Bone tissue has a significant potential for healing, which involves a significant the interplay between bone and immune cells. While fracture healing represents a useful model to investigate endochondral bone healing, intramembranous bone healing models are yet to be developed and characterized. In this study, a micro-computed tomography, histomorphometric and molecular (RealTimePCRarray) characterization of post tooth-extraction alveolar bone healing was performed on C57Bl/6 WT mice. After the initial clot dominance (0h), the development of a provisional immature granulation tissue is evident (7d), characterized by marked cell proliferation, angiogenesis and inflammatory cells infiltration; associated with peaks of growth factors (BMP-2-4-7,TGFβ1,VEGFa), cytokines (TNFα, IL-10), chemokines & receptors (CXCL12, CCL25, CCR5, CXCR4), matrix (Col1a1-2, ITGA4, VTN, MMP1a) and MSCs (CD105, CD106, OCT4, NANOG, CD34, CD146) markers expression. Granulation tissue is sequentially replaced by more mature connective tissue (14d), characterized by inflammatory infiltrate reduction along the increased bone formation, marked expression of matrix remodeling enzymes (MMP-2-9), bone formation/maturation (RUNX2, ALP, DMP1, PHEX, SOST) markers, and chemokines & receptors associated with healing (CCL2, CCL17, CCR2). No evidences of cartilage cells or tissue were observed, strengthening the intramembranous nature of bone healing. Bone microarchitecture analysis supports the evolving healing, with total tissue and bone volumes as trabecular number and thickness showing a progressive increase over time. The extraction socket healing process is considered complete (21d) when the dental socket is filled by trabeculae bone with well-defined medullary canals; it being the expression of mature bone markers prevalent at this period. Our data confirms the intramembranous bone healing nature of the model used, revealing parallels between the gene expression profile and the histomorphometric events and the potential participation of MCSs and immune cells in the healing process, supporting the forthcoming application of the model for the better understanding of the bone healing process.

Taken together, our results showed that the presence of Treg cells attenuates the severity of experimental periodontitis without impairment in the control of infection.

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti-inflammatory cytokines such as IL-10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL-10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL-10 promoter -592C/A single nucleotide polymorphism (SNP), which is associated with a decrease in IL-10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL-10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real-time-PCR. The IL-10-592 SNP CA (P=0.0012/OR=2.4/CI:1.4-4.1), AA (P=0.0458/OR=2.3/CI:1.1-4.9), and CA+AA (P=0.0006/OR=2.4/CI:1.4-3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2-2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10-592 SNP is functional in CP, being associated with lower levels of IL-10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP-3 and OPG, and influence periodontal disease outcome.

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These results associated with clinical observations suggest that diabetes mellitus can produce alterations in oral epithelial cells, detectable by microscopy and cytomorphometry, which can be used in the diagnosis of this disease.