Dent. J. 2026, 14(5), 290; https://doi.org/10.3390/dj14050290

Original title: Early Osteogenic and Stromal Marker Responses of Osteoblast-like and Bone-Marrow Stromal Cell Lines to a Hyaluronic Acid-Coated Xenogeneic Bone Graft: An Exploratory In Vitro Analysis – Yaniv Mayer, Hia Abu Sada, Hadar Zigdon Giladi, Eran Gabay, Ofri Doppelt-Flikshtain, Ofir Ginesin

In this exploratory in vitro study, osteoblast-like cells exposed to cerabone^® +HyA showed a 2.61-fold increase in RUNX2 expression compared to untreated cells, together with increased COL1A1 expression, representing the strongest early osteogenic response among the three xenografts tested. Cell metabolic activity also showed the highest numerical value for cerabone^® +HyA after 48 hours. All three xenografts, whether HA-coated or uncoated, reduced the expression of fibrotic markers in bone marrow stromal cells to a similar extent.

AIM

To investigate whether coating a xenogeneic bone graft with hyaluronic acid influences early osteogenic and fibrotic responses in vitro by comparing an HA-coated xenograft with two uncoated bovine bone graft materials.

STUDY DESIGN

Three commercially available xenografts were evaluated: cerabone^® +HyA(HA-coated), cerabone^® (its uncoated equivalent from the same manufacturer), and Bio-Oss® (an independent bovine bone substitute manufactured using a different process).

Human osteoblast-like cells (U2OS) and bone marrow stromal cells (HS5) were cultured in the presence of extracts from each material. Cell metabolic activity was assessed after 24 and 48 hours using an XTT assay. Cell adhesion to the graft surface was examined by fluorescence microscopy, while the osteogenic markers RUNX2and COL1A1, together with the fibrotic markers COL3A1 and TGF-β3, were quantified using real-time PCR. Each xenograft group was compared with untreated control cells.

The cerabone^® +HyA extract was tested at half the concentration of the extracts from the two comparator materials.

RESULTS

Metabolic activity (XTT):
Cell metabolic activity was comparable across all groups after 24 hours. At 48 hours, cerabone^® +HyA showed the highest average value (0.538 ± 0.056), followed by cerabone^® (0.450 ± 0.120) and Bio-Oss® (0.439 ± 0.073). However, after adjustment for multiple comparisons, these differences were not statistically significant.

Osteogenic markers:
Cells exposed to cerabone^® +HyA exhibited a 2.61-fold increase in RUNX2 expressioncompared with untreated controls (p = 0.01), representing the strongest early osteogenic marker response among the tested materials. COL1A1 expression was also increased in the cerabone^® +HyA group.

Cell adhesion:
Cells adhered to all three graft surfaces after 48 hours. As adhesion was assessed qualitatively rather than quantitatively, no comparison of adhesion performance between the materials could be made.

Fibrotic markers:
Expression of COL3A1 and TGF-β3 decreased by approximately 92.7% and 92.1%, respectively, in stromal cells exposed to all three xenografts (both p = 0.001), with no meaningful differences between the materials.

CONCLUSION

Compared with the uncoated material, cerabone^® +HyA induced a stronger early osteogenic response at the gene expression level while showing a similar reduction in stromal marker expression as the other tested xenografts. However, the study has several limitations. It used established cell lines rather than primary human cells, evaluated only a limited number of markers at a single early time point, and tested the cerabone^® +HyA extract at a lower concentration than the comparator materials. Further studies using primary human cells, equivalent extract concentrations, animal models, and clinical investigations are needed to confirm these findings.