Evaluation of bone volume of MAH and MAM using CBCT
A characteristic feature of periodontitis is loss of bone around the teeth. Periodontal bone loss typically reflects the degree of present inflammation. As shown in Fig. 1a, the aveolar bone around the posterior teeth in MAH was obviously resorbed. However, little bone resorption was seen surrounding the posterior teeth in MAM. Quantitative analysis of the CBCT results demonstrated that the percentage of posterior teeth’s aveolar bone loss in MAH were 20.3 ± 2.8%, 28.8 ± 5.2%, and 22.3 ± 4.6%, respectively. For MAM, the corresponding results were 6.7 ± 1.6%, 13.8 ± 3%, and 10.1 ± 2%. It is very evident that the percentage of attachment loss around posterior teeth were all significantly higher in middle-aged woman than in middle-aged Macaca fascicularis (P < 0.05, Fig. 1b). This result indicated that the average reduction in maxillary posterior alveolar bone height of MAH were more severe than that of MAM.
Inflammation level of gingival tissues from MAH and MAM via HE and IHC
For more information of gingival tissue about MAH and MAM, histological analysis were performed. When analyzing the MAH group, histological aspects of gingival samples showed the enlargement of spinocellular layer, acanthocyte edema, epithelial atrophy and rich inflammatory cells infiltrate in connective tissue. However, when analyzing microscopic aspects of gingival sections of the MAM group, histological examination of gingival samples illustrated healthy aspects of epithelium and connective tissue. In addition, histological study indicated an increasing number of inflammatory cells and blood vessles in MAH group gingival mucosal lamina propria, while no obvious histopathological changes were found in MAM gingival tissue (Fig. 2).
To understand whether the severe periodontal tissues inflammation in MAH was due to the promotion of angiogenesis and cell proliferation, expression of CD34 and CD45 in gingival tissues were analyzed. As we predicted, the number of CD34-positive and CD45-positive cells in MAH were significantly higher than in MAM group (Fig. 3a). Immunohistochemical staining revealed more neovasculature in the tissues from MAH compared with MAH (Fig. 3a). Statistical analysis further indicated dramatically increases of MVD and inflammatory cells in the tissues of MAH when compared with the MVD in the tissues of MAM (Fig. 3b). These results revealed that inflammation state of gingival tissues surrounding the dentition was more severe in the MAH group than in the MAM group. In summary, more severe periodontal disease phenotype was found in MAH than in MAM, along with significantly higher levels of aveolar bone loss and increased microvasculars and inflammation cells infiltration.
Differences of salivary microbiome of MAH and MAM with 16 s rRNA
Six known phyla were represented among the total OTUs. In the human oral samples, five phyla had relative abundances greater than 1%: Proteobacteria (67.6%), Firmicutes (31.9%), Bacteroidetes (1.98%), Actinobacteria (3.7%), and Fusobacteria (1.2%). In the MAM oral samples, five phyla had relative abundances greater than 1%: Firmicutes (34.1%), Proteobacteria (32.3%), Bacteroidetes (18.1%), Actinobacteria (5.3%), and Fusobacteria (9.6%)(Fig. 4). Both the MAH and MAM salivary microbiome were dominated by Firmicutes, followed by Proteobacteria. At the phylum level, the most striking difference was the much higher abundance of Proteobacteria in the MAH group, while the Bacteroidetes in the MAH group showed a reverse trend (P < 0.05).
In the human oral samples, the bacterial taxa with > 1% abundance were Streptococcus (26.5%), Neisseria (26.8%), Acinetobacter (11.4%), Rothia (5.2%), Granulicatella (3.2%), Yesinia(2.68%), Porphyromonas (1.7%), Gemella (1.6%), Prevotella (1.1%). In the MAM oral samples, the bacterial taxa with > 1% abundance were Streptococcus (21.1%), Porphyromonas (10.1%), Neisseria (8.3%), Fusobacterium (7.7%), Granulicatella (7.6%), Gemella (6.1%), Capnocytophaga (4.3%), Haemophilus (4.2%), Alloprevotella (2.7%), Aggregatibacter (2.6%), Leptotrichia (2.1%), Bacillus (1.1%)(Fig. 4). Moreover, Neisseria was much more dominant in MAH than in MAM. Besides, the relative abundances of Alloprevotella, Aggregatibacter, Haemophilus, Gemella and Porphyromonas in the MAH group were less than that of the MAM group obviously, while the TM7 showed the opposite result, the difference was statistically significant.
The diversity, evenness, and means of each value in MAH and MAM are presented in Fig. 5. This result indicated that observed species richness (Fig. 5a), Chao-1 index (Fig. 5b), and Shannon diversity index (Fig. 5c) of MAM oral microbiome were richer than that of MAH, and the difference was statistically significant (P < 0.05). The results of alpha diversity indices for MAH and MAM samples revealed that the MAM oral microbiota was more diverse than that in MAH group. It is accepted that this more diverse community represents a more stable and healthy ecosystem. This diversity may result from subverting defenses that would limit community composition to non-pathogenic commensals.
Difference between the salivary microbial profiles of MAH and MAM was evident as determined by principal coordinates analysis (Fig. 6). UniFrac analysis showed a certain difference in the community structure between samples. Furthermore, the MAH and MAM samples tended to cluster separately and the MAH samples indicated lower intra-sample variability in comparison with the MAM samples.