The effect of hyaluronan on osteoblast proliferation and differentiation in rat calvarial‐derived cell cultures

Abstract

Hyaluronan (or hyaluronic acid, HA) is an essential component of extracellular matrices. It interacts with other macromolecules and plays a predominant role in tissue morphogenesis, cell migration, differentiation, and adhesion. The cell signaling functions of HA are mediated through the CD-44 receptor and are dependent upon the molecular weight of the polymer. We hypothesized that an HA of appropriate molecular weight alone in optimal concentration may induce osteoblast differentiation and bone formation. Enzyme-digested calvarial-derived mesenchymal cells from 2-day-old newborn rats were cultured with the addition of HA of three different molecular weights (2300, 900, and 60 kDa). We added, 0.5, 1.0, and 2.0 mg/mL HA for each molecular weight to the medium at the first plating of cells. After 7 to 20 days in culture, cell proliferation and differentiation were evaluated by measuring thymidine incorporation, alkaline phosphatase activity, and osteocalcin gene expression. The effects of HA on bone formation were examined by using Alizarin red staining for mineralization. The results showed that low molecular weight HA (60 kDa) significantly stimulated cell growth, increased osteocalcin mRNA expression in a dose-dependent manner, but showed no apparent effects on alkaline phosphatase activity and bone mineralization. On the other hand, high-weight HA (900 and 2,300 kDa) significantly increased all the parameters examined, particularly alkaline phosphatase activity, in a dose-dependent manner and stimulated cell mineralization to 126% and 119% of the controls, respectively, in the 1.0 mg/mL dose. Our findings suggest that HA has a molecular weight-specific and dose-specific mode of action that may enhance the osteogenic and osteoinductive properties of bone graft materials and substitutes due to its stimulatory effects on osteoblasts.

Calcite Biohybrids as Microenvironment for Stem Cells

“…Our evaluation of the effect of HA concentrations on MSCs indicates that HA affected MSC proliferation in a dose-dependent manner. Data in the literature shows that the addition of small concentrations of low molecular weight HA stimulates MSC proliferation [42,43], while the same concentration of high molecular weight HA appears to inhibit the cells [44,45]. Our experiments with high molecular weight HA in a 2D system did not show any significant effect on MSC proliferation (data not shown) while cells cultured on a calcite scaffold in a 3D system in the presence of HA of various concentrations displayed a reduction in cell number.…”

Evaluation of Alginate and Hyaluronic Acid for Their Use in Bone Tissue Engineering

“…Cells cultured in direct contact with alginate hydrogels showed higher ALP activity levels than those cultured in contact with hyaluronic hydrogels at 2 and 3 % of polymer concentrations. Previous studies have described upregulation of ALP activity by alginate microbeads [36,38] or HA in a dose-dependent manner at different dosages [39]. Intracellular calcium content was also measured as an index of cell differentiation for alginate hydrogels.…”

Hyaluronan inhibits BMP‐induced osteoblast differentiation

“…HA participates in many functions via the CD44 receptor such as regulation of cartilage metabolism, including osteoclastogenesis [6][7][8]. However, the direct effects of HA on osteoblastic differentiation have rarely been studied [9].…”