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This article presents a comprehensive review of the topic of placebos, with a special focus on placebo analgesia. It includes a discussion of how placebos work (the placebo effect) and how patients react to them (the placebo response). A literature search was performed to identify relevant literature and publications related to the topic, and a qualitative assessment of papers was undertaken based on accepted rules for scientific evidence. The major finding from this review was that concepts about placebo effects and responses have changed dramatically over the years, especially in more recent years. This has occurred primarily as a result of more sophisticated experimental protocols using placebos in clinical studies of patients in pain, as well as various studies involving normal subjects. Our understanding of the biological and psychological mechanisms underlying placebo effects has expanded significantly due to recent developments in the technology of brain imaging. Based on findings from brain-imaging analyses, we now know that placebo analgesia is definitely a real (ie, biologically measurable) phenomenon. It can be pharmacologically blocked and behaviorally enhanced, and these responses have been demonstrated to be similar to those elicited by administration of 'real' analgesic substances. Psychological mechanisms involved in placebo analgesia include expectancy, meaning response, and classical conditioning. This article concludes with an emphasis on understanding therapeutic responses to various treatments for temporomandibular disorders (TMD). Acupuncture and splint therapy can be good examples of powerful placebos in the field of TMD, and both of these are discussed in detail. Present knowledge suggests that every treatment for pain contains a placebo component, which sometimes is as powerful as the so-called 'active' counterpart. While the deceptive use of placebos must be considered unethical, every health provider who is treating pain patients must be aware of this important phenomenon in order to harness its huge potential. [ABSTRACT FROM AUTHOR]

The timing of implant placement and loading following tooth extraction has recently undergone substantial reconsideration. The authors tested a protocol of immediate loading of single implants placed at the time of tooth extraction in a consecutive case series. Thirty-three patients received a single implant-supported crown to replace a maxillary anterior tooth at the time of extraction. Regular recalls were planned for the following 4 years. One implant did not integrate, and another became unstable secondary to facial trauma. Overall patient satisfaction and clinical and radiographic parameters were good. [ABSTRACT FROM AUTHOR]

Little is known about how surface topography can modulate mesenchymal cell responses to oxygen-related stress occurring with age, or during the early phases of wound healing or inflammation. To antagonize Reactive Oxygen Species (ROS), cells resort to defense mechanisms, relying on [beta]-catenin, a molecular switch between a TCF-mediated pathway, which promotes cells proliferation and commitment, and an alternative one controlled by FoxO, which induces quiescence and defenses against ROS. In the present study, we show that mesenchymal C2C12 cells are protected from H(2)O(2)-induced oxidative stress when they grow on rough (SLA) titanium surfaces. The expression of anti-ROS genes and FoxO/[beta]-catenin signaling, as measured by a reporter assay, were increased on SLA surfaces. We also show that TCF-mediated transcription was inhibited by ROS in cells growing on either smooth or SLA titanium. Our results demonstrate that surface topography modulates cell resistance to ROS and the balance between the molecular pathways regulating cell growth and cell defense against oxidative stress. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this work was to optimise printable polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) biomaterials with high percentages of β-TCP endowed with balanced mechanical characteristics to resemble human cancellous bone, presumably improving osteogenesis., Methods: PCL/β-TCP scaffolds were obtained from customised filaments for fused deposition modelling (FDM) 3D printing with increasing amounts of β-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/β-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 β-TCP. The Young's modulus of the composites was significantly higher than that of pristine PCL, indicating that the intrinsic strength of β-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/β-TCP scaffolds of up to 70 % concentrations of β-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., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article., (Copyright: © 2024 Dental Research Journal.)

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.

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., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2023 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

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., (© 2023. The Author(s).)

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.

Introduction: Aptamers are a brand-new class of receptors that can be exploited to improve the bioactivity of tissue engineering grafts. The aim of this work was to revise the current literature on in vitro and in vivo studies in order to i) identify current strategies adopted to improve scaffold bioactivity by aptamers; ii) assess effects of aptamer functionalization on cell behavior and iii) on tissue regeneration. Methods: Using a systematic search approach original research articles published up to 30 April 2022, were considered and screened. Results: In total, 131 records were identified and 18 were included in the final analysis. Included studies showed that aptamers can improve the bioactivity of biomaterials by specific adsorption of adhesive molecules or growth factors from the surrounding environment, or by capturing specific cell types. All the studies showed that aptamers ameliorate scaffold colonization by cells without modifying the physicochemical characteristics of the bare scaffold. Additionally, aptamers seem to promote the early stages of tissue healing and to promote anatomical and functional regeneration. Discussion: Although a metanalysis could not be performed due to the limited number of studies, we believe these findings provide solid evidence supporting the use of aptamers as a suitable modification to improve the bioactivity of tissue engineering constructs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Parisi, Ghezzi, Toffoli, Macaluso and Lumetti.)

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., Competing Interests: The authors declare no conflict of interest.

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.

The study of the intimate connection occurring at the interface between cells and titanium implant surfaces is a major challenge for dental materials scientists. Indeed, several imaging techniques have been developed and optimized in the last decades, but an optimal method has not been described yet. The combination of the scanning electron microscopy (SEM) with a focused ion beam (FIB), represents a pioneering and interesting tool to allow the investigation of the relationship occurring at the interface between cells and biomaterials, including titanium. However, major caveats concerning the nature of the biological structures, which are not conductive materials, and the physico-chemical properties of titanium (i.e. color, surface topography), require a fine and accurate preparation of the sample before its imaging. Hence, the aim of the present work is to provide a suitable protocol for cell-titanium sample preparation before imaging by SEM-FIB. The concepts presented in this paper are also transferrable to other fields of biomaterials research., Competing Interests: The authors have declared that no competing interests exist.

Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans.

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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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/SiO x ) nanowires with modified wettability in order to analyze cell behavior, using an in-vitro osteoblastic model. First, we synthetized SiC NWs and SiC/SiO x 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/SiO x 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/SiO x 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/SiO x 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/SiO x NWs induce a better osteoblastic cellular adhesion by increasing NWs wettability. We are therefore suggesting that hydrogen plasma treatment of SiC/SiO x can offer a suitable method to develop scaffolds for bone tissue engineering applications., (Copyright © 2020. Published by Elsevier B.V.)

The use of alloplastic materials in periodontal regenerative therapies is limited by their incapacity to establish a dynamic dialog with the surrounding milieu. The aim of the present study was to control biomaterial surface bioactivity by introducing aptamers to induce the selective adsorption of fibronectin from blood, thus promoting platelets activation in vitro and bone regeneration in vivo. A hyaluronic acid/polyethyleneglycole-based hydrogel was enriched with aptamers selected for recognizing and binding fibronectin. In vitro, the capacity of constructs to support osteoblast adhesion, as well as platelets aggregation and activation was assessed by chemiluminescence within 24 h. Matrices were then evaluated in a rat periodontal defect to assess their regenerative potential by microcomputed tomography (µCT) and their osteogenic capacity by Luminex assay 5, 15 and 30 d postoperatively. Aptamers were found to confer matrices the capacity of sustaining firm cell adhesion (p = 0.0377) and to promote platelets activation (p = 0.0442). In vivo, aptamers promoted new bone formation 30 d post-operatively (p < 0.001) by enhancing osteoblastic lineage commitment maturation. Aptamers are a viable surface modification, which confers alloplastic materials the potential capacity to orchestrate blood clot formation, thus controlling bone healing.

In vitro studies have consistently shown that titanium surface wettability affects the response of osteoprogenitors, leading to important advances in the clinical osseointegration of dental implants. However, the underlying molecular mechanisms remain unknown. Since surface conditioning by blood components initiates within milliseconds after insertion, it is reasonable to hypothesize that the amount and the type of blood proteins adsorbed influences the interaction between the implant surface and osteoprogenitors. To test this hypothesis, titanium implant surfaces with different characteristics, in terms of topography and wettability, have been conditioned with selected plasma proteins. Pure fibronectin (HFN) and albumin (HSA) solutions, or their mixture at the relative plasma concentrations were allowed to adsorb on titanium surfaces for 60 min. Protein adsorption was monitored by Bradford assay, while the contribution of HSA and HFN in forming the microfilm layer at the interface was studied by Western Blot. Subsequently, the same protein-conditioned surfaces were used to culture C2C12 cells, thus studying their capacity to adhere and to spread after 3 h. Cell viability was evaluated up to 7 days, while the expression of osteogenic genes was assessed after 3 days. Under competitive adsorption conditions, hydrophilicity promotes the selectivity of titanium for HFN regardless of the surface microtopography. As a consequence of selective HFN adsorption, cells on hydrophilic surfaces displayed enhanced adhesion and spreading, as well as increased proliferation. On the other hand, selective HFN adsorption did not appreciably affect cell differentiation. These data suggest that implant surface hydrophilicity plays a key role in guiding the selective adsorption of specific proteins from blood plasma. Moreover, the selective adsorption of HFN, as a consequence of surface hydrophilicity, was found to account for early cell responses amelioration. Thus, titanium surface hydrophilicity contributes to the clinical success of dental implant by selectively controlling protein adsorption at the interface., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The bioactivity of biomaterials is closely related to cell response in contact with them. However, shortly after their insertion, materials are soon covered with proteins that constitute the biological fluids, and which render the direct surface recognition by cells almost impossible. The control of protein adsorption at the interface is therefore desirable. Extracellular matrix proteins are of particular interest in this sense, due to their well-known ability to modulate cell behavior. Particularly, fibronectin plays a leading role, being present in both healthy and injured tissues undergoing healing and regeneration. The aim of the present work is to give an overview on fibronectin and on its involvement in the control of cell behavior providing evidence of its pivotal role in the control of cell adhesion, spreading, migration, proliferation and differentiation. A deep insight into methods to enrich biomaterials surface with fibronectin will be then discussed, as well as new cues on the possibility to design tailored platforms able to specifically retain fibronectin from the surrounding extracellular milieu., (© 2019 The Authors.)

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.

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., (© 2020 Wiley Periodicals LLC.)

Background: Calcium ions levels in bone niches have been demonstrated to severely influence new bone formation. Osteoinductive scaffolds containing calcium have been largely studied to control the release of calcium in bone regeneration and tissue engineering purpose. The aim of the present study was, firstly, to synthesize two different resorbable calcium phosphate-based powders, thought to be reservoirs of calcium ions, and secondary, to investigate their effects on human osteoblasts, in order to develop a suitable titanium coating material., Methods: Tetracalcium phosphate (A450) and biphasic tetracalcium phosphatae/tricalcium phosphate (A850) powders were prepared with an innovative method. The presence of calcium phosphate structures was chemically confirmed with XRD. Furthermore, powders macroscopic aspect was observed with a stereomicroscope. For in-vitro experiments, human osteoblastic cells were cultured in the presence of A450 and A850, and assayed for viability and metabolic activity through Crystal Violet and MTT, respectively., Results: Our synthesis led to the formation of calcium phosphates in both samples, even though A850 presented a higher level of crystallinity and a more powdery aspects than A450. Both the samples enhanced the viability of cultured cells, inhibiting cell metabolic activity in the case of A850, which furthermore showed to be internalized by cells., Conclusions: We developed two different kind of calcium phosphate-based powders and we tested their effect on human osteoblasts, underlying the possibility of use calcium phosphate-based coatings to enhance cell response on implantable materials.

Titanium surface characteristics, including microtopography, chemical composition, and wettability, are essential features to achieve osseointegration of dental implants, but the choice of a particular surface topography is still a debated topic among clinicians. An increased level of implant surface hydrophilicity has been demonstrated to ameliorate osseointegration and shorten healing times. The aim of this work is to develop and test a suitable thermal-based method to enhance titanium surface wettability without modifying other characteristics of the implant surface. For this function, titanium discs with different surface topography have been thermally treated by testing different temperatures and excluding those that led to evident chromatic and morphological modifications. The selected surface gain in wettability after the treatment was assessed through contact angle measurement, chemistry modifications through x-ray photoelectron spectroscopy (XPS) analysis, and microtopography through scanning electron microscopy (SEM). Results showed a great enhancement in hydrophilicity on the tested surfaces without any other modification in terms of surface chemical composition and topography. A possible limitation of this method could be the persistent, although relatively slow, biological aging of the surfaces after the treatment. The present findings indicate that the described treatment could be a safe and effective method to enhance dental titanium hydrophilicity and thus its biological performance.

Objectives: To investigate how a thermal treatment to increase titanium wettability influences proteins adsorption from blood serum and osteoblasts responses., Methods: Titanium discs with machined or micro-rough profiles were thermally treated to obtain hydrophilic surfaces. The adsorption kinetics of two representative serum proteins were determined by Bradford assay, while the stable protein adsorption pattern from blood serum was investigated by SDS-PAGE and Western Blot analysis. Subsequently, MC3T3-E1 cells were cultured on titanium for 24h and assayed for adhesion and morphology., Results: Thermally-induced hydrophilicity dramatically improved the capacity of titanium to selectively adsorb fibronectin and fibrinogen from blood serum, without evident influence on other representative serum proteins. The selective adsorption of fibronectin was linked to the improved capacity of MC3T3-E1 cells to adhere and spread on hydrophilic surfaces., Significance: We identified a potential method to improve selective protein adsorption on titanium by enhancing implant surface wettability through a thermal treatment. Selective fibronectin adsorption was further indicated as the responsible for improved osteoblasts adhesion. Targeting specific cell response by selective protein adsorption appears to be crucial to conceive even more performant therapies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Surface topography is one of the main factors controlling cell responses on implanted devices and a proper definition of the characteristics that optimize cell behavior may be crucial to improve the clinical performances of these implants. Substrate geometry is known to affect cell shape, as cells try to optimize their adhesion by adapting to the irregularities beneath, and this in turn profoundly affects their activity. In the present study, we cultured murine calvaria MC3T3-E1 cells on surfaces with pillars arranged as hexagons with two different spacings and observed their morphology during adhesion and growth. Cells on these highly ordered substrates attached and proliferated effectively, showing a marked preference for minimizing the inter-pillar distance, by following specific pathways across adjacent pillars and displaying consistent morphological modules. Moreover, cell behavior appeared to follow tightly controlled patterns of extracellular protein secretion, which preceded and matched cells and, on a sub-cellular level, cytoplasmic orientation. Taken together, these results outline the close integration of surface features, extracellular proteins alignment and cell arrangement, and provide clues on how to control and direct cell spatial order and cell morphology by simply acting on inter-pillar spacing., Competing Interests: The authors declare no conflict of interest.

Periodontitis is an inflammatory condition that can induce significant destruction of the periodontium, the set of specialized tissues that provide nourishment and support to the teeth. According to the guided tissue regeneration principles, the periodontium can be regenerated if the spatiotemporal control of wound healing is obtained, namely the tune control of cell response. After material implantation, protein adsorption at the interface is the first occurring biological event, which influences subsequent cell response. With the regard of this, we hypothesize that the control of selective adsorption of biological cues from the surrounding milieu may be a key-point to control selective cell colonization of scaffolds for periodontal tissue regeneration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The host-material interface is a crucial relationship dictating the possibility of successful osseointegration in implant dentistry. The aim of the present study was to characterize the effects of plasma proteins pre-adsorption on the adhesion capacity of osteoblasts, which occurs immediately after implant insertion in vivo . After having pre-adsorbed human plasma proteins on a machined and microrough titanium surface, MC3T3-E1 osteoblasts adhesion was evaluated through crystal violet cell adhesion assay, immunofluorescence staining for cytoskeleton, focal adhesions and cell nuclei, and scanning electron microscopy. The pre-adsorbed protein layer markedly affected the adhesion rate of cells, as well as their morphology and the expression of focal contacts. Moreover, protein adsorption to the underlying titanium surface was found to be correlated to surface pre-wetting. Thus, the early adsorption of serum proteins to the interface of dental implants impacts cell adhesion in terms of strength and of focal adhesions expression., Competing Interests: The authors declare no conflict of interest.

Current biomedical research is centered on the study of nanomaterials and their effects in biological environments. In particular, there is an increasing interest on TiO₂ nanostructures for biomedical applications such as drug delivery or implant materials. In this framework, we present a Chemical Vapour Deposition process to synthesize titanium dioxide nanowires (NWs) on a commercially pure titanium substrate and we test the material In Vitro as a culture substrate for murine osteoblast-like MC3T3-E1 cells. A physical-morphological, structural and optical-characterization of the inorganic samples is performed by Electron Microscopy techniques and X-ray Diffraction, showing that a mat of crystalline rutile TiO₂ NWs is obtained over the commercial substrate. In Vitro biological tests are performed by seeding MC3T3-E1 cells on the material and studying cell morphology, the cellmaterial interface and the osteoblast gene expression. These experiments show good cell adhesion to the nano-structured surface and a higher degree of early osteoblastic differentiation compared to control titanium surfaces, indicating that the present nano-structured material has good osteogenic potential for biomedical applications.

Robust cell adhesion is known to be necessary to promote cell colonization of biomaterials and differentiation of progenitors. In this paper, we propose the functionalization of Silicon Oxycarbide (SiO x C y ) nanowires (NWs) with 3-mercaptopropyltrimethoxysilane (MPTMS), a molecule containing a terminal -SH group. The aim of this functionalization was to develop a surface capable to adsorb proteins and promote cell adhesion, proliferation and a better deposition of extracellular matrix. This functionalization can be used to anchor other structures such as nanoparticles, proteins or aptamers. It was observed that surface functionalization markedly affected the pattern of protein adsorption, as well as the in vitro proliferation of murine osteoblastic cells MC3T3-E1, which was increased on functionalized nanowires (MPTMS-NWs) compared to bare NWs (control) (p < 0.0001) after 48 h. The cells showed a better adhesion on MPTMS-NWs than on bare NWs, as confirmed by immunofluorescence studies on the cytoskeleton, which showed a more homogeneous vinculin distribution. Gene expression analysis showed higher expression levels for alkaline phosphatase and collagen I, putative markers of the osteoblast initial differentiation stage. These results suggest that functionalization of SiO x C y nanowires with MPTMS enhances cell growth and the expression of an osteoblastic phenotype, providing a promising strategy to improve the biocompatibility of SiO x C y nanowires for biomedical applications.

A decisive step in cell-biomaterial interaction is represented by the adsorption of proteins at the interface, whose fine control may be useful to trigger proper cell response. To this purpose, we can selectively control protein adsorption on biomaterials by means of aptamers. Aptamers selected to recognize fibronectin dramatically enhance chitosan ability to promote cell proliferation and adhesion, but the underlying biological mechanism remains unknown. We supposed that aptamers contributed to ameliorate the adsorption of fibronectin in an advantageous geometrical conformation for cells, thus regulating their morphology by the proper activation of the integrin-mediated pathway. We investigated this possibility by culturing epithelial cells on chitosan enriched with increasing doses of aptamers in the presence or in the absence of cytoskeleton pharmacological inhibitors. Our results showed that aptamers control cell morphology in a dose dependent manner ( p < 0.0001). Simultaneously, when the inhibition of actin polymerization was induced, the control of cell morphology was attenuated ( p < 0.0001), while no differences were detected when cells contractility was challenged ( p > 0.05). Altogether, our data provide evidence that aptamers contribute to control fibronectin adsorption on biomaterials by preserving its conformation and thus function. Furthermore, our work provides a new insight into a new way to accurately tailor material surface bioactivity.

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.

Tissue engineering (TE) is a multidisciplinary science, which including principles from material science, biology and medicine aims to develop biological substitutes to restore damaged tissues and organs. A major challenge in TE is the choice of suitable biomaterial to fabricate a scaffold that mimics native extracellular matrix guiding resident stem cells to regenerate the functional tissue. Ideally, the biomaterial should be tailored in order that the final scaffold would be (i) biodegradable to be gradually replaced by regenerating new tissue, (ii) mechanically similar to the tissue to regenerate, (iii) porous to allow cell growth as nutrient, oxygen and waste transport and (iv) bioactive to promote cell adhesion and differentiation. With this perspective, this review discusses the options and challenges facing biomaterial selection when a scaffold has to be designed. We highlight the possibilities in the final mold the materials should assume and the most effective techniques for its fabrication depending on the target tissue, including the alternatives to ameliorate its bioactivity. Furthermore, particular attention has been given to the influence that all these aspects have on resident cells considering the frontiers of materiobiology. In addition, a focus on chitosan as a versatile biomaterial for TE scaffold fabrication has been done, highlighting its latest advances in the literature on bone, skin, cartilage and cornea TE.

The aim of the study was to investigate cell adhesion to micro-structured titanium. Osteoblastic MC3T3 cells were cultured on smooth (P) or sand-blasted/acid-etched (SLA) titanium discs and were observed at scanning electron microscope/focused ion beam (SEM/FIB). Myosin II and actin microfilaments were labelled for epifluorescence microscopy. FIB revealed that cell adhesion initiated centrally and expanded to the cell periphery and that cells attached on the substrate by bridging over the titanium irregularities and adhering mostly on surface peaks. Gaps were visible between concave areas and cytoplasm and areas around ridges represented preferred attachment points for cells. A different myosin distribution was observed between samples and myosin inhibition affected cell responses. Taken together our data indicate that cells attach on micro-rough titanium by bridging over its irregularities. This is likely mediated by myosin II, whose distribution is altered in cells on SLA discs.

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 μm/day ± 0.37 versus 6.50 μm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm -2 ± 2.01) to three months (28.26 n mm -2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm -2 ± 0.32 versus 1.01 n mm -2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm -2 ± 6.29 versus 6.29 n mm -2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Rough titanium surfaces enhance cell response to activation of Wnt canonical signalling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3β-inhibitors SB216763 and SB415286 on osteoblastic differentiation on titanium surfaces with different topography and wettability. Osteoblastic MC3T3 cells were plated on smooth (Pickled), sand-blasted/acid-etched (SLA) or hyper hydrophilic SLA (modSLA) titanium discs and transfected with a reporter vector sys-tem for Wnt canonical signalling. Cells were also seeded in the presence or in the absence of GSK3b-inhibitors SB216763 or SB415286 and their viability, morphology and the expression of Wnt target and osteoblast specific genes was assessed by Real Time PCR. Inhibitors altered cell morphology and mostly reduced cell viability at high concentration. SB415286 markedly increased the expression of ALP in MC3T3 cells on rough surfaces at the concentration of 100 nM before decreasing its expression at higher concentrations. OCN expression was unaffected. Increasing concentrations of SB216763 increased the expression of ALP in MC3T3 cells on rough surfaces but OCN expression was not changed at any con-centration. SB216763 and SB415286 inhibitors should be further investigated as potential tools to improve cell differentiation on titanium surfaces for endosseous implants.

Objective: Sinus floor elevation with lateral approach is probably the most frequently performed reconstructive procedure to rehabilitate posterior maxilla when a bone deficiency is present. Different graft materials have been proposed and tested, often with high clinical performances and predictable results. Histological analysis is required when evaluating new materials. We investigated human biopsies retrieved after sinus floor elevation procedure by histomorphometric evaluation to test the performance of an equine-derived bone grafting material., Study Design: Seventeen consecutive patients were enrolled and sinus lift surgeries were performed using an equine bone graft. Six months after surgery, at implant placement, bone samples were collected. Histomorphometry analysis was carried out on decalcified samples., Results: All surgeries were uneventful and no additional grafting was required prior to implant insertion. Forty percent of new bone formation was detected, which represented the most abundant tissue retrieved, followed by the residual graft material (33%) and fibrous tissue (27%). A significant reduction in particles size demonstrates a remodeling activity of the graft material., Conclusion: Within the limitations of this study, this equine-derived bone graft proved to be an effective material to induce new bone formation in the sinus floor elevation procedure.

Unlabelled: Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization., Statement of Significance: We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients' blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: No potential conflict of interest relevant to this article was reported.