In-process monitoring of a tissue-engineered oral mucosa fabricated on a micropatterned collagen scaffold: use of optical coherence tomography for quality control.

Background: We previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. Methods: A picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. Findings: Our moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. Conclusion: These findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

Heterogenous GABA(B) receptor-mediated pathways are involved in the local GABAergic system of the rat trigeminal ganglion: possible involvement of KCTD proteins.

It is well known that Gamma-aminobutyric acid (GABA) plays an important role in signal transduction in the central nervous system. However, the function of GABA in the peripheral nervous system, including sensory ganglions, is still unclear. In this study we have characterized the expression, cellular distribution, and function of GABA(B) receptor subunits, and the recently discovered GABA(B) auxiliary subunits, K(+) channel tetramerization domain-containing (KCTD) proteins, in rat trigeminal ganglion (TG) neuronal cells, which are devoid of synapses. We found heterogeneous expression of both GABA(B1) and GABA(B2) subunits, and a near-plasma membrane localization of KCTD12. In addition, we found that GABA(B2) subunits correlated with KCTD16. Whole-cell current-clamp recordings showed that responses to the GABA(B) receptor agonist, baclofen, were variable and both increases and decreases in excitability were observed. This correlated with observed differences in voltage-dependent K(+) current responses to baclofen in voltage-clamped TG neuronal cells. The functional diversity of the GABA(B)ergic regulation on the excitability of the TG neuronal cell bodies could be due to the heterogenous expression of KCTD proteins, and subsequent regulation of plasma membrane K(+) channels. Taken together with our previous demonstration of a local GABA(A) receptor-mediated system in rat TG, we provide an updated GABAergic model in the rat TG that incorporates both GABA(A)- and GABA(B)-receptor systems.

Association between mouth opening and upper body movement with intake of different-size food pieces during eating.

Head rotation is coordinated with mandibular movement during mouth opening, and the range of head rotation and mouth opening change with food size. However, past research did not include upper body movement, and no reports have related head and mandibular movement during realistic eating. The purpose of this study was to analyse head and mandibular movements with intake of different-sized food pieces during realistic eating. The test food consisted of apple cut into two different cube sizes (10mm and 20mm). Head and mandibular movements of 20 healthy young adults eating the apple pieces were simultaneously recorded in three dimensions by a wireless opto-electronic system. Reflective markers were attached to the upper lip and chin to measure the mouth opening range. Five markers were attached to eyeglasses frames to measure linear motion and rotation of the head. One marker was attached to the jugular notch of the sternum to measure linear motion of the upper body. Linear motion, and the inclination angle of the head and upper body, and mouth opening range were compared during intake of different-sized apple pieces. Mouth opening, head-neck rotation angle and the amount of upper body forward translation and inclination increased with larger apple pieces. However, isolated relative head motion was stabilized. We conclude that upper body forward motion and head-neck rotation assist mouth opening whilst stabilizing head orientation, and that the range of head-neck rotation angle, upper body translation and range of mouth opening change with food size during realistic eating.

Head motion may help mouth opening in children.

Concomitant head and mandibular movement during jaw function is well known in adults; however, its importance in children has not been studied. The brain attains 85-90% of its adult weight at 5 years of age, though the maximum rate of condylar growth is attained at approximately 14 years of age. These findings suggest that the coordination of the head and mandible may differ between children and adults. This study investigated head and mandibular movements of 19 children with complete primary dentition (average age: 5 years 5 months) and compared their functional integration of jaw and head movements to those of 16 female adults (average age: 20 years 3 months) with permanent dentition. Although the mandibular opening distance was significantly greater in the adults, the magnitude of concomitant head motion was greater in children. The results suggest that head extension in children helps increase the magnitude of mouth opening more than in adult women.

Is the reverse cycle during chewing abnormal in children with primary dentition?

The purpose of this study was to clarify whether the reverse cycle during chewing is abnormal in children with primary dentition. Children with normal primary dentition (N = 23; 5.5 +/- 0.8 years) and female adults (N = 25; 20.7 +/- 1.9 years) participated in this study. Chewing movement was recorded using an opto-electronic analysis system with six degrees-of-freedom (TRI-MET, Tokyo-shizaisha; Tokyo, Japan). Each subject was given a stick of chewing gum (new TRIDENT, WARNER-LAMBERT CO., Morris Plains, NJ, USA) and instructed to chew it normally. Starting from the maximum intercuspal position, each chewing sequence was recorded over a period of 20 s. A custom computer program identified individual chewing cycles. Frontal views were used to distinguish seven types of cycle shapes according to Ahlgren (Acta Odontol Scand, 24, 1966, 1-109; Acta Odontol Scand, 25, 1967, 3-13). Multilevel statistical models evaluated differences between children and adults. In the adults, type II cycles that initially opened towards the balancing side and then moved towards the working side were the most common, and the closing pathway was lateral to the opening pathway. In the children, type V (reverse) cycles that opened towards the working side were most common, the closing pathway was medial to the opening pathway, and there was a smaller lateral component during opening and closing than in adults (P < 0.05). We conclude that the reverse cycle is not abnormal because normal children with primary dentition have a smaller lateral component and difficulty in controlling asymmetric muscle activity.

Correlations between incisor and condylar movements during lateral excursion in children with primary dentition.

The purpose of this study was to look for associations between lower incisor movement and working and balancing condylar movement during lateral excursion in children with primary dentition and adults with permanent dentition. Mandibular movement was recorded using a TRI-MET (an optoelectronic analysis system with six degrees-of-freedom) at a sampling frequency of 100 Hz. The movement data was transferred to a graphics workstation for analysis. Subjects were categorized by age into two groups. The primary dentition group consisted of 19 children (mean age: 5 years and 5 months, s.d.: 8.7 months), and the permanent dentition group consisted of 22 women (mean age: 20 years and 5 months, s.d.: 26.3 months). The occlusion and TMJ in both groups were normal, with no history of orthodontic treatment. Three orthogonal excursive ranges and the 3D linear distance of the incisal and balancing and working condylar points, along with inter- and intra-individual correlations between the incisor and the balancing and working condyles during lateral excursion, were estimated by using multilevel statistical models. Lateral excursion in children with primary dentition was characterized by smaller incisor excursive ranges and 3D linear distance than in adults, and stronger inter- and intra-individual correlations between incisor and balancing condylar movements than in adults. In both children and adults the lateral excursion of the incisor was a good indicator of the extent of balancing condylar movement, but not working condylar movement.

A local GABAergic system within rat trigeminal ganglion cells.

We investigated the GABAergic system within the Sprague-Dawley rat (2-3-weeks old) trigeminal ganglion (TG). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed expression of glutamate decarboxylase (GAD) 65 and GAD67 mRNAs and mRNAs encoding GABA(A) receptor subunits alpha1-6, beta1-3, gamma1-3, and delta. In situ hybridization revealed that GAD65 and GAD67 mRNAs were expressed in neuronal cell bodies but not satellite cells. Immunohistochemical analysis showed that only GAD65 was expressed in all neuronal cell bodies, and approximately 70% of all neurons exhibited GABA immunoreactivity. Satellite cells were strongly immunopositive for GABA. GABA(A) receptor alpha1, alpha5, beta2/3 and gamma1/2/3 subunit immunoreactivities were observed in the majority of neurons, but no immunoreactivity for alpha2 was observed. Two types of cells were identified in TG based on cell size and morphology, type A and B. The percentage of cells expressing alpha3, alpha4, alpha6, and delta subunits appeared to be dependent on cell size, as delta and alpha6 expression were only observed in small (B-type) neurons. In whole-cell patch clamp experiments, GABA application induced inward Cl- currents in all neurons examined. The EC50 for GABA varied from 5.3 to 240 microm, and the Hill Coefficient (nH) varied between 0.98 and 2.6 at -60 mV. We found that GABA was released from TG cells by increasing extracellular K+ concentration to 100 mm. We speculate that GABA acts as a nonsynaptically released diffusible neurotransmitter, which may modulate somatic inhibition of neurons within the TG.

Characteristics of the gum chewing occlusal phase in children with primary dentition.

Understanding of the growth and development of mandibular function is indispensable to the diagnosis of functional disturbances during childhood. The purpose of this study was to clarify the characteristics of the occlusal phase during gum chewing in children with primary dentition. Chewing motion at the working molar of 14 children with primary dentition and 28 female adults was recorded optoelectrically, and the frontal and sagittal angles of their closing and following opening strokes were measured and compared. In children the closing strokes were entered more vertically and anteriorly than in adults, and the opening strokes shifted to the non-working side in adults but moved to the working side in children. The degree of variance also differed between the two groups; the variance of the frontal angle was larger than that of sagittal angle in adults, but the opposite was true in children. These results suggested that the chewing pattern in children during the occlusal phase is distinctly different from adults and the chewing movement in children is not always less stable than in adults.

Length of the occlusal glide during chewing in children with primary dentition.

Chewing is one of the most important functions of the mandible, but, to date, there are very few studies of this function in children. The purpose of this study was to quantify the length of the occlusal glide at the lower incisal point during gum chewing in children with primary dentition. Eleven girls with primary dentition were selected for this study. Mandibular excursions with occlusal contacts and gum chewing movement were measured using an optoelectronic system that can measure mandibular movement with six degrees-of-freedom at a sampling frequency of 100 Hz. A curved mesh diagram of incisor coordinates during mandibular excursions was established to calculate the length of the occlusal glide for each subject. The occlusal glide lengths of children were compared with previously reported results for adults. The estimated length of the occlusal glide during closing was significantly shorter in children than in adults, contrary to that during opening. This result suggests that children have a characteristic chewing pattern that differs from adults.

Occlusal phase of gum-chewing strokes.

The occlusal phase of chewing is especially interesting because food particles are being pulverized in this phase. For efficient chewing the upper and lower teeth must come together in a congruent fashion with less variation than in other phases. To examine this expectation the chewing motions of 28 women were recorded optoelectrically, and their frontal and sagittal angles of the closing and following opening strokes were measured at 3.0 mm (3-D linear distance) of opening. Closing strokes were more stable than opening strokes. The frontal angle was correlated with the sagittal angle during closing. The opening and closing sagittal angles were moderately correlated, and the opening and closing frontal angles were negatively correlated at the intersubject level. No direct association was found between the closing strokes and following opening strokes at the intra-subject level. These results suggest that closing strokes are more stable than opening strokes, resulting in efficient mastication.

Longitudinal observation of basic mandibular movements: report of a case.

Sound development of mandibular function during childhood is indispensable to establishing healthy function in adults. To examine this developmental process, longitudinal recordings of basic mandibular movements were done using an optoelectronic analysis. Mandibular movements were recorded on five separate occasions in one boy, from an age of six years and five months to 14 years and five months. The incisor pathways during protrusion and lateral excursion were initially shallow, with more anterior than inferior movement, but as he grew the amount of inferior movement and the amount of rotation both increased. Similarly, at his first recording there was very little hinge-like rotation during mouth closing, but rotation increased markedly after eruption of his permanent second molars. These findings suggest that mandibular movements change from being relatively simple with more translation in younger children to more complex movements with more rotation once the permanent dentition is established.

Alveolar bone loss associated with glucose tolerance in Japanese men.

AIMS: Type 2 diabetes is known to affect alveolar bone loss (ABL). The purpose of this study was to examine whether impaired glucose tolerance (IGT) is associated with ABL, as is diabetes. METHODS: A case-control study was performed with 664 Japanese men aged 46-57 years. Panoramic radiographs revealed 513 severe ABL cases, 22 moderate ABL cases, and 129 controls with good alveolar bone. Diabetes status was classified into normal glucose tolerance (NGT), impaired fasting glucose (IFG), IGT, and newly diagnosed diabetes according to the fasting plasma glucose and 75-g oral glucose tolerance test (OGTT). Diabetes under treatment was excluded. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained from ordinal logistic regression analysis. Statistical adjustment was made for total cholesterol, HDL-cholesterol, triglyceride, rank in the Self Defence Forces (SDF), cigarette-years, alcohol use, body mass index, previous 10 years' brushing habits and instrument use other than toothbrush, and history of periodontal treatment. RESULTS: A significant, approximately three-fold increase in the crude OR (crude OR=3.28; 95% CI=1.16-9.27) and non-significant 2.6-fold increase in the adjusted OR (adjusted OR=2.55; 95% CI=0.86, 7.54) of ABL was observed among men with newly diagnosed Type 2 diabetes compared with the reference group (NGT combined with IFG). However, there was no association between IGT and ABL (adjusted OR=0.99; 95% CI=0.59,1.64). CONCLUSIONS: Type 2 diabetes, but not IGT, was positively associated with ABL. Preventive maintenance against periodontitis is important in middle-aged men with diabetes.

Length of the occlusal glide at the lower incisal point during chewing.

The purpose of this study was to estimate the length of the occlusal glide during gum chewing at the lower incisal point. Mandibular excursions with occlusal contacts in 25 females with permanent dentition were recorded using an optoelectronic system that can measure mandibular movement with 6 d.f. at a sampling frequency of 100 Hz. A curved mesh diagram of incisor coordinates during maximum mandibular excursions (CMDME) was plotted. Gum chewing movement was also measured using the same system and computer software which divided the chewing movement into cycles at each maximum opening position. Each cycle was standardized at 0.1 mm intervals from the most closed position. Finally, the distance between the CMDME and each position of the incisor during a chewing cycle was calculated. Whenever, this distance was less than 0.2 mm opposing teeth were considered to be in contact. The occlusal glide was defined as the distance travelled by the lower incisal point as the mandible moved along occlusal contacts of the CMDME. The vertical coordinates at the beginning of the occlusal glide (during closing) and at the end of occlusal glide (during opening) were also calculated. The lengths of the occlusal glide pathway averaged 1.29 mm during closing and 1.55 mm during opening, a total length of 2.84 mm. Mean vertical coordinates at the beginning and end of the glide were -0.95 and -1.12 mm from intercuspal position, respectively.

The effects of bolus hardness on masticatory kinematics.

This study investigated how jaw kinematics, including cycle duration, three-dimensional (3-D) excursive ranges and velocities, and cycle shape, changed with increasing hardness of chewing gum. Twenty-six subjects (13 males and 13 females; mean age 23.6 +/- 2.5 years) with Class I normal occlusion were asked to chew two brands of gum with differing hardness. Jaw motion during chewing was tracked with an Optotrak camera at 100 Hz, and all movements were recorded as pure 3-D mandibular movements relative to Frankfort horizontal. Cycle duration did not change significantly with harder gum, but 3-D excursive ranges and velocities increased, except during the occlusal phases of the chewing. Cycle shape was similar for hard and soft gum, but the overall size of the cycle was larger with hard gum. These results suggest that greater muscular effort when chewing harder gum produces a greater acceleration of the mandible in all phases except when the harder gum slows the mandible during the occlusal phases.

Incisal and condylar paths during habitual mouth opening movement of children with anterior reverse bite in the primary dentition.

The purpose of this study was to evaluate whether the path of habitual mouth opening was influenced by anterior reverse bite malocclusion in children. Two groups of children with the primary dentition were chosen: (i) 10 children with anterior reverse bite (reverse bite group) and (ii) 10 with normal occlusion (normal occlusion group). Their habitual mouth opening movements were measured by an opto-electronic movement-analysing system with 6 d.f. at a sampling rate of 100 Hz. These movements were projected on the sagittal plane at the incisal and condylar points. To characterize the path, we used the angles between the path and the horizontal plane. In the initial stage of opening, the incisal path of the reverse bite group was displaced more posteriorly from the intercuspal position than that of the normal occlusion group. However, as opening continued, the direction of the path of the reverse bite group was more anterior than those of the normal occlusion group. During the entire opening movement, the condylar path of the reverse bite group was flatter than those of the normal occlusion group. In conclusion, it was demonstrated that anterior reverse bite in the primary dentition clearly affects the paths of habitual opening movements.

Changes in the masticatory cycle following treatment of posterior unilateral crossbite in children.

In a previous study, we established that young children with unilateral posterior crossbite have a longer mandibular ramus and more superiorly and posteriorly positioned condyles on the crossbite side. In this study, we evaluated chewing cycle shape and duration in 14 of the patients before treatment, and we looked for changes in cycle shape and duration 6 months after treatment with rapid palatal expansion. Mandibular kinematics was recorded while chewing gum using an optoelectric recording system at 100 Hz. Subjects were asked to chew normally for 20 cycles, chew on the crossbite side only for 20 cycles, and chew on the noncrossbite side only for 20 cycles. A special computer program selected the 10 most representative cycles from each series and computed an average duration and an average maximum excursion along 3 orthogonal axes. Multilevel linear models were used to generate an 8th-order polynomial describing average cycle shape and to test for statistically significant differences in shape between the patients and the controls and between the patients before and after treatment. Before treatment, the patients chewed more slowly than did the controls. Treatment shortened their cycle duration to equal control values. Before treatment, the patients also had larger maximum excursions than did the controls and exhibited a reverse-sequence cycle shape when chewing on the crossbite side. Treatment did not alter the patients' abnormal cycle shape. These results suggest that some features of the masticatory kinematics respond to orthodontic treatment alone, but others do not.

Microautoradiographic studies of glucose uptake in skeletal muscle fibers at rest.

In the present study, differences in glucose uptake by muscle fibers in deep, middle, and superficial regions of the gastrocnemius were studied at rest by 2-deoxyglucose (2-DG) microautoradiography. Expression of the glucose transporter 4 (GLUT-4) protein, an isoform of the glucose transporter family, was analyzed as well. These data were compared with the activity of succinate dehydrogenase, a marker of oxidative metabolism, a-glycerophosphate dehydrogenase, an indicator of the glycolytic capacity, and myofibrillar ATPase. In the deep regions of the muscle, most fibers (86.9%) showed high 2-DG uptake and large amounts of GLUT-4 protein, whereas in the superficial regions, all fibers showed low 2-DG uptake and GLUT-4 expression. In the middle regions, fibers dominated (80.4%) showed low 2-DG uptake and small amounts of GLUT-4 protein. Analysis of metabolic properties revealed that most fibers in the deep region were oxidative and showed the highest 2-DG uptake; in the superficial region, the fibers were anaerobic and showed the lowest 2-DG uptake. In the middle region, most fibers were of the anaerobic and fast twitch type. It is concluded that 2-DG uptake correlates with GLUT-4 expression in the plasma membrane of type I and IIx fibers rather than with oxidative enzyme activity.

Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice.

The epiphyseal growth plate, where chondrocytes proliferate and differentiate, is the major site for longitudinal bone growth, matrix synthesis and mineralization. Glucose is an important energy source for the metabolism and growth of chondrocytes. The family of facilitative glucose transporters (GLUTs) mediates glucose transport across the plasma membrane in mammalian cells. We used immunocytochemical methods with anti-GLUT antibodies to investigate the localization of GLUTs in chondrocytes of the epiphyseal growth plate in 3 age groups of rats (3, 7, and 28 days after birth). Intense immunoreactivity of GLUT isoforms 1-5 was detected in chondrocytes of 3-day and 7-day old rats, and all GLUTs were localized in the maturation zone of the hypertrophic zone. On postnatal day 28, chondrocytes in the maturation zone showed intense GLUT1, 4 and 5 immunoreactivity, and weak GLUT2 and 3 immunoreactivity. In addition to chondrocytes in the maturation zone, those in the degenerative zone and in the zone of provisional calcification showed strong GLUT4 and 5 immunoreactivity. Autoradiography of bone sections from 4-week old mice injected with 14C-2-deoxyglucose showed high silver grain density within matrix tissue in the reserve and proliferative zones but not around chondrocytes. However, in the hypertrophic zone, silver grain density was high in matrix and chondrocytes. These data indicate that chondrocytes in the hypertrophic zones use glucose as energy source. High levels of GLUT4 expression imply that glucose use in chondrocytes is regulated by insulin. Expression of GLUT5 in chondrocytes suggests that fructose is also used as an energy source.

The effects of chewing rates on mandibular kinematics.

The purpose of this study is to provide basic understanding of how the speed of chewing affects masticatory jaw kinematics. Twenty-six healthy subjects (23.6 +/- 2.5 years of age) chewed a standardized bolus of gum at fast (100 cycles s-1), habitual and slow (50 cycles s-1) rates. The rates were controlled with a metronome and the order of rates was randomized for each subject. An optoelectrical system independently recorded head and jaw movement. Special computer programs identified representative cycles for each subject and computed various aspects of jaw movement. Multilevel statistical procedures were used to compare cycle variables among the three rates, estimate variability and model jaw movements. Maximum ranges of anteroposterior (AP), vertical and lateral jaw excursions were significantly less for the fast than the habitual or slow rates. While the shape of 3-D pathway was similar for the three rates, the perimeter of the pathway was significantly shorter for fast chewing cycles. Maximum AP, vertical, lateral and total 3-D jaw velocities were significantly different among the three rates. Between cycle variation in cycle duration and jaw excursion were least during fast chewing and the greatest during slow chewing; variability in maximum velocity was similar for the three rates.

Incisor and mandibular condylar movements of young adult females during maximum protrusion and lateratrusion of the jaw.

This study evaluated the correlations between condylar translation and incisor movements during maximum protrusion and lateratrusion. The sample was 27 adult females (23--35 years old), selected for normal temporomandibular function, occlusion, and skeletal patterns. Condylar and mandibular central incisor movements [linear distances (LD) and curvilinear pathways (CP)] were recorded in three dimensions for 20 s with an optoelectric (Optotrak) jaw-tracking system while each participant performed multiple maximum protrusive and lateratrusive cycles. Masticatory analysis and multilevel statistical programs computed the three-dimensional movements of the incisors and condylar hinge axis during protrusion and lateratrusion. CP of the incisor point averaged 12.0 mm (9.3 mm LD) during protrusion, 13.0 mm (11.5 mm LD) during right excursion and 12.3 mm (11.0 mm LD) during left excursion. CP of the condyles averaged 11.9--12.9 (9.2--9.5 LD) mm during protrusion. During lateratrusion the contralateral condyles moved anteroinferiorly 11.6--14.1 mm (9.5--10.2 mm LD); the ipsilateral condyles moved posterolaterally 5.8-6.8 mm (2.3--2.5 mm LD). The left condyles demonstrated more movement than the right condyles during protrusion and than the contralateral condyles during laterotrusion. Relative variation, as measured by the coefficient of variation, was greater for the movements of the ipsilateral than contralateral condyles. Incisor movements were only moderately related to condylar movements between individuals and between replicates; LDs showed stronger correlations than CPs; and correlations were stronger for lateratrusion than protrusion. While incisor and condylar movements were not affected by repeated protrusion, incisor CP (approx. 0.2 mm/cycle) and LD (approx. 0.1 mm/cycle) increased significantly with repeated excursive movements to the left and right. It was concluded that (1) incisor protrusion and lateratrusion provide moderately reliable measures of condylar translation; (2) the linear distances that the incisors move during lateratrusion provide the best measure of contralateral condylar translation; and (3) condylar movements are not affected by repeated protrusion or lateratrusion.