IRE1α inhibits osteogenic differentiation of mouse embryonic fibroblasts by limiting Shh signaling.

OBJECTIVES: This study aimed to investigate the effect of endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) on the sonic hedgehog N-terminus (N-Shh)-enhanced-osteogenic differentiation process in mouse embryonic fibroblasts (MEFs). MATERIALS AND METHODS: Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis. RESULTS: N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers. CONCLUSIONS: Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.

Tissue-engineered bone construct promotes early osseointegration of implants with low primary stability in oversized osteotomy.

OBJECTIVES: To evaluate the histological parameters and bone mechanical properties around implants with low primary stability (PS) in grafted bone substitutes within an oversized osteotomy. MATERIALS AND METHODS: An oversized osteotomy penetrating the double cortical bone layers was made on both femora of 24 New Zealand white rabbits. Bilaterally in the femur of all animals, 48 implants were installed, subdivided into four groups, corresponding to four prepared tissue-engineering bone complexes (TEBCs), which were placed between the implant surface and native bone wall: A: tricalcium phosphate ß (TCP-ß); B: autologous adipose derived-stem cells with TCP-ß (ASCs/TCP-ß); C: ASCs transfected with the enhanced-GFP gene with TCP-ß (EGFP-ASCs/TCP-ß); D: ASCs transfected with the BMP-2 gene with TCP-ß (BMP2-ASCs/TCP-ß). Trichrome fluorescent labeling was conducted. Animals were sacrificed after eight weeks. The trichromatic fluorescent labeling (%TFL), area of new bone (%NB), residual material (%RM), bone-implant contact (%BIC), and the removal torque force (RTF, N/cm) were assessed. RESULTS: ASCs were successfully isolated from adipose tissue, and the primary ASCs were induced into osteogenic, chondrogenic, and adipogenic differentiation. The BMP-2 overexpression of ASCs sustained for ten days and greatly enhanced the expression of osteopontin (OPN). At eight weeks post-implantation, increased %NB and RTF were found in all groups. The most significant value of %TFL, %BIC and lowest %RM was detected in group D. CONCLUSION: The low PS implants osseointegrate with considerable new bone in grafted TEBCs within an oversized osteotomy. Applying BMP-2 overexpressing ASCs-based TEBC promoted earlier osseointegration and more solid bone mechanical properties on low PS implants. Bone graft offers a wedging effect for the implant with low PS at placement and promotes osteogenesis on their surface in the healing period.

The therapeutic perspective of cold atmospheric plasma in periodontal disease.

OBJECTIVES: Periodontal disease (PD) is one of the most common infectious diseases with complex inflammatory conditions, having irreversibly destructive impacts on the periodontal supporting tissues. The application of cold atmospheric plasma (CAP) is a promising adjuvant therapy modality for PD. However, the mechanism of CAP in PD treatment is still poorly understood. The review motivates to outline the latest researches concerning the applications of CAP in PD treatment. METHODS: We searched CAP-related literature through utilizing the well-established databases of Pubmed, Scopus and Web of Science according to the following keywords related to periodontal disease (periodontal, gingival, gingivitis, gingiva, periodontium, periodontitis). RESULTS: A total of 18 concerning original studies were found. These studies could be classified according to three pathophysiological perspectives of PD. The therapeutic mechanisms of CAP may be attributed to the oxidative stress-related cell death of periodontal bacteria, the suppression of periodontal inflammation and pro-inflammatory cytokine secretion, as well as the acceleration of periodontal soft tissue wound healing and hard tissue reconstruction. CONCLUSIONS: Cold atmospheric plasma has potential therapeutic effects on PD through three mechanisms: antimicrobial effect, inflammation attenuation, and tissue remodeling. This review hopefully provides a comprehensive perspective into the potential of CAP in PD therapy.

Evaluation of modified cold-atmospheric pressure plasma (MCAP) for the treatment of peri-implantitis in beagles.

OBJECTIVE: To assess the effect of adjunctive use of modified cold-atmospheric pressure plasma (MCAP) to surgically mechanical debridement (MD) on peri-implantitis (PI) in beagles. MATERIAL AND METHODS: Bilateral mandibles of beagles with PI, which induced by cotton ligature twined with steel in sub-marginal around the implant, were randomly divided into two groups: MD in conjunction with 2% CHX irrigation (control group) and MD with adjunctive intervention of MCAP (plasma group). Sulcus bleeding index (SBI), probing depth (PD) and bone height (BH) were examined before and after intervention using computed tomography and histological staining. Additionally, interleukin (IL)-1ß, IL-6 and IL-17 levels in peri-implant sulcular fluid (PISF) were measured by ELISA. RESULTS: A significant improvement in SBI, PD and BH was found in the plasma group (p < .05) when compared with the control group after three months of intervention. In addition, IL-1ß and IL-17, but not IL-6 levels decreased (p < .05) in the plasma group compared with the control group after intervention. CONCLUSIONS: The adjunctive use of MCAP to MD for PI can enhance bone formation around the implant and inhibit the inflammatory response. The application of MCAP could be considered a favourable adjunct to MD for PI.

In vitro and in vivo research of atmosphere pressure nonequilibrium plasmas on root canal disinfection: implication for alternative strategy for irrigation.

OBJECTIVE: To investigate an intracanal disinfection methodology of APNPs (atmosphere pressure nonequilibrium plasmas) or modified APNPs in root canal treatment and evaluate the antimicrobial efficiency against in vitro infected dentinal tubules and in vivo experimental apical periodontitis. MATERIALS AND METHODS: Dentine specimens were centrifugated with Enterococcus faecalis to generate 1-day-old and 3-week-old biofilms, and were treated with 2% chlorhexidine (Chx), APNP or modified APNP for 3 and 10 min (n=4). LIVE/DEAD staining was employed to analyze the ratio of deactivated bacteria. Experimental apical periodontitis in beagles was induced. Root canal therapy with APNPs or modified APNPs was performed and the antimicrobial effect was evaluated by histological and radiographical analyses. RESULTS: APNP deactivated 1-day-old and 3-week-old E. feacalis in dentinal tubules as much as 2% Chx irrigating. Modified APNP significantly deactivated more E. faecalis biofilms in dentinal tubules for 3-min and 10-min treatments, without thermal damage or dentinal destruction being observed. In beagles' apical periodontitis, significantly increased BV/TV and decreased lesion volume of apical bone were found in modified APNP group than 2% Chx irrigation group according to µCT. Fewer inflammatory cells and bacterial residual in dentine were observed in modified APNP-treated apical tissue by histology staining compared with those in the 2% Chx irrigation group. CONCLUSION: The antimicrobial effect of APNP jet irradiation was comparable to that of 2% Chx irrigation. No structural damage in dentine or tissue necrosis at the periapical region was induced upon treatment. The modified APNP demonstrated an increased antimicrobial efficacy compared with 2% Chx irrigation both in vitro and in vivo. CLINICAL RELEVANCE: The modified APNPs can be used as an alternative intracanal disinfection strategy.

Effects of miRNA-342-3p in modulating Hedgehog signaling pathway of human umbilical cord mesenchymal stem cells by down-regulating Sufu.

OBJECTIVE: Previously, we have shown that miRNA-342-3p was increased during osteogenic differentiation of human umbilical mesenchymal stem cells (hUCMSCs) via regulating the sonic hedgehog (Shh) pathway. In this study, our objective is to further investigate the role of miRNA-342-3p in activation of Shh pathway by targeting suppressor of fused protein (Sufu), a suppressor of transcriptional factor Gli, as well as the potential interaction with transforming growth factor beta (TGF-ß) signaling pathway during osteogenic induction of hUCMSCs. MATERIALS AND METHODS: HUCMSCs that stable overexpression or knockdown of miRNA-342-3p were established by infection with lentiviral vectors. mRNA and protein levels of Hedgehog signaling pathway and osteogenic genes were measured by RT-qPCR and western blot assays. Luciferase reporter assay was performed to test the direct binding site of Sufu 5'UTR targeted by miRNA-342-3p. RESULTS: Overexpression of miRNA-342-3p in hUCMSCs enhanced the expression of osteogenic genes by targeting Sufu. And the potential of osteogenic differentiation of hUCMSCs was inhibited while knocking down miRNA-342-3p. Meanwhile, induced the TGF-ß expression level was also observed upon overexpressing miRNA-342-3p, suggesting activation of TGF-ß signaling pathway was a potential mechanism of miRNA-342-3p-mediated osteogenesis in hUCMSCs. CONCLUSIONS: Our findings provide new mechanistic evidence that miRNA-342-3p might be a valuable therapeutic target in bone regeneration.

miR-342-3p elevates osteogenic differentiation of umbilical cord mesenchymal stem cells via inhibiting Sufu in vitro.

Human umbilical mesenchymal stem cells (UCMSCs) have been wildly used in tissue engineering field as a promising source because of their unlimited and noninvasive origin. To selectively induce osteogenic differentiation of UCMSCs, it's imperative to understand the regulatory molecular mechanism underlying the process of how these cells switch into osteogenic lineage path. We previously showed enhanced sonic hedgehog (Shh) signaling pathway upon osteogenic induction in mesenchymal stem cells. In this study, miRNA-seq analysis revealed substantial Shh-dependent expression of distinct miRNAs, including miR-342-3p, during ostogenesis. RT-qPCR confirmed that miR-342-3p was increased at a greater level when Shh signaling pathway was activated by N-terminal of Shh ligand compared with osteogenic induction alone, in contrast to the decreasing of suppressor-of-fused protein (Sufu). Consistently, transient overexpressing miR342-3p in UCMSCs via miR-342-3p mimics dramatically decreased Sufu, a suppressor of Gli, while osteogenic markers, including alkaline phosphate and osteocalcin, were upregulated during osteogenic induction, indicating that miR-342-3p might be involved in osteogenesis through the Shh signaling pathway. In conclusion, this study showed the potential of miR-342-3p as a therapeutic target to promote bone regeneration by modulating expression of Sufu in UCMSCs.

Effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis.

AIM: To evaluate the effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis in beagle dogs. MATERIALS AND METHODS: Six beagles received 12 implants installed in the position of the fourth mandibular premolars. Ligature-induced peri-implantitis was initiated at 3 months post-implantation. When approximately 40% of the supporting bone was lost, the ligatures were removed. The implants were subjected to the muco-periosteal scaling and chlorhexidine irrigation with or without plasma irrigation. Three months later, clinical, radiographic and microbiological analyses were performed. Block biopsies were prepared for micro-CT and histomorphometric analysis. The primary outcome was the difference in bone healing of peri-implant sites, and the secondary outcomes included changes in clinical parameters (SBI, PD) and bacterial detection. RESULTS: At baseline, no significant differences were observed between the two groups. At 3 months post-treatment, the plasma group showed a significantly higher bone level than the control group (p < 0.05), a significantly decreased detection of bacteria (Porphyromonas gingivalis and Tannerella forsythia) (p < 0.05), and a significant improvement in clinical examination (p < 0.05). CONCLUSIONS: Within the limits of this study, non-equilibrium plasma treatment as an adjunct to the conventional therapy is a feasible approach for the treatment of peri-implantitis.

Retrospective study on the clinical and radiographic outcomes of 2.8 mm diameter implants supporting fixed prostheses up to 11 years.

OBJECTIVES: This study aimed to report the clinical and radiographic results of 2.8 mm two-piece narrow diameter implant (NDI) supporting fixed restorations. MATERIALS AND METHODS: Clinical and radiographic data of 54 NDIs in 32 patients were retrospectively assessed after 2 to 11 (mean 8.17) years of follow-up. Clinical and radiographic measurements were taken. Survival rate, implant and prosthesis failure, pink aesthetic scores (PES), white aesthetic scores (WES), bleeding on probing (BOP), probing depth (PD), marginal bone loss (MBL), and mechanical and biological complications were evaluated. RESULTS: An implant failed during the follow-up period, resulting in a cumulative survival rate of 98.15% at the implant level and 96.88% in the patient. The total mean values of PES and WES for 2.8 mm NDIs were 7.09 ± 1.15 (range: 3.33-9.00) and 7.42 ± 1.03 (range: 3.67-9.33). The prevalence of sites with positive BOP was 38.14 ± 29.77%. The mean PD value was 2.46 ± 0.62 mm. The average MBL was 1.15 ± 0.74 mm (range: 0.25-4.03 mm). No implant or abutment fracture was detected. A veneer chipping was present in one patient, and a loose crown appeared in another patient. Two implants (3.7%) and two patients (6.3%) were diagnosed with peri-implantitis. CONCLUSION: Within the limitation of the study, the results indicate that the use of two-piece 2.8 mm NDI for the fixed prosthetic rehabilitation of edentulous regions with reduced interdental and/or buccal-lingual width is viable.

IRE1α pathway: A potential bone metabolism mediator.

Osteoblasts and osteoclasts collaborate in bone metabolism, facilitating bone development, maintaining normal bone density and strength, and aiding in the repair of pathological damage. Endoplasmic reticulum stress (ERS) can disrupt the intracellular equilibrium between osteoclast and osteoblast, resulting in dysfunctional bone metabolism. The inositol-requiring enzyme-1α (IRE1α) pathway-the most conservative unfolded protein response pathway activated by ERS-is crucial in regulating cell metabolism. This involvement encompasses functions such as inflammation, autophagy, and apoptosis. Many studies have highlighted the potential roles of the IRE1α pathway in osteoblasts, chondrocytes, and osteoclasts and its implication in certain bone-related diseases. These findings suggest that it may serve as a mediator for bone metabolism. However, relevant reviews on the role of the IRE1α pathway in bone metabolism remain unavailable. Therefore, this review aims to explore recent research that elucidated the intricate roles of the IRE1α pathway in bone metabolism, specifically in osteogenesis, chondrogenesis, osteoclastogenesis, and osteo-immunology. The findings may provide novel insights into regulating bone metabolism and treating bone-related diseases.

The Role of Gli1+ Mesenchymal Stem Cells in Osteogenesis of Craniofacial Bone.

Glioma-associated oncogene homolog 1 (Gli1) is a transcriptional activator of hedgehog (Hh) signaling that regulates target gene expression and several cellular biological processes. Cell lineage tracing techniques have highlighted Gli1 as an ideal marker for mesenchymal stem cells (MSCs) in vivo. Gli1+ MSCs are critical for the osteogenesis of the craniofacial bone; however, the regulatory mechanism by which Gli1+ MSCs mediate the bone development and tissue regeneration of craniofacial bone has not been systematically outlined. This review comprehensively elucidates the specific roles of Gli1+ MSCs in craniofacial bone osteogenesis. In addition to governing craniofacial bone development, Gli1+ MSCs are associated with the tissue repair of craniofacial bone under pathological conditions. Gli1+ MSCs promote intramembranous and endochondral ossification of the craniofacial bones, and assist the osteogenesis of the craniofacial bone by improving angiopoiesis. This review summarizes the novel role of Gli1+ MSCs in bone development and tissue repair in craniofacial bones, which offers new insights into bone regeneration therapy.

An in vitro investigation into retention strength and fatigue resistance of various designs of tooth/implant supported overdentures.

Previously, the choice of prosthetic implant-retained overdentures has depended on data from previous studies about the retention-fatigue strength of the attachment system selected. Little or no data have been available on the correlation between the attachment system selected and the overdenture support configuration. The purpose of the present study was to evaluate the retention force and fatigue resistance of three attachment systems and four support designs of overdenture prosthesis. Four lower edentulous acrylic models were prepared and eight combinations of attachments groups were investigated in the study. These included: O-Rings with mini-dental implants (MDIs), Dalbo elliptic with Dalbo Rotex and fabricated flexible acrylic attachments with both MDI and Dalbo Rotex. The study was divided into four test groups: groups A and B, controls, and groups C and D, experimental groups. Control group A contained three overdenture supports: two free standing MDIs in the canine region and at the midline, and one simulated tooth root with Dalbo Rotex screwed in. Control group B contained four overdenture support foundations: two free standing MDIs in the right canine region and the first premolar region, and two simulated tooth roots with Dalbo Rotex screwed in at the same MDI position, but on the left side of the model. Experimental group C contained three overdenture support foundations: two free standing MDIs in the canine region and at the midline, and one simulated tooth root with MDI screwed in. Experimental group D contained four overdenture support foundations: two free standing MDIs in the right canine region and the first premolar region, and two simulated tooth roots with MDIs screwed in at the same MDI position, but on the left side of the model. Each group was further divided into two subgroups according to attachment type used. Five samples were prepared for each group. Retention force (N) values were recorded initially (0 cycles) and after 360, 720, 1440 and 2880 insertion and removal cycles. During the tensile test a cross-head speed of 10 mm/min was applied. Values of absolute force (AF) and relative force (RF) were statistically analyzed by two-way ANOVA and multiple comparison Tukey's tests between groups and cycles periods. The results of fatigue tests showed a 50% reduction in retention force in the subgroups with flexible attachments. A triangular design of overdenture support foundations with O-Ring attachments revealed the lowest value of AF and a relatively high reduction in RF. The four overdenture support designs with flexible acrylic attachments improved the retention force and reduced the fatigue retention. Furthermore, the results of the investigation demonstrate that flexible acrylic attachments for both teeth and implant-supported overdentures offer a wide range of retention forces.

A three-dimensional finite element analysis for overdenture attachments supported by teeth and/or mini dental implants.

PURPOSE: The aim of this study was to establish the optimum design and attachment combination to support an overdenture with minimal stress and flexing produced in the alveolar bone surrounding any natural teeth and/or mini dental implants. MATERIALS AND METHODS: Twelve models were included in the study: the six main models (A, B, C, D, E, and F) were categorized according to the support designs of the overdenture prosthesis, and each model was further subdivided according to the attachment combinations into model 1: with Dalbo elliptic and/or O-ring attachments only and model 2: with flexible acrylic attachments. Vertical loads (35 N) and 17.5 N lateral loads under static conditions were applied to the models to simulate the occlusal forces following the concept of lingualized occlusion. All conditions were created using a finite element software program. Maximum von Mises stress at the level of the attachments and at the bone support foundation interfaces were compared in all 12 models. The flexing of the mandible and the attachments were also compared qualitatively. RESULTS: Stress on these models was analyzed after the given loading condition. The results showed that the model with three freestanding mini dental implants and flexible acrylic attachments showed the lowest von Mises stress and flexing, while the models with four freestanding mini dental implants and O-ring attachments showed the highest von Mises stress. CONCLUSION: Three freestanding mini dental implants with flexible acrylic attachment systems supporting an overdenture were better choices than four mini dental implants with O-ring attachment systems, which showed the maximum flexing and stress values in this qualitative comparison.

Hybrid system with stable structure of hard/soft tissue substitutes induces re-osseointegration in a rat model of biofilm-mediated peri-implantitis.

Re-osseointegration of an infected/contaminated dental implant poses major clinical challenges. We tested the hypothesis that the application of an antibiotic-releasing construct, combined with hard/soft tissue replacement, increases the efficacy of reconstructive therapy. We initially fabricated semi-flexible hybrid constructs of ß-TCP/PHBHHx, with tetracycline (TC) (TC amounts: 5%, 10%, and 15%). Thereafter, using in vitro assays, TC release profile, attachment to rat bone marrow-derived stem cells (rBMSCs) and their viability as well as anti-bacterial activity were determined. Thereafter, regenerative efficacies of the three hybrid constructs were assessed in a rat model of peri-implantitis induced by Aggregatibacter actinomycetemcomitans biofilm; control animals received ß-TCP/Bio-Gide and TC injection. Eight weeks later, maxillae were obtained for radiological, histological, and histomorphometric analyses of peri-implant tissues. Sulcus bleeding index was chronologically recorded. Serum cytokines levels of IL-6 and IL-1ß were also evaluated by enzyme-linked immunosorbent assay. Substantial amounts of tetracycline, from hybrid constructs, were released for 2 weeks. The medium containing the released tetracycline did not affect the adhesion or viability of rBMSCs; however, it inhibited the proliferation of A. actinomycetemcomitans. Osteogenesis and osseointegration were more marked for the 15% hybrid construct group than the other two groups. The height of attachment and infiltration of inflammatory cells within fibrous tissue was significantly reduced in the experimental groups than the control group. Our protocol resulted in re-osseointegration on a biofilm-contaminated implant. Thus, an antibiotic releasing inorganic/organic construct may offer a therapeutic option to suppress infection and promote guided tissue regeneration thereby serving as an integrated multi-layer substitute for both hard/soft tissues.

Effects of Atmospheric Pressure Plasma in the Treatment of Experimental Periodontitis in Beagle Dogs.

OBJECTIVE: The specific objective of this study was to evaluate the effects of atmospheric pressure plasma (APP) in the treatment of experimental periodontitis in Beagle dogs. METHODS: The APP jet was diagnosed using optical emission spectroscopy and laser-induced fluorescence spectroscopy. Six Beagles received stainless steel ligatures to establish experimental periodontitis model. The teeth in the control group were subjected to conventional root surface debridement (RSD) and chlorhexidine irrigation. The APP group also started with RSD and was then subjected to plasma irradiation. Clinical analyses including plaque index, modified sulcus bleeding index, pocket depth and attachment loss (AL), as well as cone-beam computed tomography (CBCT) analysis, were performed at baseline, 4th week, 8th week and 12th week after treatment. RESULTS: The results showed that typical reactive oxygen and nitrogen species were found in the full spectrum and the gas temperature of APP was close to room temperature. The highest concentrations of hydroxide and oxygen were obtained at 5 mm away from the nozzle. In both groups, all values in clinical examinations were significantly lower (P<0.05) at 12th week after treatment than those at baseline. At the 12th week, the AL in clinical examinations and the bone loss in CBCT images in the APP group were significantly lower than those in the control group (P<0.05). The hematoxylin-eosin staining showed more renascent alveolar bone in the APP group than in the control group. CONCLUSION: These findings suggested that APP has profound potential for use as an adjunct approach for periodontitis treatment.

Bacterial-killing effect of atmospheric pressure non-equilibrium plasma jet and oral mucosa response.

Recently, plasma sterilization has attracted increasing attention in dental community for the atmospheric pressure non-equilibrium plasma jet (APNPs), which is driven by a kilohertz pulsed DC power, may be applied to the dental and oral diseases. However, it is still in doubt whether APNPs can effectively kill pathogenic bacteria in the oral cavity and produce no harmful effects on normal oral tissues, especially on normal mucosa. The aim of this study was to evaluate the bacterial-killing effect of APNPs in the biofilms containing a single breed of bacteria (Porphyromonas gingivalis, P.g.), and the pathological changes of the oral mucosa after treatment by APNPs. P.g. was incubated to form the biofilms in vitro, and the samples were divided into three groups randomly: group A (blank control); group B in which the biofilms were treated by APNPs (the setting of the equipment: 10 kHz, 1600 ns and 8 kV); group C in which the biofilms were exposed only to a gas jet without ignition of the plasma. Each group had three samples and each sample was processed for up to 5 min. The biofilms were then fluorescently stained, observed and photographed under a laser scanning confocal microscope. In the animal experiment, six male Japanese white rabbits were divided into two groups randomly (n=3 in each group) in terms of the different post-treatment time (1-day group and 5-day group). The buccal mucosa of the left side and the mucosa of the ventral surface of the tongue were treated by APNPs for 10 min in the same way as the bacterial biofilm experiment in each rabbit, and the corresponding mucosa of the other sides served as normal control. The clinical manifestations of the oral mucosa were observed and recorded every day. The rabbits were sacrificed one or five day(s) after APNPs treatment. The oral mucosa were harvested and prepared to haematoxylin and eosin-stained sections. Clinical observation and histopathological scores were used to assess mucosal changes. The results showed the obvious P.g. biofilms were formed at 10 days, and most of the bacteria in groups A and C were alive under a laser scanning confocal microscope, but the bacteria in the group B were almost all dead. In animal experiment, no ulcers, anabrosis and oral mucositis were found in both the 1-day and 5-day groups. The average mucous membrane irritation index was -0.83 and -0.67 in the 1-day and 5-day groups, respectively, suggesting that no intense mucosal membrane irritation responses occurred. It was concluded that APNPs could effectively kill P.g. in the biofilms and did not cause any pathological changes in the normal mucosa, suggesting that the plasma jet (APNPs) may be applied to oral diseases as a novel sterilization device in the future.

Effect of bone marrow mesenchymal stem cells transfected with rAAV2-bFGF on early angiogenesis of calvarial defects in rats.

The purpose of this study was to evaluate the effect of bone marrow mesenchymal stem cells (MSCs) transfected with the basic fibroblast growth factor (bFGF)-expressing recombinant adeno-associated virus vector (rAAV2-bFGF), on early angiogenesis of calvarial defects in rats. The MSCs were cultured and transfected with rAAV2-bFGF after differential adherence isolation. The transfection efficiency was detected by RT-PCR and Western blotting. The transfected MSCs were compounded with poly-DL-lactide/hydroxyapatite (PDLLA/HA) in vitro. The cranial defect models in 36 male SD rats were created. Nothing (group A), PDLLA/HA alone (group B), PDLLA/HA combined with MSCs (group C), and PDLLA/HA combined with rAAV2-bFGF transfected MSCs (group D) were implanted in rat calvarial defects. The specimens were harvested for hematoxylin-eosin staining on the day 1, 3 and 7 after implantation. Factor VIII immunohistochemical staining and histomorphometric analysis were carried out to evaluate neovascularization around the implantation. The results indicated that MSCs could indeed be successfully transfected with the rAAV2-bFGF vector. Histological and histomorphometric analysis revealed that the angiogenesis in group D was significantly enhanced as compared with the rest groups (P<0.05). These results strongly suggest that MSCs transfected with rAAV2-bFGF in combination with PDLLA/HA can effectively promote the early angiogenesis of calvarial defects in rats, which played an important role in creating an environment suitable for the survival and activity of transplanted cells for further applications in cranio-maxillofacial bone regeneration.

Construction of adeno-associated virus system for human bone morphogenetic protein 7 gene.

To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5x10(11) vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Biomechanical Evaluation of Nano-Zirconia Coatings on Ti-6Al-7Nb Implant Screws in Rabbit Tibias.

The clinical success of dental implants can be improved by achieving optimum implant properties, such as their biomechanical and surface characteristics. Nano-structured coatings can play an important role in improving the implant surface. The purpose of the present study was to determine the most appropriate conditions for electrophoretic deposition (EPD) of nano-zirconia coatings on Ti-6Al-7Nb substrates and to evaluate the structural and biomechanical characteristics of these deposited coatings on the dental implants. EPD was used with different applied voltages and time periods to obtain a uniform layer of nano-zirconia on Ti-6Al-7Nb samples. The coated samples were weighed and the thickness of the product layer was measured. Surface analysis was performed by using optical microscopical examination, scanning electron microscope and X-ray diffraction phase analysis. For in vivo examination, 48 screw-designed implants (24 uncoated and 24 nano-zirconia coated) were implanted in both tibiae of 12 white New Zealand rabbits and evaluated biomechanically after 4- and 12-week healing intervals. Results revealed that the use of different conditions for EPD affected the final coating film properties. Increasing the applied voltage and coating time period increased the deposited nano-zirconia film thickness and weight. By selecting the appropriate coating conditions, and analyzing scanning electron microscopical examination and XRD patterns, this technique could produce a thin and continuous nano-zirconia layer with a uniform thickness of the Ti-6Al-7Nb samples. Mechanically, the nano-zirconia-coated implants showed a highly statistically significant difference in removal torque values, while histologically these coated implants enhanced and promoted osseointegration after 4 and 12 weeks of healing, compared with the uncoated ones. In conclusion, EPD is an effective technique for providing a high quality nano-zirconia coating film on dental implant surfaces. Moreover, the osseointegration of these coated dental implants is improved compared with that of uncoated ones.

Expression of matrix metalloproteinases-2, -9 and reversion-inducing cysteine-rich protein with Kazal motifs in gingiva in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Periodontitis is characterized by pathological destruction of extracellular matrix (ECM) of periodontal tissues. Matrix metalloproteinases (MMPs) promote the occurrence and development of periodontitis by degrading almost all proteins of ECM. RECK (reversion-inducing-cysteine-rich protein with kazal motifs), a novel membrane-anchored inhibitor of MMPs, could regulate the expression of MMP-2 and MMP-9 at post-transcriptional level. The study was to investigate the expression of RECK in healthy and diseased human gingival tissues and to correlate it with the production of MMP-2 and MMP-9. MATERIAL AND METHODS: Gingival biopsies were collected from chronic periodontitis patients and periodontally healthy control individuals. The protein and mRNA of RECK, MMP-2 and MMP-9 was determined by immunohistochemistry and semi-quantitative polymerase chain reaction analysis. RESULTS: The expression of RECK protein was mainly confined to the gingival epithelium in inflamed and non-inflamed gingival tissues. Expression of RECK was significantly lower in tissues from chronic periodontitis patients, while the positive expression levels of MMP-2 and MMP-9 in periodontitis specimens were significantly higher. RECK protein expression was negatively correlated to the expressions of MMP-2 and MMP-9 in periodontitis. Moreover, RECK mRNA was significanly lower in diseased gingiva than in healthy samples(P<0.05), while MMP-2 and MMP-9 mRNAs were observed overexpressed in periodontal lesions, with no significant correlation between RECK and MMP-2/MMP-9 mRNA shown in periodontally diseased group. CONCLUSION: The expression of RECK in human healthy and diseased gingiva may contribute to periodontal physiological and pathological processes; low RECK expression may be associated with the enhanced MMP-2 and MMP-9 production in inflamed gingiva.