Angiotensin(1-7) attenuates tooth movement and regulates alveolar bone response during orthodontic force application in experimental animal model.

BACKGROUND: Renin-angiotensin system and its ACE2/Ang(1-7)/Mas receptor axis regulates skeletal response to multiple physiological and pathological conditions. Recent research suggested a vital role of Ang(1-7) in regulating alveolar bone metabolism and remodeling. In this context, this study evaluated the effects of the Ang(1-7)/Mas receptor axis on orthodontic tooth movement (OTM) and the alveolar bone response to mechanical load. METHODS: A coil spring was placed between the right maxillary first molar and the anterior tooth of Wistar rats to apply bidirectional mechanical force. Ang(1-7) with or without a specific Mas receptor antagonist (A779) was infused using subcutaneous osmotic pumps (200 and 400 ng/kg/min: respectively). Animals were killed after 5 and 14 days from the OTM procedure after the clinical evaluation of tooth movement and mobility. Morphometric analysis of alveolar bone structure was conducted using micro-CT and the histological picture was evaluated after H&E staining. Moreover, collagen fiber distribution was assessed using Picro-Sirius red stain. In addition, bone samples were collected from the pressure and tension sites around the anterior tooth for gene expression analysis. RESULTS: Ang(1-7) infusion suppressed the tooth movement and mobility after 14 days of the orthodontic force application. Additionally, Ang(1-7) infusion preserved the morphometric and histological structure of the alveolar bone at pressure and tension sides. These effects were abolished by adding A779 infusion. Collagen fiber distribution was dysregulated mainly by the A779 Mas receptor blockage. Ang(1-7) affected the bone formation, remodeling- and vascularity-related genes in the pressure and tension sides, suggesting a prominent suppression of osteoclastogenesis. Ang(1-7) also improved osteoblasts-related genes on the tension side, whereas the osteoclasts-related genes were augmented by A779 on the pressure side. CONCLUSION: Collectively, the activation of Ang(1-7)/Mas receptor axis appears to hinder tooth movement and regulates alveolar bone remodeling in response to mechanical force.

On-Bone Fixation of Free Gingival Graft Induces an Osteoinductive Effect in Human Alveolar Bone.

We examined alveolar bone samples in the area of on-bone fixation of a free gingival graft performed during surgery in patients aged 37-55 years with a diagnosis of secondary partial adentia of the upper and lower jaws. Six months after fixation of the graft in the alveolar bone, foci of neoosteogenesis were found in the contact zone. They were characterized by the appearance of appositional lines, cords of basophilic osteoblasts, and growing osteons. An immunohistochemical study revealed an increase in the number of CD44+, CD29+, and osteocalcin+ cells in the layer of the outer circumferential lamellae, primary osteons, and the lining of the Haversian canals. TGF-ß1+ cells were located in the intertrabecular reticular tissue and wall of microvessels. The results indicate activation of mesenchymal stem cells in the area of localization of the graft and differentiating osteoblasts. The observed osteoinductive effect of free gingival graft is associated with its participation in reorganization in MSC and induction of morphogenetic molecules.

The Glycoprotein/Cytokine Erythropoietin Promotes Rapid Alveolar Ridge Regeneration In Vivo by Promoting New Bone Extracellular Matrix Deposition in Conjunction with Coupled Angiogenesis/Osteogenesis.

The loss of bone following tooth extraction poses a significant clinical problem for maxillofacial esthetics, function, and future implant placement. In the present study, the efficacy of an erythropoietin-impregnated collagen scaffold as an alveolar ridge augmentation material versus a conventional collagen scaffold and a BioOss inorganic bovine bone xenograft was examined. The collagen/Erythropoietin (EPO) scaffold exhibited significantly more rapid and complete osseous regeneration of the alveolar defect when compared to bone xenograft and the collagen membrane alone. The new EPO induced extracellular matrix was rich in Collagen I, Collagen III, Fibronectin (Fn) and E-cadherin, and featured significantly increased levels of the osteogenic transcription factors Runt-related transcription factor 2 (Runx2) and Osterix (Osx). Histomorphometric evaluation revealed a significant two-fold increase in the number of capillaries between the EPO and the BioOss group. Moreover, there was a highly significant 3.5-fold higher level of vascular endothelial growth factor (VEGF) in the collagen/EPO-treated group compared to controls. The significant effect of EPO on VEGF, FN, and RUNX2 upregulation was confirmed in vitro, and VEGF pathway analysis using VEGF inhibitors confirmed that EPO modulated extracellular matrix protein expression through VEGF even in the absence of blood vessels. Together, these data demonstrate the effectiveness of an EPO-impregnated collagen scaffold for bone regeneration as it induces rapid matrix production and osseoinduction adjacent to new capillaries via VEGF.

Sympathetic nervous system contributes to orthodontic tooth movement by central neural regulation from hypothalamus.

Orthodontic tooth movement (OTM) is a specific treatment of malocclusion, whose regulation mechanism is still not clear. This study aimed to reveal the relationship between the sympathetic nervous system (SNS) and OTM through the construction of an OTM rat model through the utilization of orthodontic nickeltitanium coiled springs. The results indicated that the stimulation of SNS by dopamine significantly promote the OTM process represented by the much larger distance between the first and second molar compared with mere exertion of orthodontic force. Superior cervical ganglionectomy (SCGx) can alleviate this promotion effect, further proving the role of SNS in the process of OTM. Subsequently, the ability of orthodontic force to stimulate the center of the SNS was visualized by the tyrosin hydroxylase (TH) staining of neurons in ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC) of the hypothalamus, as well as the up-regulated expression of norepinephrine in local alveolar bone. Moreover, we also elucidated that the stimulation of SNS can promote osteoclast differentiation in periodontal ligament cells (PDLCs) and bone marrow-derived cells (BMCs) through regulation of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) system, thus promoting the OTM process. In conclusion, this study provided the first evidence for the involvement of the hypothalamus in the promotion effect of SNS on OTM. This work could provide a novel theoretical and experimental basis for further understanding of the molecular mechanism of OTM.

Complement 3 mediates periodontal destruction in patients with type 2 diabetes by regulating macrophage polarization in periodontal tissues.

OBJECTIVES: Diabetes aggravates the risk and severity of periodontitis, but the specific mechanism remains confused. Complement 3 (C3) is closely related to complications of type 2 diabetes (T2DM). In the present study, we concentrated on whether C3 mediates the development of periodontitis in T2DM. MATERIALS AND METHODS: Levels of C3 in blood and gingival crevicular fluid (GCF) of patients were measured first. A C3-knockout diabetic mouse model was established, real-time PCR, Western blotting and histological investigation were performed to evaluate the progress of periodontitis. Microcomputed tomography (micro-CT) and TRAP staining were performed to detect alveolar bone resorption. Immunofluorescence was performed to detect polarization of macrophages. RESULTS: Our data showed that C3 levels were elevated in the blood and GCF of T2DM patients compared with non-diabetic individuals. Increased C3 was closely related to the upregulation of inflammatory cytokines including interleukin (IL)-1, IL-6 and tumour necrosis factor-alpha (TNF-α), as well as the decline of the bone volume density (BMD) and bone volume over total volume (BV/TV) of the alveolar bones in diabetic mice. The deletion of C3 inhibited inflammatory cytokines and rescued the decreased BMD and BV/TV of the alveolar bones. C3-mediated polarization of macrophages was responsible for the damage. CONCLUSION: T2DM-related upregulation of C3 contributes to the development of periodontitis by promoting macrophages M1 polarization and inhibiting M2 polarization, triggering a pro-inflammatory effect on periodontal tissues.

The Alveolar Ridge Splitting Technique on Maxillae: A Biomechanical Human Cadaveric Investigation.

The alveolar ridge splitting technique (ARST) offers an alternative to classic ridge augmentation techniques for successful insertion of dental implants. However, the buccal lamella is at risk of fracturing during ARST distraction. To better understand the fracture mechanisms and displacement limits of the split lamella, this study conducted biomechanical tests on human cadaveric maxilla specimens having extremely atrophied alveolar ridges treated with ARST. A total of 12 standardized alveolar splits were prepared on the maxillae of 3 elderly female donors using an oscillating piezoelectric saw. Mimicking the surgical distraction process of the lamella, each split was tested to failure using a dental osteotome attached to the crosshead of an electromechanical testing system. All specimens were scanned by means of high-resolution peripheral quantitative computed tomography prior to and post testing to evaluate split geometries and failure modes. Split stiffness, failure force, and displacement were 27.4 ± 18.7 N/mm, 12.0 ± 8.4 N, and 0.97 ± 0.31 mm, with no significant differences between anatomical sides and split locations (p ≥ 0.17). Stiffness correlated significantly with failure force (R 2 = 0.71, p < 0.01). None of the alveolar split widths correlated significantly with the outcomes from biomechanical testing (p ≥ 0.10). The results suggest that simple geometrical measures do not predict the allowed extent of lamella distraction prior to failure. More sophisticated methods are required for surgical planning to optimize the ARST outcomes. Still, the present study may advocate a clinical protocol for the maxilla where the implant site is prepared directly after osteotomy setting and immediately before full lamella dislocation, when the lamella is still stable, resistant to mechanical stress, and bone loss caused by the abrasion of the burr is minimized.

Analysis of Estrogen Effects on Eruption of Rat Molars / Análisis de los Efectos del Estrógeno en la Erupción de Molares de Rata

ABSTRACT: Tooth eruption requires resorption of the alveolar bone interposed between the tooth germ and the oral mucosa (coronal bone). The cells responsible for bone resorption are the osteoclasts and their activity can be reduced or inactivated by estrogen hormone. We aimed to investigate the effects of estrogen on the process of tooth eruption in rats. Thirty-three Wistar rats, aged two-to-17-days, were divided into control, sham and estrogen-treated groups. After daily injections with estrogen, the animals were euthanized and the jaws removed and processed for histological analysis. We performed clinical examination, morphological analysis, quantification of the number of osteoclasts on the surface of the coronal bone and immunohistochemical analysis of estrogen receptor type alpha (ERα). Estrogen therapy was effective, which could be confirmed by the higher estrogen plasma levels on treated animals. However, it had no effect on tooth development or tooth eruption. Progressive bone resorption was observed and the number of osteoclasts on coronal bone was not affected on hormoneinjected animals, allowing tooth to erupt at the same time observed in untreated animals. Immunohistochemistry for ERα confirmed the presence of this type of receptor in osteoclasts, osteoblasts and osteocytes. Taken together, our results showed that estrogen stimulation was not sufficient to decrease the number of osteoclasts on the coronal bone, supporting the idea that, although estrogen may have a protective activity on bone resorption, this may not apply to the alveolar bone that is meant to be resorbed during eruptive process.

RESUMEN: La erupción dental requiere la resorción del hueso alveolar interpuesto entre el germen dental y la mucosa oral (hueso coronal). Las células responsables de la resorción ósea son los osteoclastos y su actividad puede reducirse o inactivarse por la hormona del estrógeno. Objetivos: apuntamos a investigar los efectos del estrógeno en el proceso de la erupción dental en ratas. Treinta y tres ratas Wistar, de dos a 17 días de edad, se dividieron en grupos de control, Sham y se trataron con estrógenos. Los animales fueron eutanizados después del tratamento con estrógeno y se procesaron las mandíbulas para el análisis histológico. Se realizó el examen clínico, el análisis morfológico, la cuantificación del número de osteoclastos en la superficie del hueso coronal y el análisis inmunohistoquímico del tipo de receptor de estrógeno alfa (ERα). La terapia de estrógeno fue eficaz, lo que podría ser confirmado por los niveles plasmáticos más altos de estrógeno en los animales tratados. Sin embargo, no se observó ningún efecto sobre el desarrollo de los dientes o la erupción dental. Se observó una resorción ósea progresiva y el número de osteoclastos en el hueso coronal no se vio afectado en los animales inyectados con hormonas, permitiendo que el diente erupcionó durante el mismo período de tiempo observado en animales no tratados. La inmunohistoquímica para el ERα confirmó la presencia de este tipo de receptor en los osteoclastos, osteoblastos y osteocitos. Nuestros resultados mostraron que la estimulación del estrógeno no fue suficiente para reducir el número de osteoclastos en el hueso coronal confirmando que, si bien el estrógeno puede tener una actividad protectora en la resorción ósea, esto puede no se aplica al hueso alveolar que está destinado a ser rerecurrido durante el proceso eruptivo.

Investigation of effective intrusion and extrusion force for maxillary canine using finite element analysis.

Orthodontic tooth movement is mainly regulated by the biomechanical responses of loaded periodontal ligament (PDL). We investigated the effective intervals of orthodontic force in pure maxillary canine intrusion and extrusion referring to PDL hydrostatic stress and logarithmic strain. Finite element analysis (FEA) models, including a maxillary canine, PDL and alveolar bone, were constructed based on computed tomography (CT) images of a patient. The material properties of alveolar bone were non-uniformly defined using HU values of CT images; PDL was assumed to be a hyperelastic-viscoelastic material. The compressive stress and tensile stress ranging from 0.47 to 12.8 kPa and 18.8 to 51.2 kPa, respectively, were identified as effective for tooth movement; a strain 0.24% was identified as the lower limit of effective strain. The stress/strain distributions within PDL were acquired in canine intrusion and extrusion using FEA; root apex was the main force-bearing area in intrusion-extrusion movements and was more prone to resorption. Owing to the distinction of PDL biomechanical responses to compression and tension, the effective interval of orthodontic force was substantially lower in canine intrusion (80-90 g) than in canine extrusion (230-260 g). A larger magnitude of force remained applicable in canine extrusion. This study revised and complemented orthodontic biomechanical behaviours of tooth movement with intrusive-extrusive force and could further help optimize orthodontic treatment.

Platelet-rich fibrin and collagen membrane in the preservation of the alveolar bone: Feasibility of the elemental inorganic composition and scanning electron microscopy analysis.

The success of dental implants is related to the amount, quality, and composition of the alveolar bone. The placement of platelet-rich fibrin (PRF) clot associated with a resorbable collagen membrane (RCM) in a postextraction alveolus is a technique used for ridge preservation. This case report study analyzed the ultrastructural characteristics of cross-sectioned alveolar bone that received PRF and RCM using scanning electron microscopy and the inorganic composition using "energy dispersive X-ray spectrometry," in order to explore the feasibility of this method to clinical studies. Three alveolar bone samples from two male patients (37 and 58 years old), obtained in the procedure of placing the dental implant, were analyzed. Two bone samples previously received PRF and RCM (M37 and M58), the third sample represented a physiological bone formation without treatment (M37-control). The bone sample M37 showed irregularly shaped islets of calcified material intermingled with connective tissue. The other samples, from the 58-year-old patient with PRF and RCM (M58); and the other untreated bone sample from the same 37-year-old patient (M37-control) showed similar ultrastructural morphology with trabecular conformation without islets agglomerations. The inorganic composition analysis showed higher concentrations of calcium and phosphorus in both samples treated with PRF and RCM in comparison to the untreated bone sample. The Ca/P ratio was higher in the M37 sample compared to the others samples. The results showed morphology and inorganic composition differences among the treatments used, suggesting that this method is feasible to analyze parameters of the alveolar bone tissue.

Extracellular matrix composition during bone regeneration in the human dental alveolar socket.

Within the dental alveolar socket, the sequence of events following tooth extraction involves deposition of a provisional connective tissue matrix that is later replaced by woven bone and eventually by lamellar bone. Bone regeneration within the dental alveolar socket is unique since the space occupied by the root(s) of a tooth does not originally contain any bone. However, extracellular matrix composition of the healing alveolar socket has not previously been investigated. Here, alveolar bone biopsies representing early (7-46 months, < 4y) and late (48-60 months; 4-5y) healing periods were investigated using Raman spectroscopy, X-ray micro-computed tomography and backscattered electron scanning electron microscopy. Partially or completely edentulous individuals and those with a smoking habit were not excluded. Between < 4y and 4-5y, mineral crystallinity and bone mineral density increase, phenylalanine, proline/hydroxyproline, and bone surface-to-volume ratio decrease, while the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, and the collagen crosslink ratio remain relatively unchanged. Observed exclusively at 4-5y, hypermineralised osteocyte lacunae contain spherical and rhomboidal mineral nodules. Spearman correlation analysis reveals several significant, high (ρ = 0.7-0.9; p ≤ 0.01) and moderate (ρ = 0.5-0.7; p ≤ 0.01) correlations. Mineral crystallinity and proline/hydroxyproline, the carbonate-to-phosphate ratio and phenylalanine, mineral crystallinity and bone surface-to-volume ratio, the carbonate-to-phosphate ratio and bone surface-to-volume ratio, proline/hydroxyproline and bone mineral density, and bone mineral density and bone surface-to-volume ratio are negatively correlated. Mineral crystallinity and bone mineral density, and proline/hydroxyproline and bone surface-to-volume ratio are positively correlated. Although bone regeneration in the dental alveolar socket follows typical bone healing patterns, the compositional and microstructural patterns reveal mature bone at <4y with indications of better mechanical competence at 4-5y.

Three-dimensional finite element analysis to evaluate biomechanical effects of different alveolar decortication approaches on rate of canine retraction.

INTRODUCTION: The aim of this study was to compare different corticotomy approaches and determine their biomechanical effects on rate of canine displacement when compared to conventional orthodontics. METHOD: Three-dimensional Finite Element Models with conventional non-corticotomy approach (model 1) and three corticotomy approaches ensuing buccal and palatal vertical cuts (model 2), interseptal bone reduction (model 3), buccal vertical cuts (model 4) were fabricated. Displacement of the canine and von Mises stresses in the canine and trabecular bone were calculated and compared under a distal retraction force of 1.5N. RESULTS: The maximum displacement of canine with minimum anchorage loss was seen in model 3 followed by model 2, model 4 and model 1. The maximum equivalent (von Mises) stress was concentrated mainly on the distal side of canine in model 3 and had a uniform distribution of stresses on entire root surface. CONCLUSIONS: Corticotomy approaches effectively accelerated maxillary canine retraction, exhibiting twice the rate of canine movement with minimum anchorage loss when compared to non-corticotomy approach. Corticotomy with interseptal bone reduction was most effective in terms of canine displacement and stress distribution.

Biomechanical analysis for total distalization of the mandibular dentition: A finite element study.

INTRODUCTION: The aim of this finite element study was to analyze and clarify the mechanics of tooth movement patterns for total distalization of the mandibular dentition based on force angulation. METHODS: Long-term orthodontic movement of the mandibular dentition was simulated by accumulating the initial displacement of teeth produced by elastic deformation of the periodontal ligament. RESULTS: Displacement of each tooth was caused by movement of the whole dentition, elastic deflection of the archwire, and clearance gap between the archwire and bracket slot. The whole dentition was rotated clockwise or counterclockwise when the line of action of the force passed below or above the center of resistance. Elastic deflection of the archwire induced a lingual tipping of the anterior teeth. It became larger when increasing the magnitude of angulation. The archwire could be rotated within the clearance gap between the archwire and the bracket slot, and thereby the teeth tipped. CONCLUSIONS: Mechanics of total mandibular distalization was clarified. Selective use of force angulation with a careful biomechanical understanding can achieve proper distalization of the whole mandibular dentition.

Dental Pulp Stem Cell Transplantation with 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside Accelerates Alveolar Bone Regeneration in Rats.

INTRODUCTION: Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats. METHODS: Expressions of osteogenic messenger RNAs (including ALP, RUNX2, BGLAP, and AMBN) were examined by quantitative real-time polymerase chain reaction. Alizarin red S staining was conducted to analyze THSG-induced mineralization of hDPSCs. To investigate the regenerative effects of THSG-treated hDPSCs on dental alveolar bone, bony defects were created in male Sprague-Dawley rats. Defects were treated with Matrigel (Corning Inc, Corning, NY), hDPSCs, or hDPSCs + THSG. After 2 weeks, defect healing was evaluated by micro-computed tomographic and histologic analyses. RESULTS: In the cell model, THSG induced osteogenesis-associated genes (ALP, RUNX2, and BGLAP) and an enamel-related gene (AMBN), resulting in mineralization as detected by alizarin red S staining after 2 weeks of treatment. In the animal model, THSG increased all parameters of bone formation (the relative bone volume, trabecular thickness, trabecular number, and trabecular separation) in alveolar bony defects of rats. THSG not only improved the quality of newly formed bone but also the quantity of new bone. CONCLUSIONS: These results showed important findings in revealing the THSG-enhanced osteogenic differentiation of hDPSCs and THSG-facilitated bone regeneration, which may provide an alternative option for cell-based regenerative therapy.

Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model.

BACKGROUND: Bone morphogenetic proteins (BMPs) have played a central role in the regenerative therapies for bone reconstruction, including alveolar cleft and craniofacial surgery. However, the high cost and significant adverse effect of BMPs limit their broad application. Hydroxycholesterols, naturally occurring products of cholesterol oxidation, are a promising alternative to BMPs. The authors studied the osteogenic capability of hydroxycholesterols on human mesenchymal stem cells and the impact of hydroxycholesterols on a rodent alveolar cleft model. METHODS: Human mesenchymal stem cells were treated with control medium or osteogenic medium with or without hydroxycholesterols. Evaluation of cellular osteogenic activity was performed. A critical-size alveolar cleft was created and one of the following treatment options was assigned randomly to each defect: collagen sponge incorporated with hydroxycholesterols, BMP-2, or no treatment. Bone regeneration was assessed by means of radiologic and histologic analyses and local inflammation in the cleft evaluated. Moreover, the role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was examined. RESULTS: All cellular osteogenic activities were significantly increased on human mesenchymal stem cells treated with hydroxycholesterols relative to others. The alveolar cleft treated with collagen sponge with hydroxycholesterols and BMP-2 demonstrated robust bone regeneration. The hydroxycholesterol group revealed histologically complete bridging of the alveolar defect with architecturally mature new bone. The inflammatory responses were less in the hydroxycholesterol group compared with the BMP-2 group. Induction of hydroxycholesterol-mediated in vitro osteogenesis and in vivo bone regeneration were attenuated by hedgehog signaling inhibitor, implicating involvement of the hedgehog signaling pathway. CONCLUSION: Hydroxycholesterols may represent a viable alternative to BMP-2 in bone tissue engineering for alveolar cleft.

Effect of advanced platelet-rich fibrin on accelerating alveolar bone formation in dogs: a histological and immunofluorescence evaluation.

Several methods have been developed to regenerate lost alveolar bone. Platelet-rich fibrin (PRF) is a useful adjunct for new bone formation in dentistry. To elucidate the effect of advanced PRF (A-PRF) on bone formation, we inserted A-PRF clots in sockets after tooth extraction. Premolars were extracted from beagle dogs, and A-PRF was applied to the socket. New bone formation was assessed using histological and immunofluorescence examinations, and the bone formation ratio was evaluated 14 and 30 days postoperatively. Histological examination revealed newly formed bone filling the sockets up to the center in the A-PRF group at 14 days postoperatively, while thick and regular bone trabeculae were arranged in porous bone after 30 days. Higher expressions of osteocalcin and osteopontin were observed in newly formed bone in the A-PRF group, compared to the control group. The bone formation ratio was also higher in the A-PRF group than in the control group. Thus, A-PRF application may result in enhanced new bone formation and may aid in accelerating bone formation. A-PRF was more rapid than a self-limiting process during induction of bone formation by enhancing osteoblast activity and may be useful for bone formation in clinical medicine.

The effect of retraining hypofunctional jaw muscles on the transverse skull dimensions of adult rats.

OBJECTIVE: To experimentally investigate the effects of increased masticatory muscle function on the transverse cranial dimensions on adult rats with an earlier reduced masticatory muscle function. MATERIAL AND METHOD: Sixty young male rats were used. The experimental group received soft diet for a prolonged period, so that the animals developed weak masticatory muscles. A control group received ordinary hard food during the whole experimental period (27 weeks). After 21 weeks when the animals had nearly ceased their body growth the rats in the experimental group were divided into two groups. One group continued with soft diet until the end of the experiment (hypofunctional group). The other group received ordinary hard food to get the possibility to retrain their masticatory muscles (rehabilitation group). At week 21 and at the end of the experimental period (week 27), axial cephalograms were taken. Fourteen landmarks were defined to measure seven transverse distances of the skull. RESULTS: The increase of the anterior zygomatic arch width and interzygomatic width during the experimental period were larger in the rehabilitation group compared to both the normal and the hypofunctional group. CONCLUSION: Retraining of masticatory muscles in adult rats leads to increase of some transverse cranial dimensions.

Mineralized nanofiber segments coupled with calcium-binding BMP-2 peptides for alveolar bone regeneration.

Bone loss around tooth extraction sites can occur, thus making future placement of dental implants difficult. Alveolar bone regeneration can be guided by the application of a nanofibrous bone graft coupled with osteoinductive proteins/peptides, following tooth loss or tooth extraction. In the present study, we demonstrate the potential of mineralized nanofiber segments coupled with calcium-binding bone morphogenetic protein 2 (BMP-2) mimicking peptides for periodontal bone regeneration. Thin electrospun nanofiber membranes of PLGA-collagen-gelatin (2:1:1 wt ratios) were mineralized in 10× modified simulated body fluid (10× mSBF) and cryocut to segments of 20 µm. For predetermined weights of the mineralized nanofiber segments, it was possible to load various amounts of heptaglutamate E7-domain-conjugated BMP-2 peptide. Mineralized short fiber grafts (2 mg), with and without E7-BMP-2 peptides, were implanted into 2 mm × 2 mm (diameter × depth) critical-sized socket defects created in rat maxillae, following extraction of the first molar teeth. A sustained release profile of E7-BMP-2 from the mineralized nanofiber segments was recorded over 4 weeks. X-ray microcomputed tomography (µ-CT) analysis of peptide-loaded nanofiber graft filled defects revealed ∼3 times greater new bone volume and bone mineral density over 4 weeks in comparison to unfilled control defects. Further, histopathology data confirmed the formation of greater new osseous tissue in the BMP2 peptide-loaded, mineralized nanofiber segment group than that of fibrous connective tissue in the unfilled defect group. Altogether, the mineralized nanofiber segments coupled with E7-BMP-2 peptides may be an effective treatment option for alveolar bone loss and defects. STATEMENT OF SIGNIFICANCE: With the high incidence of dental implants/fixtures for missing teeth, the success of the surgical procedures in restorative dentistry is dictated by the quality and quantity of the supporting alveolar bone. To address the problem of alveolar bone loss and defects due to tumor, periodontitis, or even postextraction remodeling, the present study is the first report on the application of mineralized nanofiber fragments coupled with calcium-binding osteoinductive BMP-2 peptides as a synthetic graft material for oral bone regeneration. The ease of fabrication and application of cryocut mineralized nanofiber fragments as maxillofacial bone defect fillers present a promising alternative to the current dental bone graft formulations. Furthermore, the nanofiber segments may also be utilized for several biomedical applications including hemostasis, soft tissue engineering, and wound healing.

Gingival fibroblasts prevent BMP-mediated osteoblastic differentiation.

OBJECTIVES: The inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)-induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation. METHODS: Gingival fibroblasts were co-cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP-inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT-qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads. RESULTS: Co-culture experiments showed GF-secreted factors that inhibit BMP-stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well-described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity. CONCLUSIONS: Factors secreted by gingival fibroblasts inhibit BMP-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.