Enhanced Hyaline Cartilage Matrix Synthesis in Collagen Sponge Scaffolds by Using siRNA to Stabilize Chondrocytes Phenotype Cultured with Bone Morphogenetic Protein-2 Under Hypoxia

Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, L.; Goux, Didier; Chajra, Hanane; Malléin-Gerin, Frédéric; Boumediene, Karim; Galéra, Philippe; Demoor, Magali

doi:10.1089/ten.tec.2012.0508

Cited by 42 publications

(75 citation statements)

References 56 publications

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“…17,18 All patients signed an informed consent agreement form, which was approved by the Local Ethics Committee. Chondrocytes were seeded at 4 · 10 4 cells/cm 2 in plastic vessels.…”

Section: Chondrocyte Isolation and Culturementioning

confidence: 99%

“…18 At day zero, HACs were transfected with a mix of INTERFERinÔ (Polyplus-transfection SA; 3 mL), OptiMEM (Invitrogen; 100 mL) and small interfering RNA (siRNA) for 10 min at room temperature (siRNAs were used at 5 nM for the analysis of chondrocyte phenotype and at 50 nM for kinetics and in vivo studies). Then, the INTERFERin Ò -siRNA complex was supplemented with 200 mL DMEM + 2% FCS, pre-equilibrated to 3% O 2 by bubbling, with or without 50 ng/mL of BMP-2.…”

Section: Gene Silencing Experimentsmentioning

confidence: 99%

“…[10][11][12][13][14][15][16] Some factors are known to improve the chondrocyte phenotype, such as oxygen tension. 13,[17][18][19] At less than 5% oxygen, chondrocytes synthesize higher amounts of type II collagen and/or aggrecan, 13,17 which confer the two essential biomechanical properties of cartilage: tension resistance and viscoelasticity. 20 The viscoelastic properties of cartilage, due to its biphasic characteristics, are essential.…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, attention has turned to the design of an efficient biomaterial to mimic natural living conditions of chondrocytes [10][11][12][13][14][15][16] in conjunction with hypoxia (i.e., low oxygen concentrations). [16][17][18][19] Nevertheless, prochondrogenic factors, such as bone morphogenetic proteins (BMPs) or the transforming growth factor-beta (TGF-b) family, have already shown their ability to induce markers specific to cartilage. 22,23 BMP-2 is currently the best candidate among all the BMPs for improving the chondrocyte phenotype at low concentrations, although it can induce hypertrophy and osteogenesis at high concentrations.…”

Section: Introductionmentioning

confidence: 99%

“…24 In a previous study using dedifferentiated chondrocytes from OA patients, we demonstrated in vitro that the chondrocytes recover their differentiated phenotype and that the index of differentiation is higher in hypoxia than in normoxia after 7 days of culture with BMP-2 (50 ng/mL) in type I collagen sponges. 18 Furthermore, in OA cartilage, several proteases show increased expression, including the high temperature requirement factor A1 (HtrA1). [25][26][27] HtrA1 mRNAs increased seven-fold in OA cartilage compared with healthy cartilage.…”

Section: Introductionmentioning

confidence: 99%

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BMP-2, Hypoxia, and COL1A1/HtrA1 siRNAs Favor Neo-Cartilage Hyaline Matrix Formation in Chondrocytes

Ollitrault

Legendre

Drougard

et al. 2015

Tissue Engineering Part C: Methods

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Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

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“…17,18 All patients signed an informed consent agreement form, which was approved by the Local Ethics Committee. Chondrocytes were seeded at 4 · 10 4 cells/cm 2 in plastic vessels.…”

Section: Chondrocyte Isolation and Culturementioning

confidence: 99%

Section: Gene Silencing Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

BMP-2, Hypoxia, and COL1A1/HtrA1 siRNAs Favor Neo-Cartilage Hyaline Matrix Formation in Chondrocytes

Ollitrault

Legendre

Drougard

et al. 2015

Tissue Engineering Part C: Methods

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Validation of the 1,4‐butanediolthermoplastic polyurethane as a novel material for3Dbioprinting applications

Chocarro‐Wrona

Vicente

Antich

et al. 2020

Bioengineering & Transla Med

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Background Tissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage-like mechanical properties capable of sustaining high loadbearing environments to integrate into the surrounding tissue of the cartilage defect. Objective In this study we evaluated the novel 1,4-Butanediol thermoplastic polyurethane elastomer (b-TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. Methods The mechanical behaviour of b-TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad-derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. In vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analysed by Alamar blue assay, SEM, confocal microscopy, and RT-qPCR. Moreover, in vivo biocompatibility and host integration were analysed. Results This article is protected by copyright. All rights reserved. b-TPUe demonstrated a much closer compression and shear behaviour to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b-TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix (ECM) deposition and integration within the surrounding tissue. Conclusions This is the first study that validates the biocompatibility of b-TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.

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DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro

Liang

et al. 2017

Journal Cellular Physiology

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DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

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Enhanced Hyaline Cartilage Matrix Synthesis in Collagen Sponge Scaffolds by Using siRNA to Stabilize Chondrocytes Phenotype Cultured with Bone Morphogenetic Protein-2 Under Hypoxia

Cited by 42 publications

References 56 publications

BMP-2, Hypoxia, and COL1A1/HtrA1 siRNAs Favor Neo-Cartilage Hyaline Matrix Formation in Chondrocytes

BMP-2, Hypoxia, and COL1A1/HtrA1 siRNAs Favor Neo-Cartilage Hyaline Matrix Formation in Chondrocytes

Validation of the 1,4‐butanediolthermoplastic polyurethane as a novel material for3Dbioprinting applications

DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro

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