Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Cytoskeletal reorganization in skeletal muscle differentiation: from cell morphology to gene expression.

Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.

Possible role of acylphosphatase, Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload.

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells.

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Structural organization of enteric nervous system in human colon.

The organization of the Enteric Nervous System (ENS) was studied in the human colon. Fragments of the whole colonic wall were either routinely processed or Zinc-Iodide Osmium impregnated. Single-layer preparations were also obtained from some of the Zinc-Iodide Osmium-impregnated specimens. The results showed some differences in the organization of human colonic ENS from that of other mammals. In fact, the human submucous plexus was made up of three interconnected ganglionated networks arranged along three different planes. With respect to the myenteric plexus, its ganglia were large sized and irregularly shaped. Moreover, during the microdissection of the colonic wall, we found the absence of a cleavage plane between the circular and longitudinal muscle layers; on the other hand the cleavage plane between mucosa and submucosa was not immediately below the muscularis mucosae, but slightly deeper, since the innermost part of the submucosa remained adhering to overlying layers.

Vitamin E prevents neutrophil accumulation and attenuates tissue damage in ischemic-reperfused human skeletal muscle.

Neutrophil accumulation and the consequent production of oxygen-derived free radicals are involved in the pathogenesis of Ischemia-Reperfusion syndrome. In this study we investigated whether a treatment with Vitamin E, which has antioxidant properties, could attenuate the tissue damage by interfering with the influx of neutrophils within the ischemic and reperfused human skeletal muscle. To this purpose, patients undergoing aortic cross-clamping during the surgical repair of aortic abdominal aneurysm were studied as a model of ischemia-reperfusion of the lower limb muscles. Muscle biopsies from the right femoral quadriceps of patients not receiving and receiving Vitamin E pretreatment before surgery were taken: a) after the induction of anaesthesia, as control samples, and b) after a period of ischemia followed by 30 min of reperfusion. The tissue samples were either routinely processed for morphological study and immunohistochemical analysis to detect an altered expression of specific endothelial adhesion proteins, such as E-selectin and ICAM-1. The results obtained showed that Vitamin E administration was able to prevent the accumulation of neutrophils within the ischemic and reperfused muscle. This beneficial effect of Vitamin E was due to its ability to hinder the expression of E-selectin and ICAM-1, molecules known to increase the adhesiveness of endothelium to circulating neutrophils. After treatment with Vitamin E a marked attenuation of the reperfusion injury was also evident. In conclusion, Vitamin E treatment may be considered a valuable tool for protection against the ischemia-reperfusion damage of human skeletal muscle.

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus.

Unlike in mouse and hamster, the thymus of rats or guinea pigs contains measurable amounts of substance P-like immunoreactivity (SP-LI), which, in a HPLC system, eluted as authentic SP or SP sulfoxide. Ontogenetic study showed that in rats the SP-LI content of the thymus increased up to 60 days from birth, and decreased thereafter. Capsaicin, but not 6-hydroxydopamine (6-OHDA) pretreatment completely depleted thymic SP-LI content in both newborn and adult rats. Animals treated with capsaicin as newborns, but not as adults, showed lower thymus weights as compared to controls. Rats pretreated with capsaicin as adults underwent partial time-dependent recovery of thymic SP-LI content. Somatostatin-like immunoreactivity (SST-LI) of rat thymus, eluting in part as authentic SST, was unaffected both by capsaicin or 6-OHDA pretreatment. Taken together, these findings demonstrate the existence of capsaicin-sensitive structures containing SP in the rat thymus. The possible function(s) that capsaicin-sensitive structures could exert in the thymus, among which a trophic action, mediated by the efferent function of sensory neurons, remain(s) to be established.

Ultrastructural evidence of myocardial alterations in the course of heterotopic heart transplantation.

Using a novel model of heterotopic rat heart transplantation, the present study was undertaken to evaluate whether parenchymal and microvascular alterations of the ischemic and reperfused myocardium occurred and could be related to local neutrophil infiltration. In such a model, hearts were rapidly excised from donor rats, maintained in a cold saline solution at 4 degrees C and then reimplanted in recipient animals. Muscle biopsies of the ischemic and reperfused myocardium were analysed by ultrastructural and immunohistochemical techniques. Although the cold storage of the hearts provided a good protection against the ischemic insults, reperfusion with the recipient blood caused severe myocardial cell injury and microvascular damage. In particular, the microvascular endothelium showed numerous discontinuities due to the partial destruction of endothelial cell. The altered endothelial integrity was associated with aggregation and adhesion of platelets to the luminal surface. Contrary to other models of ischemia and reperfusion, where neutrophils are considered the major source of oxygen radicals and cellular dysfunctions at reperfusion, in our samples the burst of these toxic metabolites did not originate from such cells. In fact, no neutrophils were seen to accumulate within the ischemic as well as reperfused myocardium. Accordingly, the microvascular endothelium did not express E-selectin, an adhesive molecule which is responsible for the increased adherence and emigration of neutrophils in the microvasculature.

Cytocompatibility of polyurethane foams as biointegrable matrices for the preparation of scaffolds for bone reconstruction.

This work reports preliminary results on the development of biointegrable scaffolds, composed of biostable 3D polymer matrices and bioabsorbable inorganic salts, to be used for cell anchorage in bone regeneration. Three crosslinked polyurethane foams (PUFs), prepared by one-step bulk polymerisation from a polyether-polyol mixture, polymeric MDI and water as expanding agent, were tested for their ability to promote adhesion and growth of bone-derived cells. The open porosity of these foams ranged from 16 to 31% with an average pore size of 470 /600 microm, compressive strength (at 10% ε ) of 0.28/0.38 MPa and elastic moduli of 4.88/6.61 MPa. The human osteosarcoma line Saos-2, and primary cultures of normal human articular chondrocytes and bone marrow-derived (HBM) stromal cells were used for in vitro cytocompatibility tests. For cell adhesion and proliferation analysis, DNA synthesis was evaluated by 3 H-thymidine uptake. Osteoblastic differentiation of Saos-2 adherent cells was determined by measuring the enzymatic activity of alkaline phosphatase (ALP). All cell types were able to adhere to all tested PUFs and to synthesize DNA. At 48 hr culture, HBM stromal cells showed the maximal rate of adhesion with the highest rate of proliferation onto PUFs with the largest pore size, whereas both chondrocytes and Saos-2 appeared to adhere preferentially onto foams exhibiting the highest percentage of open porosity. Up to 8 days in culture Saos-2 cells were able to proliferate into all PUFs, with a time-dependent increase of DNA synthesis and ALP activity. At SEM, the morphology of cells adherent to PUF pores was spread with cytoplasmatic extroflessions, indicating a good metabolic activation. These results demonstrate a good cytocompatibility of the proposed 3D matrices, suggesting that their use in the preparation of composite scaffolds is worth further investigation. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 58-66)ABSTRACT: This work reports preliminary results on the development of biointegrable scaffolds, composed of biostable 3D polymer matrices and bioabsorbable inorganic salts, to be used for cell anchorage in bone regeneration. Three crosslinked polyurethane foams (PUFs), prepared by one-step bulk polymerisation from a polyether-polyol mixture, polymeric MDI and water as expanding agent, were tested for their ability to promote adhesion and growth of bone-derived cells. The open porosity of these foams ranged from 16 to 31% with an average pore size of 470 /600 microm, compressive strength (at 10% ε ) of 0.28/0.38 MPa and elastic moduli of 4.88/6.61 MPa. The human osteosarcoma line Saos-2, and primary cultures of normal human articular chondrocytes and bone marrow-derived (HBM) stromal cells were used for in vitro cytocompatibility tests. For cell adhesion and proliferation analysis, DNA synthesis was evaluated by 3 H-thymidine uptake. Osteoblastic differentiation of Saos-2 adherent cells was determined by measuring the enzymatic activity of alkaline phosphatase (ALP). All cell types were able to adhere to all tested PUFs and to synthesize DNA. At 48 hr culture, HBM stromal cells showed the maximal rate of adhesion with the highest rate of proliferation onto PUFs with the largest pore size, whereas both chondrocytes and Saos-2 appeared to adhere preferentially onto foams exhibiting the highest percentage of open porosity. Up to 8 days in culture Saos-2 cells were able to proliferate into all PUFs, with a time-dependent increase of DNA synthesis and ALP activity. At SEM, the morphology of cells adherent to PUF pores was spread with cytoplasmatic extroflessions, indicating a good metabolic activation. These results demonstrate a good cytocompatibility of the proposed 3D matrices, suggesting that their use in the preparation of composite scaffolds is worth further investigation. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 58-66).

Skeletal myoblasts for heart regeneration and repair: state of the art and perspectives on the mechanisms for functional cardiac benefits.

Until recently, skeletal myoblasts (SkMBs) have been the most widely used cells in basic research and clinical trials of cell based therapy for cardiac repair and regeneration. Although SkMB engraftment into the post-infarcted heart has been consistently found to improve cardiac contractile function, the underlying therapeutic mechanisms remain still a matter of controversy and debate. This is basically because SkMBs do not attain a cardiac-like phenotype once homed into the diseased heart nor they form a contractile tissue functionally coupled with the surrounding viable myocardium. This issue of concern has generated the idea that the cardiotropic action of SkMBs may depend on the release of paracrine factors. However, the paracrine hypothesis still remains ill-defined, particularly concerning the identification of the whole spectrum of cell-derived soluble factors and details on their cardiac effects. In this context, the possibility to genetically engineering SkMBs to potentate their paracrine attitudes appears particularly attractive and is actually raising great expectation. Aim of the present review is not to cover all the aspects of cell-based therapy with SkMBs, as this has been the object of previous exhaustive reviews in this field. Rather, we focused on novel aspects underlying the interactions between SkMBs and the host cardiac tissues which may be relevant for directing the future basic and applied research on SkMB transplantation for post ischemic cardiac dysfunction.

Further SEM observation of human scalp hair: statistical research.

Scalp hair of 100 somalian subjects (50 males and 50 females) were examined with SEM at standard magnification (X 1.000). On each specimen, the hair diameter, mean scale height (M.S.H.), scale index (S.I.), mean scale number (M.S.N.) and mean scale width (M.S.W.) were searched. The statistical elaboration of the obtained data indicates that: i) the hair diameter and M.S.N. are greater in the males, while the other parameters (M.S.H., M.S.W. and S.I.) are prevalent in the females; ii) the hair diameter is directly correlated with M.S.W. and M.S.N., while M.S.H. shows a direct correlation with M.S.W. and an inverse on with M.S.N.. From the comparison between these results and those previously obtained in 100 subjects of white race it results that: a) the mean hair diameter as well as M.S.W. are greater in the white subjects; b) M.S.N., M.S.H. and S.I. are prevalent in the somalians.

Scanning electron microscope study on the efficacy of root canal wall debridement of hand versus Lightspeed instrumentation.

AIM: The aim of this in vitro study was to compare the efficacy of root canal wall debridement following hand versus LightSpeed instrumentation. METHODOLOGY: Twenty recently extracted single-rooted teeth were paired and randomly placed into two treatment groups of 10 teeth each. In group 1, a step-back instrumentation without initial coronal flaring with stainless steel Hedstroem files was used; group 2 was instrumented with Ni-Ti LightSpeed instruments. Both groups had the same irrigation regimen: 2.5% NaOCl and a 15% EDTA solution. The teeth were then decoronated and each root split longitudinally into two halves to be examined using the scanning electron microscope (SEM). The presence of superficial debris and smear layer was evaluated by a standardized grading system, and the resulting scores submitted to nonparametric statistics. RESULTS: Under the conditions of this study, the removal of superficial debris was generally excellent with both canal preparation techniques. Both techniques resulted in variable presence of residual smear layer, with a canal wall covered by smear layer as the predominant characteristic. Generally, the amount of smear layer was greater in the apical than in the middle third of the root, however, this difference was statistically significant (P < 0.005) only in hand-instrumented teeth. The use of LightSpeed instruments was associated with significantly more (P < 0.05) smear layer presence in the middle region of the root when compared with hand instrumentation. In addition, less smear layer was present in the apical region following LightSpeed instrumentation than stainless steel hand files, but this difference was not statistically significant. Differences in debridement between the two halves of the same root were more evident with LightSpeed than manual instrumentation, however, there was no statistical significance. CONCLUSIONS: It may be inferred that the choice between hand and LightSpeed instrumentation should be based on factors other than the amount of root canal debridement, which does not vary significantly according to the instruments used.

Scanning electron microscopic observations on the epithelium of the human spongy urethra.

The human spongy urethras of individuals submitted to emasculation because of glans carcinoma have been studied by means of scanning electron microscopy. The luminal surface of the organ shows longitudinal folds and small glandular openings, surrounded by epithelial elements arranged in form of rosettes. Spermatozoa, partially degenerated, adhere to the epithelium. The slightly prominent apices of the superficial epithelial cells are more or less polygonal in shape and covered with short microvilli among which small granules as possible morphological expression of a secretory activity are detectable. Only seldom microplicae, intermingled with microvilli, can be observed. Additionally, some apices appear swollen and protrude into the lumen. Such a pattern may be escalated so that a cell seems to transform itself into a globular mass and then is expulsed from the epithelial surface. Sometimes an epithelial cell appears empty of its contents: only the crumpled plasmalemma remains. This may represent the morphological expression of an apocrine or holocrine secretory activity of the superficial epithelium. In few cases, the proximal portion of the spongy urethra shows small groups of ciliated elements.

The myenteric plexus of the rat colon: a methodological approach for scanning electron microscopical study.

In the present research four different procedures were tested for the Scanning Electron Microscopic identification of Auerbach's myenteric plexus in the distal colon of the rat. The specimens were processed as follows: enzymatic and chemical digestion (trypsin and HCl); fixation with glutaraldehyde; fixation and staining with zinc iodide/osmium tetroxide (ZIO); fixation and staining with zinc iodide/osmium tetroxide (ZIO) followed by maceration in NaOH solution. All the procedures required microdissection to separate the different layers of the colonic wall. Light microscopic controls were prepared with the ZIO technique. Among the methods utilized, fixation and staining with ZIO appeared to be by far the best as regards the preservation of the architectural arrangement of the myenteric plexus as well as of the fine structural details.

Endotoxin-mediated intestinal damage: protective effect of capsaicin in the rat.

BACKGROUND: Endotoxin provokes the disruption of intestinal mucosal architecture. To investigate whether afferent fibers of the enteric nervous system are involved in this damage, we have used capsaicin to induce a selective, long-lasting degeneration of these fibers in the rat. METHODS: The rats were divided in two groups, receiving subcutaneously either capsaicin or its vehicle. After 10 days the rats from each group were injected with nonlethal doses of endotoxin or with saline. The next day all the animals were killed, and the jejunum and ascending colon were collected for light and scanning electron microscopy analysis; morphometric analysis of jejunal villus height was also performed. RESULTS: The rats receiving endotoxin but not pretreated with capsaicin had severe morphologic alterations and a significant reduction in villus height. In contrast, the rats pretreated with capsaicin and then with endotoxin showed a preserved mucosa with normal mean villus height. CONCLUSIONS: Capsaicin pretreatment is able to prevent endotoxin-induced damage of intestinal mucosa; this result seems to indicate that afferent fibers of the enteric nervous system are involved in the pathogenesis of this damage.

[The development of the tongue in the chick embryo: observation under a scanning electron microscope]. / Lo sviluppo della lingua nell'embrione di pollo: osservazioni al microscopio elettronico a scansione.

The morphological features of the chick embryo tongue from the 8th day of incubation till hatching and during the early post incubation period have been investigated by means of Scanning Electron Microscope. At the SEM, it is possible to observe that already at the 8th day of incubation, the body and the root are separated by a low smooth-surfaced ridge. In the following days this ridge develops, giving rise to the so-called lingual spines, whose significance is still uncertain. As concerns the evolutive pattern of the superficial layer of the epithelium, in the first days of the considered incubation period the cells appear dome-shaped and have microvilli on their apical surface; afterwards they tend to become more flattened, and the microvilli are replaced by a thick net of microplicae. In the last days of incubation and after hatching desquamative phenomena become evident. The above described evolutive process can be regarded as a common feature of the whole dorsal lingual surface; only few regional differences are to be noted, such as the earlier development of the microplicae on the apex and borders of the tongue. In particular, the microplicae observed at the apex of the tongue show a typical aspect and arrangement; they run regularly parallel to each other. On the lingual dorsal surface taste bud-like structures have never been observed.

Insulin-like growth factor-I stimulates in vitro migration of preosteoclasts across bone endothelial cells.

Little is known about the factors and the mechanisms involved in preosteoclast emigration from the vasculature. In this study, an in vitro model of bone endothelial lining was mimicked by culturing bone endothelial (BBE) cells at confluence on a 3-microm pore polycarbonate membranes. Preosteoclastic (FLG 29.1) cells were then added on top of the BBE cell monolayer and 10 nM insulin-like growth factor-1 (IGF-I) was added below the supporting membrane. Scanning and transmission electron microscopy were used to evaluate the chemotactic responses of preosteoclastic FLG 29.1 cells towards the IGF-I generated gradient. IGF-I potently stimulated chemotaxis in the FLG 29.1 cells, as shown by the migration of the preosteoclastic cells across the underlying BBE and through the intercellular junctions between adjacent endothelial cells. Subsequently, FLG 29.1 cells penetrated the pores of the supporting membrane and reached the lower face of the membrane. Thus, IGF-I, which is abundantly present in the bone tissue microenvironment, may play a paracrine role in the recruitment of the circulating preosteoclasts from the vascular compartment into the bone tissue. This in vitro model, which mimicks the in vivo phenomenon of preosteoclast extravasation, should prove useful in elucidating the molecular mechanisms that underlie this process.

[The correlations between the echographic aspect of the perioral and masticatory muscles and dento-skeletal characteristics]. / Correlazioni tra l'aspetto ecografico dei muscoli periorali e masticatori e le caratteristiche dento-scheletriche.

INTRODUCTION: US of some muscular components of the stomatognathic system (orbicularis oris and masseter muscles) has proved to provide important information about their morphologic features. The purpose of this study is to explore the US features of the perioral and masticatory muscles in patients affected with any type of malocclusion and to analyze the correlations between dento-skeletal characteristics and muscle changes. MATERIAL AND METHODS: We examined 26 untreated patients (10 men and 16 women); we measured their cephalometric parameters on lateral skull teleradiographs and then we measured with US both the masseter and orbicular muscles dimensions. The US images were acquired with 5 MHz and 7.5 MHz probes, measuring the masseter muscle with transversal and longitudinal scans, and the two parts of the orbicularis oris with longitudinal scans. The cephalometric measures were calculated with: angular parameters useful to determine the sagittal relationships, both angular and linear parameters to define the vertical relationships and angular measures that define the incisor position. We applied the Spearman test to assess if there was a relationship between the US dimensions of the orbicularis oris and the masseter, and the dento-skeletal characteristics of these 26 patients. RESULTS: Our results show that there is a strong correlation between mandibular morphology, the palatal plane, respectively, and the masseter length. There is a direct correlation with the masseter dimension for the palatal plane obliquity, and an inverse correlation for the mandibular morphology. The incisor position is influenced by the orbicular oris muscle dimension and by the asymmetric position of the superior and inferior incisors. CONCLUSIONS: This noninvasive diagnostic examination is a useful aid to the clinical examination of the muscles, especially in orthodontic diagnosis, together with MRI and CT, respectively more expensive and unhealthier.