AB0072 A MULTICOMPONENT MEDICATION PROMOTES CHONDROGENESIS AND REDUCES MMP-13 IN PRIMARY ARTICULAR CHONDROCYTES FROM KNEE OSTEOARTHRITIS PATIENTS IN VITRO

Background:HE-1100 is a multicomponent medicinal product. Initial preclinical data potentially suggest a preventive effect on cartilage degradation.Objectives:This study aims to understand the mode of action of HE-1100 on OA chondrocytesin vitro.Methods:Primary chondrocytes were obtained from 10 knee osteoarthritis (OA) patients undergoing knee replacement surgery. The cultures were treated with 20% (v/v) HE-1100 or placebo. Samples were collected for subsequent RNA extraction using standard methods. The reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential expression analysis between HE-1100 and placebo was made in R using the DESeq2 package to identify the differentially expressed genes in the OA-associated regulatory pathways. The protein production of the selected genes was quantified by ELISA in 10 independent human OA chondrocytes cultures.Results:According to the DESeq2 analysis, HE-1100 significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1 (+93%), FOS (+87%), NR4A1 (+43%), DUSP1 (+18%), ZFP36 (+18%), ZFP36L1 (+14%), NFKBIZ (+16%) and CYR61 (+14%) were upregulated and ATF7IP (-10%), TXNIP (-11%), C10orf10 (-12%), CLEC3A (-12%) and MMP13 (-18%) were downregulated after 24h HE-1100 treatment. HE-1100 significantly increased (2.3 fold +/-1.2 after 24h, p=0.0444 and 2.3-fold +/-1.0 after 72h, p=0.0239) the CYR61 protein production by human OA chondrocytes. After 72h, HE-1100 slightly but not significantly increased aggrecan production by 14 ± 19 % (p=0.1117) and significantly increased type II collagen pro-peptide production by 27 ± 20 % (p=0.0147). For both time points CYR61 production by OA chondrocytes was positively and significantly correlated with aggrecan (r=0.66, p=0.0004) and type II collagen pro-peptide (r=0.64, p=0.0008) production. In alginate beads culture, pro-MMP-13 was significantly decreased by HE-1100 treated cultures from day 7 to day 14 (from -16 to -25 %, p<0.05) and from day 17 to 21 (-22 %, p=0.0331) in comparison to controls.Conclusion:HE-1100 significantly modified the expression of DUSP1, C10orf10, ZFP36/L1 and CLEC3A, which are pathway mediators involved in MMP-13 expression and activation. Further, long-term (28 days) treatment with HE-1100 significantly reduced the production of pro-MMP-13, the inactive precursor of the metalloproteinase MMP-13 involved in type II collagen degradation. HE-1100 also promoted extracellular matrix formation probably through CYR61 production, a growth factor well correlated with type II collagen and aggrecan production.References:/Acknowledgments:We would like to thank the staff of the GIGA ULiège Genomic Next Generation Sequencing platform for performing the RNA sequencing and Benoit Charloteaux for his help in RNAseq data analysis.Disclosure of Interests:christelle sanchez: None declared, Kathrin Hemmer Employee of: Heel, Natascha Kroemmelbein Employee of: Heel, Bernd Seilheimer Employee of: Heel, Jean-Emile Dubuc: None declared, Christophe Antoine Employee of: Artialis, Yves Henrotin Grant/research support from: HEEL, TILMAN

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Reduction of Matrix Metallopeptidase 13 and Promotion of Chondrogenesis by Zeel T in Primary Human Osteoarthritic Chondrocytes

Frontiers in Pharmacology ◽

10.3389/fphar.2021.635034 ◽

2021 ◽

Vol 12 ◽

Author(s):

Christelle Sanchez ◽

Kathrin Hemmer ◽

Natascha Krömmelbein ◽

Bernd Seilheimer ◽

Jean-Emile Dubuc ◽

...

Keyword(s):

Differential Expression Analysis ◽

Type Ii Collagen ◽

Cartilage Degradation ◽

Alginate Beads ◽

Protein Quantification ◽

Type Ii ◽

Osteoarthritic Chondrocytes ◽

Matrix Metallopeptidase ◽

Oa Chondrocytes

Objectives: Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes in vitro.Methods: Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures.Results: In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, p = 0.0444 and 72 h, p = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% (p = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, p = 0.0004) and type II collagen pro-peptide (r = 0.64, p = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7–14 (from −16 to −25%, p < 0.05) and from day 17–21 (−22%, p = 0.0331) in comparison to controls.Conclusion: Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.

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Correlation Between Muscle Strength and Functional Improvement After a Neuromuscular Electrical Strengthening Associated with Undenatured Type II Collagen in Knee Osteoarthritis

SN Comprehensive Clinical Medicine ◽

10.1007/s42399-021-00830-6 ◽

2021 ◽

Vol 3 (5) ◽

pp. 1122-1132

Author(s):

Ana Paula Costa ◽

Carlos Monteiro ◽

Verine Cunha Teixeira ◽

Bruno da Silva Schwarstzhoupt ◽

Patrícia Mota Ferreira ◽

...

Keyword(s):

Muscle Strength ◽

Knee Osteoarthritis ◽

Type Ii Collagen ◽

Functional Improvement ◽

Type Ii ◽

Undenatured Type Ii Collagen

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Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02268-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ke Zhang ◽

Zhuoying Li ◽

Yunyang Lu ◽

Linyi Xiang ◽

Jiadong Sun ◽

...

Keyword(s):

Western Blotting ◽

Planar Cell Polarity ◽

Type Ii Collagen ◽

Mapk Signaling ◽

Dependent Manner ◽

Inflammatory Microenvironment ◽

Functional Roles ◽

Protein Levels ◽

Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

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Human Umbilical Cord Mesenchymal Stem Cells Induce into Chondrocytes in 3D Cultured System

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.749.198 ◽

2013 ◽

Vol 749 ◽

pp. 198-205

Author(s):

Li Yu ◽

Jing Liu ◽

Chao Xu ◽

Er Mei Luo ◽

Ming Qiao Tang

Keyword(s):

Real Time ◽

Alcian Blue ◽

Culture System ◽

Type Ii Collagen ◽

Human Umbilical Cord ◽

Type Ii ◽

Pellet Culture ◽

Hla Dr

Objective: To investigate a better method of inducing hUC-MSCs into chondrocytes in different culture system in vitro. Method: hUC-MSCs were isolated and cultured by tissue block culture, and the cells surface antigens were identified by flow cytometry, hUC-MSCs were cultured with chondrogenic media and stained with Alcian Blue. The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method. Expressions of glycosaminoglycan (GAG), type II collagen and Sox-9 were assayed by real-time fluorescence quantitative PCR. Results: The cultured hUC-MSCs phenotype was CD105+/CD29+/CD44+/ CD31-/CD34-/ CD40-/CD45-/HLA-DR-. hUC-MSCs weakly expressed chondrocyte marker, which strongly expressed GAG and type II collagen after chondrogenic induction, and the cells were incubated in pellet culture with higher expression. Real-time PCR results demonstrated that chondrogenic induction cells were expressed GAG, type II collagen and Sox-9, and the cells were incubated in pellet culture with higher expression. Conclusion: hUC-MSCs incubated in pellet culture is more conducive to differentiate into chondrocytes than those cultured in monolayer culture system.

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Type VI collagen expression is upregulated in the early events of chondrocyte differentiation

Development ◽

10.1242/dev.117.1.245 ◽

1993 ◽

Vol 117 (1) ◽

pp. 245-251

Author(s):

R. Quarto ◽

B. Dozin ◽

P. Bonaldo ◽

R. Cancedda ◽

A. Colombatti

Keyword(s):

Suspension Culture ◽

Type I Collagen ◽

Type Ii Collagen ◽

Type I ◽

Type Ii ◽

Type Vi Collagen ◽

Full Spectrum ◽

Collagen Expression ◽

Hypertrophic Chondrocyte

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

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A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation

International Journal of Molecular Sciences ◽

10.3390/ijms19113485 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3485 ◽

Cited By ~ 10

Author(s):

Yunyun Luo ◽

Yi He ◽

Ditte Reker ◽

Natasja Gudmann ◽

Kim Henriksen ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Growth Factor ◽

Knee Osteoarthritis ◽

High Sensitivity ◽

Type Ii Collagen ◽

Cartilage Explants ◽

Type Ii ◽

Serum Samples ◽

Human Cartilage ◽

Cartilage Formation

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

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Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: From in vitro to in vivo

Analytical Biochemistry ◽

10.1016/j.ab.2006.10.034 ◽

2007 ◽

Vol 361 (1) ◽

pp. 93-101 ◽

Cited By ~ 59

Author(s):

O.V. Nemirovskiy ◽

D.R. Dufield ◽

T. Sunyer ◽

P. Aggarwal ◽

D.J. Welsch ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Type Ii Collagen ◽

Type Ii ◽

Metalloproteinase Activity

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66 A NEW SERUM-BASED ASSAY FOR TYPE II COLLAGEN HELICAL PEPTIDE (SERUM HELIX-II) IS ASSOCIATED WITH LONG-TERM RADIOLOGICAL PROGRESSION IN KNEE OSTEOARTHRITIS

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(07)61702-6 ◽

2007 ◽

Vol 15 ◽

pp. C49-C50 ◽

Cited By ~ 1

Author(s):

P. Garnero ◽

N. Charni Ben Tabassi ◽

F. Juillet ◽

R.C. Whittles ◽

M. Sharif

Keyword(s):

Knee Osteoarthritis ◽

Type Ii Collagen ◽

Type Ii ◽

Radiological Progression ◽

Helical Peptide

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Extracellular Nucleotides Selectively Induce Migration of Chondrocytes and Expression of Type II Collagen

International Journal of Molecular Sciences ◽

10.3390/ijms21155227 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5227

Author(s):

Marcin Szustak ◽

Edyta Gendaszewska-Darmach

Keyword(s):

Migration Rate ◽

Monolayer Culture ◽

Type Ii Collagen ◽

Extracellular Nucleotides ◽

Nonbridging Oxygen ◽

Nucleotide Receptors ◽

Type Ii ◽

Mrna Transcripts ◽

Chondrogenic Phenotype

The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPβS, UDPβS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPβS positively affected the expression of type II collagen, a structural component of the cartilage matrix.

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