Establishment and validation of an in vitro co-culture model for oral cell lines using human PBMC-derived osteoclasts, osteoblasts, fibroblasts and keratinocytes

Daniel Steller,Alexandra Scheibert,Tabea Sturmheit,Samer G Hakim

doi:10.1038/s41598-020-73941-0

Abstract

Indirect co-culture models with osteoclasts including oral cell lines may be influenced by M-CSF and RANKL in the common cell medium. Therefore, we investigated the viability and proliferation of osteoblasts (OB), fibroblasts (FB) and oral keratinocytes (OK) under stratified medium modification and assessed the differentiation of osteoclasts in each co-culture. The impact of M-CSF and RANKL in the common OC co-culture was assessed for OB, FB and OK via MTT assay via DAPI control. The multinuclearity and function of OC were evaluated by light microscopy, DAPI staining, resorption assay and FACS analysis. The PBMC showed the highest differentiation into OC after an incubation period of 7 days. Furthermore, co-culture with OB enhanced the number of differentiated multinucleated OC in comparison with monoculture, whereas co-culture with OK decreased PBMC multinuclearity and OC differentiation. FB did not influence the number of differentiated OC in a co-culture. RANKL and M-CSF reduction had no impact on OC differentiation in co-culture with FB or OB, whereas this medium modification for OK attenuated PBMC multinuclearity and OC differentiation in all approaches. Supplementation of RANKL and M-CSF can be modified for a co-culture of PBMC with FB or OB without disturbing OC differentiation. Thus, pathogenic processes of bone remodelling involving OB, OC, FB and OK in the oral cavity can be investigated thoroughly.

Highlights

RANKL inhibitors and bisphosphonates are used to treat osteoclast-induced disorders of bone metabolism such as osteoporosis and bone metastases[1,2]
OC differentiation was investigated by obtaining multinucleated cells from Peripheral blood mononuclear cells (PBMC), fluorescence-activated cell sorting (FACS) analysis of RANKL and calcitonin receptor expression, as well as by the functional resorption assay
Osteoclast differentiation of PBMC, evaluated as the number of multinucleated cells, increased significantly in number after 7 days of cultivation in comparison to 0 and 3 days (7 days: 17 ± 2% increase, compared to 0 days: 0%; p < 0.001; and 3 days: 10 ± 2%; p = 0.02)

Summary

Introduction

RANKL inhibitors and bisphosphonates are used to treat osteoclast-induced disorders of bone metabolism such as osteoporosis and bone metastases[1,2] Both substances stop bone turnover via multiple mechanisms, by influencing OC and OB. As a side-effect observed in experimental works, they inhibit proliferation, adhesion and cell migration of osteoblasts, fibroblasts and keratinocytes in a dose-dependent manner[4,5,6]. These events are associated with the development of the main side effect of these substances–drug-related osteonecrosis of the jaw[7]. The aim of this study was to investigate the influence of indirect co-culture cultivation on the proliferation and differentiation of PBMC-derived OC on one side and the viability of OB, FB and OK in this co-culture on the other side

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Discussion

Conclusion