Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA TargetingCol1a1for Hydrogel-Based Tissue-Engineered Cartilage

Abstract

Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-b1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-b1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-b1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte-agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA combination is effective in reinitializing correct production and assembly of the cartilage-characteristic matrix in agarose hydrogel, without production of type I collagen. Because agarose is known to favor long-term expression of the chondrocyte phenotype and agarose-based hydrogels are approved for clinical trials, this strategy appears very promising to repair hyaline cartilage.

Combination of bioactive factors and IEIK13 self‐assembling peptide hydrogel promotes cartilage matrix production by human nasal chondrocytes

“…After encapsulation, chondrocytes showed a round morphology within the peptide gel, similar to that usually observed in agarose culture . Thus, IEIK13 peptide allows intimate cell–scaffold interactions favorable for chondrocytes.…”

“…After encapsulation, chondrocytes showed a round morphology within the peptide gel, similar to that usually observed in agarose culture. [34][35][36] Thus, IEIK13 peptide allows intimate cell-scaffold interactions favorable for chondrocytes. More precisely, this round morphology was well maintained only in the presence of BIT, suggesting that the combination of the peptide and BIT is necessary to sustain the chondrocyte phenotype.…”

Efficient, Low-Cost Nucleofection of Passaged Chondrocytes

“…However, a major drawback in using passaged chondrocytes is the unstable phenotype leading to dedifferentiation and production of fibroblast matrix, rich in type I collagen (col1), rather than articular cartilage matrix, rich in type II collagen (col2) and aggrecan (acan). Studies have determined that gene targeting either through knockdown of specific molecules (such as fibroblastic matrix molecule col1, 1 or matrix degrading enzymes cathepsin k 2 ) or the overexpression of genes such as master transcriptional regulator, sox9, 3 aid in redifferentiation of passaged cells.…”

Cartilage Tissue Engineering: Recent Advances and Perspectives from Gene Regulation/Therapy

“…Meanwhile, a bicistronic plasmid encoding both BMP‐2 and SOX‐9 genes linked to the “self‐cleaving” 2A peptide sequence is constructed and delivered into de‐differentiated chondrocytes using a microporator transfection system, which enables co‐expression of BMP‐2/SOX‐9, increases matrix production and improves cartilage formation in vivo . To potentiate the re‐differentiation of de‐differentiated chondrocytes, the cells are transfected with small interfering RNA (siRNA) to silence the Col I expression, encapsulated in the agarose hydrogel and further cultured in vitro in medium containing BMP‐2 . The Col I siRNA in combination with BMP‐2 exposure is effective in re‐initializing production and assembly of the cartilage‐characteristic matrix by de‐differentiated chondrocytes in agarose hydrogel, without production of Col I …”

“…To potentiate the re‐differentiation of de‐differentiated chondrocytes, the cells are transfected with small interfering RNA (siRNA) to silence the Col I expression, encapsulated in the agarose hydrogel and further cultured in vitro in medium containing BMP‐2 . The Col I siRNA in combination with BMP‐2 exposure is effective in re‐initializing production and assembly of the cartilage‐characteristic matrix by de‐differentiated chondrocytes in agarose hydrogel, without production of Col I …”