Induction of Re-Differentiation of Passaged Rat Chondrocytes Using a Naturally Obtained Extracellular Matrix Microenvironment

Abstract

Dedifferentiated human chondrocytes severely limit successful hyaline cartilage repair in clinical practice. The primary interest of this study is to evaluate the naturally obtained cell-derived matrix (CDM) as a physical microenvironment for chondrocyte re-differentiation. Once different cell types were cultured for 6 days and decellularized using detergents and enzymes, the fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM) were obtained. From scanning electron microscope observation, each CDM was found to resemble a fibrous mesh with self-assembled fibrils. Both the FDM and PDM showed a more compact matrix structure compared to the CHDM. For compositional analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis displayed numerous matrix proteins, which were quite different from each CDM in quantity and type. Specific matrix components, such as fibronectin, type I collagen (Col I), and laminin, were detected using immunofluorescent staining. In addition, the water contact angle suggests that the FDM is more hydrophilic than the PDM or CHDM. The proliferation of rat primary chondrocytes growing on CDMs was better than those growing on a plastic coverslip (control) or gelatin. Meanwhile, synthesis of glycosaminoglycan (GAG) was more effective for passaged chondrocytes (P4) cultivated on CDMs, and the difference was significant compared to cells grown on the control or on gelatin. As for the gene expression of cartilage-specific markers, CDMs exhibited good chondrocyte re-differentiation with time: the dedifferentiating marker, Col I was restrained, whereas the ratio between Col II and Col I, and between aggrecan and Col I, as an indicator of re-differentiation, was greatly improved. In addition, immunofluorescence of Col II showed a very positive signal in chondrocytes cultivated for 2 weeks on the CDMs. In an additional study, when threedimensional cell pellets made from either plate-grown or matrix-grown dedifferentiated chondrocytes (P5) were cultured for 4 weeks, the results of Safranin-O staining, immunohistochemistry of Col II, and total GAG assay suggested that matrix-grown cells were significantly better in the induction of chondrocyte re-differentiation, than those grown on the plate. This work suggests that the naturally occurring matrix, CDM, can provide a favorable surface texture for cell attachment, proliferation, and more importantly, a chondroinductive microenvironment for the re-differentiation of dedifferentiated chondrocytes.

Nanocomposite Scaffold for Chondrocyte Growth and Cartilage Tissue Engineering: Effects of Carbon Nanotube Surface Functionalization

“…Alternately, differential integrin activation (via a4b1, a5b1, or avb3) by Fn domains may contribute to cell survival, differentiation, or matrix remodeling. [45][46][47][48][49][50] Thus, the observations demonstrated within may suggest a mechanism wherein Fn or its fragments function in an autocrine manner to promote the enhanced GAG content and E Y observed in the -COOH surface functionalized group. Further work is required to demonstrate whether altered Fn1 expression in -COOH surface functionalized groups concomitantly influences cognate integrin expression and whether this provides an instructive signal toward chondrogenic phenotype and matrix production.…”

Cell-derived matrices for tissue engineering and regenerative medicine applications

“…To evaluate the effect of different CDMs on chondrocyte dedifferentiation, rat primary chondrocytes were cultured on TCPS for 4 passages to induce dedifferentiation, marked by loss of collagen type II and aggrecan expression. When transferred to the CDMs and cultured for an additional 2 weeks, the passaged chondrocytes exhibited signs of redifferentiation, including increased GAG and collagen type II deposition and reduced collagen type I gene expression, although the gene expression levels of collagen type II and aggrecan were similar in all cultures 57 . Pellet cultures of dedifferentiated chondrocytes expanded on CDM for 2 passages also contained more GAGs, stronger peripheral staining of collagen type II and were larger in size compared to pellets formed from TCPS-expanded chondrocytes 57 .…”

“…A common limitation in expanding sensitive primary cells ex vivo is loss of the native phenotype, characterized by loss of function, senescence, dedifferentiation and/or spontaneous differentiation 57-61 . One approach to mitigate culture-induced phenotypic changes is to recapitulate the tissue-specific microenvironment, in addition to the use of soluble factors.…”

Chondroinduction from Naturally Derived Cartilage Matrix: A Comparison Between Devitalized and Decellularized Cartilage Encapsulated in Hydrogel Pastes

“…[4][5][6] These ECM materials can be obtained from cell-derived matrices secreted during in vitro culture or from native tissue, 4,[7][8][9][10][11] and they have either been decellularized to remove cellular components and nucleic acids or they have been devitalized to kill but not necessarily remove cells within the matrix.…”

“…We and other groups have already established that cartilage matrix has chondroinductive potential, 7,[13][14][15][16][17] and we recently were the first to compare the chondroinductive potential of two different types of cartilage matrix in pellet culture: devitalized cartilage (DVC), where the matrix was exposed to a freeze/thaw process to devitalize the living chondrocytes within the matrix, and decellularized cartilage (DCC), in which the cells were not only devitalized but also removed from the matrix entirely. 17 In the pellet culture study, we observed that rat bone marrow stem cells (rBMSCs) exposed to DCC outperformed those cells exposed to DVC or transforming growth factorb 3 (TGF-b 3 ) in chondroinductivity.…”