Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds.
The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.
Girotto
Davide
D
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Facoltà di Medicina, Università di Padova, Viale G, Colombo 3 35121, Italy. davide.girotto@libero.it
Urbani
Serena
S
Brun
Paola
P
Renier
Davide
D
Barbucci
Rolando
R
Abatangelo
Giovanni
G
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002642:Chick Embryo / Q000378:metabolism; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D024042:Collagen Type I / Q000737:chemistry; D024043:Collagen Type II / Q000737:chemistry; D024121:Collagen Type X / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*; D007150:Immunohistochemistry; D011108:Polymers / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D001672:Biocompatible Materials; D024042:Collagen Type I; D024043:Collagen Type II; D024121:Collagen Type X; D006025:Glycosaminoglycans; D006609:High Mobility Group Proteins; D011108:Polymers; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; C086358:hyaluronic acid benzyl ester; D006820:Hyaluronic Acid