Oral microbes have the capacity to spread throughout the gastrointestinal system and are strongly associated with multiple diseases. Given that tonsils are located between the oral cavity and the laryngopharynx at the gateway of the alimentary and respiratory tracts, tonsillar tissue may also be affected by microbiota from both the oral cavity (saliva) and the alimentary tract. Here, we analyzed the distribution and association of the microbial communities in the saliva and tonsils of Korean children subjected to tonsillectomy because of tonsil hyperplasia (n = 29). The microbiome profiles of saliva and tonsils were established via 16S rRNA gene sequencing. Based on the alpha diversity indices, the microbial communities of the two groups showed high similarities. According to Spearman’s ranking correlation analysis, the distribution ofTreponema, the causative bacterium of periodontitis, in saliva and tonsils was found to have a significant positive correlation. Two representative microbes,Prevotellain saliva andAlloprevotellain tonsils, were negatively correlated, whileTreponema 2showed a strong positive correlation between saliva and tonsils. Taken together, strong similarities in the microbial communities of the tonsils and saliva are evident in terms of diversity and composition. The saliva microbiome is expected to significantly affect the tonsil microbiome. Furthermore, we suggest that our study creates an opportunity for tonsillar microbiome research to facilitate the development of novel microbiome-based therapeutic strategies.

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated β-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

Background: Mesenchymal stem cells (MSCs) have been widely used for stem cell therapy, and serial passage of stem cells is often required to obtain sufficient cell numbers for practical applications in regenerative medicine. A long-term serial cell expansion can potentially induce replicative senescence, which leads to a progressive decline in stem cell function and stemness, losing multipotent characteristics. To improve the therapeutic efficiency of stem cell therapy, it would be important to identify specific biomarkers for senescent cells. Methods: Tonsil-derived mesenchymal stem cells (TMSCs) with 20–25 passages were designated as culture-aged TMSCs, and their mesodermal differentiation potentials as well as markers of senescence and stemness were compared with the control TMSCs passaged up to 8 times at the most (designated as young). A whole-genome analysis was used to identify novel regulatory factors that distinguish between the culture-aged and control TMSCs. The identified markers of replicative senescence were validated using Western blot analyses. Results: The culture-aged TMSCs showed longer doubling time compared to control TMSCs and had higher expression of senescence-associated (SA)-β-gal staining but lower expression of the stemness protein markers, including Nanog, Oct4, and Sox2 with decreased adipogenic, osteogenic, and chondrogenic differentiation potentials. Microarray analyses identified a total of 18,614 differentially expressed genes between the culture-aged and control TMSCs. The differentially expressed genes were classified into the Gene Ontology categories of cellular component (CC), functional component (FC), and biological process (BP) using KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. This analysis revealed that those genes associated with CC and BP showed the most significant difference between the culture-aged and control TMSCs. The genes related to extracellular matrix-receptor interactions were also shown to be significantly different (p < 0.001). We also found that culture-aged TMSCs had decreased expressions of integrin α3 (ITGA3) and phosphorylated AKT protein (p-AKT-Ser⁴⁷³) compared to the control TMSCs. Conclusions: Our data suggest that activation of ECM-receptor signaling, specifically involved with integrin family-mediated activation of the intracellular cell survival-signaling molecule AKT, can regulate stem cell senescence in TMSCs. Among these identified factors, ITGA3 was found to be a representative biomarker of the senescent TMSCs. Exclusion of the TMSCs with the senescent TMSC markers in this study could potentially increase the therapeutic efficacy of TMSCs in clinical applications.

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15–24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel^® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel^®. Furthermore, the combination of Enbrel^® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel^®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.