The role of Alk-1 and Alk-5 in the mechanosensing of chondrocytes.

We aim to demonstrate the role of Alk receptors in the response of hydrogel expansion. Chondrocytes from rat knees were cultured onto plastic and hydrogel surfaces. Alk-1 and Alk-5 were overexpressed or silenced and the effects on cells during expansion were tested and confirmed using peptide inhibitors for TGFß. Overexpression of Alk-5 and silencing of Alk-1 led to a loss of the chondrocyte phenotype, proving that they are key regulators of chondrocyte mechanosensing. An analysis of the gene expression profile during the expansion of these modified cartilage cells in plastic showed a better maintenance of the chondrocyte phenotype, at least during the first passages. These passages were also assayed in a mouse model of intramuscular chondrogenesis. Our findings indicate that these two receptors are important mediators in the response of chondrocytes to changes in the mechanical environment, making them suitable targets for modulating chondrogenesis. Inhibition of TGFß could also be effective in improving chondrocyte activity in aged or expanded cells that overexpress Alk-1.

Response of sheep chondrocytes to changes in substrate stiffness from 2 to 20 Pa: effect of cell passaging.

AIM: The influence of culture substrate stiffness (in the kPa range) on chondrocyte behavior has been described. Here we describe the response to variations in substrate stiffness in a soft range (2-20 Pa), as it may play a role in understanding cartilage physiopathology. METHODS: We developed a system for cell culture in substrates with different elastic moduli using collagen hydrogels and evaluated chondrocytes after 2, 4, and 7 days in monolayer and three-dimensional (3D) cultures. Experiments were performed in normoxia and hypoxia in order to describe the effect of a low oxygen environment on chondrocytes. Finally, we also evaluated if dedifferentiated cells preserve the capacity for mechanosensing. RESULTS: Chondrocytes showed less proliferating activity when cultured in monolayer in the more compliant substrates. Expression of the cartilage markers Aggrecan (Acan), type II collagen (Col2a1), and Sox9 was upregulated in the less stiff gels (both in monolayer and in 3D culture). Stiffer gels induced an organization of the actin cytoskeleton that correlated with the loss of a chondrocyte phenotype. When cells were cultured in hypoxia, we observed changes in the cellular response that were mediated by HIF-1α. Results in 3D hypoxia cultures were opposite to those found in normoxia, but remained unchanged in monolayer hypoxic experiments. Similar results were found for dedifferentiated cells. CONCLUSIONS: Chondrocytes respond differently according to the stiffness of the substrate. This response depends greatly on the oxygen environment and on whether the chondrocyte is embedded or grown onto the hydrogel, since mechanosensing capacity was not lost with cell expansion.

Improved Chondrogenic Capacity of Collagen Hydrogel-Expanded Chondrocytes: In Vitro and in Vivo Analyses.

BACKGROUND: The use of autologous chondrocytes in cartilage repair is limited because of loss of the cartilage phenotype during expansion. The mechanosensing capacity of chondrocytes suggests evaluating the use of soft substrates for in vitro expansion. Our aim was to test the expansion of chondrocytes on collagen hydrogels to improve their capacity for chondrogenesis after a number of passages. METHODS: Rat cartilage cells were expanded on collagen hydrogels and on plastic, and the preservation of their chondrogenic capacity was tested both in vitro and in vivo. The expression of relevant markers during expansion on each surface was measured by real-time PCR (polymerase chain reaction). Expanded cells were then implanted in focal lesions in the medial femoral condyle of healthy sheep, and the newly formed tissue was analyzed by histomorphometry. RESULTS: Compared with cells cultured on plastic, cells cultured on hydrogels had better maintenance of the expression of the Sox9, Col2 (type-II collagen), FGFR3, and Alk-5 genes and decreased expression of Alk-1 and BMP-2. Pellets also showed increased expression of the cartilage marker genes aggrecan, Sox9, and Col2, and decreased expression of Col1 and Col10 (type-I and type-X collagen). ELISA (enzyme-linked immunosorbent assay) also showed a higher ratio of type-II to type-I collagen in pellets formed from cells expanded on hydrogels. When sheep chondrocytes were expanded and implanted in cartilage lesions in the femoral condyle of healthy sheep, hydrogel-expanded cells produced histologically better tissue compared with plastic-expanded cells. CONCLUSIONS: The expansion of chondrocytes on collagen hydrogels yielded cells with an improved chondrogenic capacity compared with cells expanded on plastic. CLINICAL RELEVANCE: The study results favor the use of hydrogel-expanded cells over the traditional plastic-expanded cells for autologous chondrocyte implantation.