Bioengineering the ameloblastoma tumour to study its effect on bone nodule formation

Abstract

Ameloblastoma is a benign, epithelial cancer of the jawbone, which causes bone resorption and disfigurement to patients affected. The interaction of ameloblastoma with its tumour stroma drives invasion and progression. We used stiff collagen matrices to engineer active bone forming stroma, to probe the interaction of ameloblastoma with its native tumour bone microenvironment. This bone-stroma was assessed by nano-CT, transmission electron microscopy (TEM), Raman spectroscopy and gene analysis. Furthermore, we investigated gene correlation between bone forming 3D bone stroma and ameloblastoma introduced 3D bone stroma. Ameloblastoma cells increased expression of MMP-2 and -9 and RANK temporally in 3D compared to 2D. Our 3D biomimetic model formed bone nodules of an average surface area of 0.1 mm2 and average height of 92.37 $$\pm $$ ± 7.96 μm over 21 days. We demonstrate a woven bone phenotype with distinct mineral and matrix components and increased expression of bone formation genes in our engineered bone. Introducing ameloblastoma to the bone stroma, completely inhibited bone formation, in a spatially specific manner. Multivariate gene analysis showed that ameloblastoma cells downregulate bone formation genes such as RUNX2. Through the development of a comprehensive bone stroma, we show that an ameloblastoma tumour mass prevents osteoblasts from forming new bone nodules and severely restricted the growth of existing bone nodules. We have identified potential pathways for this inhibition. More critically, we present novel findings on the interaction of stromal osteoblasts with ameloblastoma.

Associated changes in stiffness of collagen scaffolds during osteoblast mineralisation and bone formation

“…The primary calvarial rat osteoblasts were isolated, passaged and characterised as described in [ 9 , 18 , 19 ]. The cells were cultured for 3 days until they have reached confluency prior to 3D set-up at 37 o C, 5% CO 2 in α-MEM (Gibco through Thermo Fisher Scientific, Loughborough, UK), supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine (Life Technologies).…”

“…The cells were cultured for 3 days until they have reached confluency prior to 3D set-up at 37 o C, 5% CO 2 in α-MEM (Gibco through Thermo Fisher Scientific, Loughborough, UK), supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine (Life Technologies). Different techniques were used to characterise osteoblasts and osteoblast-driven bone formation and these include analysis of osteoblast gene markers and Alizarin red staining as described in [ 9 ].…”

“…The osteoblasts were seeded in 2D (control) or 3D soft collagen scaffolds (0.2% collagen), or dense collagen scaffolds (10% collagen) in 24-well plates [ 20 ]. 3D soft and dense collagen scaffolds were prepared as described in [ 9 ] (pages 3–10). A mix of 10X Minimal Essential Medium (MEM) (Sigma-Aldrich, Dorset, UK), monomeric type I collagen, (First Link, Birmingham, UK) and the neutralising agent composed of 17% of 10 Molar (M) NaOH (Sigma-Aldrich, Dorset, UK) and 83% 10 M HEPES buffer (Gibco through Thermo Fisher Scientific, Loughborough) was prepared.…”

“…It is possible to culture neonatal rodent calvarial osteoblasts which form mature bone nodule formation in vitro in 2D [ 7 , 8 ]. This process is enhanced when culturing primary osteoblasts in dense collagen (10%) scaffolds [ 9 ]. Bakkalci et al have shown that by culturing primary osteoblast cells in 3D dense collagen, there is a significant increase in mineralisation and formation of large bone nodules compared to 2D [ 9 ].…”

“…This process is enhanced when culturing primary osteoblasts in dense collagen (10%) scaffolds [ 9 ]. Bakkalci et al have shown that by culturing primary osteoblast cells in 3D dense collagen, there is a significant increase in mineralisation and formation of large bone nodules compared to 2D [ 9 ]. This short note focuses on mineralisation and bone nodule formation and the effect these processes have on the changing stiffness of a 3D collagen scaffold.…”