Biofabrication Strategies for Oral Soft Tissue Regeneration.

Gingival recession, a prevalent condition affecting the gum tissues, is characterized by the exposure of tooth root surfaces due to the displacement of the gingival margin. This review explores conventional treatments, highlighting their limitations and the quest for innovative alternatives. Importantly, it emphasizes the critical considerations in gingival tissue engineering leveraging on cells, biomaterials, and signaling factors. Successful tissue-engineered gingival constructs hinge on strategic choices such as cell sources, scaffold design, mechanical properties, and growth factor delivery. Unveiling advancements in recent biofabrication technologies like 3D bioprinting, electrospinning, and microfluidic organ-on-chip systems, this review elucidates their precise control over cell arrangement, biomaterials, and signaling cues. These technologies empower the recapitulation of microphysiological features, enabling the development of gingival constructs that closely emulate the anatomical, physiological, and functional characteristics of native gingival tissues. The review explores diverse engineering strategies aiming at the biofabrication of realistic tissue-engineered gingival grafts. Further, the parallels between the skin and gingival tissues are highlighted, exploring the potential transfer of biofabrication approaches from skin tissue regeneration to gingival tissue engineering. To conclude, the exploration of innovative biofabrication technologies for gingival tissues and inspiration drawn from skin tissue engineering look forward to a transformative era in regenerative dentistry with improved clinical outcomes.

Modeling periodontal host-microbe interactions using vascularized gingival connective tissue equivalents.

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabeling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly,in silicoanalysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimeticin vitroplatform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.

Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip.

Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva-oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host-microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof-of-principle "gingival crevice-on-chip" microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On-chip co-culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm-like structures at the tissue-microbiome interface, long-term co-culture, and bacterial clearance secondary to simulated GCF (s-GCF) flow. Further, on-chip exposure of the gingival CTEs to the toll-like receptor-2 (TLR-2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s-GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host-microbe interactions in periodontal disease and the development of periodontal therapeutics under near-microphysiological conditions.

Differential immune responses of 3D gingival and periodontal connective tissue equivalents to microbial colonization.

Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.

Molecular insight to size and dose-dependent cellular toxicity exhibited by a green synthesized bioceramic nanohybrid with macrophages for dental applications.

Improvising bioceramics for enhancing their biocompatibility and physical properties has been a focus area for the dental industry. To further explore this area, this study reports a novel green synthesis and molecular in vitro biocompatibility of calcium aluminosilicate-chitosan nanohybrid (CAS-CH). The nanohybrids were synthesized by using a high energy ball milling (HEBM) technique and then characterized for their physiochemical properties using standard techniques including scanning electron microscopy (SEM) and dynamic light scattering (DLS). In vitro cytotoxicity evaluation of a synthesized nanohybrid was made with a RAW264.7 cell line using cell viability assays, such as, MTT, cellular morphology analysis, induction of oxidative stress, and apoptosis. CAS-CH nanohybrids were synthesized at three milling time points: 1H, 2H, and 3H. With increasing milling time, we found a reduction in sizes of particles and increased zeta potential. Viability of cells was found to be decreased with an increase in concentration. Moreover, toxic effects like ROS generation and apoptosis were reduced with increasing milling time. Computational and experimental analysis elucidated the mechanism of toxicity as a consequence of influential functionality of Sod1 and p53 proteins due to interaction and internalization of the nanohybrids with amino acid residues via hydrogen bonds and hydrophobic interactions. The detailed study depicted a novel way of synthesizing biocompatible bioceramic nanohybrids with a mechanistic insight of its cytotoxicity profile.

Mechanistic insight into ROS and neutral lipid alteration induced toxicity in the human model with fins (Danio rerio) by industrially synthesized titanium dioxide nanoparticles.

The toxicological impact of TiO2 nanoparticles on the environment and human health has been extensively studied in the last few decades, but the mechanistic details were unknown. In this study, we evaluated the impact of industrially prepared TiO2 nanoparticles on the biological system using zebrafish embryo as an in vivo model. The industrial synthesis of TiO2 nanoparticles was mimicked on the lab scale using the high energy ball milling (HEBM) method by milling bulk TiO2 particles for 5 h, 10 h, and 15 h in an ambient environment. The physiochemical properties were characterized by standard methods like field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), X-ray diffraction (XRD) and UV-Visible spectroscopy. In vivo cytotoxicity was assessed on zebrafish embryos by the evaluation of their mortality rate and hatching rate. Experimental and computational analysis of reactive oxygen species (ROS) induction, apoptosis, and neutral lipid alteration was done to study the effects on the cellular level of zebrafish larvae. The analysis depicted the change in size and surface charge of TiO2 nanoparticles with respect to the increase in milling time. In silico investigations revealed the significant role of ROS quenching and altered neutral lipid accumulation functionalised by the molecular interaction of respective metabolic proteins in the cytotoxicity of TiO2 nanoparticles with zebrafish embryos. The results reveal the hidden effect of industrially synthesized TiO2 nanoparticle exposure on the alteration of lipid accumulation and ROS in developing zebrafish embryos. Moreover, the assessment provided a detailed mechanistic analysis of in vivo cytotoxicity at the molecular level.

In Vivo Molecular Toxicity Profile of Dental Bioceramics in Embryonic Zebrafish ( Danio rerio).

The investigation of the biocompatibility of potential and commercially available dental material is a major challenge in dental science. This study demonstrates that the zebrafish model is a novel in vivo model for investigating the biocompatibility of dental materials. Two commercially available dental materials, mineral trioxide aggregate (MTA) and Biodentine, were assessed for their biocompatibility. The biocompatibility analysis was performed in embryonic zebrafish with the help of standard toxicity assays measuring essential parameters such as survivability and hatching. The mechanistic and comparative analysis of toxicity was performed by oxidative stress analysis by measuring ROS induction and apoptosis in zebrafish exposed to dental materials at different concentrations. The molecular investigation at the protein level was done by a computational approach using in silico molecular docking and pathway analysis. The toxicity analysis showed a significant reduction in hatching and survivability rates along with morphological malformations with an increase in the concentration of exposed materials. ROS and apoptosis assay results revealed a greater biocompatibility of Biodentine as compared to that of MTA which was concentration-dependent. In silico analysis showed the significant role of the tricalcium silicate-protein ( Sod1, tp53, RUNX2B) interaction in an exhibition of toxicity. The study provides a new vision and standard in dental material sciences for assessing the biocompatibility of potential novel and commercially available dental materials.

Fabrication and Appraisal of Poly (Lactic-Co-Glycolic Acid) - Moxifloxacin Nanoparticles using Vitamin E-TPGS: A Potential Intracanal Drug Delivery Agent.

INTRODUCTION: Nanotechnology is gaining momentum in the search for ideal biomaterials by dental researchers. The expansible nature of Endodontology deems exploration, learning and scrutinizing newer avenues which have the potential to be applied and investigated. The popularity of polymers as drug delivery vehicles has opened avenues for their application in the root canal system. This study utilized application of biodegradable polymers as potential drug delivery vehicles against Enterococcus faecalis, one of the main reasons for post treatment disease. AIM: This study aimed at fabricating Poly (Lactic-co-Glycolic Acid) (PLGA)-moxifloxacin nanoparticles and assessing its sustained antimicrobial efficacy with calcium hydroxide and chitosan-moxifloxacin hydrogel against Enterococcus faecalis. MATERIALS AND METHODS: PLGA (50:50) in a quantity of 100 mg was dissolved in 1 ml ethyl acetate and 45 ml of 0.3% w/v Vitamin-E Polyethylene Glycol 1000 Succinate (vitamin E-TPGS) was kept for magnetic stirring in separate beaker. Moxifloxacin (50 µl) was added to polymer PLGA following which vitamin E-TPGS was added to the polymer. Nanoparticles were fabricated using ultrasonication and collected by centrifugation. Surface characterization was assessed using scanning electron microscope. Results were obtained in the form of zone of inhibition by the nanoparticles against Enterococcus faecalis and comparisons were made with chitosan-moxifloxacin hydrogel and calcium hydroxide using Analysis of Variance (ANOVA) followed by Student t-test. RESULTS: Upon statistical analysis, the zone of inhibition against E. faecalis remained constant with PLGA-moxifloxacin nanoparticles for 14 days while it decreased with chitosan-moxifloxacin hydrogel and remained nil for calcium hydroxide (p<0.01). CONCLUSION: The study posits that PLGA encapsulated moxifloxacin nanoparticles showcased sustained antibacterial effect in low doses against the test pathogen. Its sustained and programmed release makes them unique contenders for further evaluation in Endodontics as potential intracanal medicaments.