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Atomic minerals are the smallest components of bone and the content of Ca, being the most abundant mineral in bone, correlates strongly with the risk of osteoporosis. Postmenopausal women have a far greater risk of suffering from OP due to low Ca concentrations in their bones and this is associated with low bone mass and higher bone fracture rates. However, bone strength is determined not only by Ca level, but also a number of metallic and non-metallic elements in bone. Thus, in this study, the difference of metallic and non-metallic elements in ovariectomy-induced osteoporosis tibial and maxillary trabecular bone was investigated in comparison with sham operated normal bone by laser ablation inductively-coupled plasma mass spectrometry using a rat model. The results demonstrated that the average concentrations of (25)Mg, (28)Si, (39)K, (47)Ti, (56)Fe, (59)Co, (77)Se, (88)Sr, (137)Ba, and (208)Pb were generally higher in tibia than those in maxilla. Compared with the sham group, Ovariectomy induced more significant changes of these elements in tibia than maxilla, indicating tibial trabecular bones are more sensitive to changes of circulating estrogen. In addition, the concentrations of (28)Si, (77)Se, (208)Pb, and Ca/P ratios were higher in tibia and maxilla in ovariectomised rats than those in normal bone at all time-points. The present study indicates that ovariectomy could significantly impact the element distribution and concentrations between tibia and maxilla.

Pulpal anesthesia success rates for ropivacaine following maxillary infiltration anesthesia seem to be low. We investigated the hypothesis that the addition of epinephrine would affect the pharmacokinetics of ropivacaine by retaining ropivacaine in the mucosa of the injected area through the time-dependent distribution of ropivacaine in the rat maxilla and serum following maxillary infiltration anesthesia using (3)H-labeled ropivacaine. We then examined the vasoactivity of ropivacaine with or without epinephrine on local peripheral blood flow. The addition of epinephrine to ropivacaine increased ropivacaine concentrations in the palatal mucosa and adjacent maxilla by more than 3 times that of plain ropivacaine at 20 minutes. By observing the autoradiogram of (3)H-ropivacaine, plain ropivacaine in the maxilla was remarkably reduced 20 minutes after injection. However, it was definitely retained in the palatal mucosa, hard palate, adjacent maxilla, and maxillary nerve after the administration with epinephrine. Ropivacaine with epinephrine significantly decreased labial blood flow. This study suggests that 10 μg/mL epinephrine added to 0.5% ropivacaine could improve anesthetic efficacy and duration for maxillary infiltration anesthesia over plain ropivacaine.

Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg(-1)∙day(-1), respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis.

Few studies sought to analyze the expression and function of the nonneuronal acetylcholine system in bone remodeling in vivo due to the lack of suitable models. We established a rat maxilla expansion model in which the midline palatine suture of the rat was rapidly expanded under mechanical force application, inducing tissue remodeling and new bone formation, which could be a suitable model to investigate the role of the nonneuronal acetylcholine system in bone remodeling in vivo. During the expansion, the expression pattern changes of the nonneuronal cholinergic system components and the mRNA levels of OPG/RANKL were detected by immunohistochemistry or real-time PCR. The value of the RANKL/OPG ratio significantly increased after 1 day of expansion, indicating dominant bone resorption induced by the mechanical stimulation; however after 3 days of expansion, the value of the RANKL/OPG ratio significantly decreased, suggesting a dominant role of the subsequent bone formation process. Increasing expression of Ach was detected after 3 days of expansion which indicated that ACh might play a role in bone formation. The mRNA expression levels of other components also showed observable changes during the expansion which confirmed the involvement of the nonneuronal cholinergic system in the process of bone remodeling in vivo. Further researches are still needed to figure out the detailed functions of the nonneuronal cholinergic system and its components.

This study aims to investigate the expression changes of RANKL, RUNX2, and OPG in rabbit periodontal tissues under orthodontic force and explore its effect on the remodeling of periodontal tissues. A total of 16 specific pathogen-free rabbits were used in this study. The maxillary appliance was worn on the right (experimental) side, and the appliance-free left side was used as the control. Rabbits were sacrificed after 3, 5, 7, and 14 days of treatment. Changes in the expression levels of OPG, RANKL, and RUNX2 in the periodontium were detected using real-time PCR and western blotting methods. The OPG expression levels decreased after 3 to 14 days of treatment, while the expression levels of RANKL and RUNX2 increased after 3 to 14 days. The OPG expression levels decreased while those of RANKL and RUNX2 increased during orthodontic tooth movement, which suggested that they play a role in the osteogenesis process and the reconstruction of periodontal tissue.

Background: We evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ)., Materials and Methods: Male Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies., Results: In the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group., Conclusions: This study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ.

Aim: This study aimed to determine the degree of discrepancy between clinical measurement of furcation involvement (FI) and cone beam computed tomography image analysis of multirooted teeth., Methods: FI measurements obtained from clinical records were compared to CBCT images of the same teeth to determine the degree of discrepancy between CBCT FI grading and clinical FI grading., Results: Of the hundred and fifty-four sites analysed, 22% of FI measurements from probing and CBCT were in agreement. Fifty-eight percent of clinical FI recordings were overestimated, and 20% were underestimated when compared to CBCT analysis., Conclusion: Clinical recording of FI is both over and underestimated compared to CBCT analysis. This was highest for probing recording grade I furcation involvement where it was highly overestimated. The occurrence of over and under estimation of FI will affect the assignment of prognosis to multirooted teeth, which can influence treatment planning for periodontal therapy and may result in inappropriate treatment., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Craniofacial sutures govern the shape of the craniofacial skeleton during postnatal development. The differentiation of suture mesenchymal cells to osteoblasts is precisely regulated in part by signaling through cell surface receptors that interact with extracellular proteins. Here we report that fibulin-5, a key extracellular matrix protein, is important for craniofacial skeletal development in mice. Fibulin-5 is deposited as a fibrous matrix in cranial neural crest-derived mesenchymal tissues, including craniofacial sutures. Fibulin-5-null mice show decreased premaxillary bone outgrowth during postnatal stages. While premaxillo-maxillary suture mesenchymal cells in fibulin-5-null mice were capable of differentiating into osteoblasts, suture cells in mutant mice were less proliferative. Our study provides the first evidence that fibulin-5 is indispensable for the regulation of facial suture mesenchymal cell proliferation required for craniofacial skeletal morphogenesis., (Copyright © 2015. Published by Elsevier Inc.)

Solitary Median Maxillary Central Incisor occurs in 1 of 50,000 live births. It is the mildest manifestation of the holoprosencephaly spectrum and is genetically heterogeneous. Here we report six patients with solitary median maxillary central incisor, and a range of other phenotypic anomalies with different degrees of severity, varying from mild signs of holoprosencephaly to associated intellectual disability, and with different genetic background. Using array comparative genomic hybridization, pathogenic copy number variants were found in three of the six patients. Two patients had a deletion at the 18p11 chromosomal region that includes TGIF1 while the other patient had a deletion at 7q36, including the SHH gene. In one patient, a mutation in SIX3 was detected with exome sequencing, while in the two remaining patients all known holoprosencephaly genes were excluded using multiplex ligation-dependent probe amplification and sequencing, and remain unsolved. One of the two latter patients had isolated solitary median maxillary central incisor without other visible dentofacial anomalies, while the other had clinical features not part of the known holoprosencephaly spectrum., (© 2015 Wiley Periodicals, Inc.)

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To establish a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ) that realistically mimics major clinical manifestations of the disease., Methods: Female Sprague Dawley rats received intravenous zoledronate 80 μg/kg once a week via the tail vein. Three weeks after intravenous injection, maxillary first molars were extracted under general anesthesia. Then 1, 4 and 12 weeks after tooth extraction, the rats were euthanized, and the intact maxillas were harvested en bloc. Macroscopic analysis, histological analysis and cytokine analysis were performed. Untreated rats with tooth extraction were used as controls., Results: 12 weeks after extraction, rats treated with zoledronate developed BRONJ-like disease, including characteristic features of impaired soft tissue healing, exposed necrotic bone or sequestra, increased inflammatory infiltrates, while the controls showed normal bone healing. 4 weeks after extraction, rats treated with zoledronate exhibited the decreased receptor activator of nuclear factor kappa-B ligand (RANKL) values, the increased osteoprotegerin (OPG) values and the remarkable decreased RANKL/OPG ratio when compared with the controls., Conclusion: The rats treated with zoledronate can be considered a novel, reliable and reproducible animal model to better understand the pathophysiology and pathogenesis of BRONJ and to develop a therapeutic approach.

Objective: The aim of this study was to investigate the biologic characteristics and osteogenic differentiation of mouse maxillary primordium mesenchymal cells (MPMCs) in vitro., Methods: The MPMCs were obtained from the E13.5 mouse embryos and cultured in vitro. The biologic characteristics were studied based on general observation and 5-Bromo-2-deoxyUridine (BrdU) label. The MPMCs from the first passage were cultured in the osteogenic medium for 1 week. Then, the immunofluorescence staining, alkaline phosphatase staining, Alizarin red S staining, and quantity polymerase chain reaction were used to evaluate the osteogenic capability of MPMCs., Results: The E13.5 MPMCs were successfully adherent cultured and passaged in vitro. These cells expressed Dlx2 and SMAD7, two genes that play important roles in the development of maxillary primordium. The percentage of 5-Bromo-2-deoxyUridine-labeled MPMCs was 32.1%. Osteogenic induction could promote the expression of Runx2, osteocalcin, and osteopontin, 3 osteogenic markers, in MPMCs. In addition, osteogenic induction could stimulate the secretion of alkaline phosphatase and promote the deposition of calcium nodules in MPMCs., Conclusions: The MPMCs could be cultured in vitro and could differentiate into osteoblast in vitro through osteogenic induction. It offered an alternative cell source to regenerate craniofacial bone and offered a useful cell model to study the craniofacial bone development.

Ameloblastic fibro-odontoma (AFO) is a slow-growing, expansive, benign odontogenic tumor, composed of ameloblastic epithelium embedded in an ectomesenchymal stroma resembling dental papilla, containing hard dental tissue in variable degrees of maturation, including enamel, dentin, and sometimes cementum. AFO typically affects the posterior mandible, causing bony expansion. We report a case of pigmented AFO in a 5-year-old boy, comprising clinical and histological features illustrated by immunohistochemistry using a large panel of antibodies, polarized light microscopy and scanning electron microscopy., (© The Author(s) 2014.)

Oestrogen plays major role in bone metabolism/remodelling. Despite of well-established effect of oestrogen deficiency on long bones, it remains unclear whether alveolar bone is affected. We aimed to determine the effect of oestrogen-deficiency in the alveolar bone microarchitecture. C57BL6/J and Balb/c mice were ovariectomized and implanted with oil-(OVX) or 17β-estradiol (E2)-containing (OVX+E2) capsules. Ovary-intact mice were used as controls. The dose of E2 replacement was selected based on trophic effects on the uterus and femur bone loss. As determined by maxillary alveolar bone MicroCT analysis, both C57BL6/J and Balb/c OVX mice displayed decreased trabecular thickness, bone density and bone volume, and increased trabecular separation at 15 and 30 days after ovariectomy. These effects were associated with a reduction of trabecular bone percentage and cortical thickness in the femur. A significant loss of alveolar bone crest was also associated with ovariectomy in both mice strains. The E2 replacement fully prevented ovariectomy-induced alterations in the alveolar and femoral bones. Moreover, TNF-α (tumour necrosis factor-α) levels and RANKL/OPG (receptor activator of NF-κB ligand/osteoprotegerin) ratio were increased in the maxilla after OVX, and these responses were also reversed by E2. In conclusion, oestrogen deficiency causes maxillary alveolar bone loss, which is similar to the effects found in the femur. The release of inflammatory molecules like TNF-α, RANKL and OPG is the potential mechanism to the decrease of bone quality and alveolar bone crest., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Bone regenerates mainly by periosteal and endosteal humoral and cellular activity, which is given only little concern in surgical techniques and choice of bone grafts for guided bone regeneration. This study investigates on a clinical level the biomechanical stability of augmented sites in maxillary bone when a new class of moldable, self-hardening calcium-phosphate biomaterials (SHB) is used with and without the addition of Platelet Rich Fibrin (aPRF) in the Piezotome-enhanced subperiosteal tunnel-technique (PeSPTT)., Material and Methods: 82 patients with horizontal atrophy of anterior maxillary crest were treated with PeSPTT and randomly assigned biphasic (60% HA/40% bTCP) or monophasic (100% bTCP) SHB without or with addition of aPRF. 109 implants were inserted into the augmented sites after 8.3 months and the insertion-torque-value (ITV) measured as clinical expression of the (bio)mechanical stability of the augmented bone and compared to ITVs of a prior study in sinus lifting., Results: Significant better results of (bio)mechanical stability almost by two-fold, expressed by higher ITVs compared to native bone, were achieved with the used biomaterials and more constant results with the addition of aPRF., Conclusion: The use of SHB alone or combined with aPRF seems to be favourable to achieve a superior (bio)mechanical stable restored alveolar bone.

Background: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study., Results: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, β1, β2, β4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, β1, β4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells., Conclusions: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Traumatic injuries involving the face are very common, yet the clinical management of the resulting craniofacial deficiencies is challenging. These injuries are commonly associated with missing teeth, for which replacement is compromised due to inadequate jawbone support. Using cell therapy, we report the upper jaw reconstruction of a patient who lost teeth and 75% of the supporting jawbone following injury. A mixed population of bone marrow-derived autologous stem and progenitor cells was seeded onto β-tricalcium phosphate (β-TCP), which served as a scaffold to deliver cells directly to the defect. Conditions (temperature, incubation time) to achieve the highest cell survival and seeding efficiency were optimized. Four months after cell therapy, cone beam computed tomography and a bone biopsy were performed, and oral implants were placed to support an engineered dental prosthesis. Cell seeding efficiency (>81%) of the β-TCP and survival during the seeding process (94%) were highest when cells were incubated with β-TCP for 30 minutes, regardless of incubation temperature; however, at 1 hour, cell survival was highest when incubated at 4°C. Clinical, radiographic, and histological analyses confirmed that by 4 months, the cell therapy regenerated 80% of the original jawbone deficiency with vascularized, mineralized bone sufficient to stably place oral implants. Functional and aesthetic rehabilitation of the patient was successfully completed with installation of a dental prosthesis 6 months following implant placement. This proof-of-concept clinical report used an evidence-based approach for the cell transplantation protocol used and is the first to describe a cell therapy for craniofacial trauma reconstruction., (©AlphaMed Press.)

Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/β-catenin signaling pathway. We demonstrated in vitro that WNT/β-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/β-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Epinephrine potentiates and prolongs the efficacy of local anesthetics by reducing blood flow. We investigated the effect of epinephrine on the pharmacokinetics of lidocaine and the pulpal blood volume after maxillary infiltration anesthesia in rats., Methods: We measured the (14)C-radioactivity and (14)C-distribution in the maxilla and the dental pulp after the injection of 2% (14)C-lidocaine with or without 10 μg/mL epinephrine (n = 7) into the palatine mucosa proximal to the first molar. The blood volume in the pulp was measured using (99m)Tc-pertechnetate (n = 5)., Results: When lidocaine was injected together with epinephrine, the lidocaine became widely distributed throughout the maxilla and was observed mainly in the first molar pulp. The lidocaine amount in the dental pulp at 10-60 minutes was more than 2 times higher than that after the injection of lidocaine alone. The relative pulpal blood volume after 20 minutes decreased to 63.1% of the value after the injection of lidocaine alone., Conclusions: We found that lidocaine had infiltrated into the molar pulp after infiltration anesthesia. Furthermore, our results suggested that epinephrine augmented the retention of lidocaine in the pulp., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aims: It has been accepted that bone damage and subsequent resorption occurs in cases of adverse interfacial bone strain. Fractured implants represents an important opportunity for evaluating the bone response to overstrain. The objective of the study was to analyze the bone morphology and morphometry around retrieved fractured implants in humans. Five fractured implants retrieved after 22.4 (± 2.8) years were used in the present study. The investigation was conducted in a transmitted brightfield and circularly polarized light Microscope. The parameters considered were the total bone area, total bone remodeling area, bone-remodeling rate (BRR), and bone transverse collagen fibers orientation (CFO)., Findings: For grouped samples, the BRR was 51.9% (± 10) while the transverse CFO was 13.0% (± 9.7). The interthread BRR was 58.1% (± 4.0) for level 1 and 40.4% (± 12.8) for level 2 with a significant decrease in BRR (p = 0.019) as a function of depth along the implant length., Conclusions: Both bone level and bone quality attains stability to a certain level with respect to implant neck fracture following physiologic interaction between mechanical and biological inputs. The BRR significantly decrease as function of threads depth., (© 2014 Wiley Periodicals, Inc.)

Background: We derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis., Methods: A total of 30 female Sprague-Dawley (SD) rats were randomly divided into four groups: healthy control group (Group-N, n = 5), untreated periodontitis group (Group-P, n = 5), iPS-MSCs-treated and iPSC-MSCs/TSG-6-treated periodontitis groups (Group-P1 and P2, n = 10 per group). Experimental periodontitis was established by ligature and infection with Porphyromonas gingivalis around the maxillae first molar bilaterally. MSC-like cells were generated from rat iPSCs, and transducted with TSG-6. iPSC-MSCs or iPSC-MSCs/TSG-6 were administrated to rats in Group-P1 or P2 intravenously and topically, once a week for three weeks. Blood samples were obtained one week post-injection for the analysis of serum pro-inflammatory cytokines. All animals were killed 3 months post-treatment; maxillae were then dissected for histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, and morphological analysis of alveolar bone loss., Results: Administration of iPSC-MSC/TSG-6 significantly decreased serum levels of IL-1β and TNF-α in the Group-P2 rats (65.78 pg/ml and 0.56 pg/ml) compared with those in Group-P (168.31 pg/ml and 1.15 pg/ml respectively) (p<0.05). Both alveolar bone loss and the number of TRAP-positive osteoclasts showed a significant decrease in rats that received iPSC-MSC/TSG-6 treatment compared to untreated rats in Group-P (p<0.05)., Conclusions: We demonstrated that overexpression of TSG-6 in rat iPSC-derived MSCs were capable of decreasing inflammation in experimental periodontitis and inhibiting alveolar bone resorption. This may potentially serve as an alternative stem-cell-based approach in the treatment and regeneration of periodontal tissues.

Malocclusions, such as an open bite and high canines, are often encountered in orthodontic practice. Teeth without occlusal stimuli are known as hypofunctional teeth, and numerous atrophic changes have been reported in the periodontal tissue, including reductions in blood vessels in the periodontal ligament (PDL), heavy root resorption, and reduced bone mineral density (BMD) in the alveolar bone. Low Level Laser (LLL) has been shown to have a positive effect on bone formation and the vasculature. Although the recovery of hypofunctional teeth remains unclear, LLL is expected to have a positive influence on periodontal tissue in occlusal hypofunction. The aim of the present study was to elucidate the relationship between LLL and periodontal tissue in occlusal hypofunction. Twenty-four male rats aged 5 weeks were randomly divided into control and hypofunctional groups. An anterior metal cap and bite plate were attached to the maxillary and mandibular incisors in the hypofunctional group to simulate occlusal hypofunction in the molars. LLL irradiation was applied to the maxillary first molar through the gingival sulcus in half of the rats. Rats were divided into four groups; control, control+LLL, hypofunctional, and hypofunctional+LLL. Exposure to LLL irradiation was performed for 3 minutes every other day for 2 weeks. Animals were examined by Micro-CT at 5 and 7 weeks and were subsequently sacrificed. Heads were resected and examined histologically and immunohistologically. The hypofunctional group had obvious stricture of the PDL. However, no significant differences were observed in the PDL and alveolar bone between the hypofunctional+LLL and the control groups. In addition, the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-positive cells were higher in the hypofunctional + LLL group than in the hypofunctional group. These results indicated that LLL enhanced the production of bFGF and VEGF in the periodontal tissue of hypofunctional teeth.

The development of the vertebrate face is regulated by complex interactions among several signaling pathways. Dickkopf-1 (Dkk-1), an inhibitor of the Wnt/β-catenin signaling pathway, can affect midfacial morphogenesis. The downstream target genes of the Wnt/β-catenin signaling pathway in morphogenesis of the developing upper jaw and lip remain unknown. To investigate the functional roles of Wnt/β-catenin signaling in facial development, we performed a loss-of-function experiment using local implantation of beads soaked with Dkk-1 during lip fusion at the maxillary prominence of chick embryos at stage 22(HH22). Antagonism of Wnt/β-catenin signaling by Dkk-1 induced deformities of the premaxilla and jugal bone, which are derived from the maxillary mesenchyme. Real-time and semi-quantitative RT-PCR analysis showed the significant reduction of Lhx8, Msx1 and Msx2 expression levels around the beads in the maxillary mesenchyme at 6 and 24 h after bead implantation. Time course experiments in the HH 22 embryos showed the effect of Dkk-1 on Lhx8, Msx1 and Msx2 expression was not significant after 48 h of the treatment. At HH 26 when the fusion of facial primordial started, Dkk-1 application did not exhibit any significant reduction of those genes. Our findings suggested that Dkk-1 regulates maxillary morphogenesis in chick embryos through Lhx8, Msx1 and Msx2 signals. Wnt/β-catenin signaling is responsible for intrinsic upper jaw development before the lip fusion., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteonecrosis of the jaw as a result of treatment with receptor activators of nuclear factor kappa-B ligand (RANKL) inhibitors (denosumab) is a new type of bony necrosis, the exact pathogenesis of which is unknown. Our aim was to find out whether the turnover of bone in the jaw is increased after denosumab has been given compared with other skeletal sites, and if that turnover might have a role in denosumab-related osteonecrosis of the jaw (DRONJ). Bone scintigraphic images of 45 female patients with breast cancer and bone metastases were analysed retrospectively, and divided into 3 groups: those given denosumab, those given a bisphosphonate, and a control group (n=15 in each). All patients had bone scintigraphy before treatment (T0) and during the course of treatment after 12 (T1) and 24 (T2) months. The data were analysed quantitatively using 6 preset bony regions of interest. There was similar turnover of bone in the mandible compared with other skeletal sites (such as the femur), while the maxilla showed significantly higher turnover. None of the bony regions investigated showed any significant changes after the bisphosphonate had been given. There was a tendency to increase bone turnover in those patients taking denosumab. The bone turnover of the jawbone is not overtly changed either by a bisphosphonate or denosumab, so it seems unlikely that oversuppression of bony turnover in the jawbones plays an important part either in the pathogenesis of DRONJ or in the bisphosphonate-related osteonecrosis of the jaw (BRONJ)., (Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

In the course of embryonic development skeletal elements form either through intramembranous or endochondral ossification. Wnt proteins play diverse roles during vertebrate skeletal development. Wnt16 is a key factor in developing long bones, but its exact role in craniofacial bone formation remains unclear. This study was initially undertaken to investigate the expression of Wnt16 during craniofacial bone development in mouse embryos. Wnt16 expression in the osteoid of calvaria, maxilla, and mandible started later than that of ALP and osteocalcin (OCN), but before mineralization of the craniofacial bones, suggesting that Wnt16 is involved in intramembranous ossification in the head. To confirm this, MC3T3-E1 cells were transfected with an adenovirus containing Wnt16 (Ad-Wnt16). Ad-Wnt16 cells showed decreased ALP activity and less mineralized nodule formations compared with control cells. In addition, the mRNA levels of osteogenic markers were reduced. Moreover, Wnt16 activated β-catenin signaling in MC3T3-E1 cells at both transcription and protein levels as shown by a TOPflash luciferase reporter gene assay and western blot analysis. On the other hand, Wnt/β-catenin pathway blockade by Dickkopf 1 abrogated the suppression of mineralization by Wnt16. Our findings suggest that Wnt16 is involved in intramembranous ossification and suppresses osteoblast differentiation through the Wnt/β-catenin pathway., (© 2013 Wiley Periodicals, Inc.)

We evaluated the effects of mechanical stimulation on the osteogenic differentiation of human intraoral mesenchymal stem and progenitor cells (MSPCs) using the Flexcell FX5K Tension System that mediated cyclic tensile stretch on the cells. MSPCs were isolated from human mandibular retromolar bones and characterized using flow cytometry. The positive expression of CD73, CD90, and CD105 and negativity for CD14, CD19, CD34, CD45, and HLA-DR confirmed the MSPC phenotype. Mean MSPC doubling time was 30.4 ± 2.1 hrs. The percentage of lactate dehydrogenase (LDH) release showed no significant difference between the mechanically stimulated groups and the unstimulated controls. Reverse transcription quantitative real-time PCR revealed that 10% continuous cyclic strain (0.5 Hz) for 7 and 14 days induced a significant increase in the mRNA expression of the osteogenesis-specific markers type-I collagen (Col1A1), osteonectin (SPARC), bone morphogenetic protein 2 (BMP2), osteopontin (SPP1), and osteocalcin (BGLAP) in osteogenic differentiated MSPCs. Furthermore, mechanically stimulated groups produced significantly higher amounts of calcium deposited into the cultures and alkaline phosphatase (ALP). These results will contribute to a better understanding of strain-induced bone remodelling and will form the basis for the correct choice of applied force in oral and maxillofacial surgery.

FAM20C, also known as Golgi casein kinase (G-CK), is proposed to be the archetype for a family of secreted kinases that phosphorylate target proteins in the Golgi and in extracellular matrices, but FAM20C serving an extracellular function is controversial. FAM20C phosphorylates secretory calcium-binding phosphoproteins (SCPPs), which are associated with the evolution of biomineralization in vertebrates. Current models of biomineralization assume SCPP proteins are secreted as phosphoproteins and their phosphates are essential for protein conformation and function. It would be a radical departure from current theories if proteins in mineralizing matrices were dephosphorylated as part of the mineralization mechanism and rephosphorylated in the extracellular milieu by FAM20C using ATP. To see if such mechanisms are possible in the formation of dental enamel, we tested the hypothesis that FAM20C is secreted by ameloblasts and accumulates in the enamel extracellular matrix during tooth development. FAM20C localization was determined by immunohistochemistry in day 5 mouse incisors and molars and by Western blot analyses of proteins extracted from pig enamel organ epithelia (EOE) and enamel shavings. FAM20C localized intracellularly within ameloblasts and odontoblasts in a pattern consistent with Golgi localization. Western blots detected FAM20C in the EOE extracts but not in the enamel matrix. We conclude that FAM20C is not a constituent of the enamel extracellular matrix and functions intracellularly within ameloblasts., (© 2013 American Society for Bone and Mineral Research.)

The mouse incisor is a frequently used model in studies of the molecular control of organ development. The appropriate interpretation of data on normogenesis is essential for understanding the data obtained in mutant mice. For this reason, we performed a very detailed investigation of the development of the upper incisor in wild-type mice from embryonic day (ED) 11.5 till 14.5. A combination of histology, whole mount in situ hybridization, computer-aided three-dimensional reconstructions, and fluorescent microscopy, has been used. Several sonic hedgehog (Shh) expression domains have been detected in the upper incisor region during early prenatal development. At ED11.5-13.5, there was a single Shh positive domain present in the anterior part of left or right upper jaw arches, corresponding to the epithelial thickening. More posteriorly, a new Shh expression domain appeared in the incisor bud in the developmentally more advanced ED13.5 embryos. At ED14.5, only this posterior Shh expression in the incisor germ remained detectable. This study brings new insights into the early development of the upper incisor in mice and completes the data on normal mouse incisor development. The temporal-spatial pattern of Shh expression reflects the development of two tooth generations, being detectable in two successive, antero-posteriorly located areas in the prospective incisor region in the upper jaw. The first, anterior and superficial Shh expression domain reflects the rudimentary tooth development suppressed during evolution. Only the subsequent, posterior and deeper Shh expression region, appearing at ED13.5, correlates with the prospective upper functional incisor in wild-type mice., (© 2013 Wiley Periodicals, Inc.)

Bisphosphonates are used in the treatment of hypocalcaemia, mainly in cancer and osteoporosis. Some patients experience adverse events, such as BP-related osteonecrosis of the jaw (BRONJ). DNA methylation plays a key role in gene regulation in many tissues, but its involvement in bone homeostasis is not well characterized, and no information is available regarding altered methylation in BRONJ. Using the Illumina Infinium HumanMethylation27 BeadChip assay, we performed an epigenome-wide association study in peripheral blood samples from 68 patients treated with nitrogenous BP, including 35 with BRONJ. Analysis of the estimated cumulative BP exposure distribution indicated that the exposure of the case group to BP was slightly higher than that of the control group; more severely affected cases (i.e., with BRONJ in both mandible and maxilla) were significantly more exposed to BP than were those with BRONJ only in the mandible or maxilla (one-sided Wilcoxon rank sum test, p=0.002). Logistic regression analysis confirmed the positive association between cumulative bisphosphonates exposure and risk of BRONJ (OR 1.015 per mg of cumulative exposure, 95% CI 1.004-1.032, p=0.036). Although no statistically significant differences were observed between case and control groups, methylation levels of probes mapping on three genes, ERCC8, LEPREL1 and SDC2, were strongly associated with cumulative BP exposure levels (p<1.31E-007). Enrichment analysis, combining differentially methylated genes with genes involved in the mevalonate pathway, showed that BP treatment can affect the methylation pattern of genes involved in extracellular matrix organization and inflammatory responses, leading to more frequent adverse effects such as BRONJ. Differences in DNA methylation induced by BP treatment could be involved in the pathogenesis of the bone lesion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction.

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5 days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10⁻⁷ M) treatment led to an increased number of osteoclasts, HK-1 (10⁻⁷ M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1 compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: The aim of the study was to elucidate whether a newly developed, minimally invasive procedure, piezopuncture, would be a logical modification for accelerating tooth movement in the maxilla and the mandible., Methods: Ten beagle dogs were divided into 2 groups. Traditional orthodontic tooth movement was performed in the control group. In the experimental group, a piezotome was used to make cortical punctures penetrating the gingiva around the moving tooth. Measurements were made in weeks 1 through 6. Tooth movement and bone apposition rates from the histomorphometric analyses were evaluated by independent t tests., Results: The cumulative tooth movement distance was greater in the piezopuncture group than in the control group: 3.26-fold in the maxilla and 2.45-fold in the mandible. Piezopuncture significantly accelerated the tooth movements at all observation times, and the acceleration was greatest during the first 2 weeks for the maxilla and the second week for the mandible. Anabolic activity was also increased by piezopuncture: 2.55-fold in the maxilla and 2.35-fold in the mandible., Conclusions: Based on the different effects of piezopuncture on the maxilla and the mandible, the results of a clinical trial of piezopuncture with optimized protocols might give orthodontists a therapeutic benefit for reducing treatment duration., (Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

The peripheral ossifying fibroma (POF) is a reactive gingival overgrowth occurring frequently in the anterior maxilla. It originates in the cells of the periodontal ligament and is more common in children and young adults. In the current article a case of gingival over growth, which was thought to be puberty-induced gingivitis was seen in the lower anterior maxillary gingiva. Histology of the excised tissue showed cellular, fibrous connective tissue stroma with calcified osseous calcifications indicative of POF. The definitive diagnosis is established only by histological examination, which revealed the presence of highly cellular connective tissue with focal calcifications. Surgery is the treatment of choice, though the recurrence rate can reach 20% in case of POF. After histological confirmation the recall and clinical evaluation protocol of POF varies due to its increased recurrence rate, which the general dentist should be aware of.

Active oxygens and free radicals are involved in the metabolism and clinical conditions of tissues; however, little is known about the localisation and expression levels of associated enzymes. The expressions of active oxygens, free radicals and associated enzymes are reported to be site-specific; therefore, the expression states of free radical enzymes differ between sites, even within the same cell. In particular, there has been no report concerning the catabolic enzymes of active oxygens in osteoblasts of the maxilla, other than normal osteoblasts that were weakly positive by immunohistochemical staining. We conducted this study to elucidate the relationship between osteodystrophy and radical-associated enzymes by investigating mRNAs of enzymes associated with active oxygens and free radicals using osteoblasts from the maxilla of normal and osteopetrotic model (op/op) mice. In op/op mouse maxilla osteoblasts, mRNAs of Mn-SOD, i-NOS and e-NOS were strongly positive. Mn-SOD and iNOS enzymes were considered to be highly expressed in osteoblasts of the narrowed medullary cavity of this bone., (© 2012 Blackwell Verlag GmbH.)

Considering the potential use of growth factors carried by liposomes for bone repair, this study aimed to assess the progress of bone healing process in injured alveoli of rats after administering EGF within liposomes. For this assessment we used 48 male Wistar rats that had their maxillary second molar extracted and separated into 5 groups: sockets filled with blood clot (BC), treated with empty liposome (L), PBS (P), EGF in PBS (EGF-P) and EGF in liposome (EGF-L). The animals were sacrificed after 3, 7, 14 and 21 days after surgery. Histological, histomorphometric and immunohistochemistry analysis were performed to evaluate new bone and blood vessels formation as well as the expression of fibronectin and collagen type III, two determinant proteins for early wound regeneration. Our analysis showed a continuous transformation of sockets during all stages of wound healing. Nevertheless, groups BC, L, P and EGF-P followed a regular time for regeneration significantly different from the EGF-L group, which showed faster recovering. A higher expression of fibronectin and type III collagen in the group EGF-L after 3 and 7 days of surgery was observed and might be explained by the ability of the liposome to deliver EGF in a controlled manner, stimulating mesenchymal cells migration and osteoblast differentiation. As liposome efficiently regulated the availability of EGF without risks for its function and protected the factor from early absorption and degradation, the present work indicates that liposomes can be successful used as carriers for controlled delivery of growth factors in bone healing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the correlations between clinical-radiographical aspects and histomorphometric-molecular parameters of endosseous dental implant sites in humans., Material and Methods: The study sample consisted of bone implant sites from the jawbones of 32 volunteers, which were classified according to two different systems: (1) based only on periapical and panoramic images (PP); (2) as proposed by Lekholm & Zarb (L&Z). Bone biopsies were removed using trephine during the first drilling for implant placement. Samples were stained with haematoxylin-eosin (HE), and histomorphometric analysis was performed to obtain the following parameters: trabecular thickness (Tb.Th), trabecular number, bone volume density (BV/TV), bone specific surface (BS/BV), bone surface density and trabecular separation (Tb.Sp). In addition, immunohistochemistry analysis was performed on bone tissue samples for the proteins, Receptor activator of nuclear factor kappa-B (RANK), RANK ligand (RANKL), osteoprotegerin (OPG) and Osteocalcin (OC). Also, the determination of the relative levels of gene expression was performed using Reverse transcription-real-time Polymerase Chain Reaction (RT-PCR)., Results: PP and L&Z classification systems revealed a moderate correlation with BV/TV, BS/BV, Tb.Th and Tb.Sp. L&Z's system identified differences among bone types when BV/TV, BS/BV, Tb.Th and Tb.Sp were compared. A weak correlation between PP/L&Z classifications and the expression of bone metabolism regulators (RANK, RANKL, OPG e OC) was found. The analysis of mRNA expression showed no difference between the bone types evaluated., Conclusions: Our results suggest that PP and L&Z subjective bone-type classification systems are related to histomorphometric aspects. These data may contribute to the validation of these classifications. Bone remodelling regulatory molecules do not seem to influence morphological aspects of the jawbone ., (© 2011 John Wiley & Sons A/S.)

Craniofacial skeleton mainly originate from the cranial neural crest stem cells (CNCCs), which is a subpopulation of neural crest stem cells (NCCs). Dlx2, a member of the homeodomain family of transcription factors, plays crucial roles in the development of the CNCCs derived craniofacial skeleton. Previous reports reveal that Dlx2-targeted null mutation resulted in anomalies in the skeletal derivatives of CNCCs in mice. Dlx2 overexpression in ova disturbed the migration and differentiation of affected CNCCs and induced the development of ectopic skeleton elements. However, whether Dlx2 overexpression can impair the morphogenesis of CNCCs derived craniofacial skeleton in vivo has not been explored. Here, we generated a transgenic mouse overexpressing Dlx2 in NCCs (Wnt1Cre::iZEG-Dlx2). The Wnt1Cre::iZEG-Dlx2 embryos showed decreased cell proliferation, increased cell apoptosis, abnormal chondrogenesis and impaired osteogenesis within the CNCCs population, resulting in obvious craniofacial defects that ranged from a cleft lip and midfacial clefts to neural tube defects and exencephaly. Adult Wnt1Cre::iZEG-Dlx2 mice showed nasal and premaxillary hypoplasia and spinal deformities. These findings reveal that Dlx2 overexpression in NCCs may be a new pathogenesis of facial cleft and spinal kyphosis in mammals, and may offer us a useful model organism to find suitable therapy methods for these genetic defects that may be different from the traumatic defect and resected defect., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Autologous bone is considered the gold standard for bone regeneration, even if different heterologous bone substitutes have been proposed to overcome the limits related to its use. The aim of this study was to analyze and to compare the molecular events switched on by autologous or heterologous bone graft insertion, focusing on TGFβ1 expression and OPG/RANKL ratio, to analyze resorption process, and estimating graft vascularization, new bone tissue deposition and its mineralization, through VEGF, BSP and SPARC expression evaluation, respectively. Patients needing pre-prosthetic rehabilitation of the posterior maxilla were treated using an equine-derived biomaterial (Group 1) or calvaria autologous bone (Group 2), according to the morphology of the bone defect. Bone graft integration was evaluated on bone samples obtained from the treated areas at the moment of dental implant insertion, by morphological and immunohistochemical analyses for TGFβ1, OPG, RANKL, VEGF, BSP, and SPARC expression. Morphological analysis shows the presence of biomaterial residual granules in Group 1, in parallel to a good integration between graft and host tissue. Moderate TGFβ1 expression is seen in both Group 1 and Group 2. OPG/RANKL ratio appear higher in Group 1; VEGF expression appears very strong in Group 1 and strong in Group 2, while BSP and SPARC expression results weak in Group 1 and moderate in Group 2. Results reveal the good integration between both types of graft and the host tissue, even though autologous graft seems to produce a faster regenerative process, as evidenced by the different expression of the investigated molecules. According to these observations, the clinical use of heterologous particulate equine-derived biomaterial may ensure long-term predictability of implant-prosthetic rehabilitation, comparable to that obtained with autologous bone graft.

The maxillary and mandibular arches are present before tooth eruption and will develop and mature with tooth activities. Following tooth extraction, the healing wound fills in, partly, the space occupied formerly by the natural tooth. The blood coagulum that occupies the healing wound will be replaced by the residual ridge, a scar tissue, which becomes part of the edentulous arch. After tooth loss, the resulting edentulous arches undergo extensive remodeling changes, but remain indispensable, vital oral structures. The resorptive process of the edentulous jaws is limited. The maxillary and mandibular bodies have never been known to recede completely. Furthermore, physiological spontaneous fracture of the jaws does not occur. One factor that helps preserve the jaws' integrity may be the enveloping muscle girdle attached to the external surfaces of the jawbones. The residual ridge develops after tooth extraction and continues to remodel, during the edentulous life of the individual. The ridge resorption is a chronic, progressive, irreversible process. The residual ridge may even disappear as an anatomic entity following an extended edentulous period, systemic and/or local factors. Resorption of the residual tissues seems to be hastened by, systemic affections, edentulousness time, denture wear, tobacco consumption and other unknown factors. In the edentulous patient, the maxillary ridge migration is centripetal and apical, whereas in the mandible, the remaining ridge shifts centrifugally and apically. Consequently the resulting edentulous maxillary arch is, mostly, internal or at the same vertical level with the facing toothless mandibular arch. The different individual inter arch spatial relations are to be considered for the positioning of the artificial teeth, for optional ridge augmentation procedures and for insertion of dental implants.

Objectives: To elucidate the points that require attention when interpreting fluorine-18-labelled fluoro-2-deoxy-d-glucose ((18)F-FDG)/positron emission tomography (PET) images by demonstration of (18)F-FDG accumulation in various areas of the oral cavity other than primary lesions in patients with oral cancers., Methods: (18)F-FDG accumulations with a maximal standardized uptake value of over 2.5 in various areas of the oral cavity other than primary lesions were identified in 82 patients with oral cancers., Results: (18)F-FDG/PET-positive areas, excluding primary tumours, included the front intrinsic muscles of the tongue (89.0%), upper and lower marginal parts of the orbicularis oris muscle (64.6%), sublingual glands, palatine tonsil, pharyngeal tonsil, and lingual tonsil. In addition, some areas in the jaws also showed accumulation., Conclusions: In patients with oral cancers, areas of (18)F-FDG accumulation in the oral cavity should be precisely identified and appropriately diagnosed, because accumulations can be seen in areas other than the primary tumour.

Background: Platelet growth factors obtained from platelet-rich plasma (PRP) are used in prosthetic and periodontal regenerative therapy in dentistry. Increased microvascular proliferation in the first 20 days is followed by enhanced osteoblast activity and immature osteoid formation within 3-6 weeks, improving the quality and quantity of newly formed bone tissue. The aim of this study was to evaluate, through three-dimensional X-ray monitoring, the amount of bone obtained after bone regeneration treatment with platelet gel from autologous PRP., Materials and Methods: Patients eligible for regenerative treatment of atrophic alveolar bone of the maxilla or mandible were studied. The patients' autologous whole blood was collected at the Department of Immunology and Transfusion of San Matteo Hospital for the preparation of platelet gel. The bone at the treated sites was analysed prior to and 4 months after the treatment using the three-dimensional X-ray system Galileos., Results: Over a period of 6 years, 133 patients were treated: 304 implants were inserted and there were five cases of failure. The regenerated bone area consisted of histologically immature osteoid tissue composed of thin trabeculae of vital bone and nuclei of osteocytes, associated with fibro-connective tissue., Discussion: This co-operative trial between the Transfusion Centre, for standardised production and validation of the platelet gel, and the Dental Surgery room for its application showed that the technique appears effective and safe. Although difficulties were encountered because of the small sample size and the inability to carry out long-term histological controls, the use of small amounts of PRP (5-10%) combined with autologous bone (15-20%) and alloplastic material appears to reduce the need for bone grafting.

Objective: To study the effect of Salvia miltiorrhiza on alveolar bone metabolism and variation in bone mass in diabetic rats, in order to detect whether it has an inhibitory effect on alveolar bone osteoporosis caused by diabetics., Method: Intraperitoneal injection of alloxan induced diabetes in rats. After one week of observation and maintenance of stable blood sugar level, they were treated with S. miltiorrhiza. The rats were sacrificed at the eighth week after fasting for 12 h and blood samples were collected for analysis of blood glucose and rate of bone metabolism. Meanwhile, their alveolar bones were collected for determining bone mineral density (BMD) and histological sections were made for histomorphology observation., Result: Diabetic rats showed varying degrees of abnormality in bone metabolism indicators and significant reduction in bone mineral density. After treatment with S. miltiorrhiza, their symptoms reduced to some extend and all indicators were improved especially bone density., Conclusion: S. miltiorrhiza has a certain inhibitory effect on alveolar bone osteoporosis in diabetic rats in early stage.

The effects of infiltration anesthesia with ropivacaine on the dental pulp are considered to be weak. This may be partly associated with its permeation into the oral tissue. With the objective of investigating the local pharmacokinetics of ropivacaine and lidocaine following infiltration anesthesia, we injected (3)H-ropivacaine or (14)C-lidocaine to the palatal mucosa in rats, measured distributions of radioactivity in the maxilla, and compared the local pharmacokinetics of these agents. The animals were sacrificed at various times and the maxillas were removed. The palatal mucosa and maxillary nerve were resected, and the bone was divided into 6 portions. We measured radioactivity in each tissue and calculated the level of each local anesthetic (n  =  8). Lidocaine diffused to the surrounding tissue immediately after the injection, whereas ropivacaine tended to remain in the palatal mucosa for a longer period. Lidocaine showed a higher affinity for the maxillary bone than ropivacaine. There was a correlation between the distribution level of local anesthetics in the maxillary bone and that in the maxillary nerve. The lower-level effects of infiltration anesthesia with ropivacaine on the dental pulp may be because ropivacaine has a high affinity for soft tissue, and its transfer to bone is slight.

Outgrowth and fusion of the lateral and medial nasal processes and of the maxillary process of the first branchial arch are integral to lip and primary palate development. Wnt9b mutations are associated with cleft lip and cleft palate in mice; however, the cause of these defects remains unknown. Here, we report that Wnt9b(-/-) mice show significantly retarded outgrowth of the nasal and maxillary processes due to reduced proliferation of mesenchymal cells, which subsequently results in a failure of physical contact between the facial processes that leads to cleft lip and cleft palate. These cellular defects in Wnt9b(-/-) mice are mainly caused by reduced FGF family gene expression and FGF signaling activity resulting from compromised canonical WNT/β-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. Here, we study the metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum, using RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, we tested the prediction that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, we tested hypotheses for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. Our data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles.

The use of graft materials is developed from the strong demand to support the complete bone regeneration of the empty socket and to increase the bone volume in treating the atrophies of sites already consolidated and with adverse alveolar bone conditions. A number of graft materials with different origin and mechanism of bone regeneration are available. Autologous graft materials, coming from the same patient, are defined as the gold-standard. The need of a second surgical site and the risk of morbidity and complications may make their use difficult. Human bone allografts (HBA) have been recently introduced, in order to offer an alternative to the autologous grafts. They have demonstrated to be effective in bone regeneration. Recent studies have proved the ability of HBA in bone regenerating process as they guarantee a three-dimensional structure for the re-growth of the new bone and the maintenance of inductive stimuli. In the present manuscript, Authors reviewed the evidence supporting the use of HBA in the management of the localized ridge atrophies, in the preservation of the extracted socket and in the sinus augmentation surgery, and illustrated some original case reports.

The aim of this study is to investigate the relationship between thermochemotherapy-induced apoptosis and the expressions of the Bcl-2 and Bax proteins in maxillofacial squamous cell carcinomas. Fifteen patients with maxillofacial squamous cell carcinomas were treated with microwave hyperthermia (43 °C for 40 min) following the intravenous injection of pingyangmycin (Bleomycin A5 Hydrochloride for Injection) (8 mg). Subsequently, the tumors were surgically resected. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method was used to assess the apoptosis in the carcinoma cells, and immunohistochemistry was performed using the streptavidin-peroxidase method to determine the expression levels of the Bcl-2 and Bax proteins, and quantitative analysis was employed. The number of apoptotic cells increased markedly (P < 0.001). The protein expression of Bcl-2 was downregulated (P < 0.001), while that of Bax was markedly upregulated (P < 0.001). Thermochemotherapy induces apoptosis in maxillofacial squamous cell carcinoma cells by downregulating the protein expression of Bcl-2 and upregulating that of Bax.

Teeth arise from sequential and reciprocal interactions between the oral epithelium and the cranial neural crest-derived mesenchyme. Their formation involves a precisely orchestrated series of molecular and morphogenetic events. Numerous regulatory genes that have been primarily found in organisms such as Drosophila, zebrafish, xenopus and mouse are associated with all stages of tooth formation (patterning, morphogenesis, cytodifferentiation and mineralization). Most of these genes belong to evolutionary conserved signaling pathways that regulate communication between epithelium and mesenchyme during embryonic development. These signaling molecules together with specific transcription factors constitute a unique molecular imprint for odontogenesis and contribute to the generation of teeth with various and function-specific shapes. Mutations in several genes involved in tooth formation cause developmental absence and/or defects of teeth in mice. In humans, the odontogenic molecular program is not as well known as that of mice. However, some insight can be obtained from the study of mutations in regulatory genes, which lead to tooth agenesis and/or the formation of defective dental tissues., (© 2011 John Wiley & Sons A/S.)