Establishment of a 3D-dynamic osteoblasts-osteoclasts co-culture model to simulate the jawbone microenvironment in vitro

The research on bone diseases highlights the need for reliable experimental models that may faithfully recapitulate in vitro the pathological bone microenvironment, and therefore provide a valuable tool for the development of novel strategies for bone regeneration. Taking into account that, at its simplest level, the production of bone mineralized tissue requires the presence of osteoblasts (hOBs) and osteoclasts (hOCs) enclosed in a structured matrix, nowadays much effort is focused on the set-up of specific in vitro hOBs/hOCs co-culture systems. Such simplified experimental models allow a functional characterization of the single cell types, while preserving the intimate crosstalk that naturally occurs in vivo between bone-forming and bone-resorbing cells. Our work was aimed at establishing a 3D hOBs/hOCs co-culture model to simulate the microenvironment present either in healthy or pathological jawbone, specifically for osteopenic or osteonecrotic diseases. In particular, we focused on bisphosphonate-related osteonecrosis of the jaw (BRONJ), a clinical complication found in patients treated with bisphosphonates leading to impaired jawbone turnover and tissue necrosis. We first confirmed the possibility to isolate hOBs from samples of necrotic bone (hnOB) obtained by BRONJ patients, despite the poor quality of the biological specimens. hnOB cultured in vitro maintained the typical features of osteoblastic cells, such as expression of osteogenic markers (OPN, Runx2) and mineralization capacity after culture in osteogenic medium. For the set-up of the co-colture model, our choice fell on mature and osteoprogenitor cells namely hOBs from nasal septum and MSCs from periodontal ligament (hPDLMSCs), respectively. By co-culturing hOBs or hPDLMSCs with monocytes from peripheral blood (hMCs) in transwell plates, we first demonstrated their ability to induce osteoclasts (hOCs) maturation in the absence of inducers after 7 days of co-culture. Culture in osteogenic medium for further 14 days induced the expression of OPN and deposition of mineral matrix, confirming that the co-culture system preserved the functional activity of the hOBs. We then established and compared two 3D co-culture systems, carried out in static condition using agarose-coated wells (3D-C) or dynamic condition using the Rotary Cell Culture System (3D-Dyc). After optimization of the culture parameters, we determined the lowest cell numbers that could allow the formation of viable hOBs/hOCs constructs, as the low amount of cells is a major issue when working with osteonecrotic samples. We demonstrated the presence of mature hOCs in the constructs cultured in 3D already after 7 days, in the absence of osteoclastogenic inducers. After further 14 days of culture in osteogenic medium, constructs derived from both 3D-C and 3D-Dyc culture systems were highly viable and showed the presence of mature hOCs and bone mineral matrix within the aggregates. In addition, the constructs stained positive for OPN and Col1A1. However, constructs cultured in 3D-C condition were poorly structured and showed areas with a disorganized matrix, while constructs cultured in 3D-Dyc had a very well definite structure with a uniform distribution of the mineral matrix. In conclusion, the formation of viable constructs is possible also co-culturing limited amounts of hOBs and hMCs in 3D-Dyc condition in the absence of osteoclastogenic inducers and avoiding the use of exogenous scaffolds. The finding that it is possible i. to obtain functional cells from anatomic locations with less than adequate bone quality and volume or a compromised area lacking sufficient stem and progenitor cells due to disabling conditions such as BRONJ, and ii.to create reliable 3D combinations with a few cells opens new scenarios to achieve autologous implantable constructs.