In vitro evaluation of hydroxyapatite, β-tricalcium phosphate, graphene oxide and zinc oxide for osteochondral regeneration under inflammatory conditions

The repair of osteochondral defects is challenging due to differing regenerative capacities of bone and cartilage, a challenge that is often exacerbated by chronic inflammation. This study evaluated the immunomodulatory and regenerative potential of hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP), graphene oxide (GO), and zinc oxide (ZnO) on osteoblast-like cells (Saos-2) and chondrocyte-like cells (SW1353) under IL-1β-induced inflammatory conditions. An MTT assay was used to measure cell viability at different concentrations of substances. To investigate the ability of Saos-2 cells to synthesize an inorganic extracellular matrix (ECM), mineralisation was observed via Alizarin Red staining and alkaline phosphatase (ALP) activity. The protein production of pro-COL1, pro-COL2, ACAN, MMP-9, IL-6, IL-8, BMP-2/4, RUNX2, and SOX9 was assessed using ELISA and Western blotting. The gene expression of RUNX2, SOX9, MMP-3, ADAMTS-5, COL10A1, ACAN, BGLAP, and FGF-2 was evaluated using RT-qPCR. Under inflammatory conditions, all the evaluated substances maintained basal matrix synthesis while suppressing the expression of ECM-degrading enzymes. Notably, GO exhibited a strong chondroprotective effect in SW1353 cells by significantly downregulating the expression of RUNX2 and FGF-2. Conversely, ZnO was found to impair mineralisation via the cytoplasmic trapping of RUNX2. Furthermore, their distinct impact on cellular phenotypes, ranging from GO-mediated chondroprotection to the inhibition of mineralisation by ZnO, highlights that the strategic, layer-specific integration of these substances is essential for actively guiding and sustaining the complex process of osteochondral healing. Consequently, our findings demonstrate a dual therapeutic benefit, in which the evaluated substances simultaneously suppress ECM degradation and support fundamental matrix synthesis under inflammatory stress.