J. Morris-Wiman

Temporospatial distribution of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinases during murine secondary palate morphogenesis

Extracellular matrix (ECM) molecules are known to play a pivotal role in the morphogenesis of the secondary palate. The maintenance and degradation of the ECM is mediated in part by the matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs. MMPs and TIMPs have previously been shown to be developmentally regulated within the palatal shelf during secondary palate morphogenesis. This study was conducted to examine the temporospatial distribution of these enzymes and their inhibitors within the palatal shelves using immunofluorescent localization to determine if specific changes occur in their distribution concomitant with events in palatal shelf formation and reorientation. Frontal sections through the posterior palatal shelves at gestational day (gd) 12, 13 and 14 were immunofluorescently stained for MMPs 2, 3, 9, and 13 and TIMPs 1, 2, and 3 using standard protocols and commercially available antibodies. The results demonstrated that MMPs and TIMPs were already present within the palatal shelf mesenchyme 30 h prior to reorientation and closure and that their expression within the shelf mesenchyme increased as the shelves remodeled, then decreased with closure and fusion. Increased distribution of MMPs and TIMPs within specific regions of the palatal mesenchyme and palatal epithelial basement membrane preceded decreases previously observed within these areas for their substrates, fibronectin, collagen III and collagen I. In addition, MMP-3 and TIMP-3 were immunolocalized to regions of the palatal epithelium that undergo reorganization concomitant with reorientation. The results of this study indicate that MMPs and TIMPs are developmentally regulated during palatal shelf morphogenesis and that their distribution correlates with the distribution of the ECM components of the palatal shelf they regulate. These results provide support for the idea that temporospatially controlled interactions between MMPs and their substrates may be pivotal in modulating events in palatal morphogenesis.

Effects of Echistatin and an RGD Peptide on Orthodontic Tooth Movement

We tested whether orthodontic tooth movement (OTM) could be blocked by local administration of echistatin or an arginine-glycine-aspartic acid (RGD) peptide, agents known to perturb bone remodeling, adjacent to maxillary molars in rats. These molecules were incorporated into ethylene-vinyl acetate (ELVAX), a non-biodegradable, sustained-release polymer. In vitro experiments showed that the echistatin and RGD peptide were released from ELVAX in active forms at levels sufficient to disrupt osteoclasts. Biotinylated RGD peptide was released from ELVAX into the PDL after surgical implantation. ELVAX loaded with either RGD peptide or echistatin and surgically implanted next to the maxillary molars inhibited orthodontic tooth movement (p < 0.01). The RGD peptide also reduced molar drift (p < 0.05). This study shows the feasibility of using ELVAX to deliver integrin inhibitors adjacent to teeth to limit local tooth movement in response to orthodontic forces.

Spatial Distribution of Myosin Heavy-chain Isoforms in Mouse Masseter

There is a paucity of information regarding the anatomy and muscle fiber phenotype of the masseter. The objective of this study was to characterize the distribution of each myosin heavy-chain (MyHC) isoform within different anatomical regions of male and female mouse masseters. Masseters from male and female CD-1 mice (2-4 months old) were examined for description of the anatomical partitioning of muscle fibers and endplate distribution. The spatial distribution of MyHC isoforms--embryonic, neonatal, slow, alpha-cardiac, IIa, and IIb--was determined within the defined masseter partitions by means of Western blot analysis and immunofluorescent localization. Types IIa, IIx, and IIb were the predominant MyHC isoforms observed. Distinct differences in the spatial distribution of these MyHC isoforms were found between muscle regions and varied between sexes. The regionalization of muscle fiber types in the mouse masseter is consistent with the functional compartmentalization of the masseter observed in other species.

Rapid changes in the extracellular matrix accompany in vitro palatal shelf remodelling

The sequence of events and the distribution of extracellular matrix (ECM) components was examined during mouse secondary palatal shelf elevation in an in vitro model using standard roller tube culture methods developed for the culture of early embryos. In this culture system, the morphological changes associated with remodelling and reorientation of the palatal shelves of gestational day 13 mouse fetuses were similar to those observed in vivo. However, in specimens explanted 24–30 h prior to reorientation in vivo, remodelling began rapidly after explantation, and significant reorientation was accomplished within 4 h. Midline contact between the shelves did not occur until after 18 h in vitro, concomitant with shelf growth. Therefore, in this in vitro model, events related to palatal shelf remodelling and reorientation can be distinguished from those related to shelf growth. We used this in vitro model to characterize the transient changes in ECM distribution and accumulation that occur concomitant with events in shelf remodelling. Our results show that, during rapid remodelling in vitro, the relative distributions of collagen III, fibronectin and hyaluronate, as visualized by immunofluorescent staining, decreased within specific regions of the mesenchymal compartment. In contrast, the distribution of collagen I within the mesenchyme increased, and the distribution of tenascin did not change significantly. All molecules examined, except tenascin, showed changes in distribution within the basement membrane. These patterns of distribution are similar to those observed during more gradual remodelling in vivo. During remodelling in vitro, the deposition of [3H]-glucosamine- and [3H]-proline-labelled components of the ECM, as visualized by autoradiography, was greatest during the first 3 h of culture. During this period, labelled ECM accumulated within specific regions of the mesenchyme and palatal epithelial basement membrane. Uptake was reduced dramatically during the subsequent 3 h in culture and was restricted mainly to the palatal epithelium and its underlying basement membrane. The in vitro system permitted the characterization of early events in shelf remodelling leading to reorientation. Results suggest that remodelling is accompanied by rapid, local accumulation of ECM in specific regions of the palatal shelf previously thought to play a role in the process.

Limb, Respiratory and Masticatory Muscle Compartmentalization: Developmental and Hormonal Considerations

Neuromuscular compartments are subvolumes of muscle that have unique biomechanical actions and can be activated singly or in groups to perform the necessary task. Besides unique biomechanical actions, other evidence that supports the neuromuscular compartmentalization of muscles includes segmental reflexes that preferentially excite motoneurons from the same compartment, proportions of motor unit types that differ among compartments, and a central partitioning of motoneurons that innervate each compartment. The current knowledge regarding neuromuscular compartments in representative muscles involved in locomotion, respiration, and mastication is presented to compare and contrast these different motor systems. Developmental features of neuromuscular compartment formation in these three motor systems are reviewed to identify when these compartments are formed, their innervation patterns, and the process of refinement to achieve the adult phenotype. Finally, the role of androgen modulation of neuromuscular compartment maturation in representative muscles of these motor systems is reviewed and the impact of testosterone on specific myosin heavy chain fiber types is discussed based on recent data. In summary, neuromuscular compartments are pre-patterned output elements in muscle that undergo refinement of compartment boundaries and muscle fiber phenotype during maturation. Further studies are needed to understand how these output elements are selectively controlled during locomotion, respiration, and mastication.

An in vitro model for the study of taste papillae morphogenesis using branchial arch explants.

It is generally accepted that innervation is required for the maintenance of taste papillae and taste buds, but it is not entirely clear what role, if any, innervation plays in papillae and taste bud formation. Events in taste papillae formation and differentiation take place almost entirely in utero and, therefore, the study of the role of innervation in these events requires a suitable in vitro model. In the past, investigators have made use of various culture techniques to study mammalian taste papillae development in vitro and the role of innervation in this process with varying success. All of these models examined papillae development in isolated tongue or tongue fragments and have lacked the ability to manipulate the innervation of developing taste papillae in these explants. We have established a protocol for an in vitro model of taste papillae morphogenesis using branchial arch explants and roller tube culture methodology. Our results demonstrate that this model supports the morphogenesis of the circumvallate papilla with an integrated nerve. In addition, the use of branchial arch explants allows the inclusion or exclusion of geniculate and petrosal ganglia to examine directly the effects of the presence or absence of innervation on papillae formation and maintenance.

An in vitro model for the study of the role of innervation in circumvallate papillae morphogenesis.

The following study was done to demonstrate the reliability of an in vitro model for use in the study of early events and the role of innervation in mouse circumvallate papillae development. Gestational day (gd)-11 fetuses were partially dissected to produce explants that included the mandibular, hyoid, third and fourth branchial arches and their ganglia. In ganglionectomized explants, the nodose ganglia and either the geniculate, petrosal or both ganglia were removed. Explants were cultivated in roller tube culture for 24, 48, 72, and 96 h of culture and examined for the presence of papillary structures. Innervation was verified by immunostaining for neural cell adhesion molecule (NCAM). In all control explants, circumvallate papillae had formed by 72 h in culture. These papillae were innervated by fibers originating in petrosal or nodose ganglia, although, in a small number, fibers from the geniculate also contributed. Circumvallate papillae also formed in some explants in which either the geniculate or petrosal ganglia had been removed. However, placodal structures failed to mature into papillary structures even by 96 h in explants in which both ganglia had been removed. Our results demonstrate that an in vitro model using branchial arch explants supports the morphogenesis of an epithelial placode through the formation of a definite papillary structure, the circumvallate papilla, with an integrated nerve. Our results also indicate that, whereas the initial stages in gustatory papillae formation, the formation of a placode, are nerve-independent, the maturation of the placodal structure to form a papilla requires the presence of an intact nerve.

Increased vertical dimension effects on masseter muscle fiber phenotype during maturation.

OBJECTIVE To determine changes in mouse myosin heavy chain (MyHC) protein expression that may occur with a clinically relevant vertical dimension of occlusion (VDO) increase. MATERIALS AND METHODS Six CD-1 male mice (age: 6 weeks) underwent a 10% bite opening to replicate the clinical condition using composite on the maxillary molars and were compared to six age-matched controls. Mice were sacrificed at day 7 and 14 after bite opening. A representative masseter transverse cryosection from each animal was examined in selected sampling regions (anterior, posterior, posterior-deep, and posterior-intermediate) to assay fiber phenotype proportions and fiber size. RESULTS In control masseter muscles, the proportion of muscle fibers containing MyHC IIb increased in the posterior-intermediate and posterior-deep regions between 7 and 14 days (ANOVA, P < .05). The increase in the proportion of MyHC IIb fibers in the bite opening group did not occur when compared to the control group (P < .05). In addition, after 14 days of bite opening, the proportion of fibers positive for MyHC IIa was decreased in the anterior region compared to control masseter muscles. Muscle fiber diameter remained unchanged in both groups (experimental and control) and over time (P > .10). CONCLUSION These data are consistent with a selective plasticity of the expression of MyHC IIb protein in the deep regions of the male masseter muscle in response to a clinically relevant VDO increase.