWAS) and regulatory single nucleotide polymorphism (rSNP) annotation information. Firstly, the discovery GWAS dataset and replication GWAS dataset were integrated with rSNP annotation database to obtain BMD associated SNP regulatory elements and SNP regulatory element-target gene (E-G) pairs, respectively. Then, the common genes were further subjected to HumanNet v2 to explore the biological effects. Results. Through discovery and replication integrative analysis for BMD GWAS and rSNP annotation database, we identified 36 common BMD-associated genes for BMD irrespective of regulatory elements, such as FAM3C (p. discovery GWAS. = 1.21 × 10. -25. , p. replication GWAS. = 1.80 × 10. -12. ), CCDC170 (p. discovery GWAS. = 1.23 × 10. -11. , p. replication GWAS. = 3.22 × 10. -9. ), and SOX6 (p. discovery GWAS. = 4.41 × 10. -15. , p. replication GWAS. = 6.57 × 10. -14. ). Then, for the 36 common target genes, multiple gene ontology (GO) terms were detected for BMD such as positive regulation of cartilage development (p = 9.27 × 10. -3. ) and positive regulation of chondrocyte differentiation (p = 9.27 × 10. -3. ). Conclusion. We explored the potential roles of rSNP in the genetic mechanisms of BMD and identified multiple candidate genes. Our study results support the implication of regulatory genetic variants in the development of OP. Cite this article: Bone Joint Res 2023;12(2):147–154


Bone & Joint Research
Vol. 11, Issue 10 | Pages 723 - 738
4 Oct 2022
Liu Z Shen P Lu C Chou S Tien Y

Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67. phox. was involved in suramin-enhanced chondrocyte phenotype maintenance. Results. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67. phox. ) activity and membrane translocation. Overexpression of p67. phox. but not p67. phox. AD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67. phox. with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67. phox. -induced COL2A1 and ACAN expression was significantly inhibited. Conclusion. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67. phox. activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):723–738


Bone & Joint Research
Vol. 9, Issue 11 | Pages 751 - 760
1 Nov 2020
Li Y Lin X Zhu M Xun F Li J Yuan Z Liu Y Xu H

Aims. This study aimed to investigate the effect of solute carrier family 20 member 2 (SLC20A2) gene mutation (identified from a hereditary multiple exostoses family) on chondrocyte proliferation and differentiation. Methods. ATDC5 chondrocytes were cultured in insulin-transferrin-selenium medium to induce differentiation. Cells were transfected with pcDNA3.0 plasmids with either a wild-type (WT) or mutated (MUT) SLC20A2 gene. The inorganic phosphate (Pi) concentration in the medium of cells was determined. The expression of markers of chondrocyte proliferation and differentiation, the Indian hedgehog (Ihh), and parathyroid hormone-related protein (PTHrP) pathway were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Results. The expression of SLC20A2 in MUT group was similar to WT group. The Pi concentration in the medium of cells in MUT group was significantly higher than WT group, which meant the SLC20A2 mutation inhibited Pi uptake in ATDC5 chondrocytes. The proliferation rate of ATDC5 chondrocytes in MUT group was greater than WT group. The expression of aggrecan (Acan), α-1 chain of type II collagen (COL2A1), and SRY-box transcription factor 9 (SOX9) were higher in MUT group than WT group. However, the expression of Runt-related transcription factor 2 (Runx2), α-1 chain of type X collagen (COL10A1), and matrix metallopeptidase 13 (MMP13) was significantly decreased in the MUT group. Similar results were obtained by Alcian blue and Alizarin red staining. The expression of Ihh and PTHrP in MUT group was higher than WT group. An inhibitor (cyclopamine) of Ihh/PTHrP signalling pathway inhibited the proliferation and restored the differentiation of chondrocytes in MUT group. Conclusion. A mutation in SLC20A2 (c.C1948T) decreases Pi uptake in ATDC5 chondrocytes. SLC20A2 mutation promotes chondrocyte proliferation while inhibiting chondrocyte differentiation. The Ihh/PTHrP signalling pathway may play an important role in this process. Cite this article: Bone Joint Res 2020;9(11):751–760


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_2 | Pages 117 - 117
2 Jan 2024
Hankenson K
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Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_2 | Pages 135 - 135
2 Jan 2024
Füllemann P Jörimann T Bella E Stoddart M Matthys R Verrier S
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Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control. Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain. Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_3 | Pages 33 - 33
1 Apr 2018
Timur UT Emans P van Rhijn L Welting T
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Introduction. Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation. Methods. The coding sequence of human CART-1 was custom synthesized with optimized codon usage and cloned into a p3XFLAG-CMV-7.1 expression vector. FLAG-CART-1 was transiently overexpressed in SW1353 cells by polyethyleneimine-mediated transfection (1,000 ng of plasmid/well in 12-well plates). FLAG-Empty vector was used as a negative control. FLAG-CART-1 overexpression was confirmed by means of anti-FLAG immunoblotting. To investigate a potential connection between CART-1 and established key chondrogenic pathways, TGFβ3 (10 ng/mL) was added to SW1353 cells in CART-1 overexpression cultures or their appropriate controls. Cells were harvested 48 hours after transfection and mRNA expression of several genes involved in chondrogenic differentiation was determined by qRT-PCR. Data represent three separate experiments performed in technical triplicate. Results. Overexpression of CART-1 was confirmed on protein level. CART-1 significantly upregulated the expression of hypertrophic markers MMP13 and COLX, while the expression of RUNX2, ALP and COL1 was significantly downregulated. The expression of COL2A1 and SOX9 was not altered in the presence of CART1. TGFβ3 significantly decreased MMP13 expression in SW1353 cultures, but induced the expression of COLX, RUNX2 and COL1. This TGFβ3 dependent behaviour was reversed when CART-1 was overexpressed in these cultures. Conclusion. Our results implicate a functional role for the homeodomain protein CART-1 in controlling the expression of several markers involved in chondrocyte differentiation and show important interactions with other signaling pathways involved in chondrogenic differentiation. Current efforts focus on further elucidating the connection between CART-1 and other chondrogenic pathways


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 532 - 532
1 Nov 2011
Galois L Cournil-Henrionnet C Huselstein C Mainard D Bensoussan D Stoltz J Netter P Gillet P Watrin-Pinzano A
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Purpose of the study: Monolayer cultures of chondrocytes multiply and rapidly lose their chondrocyte phenotype, limiting their potential for tissue engineering. Mesenchymatous stem cells can preserve their phenotypic characteristics after several monolayer passages, offering a promising alternative for cartilage repair. The purpose of this work was to study the influence of transforming growth factor beta-1 (TGF-beta1) and bone morphogenic protein-2 (BMP2) and/or culture supplements (hyaluronic acid) on matrix synthesis and chondrocyte differentiation of human mesenchymatous stem cells (MSC) cultured on collagen sponges. Material and methods: MSC were isolated from bone marrow harvested during hip arthroplsty. At the third passage in monolayer culture, the MSC were reseeded on collagen sponges and cultured in vitro for 28 days under seven differ conditions: insulin transferrin selenium (ITS), foetal calf serum (FCS), ITS+TGFbeta1, ITS+ hyaluronate, ITS+TGFbeta1+hyaluronate, ITS+TGFbeta1+BMP2, ITS +TGFbeta1+BMP2+hyaluronate. The phenotypic evolution was followed using the expression of different genes of interest with PCRq (collagen2, collagen1, collagen3, collagen10, agrecanne, versicanne, COMP, Sox9). Synthesis of matrix material was assessed histologically and immunohistochemically. Results: Used alone, hyaluronic acid did not trigger chondrocyte differentiation of MSC. For the additives FCS, ITS, or hyaluronate, the synthesis of matrix material in the sponge was weak and poor in major constituents of cartilage. Conversely, the other conditions in presence of TGFbeta1±BMP2 induced important expression of collagen2, agrecanne and COMP as well as increased matrix synthesis with a strong content in proteoglycans and collagen. Discussion: The usefulness of MSC is growing due to their pluripotent characteristics. The conditions leading to their differentiation into the chondrocyte phenotype remains a subject of discussion. Our results show the particular importance of TGFbeta1 in the process of differentiation. Conclusion: Chondrogenic differentiation of MSC cultured in collagen sponges as well as the synthesis of the cartilaginous matrix requires the presence of TGFbeta1 in the culture medium and to a lesser extent BMP2. These results suggest the perspective of using MSC for guided cell therapy targeting cartilage


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 363 - 363
1 Jul 2008
Tare R Forsey R Chaudhari J Oreffo R
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Cartilage is a realistic target for tissue engineering given the avascular nature and cellular composition of the tissue. Much of the work in this field has been largely empirical, indicating the need for alternative approaches to the design of cartilage formation protocols. Given the heterogeneity associated with human mesenchymal populations, continuous cell lines may offer an alternative to model and simplify cartilage generation protocols. We therefore exploited the potential of the murine chondrocytic ATDC5 cell line to, i) delineate the process of chondrocyte differentiation in monolayer culture and three-dimensional micromass pellet culture systems, and ii) model cartilage formation utilising appropriate scaffold and bioreactor (perfused and rotating) technologies. Monolayer cultures of ATDC5 cells over a 28-day period in presence of insulin demonstrated various stages of chondrocyte differentiation- proliferative, pre-hypertrophic, hypertrophic and finally, mineralisation of cartilaginous nodules. This was confirmed by gene and protein expression, by qPCR and Western blotting respectively, of chondrogenic differentiation markers- Sox-9, Bcl-2, Type II and X collagens. Pellet cultures of ATDC5 cells under chondrogenic conditions (10 ng/ml TGF-beta3, 1X ITS {insulin, transferrin, selenium}, 10 nanomolar dexamethasone, 100 micromolar ascorbate-2-phosphate) illustrated a gradual progression from an aggregation of cells at day 7, to initiation of matrix synthesis at day 14, followed by formation of well-defined cartilaginous structures at day 21. Chondrogenic differentiation at day 21 was evident by numerous proliferative/ pre-hypertrophic chondrocytes, staining for Sox-9, Aggrecan, Type II collagen and PCNA, lodged in distinct lacunae embedded in cartilaginous matrix of proteogly-cans and Type II collagen. Inclusion of TGF-beta3 in the chondrogenic medium during pellet culture beyond 21 days maintained the pre-hypertrophic phenotype, even at day 28. In contrast, removal of TGF-beta3, addition of 50 nanomolar thyroxine and reduction of dexa-methasone to 1 nanomolar in the chondrogenic medium stimulated hypertrophy at day 28, evident by down-regulation of Sox-9 expression. ATDC5 cells cultured on Polyglycolic acid fleece in the rotating bioreactor or encapsulated in chitosan /alginate and cultured in the perfused bioreactor for 21 days, formed cartilaginous explants reminiscent of hyaline cartilage. Thus, ATDC5 cells constitute an ideal cell line to elucidate the steps of chondrocyte differentiation and cartilage formation


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_4 | Pages 79 - 79
1 Apr 2018
Timur UT Caron M Welting T van Rhijn L Emans P Jahr H
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Introduction. In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions. Methods. HACs from five OA patients (passage 1) were cultured in cytokine-free medium, under 280 or 380 mOsm respectively, under standard 2D in vitro conditions. TGF-β2 knockdown (KD) by siRNA was performed in the presence or absence of the established bone morphogenetic protein (BMP) type I receptor (BMPRI) inhibitor dorsomorphin (10 μM). Expression of COL2A1 was evaluated by qRT-PCR. Results. Culturing HACs at 380 mOsm increased COL2A1 mRNA expression. Addition of dorsomorphin decreased COL2A1 mRNA expression at both 280 and 380 mOsm, but its expression was still significantly higher at 380 mOsm. In hyperosmotic 380 mOsm culture conditions, TGF-β2 KD further increased COL2A1 mRNA expression, while addition of dorsomorphin under these conditions abrogated this effect. Still, expression of COL2A1 mRNA levels remained higher as compared to 280 mOsm. Conclusion. This study confirms that BMP signalling is involved in the expression of the single best accepted key chondrocyte marker, COL2A1, in osteoarthritic HACs. However, inhibition of BMP signalling could not abrogate the increase in COL2A1 expression under hyperosmotic culture conditions. Our data suggest an inverse regulation of TGF-β2 and COL2A1, under these conditions, which may largely be dependent on increased BMPRI-mediated cell signaling. Our findings further suggest that hyperosmotic culture improves COL2A1 expression by means that are independent of TGF-β- and BMPRI-signaling. Further elucidation of the molecular network underlying this observation is ongoing


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 74 - 74
1 Jan 2017
Kuhn A Das R Pavanram P Pufe T Jahr H
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Adult chondrocytes experience a hypoxic environment in vivo. Culturing chondrocytes under oxygen tension that more closely resembles the in vivo situation, i.e. hypoxic conditions, has been shown to have positive effects on matrix synthesis. During redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances redifferentiation is still incompletely elucidated. We employed micro-bioreactor technology to elucidate the contribution of TGF-β superfamily ligands to the chondrocyte differentiation process under hypoxic conditions in vitro. Dedifferentiated chondrocytes in alginate were cultured for 48 hours under hypoxic (1% pO2) or normoxic (20%) conditions, using specialized bioreactor technology. Gene expression of chondrocyte-specific markers (SOX9, COL2A1, COL1A1, AGC1 and MMP13) as well as established hypoxia-controlled genes (GDF1-, PHD3, HAS2, VEGF, COX2) and components of the TGF-β superfamily signaling pathways were analyzed by qPCR and protein expression after 48 hours in combination with TGF-β superfamily ligand-specific siRNA as well as selected TGF-β superfamily receptor inhibitors. Hypoxic culture showed robust upregulation of the selected hypoxia-specific marker genes. In addition, well-established chondrocyte-specific markers like SOX9 and collagen type II were upregulated. TGF-β isoforms were selectively upregulated under hypoxia on both mRNA and protein level. In addition, both Activin receptor-like kinases, ALK1 and ALK5, were upregulated under hypoxia, while respective type II and III receptors were unresponsive. The hypoxia-induced COL2 expression was abrogated by TGF-β2 siRNA, as was ALK5 inhibition. Our data strongly indicates that TGF-β superfamily signaling pathways are involved in chondrocyte redifferentiation under low oxygen tension in vitro


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 16 - 16
1 Apr 2017
Beckmann R Fragoulis A Tingart M Pufe T Wruck C Jahr H
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Background. Transcription factor nuclear factor E2p45-related factor 2 (Nrf2) is crucial for controlling the antioxidant response and maintaining cellular redox homeostasis. Binding of Nrf2 to antioxidant response elements (ARE) promotes the expression of anti-oxidative stress enzymes. In osteoblasts, Nrf2 directly interacts with Runx2, a strong transcriptional activator of osteoblast-specific genes. Sox9, a key regulator of chondrocyte differentiation is dominant over Runx2 in mesenchymal chondrogenic precursors. We therefore aimed to elucidate the role of Nrf2, and its regulation of Sox9, in chondrocytes. Methods. ARE sites in SOX9 promoter fragments were inactivated and cloned into pGL3 prior to co-transfection with phRL-TK into C-28/I2 cells for dual luciferase assay (n=4). Analyses of Nrf2 and Sox9 expression (n=3), following Nrf2 RNA interference (RNAi) (Sigma-Mission shRNAs library), was performed by qPCR (Applied Biosystems) as well as by Nrf2 and Sox9 immunohistochemistry in femoral condyle cartilage of wild type (WT) and Nrf2-knockout (KO) mice with ethical approval. Results. The Sox9 promoter region contains several putative antioxidant response elements. Mutagenesis of the ARE2 site reduced SOX9 promoter activity by 50%. Successful knock-down of Nrf2 using Nrf2-specific shRNAs in C-28/I2 chondrocytes also revealed parallel suppression of Sox9 mRNA. Furthermore, Nrf2-KO mice have fewer Sox9-positive-chondrocytes in their articular cartilage compared to WT littermates. Conclusions. Successive deletion of two putative ARE sites in the SOX9 promoter region suggests that ARE2 positively regulates SOX9 transcription and is in line with Sox9 mRNA suppression upon Nrf-2-RNAi. Nrf2 binding may thus directly stimulate Sox9 expression. Nrf2-KO mice reveal reduced numbers of Sox9-positive hyaline chondrocytes, which may have important consequences for the extracellular matrix production in these animals. Our findings reveal a novel mechanism regulating extracellular matrix synthesis in chondrocytes and may improve cartilage regenerative medicine. Level of evidence. Preclinical


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 15 - 15
1 Apr 2017
Timur U van der Windt A Caron M Welting T Emans P Jahr H
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Background. Treatment of cartilage defects requires in vitro expansion of human articular chondrocytes (HACs) for autologous chondrocyte implantation (ACI). During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype (i.e. collagen type II (COL2) expression). This de-differentiation makes them inappropriate for ACI. Physiological osmolarity (i.e. 380 mOsm) improves COL2 expression in vitro, but the underlying reason is unknown. However, an accepted key regulator of chondrocyte differentiation, transforming growth factor beta (TGFβ), is known to stimulate COL2 production. In this study we aimed to elucidate if TGFβ signaling could potentially be driving the COL2 expression under physiological culture conditions. Material and methods. After informed consent was obtained, HACs were isolated from five osteoarthritis (OA) patients and cultured in cytokine-free medium of 280 or 380 mOsm, respectively, under standard 2D in vitro conditions with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, superfamily receptors and chondrocyte marker genes was evaluated by qRT-PCR, TGFβ2 protein secretion by ELISA and TGFβ bioactivity using luciferase reporter assays. Statistical significance was assessed by a student's t-test. Results. TGFβ isoform expression was differentially altered by physiological osmolarity. Specifically, 380 mOsm increased TGFβ2 expression and protein secretion, as well as TGFβ activity. Upon TGFβ2 isoform-specific knockdown COL2 expression was induced. Physiological osmolarity and TGFβ2 RNAi also induced TGFβ1, TGFβ3 and their type I receptor ALK5. Conclusions. We showed that TGFβ2 knockdown increases COL2 expression in human osteoarthritic chondrocytes in vitro, possibly through a regulatory feedback loop involving TGFβ1, TGFβ3 induction and an increased ALK5/ALK1 ratio. This study indicates that TGFβ signalling is involved in osmolarity-induced chondrocyte marker gene expression. Pharmacological targeting of this pathway holds potential to further improve future osmolarity-mediated phenotypic stabilisation in advanced cell-based cartilage repair strategies. Level of Evidence. preclinical. Disclosure. We have nothing to disclose


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 15 - 15
1 Jan 2017
Timur U Caron M Welting T Weinans H van der Windt A Emans P Jahr H
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As cartilage has poor intrinsic repair capacity, in vitroexpansion of human articular chondrocytes (HACs) is required for cell-based therapies to treat cartilage pathologies. During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. It has been shown that physiological osmolarity (i.e. 380 mOsm) increases collagen type II (COL2) expression in vitro, but the underlying molecular mechanism is unknown. Transforming growth factor beta (TGFβ) super family members are accepted key regulators of chondrocyte differentiation and known to stimulate COL2 production. In this study we aimed to elucidate the role of TGFβ superfamily member signalling as a molecular mechanism potentially driving the COL2 expression under physiological (380 mOsm) culture conditions. HACs from OA patients (p1) were cultured in cytokine-free medium of 280 or 380 mOsm, under standard 2D in vitroconditions, with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, BMPs and chondrocyte marker genes was evaluated by QPCR. TGFβ2 protein secretion was evaluated using ELISA and bioactivity was determined using an established reporter cell line. Involvement of BMP signaling was investigated by culturing OA HACs (p1) in the presence or absence of dorsomorphin (10 µM). Physiological osmolarity increased TGFβ2 and TGFβ3 mRNA expression, TGFβ2 protein secretion as well as general TGFβ activity by 380 mOsm. Upon TGFβ2 isoform-specific knockdown COL2 mRNA expression was induced. TGFβ2 RNAi induced expression of several BMPs (e.g. BMP2,-4,-6) and this induction was enhanced in culture conditions with physiological osmolarity. Dorsomorphin inhibited physiological osmolarity induced COL2 mRNA expression. TGFβ2 knockdown under 380 mOsm increases COL2 expression in human osteoarthritic chondrocytes in vitromost likely through a regulatory feedback loop via BMP signaling, which is involved in osmolarity-induced COL2 expression. Future studies will further elucidate the BMP-mediated regulatory feedback loop after TGF β2 knockdown and its influence on COL2 expression


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 348 - 349
1 May 2009
Li J Tan D Miao S Crawford R Xiao Y
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To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approaches, controllable differentiation of mesenchymal stem cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. Although bilayered scaffolds have been investigated in vitro and in vivo, no culture system is available to test BMSCs differentiation into bone and cartilage simultaneously in bilayered scaffolds. This study investigated a defined culture media, which supported osteoblast and chondrocyte differentiation depending on growth factors implemented in biomaterials. In 2-dimensional culture, BMSCs differentiated to chondrocytes when transforming growth factor-beta 3 (TGF-β3) was added to the defined media, whereas osteogenic differentiation was induced by adding bone morphogenetic protein 7 (BMP-7). BMSC differentiation to osteogenic and chondrogenic lineages was further strengthen in 3-dimensional culture. Proteoglycan formation, type II collagen, and aggrecan were upregulated in the defined media when BMSCs were mixed with fibrin gel impregnated with TGF-β3. Mineralization and the expression of osteogenic markers such as alkaline phosphatase, osteopontin, and osteoclacin were noticeable when BMSCs cultured in hydroxyapatite-tricalcium phosphate (HA/TCP) scaffolds coated with BMP-7. This study generated and tested a growth media, which could induce osteogenic and chondrogenic differentiation of BMSCs in one culture system. These results will help the development of tissue substitutes for multi-complexed tissues such as subchondral replacement


The Journal of Bone & Joint Surgery British Volume
Vol. 85-B, Issue 8 | Pages 1190 - 1195
1 Nov 2003
Martos-Rodríguez A Santos-Alvarez I Campo-Ruíz V González S García-Ruiz JP Delgado-Baeza E

Our aim was to evaluate the expression of transcription factors CCAAT/enhancer-binding protein-beta (C/EBP. β. ) and C/EBP-homologous protein (CHOP) in the growth plate. Proximal tibial epiphyseal growth plates from ten 15-day-old Wistar rats were used. Additionally, anti-proliferating cell nuclear antigen (PCNA), anti-5-bromo-2’-deoxyuridine (BrdU) immunostaining, terminal transferase dUTP nick end-labelling (TUNEL) and nucleolar organiser region-associated proteins (AgNOR) techniques were peformed. The histological morphology of the growth plate from C/EBP. β. knock-out mice was also analysed. The normal growth plate showed that C/EBP. β. and CHOP factors are expressed both in the germinative/ upper proliferative and in the lower proliferative zones. Furthermore, BdrU+ and PCNA+ cells were present exclusively in the germinative and proliferative zones, while TUNEL+ and AgNOR+ cells were seen in all three zones of the growth plate. Acellular areas, hypocellularity, the increase in cell death and anomalies in the architecture of the cell columns were observed in the growth plates of C/EBP. β. (−/ −) knockout mice. We suggest that C/EBP. β. and CHOP transcription factors may be key modulators participating in the chondrocyte differentiation process in the growth plate


Aims

To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs).

Methods

Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed.


Bone & Joint Research
Vol. 12, Issue 11 | Pages 677 - 690
1 Nov 2023
Wang X Jiang W Pan K Tao L Zhu Y

Aims

Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis.

Methods

The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene.


Bone & Joint Research
Vol. 12, Issue 7 | Pages 433 - 446
7 Jul 2023
Guo L Guo H Zhang Y Chen Z Sun J Wu G Wang Y Zhang Y Wei X Li P

Aims

To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis.

Methods

Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo.


The Journal of Bone & Joint Surgery British Volume
Vol. 81-B, Issue 6 | Pages 1076 - 1082
1 Nov 1999
Iwasaki M Jikko A Le AX

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of α1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion