title: Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.
abstract: Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.
authors.affiliation: Laboratoire Microenvironnement Cellulaire et Pathologies, MILPAT, EA 4652, SFR ICORE 146, Université de Caen Basse-Normandie, UFR de Médecine, Caen, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Legendre
authors.forename: David
authors.initials: D
authors.lastname: Ollitrault
authors.forename: Magalie
authors.initials: M
authors.lastname: Hervieu
authors.forename: Catherine
authors.initials: C
authors.lastname: Baugé
authors.forename: Laure
authors.initials: L
authors.lastname: Maneix
authors.forename: Didier
authors.initials: D
authors.lastname: Goux
authors.forename: Hanane
authors.initials: H
authors.lastname: Chajra
authors.forename: Frédéric
authors.initials: F
authors.lastname: Mallein-Gerin
authors.forename: Karim
authors.initials: K
authors.lastname: Boumediene
authors.forename: Philippe
authors.initials: P
authors.lastname: Galera
authors.forename: Magali
authors.initials: M
authors.lastname: Demoor
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D020219:Chondrogenesis / Q000187:drug effects; D002923:Cilia / Q000187:drug effects / Q000378:metabolism; D003094:Collagen / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D051457:Hyaline Cartilage / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D054457:Tissue Scaffolds / Q000737:chemistry*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D053674:Aggrecans; D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D003094:Collagen; D004247:DNA