Technical note: surgical guide for computer-aided endodontic surgery.

INTRODUCTION: CBCT combined to intra-oral scansion are the means to build an endodontic surgical guide to perform an easier and safer access to the apex in endodontic micro-surgery. . METHODS: A 38-year-old woman presented chronic apical periodontitis, which involved the three roots of 16 that was treated by endodontic therapy several years ago elsewhere. The palatine channel was retreated, the mesio- and disto- buccal roots were instead treated with apicoectomy. A surgical template was printed by a 3D printer to obtain greater precision in the surgical access. CONCLUSIONS: Endodontic microsurgery has evolved over the years. New tools have been introduced to improve therapy, even if the basic principles have not changed. In fact, according to the literature, it is necessary to cut at least 3 mm of root to be sure of eliminating the anatomical variations and the accessory channels. Several Authors have devoted themselves to creating an endodontic surgical template, some in vitro and others in vivo. The present paper introduces a new method that allows a more conservative osteotomy and greater precision the surgical access. Further investigation are needed to test and improve the effectiveness of the treatment but this technique seems very promising because it is less invasive for the patient and simplifies the work for the dentist who can perform micro-surgery in an easier and faster way.

Effect of biostimulation on oral fibroblast: a pilot study.

Bio-stimulation is a technique in aesthetic medicine in which different drugs such as nucleotides, antioxidants and glucosaminoglycans precursors are injected in the dermis to improving the anabolic function of dermal fibroblasts, i.e., protein synthesis, replication and production of extracellular matrix components. It can be achieved with multiple intra-dermal injections, using two protocols: 1) Polydeoxyribonucleotide (PDRN) plus glucosamine sulphate (Gluc); 2) N-acetylcysteine (NAC) and amino acids (Aa) (named Bio- NAC procedure). Since the role of drugs used in biostimulation on human dermal fibroblasts is not completely understood, the aim of this study is to evaluate the effect of these substances in primary cell cultures by using RT-PCR and a panel of specific genes (ELN, DSP, FN1, FBN1, ITGA1, ITGA2, ITGA5, ITGB1, COL1A1,COL3A1) to detect their effect on cell metabolism and extracellular matrix components. Both the treatments were responsible for Elastine and Desmoplakin genes activation. Only NAC plus Aa treatment enhance the expression of other genes related to tissue growth and elasticity like FBN1, ITGA1 and ITGB1. All the other genes investigated (FN1, ITGA5, ITGA2, COL1A1, COL3A1) were down-regulated by both treatments. Since the precise role of these proteins in tissue integrity and aging is not known, this study confirms the usefulness of biostimulation therapies in enhancing some of the genes responsible of cellular wellbeing. This study could be useful to consider the possibility of injective biostimulation in oral cavity, clinical applications in oral healing and in gingival atrophy as well.

Biorevitalization: an in vitro study on gingival fibroblast.

Bio-revitalization is a therapy commonly used in aesthetic medicine to improve skin quality by di¬rectly integrating hyaluronic acid alone or added to other molecules (i.e. vitamins) through intradermal injections. These injections are not aimed to fill roughness but to achieve extracellular matrix optimi¬zation. The injective medical devices used in aesthetic medicine differ for hyaluronic acid content and for the presence of additional molecules that characterize the formulation of a particular company. The aim of the present study is to compare HA with different compounds in regard to their effects on cultured fibroblasts over time by using RT-PCR and a panel of genes (ELN, DSP, FN1, FBN1, ITGA1, ITGA2, ITGA5, ITGB1, COL1A1, COL3A1) involved in connective integrity. Bio-revitalization is able to activate genes involved in tissue integrity. The reported data add new insight in the comprehension of molecular mechanism related to BR. These preliminary data have to be developed through additional experiments. However, an injective therapy seems to be effective in gingival fibroblast stimulation.

New insights in orofacial cleft: epidemiological and genetic studies on italian samples.

Cleft of the lip and/or palate (CL±P) is the most common congenital craniofacial anomaly affecting around 1 in 700 live births worldwide. Clefts of the human face can be classified anatomically as cleft lip only (CL), cleft palate only (CP), cleft lip and palate (CLP) or a combined group of cleft lip with or without cleft palate (CL±P), based on differences in embryologic development. CL±P has a genetic base and several linkage and association analyses have been performed in order to obtain important information about the role of candidate genes in its onset; not less important are gene-environment interactions that play an increasing role in its aetiology. In CL±P, several loci have been seen associated with the malformation, and, in some cases, a specific gene mapping in a locus has also been identified as susceptibility factor. In CP, one gene has been found, but many more are probably involved. In this short review the genetic studies carried out on CL±P, and the interaction with environmental factors (alcohol, smoking, drugs) are discussed.

Cleft palate only: current concepts.

Cleft palate only (CPO) is one of the most common congenital malformations worldwide. The etiopathogenesis of CPO is not completely understood. Environmental factors, such as smoking, alcohol consumption, intake of drugs during pregnancy, advanced paternal age, have been demonstrated to be a risk of CPO, but conflicting results have also been published. Insufficient intake of folic acid during the pregnancy has been suggested to increase the risk for CPO. The demonstrated risk for siblings and the higher risk for monozygotic twins suggest a genetic etiopathogenesis for CPO. In some cases of CPO a prevalent mode of inheritance has been reported, but oligogenic models with reduced penetrance, and the risk related to environmental factors have also been proved. One of the first manifestations associated with CPO is difficulty with feeding. Aerophagia is a problem in these infants with CPO and requires more frequent burping and slower feeding. The inability to generate intraoral breath pressure due to nasal air emission in CPO children frequently manifests as articulation difficulties, particularly consonant weakness, and unintelligible speech. Hearing disorders are prevalent among individuals with CPO, as a result of chronic otitis media with effusion due to eustachian tube dysfunction. A multidisciplinary team is essential to manage the many aspects of CPO. In treating CPO, the reconstructive surgeon works in cooperation with otolaryngologists, dentists and orthodontists, speech pathologists, audiologists, geneticists, psychiatrists, maxillofacial surgeons, social workers, and prosthodontists. CPO can be considered a genetically complex disease, but new knowledge and new therapeutic approaches have greatly improved the quality of life of these children. Prenatal diagnosis is an important step in the treatment of this disease.

Effects of light-emitting diode (led 640nm) on human gingival fibroblasts: a comparative in vitro study.

PURPOSE: The light-emitting diodes (LEDs) have been applied in oral surgery for tissue stimulation and wound healing. Several Authors have highlighted that fibroblasts subjected to phototherapy have an increased viability, proliferation, biomodulation of inflammatory cytokines and genes expression. It remains to be determined which are the best irradiation parameters (energy, wavelength, power) for each type of cell in order to obtain the best bio-stimulation. The aim of this study was to investigate the effects of LED irradiation on primary human gingival fibroblast cells (HGF) on DSP, ELN, HAS1, ELANE, HYAL1, RPL13 genes activation using Real Time PCR. These genes activation is directly connected with elastin protein production and HGF functionality. MATERIALS AND METHODS: Human gingival tissue biopsies were obtained from three healthy patients during extraction of teeth. The gingival pieces were fragmented with a scalpel and transferred in culture dishes for allow the cells growth. Human gingival fibroblasts at the second passage were seeded on multiple 6-well plates and were stimulated with three different light-emitting diodes (LEDs) fixture. After irradiation, the cells were trypsinized, harvested and lysed for RNA extraction. Genes expression was quantified using Real Time PCR. RESULTS: We didn't found significant differences in genes activation of HGF of the three different LEDs. The LED irradiation seems to be directly correlated with the elastin and hyaluronoglucosaminidase 1 genes activation that are directly connected with proteins production and HGF functionality. CONCLUSIONS: HGF show an increased deposition of elastin as well as enhanced expression of collagen type I, which is the main protein related to the synthesis and of the collagen-rich matrix.

Radiofrequency treatments: what can we expect?

Among non-ablative procedures in aesthetic medicine, the radiofrequency (RF) is one of the most popular for the treatment of face and body skin laxity. It can be classified as a physical bio-stimulation that produces a temperature increase on biological structures, using electromagnetic waves. The term encompasses devices having substantial differences in energy, wavelengths, handpieces dimension and structure. Moreover, for some of these, the protocols are only partially defined. The aim of this short review is to clarify some aspecst of the RF therapy starting from the physics, passing through the mechanism of action and finally, with the most suitable protocols. Contrary to mechanic waves, electromagnetic waves, physics are always transversal to the impulse and this leads to the different energy distribution in capacitive (monopolar) or resistive (bi- or multi-polar) applications. The thermal damage as therapeutic effect is a postulate that needs to be discussed and the same is true for the terms “non-surgical” and “non-ablative”, often recurrent in the scientific literature. Protocols must be optimized according to the machine and the patient, keeping in mind the possibilities of biostimulation in terms of immediate improvement and of long lasting investment in skin rejuvenation. It is mandatory to understand the possibilities and limitations of each device to perform useful, safe and correct medical treatments.

Non-ablative radio-frequency rejuvenation: a histological and bio-molecular report.

Radiofrequency machines for medical use are known to produce moderate clinical improvement of skin laxity without invasive procedures. Numerous equipment with different characteristics have been proposed after the introduction in 2002 of the first FDA approved device. This report is aimed to test if RF treatment is effective when performed at low frequency and low energy level. Two RF treatments were performed unilaterally 7 and 2 days before a planned eyebrow lifting surgery, with a radiofrequency device with 0.52 to 0.7 MHz frequencies, maximum energy of 200 W, used at 40% of its power. A bipolar handpiece with a diameter of 30 mm and a maximum power of 9-9.5 W was massaged along the temporal area for 10 min. Skin samples of treated and untreated sides were collected during surgery and processed for histologic examination and RT-PCR analysis, to test differences in gene activation in a panel of proteins that are relevant in extracellular matrix of dermal connective tissue. The histological examination of the samples showed that the treatment induced a loss of the typical oriented structure in the reticular dermis. The study through RT-PCR evidenced that ELN, the gene codifying for Elastine was strongly enhanced. Some collagen-tested genes (COL1A1, COL3A1 and COL9A1) were inhibited by the treatment, whereas COL2A1 and COL11 were activated. The genes responsible for Metallo-proteases (MMP) 2, 3 and 13 were depressed, while the MMP9 was stimulated. Gene codifying for Hyaluronic synthase 1 (HAS1), Hyluronidase 1 (HYAL1), Neutrophyl elastase (Elane), Desmoplakin (DSP) and GDF6 were inhibited. Insulin like growth factor (IGF1) gene activity was enhanced. RF treatment, with the tested non-ablative equipment, produced histological effects and change in DNA expression of some extracellular matrix related genes, confirming the biostimulatory role of this procedure.

EFFECT OF SOMATOSTATIN ON DENTAL PULP STEM CELLS.

Dental pulp stem cells (DPSCs) are multipotent stem cells with the potential to differentiate into various cell types. For this reason, they have been proposed as an alternative source for mesenchymal stem cells. Somatostatin is a peptide hormone with an inhibitory effect on several endogenous hormones. The aim of our study is to investigate whether somatostatin can promote or inhibit differentiation of DPSCs in osteoblasts and bone tissue. DPSCs were extracted from third molars of healthy subjects, and were treated with somatostatin at the concentration of 100 ng/ml for 24 and 48 h. Gene expression in treated DPSCs was compared with untreated cells (control) in order to check the effect of somatostatin on stem cell differentiation. After 24 h of treatment many genes investigated were down-regulated in treated DPSCs vs untreated DPSCs. Significantly up-regulated gene (Fold change > 2) was the Bone Morphogenetic Protein BMP4. On the contrary somatostatin induced the over-expression of bone related genes after 48 h of treatment (i.e. BMPR1B and BMPR2). TGFB family genes and their receptors were also significantly up-regulated after 48 h of treatment. Somatostatin demonstrated to promote the self-renewal of DPSCs: in our experiments somatostatin mainly acted on TGFB family genes. Further studies are needed to explore this new way of creating bone tissue.

INSULIN ACTIVITY ON DENTAL PULP STEM CELL DIFFERENTIATION: AN IN VITRO STUDY.

Insulin is a powerful and important hormone involved in the proliferation and differentiation of osteoblasts. Dental pulp stem cells (DPSCs) have the ability to self-expand and differentiate in pre-osteoblast, producing in vitro autologous bone tissue. The aim of our study is to investigate whether insulin can influence differentiation of DPSCs in osteoblast and bone tissue. Dental germ pulp was extracted from third molars of healthy subjects, following informed consent. DPSCs were treated with insulin at the concentration of 100 ng/µl for 24 and 48 h. Gene expression in treated DPSCs was compared with untreated cells (control) in order to check the effect of insulin on stem cell differentiation. After 24 h, significant up-regulated genes (Fold change > 2) in DPSCs were the Bone Morphogenetic Proteins BMP3, BMP4 and their receptor BMPR1A. BMP1 results over-expressed after 48 h of treatment. Significantly down-regulated genes were BMP4, BMP7 and TGFBR2 after 24 h of treatment and BMP5 and BMP7 after 48 h. Insulin was demonstrated to influence proliferation of DPSC, differentiation and expansion in osteoblasts. Further studies are needed to explore this new way of creating bone tissue.

HYALURONIC ACID IN DERMAL REJUVENATION: AN IN VITRO STUDY.

The purpose of this paper is to evaluate the role of hyaluronic acid in bio-revitalization by testing several extracellular matrix biological parameters in cultured dermal fibroblasts. To this aim, fibroblastic expressed genes after exposition to three hyaluronic acid medical devices were evaluated. Cells were seeded on a layer of three different medical devices containing 6.2, 10 and 20 mg/ml of hyaluronic acid for 24 h. Real Time Polymerase Chain Reaction was performed to investigate gene expressions. Genes encoding hyaluronic acid synthesis and degradation, Metalloproteinases 2 and 3 and Desmoplakin production as well as GDF6, and IGF1 were activated by hyaluronic acid products. The in vitro study showed similar effects on tested genes despite a different concentration of hyaluronic acid contained in the medical devices and the simultaneous presence of other additives. Based on the reported data, gene activations are an aspect of metabolic modulation of signalling pathways rather than the proportional production of a specific connective tissue molecule. Indeed different hyaluronic acid concentration and the presence of other additives did not change the overall effect on the studied genes. We believe that the optimization of extracellular matrix micro-environment, obtained by enhanced structural support with hyaluronic acid, leads to functional and metabolic improvement.

THE THEORIES OF AGING: REACTIVE OXYGEN SPECIES AND WHAT ELSE?

This manuscript is a short review on the theories of aging, focusing mainly on the balance between the nutrient and the oxygen intake necessary for energy metabolism and the processes for neutralizing the negative consequences of energy production. The first section entitled “Why” provides brief historical details regarding the main group of aging theories, firstly the evolutionary theories and secondly the theories of aging related to humans, cells and biomolecules are discussed. The second section entitled ‘Where’ includes brief summaries of the many cellular levels at which aging damage can occur: replicative senescence with its genetic and epigenetic implications, cytoplasmic accumulation, mitochondrial respiratory chain dysfunction, peroxisome and membrane activity. In the third section entitled ‘How’ the linking mechanisms between the caloric restriction and the antioxidant intake on lifespan and aging in experimental models are discussed. The role of ROS is evaluated in relation to the mitochondria, the AMPK activated sirtuins, the hormesis, the target of rapamicin and the balance autophagy/apoptosis.

HYALURONIC ACID: THE USE OF ITS PRECURSOR IN SKIN BIO-STIMULATION.

Bio-stimulation is an injective therapy aimed to boost the anabolic functions of dermal fibroblasts to obtain skin improvement. It can be achieved with multiple intradermal injections (0.05–0.1 ml each) of a solution of 400 mg (3 ml) of injectable glucosamine sulphate, plus 5.623 mg (3 ml) of polideoxyribonucleotide, 1 ml of lidocaine and 0.5–1 ml of sodium bicarbonate, to repeat every 7, 14, 21, and 28 days. The administration of glucosamine sulphate to skin fibroblasts is believed to lead to its incorporation in glycosaminoglycans, and thereby to the stimulation of extracellular matrix synthesis, whereas polideoxyribonucleotide possesses anti-inflammatory and regenerative capability. This study aims to elucidate the in-vitro effects of this treatment by studying what happens to several genes related to connective tissue integrity. Human dermal fibroblasts were seeded in a culture medium enriched with either two drugs alone or combined: glucosamine sulphate and/or polideoxyribonucleotide. After the end of the exposure time of 24 h, 48 h, and 72 h, the cells were trypsinized and lysed for RNA extraction. Reverse transcription to cDNA was performed directly from cultured cell lysate. Finally, the cDNA was amplified by real-time PCR and a panel of genes involved in dermal integrity was tested. Gene expression of Hyaluronan synthase 1 (HAS1), Elastine (ELN), Insulin like growth factor 1 (IGF1), Growth differentiation factor 6 (GDF6) and of a series of catabolic enzymes, such as Metalloproteases (MMP) 2, 3 and 13, the neutrophyl expressed Elastase (ELANE) and the Hyaluronidase 1 (HYAL1) were tested after 24, 48 and 72 hours of exposure to glucosamine sulphate and polideoxyribonucleotide alone or combined. All the tested genes but one were up-regulated. A negative synergism on several enzymes (particularly appreciable for Insulin-like growth factor 1 and metalloprotease 13) was observed when the two drugs were delivered together. Glucosamine sulphate acts not only as building block in the biosynthesis of glycosaminoglycan chains, but also as a booster of hyaluronan synthase 1. The association of glucosamine sulphate and polideoxyribonucleotide, used in bio-stimulation therapy protocol, has a negative synergism on catabolic genes in dermal fibroblast cultures. The present observations produce further insight into the effects of glucosamine sulphate in the biosynthesis of glycosaminoglycan chains.

Bio-Revitalization: Effects of NASHA on Genes Involving Tissue Remodeling.

BACKGROUND: The "non-animal stabilized hyaluronic acid" (NASHA) is a widely used product in bio-revitalization injective procedures in esthetic medicine. The present research aimed to quantitatively evaluate the therapeutic effect of one of the more used bio-revitalization products on cultured dermal fibroblasts. RT-PCR was used for gene expression profiling of some proteins known to be relevant in skin homeostasis. METHODS: Human dermal fibroblasts were seeded on a culture medium enriched with a product for dermal bio-revitalization, consisting of stabilized hyaluronic acid gel 20 mg/ml. After 24, 48, and 72 h of exposure, the cDNA was amplified by real-time PCR. Gene expression was quantified with the delta/delta calculation method. RESULTS: In this study, the gene of metalloproteinase (MMP)-13 is strongly expressed after NASHA incubation. The MMP-2 encoding gene instead is less expressed, but both evidence the same temporal trend, being progressively up-regulated after 24 and 48 h, thereafter the expression decreases, whereas MMP-3 maintains the same up-regulation at 72 h. Hyaluronan synthase 1 and desmoplakin are progressively up-regulated and increase at 24, 48, and 72 h. Hyaluronidase 1 and neutrophil elastase genes are overexpressed, but at 72 h they both exhibit the same behavior as the other degradative enzymes MMP-13 and MMP-2. CONCLUSIONS: Skin bio-revitalization by injecting the tested NASHA gel produces an enhancement in the expression of some genes involved in extracellular matrix degradation and organization. In this study, a time-dependent behavior, different for genes encoding degradative compared to synthesis proteins, was demonstrated.

Lateral Canthal Support in Prevention of Lower Eyelid Malpositioning in Blepharoplasty: The Tarsal Sling.

Lower blepharoplasty is a cornerstone in facial rejuvenation and improvement. Despite its popularity, several adverse effects have been described; of these, postsurgical eyelid displacement, with its aesthetic and functional consequences, is one of the more frequent complications. The tarsal sling procedure is a simplified canthopexy consisting in the fixation of the lateral portion of the septum--the canthal ligament--to the orbital wall periosteum. The aim of the current research is to demonstrate how the tarsal sling technique is effective in the prevention of lower lid malpositioning. A retrospective analysis of 40 consecutive healthy individuals was carried out. In group 1, 20 patients underwent standard blepharoplasty; in group 2, blepharoplasty was associated to the tarsal sling canthopexy. Pre- and postsurgical position of the lower eyelid margin was compared through photographic measurements at 5 time points and statistical analysis performed. Group 1 patients evidenced an increased distance between the interpupillary line and the lateral aspect of the lower lid margin. A progressive spontaneous improvement (reduction in scleral exposition) was detected. Patients who underwent the tarsal sling procedure (group 2) reported a postoperative overcorrection of scleral appearance. Two years postsurgery, progressive relapse occurred, but the lower eyelid did not reach presurgical values and maintained a slight degree of overcorrection. Tarsal sling is an easy, fast, and efficacious procedure to prevent eyelid displacement in lower blepharoplasty. Its routine use is a tool to assure further support to lower lids in the younger patients or when lid laxity is absent during presurgical examination.

Role of antioxidants in dermal aging: an in vitro study by q-RT-PCR.

BACKGROUND: Reactive oxygen species production is the final step in skin aging. These unstable molecules can damage and destroy DNA, proteins, and membrane phospholipids. The aim of this study was to test the in vitro effect of an antioxidant precursor, N-acetylcysteine (NAC), on human dermal fibroblasts. NAC alone and a solution of NAC and amino acids together, used in aesthetic medicine as intradermal injection treatment, were tested. METHODS: The expression levels of some connective related genes (HAS1, HYAL1, ELN, ELANE, DSP, GDF6, and IGF1) were analyzed on cultures of dermal fibroblasts using real-time reverse-transcription polymerase chain reaction (real time RT-PCR). RESULTS: All genes were upregulated after 24 h of treatment. CONCLUSIONS: An interesting effect of gene induction by administration of NAC and amino acids in vitro was demonstrated. Upregulation of elastin-, hyaluronic acid-, and GDF6-encoding genes supports the evidence of clinical improvement induced by NAC biostimulation in the prevention and correction of skin aging.

Effects of glucosamine and nucleotide association on fibroblast: extracellular matrix gene expression.

Glucosamine (Gluc) is a drug used as an anti-inflammatory in moderate forms of knee arthrosis. A further off label use of Gluc is in the anti-aging treatments associated with Polideoxirybonucleotide (PDRN) through intra-dermal injection for a procedure called bio-stimulation. An unexpected effect on cultured dermal fibroblasts, during an experimental study on the gene activation in aesthetic bio-stimulation, was observed. The results have potential application in orthopaedic medical therapy. Fibroblast primary cultures were carried out, seeding cells on a layer of Gluc or PDRN alone or in combination for 24 h. Real Time-PCR was performed to investigate several gene expressions. The MMP13 and the IGF-I gene expression in fibroblast cultures were strongly inhibited after 24 h of incubation with the association of Gluc and PDRN, whereas Gluc and PDRN alone produced a modest inhibition of IGF-I and an activation of MMP13. MMP13 is present in osteoarthritic cartilage and this enzyme plays a significant role in cartilage collagen degradation. IGF1 is involved in growth and development and is successfully used in tissue-engineering for cartilage repair. Based on the reported data, we infer that the association of Gluc and PDRN has a potential application in cartilage therapy. Additional basic science and clinical studies are needed to confirm this preliminary report.

Apert's syndrome: cephalometric evaluation and considerations on pathogenesis.

Apert's syndrome is a malformation characterized by abnormalities in the cranial vault, midfacial malformations, and syndactylia. The present study analyzes the lateral and frontal projections of the teleradiograms from five patients. Data were taken on the skeletal features and an attempt was made to interpret them in terms of functional matrices. We have used cephalometry as a descriptive device although the intent was also to use it to provide a perspective on the pathogenetic data derived from the literature. The hypothesis analyzed is that from the outset of craniofacial pathogenesis there may be a primitive alteration of the cartilaginous template from which are derived endochondral bones. The progressive involvement of the synchondrosis of the cranial base is subsequently transmitted to the membranous structures of the vault and face through the coronal ring and lambdoid suture systems. Although the data gained do not actually confirm this hypothesis, they do provide further support for it.

Crouzon syndrome: cephalometric analysis and evaluation of pathogenesis.

Crouzon syndrome is a craniofaciostenosis characterized by brachycephaly, ocular proptosis, and maxillary retrusion. The hypothesis has been forwarded that an alteration in anterior cranial base synchondrosis activity is responsible for the skeleton abnormalities which are associated with this disorder. The present work was aimed at assessing this pathogenetic hypothesis. Cephalometry was used as the analysis method and care was taken in determining the three-dimensional measurements of some functional spaces (e.g., orbit, rhinopharynx, and nasal cavity). The results indicate that in Crouzon syndrome the craniofacial alterations depend not only on reduced synchondrosis activity of the anterior cranial base, but also of the posterior cranial base.