A new method for oral cancer biomarkers detection with a non-invasive cyto-salivary sampling and rapid-highly sensitive ELISA immunoassay: a pilot study in humans.

Introduction: Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies and has a 5-year mortality rate close to 50%. A consistent part (70%) of all oral cancers is diagnosed at an advanced stage since available screening techniques are ineffective. Therefore, it would be urgent to improve them. The diagnostic gold standard is tissue biopsy with histological and immunohistochemical assessment. This method presents some limitations. Biopsy is invasive and the histopathological evaluation is semi-quantitative, and the absolute abundance of the target cannot be reliably determined. In addition, tissue is highly processed and may lead to loss of information of the natural state. The search for classical and new clinical biomarkers on fragments of tissue/cells collected with a cytobrush is a highly hopeful technique for early detection and diagnosis of OSCC, because of its non-invasive sampling and easy collection method. Methods: Here we analyzed cytobrush biopsies samples collected from the oral cavity of 15 patients with already diagnosed OSCC by applying an innovative high-sensitivity ELISA technique, in order to verify if this approach may provide useful information for detection, diagnosis, and prognosis of OSCC. To this end, we selected six biomarkers, already used in clinical practice for the diagnosis of OSCC (EGFR, Ki67, p53) or selected based on recent scientific and clinical data which indicate their presence or over-expression in cells undergoing transformation and their role as possible molecular targets in immunecheckpoints blockade therapies (PD-L1, HLA-E, B7-H6). Results: The selected tumor biomarkers were highly expressed in the tumor core, while were virtually negative in healthy tissue collected from the same patients. These differences were highly statistically significant and consistent with those obtained using the gold standard test clearly indicating that the proposed approach, i.e. analysis of biomarkers by a custom ELISA technique, is strongly reliable. Discussion: These preliminary data suggest that this non-invasive rapid phenotyping technique could be useful as a screening tool for phenotyping oral lesions and support clinical practice by precise indications on the characteristics of the lesion, also with a view to the application of new anti-tumor treatments, such as immunotherapy, aimed at OSCC patients.

An ELISA Test Able to Predict the Development of Oral Cancer: The Significance of the Interplay between Steroid Receptors and the EGF Receptor for Early Diagnosis.

Oral disorders including non-homogeneous leukoplakia, erythroplakia, erosive lichen planus, and many others can potentially progress to oral squamous cell carcinoma (OSCC). Currently, the late diagnosis of OSCC contributes to high mortality rates, emphasizing the need for specific markers and early intervention. In this study, we present a novel, quick, sensitive, and non-invasive method for the early detection and screening of oral cancer, enabling the qualitative assessment of neoplastic forms even before the onset of symptoms. Our method directly examines the expression of oral cancer biomarkers, such as the epithelial growth factor receptor (EGFR), and steroid receptors, including the androgen receptor (AR) and the estrogen receptor (ER). The crosstalk between sexual hormones and the EGF receptor plays a crucial role in the progression of different types of cancers, including head and neck squamous cell carcinoma. To implement our method, we developed a kit box comprising nine wells or stations, each containing buffers, lysis systems, and dried/lyophilized antibodies stored at room temperature. The kit includes instruments for sample collection and a PVDF strip (Immobilon) with specific primary antibodies immobilized on it. These antibodies capture the target proteins from cytological samples. Additionally, complementary tools are provided to ensure efficient utilization and optimal test performance. The technique can be performed outside the laboratory, either "patient side" with an instant chemocolorimetric response or with a digital reader utilizing the enzyme-linked immunosorbent assay (ELISA) method.

Comparative study of the anaesthetic efficacy of 4% articaine versus 2% mepivacaine in mandibular third molar germectomy using different anaesthetic techniques: a split-mouth clinical trial.

BACKGROUND: Currently, one of the most discouraging aspects for many patients undergoing dental procedures is the administration of local anaesthesia. Therefore, there is a constant search for new techniques to avoid the invasive and painful nature of the injection. This study aimed to compare the clinical efficacy of local anaesthetics with articaine 4% or mepivacaine 2% (both with epinephrine 1:100.000), using different anaesthetic techniques to perform germectomy of lower third molars and to assess patients' feelings and pain during surgery. METHODS: Totally 50 patients (ranged 11-16 years) who required germectomy of mandibular third molars were recruited. Each patient received local anaesthesia on one side with articaine inoculated with plexus technique while on the other side with mepivacaine using inferior alveolar nerve block technique. The patients' evaluation was performed on pre and intraoperative tactile-pressure feelings and intraoperative pain with four levels on the Visual Analogue Scale (VAS). RESULTS: Surgical operations lasted less with more efficient analgesia when articaine was used. The additional intraosseous injection was required mainly in the mepivacaine group intraoperatively. A few patients had tactile-pressure feelings while intraoperative pain sensation was absent in 90% of cases with articaine. Significant differences were found in the cases who reported "absent" and "moderate" VAS values, favoring the use of articaine. CONCLUSIONS: Articaine injected with a plexus anaesthetic technique seems to be more clinically manageable than mepivacaine for the mandibular third molar germectomy. The discomfort of tactile-pressure feelings and pain experienced was lower using articaine anaesthetic technique used.

Diagnostic Accuracy of a New Antigen Test for SARS-CoV-2 Detection.

BACKGROUND AND AIMS: Quick and reliable diagnostic tools play an important role in controlling the spread of the SARS-Cov-2 pandemic. The aim of this study was to evaluate the diagnostic accuracy of a new cyto-salivary antigen test aimed at detecting the presence of antigens for SARS-CoV-2, as compared by the gold standard RT-PCR and a lateral flow test. METHODS: A total of 433 healthy volunteers were enrolled in the study and the sensitivity and specificity of the new cyto-salivary antigen test were calculated, as compared to the RT-PCR nasopharyngeal swab and to the lateral flow test. RESULTS: A total of 433 samples were collected and tested at the Mediterranean Fair in Palermo from February 2021 until April 2021. The new cyto-salivary antigen had a sensitivity of 100% and a specificity of 94.2%. The sensitivity and the specificity of the lateral flow test were 55% and 100%, respectively. CONCLUSIONS: The new cyto-salivary antigen test detected more positive cases than the RT-PCR in a sample of asymptomatic subjects, demonstrating to be a promising tool for a more sensitive diagnosis of COVID-19. Further studies are warranted to better characterize its diagnostic accuracy.

Titanium Functionalized with Polylysine Homopolymers: In Vitro Enhancement of Cells Growth.

In oral implantology, the success and persistence of dental implants over time are guaranteed by the bone formation around the implant fixture and by the integrity of the peri-implant mucosa seal, which adheres to the abutment and becomes a barrier that hinders bacterial penetration and colonization close to the outer parts of the implant. Research is constantly engaged in looking for substances to coat the titanium surface that guarantees the formation and persistence of the peri-implant bone, as well as the integrity of the mucous perimeter surrounding the implant crown. The present study aimed to evaluate in vitro the effects of a titanium surface coated with polylysine homopolymers on the cell growth of dental pulp stem cells and keratinocytes to establish the potential clinical application. The results reported an increase in cell growth for both cellular types cultured with polylysine-coated titanium compared to cultures without titanium and those without coating. These preliminary data suggest the usefulness of polylysine coating not only for enhancing osteoinduction but also to speed the post-surgery mucosal healings, guarantee appropriate peri-implant epithelial seals, and protect the fixture against bacterial penetration, which is responsible for compromising the implant survival.

Detection of SARS-COV-2 Proteins Using an ELISA Test.

The coronavirus disease 2019 (COVID-19) global pandemic created an unprecedented public health emergency. Early recognition of an infected person and disruption of the transmission pathway are the keys to controlling this major public health threat around the world. The scientifically reliable screening method is an RT-PCR test that is performed on an ororhinopharyngeal swab in the laboratory. In the current severe SARS-CoV-2 pandemic, it is necessary to identify devices for rapid diagnosis to reduce the spread of the disease. The aim of this study was to provide a qualitative, rapid, sensitive, and specific method for a diagnosis of SARS-CoV-2 infection based on the recognition of specific antigens of the SARS-CoV-2 virus. The device was built by assembling commercially available and custom-made semi-finished products. The method was performed in environments outside the laboratory, i.e., "patient side," with an immediate chemocolorimetric response or with a digital reader using an ELISA method.

Comparative Analysis of the Chemical Composition and Microstructure Conformation Between Different Dental Implant Bone Drills.

BACKGROUND: Hardness is considered an important parameter for evaluating the clinical performance of dental implant bone drills. It is connected to the chemical composition, microstructure conformation and manufacture of the surgical drills. METHODS: Microstructure of five dental implant drills using scanning electronic microscopy (SEM) integrated with energy dispersive X-ray spectrometry. Vickers microhardness was measured using a CV 2000 microhardness tester with an indentation force of 500 g. RESULTS: Composition of the implant drills was typical of martensitic stainless steel (MSS). The drills contained 13%-17% of Cr; Mo, Si and Mn were present as minor ligands. The examined bone drills showed different external surface conformation and hardness in relation to the different industrial production processes. A rougher external surface and a higher hardness value are characteristics of the surgical bone drills produced by hot forming; the implant drills produced by machining showed mailing lines on their external surface and a lower hardness. CONCLUSIONS: Different compositions and treatments were used by the manufacturers to improve the hardness of the external layer of the dental implant drills making them prone to a diverse heat generation during the implant site preparation.

A New Controlled-Release Material Containing Metronidazole and Doxycycline for the Treatment of Periodontal and Peri-Implant Diseases: Formulation and In Vitro Testing.

BACKGROUND: Several locally administered antimicrobials have been studied in the literature as adjunctive or primary treatments for periodontitis and peri-implantitis with conflicting results. OBJECTIVE: The aim of this study was twofold: (1) the formulation of a controlled-release material containing metronidazole and doxycycline; (2) an in vitro evaluation of its antibacterial properties against planktonic and biofilm species involved in periodontal and peri-implant diseases. METHODS: Doxycycline (10 mg/ml) and metronidazole (20 mg/ml) were incorporated into a hydroxyethylcellulose-polyvinylpyrrolidone-calcium polycarbophil gel. Three milliliters of gel were dialyzed against Dulbecco's phosphate-buffered saline for 13 days. Antibiotics release at 3, 7, 10, and 13 days was determined spectroscopically. The inhibitory activity of the experimental gel was tested against A. actinomycetemcomitans, S. sanguinis, P. micra, and E. corrodens with an agar diffusion test, an inactivation biofilm test, and a confocal laser scanning microscope study (CLSMS) for S. sanguinis up to 20 days. RESULTS: After 13 days, the released doxycycline was 9.7% (at 3 days = 1.2 mg; 7 days = 0.67 mg; 10 days = 0.76 mg; 13 days = 0.29 mg), while metronidazole was 67% (30 mg, 6.8 mg, 2.5 mg, and 0.9 mg at the same intervals). The agar diffusion test highlights that the formulated gel was active against tested microorganisms up to 312 h. Quantitative analysis of biofilm formation for all strains and CLSMS for S. sanguinis showed a high growth reduction up to 13 days. CONCLUSIONS: The in vitro efficacy of the newly formulated gel was confirmed both on planktonic species and on bacterial biofilm over a period of 13 days. The controlled-release gel containing metronidazole and doxycycline had an optimal final viscosity and mucoadhesive properties. It can be argued that its employment could be useful for the treatment of periodontal and peri-implant diseases, where conventional therapy seems not successful.

Non-invasive analysis of bleaching effect of hydrogen peroxide on enamel by reflectance confocal microscopy (RCM): study of series of cases.

The aim of this study is to evaluate in vivo the effects of in-office tooth whitening hydrogen peroxide (HP) agent on enamel-microstructured surface by a reflectance confocal microscopy (RCM). Ten healthy volunteers assisted at the Dental School presenting teeth with vital pulp were selected. The 35% HP whiteness product was applied in two visits on discolored teeth, 1-week interval between, via 20-min applications. A commercially available hand-held RCM (Vivascope3000®, Lucid, Rochester, NY, USA) was used to image in vivo the dental surface of the selected tooth of each volunteer. Twenty upper central incisors' vestibular surfaces were imaged, before bleaching (T0), immediately after (T1) and 1 week later (T2). The peculiar structure of the enamel was seen at T0. After bleaching, white reflective circular bodies were found all over the teeth surfaces, which disappear 1 week later (T2). When the HP gel® was imaged, the same white circular areas were observed. Going deeper, the regular enamel architecture was preserved. Textural analysis of the images in T0 and T2 was performed: GLCM parameters were extracted. Mann-Whitney U test was performed to evaluate statistical differences between two groups of data (p > 0.05). Finally, 35 prisms were randomly selected from T0 and T2 image and diameters were measured; a paired t test was performed (p = 0.381). The RCM is a promisor tool for investigating the features of enamel in vivo, immediately after bleaching procedures, as well as longitudinally.

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering.

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration.

Human adipose stem cell differentiation is highly affected by cancer cells both in vitro and in vivo: implication for autologous fat grafting.

Recent studies showed that mesenchymal stem cells derived from adipose tissue can promote tumour progression, raising some concerns regarding their use in regenerative medicine. In this context, we co-cultured either SAOS2 osteosarcoma or MCF7 breast cancer cells with human adipose stem cells (hASCs), in order to evaluate potential effects of cancer cells on hASCs differentiation, in vitro and in vivo. In this study we observed that both SAOS2 and MCF7 cell lines induced an increase in hASCs proliferation, compared to hASCs alone, but, surprisingly, neither changes in the expression of CD90, CD29, CD324 and vimentin, nor variations in the Twist and Slug mRNAs were detectable. Noteworthy, SAOS2 and MCF7 cells induced in hASCs an upregulation of CD34 expression and stemness genes, including OCT3/4, Nanog, Sox2 and leptin, and a decrease in angiogenic factors, including CD31, PDGFα, PDGFRα, PDGFRß and VEGF. SMAD and pSMAD2/3 increased only in hASCs alone. After 21 days of co-culture, hASCs differentiated both in adipocytes and endothelial cells. Moreover, co-injection of MCF7 cells with hASCs led to the formation of a highly vascularized tumour. Taken together our findings suggest that mesenchymal stem cells, under tumour cell induction, do not differentiate in vitro or facilitate the angiogenesis of the tumour in vivo, thus opening interesting new scenarios in the relationship between cancer and stem cells. These findings may also lead to greater caution, when managing autologous fat grafts in cancer patients.

Prevalence and associated risk factors of latent tuberculosis infection among undergraduate and postgraduate dental students: A retrospective study.

To estimate the prevalence of latent tuberculosis (TB) infection (LTBI) in Italian dental students exposed to the same occupational risks as dental health care personnel and to evaluate potential risk factors, a cross-sectional study was conducted on undergraduate and postgraduate students. After clinical evaluation, students were given a tuberculin skin test; in those found positive, an interferon-γ release assay (IGRA) was conducted. Of the 281 students enrolled, 10 were only TST positive; 8 were TST or/and IGRA positive. We found that participants testing positive at TST and/or IGRA, a group in which the risk of false LTBI positives is minimal, were older and had been studying longer. Although the prevalence of LTBI among dental students in our study was low, a risk of acquiring a work-related infection exists even in a country with a low incidence of TB. Thus, dental students should be screened to catch LTBI early on.

NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression.

Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact.

Changing Paradigms in Cranio-Facial Regeneration: Current and New Strategies for the Activation of Endogenous Stem Cells.

Craniofacial area represent a unique district of human body characterized by a very high complexity of tissues, innervation and vascularization, and being deputed to many fundamental function such as eating, speech, expression of emotions, delivery of sensations such as taste, sight, and earing. For this reasons, tissue loss in this area following trauma or for example oncologic resection, have a tremendous impact on patients' quality of life. In the last 20 years regenerative medicine has emerged as one of the most promising approach to solve problem related to trauma, tissue loss, organ failure etc. One of the most powerful tools to be used for tissue regeneration is represented by stem cells, which have been successfully implanted in different tissue/organs with exciting results. Nevertheless, both autologous and allogeneic stem cell transplantation raise many practical and ethical concerns that make this approach very difficult to apply in clinical practice. For this reason different cell free approaches have been developed aiming to the mobilization, recruitment, and activation of endogenous stem cells into the injury site avoiding exogenous cells implant but instead stimulating patients' own stem cells to repair the lesion. To this aim many strategies have been used including functionalized bioscaffold, controlled release of stem cell chemoattractants, growth factors, BMPs, Platelet-Rich-Plasma, and other new strategies such as ultrasound wave and laser are just being proposed. Here we review all the current and new strategies used for activation and mobilization of endogenous stem cells in the regeneration of craniofacial tissue.

Erratum to: Vaccination against hepatitis b virus: are Italian medical students sufficiently protected after the public vaccination programme?

[This corrects the article DOI: 10.1186/s12995-015-0083-4.].

Vaccination against hepatitis b virus: are Italian medical students sufficiently protected after the public vaccination programme?

BACKGROUND: The development of a vaccine against hepatitis B virus (HBV) has been a major achievement in terms of prevention of HBV infection. For the present study, we analysed the long-term immunogenicity and effectiveness of HBV vaccination among healthcare students with different working seniorities. METHODS: A cross-sectional study of undergraduate and postgraduate students attending the Medical School of the Second University of Naples was conducted between September 2012 and December 2014. HBV serum markers were determined and multivariate logistic regression analysis was used to identify factors associated with the level of long-term immunogenicity. RESULTS: Of the 2,932 subjects evaluated, only 33 (1.1 %) declared no history of vaccination. All vaccinated subjects were HBsAg/anti-HBc negative, 459 of which had an anti-HBs titre <10 IU/L. The latter were younger, more likely to be attending a healthcare profession school (i.e., dental hygienists, nursing, paediatric nursing, radiography and midwifery) than a medical school (at either undergraduate or postgraduate level) and more likely to have been vaccinated in infancy. CONCLUSION: The results of this study suggest that assessment of HBV serum markers in workers potentially exposed to hospital infections is useful to identify small numbers of unvaccinated subjects or vaccinated subjects with low antibody titre, all of whom should be referred to a booster series of vaccinations.

Surface biocompatibility of differently textured titanium implants with mesenchymal stem cells.

OBJECTIVE: The major challenge for contemporary dentistry is restoration of missing teeth; currently, dental implantation is the treatment of choice in this circumstance. In the present study, we assessed the interaction between implants and Dental Pulp Stem Cells (DPSCs) in vitro by means of 3D cell culture in order to better simulate physiological conditions. METHODS: Sorted CD34+ DPSCs were seeded onto dental implants having either a rough surface (TriVent) or one coated with a ceramic layer mimicking native bone (TiUnite). We evaluated preservation of DPSC viability during osteogenic differentiation by an MTT assay and compared mineralized matrix deposition with SEM analysis and histological staining; temporal expression of osteogenic markers was evaluated by RT-PCR and ELISA. RESULTS: Both surfaces are equally biocompatible, preserve DPSC viability, stimulate osteogenic differentiation, and increase the production of VEGF. A slight difference was observed between the two surfaces concerning the speed of DPSC differentiation. SIGNIFICANCE: Our study of the two implant surfaces suggests that TriVent, with its roughness, is capable of promoting cell differentiation a bit earlier than the TiUnite surface, although the latter promotes greater cell proliferation.

Comparison between rapid and mixed maxillary expansion through an assessment of dento-skeletal effects on posteroanterior cephalometry.

BACKGROUND: The aim of this study is to compare the dento-skeletal effects of rapid maxillary expansion (RME) and mixed maxillary expansion (MME), assessed on posteroanterior (PA) cephalograms. METHODS: Treatment groups consisted of 42 patients; mean age in RME group (n = 21,13 female and 8 male subjects) was 8.8 years ± 1.37 at T0 and 9.6 years ± 1.45 at T1 and mean age in MME group (n = 21, 12 female and 9 male patients) was 8.9 years ± 2.34 at T0 and 10.5 years ± 2.08 at T1. Seventeen bilateral anatomic landmarks, 16 linear (12 skeletal and 4 dental) and 4 angular measurements were assessed for each patient at T0 and T1. Data from the two groups were compared using independent sample t test (p < 0.05). RESULTS: At T0, the groups were similar for all examined variables (p > 0.05). Significant and equal increase of lateronasal and maxillary and upper and lower molar widths (p < 0. 01) occurred in both groups at T1. Significant but different increases were observed for maxillary incisal, upper left first molar-lateroorbitale, and maxillary first molar angles (p < 0.001 vs. p < 0.05). Significant increases were reported for upper inter-incisal width apex (p < 0.001) and upper right first molar-lateroorbitale angle (p < 0.05) only in the RME group. At T1, differences in maxillary incisal angle (p < 0.05), upper left first molar-lateroorbitale, and maxillary first molar angles (p < 0.001) were noted. CONCLUSIONS: RME and MME were both effective to increase skeletal transverse dimensions by opening mid-palatal suture in growing patients, while MME was associated with minor dental side effects than RME.

Dental pulp stem cells: state of the art and suggestions for a true translation of research into therapy.

OBJECTIVES: Stem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies. SOURCES: An electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references. RESULTS: All research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed. CONCLUSIONS: DPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs.

Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.

Adult mesenchymal stem cells, such as dental pulp stem cells, are of great interest for cell-based tissue engineering strategies because they can differentiate into a variety of tissue-specific cells, above all, into osteoblasts. In recent years, epigenetic studies on stem cells have indicated that specific histone alterations and modifying enzymes play essential roles in cell differentiation. However, although several studies have reported that valproic acid (VPA)-a selective inhibitor of histone deacetylases (HDAC)-enhances osteoblast differentiation, data on osteocalcin expression-a late-stage marker of differentiation-are limited. We therefore decided to study the effect of VPA on dental pulp stem cell differentiation. A low concentration of VPA did not reduce cell viability, proliferation, or cell cycle profile. However, it was sufficient to significantly enhance matrix mineralization by increasing osteopontin and bone sialoprotein expression. In contrast, osteocalcin levels were decreased, an effect induced at the transcriptional level, and were strongly correlated with inhibition of HDAC2. In fact, HDAC2 silencing with shRNA produced a similar effect to that of VPA treatment on the expression of osteoblast-related markers. We conclude that VPA does not induce terminal differentiation of osteoblasts, but stimulates the generation of less mature cells. Moreover, specific suppression of an individual HDAC by RNA interference could enhance only a single aspect of osteoblast differentiation, and thus produce selective effects.