FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice.

OBJECTIVE: Fibroblast growth factors (FGFs) are a family of 22 proteins and 4 FGF receptors (FGFRs) that are crucial elements for normal development. The contribution of different FGFs and FGFRs for the homeostasis or disease of the cartilage from the mandibular condyle is unknown. Therefore, our goal was to characterize age-related alterations in the protein expression of FGF ligands and FGFRs in the mandibular condyle of mice. METHOD: Mandibular condyles of 1-, 6-, 12-, 18-, and 24-month-old C57BL/6J male mice (5 per group) were collected and histologically sectioned. Immunofluorescence for FGFs that have been reported to be relevant for chondrogenesis (FGF2, FGF8, FGF9, FGF18) as well as the activated/phosphorylated FGFRs (pFGFR1, pFGFR3) was carried out. RESULTS: FGF2 and FGF8 were strongly expressed in the cartilage and subchondral bone of 1-month-old mice, but the expression shifted mainly to the subchondral bone as mice aged. FGF18 and pFGFR3 expression was limited to the cartilage of 1-month-old mice only. Meanwhile, pFGFR1 and FGF9 were mostly limited to the cartilage with a significant increase in expression as mice aged. CONCLUSIONS: Our results indicate FGF2 and FGF8 are important growth factors for mandibular condylar cartilage growth in young mice but with limited role in the cartilage of older mice. In addition, the increased expression of pFGFR1 and FGF9 and the decreased expression of pFGFR3 and FGF18 as mice aged suggest the association of these factors with aging and osteoarthritis of the cartilage of the mandibular condyle.

Dynamic changes in transcriptome during orthodontic tooth movement.

OBJECTIVES: The objective of this study was to determine global changes in gene expression with next generation sequencing (NGS) in order to assess the biological effects of orthodontic tooth movement (OTM) on alveolar bone in a rat model. MATERIALS AND METHODS: Thirty-five Wistar rats (age 14 weeks) were used in the study. The OTM was performed using closed coil Nickel-Titanium spring to apply a mesial force on maxillary first molars of 8-10 g. Three hours, 1, 3, 7 and 14 days after the placement of the appliance, rats were killed at each time point respectively. The alveolar bone, around left maxillary first molar, were excised on compression side. The samples were immediately frozen in liquid nitrogen for subsequent RNA extraction. Total RNA samples were prepared for mRNA sequencing using the Illumina kit. RNA-Seq reads were aligned to the rat genomes using the STAR Aligner and bioinformatic analysis was performed. RESULTS: A total of 18 192 genes were determined. Day 1 has the highest number of differentially expressed genes (DEGs) observed with more upregulated than downregulated genes. A total of 2719 DEGs were identified to use as input for the algorithm. Six distinct clusters of temporal patterns were observed representing proteins that were differentially regulated indicating different expression kinetics. Principal component analysis (PCA) showed distinct clustering by time points and days 3, 7 and 14 share similar gene expression pattern. CONCLUSIONS: Distinct gene expression pattern was observed at different time points studied. Hypoxia, inflammation and bone remodelling pathways are major mechanisms behind OTM.

Anabolic Response of Intermittent Parathyroid Hormone and Alendronate on the Osteochondral Tissue of TMJ.

OBJECTIVE: To characterize the effects of parathyroid hormone (PTH) and alendronate (Alend) on the osteochondral tissue of temporomandibular joint (TMJ). MATERIALS AND METHODS: Ninety-six male and female transgenic reporter mice, 4 to 5 weeks old were divided into 6 groups: (1) Control group: Saline was injected daily for 14 days; (2) PTH: PTH was injected daily for 14 days; (3) Alend: Alend was injected every alternate days for 14 days; (4) Combined PTH and Alend: PTH was injected daily and Alend injected every alternate days for 14 days; (5) PTH then Alend: PTH was injected daily for 14 days followed by Alend injections in alternate days for 14 days; and (6) PTH wait Alend: PTH was injected daily for 14 days. There was a waiting period of 1 week before administration of Alend in alternate days for 14 days. Mice were injected with 5-ethnyl-2'-deoxyuridine (EdU), 48 and 24 hours prior to euthanization. RESULTS: There was significant increase in bone volume and decrease in osteoclastic activity in groups in which Alend was administered after PTH in both gender. There was significant increase in cartilage thickness with PTH or Alend alone in females, whereas in males, PTH alone led to increase in cartilage thickness. Chondrocyte apoptosis was significantly decreased with PTH or Alend alone in both male and female. Matrix metallopeptidase 13, and aggreganase-2 (ADAMTS5) expression were significantly decreased with PTH and Alend alone in both gender. CONCLUSION: PTH and Alend administration causes anabolic effects in the osteochondral tissue of TMJ.

Vitamin E enriched diet increases the rate of orthodontic tooth movement.

INTRODUCTION: Vitamin E is a popular antioxidant suggested to affect bone turnover. However, the effects of a vitamin E enriched diet on the rate of tooth movement are unknown. Therefore, this study aimed to evaluate tooth movement in rats receiving a vitamin E enriched diet. In addition, we examined bone remodeling in experimental and control rats. METHODS: Thirty-two 6-week-old male rats were divided into 4 groups: (1) group 1 (n = 8): orthodontic tooth movement (OTM) for 4 days + regular diet; (2) group 2 (n = 8): OTM for 14 days + regular diet; (3) group 3 (n = 8): OTM for 4 days + vitamin E diet; and (4) group 4 (n = 8) - OTM for 14 days + vitamin E diet. Maxillary alveolar bones and femurs of rats were analyzed by microcomputed tomography and histology. RESULTS: Rats fed a vitamin E diet presented an increased OTM rate at days 4 and 14. We found an increased number of osteoclasts and decreased bone volume in the vitamin E diet group at day 14 of OTM. In addition, there was increased expression of the microphthalmia-associated transcription factor in the alveolar bone of the vitamin E diet group. In contrast, there was no difference in bone remodeling in femurs or alveolar bone at the control side. CONCLUSIONS: We found that an enriched vitamin E diet increases the rate of OTM in rats, suggesting that vitamin E may be useful as an avenue to accelerate OTM.

Intermittent Parathyroid Hormone [1-34] Augments Chondrogenesis of the Mandibular Condylar Cartilage of the Temporomandibular Joint.

OBJECTIVE: To characterize the long-term effects of intermittent parathyroid hormone (I-PTH) on the mandibular condylar cartilage (MCC) and subchondral bone of the temporomandibular joint, in vivo and in vitro. MATERIALS AND METHODS: For the in vivo experiments, sixteen 10-week-old mice were divided into 2 groups: (1) I-PTH (n = 8)-subcutaneous daily injection of PTH; (2) control group (n = 8)-subcutaneous daily injection of saline solution. Experiments were carried out for 4 weeks. Mice were injected with calcein, alizarin complexone, and cell proliferation marker before euthanasia. For the in vitro experiments, primary chondrocyte cultures from the MCC of eight 10-week-old mice were treated with I-PTH for 14 days. RESULTS: There was a significant increase in bone volume, tissue density, mineral deposition, osteoclastic activity, cell proliferation in the cartilage, and cartilage thickness in the I-PTH-treated mice when compared with the control group. In addition, immunohistochemistry in cartilage revealed that I-PTH administration led to an increase in expression of vascular endothelial growth factor and to a decreased expression of sclerostin, matrix metallopeptidase 13, and aggreganase-1 (ADAM-TS4). Quantitative polymerase chain reaction analysis of the I-PTH-treated chondrocytes revealed significantly decreased relative expression of collagen type X (Col10a1), alkaline phosphatase (Alp), and Indian Hedgehog (Ihh) and remarkable increased expression of Sox9, fibroblast growth factor 2 (Fgf2), and proteoglycan 4 (Prg4). CONCLUSION: I-PTH administration causes anabolic effects at the subchondral region of the mandibular condyle while triggers anabolic and protective effects at the MCC.

The effect of differential force system and minimal surgical intervention on orthodontic tooth movement and root resorption.

OBJECTIVE: The primary objective of this study was to quantify the orthodontic tooth movement (OTM) and orthodontically induced root resorption (OIRR) with differential force system in conjunction with minimal surgical insult. MATERIAL AND METHODS: 15-week-old, 48 male Wistar rats were used in the research and were randomly divided into six groups: 1. Group 1 (8 Wistar rats): OTM for 14 days with 8-g force; 2. Group 2 (8 Wistar rats): OTM for 14 days with 25-g force; 3. Group 3 (8 Wistar rats): OTM for 14 days with 100-g force; 4. Group 4 (8 Wistar rats): OTM for 14 days with 8-g force and alveolar decortications (ADs); 5. Group 5 (8 Wistar rats): OTM for 14 days with 25-g force and ADs; 6. Group 6 (8 Wistar rats): OTM for 14 days with 100-g force and ADs. A nickel-titanium spring was used to protract the molar mesially using maxillary incisors as an anchorage. ADs (minimal surgical insult) were done using a hand piece and a round bur, adjacent to the left first maxillary molar on the palatal alveolar bone. After 14 days of OTM, Wistar rats were killed and microfocus computed tomography and histological analysis were performed. RESULTS: The 100-g group showed significant increase (P < 0.05) in OTM. However, with ADs, the OTM was significantly higher (P < 0.05) in 8 and 100 g. In addition, with ADs, there is significant increase (P < 0.05) in OIRR and significant decrease (P < 0.05) in bone volume fraction. Histological quantification of tartrate-resistant acid phosphatase indicated a significant increase (P < 0.05) in the number of osteoclasts with ADs when compared without ADs. CONCLUSIONS: Light force in conjunction with ADs are optimal to accelerate the OTM. Additionally, ADs increases the OIRR.

BMP2 Is Required for Postnatal Maintenance of Osteochondral Tissues of the Temporomandibular Joint.

OBJECTIVE: Bone morphogenetic protein 2 (BMP2) plays important roles in cartilage growth and development. Paradoxically, elevated levels of BMP2 leads to hypertrophic differentiation and osteoarthritis of cartilage. We examined the in vivo loss of BMP2 in cells expressing aggrecan of the mandibular condyle and knee. DESIGN: Three-week-old BMP2 flox/flox-CreER-positive mice and their Cre-negative littermates were treated with tamoxifen and raised until 3 or 6 months. We also investigated the direct effects of BMP2 on chondrocytes in vitro. Cells from the mandibular condyle of mice were treated with recombinant human BMP2 (rhBMP2) or rhNoggin (inhibitor of BMP2 signaling). RESULTS: Conditional deletion of BMP2 caused breakage of the cartilage integrity in the mandibular condyle of mice from both age groups, accompanied by a decrease in cartilage thickness, matrix synthesis, mineralization, chondrocyte proliferation, and increased expression of degeneration markers, while the effects at articular cartilage were not significant. In vitro results revealed that rhBMP2 increased chondrocyte proliferation, mineralization, and differentiation, while noggin induced opposite effects. CONCLUSIONS: In conclusion, BMP2 is essential for postnatal maintenance of the osteochondral tissues of the mandibular condyle.

The effect of alveolar decortication on orthodontically induced root resorption.

OBJECTIVE: To determine the effect of alveolar decortication on orthodontically induced root resorption. MATERIALS AND METHODS: A total of 24 male Wistar rats (14 week old) were used. The rats were randomly divided into one of the following three groups: group 1 (control group), orthodontic tooth movement (OTM) for 2 weeks; group 2, OTM for 2 weeks + two alveolar decortications (2AD); group 3, OTM for 2 weeks + four alveolar decortications (4AD). The first molar was moved mesially for 2 weeks. Micro computed tomography was used to analyze root volume. In addition, histological sections were stained with Tartrate Resistant Acid Phosphatase (TRAP) to quantify the osteoclast number. RESULTS: The buccal root volume in OTM + 4AD group was decreased by 8.92% and 6.11% when compared with the OTM-only group and OTM + 2AD group, respectively. Similarly, the other four root volumes in the OTM + 4AD group was decreased by 8.99% and 5.24% when compared with the OTM-only group and OTM + 2AD group, respectively. There was a decrease in buccal root density in the OTM + 4AD group by 4.66% and 3.56% when compared with the OTM-only group and the OTM + 2AD group, respectively. In addition, there was an increase in the number of osteoclasts by 195.73% and 98.74% in OTM + 4AD group in comparison with the OTM and OTM + 2AD group. CONCLUSIONS: The amount of orthodontically induced root resorption was positively correlated with the extent of surgical injury used to accelerate orthodontic tooth movement.

Raloxifene administration enhances retention in an orthodontic relapse model.

BACKGROUND AND OBJECTIVES: Orthodontic relapse is a physiologic process that involves remodelling of the alveolar bone and principle periodontal ligament fibres. Raloxifene is an Food and Drug Administration (FDA)-approved selective oestrogen receptor modulator that inhibits systemic bone loss. In our study, we examined the effects of Raloxifene on alveolar bone modelling and orthodontic relapse in a rodent model. MATERIALS AND METHODS: The efficacy of raloxifene was evaluated in 15-week-old male Wistar rats, 8 in each group (Control, Raloxifene, Raloxifene + 7-day relapse, Raloxifene + 14-day relapse) for a total of 42 days. All animals had 14 days of orthodontic tooth movement with a closed nickel-titanium coil spring tied from incisors to right first molar applying 5-8 gm of force. On the day of appliance removal, impression was taken with silicon material and the distance between first molar and second molar was filled with light-cured adhesive resin cement for retention phase. Raloxifene Retention, Raloxifene Retention + 7D, Raloxifene Retention + 14D groups received 14 daily doses of raloxifene (2.0 mg/kg/day) subcutaneously after orthodontic tooth movement during retention. After 14 days of retention, the retainer was removed and right first molar was allowed to relapse for a period of 14 days. Raloxifene injection continued for the Raloxifene + 14-day relapse group during relapse phase too. Control group received saline injections during retention. Animals were euthanized by CO2 inhalation. The outcome measure included percentage of relapse, bone volume fraction, tissue density, and histology analysis using tartrate-resistant acid phosphatase staining and determining receptor activator of nuclear factor-кB-ligand (RANKL) and osteoprotegerin expression. RESULTS: Raloxifene Retention + 14D group had significantly less (P < 0.05) orthodontic relapse when compared with other groups. There was a significant increase (P < 0.05) in bone volume fraction and tissue density in the Raloxifene Retention + 14D group when compared with other groups. Similarly, there was significant decrease in number of osteoclasts and RANKL expression in Raloxifene Retention + 14D group when compared with Raloxifene Retention + 7D group (P < 0.05). CONCLUSION: Raloxifene could decrease post-orthodontic treatment relapse by decreasing bone resorption and indirectly enhancing bone formation.

Injectable RANKL sustained release formulations to accelerate orthodontic tooth movement.

BACKGROUND: Accelerating orthodontic tooth movement (OTM) through biologically effective methods, such as increasing osteoclast-mediated alveolar resorption, could effectively shorten treatment time. OBJECTIVE: To evaluate an injectable formulation containing receptor activator of nuclear factor kappa-B ligand (RANKL) on the OTM. MATERIALS AND METHODS: We fabricated a RANKL formulation from 100 µl of 100 µg/ml RANKL adsorbed on 10 mg of poly(lactic acid-co-glycolic acid) microspheres embedded in a 10 wt% aqueous hydroxyethyl cellulose carrier gel. We characterized these formulations for the rate of RANKL release, and then tested for bioactivity using in vitro cell culture. In vivo OTM studies were conducted using 15 week old male Wistar rats for 14 days. We injected the RANKL formulations palatal to the left maxillary first molar and accomplished OTM with a nickel-titanium (NiTi) coil spring applying 5-8 g force. Control groups involved the application of NiTi coil spring with and without placebo formulation. The outcome measure included the distance of tooth movement, bone volume fraction, tissue density, and root volume determined with micro-computed tomography. We determined the amount of osteoclast activity using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: These formulations were able to sustain the release of RANKL for more than 30 days, and the released RANKL showed a positive effect on mice osteoclast precursor cells (RAW 264.7). Reported injectable RANKL formulations were effective in accelerating OTM compared with other control groups, with 129.2 per cent more tooth movement than no formulation and 71.8 per cent more than placebo formulation, corresponding with a significant increase in the amount of TRAP activity. We did not observe any significant differences in root resorption between the groups. CONCLUSION: Our study shows a significant increase in OTM with injectable formulations containing RANKL.

The effects on the mandibular condyle of Botox injection into the masseter are not transient.

OBJECTIVES: To evaluate whether the effects on the mandibular condylar cartilage (MCC) and subchondral bone are transient of botulinum neurotoxin (Botox) injection into the masseter muscle. METHODS: Botox (0.3 U) was injected into the right masseter of 6-week-old female mice (C57BL/6; n = 16). In addition, 16 mice were used as control and received no injections. Experimental and matching control mice were killed 4 or 8 weeks after the single Botox injection. Mandibles and mandibular condyles were analyzed by means of microscopic computed tomography (microCT) and histology. Sagittal sections of condyles were stained for tartrate-resistant acid phosphatase (TRAP), toluidine blue, 5-ethynyl-2'-deoxyuridine (EdU), and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. RESULTS: Bone volume fraction was significantly decreased on the subchondral bone of the Botox-injected side, compared with the control side and control mice, 4 and 8 weeks after injection. Furthermore, histologic analysis revealed decrease in mineralization, cartilage thickness, TRAP activity, and EdU-positive cells in the MCC of the Botox-injected side 4 and 8 weeks after injection. CONCLUSIONS: The effects on the MCC and subchondral bone of Botox injection into the masseter muscle persisted for 8 weeks after injection and were not considered to be transient.

The effect of the extent of surgical insult on orthodontic tooth movement.

OBJECTIVE: The primary objective of this study was to investigate how the extent of surgical insult affects the orthodontic tooth movement (OTM) and the alveolar bone modelling and remodelling in a rodent model. MATERIAL AND METHODS: 15-week-old male Wistar rats were used in the research and they were randomly divided into three treatment groups: (1) OTM only (N = 8); (2) OTM + 2 alveolar decortication (AD) (less surgical insult) (N = 8); and (3) OTM + 4 AD (more surgical insult) (N = 8). A nickel-titanium spring delivering 5-8 g of force was used to protract the molar mesially using maxillary incisors as an anchorage. AD was done using a hand piece and a round bur, adjacent to the left first maxillary molar on the palatal alveolar bone. After 14 days of OTM Wistar rats were killed and microfocus computed tomography and histological analysis were performed. RESULTS: The OTM + 4AD group presented with a significant increase (P < 0.05) in the rate of tooth movement when compared to OTM + 2AD group and OTM only group. In addition, the OTM + 4AD group had a significant decrease in bone volume and tissue density (P < 0.05) and a significant increase (P < 0.05) in the trabecular spacing and trabecular thickness when compared to OTM only. Histological quantification of tartrate-resistant acid phosphatase indicated a significant percent increase (P < 0.05) in OTM + 4AD group, when compared to OTM + 2AD and OTM only group. RESULTS: Increased surgical insult increases the rate of OTM. Additionally, increased surgical insult decreases the bone volume and the tissue density.

Temporomandibular Joint Disorders in Older Adults.

OBJECTIVES: To review the literature and summarize the evidence of temporomandibualar joint (TMJ) disorders (TMDs) in older adults, focusing on clinical manifestations of TMDs in older adults, highlighting the incidence and sexual dimorphism of TMJ degeneration and the role of sex hormones in this process, and providing potential treatment options for TMD in older adults. DESIGN: Two review authors performed the literature search, study inclusion, and data extraction. PubMed, Embase, and Google scholar were searched for literature until August 2017 (Figure ). We adopted a combination of Medical Subject Headings with related free text words for the search in PubMed and optimized the search in other search engines. RESULTS: Traditionally, it was believed that TMDs predominantly affected women of childbearing age, but recent large studies in Europe and the United States have shown that the prevalence of TMD peaks after childbearing age (45-64) and then gradually decreases with age, although not much is known about the disease in older adults. CONCLUSION: Most older adults have TMJ degeneration, which affects women more than men. In most older adults, the symptoms of TMD are mild and self-limiting and can usually be treated with self management.

Loading of the Condylar Cartilage Can Rescue the Effects of Botox on TMJ.

The purpose of this study is to evaluate whether the effects of botulinum neurotoxin (botox) injection into the masseter in the mandibular condylar cartilage (MCC) and subchondral bone could be rescued by compressive loading of the temporomandibular joint (TMJ). Twenty-four 6-week-old female mice (C57BL/6J) were used. Mice were divided in three groups: (1) Botox (n = 8); (2) Botox plus loading (n = 8); (3) Pure control (n = 8). Bone labels (3 and 1 day before sacrifice) and the proliferation marker EdU (2 and 1 day before sacrifice) were intraperitoneally injected into all groups of mice. Condyles were dissected and examined by micro-CT and histology. Sagittal sections of condyles were stained for TRAP, alkaline phosphatase, EdU, TUNEL, and toluidine blue. In addition, immunostaining for pSmad, VEGF, and Runx2 was performed. Bone volume fraction, tissue density, and trabecular thickness were significantly decreased on the subchondral bone of botox-injected side when compared to control side and control mice, 4 weeks after injection. Furthermore, histological analysis revealed decrease in mineralization, matrix deposition, TRAP activity, EdU, and TUNEL-positive cells in the MCC of the botox-injected side, 4 weeks after injection. However, compressive loading reversed the reduced bone volume and density and the cellular changes in the MCC caused by Botox injection. TMJ compressive loading rescues the negative effects of botox injection into the masseter in the MCC and subchondral bone.

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

The effects of alveolar decortications on orthodontic tooth movement and bone remodelling in rats.

Objectives: Alveolar decortication (AD) is a minimally invasive procedure that can be performed in the orthodontic office as an intervention to accelerate tooth movement. There is a gap in the literature evaluating the earlier and delayed responses after AD using lighter orthodontic forces in a rat model. Therefore, the aim of this study was to determine the effects of AD in the amount of orthodontic tooth movement and on alveolar bone remodelling in a rodent model, after 7 or 14 days. Materials and methods: A total of 32 15-week-old male Wistar rats were used in four treatment groups: (1) orthodontic spring only (7 days), (2) orthodontic spring only + AD (7 days), (3) orthodontic spring only (14 days), and (4) orthodontic spring only + AD (14 days). A closed coil nickel-titanium spring delivering 8-10 g of force was used to move the molar mesially. Alveolar decortication was done using a high speed, quarter round bur adjacent to the left first maxillary molar, on the palatal alveolar bone. At each endpoint, rats were sacrificed and microfocus computed tomography and histological analysis were performed. Results: The spring + AD group presented with a significant increase in the rate of tooth movement when compared with spring only group, 7 and 14 days after the beginning of the experiments. In addition, the spring + AD group had a significant decrease in bone volume and tissue density and a significant increase in the trabecular spacing and the number of osteoclasts at 7 and 14 days. Furthermore, a fibrous tissue was found to replace the alveolar bone in the spring + AD group at day 14. Conclusion: Alveolar decortications enhanced bone remodelling around the tooth movement region and could be used as an adjunct surgical procedure to accelerate the rate of tooth movement.

Dietary phosphate supplement does not rescue skeletal phenotype in a mouse model for craniometaphyseal dysplasia.

BACKGROUND: Mutations in the human progressive ankylosis gene (ANKH; Mus musculus ortholog Ank) have been identified as cause for craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. We previously reported a knock-in (KI) mouse model (Ank KI/KI) for CMD and showed transiently lower serum phosphate (Pi) as well as significantly higher mRNA levels of fibroblast growth factor 23 (Fgf23) in Ank KI/KI mice. FGF23 is secreted by bone and acts in kidney to promote Pi wasting which leads to lower serum Pi levels. Here, we examined whether increasing the Pi level can partially rescue the CMD-like skeletal phenotype by feeding Ank +/+ and Ank KI/KI mice with high Pi (1.7 %) diet from birth for 6 weeks. We studied the Pi metabolism in Ank KI/KI mice and CMD patients by examining the Pi regulators FGF23 and parathyroid hormone (PTH). RESULTS: High Pi diet did not correct CMD-like features, including massive jawbone, increased endosteal and periosteal perimeters and extensive trabeculation of femurs in Ank KI/KI mice shown by computed microtomography (µCT). This unexpected negative result is, however, consistent with normal serum/plasma levels of the intact/active form of FGF23 and PTH in Ank KI/KI mice and in CMD patients. In addition, FGF23 protein expression was unexpectedly normal in Ank KI/KI femoral cortical bone as shown by immunohistochemistry despite increased mRNA levels for Fgf23. Renal expression of genes involved in the FGF23 bone-kidney axis, including mFgfr1, mKlotho, mNpt2a, mCyp24a1 and m1αOHase, were comparable between Ank +/+ and Ank KI/KI mice as shown by quantitative real-time PCR. Different from normal FGF23 and PTH, serum 25-hydroxyvitamin D was significantly lower in Ank KI/KI mice and vitamin D insufficiency was found in four out of seven CMD patients. CONCLUSIONS: Our data suggests that FGF23 signaling and Pi metabolism are not significantly affected in CMD and transiently low Pi level is not a major contributor to CMD.

Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.

OBJECTIVES: To evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone. MATERIALS AND METHODS: Botox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF. RESULTS: Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side. CONCLUSION: Injection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.

The buccinator during mastication: A functional and anatomical evaluation in minipigs.

OBJECTIVE: The buccinator muscle forms the lateral wall of the oral cavity. It is presumed to aid mastication by maintaining bolus position. Such a function would involve thickening the cheek, possibly compressing the alveolar bone and contributing to malocclusions. However, neither buccinator deformation nor its effect on pressure has been demonstrated. Our objective was to evaluate buccinator EMG during feeding, its changes in length and thickness, and the pressure exerted on its alveolar attachment, using miniature pigs as an animal model. METHODS: EMG of the buccinator and other oral muscles was recorded with fine-wire electrodes. Anteroposterior length and mediolateral thickness of the buccinator were evaluated with implanted sonomicrometry crystals, and pressure was measured by flat transducers placed beneath the mandibular origin of the buccinator. Recordings were made during feeding and muscle stimulation. Tissues were collected postmortem for histology. RESULTS: During mastication, buccinator EMG showed regular peaks that preceded those of the jaw closers. Pattern differences clearly distinguished working and balancing sides. The buccinator shortened and thickened when it contracted. Positive pressures were observed at the mandibular attachment of the buccinator, increasing when the muscle was active. Histological evaluation showed a complex interweaving of fibres closely associated with salivary tissue. CONCLUSIONS: Buccinator contraction does thicken the cheek, and during mastication this activity takes place just as the closing stroke begins. In addition to controlling the bolus, there may be an effect on salivation. Despite the fact that the muscle pulls on its attachment, the local mechanical environment at the alveolar bone is one of positive pressure.