Composite PCL Scaffold With 70% ß-TCP as Suitable Structure for Bone Replacement.

OBJECTIVES: The purpose of this work was to optimise printable polycaprolactone (PCL)/ß-tricalcium phosphate (ß-TCP) biomaterials with high percentages of ß-TCP endowed with balanced mechanical characteristics to resemble human cancellous bone, presumably improving osteogenesis. METHODS: PCL/ß-TCP scaffolds were obtained from customised filaments for fused deposition modelling (FDM) 3D printing with increasing amounts of ß-TCP. Samples mechanical features, surface topography and wettability were evaluated as well as cytocompatibility assays, cell adhesion and differentiation. RESULTS: The parameters of the newly fabricated materila were optimal for PCL/ß-TCP scaffold fabrication. Composite surfaces showed higher hydrophilicity compared with the controls, and their surface roughness sharply was higher, possibly due to the presence of ß-TCP. The Young's modulus of the composites was significantly higher than that of pristine PCL, indicating that the intrinsic strength of ß-TCP is beneficial for enhancing the elastic modulus of the composite biomaterials. All novel composite biomaterials supported greater cellular growth and stronger osteoblastic differentiation compared with the PCL control. CONCLUSIONS: This project highlights the possibility to fabricat, through an FDM solvent-free approach, PCL/ß-TCP scaffolds of up to 70 % concentrations of ß-TCP. overcoming the current lmit of 60 % stated in the literature. The combination of 3D printing and customised biomaterials allowed production of highly personalised scaffolds with optimal mechanical and biological features resembling the natural structure and the composition of bone. This underlines the promise of such structures for innovative approaches for bone and periodontal regeneration.

Relationship between periodontal disease and obstructive sleep apnea in adults: A systematic review.

The purpose of this systematic review was to evaluate whether there are scientific evidence regarding the association between periodontitis and obstructive sleep apnea (OSA) in adults. An electronic search was performed on MEDLINE/PubMed for prospective and retrospective longitudinal studies, cohort studies, and case-control studies conducted in human adults affected by both OSA and periodontitis. Two reviewers extracted the data using a custom Excel spreadsheet. A methodological assessment of the quality of the studies was performed using the Newcastle-Ottawa Scale. Fourteen studies were included. All studies evaluated the association between periodontitis and OSA. None of the studies evaluated the cause-effect relationship. Eleven studies found a significant positive relationship between periodontitis and OSA, whereas three found no statistically significant association. Several study limitations were observed, such as lack of standardization of study groups, diagnosis of periodontitis and OSA, and differences in study design. Evidence of a plausible association between periodontitis and OSA was found. The possible relationship could be explained by systemic inflammation, oral breathing, and the comorbid relationship attributable to common risk factors. Observational and randomized controlled studies are needed to clarify the mechanism of interaction between the two conditions.

Anti-Fibronectin Aptamer Modifies Blood Clot Pattern and Stimulates Osteogenesis: An Ex Vivo Study.

BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation. METHODS: 20 µg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points. RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT. CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro.

Background/purpose: Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. Materials and methods: We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Results: Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. Conclusion: These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application.

A Novel Self-Assessment Method for Training Access Cavity on 3D Printed Endodontic Models.

BACKGROUND: New technologies can facilitate the transition from pre-clinical to clinical settings. We investigate students' satisfaction with a novel learning method adopted in access cavity exercises. METHODS: Students performed their access cavity on inexpensive, in-house 3D printed teeth. Their performances were evaluated by scanning the prepared teeth with an intraoral scanner and visualized using a mesh processing software. Then, the same software was used to align the tooth prepared by the student and the teacher's one for self-assessment purposes. Students were asked to answer a questionnaire about their experiences with this new learning method. RESULTS: From the teacher's perspective, this novel learning approach was easy, straightforward and affordable. Overall, student feedback was positive: 73% found that access cavity assessment by scanning was more useful compared to a visual inspection under magnification and 57% reported that they had a better understanding of errors and mishaps. On the other hand, students pointed out that the material used to print teeth was too soft. CONCLUSION: The use of in-house 3D printed teeth in pre-clinical training is a simple way to overcome some of the drawbacks associated with extracted teeth, such as limited availability, variability, cross-infection control, and ethical constraints. The use of intraoral scanners and mesh processing software could improve student self-assessment.

Predictability of intraoral scanner error for full-arch implant-supported rehabilitation.

OBJECTIVES: The present study aimed to analyze the behaviors of three intraoral scanners (IOSs): evaluating the interdistance and axial inclination discrepancies in full-arch scans, predictable errors were searched. MATERIALS AND METHODS: Six edentulous sample models with variable numbers of dental implants were used; reference data were obtained with a coordinate-measuring machine (CMM). Each IOS (i.e., Primescan, CS3600, and Trios3) performed 10 scans per model (180 total scans). The origin of each scan body was used as a reference point to measure interdistance lengths and axial inclinations. Precision and trueness of interdistance measurements and axial inclinations were evaluated to address error predictability. Bland-Altman analysis, followed by linear regression analysis and Friedman's test (plus Dunn's post hoc correction), was performed to evaluate the precision and trueness. RESULTS: Regarding interdistance, Primescan showed the best precision (mean ± SD: 0.047 ± 0.020 mm), while Trios3 underestimated the reference value more than the others (p < 0.001) and had the worst performance (mean ± SD: -0.079 ± 0.048 mm). Concerning the inclination angle, Primescan and Trios3 tended to overestimate angle values, while CS3600 underestimated them. Primescan had fewer inclination angle outliers, but it tended to add 0.4-0.6° to the measurements. CONCLUSIONS: IOSs showed predictable errors: they tended to overestimate or underestimate linear measurements and axial inclinations of scan bodies, one added 0.4-0.6° to the angle inclination values. In particular, they showed heteroscedasticity, a behavior probably related to the software or the device itself. CLINICAL SIGNIFICANCE: IOSs showed predictable errors that could affect clinical success. When performing a scan or choosing a scanner, clinicians should clearly know their behaviors.

How Is Endodontics Taught in Italy? A Survey of Italian Dental Schools.

The aim of our study was to investigate how endodontics is taught in Italian universities. An online survey was conducted from August to December 2021. A comparison between courses led by full or associate professors (Group 1) versus courses led by other figures, such as researchers or temporary lecturers (Group 2), was made. A total of 28 out of 36 schools participated (78%). In most schools, endodontics is taught in the fifth year to 15-29 students. All schools planned pre-clinical endodontic training, and in 25/28 schools (89.3%), clinical endodontic training was also provided. The course programs varied among schools, and significantly more hours were allocated to teaching nonsurgical root canal treatment in Group 1 schools than in Group 2 schools. The average numbers of hours of preclinical and clinical training were 34.3 ± 23.6 and 84.1 ± 76.7, respectively. All schools used rotary NiTi files in their clinical training, and the vertical condensation of hot gutta-percha was the most frequently taught obturation technique. As expected, the scenario of endodontic education in Italian universities was variable and needs harmonization. Courses led by full or associate professors seem to be better structured.

Testosterone Enanthate: An In Vitro Study of the Effects Triggered in MG-63 Cells.

The aim of this study was to investigate the effects of the androgenic hormone testosterone enanthate (TE) on human MG-63 cells. MG-63 were cultured for 24 h in the presence of TE at increasing concentrations to assess its lethal dose. Therefore, the suitable concentration for a prolonged use of TE in vitro was assessed by viability assay over 9 days. Finally, MG-63 were exposed to TE for 14 days and assayed for differentiation by qPCR and Alizarin Red S staining. TE in the amount of 100 µM resulted as the maximum dose tolerated by MG-63 cells after 24 h. However, a prolonged exposure in culture TE in the amount of 100 µM showed a cytostatic effect on cell proliferation. On the contrary, TE 10 µM was tolerated by the cells and did not boost cell proliferation, but did enhance new bone formation, as revealed by COL1A1, ALPL, BGLAP, and IBSP gene expression after 3, 7, and 14 days, and calcium deposition by Alizarin Red S staining after 14 days. Based on the current study, 10 µM is the critical dose of TE that should be used in vitro to support bone differentiation of MG-63 cells.

3D Printed Masks for Powders and Viruses Safety Protection Using Food Grade Polymers: Empirical Tests.

The production of 3D printed safety protection devices (SPD) requires particular attention to the material selection and to the evaluation of mechanical resistance, biological safety and surface roughness related to the accumulation of bacteria and viruses. We explored the possibility to adopt additive manufacturing technologies for the production of respirator masks, responding to the sudden demand of SPDs caused by the emergency scenario of the pandemic spread of SARS-COV-2. In this study, we developed different prototypes of masks, exclusively applying basic additive manufacturing technologies like fused deposition modeling (FDM) and droplet-based precision extrusion deposition (db-PED) to common food packaging materials. We analyzed the resulting mechanical characteristics, biological safety (cell adhesion and viability), surface roughness and resistance to dissolution, before and after the cleaning and disinfection phases. We showed that masks 3D printed with home-grade printing equipment have similar performances compared to the industrial-grade ones, and furthermore we obtained a perfect face fit by customizing their shape. Finally, we developed novel approaches to the additive manufacturing post-processing phases essential to assure human safety in the production of 3D printed custom medical devices.

Hydrogen plasma treatment confers enhanced bioactivity to silicon carbide-based nanowires promoting osteoblast adhesion.

Nanomaterials play a pivotal role in modern regenerative medicine and tissue engineering, due to their peculiar physical, optical and biological properties once they are used in the nanometric size. Many evidences are showing the importance of biomaterial micro- and nano-topography on cellular adhesion, proliferation and differentiation, and hence, tissue regeneration. It is well known that nanowires (NWs) can mimic many different tissues as a result of their shape and their surface characteristics, and that surface hydrophilicity affects early protein adsorption and cellular adhesion. Therefore a material able to induce bone regeneration might be obtained by combining optimal surface topography and hydrophilicity. Based on these evidence, we designed silicon carbide (SiC) and core/shell silicon carbide/silicon dioxide (SiC/SiOx) nanowires with modified wettability in order to analyze cell behavior, using an in-vitro osteoblastic model. First, we synthetized SiC NWs and SiC/SiOx NWs through a chemical-vapour-deposition (CVD) process, and then we used hydrogen plasma to modify their hydrophilicity. Subsequently we evaluated the four types of NWs in terms of their morphology and contact angle, and we studied their behavior in the presence of MC3T3-E1 murine osteoblasts. Cell metabolic activity, viability, morphology and focal adhesions formation were considered. Morphological data showed different dimensions between SiC and SiC/SiOx NWs. SiC NWs before the hydrogen plasma treatment showed a very low contact angle, that was absent after the treatment. Osteoblastic cells appeared healthy on all of the samples. Interestingly, both hydrophilic SiC NWs and SiC/SiOx NWs generated a favorable distribution of focal adhesions around the cell body confirmed also by scanning electron microscopy images. Moreover, osteoblasts grown on hydrogen plasma treated SiC/SiOx NWs showed an increased metabolic activity testified by a significantly higher cell number. In conclusion, we are here demonstrating that hydrogen plasma treatment of SiC and SiC/SiOx NWs induce a better osteoblastic cellular adhesion by increasing NWs wettability. We are therefore suggesting that hydrogen plasma treatment of SiC/SiOx can offer a suitable method to develop scaffolds for bone tissue engineering applications.

The perceived impact of the COVID-19 pandemic on dental undergraduate students in the Italian region of Emilia-Romagna.

INTRODUCTION: The outbreak and diffusion of the novel SARS-CoV2 coronavirus have caused an emergency status in the dental education system. MATERIALS AND METHODS: An anonymous survey composed of 34 questions was delivered to students of the Master Degree Programme in Dentistry and Dental Prosthodontics of the Universities of Emilia-Romagna, the fifth Italian region most affected by the pandemic. The psychological impact of COVID-19 was assessed by means of the Generalised Anxiety Disorder-7 scale (GAD-7). Numerically recoded data were analysed using the Analysis of Variance (ANOVA), whilst to investigate the association between quantitative variables, the Pearson correlation coefficient (R) was computed. RESULTS: The questionnaire was completed by 399 students (75%) out of 532. Most students experienced difficulties in working at the thesis during the COVID-19 emergency. For over half of them, online teaching could only partially replace traditional face-to-face lessons. The negative impact on the study career was judged as particularly high by sixth-year students. Clinical training activities were considered as exposing to the risk of contracting COVID-19 infection by the majority of the students. The level of concern of contracting COVID-19 infections during future university activities was positively correlated to risk perception related to clinical training. CONCLUSION: The results of this survey could be used to train students to a correct risk assessment. Students reported experiencing concern whilst thinking of COVID-19 and 6.5% of them showed symptoms related to high levels of anxiety. These data may guide Universities in trying to reduce students' anxiety by means of correct communication strategies.

Correction to: 3D printed bone models in oral and craniomaxillofacial surgery: a systematic review.

An amendment to this paper has been published and can be accessed via the original article.

3D printed bone models in oral and cranio-maxillofacial surgery: a systematic review.

AIM: This systematic review aimed to evaluate the use of three-dimensional (3D) printed bone models for training, simulating and/or planning interventions in oral and cranio-maxillofacial surgery. MATERIALS AND METHODS: A systematic search was conducted using PubMed® and SCOPUS® databases, up to March 10, 2019, by following the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) protocol. Study selection, quality assessment (modified Critical Appraisal Skills Program tool) and data extraction were performed by two independent reviewers. All original full papers written in English/French/Italian and dealing with the fabrication of 3D printed models of head bone structures, designed from 3D radiological data were included. Multiple parameters and data were investigated, such as author's purpose, data acquisition systems, printing technologies and materials, accuracy, haptic feedback, variations in treatment time, differences in clinical outcomes, costs, production time and cost-effectiveness. RESULTS: Among the 1157 retrieved abstracts, only 69 met the inclusion criteria. 3D printed bone models were mainly used as training or simulation models for tumor removal, or bone reconstruction. Material jetting printers showed best performance but the highest cost. Stereolithographic, laser sintering and binder jetting printers allowed to create accurate models with adequate haptic feedback. The cheap fused deposition modeling printers exhibited satisfactory results for creating training models. CONCLUSION: Patient-specific 3D printed models are known to be useful surgical and educational tools. Faced with the large diversity of software, printing technologies and materials, the clinical team should invest in a 3D printer specifically adapted to the final application.

The effect of a 2-mm inter-implant distance on esthetic outcomes in immediately non-occlusally loaded platform shifted implants in healed ridges: 12-month results of a randomized clinical trial.

BACKGROUND: Three millimeter is considered as the minimum distance to obtain soft and bone tissue stability in case of adjacent implants. The possibility to preserve peri-implant bone level using a platform switching connection has questioned this concept. PURPOSE: The study evaluates soft tissue maintenance and marginal bone stability around implants, placed at 2 or 3 mm of distance. MATERIALS AND METHODS: Thirty patients received two immediately loaded implants either at 2-mm (test) or at 3-mm (control) of distance in the premolar area. Soft tissue esthetics (papilla height and fill, keratinized tissue, recession) and radiographic peri-implant bone level changes were measured at 3, 6, and 12 months. RESULTS: No significant differences between the two groups were detected neither for all soft tissue esthetic outcomes nor for bone level modifications up to 12 months. CONCLUSION: The results suggested that up to 12 months post-loading, both 2- and 3-mm inter-distance platform-switched implants in healed site, supported adequate esthetic outcomes and peri-implant bone stability.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. OBJECTIVE: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. MATERIAL AND METHODS: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. RESULTS: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. CONCLUSIONS: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

"Over-inlay" block graft and differential morphometry: a novel block graft model to study bone regeneration and host-to-graft interfaces in rats.

PURPOSE: The aim of this study was to present new a model that allows the study of the bone healing process, with an emphasis on the biological behavior of different graft-to-host interfaces. A standardized "over-inlay" surgical technique combined with a differential histomorphometric analysis is presented in order to optimize the use of critical-size calvarial defects in pre-clinical testing. METHODS: Critical-size defects were created into the parietal bone of 8 male Wistar rats. Deproteinized bovine bone (DBBM) blocks were inserted into the defects, so that part of the block was included within the calvarial thickness and part exceeded the calvarial height (an "over-inlay" graft). All animals were sacrificed at 1 or 3 months. Histomorphometric and immunohistochemical evaluation was carried out within distinct regions of interest (ROIs): the areas adjacent to the native bone (BA), the periosteal area (PA) and the central area (CA). RESULTS: The animals healed without complications. Differential morphometry allowed the examination of the tissue composition within distinct regions: the BA presented consistent amounts of new bone formation (NB), which increased over time (24.53%±1.26% at 1 month; 37.73%±0.39% at 3 months), thus suggesting that this area makes a substantial contribution toward NB. The PA was mainly composed of fibrous tissue (71.16%±8.06% and 78.30%±2.67%, respectively), while the CA showed high amounts of DBBM at both time points (78.30%±2.67% and 74.68%±1.07%, respectively), demonstrating a slow remodeling process. Blood vessels revealed a progressive migration from the interface with native bone toward the central area of the graft. Osterix-positive cells observed at 1 month within the PA suggested that the periosteum was a source of osteoprogenitor elements. Alkaline phosphatase data on matrix deposition confirmed this observation. CONCLUSIONS: The present model allowed for a standardized investigation of distinct graft-to-host interfaces both at vertically augmented and inlay-augmented sites, thus possibly limiting the number of animals required for pre-clinical investigations.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study.

BACKGROUND: Osteochondral defects significantly affect patients' quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically. RESULTS: The retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage. CONCLUSIONS: In conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.

RhoA controls Wnt upregulation on microstructured titanium surfaces.

Rough topography enhances the activation of Wnt canonical signaling in vitro, and this mediates its effects on cell differentiation. However, the molecular mechanisms underlying topography-dependent control of Wnt signaling are still poorly understood. As the small GTPase RhoA controls cytoskeletal reorganization and actomyosin-induced tensional forces, we hypothesized that RhoA could affect the activation of Wnt signaling in cells on micropatterned titanium surfaces. G-LISA assay revealed that RhoA activation was higher in C2C12 cells on rough (SLA) surfaces under basal conditions than on smooth (Polished) titanium. Transfection with dominant negative RhoA decreased Wnt activation by normalized TCF-Luc activity on SLA, whilst transfection with constitutively active RhoA increased TCF-Luc activation on Polished titanium. One mM Myosin II inhibitor Blebbistatin increased RhoA activation but decreased Wnt activation on SLA surfaces, indicating that tension-generating structures are required for canonical Wnt modulation on titanium surfaces. Actin inhibitor Cytochalasin markedly enhanced RhoA and TCF-Luc activation on both surfaces and increased the expression of differentiation markers in murine osteoblastic MC3T3 cells. Taken together, these data show that RhoA is upregulated in cells on rough surfaces and it affects the activation of Wnt canonical signaling through Myosin II modulation.