Effect of Hyaluronan on Gene Expression in Osteoblastic Cells

Objectives: We hypothesized that the application of hyaluronan (HA) to the site of injury can provide a potential therapy for bone repair. Collagen gel culture is considered a model of tissue reorganization during wound healing and tissue remodeling in vitro. We previously demonstrated that HA enhanced collagen gel contraction. In this study, we analyzed the effect of HA on the gene expression of osteoblast-like cells embedded in collagen gel using microarray and real-time PCR. Methods: Osteoblastic MC3T3-E1 cells were embedded in type I collagen gels in culture medium with or without 1.0 mg/ml of HA (molecular weight: 6-12x10⁵). The gels were allowed to float or remained attached to the culture dish. Total RNA was extracted from cells embedded in the gels, reverse transcribed and double stranded cDNA synthesized. cDNAs were labeled with either fluorescent Cy3-UTP or Cy5-UTP, and hybridized on the DNA array. The hybridized arrays were visualized using an array scanner. The gene expressions of receptors (CD44 and ß₁ integrin) were determined using real-time PCR. Results: Of the 9,288 reliable genes detected, 303 were differentially expressed by MC3T3-E1 cells in the presence of HA. Among the 303, 102 were found to be up-regulated, whereas 201 genes demonstrated down-regulation. Cells on plastic plates showed a slight decrease in the receptors mRNA expression in the presence of HA. On the other hand, HA increased the receptors mRNA expression in anchored gels (P<0.05) while the expression levels increased slightly in floating gels with HA. Conclusion: It is suggested that HA activated signal transduction pathways through receptors when intracellular tension developed. Our data provides a comprehensive gene activity related to HA in osteoblasts. Supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (No.16791221) and Osaka Dental University Joint Research Funds (B03-01).