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.

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.

Occlusal contacts during lateral excursions in children with primary dentition.

The presence of non-working occlusal contacts in adults is considered abnormal and may initiate parafunctional activity. Few studies have looked for non-working occlusal contacts in children with primary dentition. The purposes of this study were (1) to prove the existence of non-working-side occlusal contacts, and (2) to quantify their area during lateral excursion in children with primary dentition. To achieve this purpose, we developed a measurement system that combined a tracking system for mandibular movements with a three-dimensional digitizer for tooth shape. Ten children were selected for this study. Estimated occlusal contact area of the primary second molar on the non-working side was 0.8 mm2, in contrast to 2.0 mm2 on the working side, at 3.0 mm of movement of the lower incisor. All children examined had some occlusal contacts on the non-working side during the first part of lateral excursion.

Quantification of human chewing-cycle kinematics.

This study introduces new methods of quantifying and evaluating the human chewing cycle. These methods were validated on a sample of 26 young adults (11 women and 15 men) between 20-35 years of age. Movements of the mandibular central incisors were recorded (100 Hz) using an optoelectric computer system while the participants chewed gum. A subsample of 10 cycles was automatically selected, based on multiple objective criteria to ensure representative cycles for each individual. Once representative cycles had been identified, multilevel statistical models were used to evaluate and describe the sample's kinematic patterns. The multilevel procedures allow for missing observations, they do not assume equal intervals, and variation can be partitioned hierarchically. Two-level models showed significantly shorter cycle duration for males (835 msec) than females (973 msec). Inferior-superior (IS) cycle range was 2.6 mm larger and maximum IS velocity was 19.6 mm/sec faster in males than females. There were no significant differences in medial-lateral (ML) and anteroposterior (AP) excursive ranges or velocities. With the exception of cycle duration and ML ranges of motion, random variation was three to five times larger between individuals than between cycles. The three-level models showed that eighth-order polynomials were necessary to describe IS, AP, and ML chewing movements of the entire cycle. The models identified highly significant sex differences in cycle kinematics (excursions, velocities, accelerations, etc.) for each aspect of movement (AP, IS, and ML). It is concluded that this approach provides several important advantages over existing methods, including (a) its objectivity, (b) a more complete description of kinematic patterns, (c) a hierarchical description of variation, and (d) its ability to test hypotheses statistically.

Associations between incisor and mandibular condylar movements during maximum mouth opening in humans.

This study evaluated the common clinical assumption that condylar translation and mouth opening at the incisor are closely related. The sample studied comprised 27 adult females (23-35 years), selected for normal temporomandibular function, occlusion, and skeletal patterns. Condylar and mandibular central incisor movements (straight-line distances and curvilinear pathways) were recorded in three dimensions (3D) for 20 s with an optoelectric (Optotrak(R)) jaw-tracking system while each participant performed multiple maximum opening cycles. Masticatory analysis and multilevel statistical programs computed the 3D movements of the incisors and condylar hinge axis during opening and closing. The incisor point moved an average straight-line distance of 46.6 mm during opening and 45.9 mm during closing; the lengths of the opening and closing curvilinear pathways were 48.6 and 47.7 mm, respectively. The condyles moved average straight-line distances of 11.9 and 12.2 mm during opening and closing, respectively. The condyles' curvilinear pathways during opening and closing were 14 and 14.6 mm, respectively. Ranges of condylar movement varied widely between individuals. The straight-line distances and curvilinear pathways were highly correlated for the incisors (R=0.98) and the condyles (R=0.98). Neither the straight-line distances nor curvilinear pathways of the incisors were correlated with those of the condyles. Incisor straight-line distances and curvilinear pathways were moderately correlated with mandibular rotation (R(between subjects)=0.82 and R(between repeats)=0.88). During repeated openings, both condylar and incisor excursions increased, but during repeated closings only incisor excursions increased. It is concluded that (1) maximum incisor opening does not provide reliable information about condylar translation and its use as a diagnostic indicator of condylar movement should be limited, (2) healthy individuals may perform normal opening with highly variable amounts of condylar translation, (3) the straight-line distances of the incisor and condyles provide adequate information about the length of the curvilinear pathway, and (4) variation in maximum incisor opening is largely explained by variation in the amount of mandibular rotation.

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.

Histologic distribution of insulin and glucagon receptors.

Insulin and glucagon are the hormonal polypeptides secreted by the B and A cells of the endocrine pancreas, respectively. Their major physiologic effects are regulation of carbohydrate metabolism, but they have opposite effects. Insulin and glucagon have various physiologic roles, in addition to the regulation of carbohydrate metabolism. The physiologic effects of insulin and glucagon on the cell are initiated by the binding of each hormone to receptors on the target cells. Morphologic studies may be useful for relating biochemical, physiologic, and pharmacologic information on the receptors to an anatomic background. Receptor radioautography techniques using radioligands to label specific insulin and glucagon receptors have been successfully applied to many tissues and organs. In this review, current knowledge of the histologic distribution of insulin and glucagon receptors is presented with a brief description of receptor radioautography techniques.

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.

Translation and rotation of the mandible during habitual mouth opening movement in children with anterior reverse bite in the primary dentition.

Translation and rotation of the mandible during habitual mouth opening movements were studied in 13 children with skeletal-based anterior reverse bite (reverse bite group) and in 13 children with normal occlusion (normal occlusion group) whose dental stage was the primary dentition. Movements were recorded by an opto-electronic movement-analyzing system that could measure mandibular movements with six degrees of freedom. Inferior translation of the mandible was analyzed at the left primary central incisor, both of the primary canines, and both of the primary second molars. Anterior translation of the mandible was analyzed at both of the condyles. Rotation of the mandible was measured in the sagittal plane. The results showed that the associations between the translation and rotation of the mandible during habitual mouth opening in the reverse bite group differed from those in the normal occlusion group. The reverse bite group had greater anterior translation of both of the condylar points than did the normal occlusion group. No significant differences were found in the inferior translation or rotation of the mandible between the two groups.

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.

Changes in the spine density on apical dendrites of pyramidal neurons in the motor area of the cerebral cortex after callosotomy: a study by a modified Golgi-Cox method in the mouse.

Changes in the number of spines on apical dendrites of pyramidal neurons in layers III and V of the motor area of the cerebral cortex were examined in the young adult mice (30-60 days old) by a modified Golgi-Cox method and laser scanning microscopy on the 1st, 6th, 10th and 12th days after callosotomy. The anterior part of the callosal body, including rostrum, genu and truncus corporis callosi, was sectioned with a razor blade. The numbers of spines on apical dendrites and their oblique branches of pyramidal neurons were counted at the level of layer II for layer III pyramidal neurons and at the level of layer III for layer V pyramidal neurons. The density of spines was increased on all these dendritic parts during 10 days after callosotomy; the diameters of spine stems and dendrites were also increased. These changes, however, appeared to be transient as they were decreased almost to normal level on the 12th day after callosotomy.

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.

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.