Abnormal Extracellular Matrix Environment And Bone Invasion In Ameloblastoma Of The Jaw Bone

Objective: To investigate the effects of core genes involved in the development of jaw ameloblastoma(AB)and its associated extracellular matrix(ECM)on osteogenic and osteoclastic differentiation in order to provide a theoretical foundation for clinical treatment and future research into bone aggressiveness of AB.Methods: The core genes were identified by five AB patients’ whole-exome sequencing and the microarray datasets GES38494 and GES132472.Moreover,q PCR and Western blot were used to detect the expression of core genes and proteins in AB tissues.Immunohistochemical were used to observe the expression sites of core genes in AB tissues.According to the location of core genes,decellularized extracellular matrix(d ECM)derived from jaw ameloblastoma and gingival tissue were prepared to simulate the effect of AB tumor microenvironment on osteogenic differentiation of periodontal ligament stem cells and osteoclastic differentiation of bone marrow macrophages.Results: The AB essential genes including COL1A2、COL4A2、FBN1 and HPSE were discovered.Among them,the expression of HPSE was down-regulated,while that of COL1A2、COL4A2 and FBN1 were noticeably up-regulated in AB compared with normal gingival tissues of the jaws.Immunohistochemical results showed that COL1A2、COL4A2 and FBN1 were expressed in the connective tissues surrounding the epithelial islands of tumors,while HPSE is mostly in the nucleus and cytoplasm.According to the expression locations and functions of core genes,ABECM was studied.After decellularized treatment,the tissue became translucent milky white tissue.HE staining showed that the cell nucleus disappeared and the DNA removal rate was up to 99%(P<0.0001).The primary periodontal ligament stem cells(PDLLS)were cultured from the periodontal membrane tissues of the third molar,which were proved to be the mesenchymal source of PDLSC by immunofluorescence.Cloning and formation experiments showed that a single PDLSC had the proliferation ability to clone into clusters.ALP staining showed no difference among the induction groups,alizarine red staining showed that bone differentiation of ABd ECM was inhibited.The expression of ALP,RUNX2,OSX,OPN and OCN markers of osteogenic differentiation in ABd ECM group were significantly lower than those in the positive control group(P<0.0001).Bone marrow macrophages(BMMC)were induced by osteoclast with M-CSF and RANKL,TRAP staining showed that osteoclast differentiation was inhibited in the ABd ECM group,and the expression of CTSK and TRAP osteoclast differentiation marker protein in the ABd ECM group was significantly lower than that in the positive control group(P<0.0001).Conclusions: Abnormal ECM proteins encoded by COL4A2,COL1A2,FBN1 and HPSE genes can cause disturbance in the ECM environment,which affects bone cell differentiation to promote bone resorption.