Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

Antimicrobial and immunomodulatory in vitro profile of double antibiotic paste.

AIM: To evaluate the antimicrobial and immunomodulatory activity of double antibiotic paste (DAP) in an in vitro infection model. METHODOLOGY: The minimum inhibitory and bactericidal concentrations (MIC and MBC) and the antibiofilm activities (TTC assay) of DAP and its components (ciprofloxacin and metronidazole) were evaluated against Staphylococcus aureus and Enterococcus faecalis compared with triple antibiotic paste (TAP). The cellular viability of RAW 264.7 macrophages (24 and 72 h) and L929 fibroblasts (48 and 72 h) was evaluated by MTT. Furthermore, the production of TNF-α, IL-12, IL-6, IL-1α, IL-10 and NO (on RAW 264.7), besides IL-6, TGF-ß and NO (on L929), stimulated with DAP in baseline and associated with heat-killed microbial-antigen conditions was measured by ELISA and Griess reaction. Data were analysed using the one-way ANOVA test with Bonferroni's corrections. RESULTS: The MBC of pharmacopoeia DAP was similar to TAP for E. faecalis (0.25 µg.  mL-1 ) and lower for S. aureus (DAP 1 µg. mL-1 and TAP 2 µg. mL-1 ; p < .001). Ciprofloxacin was the most effective antibiofilm drug from the pastes (35% of reduction for E. faecalis and S. aureus; p < .0001), and both pastes had a similar antibiofilm eradication against both biofilm species (29% and 35% for S. aureus and 76% and 85% for E. faecalis; p < .0001). DAP was cytotoxic against the tested cells. DAP significantly upregulated IL-1α (p < .001), IL-6 (p < .0001), TNF-α (p < .01) and IL-12 (p < .05; in the absence of antigens) and significantly reduced IL-6 (p < .0001; in the presence of HK-S. aureus) and IL-10 (p < .05; in the presence of both antigens) on macrophages. Furthermore, DAP upregulated IL-6 (p < .001) and NO (p < .05; in the absence of antigens), IL-6 (p < .001; in the presence of HK-S. aureus) and reduced NO (p < .001; in the presence of HK-S. aureus). CONCLUSIONS: Double antibiotic paste and TAP had similar antimicrobial activity against S. aureus and E. faecalis. DAP upregulated pro-inflammatory cytokines mainly in the absence of antigens and had pro- and anti-inflammatory activity in RAW 264.7 macrophages and L929 fibroblasts in the presence of antigens involved in pulp infections.

Mineral trioxide aggregate (MTA) inhibits osteoclastogenesis and osteoclast activation through calcium and aluminum activities.

OBJECTIVE: To evaluate the effect(s) of mineral trioxide aggregate (MTA) on in vitro RANKL-mediated osteoclast-dependent bone resorption events and the influence of Ca2+ and Al3+ on the osteoclastogenesis inhibition by MTA. MATERIALS AND METHODS: Two types of osteoclast precursors, RAW 264.7 (RAW) cell line or bone marrow cells (obtained from BALB/c mice and stimulated with recombinant (r) macrophage colony stimulation factor (M-CSF), were stimulated with or without recombinant (r) activator of nuclear kappa B ligand (RANKL), in the presence or absence of MTA for 6 to 8 days. White Angelus MTA and Bios MTA (Angelus, Londrina, Paraná, Brazil) were prepared and inserted into capillary tubes (direct contact surface = 0.50 mm2 and 0.01 mm2). Influence of MTA on these types of osteoclast precursors was measured by the number of differentiated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (RAW and bone marrow cells), TRAP enzyme activity (RAW cells), cathepsin K gene expression (RAW cells), and resorptive pit formation (RAW cells) by mature osteoclasts. Besides, RAW cells were also stimulated with Ca2+ and Al3+ to evaluate the influence of these ions on MTA anti-osteoclastogenic potential. RESULTS: In bone marrow and RAW cells, the number of TRAP-positive mature osteoclast cells induced by rRANKL was significantly inhibited by the presence of MTA compared with control rRANKL stimulation without MTA (p < 0.05), along with the reduction of TRAP enzyme activity (p < 0.05) and the low expression of cathepsin K gene (p < 0.05). In contrast, to control mature osteoclasts, the resorption area on dentin was significantly decreased for mature osteoclasts incubated with MTA (p < 0.05). rRANKL-stimulated RAW cells treated with Ca2+ and Al3+ decreased the number of osteoclasts cells. Besides, the aluminum oxide was the dominant suppressor of the osteoclastogenesis process. CONCLUSIONS: MTA significantly suppressed RANKL-mediated osteoclastogenesis and osteoclast activity and, therefore, appears able to suppress bone resorption events in periapical lesions. This process might be related to Ca2+ and Al3+ activities. CLINICAL RELEVANCE: MTA is an important worldwidely acknowleged biomaterial. The knowledge about its molecular activities on osteoclasts might contribute to improving the understanding of its clinical efficacy.

Enterococcus faecalis and Staphylococcus aureusstimulate nitric oxide production in macrophages and fibroblasts in vitro

Aim: Nitric oxide (NO) is an important mediator related to damage of the pulp tissue and at the same time to regenerative pulp processes. However, it is not clear how common endodontic microorganisms can regulate this mediator. This study aimed to investigate NO production by macrophages and fibroblasts against Enterococcus faecalis- and Staphylococcus aureus-antigens. Methods: RAW 264.7 macrophages and L929 fibroblast cell lines were stimulated with different heat-killed (HK) antigen concentrations (105-108 colony forming units - CFU) from E. faecalis and S. aureus with or without interferon-gamma (IFN-γ). Cell viability by MTT colorimetric assay and NO production from the culture supernatants were evaluated after 72 h. Results: Data here reported demonstrated that none of the antigen concentrations decreased cell viability in macrophages and fibroblasts. The presence of HK-S. aureus and HK-E. faecalis antigen- stimulated NO production with or without IFN-γ on RAW 264.7. The HK-S. aureus antigen stimulated NO production in L929 fibroblasts with or without IFN-γ, and the highest concentration of HK-E. faecalis with IFN-γ also stimulated NO production by these cells. Conclusion: The amount of NO produced by macrophages and fibroblasts may be involved in the concentration and type of prevalent endodontic microorganisms, generating new answers for the understanding of pulpal revascularization/regeneration processes

Immune Response Profile against Persistent Endodontic Pathogens Candida albicans and Enterococcus faecalis In Vitro.

INTRODUCTION: Persistent microorganisms such as Candida albicans and Enterococcus faecalis might be directly related to endodontic treatment failure. The host response to these microorganisms impairs the reestablishment of intraradicular and periradicular health. METHODS: The present investigation evaluated the expression of inflammatory mediators produced by RAW 264.7 cells in the presence of heat-killed antigens (HK) C. albicans and E. faecalis. Cultures of RAW cells were stimulated with both antigens in the presence or absence of recombinant interferon (rIFN)-γ. Parameters of cell viability, production of nitric oxide (NO), as well as the synthesis of interleukin (IL)-1α, IL-6, IL-10, IL-12, monocyte chemotactic protein-1, and tumor necrosis factor (TNF)-α were analyzed. RESULTS: Results demonstrated that cell viability was especially reduced in antigens and rIFN-γ-stimulated groups. Groups stimulated with HK C. albicans upregulated IL-10 production. Otherwise, the addition of rIFN-γ to HK C. albicans upregulated TNF-α and NO production. Groups stimulated with HK E. faecalis upregulated TNF-α production. HK E. faecalis and rIFN-γ upregulated TNF-α and NO synthesis. The production of other cytokines remained unchanged by all stimuli. CONCLUSIONS: Knowledge regarding the host mechanism of response to microorganisms that perpetuate endodontic infection and the periradicular lesions can contribute to optimization of endodontic therapy. The mentioned inflammatory mediators and virulence factors involved in endodontic failure might guide lesion progression and also be targets in the development of disinfectant and immunomodulatory agents.

Antimicrobial peptide-based treatment for endodontic infections--biotechnological innovation in endodontics.

The presence/persistence of microorganisms in the pulp and periapical area corresponds to the maintenance of an exacerbated immune response that leads to the start of periradicular bone resorption and its perpetuation. In endodontic treatment, the available intracanal medications do not have all the desirable properties in the context of endodontic infection and apical periodontitis; they need to include not only strong antimicrobial performance but also an immunomodulatory and reparative activity, without host damage. In addition, there are various levels of resistance to root canal medications. Thus, antimicrobial agents that effectively eliminate resistant species in root canals could potentially improve endodontic treatment. In the emergence of new therapies, an increasing number of studies on antimicrobial peptides (AMPs) have been seen over the past few years. AMPs are defense biomolecules produced in response to infection, and they have a wide spectrum of action against many oral microorganisms. There are some studies that correlate peptides and oral infections, including oral peptides, neuropeptides, and bacterial, fish, bovine and synthetic peptides. So far, there are around 120 published studies correlating endodontic microbiota with AMPs but, according to our knowledge, there are no registered patents in the American patent database. There are a considerable number of AMPs that exhibit excellent antimicrobial activity against endodontic microbiota at a small inhibitory concentration and modulate an exacerbated immune response, down-regulating bone resorption. All these reasons indicate the antimicrobial peptide-based endodontic treatment as an emerging and promising option.