Titanium Dioxide Nanowires Grown on Titanium Disks Create a Nanostructured Surface with Improved In Vitro Osteogenic Potential.

Current biomedical research is centered on the study of nanomaterials and their effects in biological environments. In particular, there is an increasing interest on TiO2 nanostructures for biomedical applications such as drug delivery or implant materials. In this framework, we present a Chemical Vapour Deposition process to synthesize titanium dioxide nanowires (NWs) on a commercially pure titanium substrate and we test the material In Vitro as a culture substrate for murine osteoblast-like MC3T3-E1 cells. A physical-morphological, structural and optical-characterization of the inorganic samples is performed by Electron Microscopy techniques and X-ray Diffraction, showing that a mat of crystalline rutile TiO2 NWs is obtained over the commercial substrate. In Vitro biological tests are performed by seeding MC3T3-E1 cells on the material and studying cell morphology, the cellmaterial interface and the osteoblast gene expression. These experiments show good cell adhesion to the nano-structured surface and a higher degree of early osteoblastic differentiation compared to control titanium surfaces, indicating that the present nano-structured material has good osteogenic potential for biomedical applications.

Maxillary Sinus Floor Augmentation Using an Equine-Derived Graft Material: Preliminary Results in 17 Patients.

OBJECTIVE: Sinus floor elevation with lateral approach is probably the most frequently performed reconstructive procedure to rehabilitate posterior maxilla when a bone deficiency is present. Different graft materials have been proposed and tested, often with high clinical performances and predictable results. Histological analysis is required when evaluating new materials. We investigated human biopsies retrieved after sinus floor elevation procedure by histomorphometric evaluation to test the performance of an equine-derived bone grafting material. STUDY DESIGN: Seventeen consecutive patients were enrolled and sinus lift surgeries were performed using an equine bone graft. Six months after surgery, at implant placement, bone samples were collected. Histomorphometry analysis was carried out on decalcified samples. RESULTS: All surgeries were uneventful and no additional grafting was required prior to implant insertion. Forty percent of new bone formation was detected, which represented the most abundant tissue retrieved, followed by the residual graft material (33%) and fibrous tissue (27%). A significant reduction in particles size demonstrates a remodeling activity of the graft material. CONCLUSION: Within the limitations of this study, this equine-derived bone graft proved to be an effective material to induce new bone formation in the sinus floor elevation procedure.

Study of GSK3b inhibitors SB415286 and SB216763 to improve osteoblastic differentiation on microstructured titanium.

Rough titanium surfaces enhance cell response to activation of Wnt canonical signalling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3ß-inhibitors SB216763 and SB415286 on osteoblastic differentiation on titanium surfaces with different topography and wettability. Osteoblastic MC3T3 cells were plated on smooth (Pickled), sand-blasted/acid-etched (SLA) or hyper hydrophilic SLA (modSLA) titanium discs and transfected with a reporter vector sys-tem for Wnt canonical signalling. Cells were also seeded in the presence or in the absence of GSK3b-inhibitors SB216763 or SB415286 and their viability, morphology and the expression of Wnt target and osteoblast specific genes was assessed by Real Time PCR. Inhibitors altered cell morphology and mostly reduced cell viability at high concentration. SB415286 markedly increased the expression of ALP in MC3T3 cells on rough surfaces at the concentration of 100 nM before decreasing its expression at higher concentrations. OCN expression was unaffected. Increasing concentrations of SB216763 increased the expression of ALP in MC3T3 cells on rough surfaces but OCN expression was not changed at any con-centration. SB216763 and SB415286 inhibitors should be further investigated as potential tools to improve cell differentiation on titanium surfaces for endosseous implants.

Anti-fibronectin aptamers improve the colonization of chitosan films modified with D-(+) Raffinose by murine osteoblastic cells.

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.

Stanozolol-soaked grafts enhance new bone formation in rat calvarial critical-size defects.

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 µm/day ± 0.37 versus 6.50 µm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm-2 ± 2.01) to three months (28.26 n mm-2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm-2 ± 0.32 versus 1.01 n mm-2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm-2 ± 6.29 versus 6.29 n mm-2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Improved scaffold biocompatibility through anti-Fibronectin aptamer functionalization.

UNLABELLED: Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization. STATEMENT OF SIGNIFICANCE: We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients' blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule.

Chitosan scaffold modified with D-(+) raffinose and enriched with thiol-modified gelatin for improved osteoblast adhesion.

The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml(-1) BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties.

Tardive Dyskinesia, Oral Parafunction, and Implant-Supported Rehabilitation.

Oral movement disorders may lead to prosthesis and implant failure due to excessive loading. We report on an edentulous patient suffering from drug-induced tardive dyskinesia (TD) and oral parafunction (OP) rehabilitated with implant-supported screw-retained prostheses. The frequency and intensity of the movements were high, and no pharmacological intervention was possible. Moreover, the patient refused night-time splint therapy. A series of implant and prosthetic failures were experienced. Implant failures were all in the maxilla and stopped when a rigid titanium structure was placed to connect implants. Ad hoc designed studies are desirable to elucidate the mutual influence between oral movement disorders and implant-supported rehabilitation.

Pharmacological GSK-3beta inhibition improves osteoblast differentiation on titanium surfaces.

Rough titanium surfaces enhance the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3b-inhibitor (2'Z,3'E)- 6-Bromoindirubin-3'-oxime (BIO) on osteoblastic differentiation on titanium surfaces with different topography and wettability. C2C12 cells were plated on pickled, acid-etched/sand-blasted (SLA), modified hydrophilic SLA titanium discs (modSLA) and stimulated with increasing doses of BIO. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by Real Time PCR. Osteoblastic MC3T3 cells were then plated on discs with or without BIO and the expression of osteoblast specific genes was assessed by Real Time PCR. One mM BIO activated Wnt canonical signaling in C2C12 cells on all surfaces, and the highest effect was on rough surfaces. BIO markedly increased the expression of Osteoprotegerin and Osteocalcin in MC3T3 cells on rough surfaces at the concentration of 100 nM, and on all surfaces at the concentration of 1 mM. BIO enhances Wnt signaling activation and the expression of osteoblastic genes on rough surfaces and could be a viable approach to improve cell response to implant surfaces.

Periostin improves cell adhesion to implantable biomaterials and osteoblastic differentiation on implant titanium surfaces in a topography-dependent fashion.

Periostin is a matricellular protein highly expressed in periodontal ligament and periostium and has been shown to be required for tissue development and maintenance. We showed that the adhesion of murine osteoblastic MC3T3 cells to thiolated hyaluronic acid/polyethyleneglycol hydrogels was greatly improved by enrichment with periostin. Polished or sand-blasted/acid-etched (SLA) commercially pure titanium surfaces were also coated with this protein and periostin ameliorated cell adhesion and dramatically affected cell morphology on both surfaces, as assessed at fluorescence microscopy, scanning electron microscopy, and chemiluminescence-based viability assay. Moreover, periostin increased the expression of alkaline phosphatase, osteoprotegerin, connective tissue growth factor, collagen 1a1, osteocalcin, Runx2, and osterix transcription factors on smooth surfaces. However, it did not affect, or even decreased, the expression of these genes on SLA discs. Transcript levels for connexin 43 were greatly increased on both surfaces in the presence of periostin. Taken together, these results show that periostin coatings can be a viable approach to improve cell adhesion and differentiation on implantable biomaterials.

GSK3b-inhibitor lithium chloride enhances activation of Wnt canonical signaling and osteoblast differentiation on hydrophilic titanium surfaces.

AIMS: Promoting bone formation at the tissue interface is an important step to improve implant success. This study investigated whether stimulation of Wnt signaling by GSK3b inhibitor lithium chloride (LiCl) could affect the response of mesenchymal or osteoblastic cells growing on titanium surfaces with different topography and wettability, and improve their differentiation along the osteoblastic lineage. MATERIAL AND METHODS: Murine mesenchymal C2C12 cells were plated on Pickled, acid-etched/sand-blasted (SLA), and hydrophilic SLA titanium disks (modSLA) and stimulated with increasing doses of LiCl. Cell viability was measured using chemiluminescence-based ATP quantitation and activation of Wnt canonical signaling was measured using a Luciferase-based reporter assay. Gene expression was measured using real time PCR in C2C12 cells, murine osteoblastic MC3T3 cells or murine primary bone marrow cells. RESULTS: LiCl stimulated Wnt activation and expression of Wnt markers in C2C12 cells on modSLA. Addition of 1 mM LiCl increased levels for bone marker Osteocalcin in MC3T3 cells on modSLA surfaces. Similarly, LiCl potently enhanced Osteoprotegetrin levels in MC3T3 cells on modSLA. When primary bone marrow cells were stimulated with LiCl, the expression of Wnttarget genes and osteoblastic differentiation markers was increased on modSLA surfaces. CONCLUSIONS: Stimulation of the canonical Wnt pathway promoted osteoblast differentiation on hydrophilic modSLA surfaces. Taken together, these results demonstrate that Wnt activators such as LiCl should be further tested as a possible approach to improve implant osseointegration.

The importance of WNT pathways for bone metabolism and their regulation by implant topography.

Endosseous implants are important tools to replace missing teeth or damaged tissue segments. Their clinical success depends on their integration in bone and, thus, on the response of bone cells to material and surface characteristics. Recent evidence has shown that surface topography and chemistry affect WNT signalling, a pivotal pathway for the commitment of mesenchymal progenitors to the osteoblast lineage and for bone homeostasis. WNT signalling comprises several cascades that, acting through different effectors, modulate several aspects of cell behaviour. It has been shown that cells growing on rough titanium surfaces display a different expression profile for WNT factors, and that surface features can alter the response of bone cells to WNT factors. Although the underlying mechanisms to this regulation are still poorly understood, the present review reports intriguing evidence that that cell cytoskeletal signalling is involved in activating WNT signalling in cells growing on rough implant surfaces.

Actin cytoskeleton controls activation of Wnt/ß-catenin signaling in mesenchymal cells on implant surfaces with different topographies.

Surface topography affects cell function and differentiation. It has been previously shown that rough surfaces can enhance the activation of canonical Wnt signaling, an important pathway for osteoblast differentiation and bone maintenance, but the underlying mechanisms are still poorly understood. The present paper investigates whether cytoskeletal organization contributes to regulating this pathway. Rho-associated protein kinase (ROCK), an important controller of actin microfilaments, was inhibited with 2mM specific antagonist Y-27632 in mesenchymal and osteoblastic cells growing on titanium discs with a polished or acid-etched, sand-blasted (SLA) surface. Y-27632 subverted the morphology of the cytoskeleton on polished and, to a lesser extent, on SLA surfaces, as evidenced by fluorescence microscopy. Although ROCK inhibition did not affect cell viability, it increased activation of Wnt signaling in uncommitted C2C12 mesenchymal cells on polished surfaces but not on SLA discs upon reporter assay. Consistently with this, real-time polymerase chain reaction analysis showed that MC3T3 cells on polished surfaces expressed higher mRNA levels for ß-catenin and alkaline phosphatase, a known Wnt target gene, and for the osteoblastic differentiation marker osteocalcin after ROCK inhibition. Taken together, these data demonstrate that cytoskeletal organization mediates activation of Wnt canonical signaling in cells on titanium surfaces with different topographies.

Titanium topography controls FoxO/beta-catenin signaling.

Little is known about how surface topography can modulate mesenchymal cell responses to oxygen-related stress occurring with age, or during the early phases of wound healing or inflammation. To antagonize Reactive Oxygen Species (ROS), cells resort to defense mechanisms, relying on ß-catenin, a molecular switch between a TCF-mediated pathway, which promotes cells proliferation and commitment, and an alternative one controlled by FoxO, which induces quiescence and defenses against ROS. In the present study, we show that mesenchymal C2C12 cells are protected from H2O2-induced oxidative stress when they grow on rough (SLA) titanium surfaces. The expression of anti-ROS genes and FoxO/ß-catenin signaling, as measured by a reporter assay, were increased on SLA surfaces. We also show that TCF-mediated transcription was inhibited by ROS in cells growing on either smooth or SLA titanium. Our results demonstrate that surface topography modulates cell resistance to ROS and the balance between the molecular pathways regulating cell growth and cell defense against oxidative stress.

Sleep bruxism and sleep arousal: an experimental challenge to assess the role of cyclic alternating pattern.

Rhythmic masticatory muscle activity (RMMA) is the characteristic electromyographic pattern of sleep bruxism (SB), a sleep-related motor disorder associated with sleep arousal. Sleep arousals are generally organised in a clustered mode known as the cyclic alternating pattern (CAP). CAP is the expression of sleep instability between sleep maintaining processes (phase A1) and stronger arousal processes (phases A2 and A3). This study aimed to investigate the role of sleep instability on RMMA/SB occurrence by analysing CAP and electroencephalographic (EEG) activities. The analysis was performed on the sleep recordings of 8 SB subjects and 8 controls who received sensory stimulations during sleep. Baseline and experimental nights were compared for sleep variables, CAP, and EEG spectral analyses using repeated measure ANOVAs. Overall, no differences in sleep variables and EEG spectra were found between SB subjects and controls. However, SB subjects had higher sleep instability (more phase A3) than controls (P= 0·05). The frequency of phase A3 was higher in the pre-REM sleep periods (P < 0·001), where peaks in RMMA/SB activity were also observed (P = 0·05). When sleep instability was experimentally increased by sensory stimuli, both groups showed an enhancement in EEG theta and alpha power (P = 0·04 and 0·02, respectively) and significant increases in sleep arousal and all CAP variables. No change in RMMA/SB index was found within either groups (RMMA/SB occurred in all SB subjects and only one control during the experimental night). These findings suggest that CAP phase A3 may act as a permissive window rather than a generator of RMMA/SB activity in predisposed individuals.

FoxOs, Wnts and oxidative stress-induced bone loss: new players in the periodontitis arena?

BACKGROUND AND OBJECTIVE: Chronic periodontitis is a widespread disease affecting tooth-supporting structures that can lead to extensive loss of periodontal ligament and bone, ultimately resulting in tooth loss. Extensive evidence has demonstrated a strong association between age, metabolic disorders such as type II diabetes, oxidative stress and alveolar bone loss. The molecular players controlling bone maintenance and underlying age-related bone loss and its links to the general metabolism are currently the object of intense research. MATERIAL AND METHODS: Recent findings are summarized to elucidate the molecular mechanisms linking oxidative stress, bone loss and metabolic factors. RESULTS: It is well known that reactive oxygen species are an inevitable consequence of cellular respiration and that organisms have developed an efficient array of defenses against them. The core of this complex defense line is a family of transcription factors, known as FoxOs, which can bind to ß-catenin and initiate a transcriptional programme regulating cell apoptosis, DNA repair and degradation of reactive oxygen species. An increase in reactive oxygen species due, for example, to age or insulin resistance, generates a situation in which bone formation is impaired by activation of FoxO, and a decrease in Wnt signaling and bone resorption are promoted. CONCLUSION: The balance between FoxO and the Wnt pathway is finely tuned by systemic and local factors, creating a far-reaching mechanism that dictates the fate of mesenchymal progenitors and regulates the homeostasis of bone, providing a rationale for the impairment of systemic and alveolar bone maintenance clinically observed with age and metabolic diseases.

Osteocytes and WNT: the mechanical control of bone formation.

Mechanical loading is of pivotal importance in the maintenance of skeletal homeostasis, but the players involved in the transduction of mechanical stimuli to promote bone maintenance have long remained elusive. Osteocytes, the most abundant cells in bone, possess mechanosensing appendices stretching through a system of bone canaliculi. Mechanical stimulation plays an important role in osteocyte survival and hence in the preservation of bone mechanical properties, through the maintenance of bone hydratation. Osteocytes can also control the osteoblastic differentiation of mesenchymal precursors in response to mechanical loading by modulating WNT signaling pathways, essential regulators of cell fate and commitment, through the protein sclerostin. Mutations of Sost, the sclerostin-encoding gene, have dramatic effects on the skeleton, indicating that osteocytes may act as master regulators of bone formation and localized bone remodeling. Moreover, the development of sclerostin inhibitors is opening new possibilities for bone regeneration in orthopedics and the dental field.

Tongue lesions and isolated hypoglossal nerve palsy: a case report.

We report the case of a patient with oral lesions on the upper surface of his tongue. Intraoral examination revealed marked right-sided hemiatrophy of the tongue with fasciculation, partial deviation of the tongue on the right side, and inability of the patient to completely deviate the tongue toward the left side of his mouth on protrusion. A magnetic resonance image showed presence of a tumor lesion characterized by irregular margins localized in the intradural space, with a lateral extension along the omolateral hypoglossal canal. This was consistent with the diagnosis of a hypoglossal neurinoma, later confirmed by the histopathologic examination.

Effects of local and remote muscle pain on human jaw reflexes evoked by fast stretches at different clenching levels.

Muscle pain imposes significant changes on natural motor tasks, but the consequences for stretch reflexes are still disputed. The present study examined the jaw reflexes to fast (10 ms) stretches of the mandible in an experimental model with local pain in the masseter muscle and remote pain in the tibialis anterior muscle. The stretch reflexes were elicited in healthy volunteers (n=13) before, during, and after periods with constant levels of experimental pain and while the subjects clenched at 0%, 15%, 30%, and 45% of the maximal voluntary contraction (MVC) levels. Surface electromyography (EMG) was used to record the reflex responses. Pain in the masseter muscle (mean +/- SEM, 3.8+/-0.4 on a 10-cm visual analogue scale), but not in the tibialis anterior muscle (3.4+/-0.3; paired t-test, P=0.318) was associated with significant changes in both prestimulus EMG activity (ANOVA, P=0.002) and in peak-to-peak amplitudes of the stretch reflex (ANOVA, P=0.022). However, when the changes in prestimulus EMG activity were taken into consideration a significant increase in the stretch reflex persisted in the painful muscle at 15% and 30% MVC. Local circuits at the trigeminal level involving the fusimotor system are proposed to mediate a significant part of this modulatory effect.

Conditioning of heteronymous H reflex in human temporalis muscle by stimulation of perioral afferents.

A heteronymous H reflex in the temporalis muscle can be elicited by selective stimulation of the masseteric nerve. The present study aimed at defining the optimal amplitude of the H reflex to detect inhibitory changes induced by stimulation of the perioral afferents and at providing new information on the control of masticatory muscles. Sixteen healthy volunteers participated in the experiment. A conditioning stimulus (CS) to the perioral skin was applied at various delays before an ipsilateral selective masseteric nerve stimulation (test stimulus: TS) while the subject was clenching the teeth at 25% of the maximal voluntary contraction. Two intensities of CS and TS were employed, high and low. The peak-to-peak amplitude of the H reflex (TS) and the root-mean-square value of the preceding electromyography were measured and the data analyzed by three-way analysis of variance and Tukey's posthoc tests. For both intensities used the heteronymous H reflex in the temporalis muscle was significantly decreased by prior activation of perioral afferents for delays from 5 to 60 ms. With a delay of 5 and 35 ms the preceding EMG level was not changed, while it was reduced at 20 and 60 ms delay. The intensities used to elicit the heteronymous H reflex of the temporalis muscle were appropriate to detect a reduction in motoneuron excitability. The reduction in the H reflex without a change in the preceding EMG at 5 and 35 ms delays could be due to presynaptic inhibition of the masseteric afferents exerted by the ipsilateral perioral afferents.