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Background NETosis is a neutrophil-mediated defense mechanism during which DNA and enzymes are extruded forming a network (NETs) trapping and killing different pathogens. NETosis is reduced in both mice and humans during aging. Aims We explored the difference in the efficacy of NETs released in elderly (> 65 years) versus adults (20–50 years) subjects in inhibiting Staphylococcus aureus growth and activating the growth of keratinocytes. Methods Neutrophil granulocytes, obtained from venous blood both in healthy elderly and adult subjects, were stimulated by LPS (0–250 µg/ml) to induce the formation of NET. NETs were quantified by SYBR Green staining and growth inhibition of S. aureus was evaluated by disk diffusion test. Furthermore, NETs (0–500 ng/ml) were added to immortalized human keratinocytes (HaCaT cells), and their proliferation was evaluated by MTT assay after 24 h. Finally, the DNA size of NETs was evaluated by flow cytometry after SYBR Green staining. Results Greater production of NETs was observed in elderly subjects than in adults, but these NETs showed reduced bactericidal capacity and HaCaT cells’ proliferation stimulation. The activities of the NETs are related to the size of the extruded DNA threads, and when NETs size was analyzed, DNA from elderly showed a higher size compared to that obtained by adults. Discussion Unexpected results showed aging-related NETs structural modification resulting in both a lower antimicrobial activity and keratinocyte proliferation stimulation compared to NETs obtained from adults. Conclusions The NETs DNA size observed in elderly subjects has not been previously reported and could be part of other pathogenic mechanisms observed in aging.

Although a growing body of literature has been emphasizing the role of vitamin D in oral health, there is still a gap of knowledge regarding the correlation between temporomandibular disorders (TMDs) and vitamin D. Therefore, the aim of this systematic review was to assess the linkage between hypovitaminosis D and TMDs to map the current literature in this field. On 10 September 2022, PubMed, Scopus, and Web of Science databases were systematically searched from the date of their inception to identify the studies that had assessed patients with TMDs. The primary outcome assessed in this review was the relationship between hypovitaminosis D and TMDs. Out of the 329 studies identified, 13 studies met the eligibility criteria and were included in the present work. Seven studies assessed the relationship between vitamin D and TMDs, reporting that vitamin D serum levels are lower in patients with TMDs. Our results suggested that vitamin D receptor (VDR) polymorphisms might have a role in TMDs’ development. However, the quality assessed underlined that only one study did not present a serious risk of bias. Further good-quality studies are needed to clarify the linkage between vitamin D deficiency and TMDs, but the evidence currently available has suggested potential correlations.

The goal of this review is to provide a comprehensive overview of (i) bone and muscle tissue modifications pathophysiology in spinal cord injury (SCI), (ii) experimental data on the physiopathological mechanisms underpinning these modifications and their similarities with the aging process, and (iii) potential clinical implications in the management of the disabling sequelae of SCI. Several studies attempted to describe the biology underpinning the links between bone and muscle tissues in the setting of highly disabling conditions, such as osteoporosis, sarcopenia, and neurodegenerative disorders, although these bidirectional connections remain still unclear. SCI could be considered an in vivo paradigmatic model of the bone muscle interactions in unloading conditions that might be expanded in the field of neurodegenerative disorders or cancer studies. Future studies should take into consideration the newer insights into bone muscle crosstalk in order to develop multitargeted and therapeutic interventions.

Breast cancer (BC) survivors treated with aromatase inhibitors (AIs) commonly show several pathological issues, including poor oral health, bone health impairment, and vitamin D deficiency. However, to date, oral health issues in BC survivors treated with AIs have been poorly investigated and their relationship with vitamin D deficiency are far from being understood. This study aimed to evaluate the correlation between oral health and vitamin D status in BC survivors undergoing treatment with AIs through a machine learning approach. In this cross-sectional study, we included post-menopausal BC women with vitamin D deficiency undergoing AIs therapy. The outcome measures were the following: oral health indexes as the Decayed, Missing, and Filled Permanent Teeth Index (DMFT); serum levels of 25(OH)D3; Bone Mineral Density (BMD); and the diagnosis of osteoporosis. We included 41 post-menopausal BC women, mean aged 66.10 ± 8.47 years, with mean serum levels of vitamin D of 14.63 ± 6.62 ng/mL. Furthermore, 56.10% of patients had a diagnosis of osteoporosis and 36.59% were osteopenic. DMFT was significantly related to smoking (p-value = 0.005) and dental floss use (p-value = 0.001). There was a significant correlation between DMFT and vitamin D levels (Pearson’s r: −0.73; p-value = 0.001). The regression machine learning model showed that vitamin D status and the use of dental floss were the most relevant variables in terms of correlation with DMFT. In conclusion, vitamin D deficiency, inadequate use of dental floss, and smoking had a negative impact on oral health in BC women. Thus, vitamin D deficiency screening and supplementation and a prompt oral rehabilitation plan should be suggested and implemented in the complex treatment framework of BC survivors undergoing treatment with AIs.

Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5 – 16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host’s DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from “old” (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics.

Rapid and efficient wound healing response is essential to maintain whole organism integrity and homeostasis. Such process could be influenced by a plethora of factors that when interfering with the different healing phases finally result in impaired tissue repair. Furthermore, defective healing process is often associated with chronic and difficult to heal wounds, both conditions deeply affecting patients’ quality of life. Advanced wound management solutions are the new standard in difficult to heal treatment and their key feature is represented by their ability to protect the wound bed but also to direct cellular behavior. The aim of the present work was to evaluate the effectiveness of a bioactive hydrogel in modulating wound healing in a rat excisional wound model. Surgical wounds were treated with hydrogel matrix enriched with a keratinocyte specific growth factor (epiregulin), saline (control condition) or the different hydrogel components alone. Results showed from both a macroscopic and microscopic point of view a faster wound closure in hydrogel treated wounds associated with a better quality of the regenerated tissue. Such results thus support the use of the proposed hydrogel matrix as a springboard to develop innovative advanced wound dressings to treat difficult to heal wounds.

All patients (n=114) sents from Outpatient Clinic of Physical and Rehabilitative Medicine, “Maggiore della Carità” Hospital, Novara, Italy the underwent a dental examination, in anticipation of taking Denosumab. Patients undergoing a dental check-up were on adjuvant therapy with hormone therapy drugs due to previous breast and prostate cancer. Good oral health is a prerequisite for starting denosumab treatment.

The neutrophils extracellular traps (NETs) are a meshwork of chromatin, histonic and non-histonic proteins, and microbicidal agents spread outside the cell by a series of nuclear and cytoplasmic events, collectively called NETosis. NETosis, initially only considered a defensive/apoptotic mechanism, is now considered an extreme defensive solution, which in particular situations induces strong negative effects on tissue physiology, causing or exacerbating pathologies as recently shown in NETs-mediated organ damage in COVID-19 patients. The positive effects of NETs on wound healing have been linked to their antimicrobial activity, while the negative effects appear to be more common in a plethora of pathological conditions (such as diabetes) and linked to a NETosis upregulation. Recent evidence suggests there are other positive physiological NETs effects on wound healing that are worthy of a broader research effort.

Human skin hosts a variety of microbes that can be transferred to surfaces (“touch microbiome”). These microorganisms can be considered as forensic markers similarly to “touch DNA”. With this pilot study, we wanted to evaluate the transferability and persistence of the “touch microbiome” on a surface after the deposition of a fingerprint and its exposure for 30 days at room temperature. Eleven volunteers were enrolled in the study. Skin microbiome samples were collected by swabbing the palm of their hands; additionally, donors were asked to touch a glass microscope slide to deposit their fingerprints, that were then swabbed. Both human and microbial DNA was isolated and quantified. Amelogenin locus and 16 human STRs were amplified, whereas the V4 region of 16 S rRNA gene was sequenced using Illumina MiSeq platform. STR profiles were successfully typed for 5 out of 22 “touch DNA” samples, while a microbiome profile was obtained for 20 out of 22 “touch microbiome” samples. Six skin core microbiome taxa were identified, as well as unique donor characterizing taxa. These unique taxa may have relevance for personal identification studies and may be useful to provide forensic intelligence information also when “touch DNA” fails. Additional future studies including greater datasets, additional time points and a greater number of surfaces may clarify the applicability of “touch microbiome” studies to real forensic contexts.

13 p.-7 fig.-1 tab., Inducible T-cell costimulator (ICOS) upon binding to its ligand (ICOSL) mediates adaptive immunity and antitumor response. Thus, antitumor therapies targeting the ICOS/ICOSL pathway hold great promise for cancer treatment. In this regard, ICOSL triggering by a soluble recombinant form of ICOS (ICOS-Fc) hampered adhesiveness and migration of dendritic, endothelial, and tumor cells in vitro. Furthermore, in vivo treatment with ICOS-Fc previously showed the capability to inhibit lung metastatization of ICOSL+ B16-F10 melanoma cells when injected intravenously in mice, but it failed to block the growth of established subcutaneous B16-F10 murine tumors. Thus, we asked whether passive targeting of solid tumors with ICOS-Fc-loaded biocompatible and biodegradable nanoparticles (NPs) could instead prove effectiveness in reducing tumor growth. Here, ICOS-Fc was loaded in two types of polymer nanoparticles, i.e. cross-linked β-cyclodextrin nanosponges (CDNS) and poly(lactic-co-glycolic acid) (PLGA) NPs and in vitro characterized. In vivo experiments showed that treatment of C57BL6/J mice with ICOS-Fc loaded into the two nanoformulations inhibits the growth of established subcutaneous B16-F10 tumors. This anticancer activity appears to involve both anti-angiogenic and immunoregulatory effects, as shown by decreased tumor vascularization and downmodulation of IL-10 and Foxp3, two markers of regulatory T cells (Tregs). Overall, the substantial in vivo anticancer activity of ICOS-Fc-loaded CDNS and PLGA NPs against different components of the tumor microenvironment makes these nanoformulations attractive candidates for future combination cancer therapy., This work was supported by the Associazione Italiana Ricerca sul Cancro (IG 20714, AIRC, Milano), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017–0535).

Aims Monocytes/macrophages are essential in innate immune response against pathogens also because their ability to release extracellular traps named METs (monocytes/macrophages extracellular traps). These structures are composed of DNA fibers decorated with nuclear and cytoplasmic proteins and their production process is called METosis. In this study attention has been focused on the ability of differently charged molecular systems (polyhedral oligomeric silsesquioxanes, POSS positively or negatively charged) to induce METosis. Main methods METs formation was induced by lipopolysaccharide (250 μg/ml, positive control) and POSS positive and negative (0.05–1 mg/ml) treatment. METs were visualized and quantified by confocal microscopy using Sytox green staining. Oxidative stress, autophagy, as well as endocytosis involvement in the POSS induced METosis was evaluated. Key findings Results obtained indicate a POSS positive or negative dose dependent ability in inducing MET release independently to their charge and that this phenomenon is a consequence of POSS +/− internalization. Moreover, studies using many reactive oxidative species (ROS) blockers and autophagy inhibitor showed a strong reduction in POSS induced METosis indicating their involvement. Significance POSS +/− induce extracellular traps production in human monocytes/macrophages by oxidative and autophagic pathway.

Introduction Granulocytes play a pivotal role in innate immune response, as pathogen invasion activates neutrophils, a subclass of granulocytes, inducing the production of neutrophil extracellular traps (NETs). In this study, it has been evaluated how NETs could affect human keratinocytes (HaCaT cells) behaviour. Materials and methods HaCaT cells were treated with increasing NETs concentrations (0.01–200 ng/ml) and the effect on cell proliferation was evaluated by MTT assay. Inhibition studies were performed by pre-treating cells with dexamethasone, chloropromazine or amiloride. NF-kB pathway activation was evaluated by western blot. Results HaCaT cells stimulation with increasing concentrations of NETs (0.01–50 ng/ml) for 48 h resulted in a modulation of cell proliferation with a maximum increase corresponding to 0.5–1 ng/ml stimulation. NETs low concentrations not only increased cell proliferation, but were also able to induce a faster wound closure in an in vitro scratch assay. NETs scaffold, composed by histone proteins and DNA, is recognized by Toll Like Receptor 9 (TLR 9) that, in turn, activates the NF-kB pathway. In fact, NETs induced proliferation was inhibited by chloropromazine (1 nM), that blocks chlatrin vesicles formation, and by amiloride (50 nM) that inhibits macropinocytosis. Moreover, dexamethasone, an inhibitor of NF-kB, was able to abolish the NETs effect. Discussion This study thus demonstrates that low NETs concentrations undergo internalization finally resulting in a quick NF-kB pathway activation and HaCaT cells proliferation increase, suggesting a close relationship between first immune response and wound healing onset.

Photodinamic therapy (PDT) has gained an increasing interest as a new tool to treat skin cancers such as melanoma. This clinical approach take advantage from the combination of a photosensitizer and a specific light wavelength able to induce singlet oxygen production. Mesoporous silica nanoparticles (MSNs) have been widely investigated as drug nanocarriers as their structure and morphology could be customized to produce suitable nanoplatforms enabling high cargo capacity. In the present study MSNs were successfully conjugated with the second generation photosensitizer verteporfin and the resulting nanoplatform (Ver-MSNs) was tested in an in vitro PDT model as a potential tool for melanoma treatment. Ver-MSNs based PDT did not affect cell proliferation of neither a normal human keratinocyte cell line (HaCaT) or a low mestastatic melanoma cell line (A375P). On the other hand Ver-MSNs based PDT deeply affect the highly invasive SK-MEL-28 melanoma cell line behavior, as testified by the strong reduction in cell proliferation along with the dramatic change in cellular morphology, through a nanoparticle internalization dependent mechanism. In fact, experiments performed in the presence of endocytosis inhibitors (chlorpromazine and amiloride) resulted in an attenuation of Ver-MSNs based PDT induced cell death, along with a recover in cellular morphology. MSN doped with verteporfin could thus represent a promising and useful tool for PDT treatment of highly invasive melanoma.

The gold standard to arrange impacted teeth in the dental arch is represented by a surgical approach followed by orthodontic traction force application. In the literature, many surgical approaches are proposed to reach such a scope. The aim of the present study is to demonstrate how laser technique could positively assist surgical approaches.Study population was composed by 16 patients undergoing orthodontic treatment of 20 impacted teeth. In 10 patients (population A) surgical exposure of the impacted teeth was performed using a 980 nm diode laser, while in the other 10 patients (population B), surgical incision was performed using a traditional lancet.Only 3 patients of the population A needed local anesthesia for surgical procedure while the remaining 7 patients reported only faint pain during surgery. Two patients referred postsurgical pain (numerical rating scale average value = 2) and needed to take analgesics. None of the patients showed other postsurgical side effects (bleeding, edema).All population B patients needed infiltrative anesthesia and referred postsurgical pain (numerical rating scale average value >4) treated with analgesics. Moreover, in such population, 4 patients referred lips edema while 4 showed bleeding and 6 needed surgical sutures of soft tissues.The lack of side effects of laser surgical approach to expose impacted teeth must persuade dental practitioners to choose such a clinical approach to closed surgical approach every time it is possible.

Melanoma is one of the most lethal tumors among the skin cancers, arising from complex genetic mutations in melanocyte. Melanoma microenvironment is very heterogeneous, showing complex vascular networks and immunogenicity, as well as induced acquired resistance to treatments by upregulation of multidrug resistance (MDR) mechanisms. Different studies have showed that Photodynamic Therapy (PDT) could be considered a new potential approach for melanoma treatment. PDT combines a light with a specific wavelength and a photosensitizer: when these two elements interact reactive oxygen species (ROS) are generated leading to tumor cell destruction. In this study verteporfin (Ver), a second-generation photosensitizer, has been conjugated with mesoporous silica nanoparticles (MSNs): the resulting Ver-MSNs are an efficient nanoplatforms used to enhance cargo capacity and cellular uptake. Our in vitro and in vivo studies investigated whether Ver-MSNs were able to reduce or inhibit melanoma growth. In vitro experiments performed using B16F10 mouse melanoma cells showed that Ver-MSNs stimulated by red light (693 nm) significantly decreased in vitro cells proliferation in a range of concentration between 0.1 μg/ml to 10 μg/ml. When Ver-MSNs (5 μg/ml in glycerol) were topically administrated to melanoma tumor mass developed in mice and stimulated by red light for four times in 16 days, they were able to reduce the tumor mass of 50.2 ± 6,6% compared to the untreated (only glycerol) mice. In the light of this information, PDT performed using Ver-MSNs could be considered a new promising and potential approach to treat melanoma.

The deposition of organic functionalities on biomaterials to immobilize biomolecules is a research area of great interest in the medical field. The surface functionalization of a 3D porous scaffolds of PDLLA with carboxyl (-COOH) and amino (-NH2) groups by cold plasma treatment at atmospheric pressure is described in this paper. Two methods of continuous and pulsed plasma deposition were compared to assess the degree of functionalization of the internal porous 3D scaffold. In particular, the pulsed plasma treatment was found to functionalize uniformly not only the sample surface but also inside the open cavities thanks to its permeability and diffusion in the porous 3D scaffold. The species developed in the plasma were studied by optical emission spectroscopy (OES) technique, while the functionalization of the sponges was evaluated by the Diffuse Reflectance Fourier-Transform Infrared Spectroscopy (DR-FTIR) technique using also the adsorption of ammonia (NH3) and deuterated water (D2O) probe molecules. The functional groups were deposited only on the front of the sponge, then the structural characterization of both front and back of the sponge has demonstrated the uniform functionalization of the entire scaffold.

Background: Physical exercise and nutrition seem to have a key role in the management of hip fracture patients. Aim: To evaluate the impact of a 2-month rehabilitative protocol combined with dietetic counseling, with or without essential amino acid supplementation, on functioning in hip fracture patients. Methods: In this pilot randomized controlled study, we recruited patients aged more than 65 years, at 3 months after hip fracture. We randomly assigned the participants into two groups (A and B). Both groups performed a physical exercise rehabilitative programme (five sessions of 40 min/week for 2 weeks, followed by a home-based exercise protocol) and received a dietetic counseling; only group A was supplemented with two sachets of 4 g/day of essential amino acids (Aminotrofic®). We evaluated at baseline and after 2 months of intervention (T1): hand grip strength, Timed Up and Go, and Iowa Level of Assistance scale (ILOA). Results: The 32 hip fracture patients (mean aged 79.03 ± 7.80 years) were allocated into two groups: group A (n = 16) and group B (n = 16). All the participants showed significant differences in all outcomes at T1 (p < 0.017). Sarcopenic patients in group A (n = 10) showed statistically significant differences in all the primary outcomes at T1 (p < 0.017), whereas sarcopenic patients in group B (n = 13) showed a significant reduction of ILOA only. In non-sarcopenic patients, we found no differences at T1 in all outcome measures. Discussion: Hip fractures are a complex multifactorial condition of the elderly that determines devastating effects on functioning and independence. Conclusion: A multidisciplinary rehabilitative and nutritional intervention seems to be effective on functioning in hip fracture patients, in particular sarcopenic ones.

A highly efficient nanoplatform for photodynamic therapy was obtained by covalent conjugation of verteporfin with mesoporous silica nanoparticles. Verteporfin has an intense absorption band in the red region of the Vis spectrum ensuring a deeper tissue penetration; this feature makes verteporfin-based formulations particularly attractive for in vivo PDT applications. The verteporfin loading, dispersion and efficiency in the photoproduction and delivery of singlet oxygen (1O2) were carefully evaluated. In vitro tests have evidenced that the optimized nanoplatform is able to largely reduce HeLa cell proliferation after red-light irradiation.

Recently, Vitamin D (Vit. D) has gained importance in cellular functions of a wide range of extraskeletal organs and target tissues, other than bone. In particular, Vit. D has displayed important beneficial effects in the cardiovascular system. Although little is known about the mechanism by which this response is exerted, a Vit. D-induced eNOS-dependent nitric oxide (NO) production in endothelial cells (EC) has been reported. The aim of this study was to evaluate whether Vit. D administration could affect human EC proliferation and/or migration through NO production. For this purpose, HUVEC (human umbilical vein endothelial cells) were used to evaluate Vit. D effects on cell proliferation and migration in a 3D matrix. Experiments were also performed in the presence of the specific VDR ligand ZK159222 and eNOS inhibitor L-NAME. This study demonstrated that Vit. D can promote both HUVEC proliferation and migration in a 3D matrix. These effects were NO dependent, since HUVEC proliferation and migration were abrogated along with Vit. D induced MMP-2 expression by inhibiting eNOS activity by L-NAME. These findings support the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in tissue repair and wound healing.

Case-control study.To assess serum myostatin levels, bone mineral density (BMD), appendicular skeletal muscle mass (ASMM) and serum sclerostin levels in chronic spinal cord injured (SCI) patients and healthy controls.SCI centre in Italy.Blood samples, whole-body bioelectrical impedance analysis and BMD measurement with the ultrasound technique at the calcaneus level were taken from patients suffering from chronic SCI (both motor complete and incomplete) and healthy control subjects.A total of 28 SCI patients and 15 healthy controls were enrolled. Serum myostatin levels were statistically higher (P0.01) in SCI patients compared with healthy controls. Similar results were found comparing both the motor complete and the motor incomplete SCI subgroups to healthy controls. Serum sclerostin was significantly higher in patients with SCI compared with healthy controls (P0.01). BMD, stiffness and mean T-score values in SCI patients were significantly lower than those in healthy controls. Serum myostatin concentrations in the motor complete SCI subgroups correlated only with serum sclerostin levels (r(2)=0.42; P=0.001) and ASMM (r(2)=0.70; P=0.002) but not in healthy controls.Serum myostatin and serum sclerostin are significantly higher in chronic SCI patients compared with healthy controls. They are potential biomarkers of muscle and bone modifications after SCI. This is the first study reporting an increase in serum myostatin in patients suffering from chronic SCI and a correlation with ASMM.

Photobiomodulation is a widely used tool in regenerative medicine thanks to its ability to modulate a plethora of physiological responses. Wound re-epithelialization is strictly regulated by locally produced chemical mediators, such as nitric oxide (NO), a highly reactive free radical generated by the nitric oxide synthase (NOS) enzymatic family. In this study, it has been hypothesized that a 980-nm low-level laser stimulation could increase NO production in human keratinocytes and that such event might be directly related to the re-epithelialization process. Human keratinocytes were irradiated with increasing energy outputs (10–75 J) in the absence or presence of L-NAME, a NOS inhibitor. Laser stimulation induced an increase in NO production, resulting in an energy-dependent increase in both keratinocytes proliferation and re-epithelialization ability. The direct link between increased NO production and the observed physiological responses was confirmed by their inhibition in L-NAME pre-treated samples. Since NO production increase is a quick event, it is conceivable that it is due to an increase in existing NOS activity rather than to a de novo protein synthesis. For this reason, it could be hypothesized that photobiomodulation-derived NO positive effects on keratinocytes behavior might rely on a near infrared mediated increase in NOS conformational stability and cofactors as well as substrate binding ability, finally resulting in an increased enzymatic activity.

Osteonecrosis of the jaws (ONJ) in osteoporosis patients has been defined as rare, but the number of reported cases is increasing. We report a case of delayed ONJ diagnosis in a patient, who was being treated with alendronate, developing bone alterations both in maxilla and in mandible. Underestimation of ONJ incidence and missed or delayed ONJ diagnosis in osteoporosis patients might derive from lack of awareness of health providers as well as from an ONJ definition that is too restricted. The more recent definition of medication-related osteonecrosis of the jaws (MRONJ) released in 2014 by the American Association of Oral Maxillofacial Surgeons (AAOMS) accept fistula, besides bone exposure, as a major sign of disease, but it seems to be insufficient since it excludes all cases of ONJ disease without bone exposure. A new MRONJ definition is needed to avoid missing or delayed diagnosis.

Organically modified mesoporous silica nano- particles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen ( 1 O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.

Low-level laser therapy (LLLT) is widely used in regenerative medicine and in dental therapy by virtue of its beneficial effects in a plethora of pathological conditions. In this study, the effect of a 980 nm diode laser on pre-osteoblasts proliferation has been evaluated, along with reactive oxygen species (ROS) production. We hypothesized that ROS were a key factor in LLLT-induced pre-osteoblasts proliferation, as it is known that ROS can induce the activation of many biological pathways, leading to cell proliferation, differentiation or apoptosis. Murine pre-osteoblasts MC3T3 cells were irradiated with different energy outputs (1-50 J) in the absence or presence of the antioxidant N-Acetyl-L-cysteine (NAC). Laser treatment, in the absence of NAC, was able to induce a fluence-dependent statistically significant increase in ROS generation, while the presence of NAC strongly inhibited it. Cell proliferation, measured after laser stimulation, was significantly increased both at low and higher energy, with a peak at 10 J in the absence of the antioxidant. On the contrary, in the presence of NAC, laser irradiation was not able to induce any cell proliferation, suggesting a crucial role of ROS in this laser-induced cell effect. These results suggest that LLLT may be a useful tool for bone regeneration therapy and an effective range of fluences to be used is indicated.

Background/Aims: The 1α,25-dihydroxycholecalciferol (Vit. D) induces eNOS dependent nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). To our knowledge, there are no reports directly relating Vit. D induced NO production to proliferation and/or migration in endothelial cells (EC). The aim of this study was to evaluate whether Vit. D addition to porcine EC could affect their proliferation and/or migration in a three-dimensional matrix via NO production. Materials and Methods: Porcine aortic endothelial cells (PAE) were used to evaluate Vit. D effects on cell proliferation and migration in a three-dimensional matrix. Results: Vit. D induced NO production in PAE cells. Moreover, it induced a significant increase in cellular proliferation and migration in a three-dimensional matrix. These effects were NO dependent, as inhibiting eNOS activity by L-NAME PAE migration was abrogated. This effect was strictly related to MMP-2 expression and apparently dependent on Vit. D and NO production. Conclusions: Vit. D can promote both endothelial cells proliferation and migration in a three-dimensional matrix via NO-dependent mechanisms. These findings cast new light on the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in such fields as tissue repair and wound healing.

Aim: To evaluate, in an in vitro wound healing model, the effect of zoledronate on keratinocyte cellular behavior. -Materials and Methods: Human spontaneously immortalized keratinocytes (HaCaT) were treated with low zoledronate concentrations (10 nmol/l to 10 µmol/l) and its effect on cell proliferation was evaluated by means of a fluorescent assay (Tox-8), along with the analysis of cytokeratin 5 and filaggrin gene expression. Moreover, zoledronate effects on cell migration were evaluated by in vitro wound healing, and matrix metalloproteinase (MMP) activity was investigated by gelatin zymography. Results: At the tested concentrations, zoledronate stimulated keratinocyte proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression and wound healing ability, without any significant effect on MMP-9 activity. The lack of zoledronate effects on MMP-9 activity indicates that, in our experimental model, wound closure is mainly due to an increase in cell proliferation rather than an increase in cell migration. Moreover, the observed increase in cell proliferation could be ascribed to a zoledronate-mediated reduction of farnesyl pyrophosphate endogenous levels. Conclusions: These results could foster new clinical applications for this ‘old' drug in the field of epithelial regeneration.

Biocompatible and resorbable polylactic acid (PLA) derived polymers are used for clinical applications. Antioxidant vitamin E (Vit.E) and vitamin E acetate (Vit.E Ac), a Vit.E commercial analog, were added to PLA during films preparation. Wettability, protein adsorption analysis, platelet adhesion and whole blood coagulation experiments highlighted that Vit.E incorporation improved PLA hemocompatibility. NMR analysis showed that Vit.E and Vit.E Ac blending differentially altered PLA polymer organization with Vit.E leading to the formation of microcavities, as observed by SEM analysis, while reducing surface hydrophobicity. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Novel hydrogel composites based on natural gelatin matrix and containing different loading of a water soluble octa-am- monium POSS (POSS-NH3 þ ), (from 0.5 to 2 wt%) have been prepared in this work and their biocompatibility and mechanical behavior have been tested. The presence of 1 wt% of POSS-NH3 þ molecules in the hydrogel matrix increased the amount of human plasma proteins adsorption and human keratinocyte adhesion at 24 hours. In addition, the presence of increasing amounts of POSS in the hydrogel matrix allowed to a significantly increase of the matrix swelling degree at acid and neutral pH. Finally, a significant modulation of the mechanical characteristics (Young modulus, strain at break) of the hydrogel matrix induced by the presence of dif- ferent amounts of POSS-NH3 þ was observed. The novel ability of POSS-NH3 þ to modulate both biological and mechanical proper- ties of a natural hydrogel could be useful for the production of new composites of interest for soft tissue engineering. V C 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 129: 699-706, 2013

Our skin is in close contact with clothes most of the time thus risking potentially noxious chemicals contact. One of the potentially harmful manufacturing by-products that can be released by textiles when sweating is formaldehyde, used as an anti-crease treatment. As it is known to be carcinogenic to humans and a potent skin sensitizer, the aim of this study was to investigate its effects on both normal human keratinocytes (HaCaT cells) and on a highly invasive malignant melanoma cell line (SK-MEL-28) in order to contribute to the definition of safety cut-off to be applied to the production processes. Formaldehyde concentrations below the commonly accepted limits (10-50μM) were obtained by diluting formaldehyde in simulated sweat (UNI EN ISO 105-E04). The effects on cell proliferation were evaluated by cell counting, while ERK pathway activation was evaluated by western blot. Low concentrations of formaldehyde (10μM) in both acidic and alkaline simulated sweat were able to increase malignant melanoma cell proliferation, while not affecting normal keratinocytes. Melanoma proliferation increase was greater in acidic (pH=5.5) than in alkaline (pH=8) conditions. Moreover, formaldehyde stimulation was able to induce ERK pathway activation. The data obtained suggest the need for an even increasing attention to the potentially harmful effects of textile manufacturing by-products.

This in vitro experimental study has been designed to assess the effects of photodynamic antimicrobial chemotherapy (PACT) on human keratinocytes proliferation. Human keratinocytes (HaCaT) monolayers (∼0.5 cm(2)) have been irradiated with 635 nm red laser light with a fluence of 82.5 or 112.5 J/cm(2) in the absence or presence of toluidine (TB). Cell proliferation, monolayer area coverage, cytokeratin 5 (K5) and filaggrin (Fil) expression, and metalloproteinase (MMP)-2 and MMP-9 activity were measured after 72 h from laser irradiation. HaCaT proliferation was reduced by TB staining. Cell exposure to both low- and high-fluence laser irradiation in both presence and absence of TB staining reduced their proliferation and monolayer area extension. Moreover both laser treatments were able to reduce K5 and Fil expression and MMP-9 production in keratinocytes not treated with TB. These data indicate that PACT could exert toxic effects on normal proliferating keratinocytes present around parodontal pockets. The observed reduced cell proliferation along with a reduced production of enzymes involved in wound healing could alter the clinical outcome of the patients treated with PACT.

Ionic hydrogels are biocompatible interesting candidates for tissue-engineering applications, such as the creation of artificial skin, as they can also be used, along with growth factors and cells grown in vitro, for developing bioengineered tissues to be implanted. Among the growth factors that can be used to induce keratinocytes growth in vitro, epiregulin, a broad-specificity epidermal growth factor (EGF) family member, has been shown to be more effective than EGF and transforming growth factor-alpha (TGF-α) in promoting re-epithelization in vitro. To produce a drug-delivery hydrogel for epiregulin, bovine gelatin was cross-linked with poly(glutamic acid) (PLG) in the presence of epiregulin (5-50 ng/ml). Spontaneously immortalized human keratinocytes (HaCaT) were seeded on unloaded and epiregulin-loaded hydrogels and cell adhesion was evaluated after 6 h. Moreover, cell proliferation and stratification, cytokeratins (K5, K10), differentiation markers (filaggrin and transglutaminase-1 (TG-1)) and matrix metalloproteinases (MMP-2, MMP-9 and MMP-28) expression were evaluated after 7 days. The presence of epiregulin induced an increase in cell proliferation, stratification and K5 expression along with MMP-9 and MMP-28 expression, while all differentiation markers expression (K10, filaggrin, TG-1) was decreased. These data indicated that a simple hydrogel loaded with epiregulin could be an effective tool for skin tissue engineering.

Polylactic acid (PLA)-derived polymers are widely used in many biotechnological fields, thanks to their biocompatibility and resorbability through natural pathways. Moreover, PLA is one of the few polymers in which both the properties and the stereochemical structure can be easily modified, making possible a specific ‘tailoring’ of the final polymer according to the desired use. In this study, we obtained, by salt leaching technique, P(d,l)LA sponges in which various concentrations of α-tocopherol (Vit. E 10–40%, w/w) were incorporated. Vit.E is a natural biological antioxidant, also known to have anti-inflammatory activity, which has been extensively used to improve biostability and biocompatibility of different biomaterials. To assess whether Vit.E could modify the main physical–chemical properties of P(d,l)LA sponges, their morphology, water uptake and hydrolytic degradation kinetics, along with protein loading and releasing attitudes, were investigated. Our results highlighted that incorporation of Vit.E into P(d,l)LA sponges modified the sponge morphology, decreased P(d,l)LA water uptake and degradation, and modified protein releasing kinetics. These Vit.E-related effects could make P(d,l)LA more suitable as drug delivery system and tissue engineering scaffold.

J Oral Pathol Med (2011) 40: 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn-dependent enzyme involved in cell migration. Among them, gelatinase B (MMP-9) and epilysin (MMP-28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP-28 expression has been analyzed in epithelium sections using an anti-MMP-28 antibody, whereas MMP-9 presence and activity has been measured in cell-conditioned medium analyzed by gelatine zymography. The expression of MMP-9 was reduced by Nic in a dose-dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP-28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP-9 and MMP-28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.

Lasers devices are widely used in various medical fields (eg, surgery, dermatology, dentistry, rehabilitative medicine, etc.) for different applications, ranging from surgical ablation of tissues to biostimulation and pain relief. Laser is an electromagnetic radiation, which effects on biological tissues strongly depends on a number of physical parameters. Laser wavelength, energy output, irradiation time and modality, temperature and tissue penetration properties have to be set up according to the clinical target tissue and the desired effect. A less than optimal operational settings, in fact, could result in a null or even lethal effect. According to the first law of photobiology, light absorption requires the presence of a specific photoacceptor that after excitation could induce the activation of downstream signaling pathways. Low-level lasers operating in the red/near infrared portion of the light spectra are generally used for biostimulation purposes, a particular therapeutic application based on the radiant energy ability to induce nonthermal responses in living cells. Biostimulation process generally promotes cell survival and proliferation. Emerging evidences support a low-level laser stimulation mediated increase in "good" reactive oxygen species, able to activate redox sensitive signal transduction pathways such as Nrf-2, NF-kB, ERK which act as key redox checkpoints.