Fibrocyte: A missing piece in the pathogenesis of fibrous epulis.

OBJECTIVES: To explore the role of fibrocytes in the recurrence and calcification of fibrous epulides. METHODS: Different subtypes of fibrous epulides and normal gingival tissue specimens were first collected for histological and immunofluorescence analyses to see if fibrocytes were present and whether they differentiated into myofibroblasts and osteoblasts upon stimulated by transforming growth factor-ß1 (TGF-ß1). Electron microscopy and elemental analysis were used to characterize the extracellular microenvironment in different subtypes of fibrous epulides. Human peripheral blood mononuclear cells (PBMCs) were subsequently isolated from in vitro models to mimic the microenvironment in fibrous epulides to identify whether TGF-ß1 as well as the calcium and phosphorus ion concentration in the extracellular matrix (ECM) of a fibrous epulis trigger fibrocyte differentiation. RESULTS: Fibrous epulides contain fibrocytes that accumulate in the local inflammatory environment and have the ability to differentiate into myofibroblasts or osteoblasts. TGF-ß1 promotes fibrocytes differentiation into myofibroblasts in a concentration-dependent manner, while TGF-ß1 stimulates the fibrocytes to differentiate into osteoblasts when combined with a high calcium and phosphorus environment. CONCLUSIONS: Our study revealed fibrocytes play an important role in the fibrogenesis and osteogenesis in fibrous epulis, and might serve as a therapeutic target for the inhibition of recurrence of fibrous epulides.

Early growth response 1 reduction in peripheral blood involving condylar subchondral bone loss.

OBJECTIVES: To detect whether early growth response 1 (EGR1) in peripheral blood leucocytes (PBLs) indicates temporomandibular joint (TMJ) osteoarthritis (OA) lesions. MATERIALS AND METHODS: Egr1 mRNA expression levels in PBLs were detected in eight malocclusion patients without temporomandibular disorder (TMD) signs and 16 malocclusion patients with clinical TMD signs with (eight) or without (eight) imaging signs of TMJ OA. Twelve 6-week-old rats were randomized to a control group and a unilateral anterior crossbite (UAC) group and were sampled at 4 weeks. The Egr1 mRNA expression levels in PBLs and protein expression levels in different orofacial tissues were measured. RESULTS: Patients with TMD signs with/without TMJ OA diagnosis showed lower Egr1 mRNA expression levels in PBLs than patients without TMD signs. The lower Egr1 mRNA expression was also found in the PBLs of UAC rats, which were induced to exhibit early histo-morphological signs of TMJ OA lesions. In subchondral bone of UAC rats, EGR1 protein expression was decreased, co-localization of EGR1 with osterix or dentin matrix protein-1 was identified, and the number of EGR1 and osterix double-positive cells was reduced (all p < .05). CONCLUSION: Egr1 reduction in PBLs potentially indicates subchondral bone OA lesions at an early stage.

Bacteria-mediated resistance of neutrophil extracellular traps to enzymatic degradation drives the formation of dental calculi.

Dental calculi can cause gingival bleeding and periodontitis, yet the mechanism underlying the formation of such mineral build-ups, and in particular the role of the local microenvironment, are unclear. Here we show that the formation of dental calculi involves bacteria in local mature biofilms converting the DNA in neutrophil extracellular traps (NETs) from being degradable by the enzyme DNase I to being degradation resistant, promoting the nucleation and growth of apatite. DNase I inhibited NET-induced mineralization in vitro and ex vivo, yet plasma DNases were ineffective at inhibiting ectopic mineralization in the oral cavity in rodents. The topical application of the DNA-intercalating agent chloroquine in rodents fed with a dental calculogenic diet reverted NET DNA to its degradable form, inhibiting the formation of calculi. Our findings may motivate therapeutic strategies for the reduction of the prevalence of the deposition of bacteria-driven calculi in the oral cavity.

Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.

Combined degenerative and regenerative remodeling responses of the mandibular condyle to experimentally induced disordered occlusion.

INTRODUCTION: The purposes of this research were to investigate the long-term responses of mandibular condylar cartilage to experimentally induced disordered occlusion and to evaluate changes in the expression of the SDF-1/CXCR4 axis. METHODS: Experimentally induced disordered occlusions were created in 8-week-old female Sprague-Dawley rats by orthodontic methods. After 24 weeks, remodeling of the mandibular condylar cartilage was assessed by hematoxylin and eosin staining. Protein and mRNA expression of SDF-1, CXCR4, MMP9, IL6, OPG, and RANKL were investigated by means of immunohistochemical staining and real-time polymerase chain reaction. RESULTS: Obvious cartilage degenerative remodeling responses were observed; they appeared as uneven distributions of cellular disposition, loss of cartilage surface integrity, and cell-free areas. Regenerative responses presenting as thickening of the whole and the calcified cartilage layers in the experimental group were also observed. Compared with the age-matched controls, the protein and mRNA levels of SDF-1, CXCR4, MMP9, IL6, and OPG, but not RANKL, were increased in the experimental group (all, P <0.05). In addition, the mRNA level of RANKL/OPG showed a decreasing trend in the experimental group compared with the age-matched controls (P = 0.052). CONCLUSIONS: This study demonstrated that long-term experimentally induced disordered occlusion leads to a combined response in degeneration and regeneration of mandibular cartilage, accompanied by active interaction of the SDF-1/CXCR4 axis and local upregulation of MMP9, IL6, and OPG.

Optimization of Lactoferrin-Derived Amyloid Coating for Enhancing Soft Tissue Seal and Antibacterial Activity of Titanium Implants.

A poor seal of the titanium implant-soft tissue interface provokes bacterial invasion, aggravates inflammation, and ultimately results in implant failure. To ensure the long-term success of titanium implants, lactoferrin-derived amyloid is coated on the titanium surface to increase the expression of cell integrins and hemidesmosomes, with the goal of promoting soft tissue seal and imparting antibacterial activity to the implants. The lactoferrin-derived amyloid coated titanium structures contain a large number of amino and carboxyl groups on their surfaces, and promote proliferation and adhesion of epithelial cells and fibroblasts via the PI3K/AKT pathway. The amyloid coating also has a strong positive charge and possesses potent antibacterial activities against Staphylococcus aureus and Porphyromonas gingivalis. In a rat immediate implantation model, the amyloid-coated titanium implants form gingival junctional epithelium at the transmucosal region that resembles the junctional epithelium in natural teeth. This provides a strong soft tissue seal to wall off infection. Taken together, lactoferrin-derived amyloid is a dual-function transparent coating that promotes soft tissue seal and possesses antibacterial activity. These unique properties enable the synthesized amyloid to be used as potential biological implant coatings.

A biomaterial-based therapy using a sodium hyaluronate/bioglass composite hydrogel for the treatment of oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic, inflammatory and potentially malignant oral disorder. Its pathophysiology is extremely complex, including excessive collagen deposition, massive inflammatory infiltration, and capillary atrophy. However, the existing clinical treatment methods do not fully take into account all the pathophysiological processes of OSF, so they are generally low effective and have many side effects. In the present study, we developed an injectable sodium hyaluronate/45S5 bioglass composite hydrogel (BG/HA), which significantly relieved mucosal pallor and restricted mouth opening in OSF rats without any obvious side effects. The core mechanism of BG/HA in the treatment of OSF is the release of biologically active silicate ions, which inhibit collagen deposition and inflammation, and promote angiogenesis and epithelial regeneration. Most interestingly, silicate ions can overall regulate the physiological environment of OSF by down-regulating α-smooth muscle actin (α-SMA) and CD68 and up-regulating CD31 expression, as well as regulating the expression of pro-fibrotic factors [transforming growth factor-ß1 (TGF-ß1), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and tissue inhibitors of metalloproteinase-1 (TIMP-1)] and anti-fibrotic factors [interleukin-1ß (IL-1ß)] in macrophage. In conclusion, our study shows that BG/HA has great potential in the clinical treatment of OSF, which provides an important theoretical basis for the subsequent development of new anti-fibrotic clinical preparations. STATEMENT OF SIGNIFICANCE: : Oral submucous fibrosis (OSF) is a chronic, inflammatory and potentially malignant mucosal disease with significant impact on the quality of patients' life. However, the existing clinical treatments have limited efficacy and many side effects. There is an urgent need for development of specific drugs for OSF treatment. In the present study, bioglass (BG) composited with sodium hyaluronate solution (HA) was used to treat OSF in an arecoline-induced rat model. BG/HA can significantly inhibit collagen deposition, regulate inflammatory response, promote angiogenesis and repair damaged mucosal epithelial cells, and thereby mitigate the development of fibrosis in vivo.

The immunomodulatory effects of RNA-based biomaterials on bone regeneration.

The use of RNA as therapeutic agents is a visionary idea in contemporary medicine. Some forms of RNA can modulate the immune response of the host to enhance tissue regeneration events such as osteogenesis. Herein, RNA molecules commercially available for immunomodulatory applications (imRNA) were used to prepare biomaterials for bone regeneration. The polyanionic imRNA stabilized calcium phosphate ionic clusters to produce imRNA-ACP that had the capacity to mineralize the intrafibrillar compartments of collagen fibrils. For the first time, it was shown that incorporating imRNA-ACP into collagen scaffolds resulted in rapid new bone formation in cranial defects of mice. Both in vivo and in vitro results demonstrated that macrophage polarization was highly-sensitive to the imRNA-ACP containing collagen scaffolds. Macrophages were polarized into the anti-inflammatory M2 phenotype that produced anti-inflammation cytokines and growth factors. The favorable osteoimmunological microenvironment created by the scaffolds prevented their immunorejection and facilitated osteogenesis. The potential of RNA in creating immunomodulatory biomaterials has been underestimated in the past. The overall aim of this study was to explore the potential application of imRNA-based biomaterials in bone tissue engineering, with the competitive edge of facile synthesis and excellent biocompatibility. STATEMENT OF SIGNIFICANCE: In this work, commercially available RNA extracted from bovine spleens for immunomodulatory applications (imRNA) were used to stabilize amorphous calcium phosphate (ACP) and induce mineralization within collagen fibrils. Incorporation of imRNA-ACP into collagen scaffolds regenerated new bone in-situ. Because of its immunomodulatory effects, the imRNA-ACP that was incorporated into collagen scaffolds modulated the local immune environment of murine cranial defects by altering the macrophage phenotype through JAK2/STAT3 signaling pathway. The novelty of this work existed in the discovery of RNA's potential in creating immunomodulatory biomaterials. With the competitive edge of facile synthesis and excellent biocompatibility, the imRNA-based biomaterials are potentially useful for future bone tissue engineering applications.

Changes in the expression of aromatase, estrogen receptor α and ß in mandibular condylar cartilage of rats induced by disordered occlusion.

BACKGROUND: Estrogens play an important role in modulating the morphology and function of temporomandibular joints (TMJs), which is suggested to act via estrogen receptors (ERs). The present study was to investigate the expression of aggrecan, collagen type II (Col II), Col X, aromatase, ERα and ERß in degenerative changes of mandibular condylar cartilage. METHODS: Forty male and 40 female 8-week-old rats were enrolled in this study. In experimental groups, the disordered occlusion was created by moving the first molars mesially and the third ones distally. Immunohistochemistry and real-time PCR were performed at the end of the second or fourth week. RESULTS: Degenerative changes, characterized by interrupted continuity of hypertrophic layer, pyknotic and eosinophilic lesion with few nuclei, areas filled with eosinophilic nuclei, were observed in more joints from female experimental groups than male ones. However, thickening changes in hypertrophic layer were only found in male experimental groups. The gene expression of Col II, Col X and aggrecan increased in 4-wk male experimental subgroup (both P < 0.01), but decreased in 2-wk and 4-wk female subgroups (P < 0.05). The gene expression of ERα decreased in 2-wk male and female experimental subgroups (both P < 0.01), however, that of ERß increased except the 2-wk female experimental subgroup (all P < 0.01). The expression of aromatase decreased in both male and female experimental subgroups (all P<0.01). CONCLUSIONS: Mandibular condylar cartilage responses differently to the disordered occlusion in male and female rats. The levels of locally synthesized estrogen, ERα and ERß may have limited attribution, if any, to the sex-specific cartilage response.

Degenerative changes in rat condylar cartilage induced by non-matching occlusion created by scattered orthodontic teeth-moving.

The effect of occlusion on the temporomandibular joint (TMJ) is debated. By inserting rubber-bands that were replaced by self-curing resin one week later, the left maxillary and the right mandibular first-molars were moved and kept mesially in Sprague-Dawley (SD) rats in both experimental I (EXP-I) and II (EXP-II) groups, aiming to establish a non-matching cusp-to-fossa occlusal relation. Four weeks later, the left maxillary and the right mandibular third-molars were moved and kept distally in the EXP-II group. Degenerative changes, typically as a cell-free area, were observed in TMJs of the EXP groups. Binary logistical analysis indicated that the odds ratio of EXP group, EXP-II vs. EXP-I, on the incidence of a cell-free area, was 2.8 (p=.036). Time point, gender, and side did not have such effects (p>0.05). The results indicate that the persistence of more scattered non-matching cusp-to-fossa occlusion is more harmful to the condylar cartilage in terms of the incidence of degenerative changes.

Clinical and pathologic factors associated with the relapse of fibrous gingival hyperplasia.

BACKGROUND: The association between clinicopathologic characteristics and the relapse of fibrous gingival hyperplasia is unknown. METHODS: The records of 211 consecutive patients with a clinicopathologic diagnosis of fibrous gingival hyperplasia were retrieved. Patients who experienced relapse after surgical excision of the lesion were considered case patients (n = 30). All control patients were informed that there was no recurrence (n = 181). Logistic regression was used to evaluate the associations among different characteristics and the recurrence. Stratified analyses on sex was applied to identify the different associations. RESULTS: Binary logistic regression showed that patients with ulcer (odds ratio [OR], 3.23; 95% CI, 1.18 to 8.83) or mechanical stimulation (OR, 2.42; 95% CI, 1.03 to 5.68) had a higher risk of experiencing recurrence. Stratified analysis of sex identified significant association in females (ulcer: OR, 4.04; 95% CI, 1.14 to 14.34; mechanical stimulation: OR, 3.30; 95% CI, 1.15 to 9.42). No significant difference was observed in males (ulcer: OR, 2.44; 95% CI, 0.40 to 15.06; mechanical stimulation: OR, 1.62; 95% CI, 0.28 to 9.40). Male patients with larger epulides had fewer recurrence (OR, 0.13; 95% CI, 0.02 to 0.74). There was no significant difference in pathologic calcification between case and control patients (P > .05). CONCLUSIONS: Patients with ulcer and mechanical stimulation may have a high risk of experiencing recurrent epulis. PRACTICAL IMPLICATIONS: More attention should be paid to patients with ulcer and mechanical stimulation. Apart from complete surgical removal, it is important to remove local stimulation to prevent recurrence of these lesions.

Regulation of biomineralization by proteoglycans: From mechanisms to application.

Proteoglycans consist of core proteins and one or more covalently-linked glycosaminoglycan chains. They are structurally complex and heterogeneous. Proteoglycans bind to cell surface receptors, cytokines, growth factors and have strong affinity for collagen fibrils. Together with their complex spatial structures and different charge densities, proteoglycans are directly or indirectly involved in biomineralization. The present review focused on the potential mechanisms of proteoglycans-mediated biomineralization. Topics covered include the ability of proteoglycans to influence the proliferation and differentiation of odontoblasts and osteoblasts through complex signaling pathways, as well as regulate the aggregation of collagen fibrils and mineral deposition. The functions of proteoglycans in mineralization regulation and biomimetic properties render them important components in bone tissue engineering. Hence, the integrated impact of proteoglycans on bone formation was also succinctly deliberated. The potential of proteoglycans to function therapeutic targets for relieving the symptoms of ectopic mineralization and mineralization defects was also comprehensively addressed.

Polyphosphate-crosslinked collagen scaffolds for hemostasis and alveolar bone regeneration after tooth extraction.

Post-extraction bleeding and alveolar bone resorption are the two frequently encountered complications after tooth extraction that result in poor healing and rehabilitation difficulties. The present study covalently bonded polyphosphate onto a collagen scaffold (P-CS) by crosslinking. The P-CS demonstrated improved hemostatic property in a healthy rat model and an anticoagulant-treated rat model. This improvement is attributed to the increase in hydrophilicity, increased thrombin generation, platelet activation and stimulation of the intrinsic coagulation pathway. In addition, the P-CS promoted the in-situ bone regeneration and alveolar ridge preservation in a rat alveolar bone defect model. The promotion is attributed to enhanced osteogenic differentiation of bone marrow stromal cells. Osteogenesis was improved by both polyphosphate and blood clots. Taken together, P-CS possesses favorable hemostasis and alveolar ridge preservation capability. It may be used as an effective treatment option for post-extraction bleeding and alveolar bone loss. Statement of significance: Collagen scaffold is commonly used for the treatment of post-extraction bleeding and alveolar bone loss after tooth extraction. However, its application is hampered by insufficient hemostatic and osteoinductive property. Crosslinking polyphosphate with collagen produces a modified collagen scaffold that possesses improved hemostatic performance and augmented bone regeneration potential.

Mandibular condylar cartilage response to moving 2 molars in rats.

INTRODUCTION: The purpose of this study was to investigate the responses of mandibular condylar cartilage to moving 2 molars in different combinations. METHODS: Rats were assigned to male and female control and experimental groups (each, n = 5). Elastic rubber bands were used to move medially the maxillary left and the mandibular right first molars in experimental group I. The same method was used to distally move the maxillary left and the mandibular right third molars, 2 mandibular third molars, and 2 maxillary third molars in experimental groups II, III, and IV, respectively. At the end of the eighth week, all condyles were examined histologically. The areas of histologic change as a percentage of total cartilage area were compared by using the Mann-Whitney U test. RESULTS: Cartilage degenerative remodeling was observed in experimental groups II, III, and IV. The percentage areas of degenerative remodeling were higher in female experimental groups II and III than in the female control group, and in female experimental group II than in female experimental group IV and male experimental group II (all, P <0.05). CONCLUSIONS: The mandibular condylar cartilage of female rats responded variously to different combinations of molar movement; the most obvious remodeling was observed in groups in which the maxillary left and mandibular right third molars were moved.

Conditional deletion of Adrb2 in mesenchymal stem cells attenuates osteoarthritis-like defects in temporomandibular joint.

ß2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of ß2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2-/-), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2+/+) and Adrb2-/- mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2+/+ and Adrb2-/- mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2+/+ UAC mice; the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression; the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA.

Death and proliferation of chondrocytes in the degraded mandibular condylar cartilage of rats induced by experimentally created disordered occlusion.

OBJECTIVE: To investigate the effect of experimentally created disordered occlusion (ECDO) on cell death and proliferation in rat mandibular condylar cartilage. METHODS: Sprague-Dawley rats were randomly assigned to experimental and control groups. In the experimental groups, ECDO was created by the dental orthodontic method. By means of histological evaluation, immunohistochemistry and TUNEL staining, we studied the histomorphological changes, the death and proliferation of chondrocytes. RESULTS: Time- and sex-related progressive histologic degradation was observed in the condylar cartilage of ECDO rats, accompanied with diminished chondrocyte proliferation in the female 12-week ECDO subgroup (P < 0.05). An increase in the number of apoptotic chondrocytes was seen in both the female 8- and 12-week ECDO subgroups and in the male ECDO 12-week subgroup (all P < 0.05), but not in the male ECDO 8-week subgroup (P > 0.05). CONCLUSION: ECDO induces degradation in the rat condylar cartilage accompanied by an increase in chondrocyte death.

Activation of α2A-adrenergic signal transduction in chondrocytes promotes degenerative remodelling of temporomandibular joint.

This study tested whether activation of adrenoreceptors in chondrocytes has roles in degenerative remodelling of temporomandibular joint (TMJ) and to determine associated mechanisms. Unilateral anterior crossbite (UAC) was established to induce TMJ degeneration in rats. Saline vehicle, α2- and ß-adrenoreceptor antagonists or agonists were injected locally into the TMJ area of UAC rats. Cartilage degeneration, subchondral bone microarchitecture and the expression of adrenoreceptors, aggrecans, matrix metalloproteinases (MMPs) and RANKL by chondrocytes were evaluated. Chondrocytes were stimulated by norepinephrine to investigate signal transduction of adrenoreceptors. Increased α2A-adrenoreceptor expression was observed in condylar cartilage of UAC rats, together with cartilage degeneration and subchondral bone loss. Norepinephrine depresses aggrecans expression but stimulates MMP-3, MMP-13 and RANKL production by chondrocytes through ERK1/2 and PKA pathway; these effects were abolished by an α2A-adrenoreceptor antagonist. Furthermore, inhibition of α2A-adrenoreceptor attenuated degenerative remodelling in the condylar cartilage and subchondral bone, as revealed by increased cartilage thickness, proteoglycans and aggrecan expression, and decreased MMP-3, MMP-13 and RANKL expressions in cartilage, increased BMD, BV/TV, and decreased Tb.Sp in subchondral bone. Conversely, activation of α2A-adrenoreceptor intensified aforementioned degenerative changes in UAC rats. It is concluded that activation of α2A-adrenergic signal in chondrocytes promotes TMJ degenerative remodelling by chondrocyte-mediated pro-catabolic activities.

Revival of nitrogen-containing bisphosphonate-induced inhibition of osteoclastogenesis and osteoclast function by water-soluble microfibrous borate glass.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious skeletal complication associated with the long-term oral or intravenous use of nitrogen-containing bisphosphonates (N-BPs). Here, we investigated the effects of an ionic cocktail prepared from water-soluble microfibrous borate glass on neutralizing the inhibitory effects of two heterocyclic N-BPs, risedronate or zoledronic acid, on osteoclastogenesis, apoptosis of differentiated osteoclasts and osteoclast function. Cell growth and proliferation assays were first performed on RAW 264.7 cells to optimize the concentrations of the ionic cocktail and N-BPs to be used for static cell culture. The pre-osteoclasts were then stimulated with RANKL to differentiate into osteoclasts. The effects of the ionic cocktail and N-BPs on osteoclast differentiation, apoptosis and function were subsequently examined using 3 series of experiments conducted at the gene, protein, morphological and functional levels. After concentration optimization, the ionic cocktail was found to partially reverse N-BP-induced inhibition of osteoclastogenesis, stimulation of osteoclasts apoptosis and reduction of osteoclast resorptive activity. Ultrastructural examination of osteoclasts that had been exposed to either N-BP identified classical features of late apoptosis and secondary necrosis, while osteoclasts exposed simultaneously to the concentration-optimized ionic cocktail and N-BPs exhibited only signs of early apoptosis that were possibly reversible. Taken together, the results of the 4 series of experiments indicate that the ionic cocktail produced from dissolution of borate glass dressings has the potential to rescue the adverse effects of heterocyclic N-BPs on osteoclast differentiation and function. These results warrant further confirmation using dynamic cell culture and small animal BRONJ models. STATEMENT OF SIGNIFICANCE: Long-term oral and intravenous use of nitrogen-containing bisphosphonates (N-BPs) may result in bisphosphonate-related osteonecrosis of the jaw (BRONJ) due to the suppression of normal bone turnover. There is no effective treatment for such a complication to date. This work reported the use of an ionic cocktail derived from water-soluble microfibrous borate glass to revert heterocyclic N-BP-induced inhibition of osteoclastogenesis, stimulation of osteoclasts apoptosis and reduction of osteoclasts resorption in static cell culture condition. This ionic cocktail may have the potential to be further developed into a new adjunctive treatment for BRONJ.

Caries-resistant bonding layer in dentin.

The present study examined the mechanism for caries resistance and the pulp responses in vital teeth following the use of the augmented-pressure adhesive displacement technique. Dentin adhesives were applied to the surface of sound dentin disks in 4 experimental groups: non-antibacterial adhesive and gentle adhesive displacement (N-G), non-antibacterial adhesive and augmented-pressure adhesive displacement (N-H), antibacterial adhesive and gentle adhesive displacement (A-G), antibacterial adhesive and augmented-pressure adhesive displacement (A-H). The depth of demineralization induced by biological or chemical demineralization models was measured using confocal laser scanning microscopy and analyzed with two-way ANOVA. Pulp responses of vital dog's teeth to the augmented-pressure adhesive displacement technique were evaluated using light microscopy. Depth of demineralization was significantly affected by "adhesive type" and "intensity of adhesive displacement" for biological demineralization. For chemical demineralization, only "intensity of adhesive displacement" showed significant influence on lesion depth. Pulp response of 0.1, 0.2 and 0.3 MPa groups showed only moderate disorganization of the odontoblast layer at 24 hours that completely re-organized after 3 weeks. Augmented-pressure adhesive displacement improves the caries resistance property of bonded dentin and does not cause irreversible pulpal damage to vital teeth when the air pressure employed is equal or smaller than 0.3 MPa.