Sunil Wadhwa

Increased mandibular condylar growth in mice with estrogen receptor beta deficiency.

Temporomandibular joint (TMJ) disorders predominantly afflict women of childbearing age, suggesting a role for female hormones in the disease process. In long bones, estrogen acting via estrogen receptor beta (ERβ) inhibits axial skeletal growth in female mice. However, the role of ERβ in the mandibular condyle is largely unknown. We hypothesize that female ERβ‐deficient mice will have increased mandibular condylar growth compared to wild‐type (WT) female mice. This study examined female 7‐day‐old, 49‐day‐old, and 120‐day‐old WT and ERβ knockout (KO) mice. There was a significant increase in mandibular condylar cartilage thickness as a result of an increased number of cells, in the 49‐day‐old and 120‐day‐old female ERβ KO compared with WT controls. Analysis in 49‐day‐old female ERβ KO mice revealed a significant increase in collagen type X, parathyroid hormone–related protein (Pthrp), and osteoprotegerin gene expression and a significant decrease in receptor activator for nuclear factor κ B ligand (Rankl) and Indian hedgehog (Ihh) gene expression, compared with WT controls. Subchondral bone analysis revealed a significant increase in total condylar volume and a decrease in the number of osteoclasts in the 49‐day‐old ERβ KO compared with WT female mice. There was no difference in cell proliferation in condylar cartilage between the genotypes. However, there were differences in the expression of proteins that regulate the cell cycle; we found a decrease in the expression of Tieg1 and p57 in the mandibular condylar cartilage from ERβ KO mice compared with WT mice. Taken together, our results suggest that ERβ deficiency increases condylar growth in female mice by inhibiting the turnover of fibrocartilage.

Murine TMJ Loading Causes Increased Proliferation and Chondrocyte Maturation

The purpose of this study was to examine the effects of forced mouth opening on murine mandibular condylar head remodeling. We hypothesized that forced mouth opening would cause an anabolic response in the mandibular condylar cartilage. Six-week-old female C57BL/6 mice were divided into 3 groups: (1) control, (2) 0.25 N, and (3) 0.50 N of forced mouth opening. Gene expression, micro-CT, and proliferation were analyzed. 0.5 N of forced mouth opening caused a significant increase in mRNA expression of Pthrp, Sox9, and Collagen2a1, a significant increase in proliferation, and a significant increase in trabecular spacing in the subchondral bone, whereas 0.25 N of forced mouth opening did not cause any significant changes in any of the parameters examined. Forced mouth opening causes an increase in the expression of chondrocyte maturation markers and an increase in subchondral trabecular spacing.

Effect of cyclical forces on the periodontal ligament and alveolar bone remodeling during orthodontic tooth movement

OBJECTIVE To investigate the effect of externally applied cyclical (vibratory) forces on the rate of tooth movement, the structural integrity of the periodontal ligament, and alveolar bone remodeling. METHODS Twenty-six female Sprague-Dawley rats (7 weeks old) were divided into four groups: CTRL (unloaded), VBO (molars receiving a vibratory stimulus only), TMO (molars receiving an orthodontic spring only), and TMO+VB (molars receiving an orthodontic spring and the additional vibratory stimulus). In TMO and TMO+VB groups, the rat first molars were moved mesially for 2 weeks using Nickel-Titanium coil spring delivering 25 g of force. In VBO and TMO+VB groups, cyclical forces at 0.4 N and 30 Hz were applied occlusally twice a week for 10 minutes. Microfocus X-ray computed tomography analysis and tooth movement measurements were performed on the dissected rat maxillae. Tartrate-resistant acid phosphatase staining and collagen fiber assessment were performed on histological sections. RESULTS Cyclical forces significantly inhibited the amount of tooth movement. Histological analysis showed marked disorganization of the collagen fibril structure of the periodontal ligament during tooth movement. Tooth movement caused a significant increase in osteoclast parameters on the compression side of alveolar bone and a significant decrease in bone volume fraction in the molar region compared to controls. CONCLUSIONS Tooth movement was significantly inhibited by application of cyclical forces.

Effect of corticision and different force magnitudes on orthodontic tooth movement in a rat model

INTRODUCTION The aims of this study were to evaluate the effect of 2 distinct magnitudes of applied force with and without corticision (flapless corticotomy) on the rate of tooth movement and to examine the alveolar response in a rat model. METHODS A total of 44 male rats (6 weeks old) were equally divided into 4 experimental groups based on force level and surgical intervention: light force, light force with corticision, heavy force, and heavy force with corticision. The forces were delivered from the maxillary left first molar to the maxillary incisors using prefabricated 10-g (light force) or 100-g (heavy force) nickel-titanium springs. The corticision procedure was performed at appliance placement and repeated 1 week later on the mesiopalatal aspect of the maxillary left first molars, with the right sides serving as the untreated controls. Microcomputed tomography was used to evaluate tooth movement between the maxillary first and second molars, and the alveolar response in the region of the maxillary first molar on day 14. Osteoclasts and odontoclasts were quantified, and the expression of receptor activator of nuclear factor kappa ß ligand was examined. RESULTS Intragroup comparisons of bone volume fraction (BVF) and tissue density were found to be significantly less on the loaded sides, with the exception of BVF in the light force group. Intergroup comparisons evaluating magnitude of tooth movement, BVF, apparent density, and tissue density showed no significant differences. Histomorphometric analysis indicated that BVF was decreased in the light force group. No significant differences in the total numbers of osteoclasts and odontoclasts and the expression of receptor activator of nuclear factor kappa ß ligand were found between the groups. CONCLUSIONS No differences in tooth movement or alveolar response were observed with microcomputed tomography based on force level or corticision procedure. A flapless surgical insult in the mesiopalatal aspect of the first molar with a single-site corticision was unable to induce clinical or histologic changes after 2 weeks of orthodontic tooth movement regardless of the force magnitude. Histologic analysis of the furcation area showed that light force significantly decreased BVF.

Isolation and characterization of murine mandibular condylar cartilage cell populations.

Objectives: The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. Methods: Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. Results: Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. Conclusion: Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway.

Osteocyte death during orthodontic tooth movement in mice

OBJECTIVE To investigate the time course of osteocyte death in a mouse model of orthodontic tooth movement (OTM) and its association to the caspase-3 activation pathway and osteoclast formation. MATERIALS AND METHODS Twenty-five male wild type CD-1 mice (8-12 weeks old) were loaded with an orthodontic appliance. A spring delivering 10-12 g of force was placed between the right first molar and the incisor to displace the first molar mesially. The contralateral unloaded sides served as the control. The animals were equally divided into five different time points: 6, 12, 24, and 72 hours and 7 days of orthodontic loading. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3 immunostaining, and tartrate-resistant acid phosphatase (TRAP) staining was performed on histologic sections of the first molars. The labeling was quantified in osteocytes on the compression side of the alveolar bone at each time point. RESULTS TUNEL labeling significantly increased at 12, 24, and 72 hours after orthodontic loading; the peak was observed at 24 hours. Elevated caspase-3 labeling was noted at 12, 24, and 72 hours and 7 days after loading, although the increase was not significant. Significant osteoclast formation was initially evident after 72 hours and progressively increased up to 7 days. CONCLUSIONS Osteocyte death during OTM peaks at 24 hours, earlier than initial osteoclast activation. However, only a slight trend for increased caspase-3 activity suggests that other mechanisms might be involved in osteocyte death during OTM.

Early effects of orthodontic forces on osteoblast differentiation in a novel mouse organ culture model.

OBJECTIVE To develop a mouse orthodontic organ culture model and examine early-induced changes in osteoblast differentiation markers within the periodontal ligament (PDL) and alveolar bone. METHODS Mandibles from 4- to 12-week-old transgenic mice were dissected and hemisected. A conventional superelastic orthodontic spring (25 grams) was bonded to the incisor and first molar on one side of the mandible; the other side served as a control. Dissected mandibles were cultured for 6 hours and then were histologically analyzed for proliferation (BrdU immunostaining) and fluorescent protein expression. Additionally, an in vivo model using the same methods was applied to 3.6 Col1-GFP transgenic mice. RESULTS In vitro, after 6 hours of orthodontic loading, a significant increase was noted in 3.6Col1-GFP- and BSP-GFP-positive cells within the tension side of the PDL compared with unloaded controls. On the compression side, a significant decrease in positive cells in 3.6Col1-GFP mice was observed in the PDL compared with unloaded controls. In vivo, the same tendencies were found. CONCLUSION This novel in vitro mandibular tooth movement organ culture model coupled with transgenic mouse technology provides a powerful tool for delineating initial cellular and molecular events of orthodontic tooth movement.

Progenitor Cells of the Mandibular Condylar Cartilage

The secondary cartilage of the mandibular condyle is unique as it undergoes endochondral ossification during growth and robustly remodels in response to changes in its mechanical loading environment. This cartilage is derived from mesenchymal progenitor cells that express markers of early osteoblast differentiation, namely alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2). Interestingly, these progenitor cells then differentiate into cartilage with appropriate mechanical loading. Our laboratory has determined that these cells can be labeled by osteoblast progenitor cell markers, including the 3.6 fragment of the rat collagen type 1. However, the role these mesenchymal progenitor cells play in adult mandibular condylar cartilage maintenance and adaptation, as well as the existence of a more potent progenitor cell population within the mandibular condylar cartilage, remains in question. Further characterization of these cells is necessary to determine their potency and regenerative capacity to elucidate their potential for regenerative therapy.

Evaluation of BSP expression and apoptosis in the periodontal ligament during orthodontic relapse: a preliminary study

OBJECTIVE To examine the expression of bone sialoprotein (BSP) and apoptosis in an in vivo orthodontic relapse model. MATERIALS AND METHODS Male mice (10-12 weeks old), either transgenic [green fluorescent protein (GFP) driven by the BSP promoter] or wild type, were used in this study. To achieve orthodontic tooth movement (OTM), maxillary right first molars were moved mesially using closed-coil springs. Animals were divided into an OTM group (14 days continuous orthodontic force - 11 animals) or Relapse group (10 days of force application followed by 4 days of relapse - 8 animals). The control group was comprised of the contralateral maxillary molars. The periodontal ligament (PDL) was analyzed in areas of compression and tension for transgenic expression, osteoclast localization, and the presence of apoptotic cells. RESULTS There was a significant decrease in GFP-labeled cells on the compression and tension sides of the PDL in the OTM group compared with control. In the relapse group, GFP-labeled cells were significantly decreased only on the old compression side. Osteoclasts were localized on the compression side of the OTM group, whereas in the Relapse group, they were present on both sides. PDL apoptosis significantly increased on the compression side in OTM and Relapse groups. CONCLUSION Both OTM and Relapse groups exhibited a decreased number of GFP-labeled cells in areas of compression and tension. There was significant PDL apoptosis in regions under compressive forces following OTM and to a lesser extent following relapse.

Sex differences in the estrogen-dependent regulation of temporomandibular joint remodeling in altered loading

OBJECTIVE Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role of estrogen in the disease etiology. Previously, we determined that decreased occlusal loading (DOL) inhibited collagen type II (Col2) expression in the mandibular condylar cartilage (MCC) of female wild-type (WT) mice whereas no change was observed in males. This decrease in chondrogenesis was abolished by estrogen receptor beta (ERβ) deficiency in females. Therefore, the goal of this study was to examine the role of estradiol - ERβ signaling in mediating DOL effects in male mice to further decipher sex differences. METHODS Male 21 day-old WT and ERβKO male mice were treated with either placebo or estradiol and exposed to normal or DOL for 4 weeks. Cartilage thickness and cell proliferation, gene expression and immunohistochemistry of chondrogenic markers and estrogen receptor alpha (ERα), and analysis of bone histomorphometry via microCT were completed to ascertain the effect of estradiol on DOL effects to the TMJ. RESULTS ERβKO male mice lack a MCC phenotype. In both genotypes, estradiol treatment increased Col2 gene expression and trabecular thickness. DOL in combination with estradiol treatment caused a significant increase in Col2 gene expression in both genotypes. CONCLUSIONS The sex differences in DOL-induced inhibition of Col2 expression do not appear to be mediated by differences in estradiol levels between male and female mice. Greater understanding on the role of estrogen and altered loading are critical in order to decipher the sex dimorphism of TMJ disorders.