Here, we reveal a previously unknown, biphasic mode of action of Mg2+ in bone repair. During the early inflammation phase, Mg2+ contributes to an upregulated expression of transient receptor potential cation channel member 7 (TRPM7), and a TRPM7-dependent influx of Mg2+ in the monocyte-macrophage lineage, resulting in the cleavage and nuclear accumulation of TRPM7-cleaved kinase fragments (M7CKs). This then triggers the phosphorylation of Histone H3 at serine 10, in a TRPM7-dependent manner at the promoters of inflammatory cytokines, leading to the formation of a pro-osteogenic immune microenvironment. In the later remodeling phase, however, the continued exposure of Mg2+ not only lead to the over-activation of NF-κB signaling in macrophages and increased number of osteoclastic-like cells but also decelerates bone maturation through the suppression of hydroxyapatite precipitation. Thus, the negative effects of Mg2+ on osteogenesis can override the initial pro-osteogenic benefits of Mg2+. Taken together, this study establishes a paradigm shift in the understanding of the diverse and multifaceted roles of Mg2+ in bone healing.Despite the widespread observations on the osteogenic effects of magnesium ion (Mg Supplementation of magnesium (Mg2+) or its inclusion in biomaterials has beneficial effects for bone formation, but it has also been reported that it can have detrimental effects. Here, the authors analyse dose- and time-dependent effects of Mg2+ on bone regeneration and show that it can stimulate monocyte-macrophage lineage cells to support bone formation in the early phases of repair, but inhibit bone repair and mineralization in later stages by promoting a pro-inflammatory environment. Thus, clinical interventions using orthopedic biomaterials are often required to accelerate bone healing while maintaining the amount and quality of bone mass. Magnesium ion (Mg2+) is integral to bone homeostasis and metabolism. Deficiency in Mg2+ is known to disrupt systemic bone metabolism, characterized by the inadequate bone formation and deregulated bone resorption8. In contrast, Mg2+ supplement is beneficial to patients with osteoporosis9. Magnesium, its alloys13 and derivatives16 have been extensively studied as replacements of non-degradable metallic implants e.g., titanium alloys in bone surgeries. Mg2+ modified biomaterials have shown a superior osteogenic capacity in many reports21. However, detrimental effects of Mg2+ released upon degradation have also been observed24. These conflicting results may reflect the incomplete understanding of the roles of Mg2+ in the complex biological process of bone healing.Bone tissue has a substantial capacity for repair and regeneration after injury or surgical treatment. However, the natural healing of bone can be a slow process that often fails to restore the bone to its original strength and structure2+ in polycaprolactone (PCL) implant18 and poly(lactic-co-glycolic acid) (PLGA) microsphere19 can promote bone formation in a rat femoral defect model. We identified ~50–200 ppm as the optimal Mg2+ concentration for promoting osteogenic activities of osteoblasts in vitro, as well as new bone formation in vivo18. Moreover, by using customized biomaterials that enable the controlled release of Mg2+ at different stages of bone healing, we demonstrated that the bone regeneration rate and the quality of newly formed bone tissues depend on the release profile of Mg2+19. Bone healing is a complex process that involves the precise coordination of osteoclastogenesis and osteogenesis, through the interplay of multiple types of cells in a dynamic microenvironment. It is possible that the different cell types involved in various phases of bone healing, from early inflammation to the later bone formation and remodeling, may respond to Mg2+ in different ways.Our group has recently reported that the incorporation of Mg25. The pro-inflammatory cytokines caused by Mg2+ deficiency can contribute to osteoclastogenesis27, whereas the Mg2+-induced anti-inflammatory cytokines and tissue repair factors benefit tissue regeneration29. The doping of Mg2+ into titanium30 and calcium phosphate cement31 was demonstrated to promote the M2 polarization of macrophages. However, there is yet a consensus on which macrophage phenotype is more beneficial to bone regeneration because both M133 and M235 phenotypes have been reported to contribute to osteogenesis. Moreover, the conventional M1/M2 classification of macrophages has been challenged by a more heterogeneous grouping method, which suggests there may exist a continuum between M1 and M2 phenotypes yet to be identified36. Thus, the complexity of the Mg2+-induced immunomodulation on macrophages, as well as its specific effects on the bone healing process in the complicated in vivo scenario requires further investigation.The monocyte-macrophage cell lineage has been recognized as a major player in bone regeneration and in acute inflammation responses to biomaterials, mainly due to their high plasticity in response to environmental cues and their multiple roles in bone homeostasis. According to their distinct functional properties, surface markers, and inducers, macrophages are characterized into several phenotypes 2+ on the monocyte-macrophage-osteoblast axis in bone healing and investigated the underlying mechanisms behind the action of Mg2+. We demonstrated a previously unknown immunomodulatory role of Mg2+ at the early phase of bone healing, in which macrophages are stimulated, through a signaling pathway that involves transient receptor potential cation channel member 7 (TRPM7), to generate a specific pro-osteogenic immune microenvironment. At the later bone repair/remodeling phase, the prolonged presence of Mg2+ impacts bone healing in another way, through activation of NF-κB signaling and inhibition on mineralization of extracellular matrix. We believe that these results will inspire the development of next-generation of Mg2+-based degradable biomaterials for clinical uses that can better harness the healing power of Mg2+.In this study, we systematically analyzed the dose-dependent and time-dependent effects of Mg2+ on bone healing, we developed an alginate-based hydrogel that allows a transient release of MgCl2, at a concentration of ~10 mM, over one week , we demonstrated that the Mg2+ releasing hydrogel significantly increased the magnesium content while decreasing the calcium content in the defects on day 7 post-injury  positive osteoblasts increased in the Mg2+ treated group compared to the control and sham groups  of bone healing . An increase of Mg2+ concentration in the culture medium significantly promoted the maturation of suspension monocytes into adhered macrophages , the murine structural and functional homolog of human IL-842. We found that 10× Mg2+ treatment led to an ~1.5-fold increase in the expression of MIP-2 compared to the control. These in vitro findings were supported by our immunostaining and ELISA data in vivo showing that Mg2+ released from the hydrogel led to a significant increase in the level of IL-8  was found to be increased in THP1-derived macrophages  other than Mg2+46, it is possible that the inhibition of TRPM7 may alter macrophage functions in multiple ways.To elucidate the role of TRPM7 in MgRNA Fig. , S6g or RNA Fig. . ConsequRNA Fig. . Moreovehed Fig. . Interes2+ led to an increased expression of IKK-α and IKK-β in THP1-derived macrophages , IκB phosphorylation, and nuclear p65  were elevated by the addition of Mg2+, whereas the late osteoclastic markers , which indicates the maturation of osteoclasts, were downregulated , which both play a crucial role in the terminal maturation of osteoclast  to Mgges Fig. , which cges Fig. , S6h. Thp65 Fig. . In conslls Fig. , and extlls Fig.  during tted Fig. . Supportast Fig. . Also, oury Fig. . Hence, 2+ . The concentrations of Mg2+in all CM were adjusted to 1× before use (see “Methods” section). As expected, CM-10× (CM harvested from THP1-derived macrophages pre-incubated in 10× Mg2+) significantly increased the number  with conditioned media (CM) harvested from THP1-derived macrophages pre-incubated in different concentrations of Mgber Fig.  and osteber Fig.  and the RNA Fig.  and protRNA Fig. . AlizariSC Figs. , S8a. ThMSC Fig. .Fig. 7Th2+, we tested the effect of these two factors, individually and in combination, on MSC differentiation. The results indicated that human recombinant IL-8 replicated the effect of CM-10×, as evidenced by the significant increase in MSC proliferation  significantly reduced the ALP activity and the expression of osteogenic marker genes of MSC  only contributed to a < 10% increase in MSC proliferation  could suppress the spontaneous mineralization . The typical TEM images and selected area electron diffraction (SAED) patterns of the precipitated calcium salts showed that the formation of crystallized apatite was significantly inhibited by the increase of Mg2+ level in the medium  and bone morphogenic protein (BMP) signaling, who plays fundamental roles in regulating MSC differentiation during skeletal development, is associated with the stimulation of Mg2+23. Although our in vitro data in this study verified the direct osteogenic action of Mg2+ on MSC, especially at the initiation of osteogenic differentiation, we noticed excessive Mg2+ did not contribute to late osteoblastic activities, especially the mineralization of the extracellular matrix. Indeed, previous studies suggest that the addition of Mg2+ needs to be tailored to an optimal range (around 2–8 mM) to achieve its osteogenic effects, otherwise, the presence of high levels of Mg2+ can be detrimental to the osteogenic differentiation of bone-forming cells50. Hence, the conventional approach that focuses on the effect of Mg2+ on the osteoblast lineage cannot fully address the osteogenic effect of Mg and its alloy, especially in scenarios where bone-forming cells are continuously challenged by high concentrations of Mg2+ over a long period51. Recently, implant-derived Mg2+ is demonstrated to trigger the release of neuronal calcitonin gene-related polypeptide-α from dorsal root ganglia, resulting in enhanced osteogenic differentiation of periosteum-derived stem cells and improved bone-fracture healing52. This finding suggests that Mg2+ may contribute to the osteogenic differentiation of osteoblast lineage through the activation of various other cell types in the bone microenvironment.Mg2+ releasing hydrogel at different stages of bone healing, our animal studies revealed an effective window for the administration of Mg2+ to achieve bone healing: the initial inflammation stage is more important than the subsequent active bone repair stage. As macrophages are known for their pivotal roles in the innate inflammatory response and their regulatory effects on bone homeostasis in tissue regeneration54, we selectively depleted the phagocytes in the early inflammation stage using liposome clodronate and showed that the osteogenic effect of Mg2+ was abolished. This suggests macrophages are the primary target cells responsible for Mg2+-induced bone healing, which corroborates with numerous studies showing macrophages play important roles in the healing of bone tissues58. In this study, our in vivo and in vitro data have provided compelling evidence showing the cellular activities of macrophages, especially the cytokine release profiles, to be significantly affected by the presence of Mg2+. First, the presence of Mg2+ facilitates the recruitment and activation of monocyte towards matured macrophages. Interestingly, although the upregulation of CD163 and CD206 suggest an M2 polarization of the Mg2+-treated macrophages, their cytokine secretion profile was distinct from the classical M1 and M2 regimes59, as inflammatory genes typically observed in M1 macrophages  and in M2 macrophages  were not significantly upregulated. Moreover, genes that are known to be involved in macrophage-mediated osteogenesis  were not dramatically altered by Mg2+ treatment. Instead, Mg2+ triggers a previously unknown cytokine profile, characterized by an increased level of CCL5, IL-8, and IL-1ra and the reduction of IL-1β. CCL5 and IL-8 are traditionally recognized as pro-inflammatory factors responsible for cell recruitment to the sites of injuries and inflammation62, however, both of them have been demonstrated capable of recruiting MSCs to bone healing site64. Moreover, IL-8 has been shown as a crucial mediator for angiogenesis65, the commitment of MSCs to bone regeneration66, and restricting bone resorption activity of osteoclasts67. IL-1β is one of the major pro-inflammatory cytokines that stimulate osteoclastogenesis68. A reduction of IL-1β in response to the addition of Mg2+, possibly due to the anti-inflammatory effect of Mg2+ and the IKK-dependent activation of NF-κB69, contributes to the generation of new bone. In addition, Mg2+ also upregulated the expression of IL-1 receptor antagonist (IL1-ra), which further limits IL-1β functions70. Collectively, our observations suggest that Mg2+ stimulated macrophages to a cytokine mixture tailored for bone regeneration.By grafting Mg2+ channel in mammalian cells. Conditional gene deletion experiments suggest that this transporter is involved in a variety of immune-related functions, including macrophage activation and polarization in a current dependent manner71, but the implications of these functions in tissue regeneration have not been well explored. Moreover, the intracellular Ser/Thr kinase domain located at the carboxyl terminus of the membrane channel is reported to modulate cellular functions by directly modifying channel activity72 or interacting with nuclear chromatin-remodeling complexes44. We observed that an increase in extracellular Mg2+ contributed to the upregulation of TRPM7 expression, as well as the cleavage and nuclear translocation of TRPM7 kinase fragments (M7CKs). Consequently, in consistent with a previous study44, these chromatin-bound M7CKs induced the phosphorylation of Histone H3 at Ser 10 (p-H3S10), which has been reported to be critical for the recruitment of IKK to NF-κB signaling responsive promotors73 and the binding of IKK to a variety of inflammatory gene promoters, such as CCL5 and IL-875. Our ChIP assay demonstrated that Mg2+ led to an increased association of p-H3S10 with the promoters of IL-8 and IκBα, thus establishing a role of Mg2+ in activating NF-κB signaling and the production of inflammatory cytokines like IL-8.TRPM7 is a predominant Mg2+-induced activation of the NF-κB signaling pathway, which is crucial in inflammation responses76. However, our experiments with the TRPM7 blocker FTY720 revealed that the channel activity of TRPM7 does not account for the entire spectrum of defects seen in TRPM7 siRNA-treated macrophages, especially in terms of the phosphorylation of H3S10 and the activation of NF-κB signaling. This implies the cleaved kinase domain may play a more important role in the regulation of inflammatory cytokines in response to Mg2+. Recent evidence suggests that divalent cations like Ca2+ may play dual roles in macrophages through TRPM7: a rapid role in jump-starting TLR4 endocytosis and possibly a slower role in tailoring an appropriate inflammatory response by modifying the activity of NF-κB77. Therefore, we propose that the TRPM7-mediated inflammatory response to Mg2+ can be time-dependent: an initial M7CK mediated phosphorylation of H3S10 located at specific gene promoters followed by a better-tailored activation of NF-κB signaling. This idea is supported by our observation that the promoting effect of Mg2+ on IL-8 secretion and the inhibitory effect on IL-1β was abrupted between 24–48 h after the treatment. Moreover, since IL-8 also serves as a strong inducer for the activation of NF-κB signaling, the increased IL-8 in the microenvironment resulting from the M7CK-mediated histone modification can enhance the NF-κB signaling cascade through the IL-8 receptor (CXCR1/2) expressed on the membrane of macrophage79. This explains why prolonged exposure to Mg2+ leads to unnecessary over activation of NF-κB signaling and osteoclastogenesis.This study demonstrates the role of TRPM7 in the Mg+ and TRAP+ osteoclastic-like cells in the wound, which peaked on day 7 after the delivery of Mg2+. Unlike the TRAP+ multinucleated cells observed in the bone remodeling stage, osteoclastic-like cells at an early stage of bone healing have been suggested to represent inflammatory responses to biomaterials instead of bone resorption80. Indeed, they are actively involved in bone formation by the secretion of inflammatory cytokines and chemokines81. We noticed that a short-term delivery of Mg2+ contributed to an increased number of TRAP+ osteoclastic-like cells at the inflammation stage but ended with a lower number of bone-resorbing osteoclasts in the remodeling stage than the control or sham group. By contrast, when the delivery of Mg2+ was extended, the inhibitory effect of Mg2+ on osteoclastogenesis at the remodeling stage disappeared. Given the above-mentioned influence of Mg2+ on the activation of NF-κB signaling, which plays a central role in RANKL-induced osteoclastogenesis82, the administration of Mg2+ beyond the early inflammation stage may be detrimental for bone regeneration.We detected a group of CD682+ could also interfere with biomineralization. While a high level of Mg2+ (10×) administrated at the beginning of the osteogenic induction of MSC could promote extracellular matrix formation and mineralization, the supplementation of Mg2+ at the late stage dramatically impaired the mineralization of MSC. This finding recapitulated our in vivo data that the prolonged Mg2+ treatment could reduce BMD, as extensively reported elsewhere52. The formation of calcified tissue in the biological environment starts with the nucleation and growth of calcium phosphate precursors. The presence of Mg2+ may inhibit this process by selectively stabilizing more acidic hydrated precursor phases, like dicalcium phosphate84. Moreover, the adsorption of excessive Mg2+ on the surface of precursor and nuclei could further lead to the blocking of the active growth sites85. Thus, this study reveals the biphasic nature of Mg2+ effect on biological mineralization: during the early phase of MSC differentiation, Mg2+ itself and, to a much larger extent, osteogenic factors secreted by macrophage upon the stimulation of Mg2+, effectively promote osteogenic differentiation. However, as MSC is committed to osteoblast lineage, they become less responsive to Mg2+ and the Mg2+-induced immune factors. Instead, the persistent presence of Mg2+ beyond this stage suppresses mineralization and promotes osteoclastic bone resorption.In addition to the osteoclastogenic effect, we observed that the prolonged exposure to Mg2+ in bone healing  was used for the preparation of magnesium cross-linked alginate . The gel was then loaded in a syringe and sterilized by gamma rays (Co-60) irradiation at a dose of 25 kGy. The as-prepared gel was immersed in 1× Phosphate-buffered saline (PBS) at a ratio of 0.1 g/mL and kept in an incubator at 37 °C for five days. At the designated time point, the Mg2+ concentration in the PBS was measured by inductively coupled plasma optical emission spectrometry .20% alginate gel (Alg) was prepared by mixing sodium alginate powder  in deionized water, while 10% magnesium chloride  injection at day 7 post-injury and (3) injection at both day 0 and day 7 post-injury. Controls for each treatment regimen were animals injected with an equivalent dose of alginate hydrogel without Mg2+. After the layer-by-layer closure of the wound, the rats immediately received subcutaneous injections of terramycin (1 mg/kg) and ketoprofen (0.5 mg/kg).All the animal procedures were performed in accordance with a protocol approved by the Committee on the Use of Live Animals in Teaching and Research . 6-8-week old Sprague Dawley (SD) rats, weighing 200–250 g, were purchased from Charles River Lab (USA) and maintained in the specific pathogen-free facilities . After the rats were anesthetized via intraperitoneal injections of ketamine hydrochloride  and xylazine hydrochloride . A tunnel defect, 2 mm in diameter, was prepared at the lateral epicondyle of each rat using a hand driller. Animals were randomly assigned into one of three treatment regimens; (1) injection of Mg3 were scanned with each sample for calibration. The resolution was set at 17.3 µm/pixel. Data reconstruction was done using the NRecon software (Skyscan Company), and image processing and analysis were done using CTAn software (Skyscan Company).In order to monitor the healing process and examine new bone formation around the gel, micro-CT scans were conducted at serial time points after the surgery . After the rats were anesthetized, the grafted site was scanned using a live animal scanning device . Two phantom contained rods with the standard densities of 0.25 and 0.75 g/cmTwo fluorochrome labels were used sequentially to evaluate bone regeneration and remodeling within the defects. Calcein green  was subcutaneously injected into rat femora one week after the surgery, while xylenol orange  was injected three weeks after the surgery. The fluorochrome labels were visualized under fluorescence microscopy . The intensity of fluorescence was analyzed by ImageJ software .After euthanasia, the femora were collected and fixed in 4% paraformaldehyde solution. For non-decalcified sections, the samples were dehydrated with gradient ethanol and embedded with methyl-methacrylate  after using xylene as a transition. Then, the embedded samples were cut into slices with a thickness of 200 µm and micro-ground to a thickness of 50–70 µm. The selected sections were stained with Goldner’s trichrome (Sigma-Aldrich) staining. For decalcified sections, the samples were decalcified with 12.5% ethylenediaminetetraacetic acid  for 6 weeks. The specimens were then dehydrated in ethanol, embedded in paraffin, and cut into 5 µm-thick sections using a rotary microtome . Haematoxylin and eosin (H&E) staining and TRAP staining (Sigma-Aldrich) was performed on selected sections from each sample following the manufacturer’s instructions. Images were captured using polarizing microscopy . The quantification of osteoblasts and osteoclasts in the grafted defect area was done on ×10 histological images using ImageJ software .MMA embedded bone specimens were sliced and polished to ~100 µm before observation. The elemental composition and distribution in the grafted area were determined by SEM-EDS .2O2 for the elimination of endogenous peroxidase activity. After blocking with normal goat serum, the slides were incubated with primary antibody overnight at 4 °C, respectively. The primary antibodies used in this study include: rabbit anti-OCN (Abcam), rabbit anti-IL-8 (Abcam), rabbit anti-CCL5 (Abcam), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1ra (Abcam), anti-CD68 (Abcam), rabbit anti-Phospho-Histone H3S10 (Abcam), and rabbit anti-TRPM7 (Abcam). The slides were then incubated with goat anti-rabbit secondary antibody and visualized using Diaminobenzidine (DAB) staining kit  following the manufacturer’s instruction. Immunofluorescent staining was done using Alexa-Fluor 488 conjugated anti-rabbit IgG or Alexa-Fluor 647 conjugated anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) and Hoechst 33324 (Thermo Fisher Scientific). Immunofluorescent images were captured using an LSM 780 confocal microscopy The dewaxed slide was treated with Proteinase K (Sigma-Aldrich) for proteolytic digestion and 3% HThe femora of the rats grafted with pure alginate or Mg-crosslinked alginate were harvested. The bone tissues in and around the grafted defects, around 1 cm in length, were ground into mud using a ceramic mortar and pestle. The mud of bone tissue was then homogenized in pre-cooled RIPA Lysis and Extraction Buffer (ThermoFisher Scientific) for 1 h. The buffer solution was centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was collected for protein concentration quantification with BCA Protein Assay Kit (ThermoFisher Scientific). An equal amount of protein from each sample was subjected to quantitative analysis of IL-8 and IL-1β using an ELISA kit (R&D system) following the manufacturer’s instructions.2. Human mesenchymal stem cells (MSC) was kindly provided by Prof. D. Campana , and maintained in Dulbecco’s modified Eagle medium  supplemented with 10% FBS (Thermo Fisher Scientific) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C under 5% humidified CO2. Macrophages were differentiated from THP1 cells using serum-free DMEM supplemented with 10 ng/mL phorbol 12-myristate 13-acetate . After 48 h, the THP1-derived macrophages were further cultured in customized Mg-free DMEM (Thermo Fisher Scientific) supplemented with different concentrations of Mg2+  for another 72 h to allow full differentiation and polarization. Mouse bone marrow macrophages (BMM) were obtained by incubating bone marrow cells isolated from both femurs of C57BL/6J mice in a culture medium supplemented with 20 ng/mL macrophage colony-stimulating factor  for 6 days86.The human monocyte cell line THP1 was obtained from the American Type Culture Collection , and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum  and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C under 5% humidified CO2+ for three days were washed and incubated with fresh serum-free DMEM (contained 1× Mg2+) for 24 h. These media were then collected, mixed 1:2 (v/v) with fresh serum-free DMEM  for the culture of MSC. For osteogenic differentiation of MSC, osteogenic supplements  were added to the serum-free DMEM. For osteoclastic differentiation of THP1-derived macrophages, RANKL (50 ng/mL), and M-CSF (30 ng/mL) were supplemented in the medium for a 14-day osteoclastic induction.To collect conditional medium (CM), THP1-derived macrophages that have been cultured at different concentrations of MgFlow cytometric assay was performed using FACSCantoII Analyzer . Differentiated macrophages were detached with trypsin and washed with 1× PBS. For the detection of macrophage surface markers, cells were incubated with monoclonal mouse anti-human antibodies CD163-Cy5.5, CD206-FITC, and CD80-FITC , or relevant isotypes (BD Biosciences) for 1 h at 4 °C in dark. After washing with 1× PBS, the fluorescence was compared to isotypes with 10,0000 events recorded. All flow cytometric data were analyzed using Flowjo software, version 10 .2+ on the attachment of THP1-derived macrophages and the proliferation of MSC was assessed using a cell counting kit-8 . At designated time points, the culture medium was replaced by a fresh serum-free medium containing 10% CCK-8. The optical density (OD) value at the wavelength of 450 nm was measured using a microplate spectrophotometer . Cell viability was presented in percentage by comparing the OD value of the tested group to that of the control group.The effect of Mg2+, the macrophages were lysed, and the intracellular ATP concentrations were determined using a luminescence ATP detection assay  following the manufacturer’s instructions.After the stimulation of Mg2+ were determined by Proteome Profiler antibody arrays (R&D System) following the manufacturer’s instructions. The concentration of IL-8 and IL-1β was further confirmed by specific ELISA kits (R&D System).The cytokines produced by THP1-derived macrophages after the stimulation of different concentrations of Mg2+ or conditional medium from macrophages on the ALP activity of MSC were evaluated using the p-NPP method. At the designated time points, the cells were lysed with 0.2% Triton X-100 at 4 °C for 2 h. The supernatant of the lysis after centrifugation was collected and assayed using an ALP detection kit (Sigma-Aldrich) following the manufacturer’s instructions. The total protein content was measured using BCA Protein Assay Kit (ThermoFisher Scientific). The relative ALP activity was normalized to total protein content and expressed as units/g·protein.The effects of MgSirius Red staining was used to study the formation of the extracellular matrix of MSC. At the designated time points, cells were fixed with 75% ethanol, and the collagen was stained with Sirius Red solution (Sigma-Aldrich) for 1 h. Alizarin Red staining was used to study the mineralization of MSC. At the designated time points, cells were fixed with 4% paraformaldehyde, and the mineralization nodules were stained with Alizarin Red solution (Sigma-Aldrich) for 5 min. After a thorough wash with Millipore water, the sample was dried in the air before photo taking.http://pga.mgh.harvard.edu/primerbank/, Table The total RNA of the cells was extracted and purified using the RNeasy Plus kit  following the manufacturer’s instructions. For the reverse transcript, complementary DNA was synthesized using Takara RT Master Mix  following the manufacturer’s instructions. The primers used in the RT-qPCR assay were synthesized by Life Technologies (ThemoFisher Scientific) based on sequences retrieved from Primer Bank . Cytosolic and nuclear extracts were prepared using NE-PER reagents (ThermoFisher Scientific) following the manufacturer’s instructions. A total of 30 µg of protein from each sample were subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane . Then the membrane was blocked in 5% w/v bovine serum albumin  and incubated with blocking buffer diluted primary antibodies overnight at 4 °C. The primary antibodies used include rabbit anti-TRPM7 (Abcam), rabbit anti-IL-8 (Abcam), rabbit anti-CCL5 (Abcam), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1ra (Abcam), rabbit anti-OPN (Abcam), mouse anti-ALP , rabbit anti-OCN (Abcam), rabbit anti-IKKβ , mouse anti-IKKα (CST), rabbit anti-NF-κB p65 (CST), rabbit anti-Phospho-IκBα (CST), mouse anti-IκBα (CST), rabbit anti-Phospho-Histone H3S10 (Abcam), rabbit anti-Histone H3 (Abcam), rabbit anti-Phospho-JNK (CST), rabbit anti-JNK (CST), mouse anti-NFATc1 (Santa Cruz), and mouse anti-β-actin (CST). The protein bands were visualized by ECL substrate (ThermoFisher Scientific) and exposed under ChemiDoc XRS System .At the designated time points after the treatment, the cells were rinsed with ice-cold PBS and lysed with RIPA Lysis and Extraction Buffer (ThermoFisher Scientific). After centrifugation at 15,000×2+, cells were washed with 1× PBS three times, permeabilized with 0.2% Triton X-100 for 10 min either before or after fixation with 4% paraformaldehyde. The primary antibodies used include anti-TRPM7 (Abcam), anti-NF-κB p65 (CST), and anti-Phospho-H3S10 (Abcam). The secondary antibodies used include Alexa-Fluor 488 conjugated anti-rabbit IgG (Thermo Fisher Scientific), Alexa-Fluor 647 conjugated anti-mouse IgG (Thermo Fisher Scientific). FITC-Phallotoxins (Sigma-Aldrich) and Hoechst 33342 (ThermoFisher Scientific) were used for counterstain. The fluorescent images were captured using a Carl Zeiss LSM 780 confocal microscopy .Following the stimulation of DMEM containing different concentrations of Mg2+-free DMEM (ThermoFisher Scientific) supplemented with 4 µM Mag-Fluo-4 (ThermoFisher Scientific) and 1 µM F-127 (ThermoFisher Scientific) for 30 min. After that, cells were washed with Mg2+-free DMEM and counterstained with Hoechst 33324. Real-time images were captured using a spinning confocal microscope  immediately after the addition of 8.0 mM MgCl2 into the Mg2+-free DMEM. For visualization of mitochondrial, cells were pretreated with MitoTracer (ThermoFisher Scientific) for 1.5 h before observation.THP1-derived macrophages were incubated in MgTHP1-derived macrophages were seeded on coverslips with locking beads. After the treatment, cells were washed with 1× PBS three times, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Then, the cells were incubated with mouse anti-TRPM7 and rabbit anti-Histone H3 primary antibodies overnight. After thorough washing steps with PBS, they were stained with Alexa-Fluor 647 and Alexa-Fluor 488 conjugated secondary antibody (Thermo Fisher Scientific) before being mounted to a Stochastic Optical Reconstruction Microscopy .For TRPM7 silencing, THP1-derived macrophages were transfected with 10 nM siRNA targeting human TRPM7  following the manufacturer’s instructions using siTran1.0  as the agent. Cells transfected with nonspecific control siRNA  were used as the control. siRNA transfection efficiency was verified 72 h after the transfection by western blots. For the inhibition of TRPM7 activity, cells were pretreated with 3 µM FTY720 (Sigma-Aldrich) for 2 h, washed with 1× PBS, and subjected to the other assays.After the stimulation, chromatin immunoprecipitation (ChIP) assay was performed using a ChIP kit (Abcam) as described by the manufacturer’s instruction. In brief, chromatin from crosslinked macrophages was sheared by sonication  and incubated overnight with rabbit anti-Phospho-Histone H3S10 (Abcam) at 4 °C. Precipitated DNAs were analyzed by RT-qPCR with SYBR Green Premix Ex Taq (Takara) and primers for human IL-8 promoter (−121 to +61) 5′-GGGCCATCAGTTGCAAATC-3′ and 5′-TTCCTTCCGGTGGTTTCTTC-3′, human IκBα promoters (−316 to −15) 5′-GACGACCCCAATTCAAATCG-3′ and 5′-TCAGGCTCGGGGAATTTCC-3′, as well as a human β-actin promoter (−980 to −915) 5′-TGCACTGTGCGGCGAAGC-3′ and 5′-TCGAGCCATAAAAGGCAA-3′.t-test was performed with SPSS ver.13.0 . The level of significant difference among groups was defined and noted as *p < 0.05 and **p < 0.01.Each experiment was performed at least three times, and the results were expressed as means ± standard deviations (s.d.). For the data analysis, either one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc test or two-sample Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


HNC risk also increased according to daily amount of alcohol consumption (p-trend < 0.01), but plateaued from 5–7 units/occasion  to >14 units/occasion . When stratified by average alcohol consumption, drinking frequency, but not daily amount, showed a linear relationship with HNC risk in moderate and heavy drinkers. When comparing the HNC subtypes, similar tendencies were observed in cancers of the oral cavity, pharynx, and larynx, but not in the salivary gland. In conclusion, drinking frequency is a stronger risk factor for HNC, especially for cancer of the oral cavity, pharynx, and larynx, than the daily amount of alcohol consumption.Alcohol consumption is a major risk factor for head and neck cancer (HNC), yet little data exist examining drinking patterns and HNC risk. In this population-based, retrospective cohort study, 11,737,467 subjects were recruited from the Korean National Health Insurance Service database. The risks of overall HNC and HNC subtypes according to average alcohol consumption, drinking frequency, and daily amount were examined using Cox proportional hazard models. Over the median follow-up of 6.4 years, 15,832 HNC cases were identified. HNC risk linearly increased with drinking frequency ( As global cancer incidence and cancer death are rapidly growing, identifying modifiable cancer risk factors is becoming more important ,2. Therep for trend = 0.03) [p for trend = 0.01) [n = 187) and drinking frequency in a study based on the same data [Previous studies of HNC risk have mostly focused on the average alcohol consumption level ,6,7,8. T = 0.03) . In anot = 0.03) . Drinkin = 0.03) . However = 0.03)  and 49 H = 0.03) , respect = 0.03) . This st = 0.01) , whereasame data . There aame data . FrequenIn our study, we aimed to investigate the association between alcohol drinking patterns and the risk of overall HNC or HNC subtypes using a large population-based cohort. The effects of two components of drinking patterns -drinking frequency and daily amount- were compared using stratified analyses by average alcohol consumption.The Korean National Health Insurance Service (NHIS) database was used for this study. The National Health Insurance Service is a mandatory social insurance service managed by the Korean government that provides universal health coverage to nearly the whole population of South Korea . NHIS aln = 303,424), who had any cancer within one year from the health screening , or who were deceased within one year from the health screening  were excluded. Subjects who had any missing data  were also excluded. The final study population comprised 11,737,467 subjects.From this database, 12,724,396 subjects who were aged over 40 years and who received national health screening in 2009–2010 were initially included. Subjects who were diagnosed with any cancer before the health screening , whichever came first. The median follow-up time was 6.4 years, after a one-year lag period.This study was approved by the Institutional Review Board of Samsung Medical Center (IRB File No. SMC 2019-02-059). The requirement for written informed consent from patients was waived because the data were anonymized under confidentiality guidelines.Data on alcohol consumption was collected from the self-reported questionnaires used for the national health examination in 2009–2010. This questionnaire includes questions about the average drinking frequency (number of days per week) during the last year and the daily amount (standard units per occasion) of drinking, regardless of the type of alcoholic beverage.Based on this data, we calculated the pure alcohol intake per occasion first. Standard drink size seems to vary among countries, possibly due to cultural differences. ,19. One The average weekly alcohol consumption (g/week) was calculated by multiplying the pure alcohol intake per occasion and drinking frequency. Since even a light-to-moderate amount of drinking might increase the risk of cancer , the ave2. Baseline comorbidities, including hypertension, diabetes mellitus, and dyslipidemia, were defined based on a combination of health examination records, disease diagnosis codes, and prescription records.Information on potential confounders, including age, sex, income level, smoking status, physical activity, body mass index (BMI), and comorbidities, were collected from the NHIS claims database and the national health screening database. The level of income was categorized into quartiles. Smoking status was categorized into never smoker, ex-smoker, and current smoker; ex-smoker and current smoker were each further categorized into <20 pack-years of smoking history and ≥20 pack-years of history. Physical activity level was categorized into non-regular and regular exercise; the regular exercise group was defined as performing either >30 min of moderate physical activity at least five times/week or >20 min of strenuous physical activity at least three times/week. BMI was calculated by kg/mDiagnosis of new HNC was the endpoint of this study. Incidence of HNC was identified using claims records with given diagnosis codes, according to the International Classification of Disease, 10th Revision (ICD-10): cancers of the oral cavity (C00–C06), salivary gland , pharynx (C09–C13), larynx (C32), and other sites in the head and neck  were defined as HNC. In addition, a diagnosis of cancer was also identified based on the receipt of a special co-payment reduction program for critical illness. In Korea, cancer patients who possess a medical certificate of diagnosis can apply for the special co-payment reduction program. The enrollees of this program pay only 5% of medical expenses for cancer work-up and cancer treatment, whereas other patients pay usually 20–30% of expenses for common diseases. Therefore, nearly all cancer patients are registered in this program in Korea, which makes our information on HNC diagnosis extremely reliable.t-test was performed to compare continuous variables, and a chi-squared test was performed to compare categorical variables.Descriptive statistics were used to examine the baseline characteristics of the study population according to average alcohol consumption level. A Student’s Incidence rates of overall HNC and HNC subtypes were calculated as the sum of new cases during the follow-up period divided by the sum of the person-years . To evaluate the effect of each independent variable, including average alcohol consumption, drinking frequency, and daily amount, on the risk of developing overall HNC or one of the HNC subtypes, Cox proportional hazard regression analyses were conducted. For all analyses, non-drinkers were used as the reference group. We also adjusted for all of the following covariates in gradual modeling: (1) Model 1 was adjusted for age and sex as a basic analysis; (2) Model 2 was additionally adjusted for variables that generally show associations with HNC or were commonly considered in previous studies, including income, smoking status, physical activity level, BMI and diabetes mellitus ,14,22,23The risks of overall HNC and HNC subtypes were stratified by average alcohol consumption level. The impacts of drinking frequency (days per week) and daily amount (standard units per occasion) were examined.p < 0.05.All analyses were performed using SAS version 9.4 , and the two-sided significance levels were set at n = 4,727,135) were drinkers. When compared with non-drinkers, drinkers were younger, male, in lower socioeconomic status, and had a smoking history (ex- or current smoker). The proportion of ever-smokers increased with increasing average alcohol consumption level (p < 0.0001).p-trend < 0.01): the risk began to significantly increase in subjects who drank 2 days/week  compared to non-drinkers and was highest in subjects who drank 7 days/week . HNC risk was also increased according to the daily amount per occasion (p-trend < 0.01): it continuously increased in subjects who drank ≤5–7 standard units per occasion when compared to non-drinkers, and then plateaued afterwards . Regarding the adjustment model, the addition of comorbidities of hypertension and dyslipidemia (Model 3) did not produce a substantially different result from that of Model 2.15,832 HNC cases were identified during the follow-up period. The risk of developing HNC according to average alcohol consumption, drinking frequency (days per week), and daily amount (standard units per occasion) are presented in The results of analyses on HNC subtypes and drinking status are presented in Moderate drinkers showed a nearly linear increase in HNC risk according to drinking frequency , whereas HNC risk decreased with increasing daily amount among subjects who drank at least 3–4 units/occasion.In heavy drinkers, HNC risk linearly increased according to drinking frequency , whereas no definite difference in HNC risk was observed according to the daily amount.When comparing the associations between drinking patterns and risks of developing one of the HNC subtypes, similar tendencies were observed in cancers of the oral cavity, pharynx, and larynx, but not salivary gland cancers .In this large-scale, nationwide, cohort study, average alcohol consumption was associated with an increased risk of overall HNC. The frequency of drinking was almost linearly associated with the risk of overall HNC. The daily amount of drinking also showed a positive association with overall HNC risk, but the level of association was weaker than that observed with drinking frequency. When comparing HNC subtypes, cancers of the oral cavity, pharynx, and larynx showed similar patterns as those of overall HNC, but salivary gland cancer showed no definite tendency according to drinking status.We found that average alcohol consumption was positively associated with HNC risk, which is consistent with previous research. Numerous studies have reported that drinking is an independent risk factor of HNC and that average alcohol consumption level is strongly associated with HNC risk in a dose-response manner ,26,27,28When comparing HNC subtypes, associations of similar magnitudes were observed in cancers of the oral cavity, pharynx, and larynx  in our study. Results regarding the differential strength of associations between HNC subtypes and alcohol consumption have varied between studies ,26. A meThe risk of salivary gland cancer showed no definite association with alcohol consumption in our study. Because of its rarity , little The most intriguing finding of our study was that drinking frequency is a more important risk factor of HNC than the daily amount of alcohol consumption. When stratified by average alcohol consumption, drinking frequency, but not daily amount, showed a linear relationship with HNC risk in moderate and heavy drinkers. The underlying mechanism for this finding remains unclear, but one possible explanation is that repetitive contact between a carcinogen  and the mucosa of the upper aerodigestive tract might lead these cells to increasingly undergo malignant transformations ,45,46,47In our study, mild and moderate drinkers who drank 5–7 days/week showed a slightly lower risk of HNC compared with subjects who drank 3–4 days/week. Considering that the average amount of alcohol per occasion would have been less than 21 g and 42 g, respectively, among these subjects, salivary AA concentration may have been insufficient, at least for some time, to promote local carcinogenic activity.Our study has important clinical and public health implications. Recent guidelines on drinking habits for the prevention of cancer and chronic diseases mostly advise individuals to reduce their overall consumption or daily amount without mentioning drinking frequency ,52. Howen = 15,832) were included than in previous investigations, allowing us to perform detailed analyses according to drinking patterns and cancer subtypes.Strengths of our study include being the only study to examine impacts of both drinking frequency and daily amount on HNC risk, specifically. Also, our study was based on a large, population-based, representative sample. A much higher number of HNC cases (However, there are also some limitations in our study. First, due to the retrospective nature of our study, we could not obtain information on other components of drinking habits, such as the total duration of alcohol drinking or changes in drinking habits. As we were not able to exclude former drinkers who quit drinking recently, HNC risks among non-drinkers could have been exaggerated. Information on human papillomavirus infection, which is increasingly reported to increase the risk of oropharyngeal and oral cavity cancer , was alsIn conclusion, this nationwide cohort study suggests that the frequency of drinking is a stronger risk factor for the development of HNC, especially for cancers of the oral cavity, pharynx, and larynx than the daily amount. Further research is needed on cancer prevention strategies with regard to drinking patterns.


This study assesses and compares the knowledge level of endodontists (ENDs) and general dental practitioners (GPs) from Brazil and United States of America (USA) in the diagnosis and treatment of internal and external inflammatory root resorptions through periapical radiographic (PA) and cone beam computed tomography (CBCT) examinations. A cross-sectional online questionnaire-based survey was presented to the volunteers containing questions regarding personal and professional profile, as well as three clinical cases of internal and external inflammatory root resorption. A series of multiple-choice questions about the diagnosis and treatment options were surveyed. The data collected was analysed by the Chi-square test with Yates correction with a significance level of 5 %. p<0.05); USA presented higher level of adequate responses than Brazil (p<0.05). Most answers were considered adequate when all three questions about the diagnosis and all two questions relating to the treatment were answered accurately. A total of 374 dentists answered the survey (n: 229 from Brazil vs. 145 from USA) being 41% END and 59% GP. END presented higher level of knowledge than GP regarding to diagnosis and treatment of inflammatory root resorptions both in Brazil and USA (END achieved a level of knowledge of the diagnosis and treatment of root resorption superior to the GP. Comparing the results obtained in both countries, it was observed that the USA had a higher correct response rate than Brazil. Key words:Internal root resorption, external root resorption, management, diagnosis, treatment. Root resorption occurs especially as a result of osteoclastic cell activity . In permInternal root resorption is an inflammatory process initiated within the pulp space that might lead to a possible cementum invasion. It is followed by a multinucleated giant cells accumulation and, consequently, a granular tissue formation . ClinicaOn the other hand, external inflammatory root resorption occurs as an imbalance between osteoblasts and osteoclasts present in the periodontal ligament . Since iAs a result of the similarities between internal and external inflammatory root resorptions and their detection being based on radiographic findings, the differential diagnosis should be achieved with multiple radiographs with different angulation for comparison and analysis of the lesions. In addition, dentists are encouraged to use CBCT exams to diagnose and assess the lesions extent and involvement .Proper diagnosis is paramount for their proper treatment. Therefore, the purpose of this study was to assess and compare the knowledge level of endodontists (ENDs) and general dental practitioners (GPs) from Brazil and United States of America (USA) in the diagnosis and treatment of internal and external inflammatory root resorptions through periapical radiographic (PA) and cone beam computed tomography (CBCT) examinations.-Research design The project was approved by the Research Ethics Committee of the University of Oeste Paulista - UNOESTE, CAAE 87440618.1.0000.5515 and by the University of Detroit Mercy Institutional Review Board under IRB protocol number 1819-36.In order to access the study population knowledge, an online questionnaire was designed with a mixture of qualitative and quantitative questions. Dentists were approached through an email database, social networking groups and personal contacts to participate in the research through an open google document. The online survey was kept available for 60 days.The inclusion criteria were being a private practice dentist in Brazil or USA. If the dentist was a specialist, only endodontist graduated from Brazil or USA was accepted. Subjects not included in these criteria, was excluded from the study.The questionnaire included a personal and professional profile, such as age, state of practice, experience as a dentist and/or an endodontist, judgment about the quality of endodontic teaching during graduation and the number of cases including internal and/or external resorption observed throughout their professional experience. Subsequently, the volunteer was presented with PA images of clinical cases of internal and external inflammatory root resorption, as well as a CBCT for the internal root resorption case. A series of multiple-choice questions about the diagnosis and treatment of both cases were asked. The survey is shown in -Statistical analysis The null hypothesis tested is that the knowledge level of general dentists is equal to the endodontists regarding the diagnosis and treatment of inflammatory root resorptions, both in Brazil and USA. The data collected was tabulated and analysed by the Chi-square test with Yates correction by the R software with a significance level of 5 % to compare the differences between specialists or not and within Brazil and USA.-Demographics Due to the questionnaire distribution, through an email database, social networking groups and personal contacts, we do not know how many subjects had access to the survey. A total of 374 dentists answered the survey (n: 229 from Brazil vs. 145 from USA). Of those, 154 (41%) were ENDs , with a mean age of 42.4±11.8 (41.2 vs. 45) years old, and with an average of 19±12.1 (19.2 vs. 18.8) years of experience. In addition, 220 (59%) were general dentists , with a mean age of 36.3±13.6 (31.3 vs. 44.5) years old, and with an average of 13.1±12.9 (9.7 vs. 18.7) years of experience. All results presented in parenthesis are Brazil vs. USA, respectively .A total of 293  dentists reported having adequate endodontic training during their dental school. When they were asked about their weaknesses, both groups pointed out that lack of self-interest, lack of patients, and outdated instructors were important factors (data not-shown).Most professionals never diagnosed or diagnosed few cases of internal and external inflammatory root resorption , with 82Diagnosis and treatment results Case 1, external root resorption Fig. .Case 2, internal root resorption Fig. , Q.9: OvCase 3 internal root resorption Fig. , Q.14: TWhen asked if any additional exam should be requested in cases of internal and external root resorptions, 5% of all surveyed population (6% vs. 5%) answered no, 28% (25% vs. 32%) did not know and would refer to another professional, and 144  would request additional exams. Regarding the ENDs cohort, all 154 ENDs from both countries would request additional exams. The most common exam would be CBCT, however some professionals would request angled PAs and pulp vitality tests. All results presented in parenthesis are Brazil vs. USA within the respective cohort.p <0.01, p>0.05).In this study, it was considered adequate when all three questions about the diagnosis and all two questions relating to the treatment were answered accurately. Statistically significant difference between the general dentists and ENDs was observed for both diagnosis and treatment of inflammatory root resorption p <0.01, . Same reP<0.05). The same was observed for ENDs from USA related to treatment (p<0.05); however, when general dentists were compared, no differences were found between countries (When the responses between Brazil and the USA were compared, it was observed that both general dentists and ENDs from USA had higher adequate answers related to diagnosis (ountries .This is the first article to address the difference between Brazilian and American dentists and ENDs on the diagnosis and treatment decision of inflammatory root resorptions. Our findings indicate that ENDs presented higher level of knowledge than GPs in both countries. In terms of region, USA presented higher level of knowledge than Brazil.The better outcome observed in ENDs were expected, since they have more years of training and experience than GPs. We also observed that GPs diagnosed and chose the treatment more appropriately for internal inflammatory root resorption compared with external resorption. This can be explained by the case chosen for this study, i.e., a severe internal root resorption case, making it easier to give a proper diagnosis.Due to a similar clinical and radiographical presentations of internal and external inflammatory resorptions, a provider should request multiple radiographs with different incidence angles . If a diet al. in 2009 observed that although a PA radiograph is an acceptable diagnostic method for internal and external root resorption, CBCT provides a more precise information, thus, increasing the probability of a proper lesion management (et al. in 2016 demonstrated the effectiveness of CBCT imaging in root resorptions diagnosis (et al. in 2016 compared the diagnostic efficacy of CBCT with parallax periapical radiographs for the detection and classification of simulated external cervical resorption lesions (Interestingly, the majority of GPs presented the appropriate diagnosis of internal inflammatory root resorption using PAs, however, when the CBCT was presented, higher failure of diagnosis was observed. This raises the question whether GPs know when the CBCT should be requested and, consequently, how to interpret it. Although the aim of the present research was not to compare the exams - CBCT and periapical radiograph, this surprising result was observed. Patel nagement . Similariagnosis . Also, V lesions . The per lesions . It is iThe literature shows the need of complementary exams - such as CBCT - to correctly diagnosis. Currently, the European Society of Endodontology statement highlights the relevance of CBCT for the management of potentially restorable external resorption lesions, especially when the diagnosis is unclear, and/or treatment is being planned, beyond the follow-up. This statement is also supported by American Association of Endodontists and American Academy of Oral & Maxillofacial Radiology ,17. Howeet al. in an ex-vivo study observed that 74% of teeth with periapical lesions presented some degree of internal inflammatory resorption (When the participants were questioned about the frequency of root resorption cases seen in their careers, both GPs and ENDs reported low numbers of cases overall. Epidemiological data on internal inflammatory root resorption are scarce. This can be due to its low prevalence in general population . Howeversorption . Externasorption . With thet al., focused on comparing the knowledge of Brazilian GPs and ENDs on the emergency management of dento-alveolar trauma, observed an overall poor knowledge and management of the cases, and correlated that with years of graduation from dental school (The difficulties in diagnosing these lesions may contribute to lower incidence reported , raisingl school . Brazil l school . Most ofl school . The lowIn conclusion, regardless of the country, END specialists possess a level of knowledge of the diagnosis and treatment of root resorption superior to the general practitioners. Knowledge levels of external inflammatory resorption were low for general practitioners. Comparing the results obtained in both countries, it was observed that the USA had a higher knowledge regarding these lesions that than Brazil.


Tannin-furanic rigid foams are bio-based copolymers of tannin plant extract and furfuryl alcohol, promising candidates to replace synthetic insulation foams, as for example polyurethanes and phenolics, in eco-sustainable buildings thanks to their functional properties, such as lightness of the material and fire resistance. Despite their relevance as environmental-friendly alternatives to petroleum derivatives, many aspects of the polymerization chemistry still remain unclear. One of the open issues is on the spatial heterogeneity of the foam, i.e., whether the foam constituents prevalently polymerize in spatially segregated blocks or distribute almost homogenously in the foam volume. To address this matter, here we propose a multiscale FTIR study encompassing 1D FTIR spectroscopy, 2D FTIR imaging and 3D FTIR micro-tomography (FTIR-μCT) on tannin-furanic rigid foams obtained by varying the synthesis parameters in a controlled way. Thanks to the implementation of the acquisition and processing pipeline of FTIR-μCT, we were able for the first time to demonstrate that the polymer formulations influence the spatial organization of the foam at the microscale and, at the same time, prove the reliability of FTIR-μCT data by comparing 2D FTIR images and the projection of the 3D chemical images on the same plane. Fourier Transform InfraRed (FTIR) spectroscopy is a very informative analytical technique that allows to identify chemical moieties of a sample in a non-destructive and label-free way ,2. With Nevertheless, FTIR imaging projects a 3D object onto a 2D plane, limiting diagnostic capabilities of those samples that are not chemically homogeneous along the direction of the optical path. In order to obtain a better representation of the system under analysis, a tridimensional reconstruction might be the solution, achievable by FTIR micro-computed tomography (FTIR-µCT). This relatively new imaging modality, as presented by Martin et al. in 2013 , combineTo date, just few examples of application of FTIR-µCT have been published, that are mostly proof-of-concept application of the technique to characterize biological samples such as tissues  or cellsHighly-porous materials can be indeed investigated with this approach, even when they are relatively thick and, as we have recently proven by presenting the applicability of the technique for the analysis of tannin-furanic rigid foams embedded in paraffin oil . In the From a chemical point of view tannin-furanic foams are copolymers of two units: the polyphenolics of the tannin plant extract and the furanics derived from the acid-catalyzed polymerization of furfuryl alcohol (FOH). In Specifically, if the reaction conditions favor the prevalent homopolymerization of FOH to PFA, extended and almost segregated furanic blocks, rich of furanic rings and carbonyl groups, and extended tannin blocks, rich of phenolics and hydroxy groups, are expected. Conversely, if the alternated copolymerization is favored, an alternation of these blocks is more likely. Of course, all possible shades between these extremes are possible and indeed very probable. The spatial distribution of these polymer blocks in the foam structure will depend not only from its formulation, but also from other reaction parameters such as stirring condition, temperature distribution and reagent’s dispersion. In this study, we focused on the importance of the chemical formulation, by varying the relative proportion of furfuryl alcohol (FOH High and FOH Low formulations) and sulphuric acid (Acid High and Acid Low formulations) with respect to a standard formulation (Reference from here on), as reported in In order to verify if the foams are in line with the expectations, we exploited a multiscale approach, based on 1D ATR-FTIR spectroscopy, 2D FTIR imaging and 3D FTIR-µCT.−1 spectral region. In −1 spectral range are plotted in −1 spectral region can be considered diagnostic of furanic moieties, such as the ones at ~1748, ~1718 and ~1703 cm−1, associated to carbonyl stretching, while the broader signal centered at ~1610 cm−1, associated to aromatic -C=C-, is mostly related to the phenolic nature of tannins. Therefore, under these assumptions, FTIR spectroscopy can provide evidences of the relative abundance of tannin and furanic blocks in the foam, but clear spectroscopy evidences of the extent of the copolymerization are not directly deducible. Indeed, methylene bridges are expected to link the copolymer blocks, but their quantification is actually prevented by the complex architecture of tannin rigid foam spectrum, being stretching and bending modes of methylene moieties also part of the tannin and PFA, both for the linear  and cyclic (III) forms.Average ATR-FTIR absorbance spectra of the crushed powders of the five considered formulations  are plotted in −1 spectral region was exploited to highlight the most relevant differences in the 5-formulations dataset. The resulting PC1 vs PC3 scatter plot, shown in −1, related to C-O-C vibrations of Diels Alder arrangement of PFA, at 1560 cm−1, assigned to the C=C stretching of the 2,5-disubstituted furan ring, at the spectral frequencies associated to carbonyl moieties of PFA, and at 1453 and 1505 cm−1 due to the C=C aromatic stretching of different constituents of the foam, mainly of tannin origin. The trend reveals that for higher concentrations of the acid catalyst, the reaction path favors FOH polymerization with respect to the Reference formulation, while the aromaticity of polyphenols is reduced, possibly due to side reactions initiated by the strong acid catalyzer that induces the aromatic ring opening. When considering FOH diversified formulations, PC3 presents the most intense contributions at 1560 cm−1, at around 1703 cm−1 and 1714 cm−1, due to C=O of PFA, and at 1080 cm−1, associated to alicyclic secondary alcohol (pyranic ring). Therefore, at higher concentrations of FOH, the reaction conditions also favor the FOH polymerization, but without affecting the aromaticity of tannin.Principal Component Analysis (PCA) of absorbance spectra in the 900–1800 cmNevertheless, since clear spectroscopic evidences of the copolymerization are not directly achievable, it is impossible by the sole 1D chemical information to speculate on the polymer block’s alternation/graft in the copolymer structure. Therefore, 2D analysis was performed in order to understand if the spatial localization of furanic and tannin moieties at the micron-scale was comparable or not, being the spatial-heterogenicity an indirect consequence of prevalent segregation, while homogeneity of alternated copolymerization. −1), ROI_1, and the -C=C- chemical moieties (1580–1635 cm−1), ROI_2, respectively. The considered images are the first of the data set acquired for each sample for FTIR μ-CT purposes, and they will be named images at zero angle projection from here on. In FTIR images of the five formulations were collected on minutes foam pieces attached into nylon loops, as better specified in Considering the Reference foam, distinct and extended tannin rich areas can be seen as blue and violet regions. Furanic and tannin moieties only partially colocalizes (black to gray pixels), letting to postulate the presence of variable-size furanic and tannin blocks, with a predominance of tannin ones. A mostly graft tannin-foam copolymer can be hypothesized also in the case of the Acid Low formulation: the large predominance of yellow areas allows to speculate that a dominant homopolymerization of FOH to PFA could have taken place. When considering the Acid High formulation, a tannin-rich area can be highlighted  with respect to a fragmented distribution of small tannin and furanic hot-spots in the inner part of the sample, and more extended furanic hot spots in bright yellow. The present results do not support our initial hypothesis of a prevalent grafted copolymerization resulting in a chemically homogenous foam in the case of Acid High formulation. For FOH High formulation, a clear co-localization of furanic and tannin hot-spots exists which appears in grayscale colour zones. Only sporadic segregated tannin and furanic richer areas can be highlighted as bluish and yellowish hot spots, differently from our initial hypothesis of more extended furanic blocks for this formulation. Conversely, for FOH Low formulations, a clear segregation between furanic and tannin blocks can be clearly distinguished (bluish and yellowish sample regions).z-axis.Nevertheless, the investigated samples are inhomogeneous in shape, both lateral size and thickness. It is therefore questionable if the observed hot spots derive solely from a different chemistry or they are also affected by polymer blocks segregation also along the In order to further investigate the diverse formulations and clarify the doubts generated by the analysis of 2D images, FTIR µ-CT experiments were performed on the five foam formulations. In Overall, the progression from 1D to 3D analysis confirms the hypothesis on the effects of the diverse formulation on the final chemical composition and spatial organization of the tannin foam, both characteristics that could not be obtained by 2D analysis alone. Indeed, with respect to 2D analysis, the 3D reconstructions provide also the first evidences on the fact that average foam formulation is not the same at each foam location, and that the variability seems independent to the foam morphology since tannin and furanic regions segregate or coexist in the foam cells at unspecific locations. This information could be clearly deduced only from 3D µCT, since the projection of a 3D object on a 2D plane could lead to partial conclusions. Nevertheless, one might doubt on the consistency of the results inferred from 3D chemical images, considered the heavy data processing applied for their reconstruction. Therefore, to confirm the limited effect of the data treatment on the chemical information contained in the original data, the spectral band integrals of ROIs along the projection axis were computed at zero projection angle  with water and furfuryl alcohol . Sulfuric acid  was added to the reagents’ mixture until it was homogeneous and was then placed in the oven for 30 min at 90 °C. Afterwards, the mixture was kept at 20 °C and 65% relative humidity to fully react, until achieving the classical appearance: a dark-brown rigid foam. The complete recipe for reference tannin foam is reported elsewhere , whereasn = 5 different samples were measured in the 50–6000 cm−1 range, averaging 256 scans at 2 cm−1 spectral resolution. The recorded spectra were cut in the 400–4000 cm−1 range, baseline corrected , vector normalized and offset corrected in the whole range. FTIR-ATR spectra analysis was performed using Quasar [−1.The foams were crushed into powder, and then measured using the monolithic diamond ATR accessory Platinum (Bruker Optics) of the Vertex 70v interferometer (Bruker Optics) in vacuum, equipped with the FIR-MIR DTGS detector and beamsplitter. For each formulation, g Quasar . Second 2. FTIR-µCT measurements were done both at the Chemical and Life Science branch of SISSI beamline  and the Before the experiment, a single small piece of the sample was attached to a nylon micro-loop (50 µm aperture size and 20 µm nylon rod diameter): the fragment was fished from a NaCl (0.9%) solution and, by exploiting the evaporation of the solvent and the crystallization of the NaCl salt, the specimen was anchored onto the loop. The tomographic setup is shown in −1 spectral resolution in the 900–4000 cm−1 spectral range, in order to limit the acquisition time to 80 s per image. For the same reason, only the sample interferogram was recorded and the Fourier Transform (FT) and the rationing with an air background in order to obtain the absorbance spectrum was performed afterwards. Spectral acquisition and FT  were done by using the Bruker proprietary software OPUS 7.5.Each hyperspectral projection was acquired averaging 128 scans at 16 cmBriefly, once recorded a single FTIR-FPA hyperspectral image, the sample was rotated by 1°, until having acquired the whole dataset: 360 projections were collected. Each sample experiment took about 8 hours, although, being the procedure completely automated and motion and acquisition software fully synchronized  - stretching mode of carbonyl groups); ROI_2: 1580–1635 cm−1 (ν(C=C) - stretching of the aromatic rings). All the procedures were executed by an in-house code written in Python 3.5 using the routine matplotlib [The data processing workflow is summarized in tplotlib , and scitplotlib  packagesFinally, for each rendering, the sub-volume of each ROI was projected along the axis corresponding to the optical path of the zero-angle projection, to generate a 2D image . These images were then compared with the FTIR-µCT pre-processed images at 0 degrees through PCA analysis.


A growing number of children and adolescents play video games (VGs) for long amounts of time. The current outbreak of the Coronavirus pandemic has significantly reduced outdoor activities and direct interpersonal relationships. Therefore, a higher use of VGs can become the response to stress and fear of illness. VGs and their practical, academic, vocational and educational implications have become an issue of increasing interest for scholars, parents, teachers, pediatricians and youth public policy makers. The current systematic review aims to identify, in recent literature, the most relevant problems of the complex issue of playing VGs in children and adolescents in order to provide suggestions for the correct management of VG practice. The method used searches through standardized search operators using keywords related to video games and the link with cognition, cognitive control and behaviors adopted during the pandemic. Ninety-nine studies were reviewed and included, whereas twelve studies were excluded because they were educationally irrelevant. Any debate on the effectiveness of VGs cannot refer to a dichotomous approach, according to which VGs are rigidly ‘good’ or ‘bad’. VGs should be approached in terms of complexity and differentiated by multiple dimensions interacting with each other. In the last decades, a very large body of literature has shown an increasing interest in video games (VGs) and their impact on the brain, cognition and behavior, especially in children and adolescents . Indeed,The current systematic review focuses on some crucial outstanding issues within the debate on the effects of VGs on cognition and behavior in order to provide suggestions for parents, pediatricians, health providers and educators dealing with pediatric ages, especially in the complex pandemic period. Namely, it analyzes the most debated and educationally relevant problems on the relationship between video games, cognition and behavior: 1. video games’ effects on cognitive function; 2. video games’ effects on attention and addictive behaviors; 3. video games and prosocial or aggressive behavior. Therefore, the current analysis may be accounted as an original contribution to the practical dimension in the educational and rehabilitation field for parents and educators.Early common predominant opinions mainly focused on VGs according to dichotomous thinking, as enjoyable entertainment or harmful tools . The recMondéjar and colleagues , in a grSimilarly, Lee et al. , found aRecently, Kwak et al.  longitudThe current systematic review was structured according to the guidelines and recommendations contained in the PRISMA statement .Eligibility CriteriaBoth experimental and correlational studies and meta-analyses between the years of 2000 and 2020 that investigated outcomes of VG exposure were included. They were considered children and adolescents. Studies employing different methodologies were included: studies in which naive participants were trained to use a VG versus a control group and studies comparing experienced versus non-gamers, or inexperienced players. Primary outcome measures were any type of structural and functional data obtained using neuroimaging techniques and behavioral testing.Information SourcesOne hundred and twenty-two studies were identified through electronic database searching in Ovid MEDLINE, Embase, PsycINFO, PubMed, Scopus (Elsevier) and Web of Sciences. The final database search was run on January 2021 using the following keywords: video games; video games and cognition; video games and epidemic; cognitive control; behavior control; brain and video games; spatial cognition; prosocial behavior; violence in video games; aggressive behavior; addictions in adolescents; children and video games. Study SelectionInclusion criteria: written in English; published since 2000; deals in depth with cognitive skills, attention, executive functions, or cognitive control; follows a high methodological rigor.Exclusion criteria: does not refer to key topics directly; the full text could not be obtained; lack of transparency due to missing methodology information. Ninety-nine studies were reviewed and included, whereas twelve studies were excluded because they were irrelevant to the topic or because the full text was not obtained. General communication materials, such as pamphlets, posters and infographics, were excluded as they do not provide evidence about their effectiveness.Any modern VG requires an extensive repertoire of attentional, perceptual and executive abilities, such as a deep perceptual analysis of complex unfamiliar environments, detecting relevant or irrelevant stimuli, interference control, speed of information processing, planning and decision making, cognitive flexibility and working memory. Literature data in the last years have proven that VGs may improve a variety of cognitive domains ,25 as, fHomer et al.  examinedSimilarly, Oei and Patterson  examinedMoreover, several meta-analytic studies provide evidence that action VG training may become an efficient way to improve the cognitive performance of healthy adults. Wang et al. , in a meIn summation, the studies on VG effects, by different methodologies, document both in adults and in children significant positive outcomes in different cognitive domains. Such performance improvements may be paralleled by functional brain remodelling .Attentional problems are accounted as a crucial area of focus on outcomes of intensive game-play practices in children and adolescents. However, literature on the topic appears inconsistent. While some research has found mixed results  or a posGentile et al. , examiniActually, any modern VG is a highly engaging activity with a variety of attractive cues, such as, for example, violence, rapid movement, fast pacing and flashing lights ,61. AccoProbably, in line with the bidirectional causality framework , such reIn such a discussion of the pathological nature of VGs, another outstanding question is whether pathological play is a major problem, or if it is the phenomenological manifestation of another pathological condition. Several studies have suggested that video game play can become harmful enough to be categorized as a psychiatric disorder, or it could be a symptom of an underlying psychopathological condition, such as depression or anxiety. Moreover, the functional impairments observed in individuals with game addictions are also thought to be similar to the impairments observed in other addictions. Neuroimaging studies have shown that the brain reward pathways which are activated during video game playing are also activated during cue-induced cravings of drug, alcohol or other type of substances abuse ,73,74.Some longitudinal studies ,75,76 prIn summary, attention problems and addictive behaviors in the context of VGs should be addressed in a circular and bidirectional way in which each variable can influence the others.The positive impact of video games also concerns the social and relational dimension, as occurs in the VG training of prosocial or educational skills. Several studies have reported that playing prosocial VGs, even for a short time, increases prosocial cognition , positivIn four different experiments , playingFollowing experimental, correlational, longitudinal and meta-analytic studies provided further evidence that playing a prosocial VG results in greater interpersonal empathy, cooperation and sharing and subsequently in prosocial behavior ,88,89,90Such literature’s data are consistent with the General Learning Model ,92, accoIn the same conceptual framework, educational video games have been found to positively affect behaviors in a wide range of domains , school Violence in VGs is a matter of intense debate, both in public opinion and in the scientific context ,102. A vSimilarly, experimental, correlational and longitudinal studies supported the causal relationship between violent VGs and aggression, in the short- and long-term, both in a laboratory and in a real-life context. A greater amount of violent VGs, or even a brief exposure, were significantly associated with more positive attitudes toward violence , higher In summation, the overall literature data support the opinion that violent video games, over time, affect the brain and activate a greater availability to aggressive behavior patterns, although some researchers have pointed out that the negative effects of violent VGs are small and may be a publication bias ,111. The focus of the current overview was to identify, from a functional point of view, the most significant issues in the debate on the impact of VGs on cognition and behavior in children and adolescents, in order to provide suggestions for a proper management of VG practice. Overall, the reviewed literature agrees in considering the practice of VGs as much more than just entertainment or a leisure activity. Moreover, research agrees that any debate on the effectiveness of VGs cannot refer to a unitary construct , nor to The term ‘video game’ should be viewed as an ‘umbrella term’ that covers different meanings, far from a single unitary construct ,114. FurGentile and colleagues ,118,119 However, the content effects, individually focused, are frequently overemphasized. According to the General Learning Model, children would learn the contents of the specific games and apply them to their lives. Nevertheless, a violent game using a team-based game modality may have different impacts than a violent game using a ‘free for all’ game modality. Although both are equally violent games, the former could suggest teamwork and collaborative behaviors, while playing in an ‘everyone for oneself’ mode could foster less empathy and more aggressive thoughts and behaviors ,88. Likewise, the outside social context can have different effects and it may even mitigate or reinforce the effects of the content. Playing violent games together with others could increase aggression outcomes if players reinforce each other in aggressive behavior. Instead, it could have a prosocial effect if the motivations to play together are to help each other . According to the dominant literature, the psychological appeal of video games may be related to an operant conditioning that reinforces multiple psychological instances, including the need for belonging and social interaction ,58. On sVGs can also have a different psychological appeal in relation to their structural organization and the way they are displayed. Many structural features can affect playing behavior, regardless of the individual’s psychological, physiological, or socioeconomic status , such asThe more or less realistic mechanics can also configure the game differently and affect fine or gross motor skills, hand-eye coordination or even balance skills, depending on the type of controller, such as a mouse and keyboard, a game control pad, a balance board, or a joystick.Therefore, VGs may differ widely in multiple dimensions and, as a result, in their effects on cognitive skills and behavior ,33. MoreThe current analysis of the literature, therefore, supports the need for further experimental and longitudinal research on the role of multiple characteristics of video games and their interactions. A wide-ranging approach dynamically focused on the multiple dimensions will allow a deeper theoretical understanding of the different aspects of video games.Nevertheless, according to common opinion, the violence would always have a negative impact on behavior, especially in pediatric subjects. However, a strictly causal relationship between violent VGs and aggressive behavior appears rather reductive ,127. AggAn alternative theoretical framework ,127 assuConversely, playing prosocial VGs, even for a short time, increases prosocial cognition, affect and behaviors in children and adolescents ,84,85,89Moreover, several studies emphasize the educational and academic potential of VGs that may become effective and ‘exemplary teachers’ ,82 proviStudies focused on the attentional functions in VG playing reported inconsistent data. Playing action games may improve attention skills implied in a specific game. However, according to the attractive hypothesis  and operSimilarly, since VGs and their cues appear more pleasant and desirable, a large amount of attractive VG exposure can lead to addiction and impair ability to focus on effortful goal oriented behavior . HoweverIn the fifth edition appendix of the Diagnostic and Statistical Manual of Mental Disorders , the diaThe debate on the relationship between pure game addiction behaviors and game addiction in comorbidity with other psychiatric disorders appears still on. Some researchers have argued that game addiction, as a standalone clinical entity, does not exist , but it Likewise, the relationship between clinical symptoms and changes in brain activity and the dynamics by which video games triggers such widespread brain plasticity needs to be more clearly defined. The current analysis of the literature provides strong evidence on the power of video games as highly motivating and engaging tools in the broader context of cognitive, emotional and relational development of children and adolescents. However, the effectiveness of such tools does not arise exclusively from their content, but it results from a set of variables interacting each other. Video games, beyond their content, can favor pathological aggression, withdrawal, escape from reality and reduction of interests. Virtual reality becomes more attractive than the real one and can become the ‘non-place’ to escape from the complexity of everyday life. Recently, to contain the spread of the COVID-19 pandemic, health authorities have forced populations to stay home and children and adolescents may experience an exacerbation of exposure to video games. Parents, educators and teachers should ensure an educational presence, monitoring times and modalities of VG practice in a broader context in which children and adolescents live with a wider repertoire of interests, without losing social and relational engagement. Moreover, pediatric health care visits may be a great opportunity to support parents helping children to deal with media and video games. On these assumptions, as practical suggestions to prevent or mitigate addictive behaviors, parents and educators should enforce the golden rule as the educational presence of the adult. Moreover, in line with the literature, the core values to prevent a negative impact of video games should be focused on a few rules to be proposed with assertiveness and authority: 1. set a clear time limit to play, 2. prefer games that can also be played with family, 3. alternate video games with other games and activities, 4. avoid highly addictive games, 5. keep a social life in the real world.


Photo Response Non-Uniformity (PRNU) is reputed the most successful trace to identify the source of a digital video. However, its effectiveness is mainly limited by compression and the effect of recently introduced electronic image stabilization on several devices. In the last decade, several approaches were proposed to overcome both these issues, mainly by selecting those video frames which are considered more informative. However, the two problems were always treated separately, and the combined effect of compression and digital stabilization was never considered. This separated analysis makes it hard to understand if achieved conclusions still stand for digitally stabilized videos and if those choices represent a general optimum strategy to perform video source attribution. In this paper, we explore whether an optimum strategy exists in selecting frames based on their type and their positions within the groups of pictures. We, therefore, systematically analyze the PRNU contribute provided by all frames belonging to either digitally stabilized or not stabilized videos. Results on the VISION dataset come up with some insights into optimizing video source attribution in different use cases. Video source attribution is commonly addressed by extracting the traces left into the content by the Photo Response Non-Uniformity (PRNU), originated by manufacturing processes in the form of slight imperfections in light response of pixels, and by comparing them to a reference trace characterizing the device. This methodology, firstly applied for image source attribution ,2, proviFew years later, the same technology was extended to digital videos ,9. The fSeveral solutions have been proposed to cope with video compression: in , the autHowever, all the mentioned works do not consider devices equipped with electronic image stabilization (EIS), which aimes at compensating for camera shake. The technical specifics of this compensation are generally based on proprietary software, and their implementation details usually vary among brands. In few cases, some differences among the existing EIS implementations have been studied in controlled environments and pointed out . HoweverNote that when EIS is applied, PRNU is transformed accordingly, being solid with respect to the transformation, and this issue has to be considered during PRNU comparison. Starting from this consideration, in , the autThe paper is organized as follows: Before going into the details of source camera attribution based on PRNU, we briefly recall some basics of video compression. Any digital video is essentially a temporal sequence of pictures  acquired at a given frame rate. Such a data stream can be compressed to save storage or transmission bandwidth by exploiting spatial and temporal redundancies and, at the same time, by considering the characteristics of the Human Vision System (HVS) in order to minimize visual degradation. To achieve this goal, the most common video compression standards such as Advanced Video Coding  ,25, or tWhen a frame is compressed, the encoder divides it into macroblocks (MBs), and encodes each MB individually: MBs belonging to I frames are always encoded without referring to other frames, by means of a DCT quantization strategy. In this sense, I frames are encoded in a similar way as JPEG images. The main difference resides in the MB size, bigger than the typical On the contrary, MBs belonging to predictive-coded frames may be encoded referring to previous frames (this is the only possibility in P frames) or even referring to the following frames . Besides predicted MBs, the encoder embeds the motion vectors associated to each MB. In some configurations, the encoder has also the possibility to skip a MB in a predictive-coded frame, if this MB can be directly copied from a previous frame . Finally the numeric stream is further compressed by means of lossless compression.For a more detailed explanation, reader might refer to ,25,26.When dealing with digitally stabilized videos, it is convenient to estimate the camera fingerprint from still images because they are not affected by EIS .In the proposed analysis, we thus exploit still images to generate fingerprints of all available devices. The workflow for generating camera fingerprints is shown in N images Given a set g filter  is applistimator :(1)K˜I=∑rtifacts .In parallel, the same denoising filters and processing are used to extract the noise patterns from video frames originated by the same camera as the set of images ested in . FinallyThe testing procedure is described in CE ratio  is derivN frames, we compute the test statisticIn general, a query video Equation  for all For a given threshold Algorithm 1 Fingerprint Generation.1:input data: for each device, 2:                   neighbourhood size 3:                   minimum scale 4:result: Fingerprint of the inquired camera;5: 6:fordo7:    extract PRNU 8:end for9: 10:apply Equation (1) among 11: 12:for do13:    extract PRNU 14:    inizialize 15:for do    16:          17:s;          compute 18:          19:end for    20:if max and testing the video frames respectively (Algorithm 2).The experiments are performed on a computer with an Intel64 Family 6 Model 158 Stepping 9 GenuineIntel @ 3.601 GHz CPU and 32 GB RAM. The code for PRNU extraction and matching is developed in MATLAB.indoor and outdoor according to VISION names convention.Tests were performed on the VISION dataset , includiffprobe, a tool provided by FFmpeg (https://ffmpeg.org/ accessed on 12 February 2022). To be independent of the resolution, we computed the PCE between the reference and the frame PRNU by cropping a central patch of We systematically analyzed the PRNU contribution of each frame type. To do so, we extracted the PRNU from all video frames, and we determined the frame types by using asonable .whether the frame is an intra or a predicted coded one;whether the video is acquired in presence of EIS or not;whether the frame belongs or not to the first GOP.Afterward, we performed a distinctive analysis in function of:The last distinction was made to reach a deeper understanding of some devices where EIS eventually is not applied to the first (intra-coded) video frame .First, we evaluated the PCE distributions grouped by devices. Results are shown in Globally, in non-stabilized videos, frames tend to match in a position very close to the same PCE location see a,b, exceThe situation rather changes for stabilized recordings d–f. CommThe previous analysis provides useful insights at the level of a single frame. At the video level, we applied Equation , and theA first observation rises: performance over stabilized videos seems better than non-stabilized videos over the VISION dataset, independently from the type of frame considered. This outcome is quite unexpected because the PRNU footprint of non-stabilized videos should not be misaligned, and we should have observed higher performance for non-stabilized videos. Either can explain this phenomenon by poor quality reference PRNU for some devices (particularly in devices D023 and others) or stronger compression for the oldest devices, which usually need to produce more compressed video files to address their limited storage capabilities. Nevertheless, by analyzing the two figures separately, we still notice some interesting insights. In Regarding stabilized videos, we remind to We extended the previous analysis to frames in other positions see . We showAs a the last experiment, we compared two different strategies: the first (suggested in ) is to uThe above analysis comes up with some findings and insights that can be exploited to design a PRNU-based source video attribution task. In the following, we summarize them.Finding #1a strategy that could be optimal both in presence of EIS and not is difficult to be achieved. In fact, the literature ,31 showsFinding #2from the previous finding, we can derive that a system able to classify whether a video is stabilized or not reliably could lessen the problem. Some techniques exist, such as , but if Finding #3the overall performance of source attribution based on PRNU heavily depends on the device model and/or brand.Finding #4I frames indeed convey a less attenuated and distorted PRNU independently from the presence/absence of EIS and the source brand.Finding #5In presence of EIS, the first I frame usually provides the most significant PRNU information. Depending on the device, it can be affected or not by EIS.Finding #6P frames could contain a weakly distorted-attenuated PRNU in both cases. In presence of EIS, we found that P frames within the first GOP generally provide the most reliable PRNU information.Finding #7When possible, P frames that follow an I frame should be preferred. Except for this, the contribution of P frames does not depend on their position within the GOP.By following the previous findings, we provide some possible strategies in function of the use cases.Insight #1If a reliable system for classifying a video as stabilized or not is provided, it should be adopted in order to perform two different matching strategies . The final performance should take into consideration the accuracy of the whole system.Insight #2If the video duration is long enough and with a sufficient number of I frames , the choice of using only I frames is a good trade-off between performance and complexity. The first I frames should be privileged in the analysis.Insight #3If the video is short and the number of I frames is limited, P frames could be exploited to improve performance. In particular, the first P frames following an I frame are proved to be more suitable than the others.Insight #4When deciding a threshold for the binary decision, a training with a control dataset should be adopted. The dataset should be as close as possible to the target devices in terms of EIS and video encoding to avoid unexpected classification errors.In this paper, we evaluated which type of frame is more useful to perform PRNU-based video source attribution, even in presence of electronic image stabilization. We systematically analyzed the PRNU contribution of each video frame in order to assess if an optimum unified strategy could be performed, independently from the adoption of electronic image stabilization and the device model. The analysis shows that such a strategy still remains far from being defined, since we discovered that video compression behavior and EIS implementations can strongly vary among brands. At the same time, we provided some findings and insights that the forensic practitioner should take into account to optimize the source attribution strategy depending on the investigated device and the available information.