GPDPLQ1237—A Type II Collagen Neo-Epitope Biomarker of Osteoclast- and Inflammation-Derived Cartilage Degradation in vitro

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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The Role of MicroRNA-381 in Chondrogenesis and Interleukin-1-β Induced Chondrocyte Responses

Cellular Physiology and Biochemistry ◽

10.1159/000430148 ◽

2015 ◽

Vol 36 (5) ◽

pp. 1753-1766 ◽

Cited By ~ 26

Author(s):

Changhe Hou ◽

Fangang Meng ◽

Zhiqi Zhang ◽

Yan Kang ◽

Weishen Chen ◽

...

Keyword(s):

Interleukin 1 ◽

Type Ii Collagen ◽

Cartilage Degeneration ◽

Cartilage Degradation ◽

Luciferase Assay ◽

Type Ii ◽

Osteoarthritic Cartilage ◽

Enhanced Expression

Aim: The molecular pathways regulating cartilage degradation are unclear. miR-381 was identified as a putative regulator of chondrogenesis related genes. Here, we examined its role in chondrogenesis and osteoarthritic cartilage degeneration. Methods: miR-381 expression was assessed in vitro in response to IL-1β stimulation in primary human (PHC) and mouse (PMC) chondrocytes, and ATDC5 derived chondrocytes; and in vivo in mouse embryos and human osteoarthritic cartilage. The effects of miR-381 on chondrogenesis and NF-kB signaling were assessed using a synthetic RNA mimic or inhibitor and luciferase assay, respectively. Upstream regulators of miR381 were probed using siRNA or overexpression plasmids for Sox9 and Runx2. Results: miR-381 expression was elevated in chondrogenic and hypertrophic ATDC5 cells. miR-381 was induced in vitro by IL-1β in ATDC5 cells, PMCs, and PHCs, and was expressed in areas of cartilage degradation or absorption in vivo. Overexpression of Runx2 or Sox9 increased miR-381 expression in ATDC5 cells. miR-381 suppressed expression of collagen, type II, alpha 1, and enhanced expression of metalloproteinase-13 (MMP-13), but did not regulate NFKBIA and NKRF activity. Conclusion: miR-381 was highly expressed during chondrogenesis and in arthritic cartilage. It may contribute to absorption of the cartilage matrix by repressing type II collagen and inducing MMP-13.

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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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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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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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Levels of matrix metalloproteinase-2 in committed differentiation of bone marrow mesenchymal stem cells induced by kartogenin

Journal of International Medical Research ◽

10.1177/0300060519853399 ◽

2019 ◽

Vol 47 (7) ◽

pp. 3261-3270

Author(s):

Cheng Wang ◽

Qiaohui Liu ◽

Xiaoyuan Ma ◽

Guofeng Dai

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Matrix Metalloproteinase ◽

Type Ii Collagen ◽

Matrix Metalloproteinase 2 ◽

Type Ii ◽

Proliferation And Differentiation ◽

Marrow Mesenchymal Stem Cells

Objective To measure the inductive effect of kartogenin on matrix metalloproteinase-2 levels during the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes in vitro. Methods In vitro cultured bone marrow hMSCs were grown to the logarithmic phase and then divided into three groups: control group (0 µM kartogenin), 1 µM kartogenin group and 10 µM kartogenin group. After 72 h of culture, cell proliferation and differentiation were observed microscopically. Matrix metalloproteinase-2 (MMP-2) in the cell supernatant and type II collagen levels in the cells were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively. Results Kartogenin induced the proliferation and differentiation of hMSCs. With the increase of kartogenin concentration, the level of type II collagen was increased, while the level of MMP-2 decreased. Conclusion These findings indicate that kartogenin can induce hMSCs to differentiate into chondrocytes, and with the increase of kartogenin concentration, degeneration of the cartilage extracellular matrix may be inhibited.

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Effects of bio-mimic collagen II-g-hyaluronic acid copolymers on chondrocyte maintenance

Journal of Bioactive and Compatible Polymers ◽

10.1177/0883911519876069 ◽

2019 ◽

Vol 34 (4-5) ◽

pp. 373-385

Author(s):

Kuan Wei Lee ◽

Tang-Ching Kuan ◽

Ming Wei Lee ◽

Chen Show Yang ◽

Lain-Chyr Hwang ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Proliferation ◽

Hyaluronic Acid ◽

Specific Binding ◽

Type Ii Collagen ◽

Polymerase Chain Reaction Analysis ◽

Collagen Fibrils ◽

Type Ii ◽

Human Cartilage

Extracellular matrix has an important part of the role in tissue engineering and regenerative medicine, so it is necessary to understand the various interactions between cells and extracellular matrix. Type II collagen and hyaluronic acid are the major structural components of the extracellular matrix of articular cartilage, and they are involved in fibril formation, entanglement and binding. The aim of this study was to prepare type II collagen fibrils with surface grafted with hyaluronic acid modified at the reducing end. The topographic pattern of type II collagen fibrils showed a significant change after the surface coupling of hyaluronic acid according to atomic force microscopy scanning. The presence of hyaluronic acid on the type II collagen fibrillar surface was confirmed by the specific binding of nanogold labelled with lectin. No significant increase in cell proliferation was detected by a WST-1 assay. According to histochemical examination, the maintenance of the round shape of chondrocytes and increased glycosaminoglycan secretion revealed that these cell pellets with Col II- g-hyaluronic acid molecules contained un-dedifferentiated chondrocytes in vitro. In the mixture with the 220-kDa Col II- g-hyaluronic acid copolymer, the expression of type II collagen and aggrecan genes in chondrocytes increased as demonstrated by real-time polymerase chain reaction analysis. Experimental results show that the amount of hyaluronic acid added during culturing of chondrocytes can maintain the functionality of chondrocytes and thus allow for increased cell proliferation that is suitable for tissue repair of human cartilage.

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