Ameloblastoma (AM) is a benign, epithelial tumour of the jawbones originating from the residual epithelium of the tooth germ. AM shows a locally aggressive behaviour characterised by progressive bone resorption. The precise molecular mechanisms behind the disruption of bone homeostasis are unclear. We developed a novel 3D model composed of an AM tumour mass and a bone stroma to recreate the tumour microenvironment.
Type I collagen was used to fabricate 3D models. The collagen/cell mix was plastic compressed using Lonza RAFTTM 3D cell culture system using AM-1 (plexiform ameloblastoma) and AM-3 (follicular ameloblastoma). We engineered an active bone forming stroma using primary osteoblasts in stiff collagen matrix. The gene analysis was completed through reverse transcriptase- quantitative polymerase chain reaction (RT-qPCR) and protein levels were measured using Enzyme-Linked Immunoabsorbent Assay (ELISA). The bone nodules formed in the bone-stroma were characterised by nano-computed tomography (CT), transmission electron microscopy (TEM), Raman spectroscopy and gene analysis. The images were analysed using Fiji ImageJ and statistical analyses were performed using GraphPad Prism with p < 0.05 was considered as statistical significance.
AM cells expressed higher levels of invasion and bone resorption markers in 3D compared to 2D. Matrix-metalloproteinase (MMP)-2 was 2-times upregulated in 3D (p = 0.03) and MMP-9 was 3-fold upregulated in 3D. The bone nodules had an average surface area of 0.1 mm2 and average height of 92.37 ± 7.96 mm over 21 days in 3D. Our 3D biomimetic model presented a woven bone phenotype with mineral and matrix components. When the gene correlation between bone forming 3D stroma and AM introduced 3D bone stroma, we observed downregulation of bone formation genes such as RUNX2. AM cells inhibited osteoblasts from forming new bone nodules and limited the growth of existing bone nodules.
CONCLUSION & DISCUSSION
The biomimetic 3D tumouroid model accurately mimicked native subtype cell morphology. A novel bone-stroma model was established and used to study the AM tumour microenvironment. This study is the first to report that AM cells inhibit bone nodule formation by osteoblasts. It paves the way to further studies exploring the pathways of crosstalk between AM cells and the surrounding bone microenvironment. The model could represent a unique opportunity to test the effects of medications upon bone formation and resorption subject to perturbation by AM.
Dr. Liebert Parreiras Noguiera performed the Nano-CT scans. Dr. Mark Turmaine conducted TEM sample preparation and imaging at University College London.