Engineering models of head and neck and oral cancers on-a-chip.

Head and neck cancers (HNCs) rank as the sixth most common cancer globally and result in over 450 000 deaths annually. Despite considerable advancements in diagnostics and treatment, the 5-year survival rate for most types of HNCs remains below 50%. Poor prognoses are often attributed to tumor heterogeneity, drug resistance, and immunosuppression. These characteristics are difficult to replicate using in vitro or in vivo models, culminating in few effective approaches for early detection and therapeutic drug development. Organs-on-a-chip offer a promising avenue for studying HNCs, serving as microphysiological models that closely recapitulate the complexities of biological tissues within highly controllable microfluidic platforms. Such systems have gained interest as advanced experimental tools to investigate human pathophysiology and assess therapeutic efficacy, providing a deeper understanding of cancer pathophysiology. This review outlines current challenges and opportunities in replicating HNCs within microphysiological systems, focusing on mimicking the soft, glandular, and hard tissues of the head and neck. We further delve into the major applications of organ-on-a-chip models for HNCs, including fundamental research, drug discovery, translational approaches, and personalized medicine. This review emphasizes the integration of organs-on-a-chip into the repertoire of biological model systems available to researchers. This integration enables the exploration of unique aspects of HNCs, thereby accelerating discoveries with the potential to improve outcomes for HNC patients.

Self-assembly peptide P11-4 induces mineralization and cell-migration of odontoblast-like cells.

OBJECTIVES: Self-assembling peptide P11-4 is amphiphilic and pH-triggered, effective on repairing early enamel carious lesions and dentin remineralization. However, P11-4 effects on dentin biomineralization and repair ability remain unexplored. Thus, cytocompatibility and effectiveness of P11-4 on inducing mineralization and migration of odontoblast-like cells (MDPC-23) were investigated. METHODS: MDPC-23 were seeded in contact with P11-4 (0.5 and 1 µg/ml), Dentin Matrix Protein 1 (DMP1 0.5 and 1 µg/ml) or Calcium hydroxide (Ca(OH)2 100 µg/ml) solutions. Cell viability was verified using MTT (n = 6/group). Mineral deposition was tested using Alizarin Red (n = 4/group). Cell migration was assessed by light microscopy (n = 2/group). MTT and Alizarin Red data were compared using Kruskal-Wallis and Mann-Whitney (α=0.01). RESULTS: P11-4 (0.5 and 1 µg/ml) and DMP1 (0.5 and 1 µg/ml) resulted the highest cell viability; Ca(OH)2 presented the lowest. 1 µg/ml DMP1 and 1 µg/ml P11-4 promoted the highest mineral deposition. Ca(OH)2 presented lower values of mineral deposits than DMP1 1 µg/ml (p < 0.01), but similar to P11-4 1 µg/ml. P11-4 and DMP1 at 0.5 µg/ml induced lesser mineral precipitation than P11-4 and DMP1 at 1 µg/ml (p < 0.01), with no difference to Ca(OH)2. All materials stimulated cell migration, however, lower concentrations of DMP1 and P11-4 demonstrated a higher migration potential. CONCLUSION: P11-4 did not affect cell viability, induces mineral deposition and MDPC-23 migration like DMP1. CLINICAL SIGNIFICANCE: Self-assembling peptide P11-4 does not affect the cell viability and induces mineral deposition comparable to native protein involved in biomineralization. Combined with its ability to bind type I collagen, P11-4 is a promising bioinspired molecule that provides native-tissue conditions and foster further studies on its ability to form dentin bridges in pulp-capping strategies.

Impact of biomineralization on resin/biomineralized dentin bond longevity in a minimally invasive approach: An "in vitro" 18-month follow-up.

OBJECTIVES: To determine the impact of treating caries-affected dentin (CAD) with: 0.2% sodium fluoride (NaF), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP/MI Paste™) or peptide P11-4 (Curodont™ Repair) on the longevity of resin/CAD interface at storage times of 24 -h, 6- and 18-month. METHODS: 255 caries-free third molars were used, and CAD was produced by a biological method. The teeth were randomly distributed into: G1- Sound dentin (SD); G2- CAD; G3- CAD + 0.2% NaF (CAD/NaF); G4- CAD + CPP-ACP (CAD/ACP); G5- CAD + Curodont™ Repair (CAD/P11-4). The Filtek Z350 composite resin block was bonded to dentin using Adper™ Single 2 (4 mm/height). Resin/dentin blocks were stored in a solution of Simulated Body Fluid at 37 °C, pressures were modified to simulate natural pulpal pressures. Specimens were investigated by microtensile bond strength (µTBS) (n = 8), Scanning Electron Microscopy (to assess the failure mode) (n = 8), nanoinfiltration (to assess the interface sealing) (n = 3), in situ zymography (to assess the gelatinolytic activity) (n = 3) and micro-computed microtomography (µ-CT) (to assess the mineralization) (n = 3). Data from µTBS, µ-CT and, nanoinfiltration and hybrid layer formation/degradation were submitted to two-way ANOVA and Tukey tests, and failure patterns and in situ zymography to Kruskal-Wallis and Dunn tests (α = 5%). RESULTS: The highest mineral density change by µ-CT, smallest silver nitrate infiltration and proteolytic activity in the adhesive layer were obtained significantly for the groups SD, CAD/ACP and CAD/P11-4, with most mixed fractures at 18-month (p < 0.001). CAD/NaF showed significantly similar values to CAD, CAD and CAD/NaF which presented a high percentage of adhesive fracture (p < 0.001) at all time periods. SIGNIFICANCE: Treating caries-affected dentin with remineralizing agents CPP-ACP and Curodont™ Repair, has the potential to be a clinically relevant treatment protocol to increase the longevity of adhesive restorations.

3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy.

Direct visualization of the spatial relationships of the dental pulp tissue at the whole-organ has remained challenging. CLARITY (Clear Lipid-exchanged Acrylamide Tissue hYdrogel) is a tissue clearing method that has enabled successful 3-dimensional (3D) imaging of intact tissues with high-resolution and preserved anatomic structures. We used CLARITY to study the whole human dental pulp with emphasis on the neurovascular components. Dental pulps from sound teeth were CLARITY-cleared, immunostained for PGP9.5 and CD31, as markers for peripheral neurons and blood vessels, respectively, and imaged with light sheet microscopy. Visualization of the whole dental pulp innervation and vasculature was achieved. Innervation comprised 40% of the dental pulp volume and the vasculature another 40%. Marked innervation morphological differences between uni- and multiradicular teeth were found, also distinct neurovascular interplays. Quantification of the neural and vascular structures distribution, diameter and area showed that blood vessels in the capillary size range was twice as high as that of nerve fibers. In conclusion whole CLARITY-cleared dental pulp samples revealed 3D-morphological neurovascular interactions that could not be visualized with standard microscopy. This represents an outstanding tool to study the molecular and structural intricacies of whole dental tissues in the context of disease and treatment methods.

No additional benefit of using a calcium hydroxide liner during stepwise caries removal: A randomized clinical trial.

BACKGROUND: Clinicians often use calcium hydroxide liners during stepwise treatment of advanced caries. In this randomized clinical trial, the authors compared the short-term outcome of stepwise caries removal with and without use of a calcium hydroxide liner in conjunction with provisional resin-modified glass ionomer (RMGI) restorations. METHODS: The authors included in the trial 98 patients aged 15 to 30 years who had a deep carious lesion in a posterior tooth. The authors measured the dentin thickness radiographically and recorded its color, consistency, and moisture, as well as the bacterial count of the lesions. After partial caries removal, the authors assigned patients randomly to have their caries provisionally restored using RMGI with (control group) or without (test group) a calcium hydroxide liner. The primary outcome measure was tooth vitality after 90 days. Secondary outcomes included changes in dentinal, radiographic, and microbiological characteristics of the lesions. RESULTS: The authors found no statistically significant difference between the test and control groups in tooth vitality after 90 days. Irrespective of calcium hydroxide liner use, the authors observed darker, harder, drier, and less contaminated dentin after the provisional restorations, but dentin thickness remained unchanged. CONCLUSIONS: On the basis of this 3-month clinical trial's results, the use of a calcium hydroxide liner during stepwise caries excavation and provisional restoration did not provide any additional benefit. PRACTICAL IMPLICATIONS: After 3 months, using a calcium hydroxide liner does not appear to offer any additional benefit when clinicians use RMGI provisional restorations during stepwise caries removal. Longer studies are needed to confirm these results.

Surface roughness of restoration margin preparations: a comparative analysis of finishing techniques.

This study compared the margin profile and surface roughness created by the tips of four different finishing instruments: fine diamond, dura white stone, tungsten carbide, and ultrasonic diamond-coated tips (UDTs). The aim was to determine which of these instruments produced the smoothest finish and created the most evenly contoured margin characteristics. It was hypothesized that UDTs would produce a rougher dentin surface than a fine diamond bur, that a tungsten carbide bur would provide a smoother finish than a fine diamond, and that the dura white stone would produce an intermediate finish. Forty extracted premolars were divided into two groups. For the first group, a 1.5 x 3.0-mm dentin slot was prepared in 30 teeth using a control 50-µm diamond bur, followed by one of the four finishing instruments. The surface roughness (Ra) was then measured using a surface profilometer and a one-way analysis of variance followed by a post hoc Bonferroni test to assess whether any statistical difference existed among the Ra values. For the second group, shoulder margins were prepared in 10 teeth. They were then refined with one of the four finishing instruments and examined with scanning electron microscopy (SEM). The fine diamond bur created a significantly smoother surface than the control diamond (P < .001), UDTs (P < .007), and tungsten carbide bur (P < .010). The fine diamond was not found to be significantly smoother than the dura white stone. SEM images of the fine diamond showed divoting on the margin floor. The dura stone showed a well-defined, undamaged margin. The tungsten carbide bur created frequent chipping in enamel margins. The UDT specimens showed an inconsistent finish and discrete patches of open dentinal tubules. The fine diamond created the lowest Ra values; however, the dura stone offered efficient finishing and less damage to the margin profile.

Influence of hydration on nanoindentation induced energy expenditure of dentin.

Improved understanding of the effects of hydration and drying in mineralized tissues is highly desirable, particularly for physiologically hydrated biological materials such as dentin. We investigated the influence of hydration on the nanomechanical properties of healthy dentin and hypothesized that drying leads to an increase in indentation induced energy expenditure and hardness. Hydrated and dry dentin were tested with a UMIS set up with a Berkovich indenter at a maximum load of 50 mN. Values representative of the energy expenditure behavior were presented as dissipated energy, U(d), recovered energy, U(e), normalized energy expenditure index, ψ, and hardness, H. Energy expenditure index results, which normalize the energy expenditure for each test and describe the relative energy dissipation-recovery behavior of a material, suggested that, for the relatively severe contact strains about a sharp Berkovich indenter, dissipation dominates the mechanical response of both the hydrated and dry dentin. In support of our initial hypothesis, dry dentin presented a significantly higher energy expenditure index than hydrated dentin (p<0.0001). These results were primarily associated with a lower U(e) that was found upon drying. Hydration also decreased H significantly (p<0.0001). In summary, this study presents the first direct measurements of the energy expenditure behavior of hydrated and dry dentin using instrumented nanoindentation.

Insights into the structure and composition of the peritubular dentin organic matrix and the lamina limitans.

Dentin is a mineralized dental tissue underlying the outer enamel that has a peculiar micro morphology. It is composed of micrometer sized tubules that are surrounded by a highly mineralized structure, called peritubular dentin (PTD), and embedded in a collagen-rich matrix, named intertubular dentin. The PTD has been thought to be composed of a highly mineralized collagen-free organic matrix with unknown composition. Here we tested the hypothesis that proteoglycans and glycosaminoglycans, two important organic structural features found in dentin, are key participants in the microstructure and composition of the PTD. To test this hypothesis dentin blocks were demineralized with 10 vol% citric acid for 2 min and either digested with 1mg/ml TPCK-treated trypsin with 0.2 ammonium bicarbonate at pH 7.9 (TRY) or 0.1 U/mL C-ABC with 50mM Tris, 60mM sodium acetate and 0.02% bovine serum albumin at pH 8.0 (C-ABC). TRY is known to cleave the protein core of dentin proteoglycans, whereas C-ABC is expected to selectively remove glycosaminoglycans. All specimens were digested for 48 h in 37°C, dehydrated in ascending grades of acetone, immersed in HMDS, platinum coated and imaged using an FE-SEM. Images of demineralized dentin revealed a meshwork of noncollagenous fibrils protruding towards the tubule lumen following removal of the peritubular mineral and confirmed the lack of collagen in the peritubular matrix. Further, images revealed that the peritubular organic network originates from a sheet-like membrane covering the entire visible length of tubule, called lamina limitans. Confirming our initial hypothesis, after the digestion with C-ABC the organic network appeared to vanish, while the lamina limitans was preserved. This suggests that glycosaminoglycans are the main component of the PTD organic network. Following digestion with TRY, both the organic network and the lamina limitans disappeared, thus suggesting that the lamina limitans may be primarily composed of proteoglycan protein cores. In summary, our results provide novel evidence that (1) PTD lacks collagen fibrils, (2) PTD contains an organic scaffold embedded with mineral and (3) the PTD organic matrix is manly composed of glycosaminoglycans, whereas the lamina limitans is primarily made of proteoglycans protein cores.

Evaluation of surface structural and mechanical changes following remineralization of dentin.

This study sought to gain insights into the surface structural and mechanical changes leading to remineralization of dentin. Remineralization was compared between a continuous remineralization approach and a nonbuffered static approach using solutions of the same initial composition. Artificial carious lesions were treated for 5 days and analyzed every 24 h using nanoindentation in water, SEM, and AFM. The continuous approach yielded a recovery of mechanical properties of up to 60% of normal dentin, whereas the static approach led to recovery of only 10%. Image analysis revealed that the static approach yielded the formation of areas suggestive of an apatite precipitate on the surface of the dentin matrix. In contrast, surface precipitate was absent using the continuous approach, suggesting that mineral formed within the lesion and re-associated with the collagenous matrix. This study provided evidence that mechanical recovery of dentin in near physiological conditions is attainable through the continuous delivery of calcium and phosphate ions.

Effect of additives on the compressive strength and setting time of a Portland cement.

Improvements in strength and setting time of Portland cements (PC) are needed to enhance their performance as endodontic and load bearing materials. This study sought to enhance the compressive strength and setting time of a PC by adding one of the following additives: 20% and 30% poly-methylmethacrylate (PMMA), 20% and 30% irregular and spherical amalgam alloys, and 10% CaCl(2). The control consisted of unreinforced PC specimens. Setting time was determined using a Gillmore apparatus according to standardized methods while compressive strength was measured using a universal testing machine after 21 hours or 60 days of water storage. Data were analyzed by ANOVA, Tukey and Games-Howell tests (alpha = 5%). All additives significantly decreased both initial and final setting times as compared with the PC-control (p < .05). 30% PMMA and 30% irregular alloy had the lowest values of initial setting time. 30% irregular alloy also produced the lowest values of final setting time while 30% spherical alloy yielded the highest (p < .05). No differences were detected between the compressive strength values of 21 hours and 60 days. While 10% CaCl(2), 20% and 30% PMMA produced values significantly lower than the PC-control, 30% spherical alloy significantly improved the compressive strength of the reinforced PC (p < .05). In summary, all additives significantly reduced the setting time and 30% spherical amalgam alloy yielded a significant increase in compressive strength for the tested PC, which might represent an improved composition for PCs to expand their use as endodontic and potentially load bearing materials.

Effect of additives on the compressive strength and setting time of a Portland cement

Improvements in strength and setting time of Portland cements (PC) are needed to enhance their performance as endodontic and load bearing materials. This study sought to enhance the compressive strength and setting time of a PC by adding one of the following additives: 20 percent and 30 percent poly-methylmethacrylate (PMMA), 20 percent and 30 percent irregular and spherical amalgam alloys, and 10 percent CaCl2. The control consisted of unreinforced PC specimens. Setting time was determined using a Gillmore apparatus according to standardized methods while compressive strength was measured using a universal testing machine after 21 hours or 60 days of water storage. Data were analyzed by ANOVA, Tukey and Games-Howell tests (á = 5 percent). All additives significantly decreased both initial and final setting times as compared with the PC-control (p < .05). 30 percent PMMA and 30 percent irregular alloy had the lowest values of initial setting time. 30 percent irregular alloy also produced the lowest values of final setting time while 30 percent spherical alloy yielded the highest (p < .05). No differences were detected between the compressive strength values of 21 hours and 60 days. While 10 percent CaCl2, 20 percent and 30 percent PMMA produced values significantly lower than the PC-control, 30 percent spherical alloy significantly improved the compressive strength of the reinforced PC (p < .05). In summary, all additives significantly reduced the setting time and 30 percent spherical amalgam alloy yielded a significant increase in compressive strength for the tested PC, which might represent an improved composition for PCs to expand their use as endodontic and potentially load bearing materials.

Nanotechnology in Dental Sciences: Moving towards a Finer Way of Doing Dentistry.

Nanotechnologies are predicted to revolutionize: (a) the control over materials properties at ultrafine scales; and (b) the sensitivity of tools and devices applied in various scientific and technological fields. In this short review, we argue that dentistry will be no exception to this trend. Here, we present a dynamic view of dental tissues, an adoption of which may lead to finer, more effective and minimally invasive reparation approaches. By doing so, we aim at providing insights into some of the breakthroughs relevant to understanding the genesis of dental tissues at the nanostructural level or generating dental materials with nanoscale critical boundaries. The lineage of the progress of dental science, including the projected path along the presumed nanotechnological direction of research and clinical application is mentioned too. We conclude by claiming that dentistry should follow the trend of probing matter at nanoscale that currently dominates both materials and biological sciences in order to improve on the research strategies and clinical techniques that have traditionally rested on mechanistic assumptions.