Mechanical properties of 3D-printed prosthetic materials compared with milled and conventional processing: A systematic review and meta-analysis of in vitro studies.

STATEMENT OF PROBLEM: Three-dimensional (3D) additive manufacturing (AM) is an evolving technology in dentistry, proposed as an alternative to subtractive milling manufacture (MM) or conventional processing. However, a systematic review of the use of AM technology instead of milling or conventional processing is lacking. PURPOSE: The purpose of this systematic review and meta-analysis was to evaluate the mechanical properties of 3D-printed prosthetic materials compared with MM and conventional techniques. MATERIAL AND METHODS: An electronic search of the literature was conducted on the MEDLINE (via PubMed), Scopus, and Web of Science databases. The inclusion criteria were in vitro studies published in the last 5 years, in English or Italian, and with 3D AM printed dental prosthetic materials. Data extraction was focused on dental prosthetic materials (ceramics, polymers, and metals) and their mechanical properties: flexural strength, fracture load, hardness, roughness, removable partial denture (RPD) fit accuracy, trueness, marginal discrepancy, and internal fit. Data considered homogenous were subjected to meta-analysis using the Stata17 statistical software program (95% confidence interval [CI]; α=.05). Since all variables were continuous, the Hedge g measure was calculated. A fixed-effects model was used for I2=0%, while the statistical analysis was conducted using a random-effects model with I2>0%. RESULTS: From a total of 3624 articles, 2855 studies were selected, and 76 studies included after full-text reading. The roughness of AM-printed ceramics generally increased compared with that of conventional processing while the marginal discrepancy was comparable both for ceramics and polymers. The flexural strength, hardness, and fracture load of AM-printed polymers were statistically lower than those of the conventional group (P<.05). No significant difference was detected in terms of hardness, roughness, marginal discrepancy, fracture load, trueness, or internal fit between the AM and MM techniques (P>.05). Milling techniques showed significantly higher values of flexural strength (Hedge g=-3.88; 95% CI, -7.20 to -0.58; P=.02), also after aging (Hedge g=-3.29; 95% CI, -6.41 to -0.17; P=.04), compared with AM printing. CONCLUSIONS: AM is comparable with MM in terms of mechanical properties, in particular with polymeric materials. The flexural strength of AM-printed prostheses is lower than with conventional and MM techniques, as are the parameters of hardness and fracture load, while the marginal discrepancy is similar to that of MM and conventional techniques. AM prostheses are commonly used for interim crowns and fixed partial dentures, as their rigidity and fracture resistance cannot support mastication forces for extended periods. More comparative studies are needed.

Heat-not-burn tobacco (IQOS), oral fibroblasts and keratinocytes: cytotoxicity, morphological analysis, apoptosis and cellular cycle. An in vitro study.

OBJECTIVES: The aim of this work is to investigate the biological effects of IQOS smoking on human gingival fibroblasts and human keratinocytes analysing cell viability, morphology, migration, apoptosis and cell cycle. BACKGROUND: Electronic cigarettes and tobacco heating systems have been marketed to reduce smoking damages caused by combustion. METHODS: Human gingival fibroblasts and human keratinocytes viability was determined by a colorimetric assay measuring mitochondrial dehydrogenase activity (MTT assay); after an in vitro exposure of 24 h, cell morphology was analysed with scanning electron microscope and cell migration was tested by Scratch assay, a method to mimic the migration of the cells during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry, and the expression of related genes (p53, Bcl2, p16 and p21) was indagated using real-time polymerase chain reaction. RESULTS: IQOS extracts increased both cell viability (23%-41% with fibroblasts and 30%-79% with keratinocytes) and migration. No morphological alterations were observed. IQOS extracts did not induced an increase in cell death, but rose the number of S- and G2/M-phase cells. IQOS extracts also significantly increased p53 expression by fibroblasts (undiluted and 6.25% dilution, 2- and 3.6-fold higher, respectively) and reduced both Bcl2 (about two- and fivefold, respectively) and p21 expressions (about twofold with both extracts), while on keratinocytes both undiluted and 6.25% dilution extracts increased Bcl2 expression (about four- and threefold higher, respectively) and reduced p53 expression (about two- and fivefold, respectively). CONCLUSION: IQOS smoke seemed to induce proliferation as highlighted by a viability assay, and migration and cell cycle analysis. The increased cell proliferation induced by IQOS devices must be carefully investigated for its possible clinical effects on oral cell populations.

Morpho-functional effects of different universal dental adhesives on human gingival fibroblasts: an in vitro study.

To analyze the effects of four universal adhesives (Optibond Solo Plus-OB, Universal Bond-UB, Prime&Bond Active-PBA, FuturaBond M + -FB) on human gingival fibroblasts in terms of cytotoxicity, morphology and function. After in vitro exposure for up to 48 h, fibroblast viability was determined by the MTT assay determined, morphology by phase-contrast microscopy and migration by the scratch wound assay. Expression levels of IL1ß, IL6, IL8, IL10, TNFα and VEGF genes were assessed by RT-PCR and their protein production by Western blot analysis. Apoptosis and cell cycle were analyzed by flow cytometry. OB and UB induced early morphological changes on fibroblasts (3 h) with extended cell death at 24 h/48 h. Gene expression of collagen type I and fibronectin increased fivefold compared with controls, elastin disappeared and elastase increased threefold, indicating gingival tissue tended to become fibrotic. Only UB and OB increased gene expression of inflammatory markers: IL1ß at 3 and 48 h (up to about three times), IL6 and IL8 at 3 h (up to almost four times) which corresponded to the increase of the activated form NF-kB. All adhesives showed an effect on the functionality of fibroblasts with cytotoxic effect time and concentration dependent. Among all the OB and UB adhesives, they showed the greatest cell damage. The in-depth analysis of the effects of universal adhesives and possible functional effects represents an important information for the clinician towards choosing the most suitable adhesive system.

Systematic Review of Zinc's Benefits and Biological Effects on Oral Health.

BACKGROUND: This review was based on the following question: "What is the state-of-the-art regarding the effect of zinc exposure in the oral cavity on a population of adults and children, compared to dental products containing materials other than zinc, considering in vivo (clinical trials and observational studies) and in vitro studies?" according to a PICOS strategy format. This study aims to analyze zinc application in dental materials, with different compositions and chemical formulations, considering how mechanical and biological properties may influence its clinical applicability. METHODS: In vivo (clinical trials: controlled clinical trials (CCTs) and randomized controlled trials (RCTs); and observational studies: case control and cohort studies) trials or in vitro studies published in English or Italian during the last 10 years on children and adult patients with zinc exposure were included by three different reviewers using the MEDLINE (via PubMed), Scopus, and Web of Science electronic databases. RESULTS: Titles and abstracts were evaluated following the eligibility criteria. The full texts of eligible studies were then reviewed against the inclusion/exclusion criteria. Scientific and technical information of the 33 included studies were collected into evidence tables, reporting data on in vivo and in vitro studies. A narrative approach was adopted. CONCLUSIONS: Antibacterial activity was found to be the most studied property of zinc, but further investigations are needed to establish adjuvant zinc therapies in patients with oral disease.

Acute and chronic cannabidiol treatment: In vitro toxicological aspects on human oral cells.

Cannabidiol is gaining increasing interest for its potential anti-inflammatory, immunomodulatory, and antineoplastic effects. The purpose of this study is to investigate the biological effects of acute and chronic CBD administration on gingival fibroblasts and oral keratinocytes. Viability, morphology, migration, apoptosis and cell cycle, and expression of related genes (p53, BCL2, p21, and BAX) and of endocannabinoid system receptors (CB1, CB2 and GPR55) with real-time PCR and DNA damage with phospho-γ-H2AX immunofluorescence detection were analyzed. Concentrations between 100 µM and 0.001 µM were used: 50 µM (toxic dose), 25 µM (viability promoter), and 1 µM (nontoxic), were selected for subsequent chronic analysis. Acute treatment reveals significant effects than chronic, in particular in fibroblasts: concentrations ≥50 µM are highly cytotoxic, with increased apoptosis and reduced migration. Cell death correlates with increased p53 and BAX, followed by arrest in G0/G1 phase, with elevated p21 levels, suggesting a time- and dose-dependent damage. An increase in H2AX phosphorylation was observed with 25 µM and 50 µM, while 1 µM was biocompatible. Keratinocytes showed less cytotoxic effect than fibroblasts. Induced cell damage was dose- and time-related, with less damage after chronic treatment. Further investigations are needed with longer time frames to evaluate CBD dose- and time-dependent effects to identify an effective therapeutic dose.

In Vitro effects of alternative smoking devices on oral cells: Electronic cigarette and heated tobacco product versus tobacco smoke.

OBJECTIVE: To investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). DESIGN: Human gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. RESULTS: Undiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. CONCLUSIONS: Comparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations.

Use of the Er:YAG Laser in Conservative Dentistry: Evaluation of the Microbial Population in Carious Lesions.

The aim of this study is to investigate the Erbium:Yttrio-Aluminum-Granate (Er:YAG) laser photothermal and mechanical effects on cariogenic species concentration and on the microbial load composition of therapeutic cavities, in order to evaluate the possible micro-organisms reduction and make a comparison with manual and rotating conventional therapy (CT). A clinical trial was designed, including adults with active deep carious lesions on permanent teeth who were divided into two groups, i.e., control group and intervention group treated with CT and Er:YAG therapy, respectively. Before and after any conservative treatment, two oral samples were collected using a small sterile microbrush scrubbed within the base of the dentinal cavity tissue. The percentage of reduction and the colony-forming units (CFUs) count after Er:YAG and conventional treatments were compared for total microorganisms, including Candida spp., Streptococcus spp., and Lactobacillus spp. The microbial reduction varied from 90.2% to 100% and was significantly observed for total microorganisms and Streptococcus spp. (p < 0.05). The Er:YAG laser shows the potential for clinical applications, especially with paediatric and complicated patients, thanks to its minimally invasive properties and its effect on the reduction of microbial load.

Bio-mechanical characterization of a CAD/CAM PMMA resin for digital removable prostheses.

OBJECTIVE: To compare the mechanical and biological features of a polymethylmethacrylate (PMMA) disc for CAD/CAM prostheses (test samples, TG) with a traditional resin (control samples, CG). METHODS: Mechanical analysis was performed using Dynamic Mechanical Analysis (DMA) and Brillouin's micro-spectroscopy. Human keratinocyte morphology and adhesion were analyzed by scanning electronic microscopy (SEM), cytotoxicity by the MTT assay, apoptosis by flow cytometry and p53, p21 and bcl2 gene expression by real time PCR. RESULTS: TG exhibited a higher elastic modulus than CG (range 5100-5500 ± 114.3 MPa vs 3000-3300 ± 99.97 MPa). The Brillouin frequency was found at ωB= (15.50 ± 0.05) GHz for TG and at ωB_1 = (15.50 ± 0.05) GHz and ωB_2 = (15.0 ± 0.1) GHz for CG where two peaks were always present independently of the sample point. SEM analysis revealed that keratinocytes on TG disks appeared to be flattened with lamellipodia. Keratinocytes on CG disks rose above the substrate with cytoplasmatic filaments. MTT viability data at 3 h and 24 h showed TG was significantly less cytotoxic than CG (p < 0.001). No significant differences emerged in apoptosis on CG and TG. Real-time PCR showed p53 expression increased after 3 h by about 9-fold in keratinocytes on TG (p < 0.001) and about 5-fold in those on CG (p < 0.001). High p53 expression persisted after 24 h on both disks. No significant variations were observed in p21 and bcl2 expression at any time-point. SIGNIFICANCE: PMMA resins, as used in CAD/CAM technology, displayed suitable biocompatible and mechanical properties for removable prostheses.

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture.

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.

Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.

PURPOSE: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. MATERIALS AND METHODS: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. RESULTS: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. CONCLUSION: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.

Cytotoxicity of universal dental adhesive systems: Assessment in vitro assays on human gingival fibroblasts.

Universal adhesives are the most important innovation in restorative dentistry. They are composed of different monomers, solvents and fillers. The potential cytotoxic effect of these materials is an important scientific aspect in recent literature. The aim of this study was to determine, using different in vitro techniques, the cytotoxicity evaluation of seven universal enamel-dental adhesives on human gingival fibroblasts. For this purpose, seven universal dental enamel adhesives have been evaluated by in vitro cytotoxicity tests using direct contact tests (an unpolymerized and a polymerized method) and an indirect contact test: preparation of extracts. The polymerized method showed a cytotoxicity range from 36% (G-PremioBond, GPB) to 79% (FuturaBond M+, FB). With the unpolymerized direct methods the range was from 4% (Prime&Bond Active, PBA) to 40% (Ibond Universal, IB) for undiluted adhesives; generally passing to the major dilutions the test showed a strong inhibitory activity by all the adhesives. Whereas with the indirect method by diluting the extracts of all dental adhesives the cell viability increased. The data obtained from the work has shown a lower cytotoxic effect of Optibond Solo Plus (OB) and Adhesive Universal (AU) with more reliable results with the extracts technique. The choice of reliable in vitro cytotoxic technique could represent, in dental practice, an important aid for clinical procedures in the use of adhesive systems.

Biological, thermal and mechanical characterization of modified glass ionomer cements: The role of nanohydroxyapatite, ciprofloxacin and zinc l-carnosine.

The study evaluated the effects of 4 wt% nanohydroxyapatite (HA), 6 wt% zinc l-carnosine (MDA) and 1.5 wt% Ciprofloxacin (AB) on the mechanical, thermal and biological properties of glass ionomer cements (GIC). Filler and additive concentrations were selected after a previous study had tested single components and different percentages. Specimens included five silicon molds of each GIC cement for all tests. They were stored at room temperature for 24 h from specimen collection to analysis. Mechanical tests, calorimetric analysis, morphological investigation, antibacterial and cell viability assays were conducted. One-way analysis of variance (ANOVA) was used for data analysis with significance set at p < 0.05. Adding HA, MDA and AB to GICs modified their thermal, mechanical and microbiological properties. Polymerization increased. A slight decrease in the compressive strength of modified GICs was observed in dry condition (p < 0.05). Cement extracts affected cell viability in relation to extract dilution. Mechanical behavior improved in modified glass ionomer cements, especially with the powder formulated antibiotic. Overall cytotoxicity was reduced. Therefore adding nanohydroxyapatite, antibiotic and a mucosal defensive agent to conventional glass ionomer cement in special need patients could improve the clinical, preventive and therapeutic performance of the cements, without altering their mechanical properties.

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Cytotoxicity of three commercial mouthrinses on extracellular matrix metabolism and human gingival cell behaviour.

This study evaluated the effects of commercially available antiseptic mouthrinses on human gingival fibroblast and keratinocyte behaviour and metabolism. Three mouthrinses containing essential oil (EO), chlorhexidine (CHX) and amine fluoride/stannous fluoride (AFSF), were tested in an in vitro study. Human gingival fibroblasts and keratinocytes were washed with 10% or 30% concentration of the commercial mouthrinses and their effects on cell adhesion and proliferation were investigated as well as the specific gene expression of markers involved in oral mucosa metabolism. As markers of cell metabolism, type I and IV collagens, laminin, fibronectin, fibromodulin and integrins were studied with real-time PCR. Moreover, interleukin-1 secretion, one of the major pro-inflammatory cytokines, was evaluated. The results showed that CHX significantly reduced fibroblast and keratinocyte substrate adhesion capacities and CHX and EO inhibited cell proliferation better than AFSF rinse. The gene expression of several matrix components and cell adhesion receptors was downregulated in cells washed with CHX and EO compared with those washed with AFSF rinse. In conclusion, the AFSF mouthrinse does not induce or induces to a lesser extent the onset of irritation and/or cytotoxicity than CHX or EO. These findings and those of future studies will enable us to gain further insight into the clinical significance and effects of commercial mouthrinses. Pending further investigations, clinicians should be aware of the potentially adverse effects of mouthrinses and warn their patients against making improper use of these products.

Biocompatibility of collagen membranes crosslinked with glutaraldehyde or diphenylphosphoryl azide: an in vitro study.

Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen.

Effects of hydroxyapatite and Biostite on osteogenic induction of hMSC.

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.