Down regulation of degenerative cartilage molecules in chondrocytes grown on a hyaluronan-based scaffold.
Hyaluronic-acid-based biomaterials used for cartilage repair allow the expression of specific extracellular matrix molecules by human chondrocytes grown onto them. We investigated whether these biomaterials could also create an environment in which the cells downregulate the expression of some catabolic factors. Chondrocytes were isolated from human articular cartilage obtained from the knees of patients with a history of trauma. First, the cells were expanded in monolayers and then they were seeded on a hyaluronic-acid derivative scaffold. Constructs and surnatants were collected and analysed at 1, 3, 7, 14 and 21 days after seeding. Immunohistochemical analysis for CD44 and caspase was carried out on paraffin-embedded sections. The Tunel method was used to identify chondrocyte apoptosis status. Secretion of MMP-1 and MMP-13 in the surnatants of the cells grown onto the biomaterial was measured by enzyme-linked immunosorbent assay. Nitric oxide (NO) production was evaluated by estimating the stable NO metabolite nitrite by the Griess method. A real-time RT-PCR analysis was performed on the constructs to evaluate the expression of type I and II collagens, aggrecan, Sox-9, MMP-1 and MMP-13 mRNAs at the different experimental times evaluated. Decreased levels of metalloproteinases and nitric oxide were observed in the surnatants of chondrocytes grown onto the hyaluronan-based scaffold. This was also confirmed by real-time PCR analysis which showed that the cells expressed the specific differentiated phenotype downregulating the expression of some catabolic molecules. Cells apoptosis decreased during the culture period, which further supported the biochemical data. The ability of the hyaluronan scaffold to reduce the expression and production of molecules involved in cartilage degenerative diseases indicates its use to treat early lesions of osteoarthritic patients.
Grigolo
Brunella
B
Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy. grigolob@alma.unibo.it
De Franceschi
Luciana
L
Roseti
Livia
L
Cattini
Luca
L
Facchini
Andrea
A
D000293:Adolescent; D000328:Adult; D017209:Apoptosis; D002358:Cartilage, Articular / Q000293:injuries / Q000378:metabolism* / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology*; D015536:Down-Regulation; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*
D016326:Extracellular Matrix Proteins; D009569:Nitric Oxide; D006820:Hyaluronic Acid; D020782:Matrix Metalloproteinases