Deep Learning for Microstructural Characterization of Synchrotron Radiation-Based Collagen Bundle Imaging in Peri-Implant Soft Tissues

The study of the organizational kinetics in the area surrounding the transmucosal part of dental implants promises to ensure an accurate diagnosis of the healing process, in terms of osseointegration and long-term implant success. In this demonstrative work, the morphological, qualitative and quantitative characteristics of 3D images of collagen bundles obtained by synchrotron-based high-resolution X-ray tomography were analyzed. Data analysis was performed using deep learning algorithms, neural networks that were applied on multiple volumes extracted from connective portions of different patients. The neural network was trained with mutually consistent examples from different patients; in particular, we used a neural network model, U-Net, well established when applying deep learning to datasets of images. It was trained not only to distinguish the collagen fibers from the background, but also to subdivide the collagen bundles based on the orientation of the fibers. In fact, differently from conventional thresholding methods, deep learning semantic segmentation assigns a label to each pixel, not only relying on grey level distribution but also on the image morphometric (shape or direction) characteristics. With the exception of Pt2 biopsies that, as confirmed by the polarized light investigation, were shown to present an immature tissue condition, the quantity, the anisotropy degree and the connectivity density of transverse bundles were always demonstrated to be higher than for longitudinal ones. These are interesting and new data; indeed, as collagen bundles are organized in an intertwining pattern, these morphometric and 3D complexity parameters, distinguished in transversal and longitudinal directions, give precise indications on the amount and distribution of connective tissue forces exerted during the healing process

Hemostatic Collagen Sponge with High Porosity Promotes the Proliferation and Adhesion of Fibroblasts and Osteoblasts

The use of biomaterial for tissue repair involves the interaction between materials and cells, and the coagulum formation represents the first step of tissue healing. This process is particularly critical in the oral cavity, where the wounds are immediately subjected to the masticatory mechanical stress, saliva invasion, and bacterial attack. Therefore, the present study aimed to explore the structural features and the biological activities of a hemostatic collagen sponge on human gingival fibroblasts (HGFs) and human oral osteoblasts (HOBs). The microstructure of the collagen sponge was characterized by a scanning electron microscope (SEM) and histological analysis. The porosity was also calculated. To investigate biological activities, HGFs and HOBs were cultured on the collagen sponges, and their adhesion was observed at SEM on the third day, while cell viability was investigated at the third and seventh days by Tetrazolium (MTT) assay. For osteoblasts seeded on collagen sponge the mineralization ability was also evaluated by alkaline phosphatase (ALP) assay at the seventh day, and by Alizarin red staining on the 14th. Furthermore, the gene expression of ALP and osteocalcin (OCN) was investigated after 3, 7 and 14 days. SEM images of the sponge without cells showed a highly porous 3D structure, confirmed by the measurement of porosity that was more than 90%. The samples cultured were characterized by cells uniformly distributed and adhered to the sponge surface. Proliferation ended up being promoted, as well as the mineralization ability of the osteoblasts, mainly at the mature stage. In conclusion, this collagen sponge could have a potential use for tissue healing

Nanoporous Titanium Enriched with Calcium and Phosphorus Promotes Human Oral Osteoblast Bioactivity

Implant surfaces are known to influence the osseointegration process; therefore, their modifications represent an important subject of investigation. On this basis, the purpose of this study was to evaluate the response of human oral osteoblasts (hOBs) to three different GR4 titanium discs: Machined, double-etched (Osteopore), and double-etched, surface-enriched with calcium and phosphorus (CaP) (Nanopore). The superficial topography was investigated with scanning electron microscopy (SEM) and the sessile drop technique. To test cellular response and osteoinductive properties, the following points were evaluated: (i) proliferation by MTS assay after 2 and 5 days; (ii) adhesion by multiphoton microscopy at day 2; (iii) the interaction with Ti discs by blue toluidine staining at day 5; (iv) alkaline phosphatase (ALP) activity by ALP assay after 14 days; (v) calcium deposition by alizarin red staining and by cetylpyridinium chloride after 14 days. The SEM analysis showed that Nanopore and Osteopore surfaces were characterized by the same micro-topography. Nanopore and Osteopore discs, compared to Machined, stimulated higher osteoblast proliferation and showed more osteoinductive properties by promoting the ALP activity and calcium deposition. In conclusion, the CaP treatment on DAE surfaces seemed to favor the oral osteoblast response, encouraging their use for in vivo applications

What Is the Impact of Antimicrobial Photodynamic Therapy on Oral Candidiasis? An In Vitro Study

This study aimed to evaluate the ability of photodynamic therapy, based on the use of a gel containing 5% delta aminolaevulinic acid (ALAD) for 45′ followed by irradiation with 630 nm LED (PDT) for 7′, to eradicate Candida albicans strains without damaging the gingiva. C. albicans oral strains and gingival fibroblasts (hGFs) were used to achieve these goals. The potential antifungal effects on a clinical resistant C. albicans S5 strain were evaluated in terms of biofilm biomass, colony forming units (CFU/mL) count, cell viability by live/dead analysis, and fluidity membrane changes. Concerning the hGFs, viability assays, morphological analysis (optical, scanning electronic (SEM), and confocal laser scanning (CLSM) microscopes), and assays for reactive oxygen species (ROS) and collagen production were performed. ALAD-mediated aPDT (ALAD-aPDT) treatment showed significant anti-biofilm activity against C. albicans S5, as confirmed by a reduction in both the biofilm biomass and CFUs/mL. The cell viability was strongly affected by the treatment, while on the contrary, the fluidity of the membrane remained unchanged. The results for the hGFs showed an absence of cytotoxicity and no morphological differences in cells subjected to ALAD-aPDT expected for CLSM results that exhibited an increase in the thickening of actin filaments. ROS production was augmented only at 0 h and 3 h, while the collagen appeared enhanced 7 days after the treatment

The Effects of 5% 5-Aminolevulinic Acid Gel and Red Light (ALAD-PDT) on Human Fibroblasts and Osteoblasts

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT), consisting of 5% 5-aminolevulinic acid-gel and 630 nm-LED, already used for antibacterial effects in the treatment of periodontitis, on human gingival fibroblasts (HGF) and primary human osteoblasts (HOB). HGF and HOB were incubated with different ALAD concentrations for 45 min, and subsequently irradiated with 630 nm-LED for 7 min. Firstly, the cytotoxicity at 24 h and proliferation at 48 and 72 h were assessed. Then the intracellular content of the protoporphyrin IX (PpIX) of the ROS and the superoxide dismutase (SOD) activity were investigated at different times. Each result was compared with untreated and unirradiated cells as the control. Viable and metabolic active cells were revealed at any concentrations of ALAD-PDT, but only 100-ALAD-PDT significantly enhanced the proliferation rate. The PpIX fluorescence significantly increased after the addition of 100-ALAD, and decreased after the irradiation. Higher ROS generation was detected at 10 min in HGF, and at 30 min in HOB. The activity of the SOD enzyme augmented at 30 min in both cell types. In conclusion, ALAD-PDT not only showed no cytotoxic effects, but had pro-proliferative effects on HGF and HOB, probably via ROS generation

A Systematic Review on Organ-on-a-Chip in PDMS or Hydrogel in Dentistry: An Update of the Literature

Organs-on-a-chip (OoCs) are microfluidic devices constituted by PDMS or hydrogel in which different layers of cells are separated by a semipermeable membrane. This technology can set many parameters, like fluid shear stress, chemical concentration gradient, tissue–organ interface, and cell interaction. The use of these devices in medical research permits the investigation of cell patterning, tissue–material interface, and organ–organ interaction, mimicking the complex structures and microenvironment of human and animal bodies. This technology allows us to reconstitute in vitro complex conditions that recapitulate in vivo environments. One of the main advantages of these systems is that they represent a very realistic model that, in many cases, can replace animal experimentation, eliminating costs and related ethical issues. Organ-on-a-chip can also contain bacteria or cancer cells. This technology could be beneficial in dentistry for testing novel antibacterial substances and biomaterials, performing studies on inflammatory disease, or planning preclinical studies. A significant number of publications and reviews have been published on this topic. Still, to our knowledge, they mainly focus on the materials used for fabrication and the different patterns of the chip applied to the experimentations. This review presents the most recent applications of organ-on-a-chip models in dentistry, starting from the reconstituted dental tissues to their clinical applications and future perspectives

Comparison between Single and Multi-LED Emitters for Photodynamic Therapy: An In Vitro Study on Enterococcus faecalis and Human Gingival Fibroblasts

Aim of the study: The aim was to evaluate the effects of two LED devices, TL-01 and TL-03 in photodynamic therapy (PDT), on Enterococcus faecalis and on human gingival fibroblasts (HGFs). TL-01, characterized by a single emitter, irradiates one periodontal site at a time, whereas the multi-led device (TL-03) irradiates all vestibular sites of a single arch at a time. Methods: E. faecalis bacterial suspensions and HGFs were incubated for 45 min with Aladent gel (ALAD) containing 5-aminolevulinic acid and then exposed to LED devices (ALAD-PDT), having different distance and timing of irradiation (TL-01 N (0.5 mm, for 7 min), TL-03 N (0.5 mm, 15 min) and TL-03 F (30.0 mm, 15 min)). For bacterial suspension, the colony forming units and the live/dead staining were evaluated after 24 h, while the protoporphyrin IX (PpIX) content was monitored in all phases of the experimentation. For HGFs, the cell viability, proliferation, cell morphology, and adhesion were evaluated at 24 h. Results: Both TL-01 and TL-03 showed a significant reduction of bacterial load. The photoinactivation was inversely proportional to the PpIX accumulation. TL-01 and TL-03 promoted proliferation and adhesion of HGFs. Conclusions: Both tested devices for ALAD-PDT were equally effective in significantly reducing Enterococcus faecalis growth and in promoting HGFs proliferation and adhesion, in vitro

Efficacy of 5% Aminolaevulinic Acid and Red Light on <i>Enterococcus faecalis</i> in Infected Root Canals

Background: In this ex vivo study, the aim was to evaluate the effects of ALAD and red light on Enterococcus faecalis in infected root canals using a special intracanal fiber. Methods: A total of 70 extracted, single-rooted teeth were used. The teeth were decoronated at the length of the roots to approximately 15 mm and then instrumented. The apical foramen was sealed by composite resin, and the root canals were infected with a pure culture of E. faecalis ATCC 29212 for eight days at 37 °C. Following the contamination period, the roots were divided into seven groups, including the positive and negative control groups, and treated as follows: ALAD 45 min; red light activation 7 min; ALAD 45 min and red-light activation 7 min; sodium hypochlorite 2.5% 15 min; sodium hypochlorite 1% 15 min. The samples were taken by three sterile paper points, transferred to tubes containing 1 mL of PBS, and immediately processed for the number of colony-forming units and the cell viability by using live/dead. Results: The best treatment is obtained with 2.5% NaOCl. Except for ALAD + red light vs. 1% NaOCl, a statistically significant difference is recorded for all treatments. The combination of 2.5% NaOCl and ALAD + 7 min irradiation produces an evident killing effect on the E. faecalis cells. On the other hand, 1% NaOCl is ineffective for the viability action, with 25% of dead cells stained in red. Conclusions: This ex vivo study shows that ALAD gel with light irradiation is an efficacious protocol that exerts a potent antibacterial activity against E. faecalis in infected root canals

5-Aminolevulinic Acid and Red Led in Endodontics: A Narrative Review and Case Report

The present study aims to discuss the main factors involving the use of 5-aminolevulinic acid together with red LED light and its application in endodontic treatment through a narrative review and a case report. Persistence of microorganisms remaining on chemical-mechanical preparation or intracanal dressing is reported as the leading cause of failure in endodontics. Photodynamic therapy has become a promising antimicrobial strategy as an aid to endodontic treatment. Being easy and quick to apply, it can be used both in a single session and in several sessions, as well as not allowing forms of microbial resistance. 5-aminolevulinic acid in combination with red LED light has recently been studied in many branches of medicine, with good results against numerous types of bacteria including Enterococuss faecalis. The case report showed how bacterial count of CFU decreased by half (210 CFU/mL), after 45 min of irrigation with a gel containing 5% of 5-aminolevulinic acid compared to the sample before irrigation (420 CFU/mL). The subsequent irradiation of red LED light for 7 min, the bacterial count was equal to 0. Thus, it is concluded that the use of 5-aminolevulinic acid together with red LED light is effective in endodontic treatment

Oxidative stress induces Wnt canonical/non-canonical pathways modulation in colon cancer cell models

BACKGROUND-AIM. Increased reactive oxygen species (ROS) levels play critical roles in chronic inflammation, and predispose to colon carcinogenesis. Wnt signaling is essential for gut morphogenesis, tissue homeostasis and self-renewal, but its aberrant activation may drive the colorectal cancer (CRC). The ROS production seems to induce the Wnt/β-Catenin pathways, but the molecular mechanisms involved in CRC progression are still undefined. To evaluate the molecular relationship among oxidative stress and canonical/non-canonical Wnt pathways, we analyzed the response to ROS exposure in CRC cell lines with different Wnt signaling behaviour. METHODS. HCT116 (MSI) and SW480 (MSS) cells were treated with H2O2 [2 mM and 10 mM] for 15’and 30’. We assayed cell viability by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR, and protein expression was analyzed by IHC. Statistical analysis was performed by T-test (p value<0.05). RESULTS. MTS revealed different inhibition rates of cell growth at H2O2 concentrations. Acute stress induced by H2O2 [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non canonical ROR2 and JUN/AP1 molecules in SW480, while reduced ROR2 and LRP6 expression in HCT116. Both pathways showed a dose dependent increase in SW480, at H2O2 [10mM]. In HCT116 down-regulated gene expression of APC, LRP6, LEF1, and p65-NFkB was dependent on treatment time, in opposition to non-canonical ROR2. MUTYH, OGG1, NRF2, COX2 and JUN/AP1 expression significantly increased. H2O2 treatment induced FZD6 protein expression in HCT116 cytoplasm and E-cadherin protein expression in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly we relieved a de novo APC expression in both cell lines cytoplasm. FACS analysis of cell cycle showed time dependent changes: upon H2O2 [2mM] treatment at 15’, SW480 increased in G1 and G2 and decreased in S, whereas HCT116 increased in G1 and slightly reduced in G2; after 30’, SW480 enhanced in G1 and S, and reduced in G2 while HCT116 diminished in G1 and increased in S/G2. CONCLUSIONS. In MSI and MSS CRC cells, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel a new scenario for innovative CRC therapeutic approaches