Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae.

The current study aimed to evaluate bone tissue regeneration using a combination of ß-tricalcium phosphate (ßTCP) and phosphorylated pullulan (PPL, a phosphate-rich polysaccharide polymer consisting of maltotriose units). Round defects of 2 mm diameter were created in the arterial center of rat tibiae, which were further treated with vehicle (control group), ßTCP (ßTCP group), or ßTCP + PPL (ßTCP + PPL group) grafts. The control specimens without bone grafts exhibited rapid bone formation after 1 week; however, the regenerated bone was not resorbed until 4 weeks. In contrast, ßTCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the ßTCP + PPL group displayed many fine trabeculae at 4 weeks. In the ßTCP + PPL group, new bone was associated with the ßTCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the ßTCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and ßTCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin αv, Osteopontin, Osteocalcin, and Dmp-1 in PPL-treated MC3T3-E1 osteoblastic cells, suggesting that PPL did not facilitate osteoblastic differentiation. However, von Kossa staining identified abundant needle-like calcified structures extending inside the PPL. Furthermore, transmission electron microscopy (TEM) revealed many globular structures identical to calcified nodules. In addition, calcified collagen fibrils were observed in the superficial layer of the PPL. Thus, PPL may serve as a scaffold for osteoblastic bone formation and promotes calcification on its surface. In conclusion, we speculated that ßTCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.

Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial.

Objectives: The aims of this study were to select heat-killed lactic acid bacteria (HKL) with antibiotic activity and investigate the efficacy of this bacteria in maintaining periodontal parameters in healthy participants. Materials and methods: An in vitro evaluation was conducted to assess the inhibitory efficacy of lactic acid bacteria against Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum. The effects of HKL administration on various parameters (plaque control record, bleeding on probing, and probing pocket depth) were assessed in a randomized, placebo-controlled trial. Participants in the test and placebo groups (n = 32) consumed oral tablets containing placebo or HKL daily for 8 weeks. Oral bacteria in supra-plaque and saliva were identified using 16S rRNA gene community profiling analysis. Results: Heat-killed Ligilactobacillus salivarius CP3365 significantly (p < 0.05) decreased the viability of oral bacteria and was selected for clinical trials. Administration of HKL CP3365 significantly (p < 0.05) inhibited increases in each parameter. No changes in the relative abundance of P. gingivalis or F. nucleatum subsp. nucleatum were detected by HKL CP3365, but the relative abundance of oral bacteria (genera Porphyromonas, Fusobacterium, and Haemophilus) was significantly (p < 0.05) decreased. Conclusion: HKL CP3365 effectively inhibited oral bacteria growth and was useful for maintaining periodontal health. Clinical Trial Registration: [https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045656].

Cauterization of Narrow Root Canals Untouched by Instruments by High-Frequency Current.

The mechanical removal of bacteria is fundamental to the treatment of infected root canals, but complete sterilization of biofilms tends not to extend to uninstrumented areas. However, during electrical conduction to a root canal filled with a conductor, the higher impedance where the root canal is narrower generates Joule heat that may result in a large temperature increase and sterilization. The effect of a high-frequency electric current on the wall of a simulated narrow root canal was investigated by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). Simulated root canals, 0.1 mm in diameter, were prepared in dentine blocks. The root canal wall was treated with Plank-Rychlo solution for 5 min to create a decalcified layer. The simulated root canal was filled with either saline or NaClO, and 150 or 225 V at 520 kHz was applied for 0 s, 1 s, or 5 s. As the conduction time increased, and when the saline was replaced with NaClO, the proportion with a flat decalcified surface decreased, dentinal tubules and a lava-like morphology were significantly more evident on SEM (p < 0.01), and EDS showed significant decreases in carbon and oxygen and increases in calcium (p < 0.01). It was concluded that filling uninstrumented root canals with NaClO and using electrical conduction for 5 s could incinerate and eliminate the organic material of the root canal wall. The application of high-frequency electric current may lead to the cure of many cases of persistent apical periodontitis.

Can a low dosage of recombinant human bone morphogenetic protein-2 loaded on collagen sponge induce ectopic bone?

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is one of the growth factors that may induce the formation of new bone. The aim was to determine the efficacy of low doses of rhBMP-2 for bone regeneration using a collagen sponge as a carrier. Three doses of rhBMP-2 (1.167, 0.117, and 0.039 mg/mL) were combined with an absorbable collagen sponge (ACS) as a delivery vehicle. The rhBMP-2/ACS implants were placed in the subcutaneous tissues of rat backs. X-ray microcomputed tomography (micro-CT) and histological analysis were used to evaluate bone formation. The samples treated with 1.167 mg/mL of rhBMP-2 showed greater bone formation than the samples treated with 0.117 mg/mL of rhBMP-2 four weeks after surgery. However, there was no evidence of bone formation in the samples that were treated with 0.039 mg/mL of rhBMP-2. It was found that rhBMP-2 was osteogenic even at one-tenth of its manufacturer's recommended concentration (1.167 mg/mL), indicating its potential for clinical use at lower concentrations.

Water-resistant antibacterial properties of a graphene oxide/cetylpyridinium chloride complex formed on medical gauze fibers.

OBJECTIVES: Graphene oxide (GO) is a nanocarbon material with a high aspect ratio (width:thickness) and abundant anionic functional groups on its surface. In this study, we attached GO to the surface of medical gauze fibers, constructed a complex with a cationic surface active agent (CSAA), and demonstrated that the treated gauze exhibits antibacterial activity even after rinsing with water. METHODS: Medical gauze was immersed in GO dispersion (0.001%, 0.01%, and 0.1%), rinsed with water, dried, and subjected to the Raman spectroscopy analysis. Subsequently, the gauze treated with 0.001% GO dispersion was immersed in 0.1% cetylpyridinium chloride (CPC) solution, immediately rinsed with water, and dried. Untreated, GO-only, and CPC-only gauzes were prepared for comparison. Each gauze was placed in a culture well, seeded with Escherichia coli or Actinomyces naeslundii, and turbidity was measured after 24 h of incubation. RESULTS: The Raman spectroscopy analysis of the gauze after immersion and rinsing showed a G band peak, indicating that GO remained on the surface of the gauze. The turbidity measurements indicated that GO/CPC-treated gauze (GO-treated and rinsed, followed by CPC-treatment and rinsing) significantly decreased turbidity compared to the other gauzes (P < 0.05), suggesting that the GO/CPC complex remained on the gauze fibers even after water rinsing and showed antibacterial activity. CONCLUSIONS: The GO/CPC complex imparts water-resistant antibacterial properties to gauze and has the potential to be widely used for the antimicrobial treatment of clothes.

Periodontal tissue regeneration by recombinant human collagen peptide granules applied with ß-tricalcium phosphate fine particles.

OBJECTIVES: Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in arginine-glycine-aspartic acid sequences with good processability whose use for regenerative medicine applications is under investigation. The biocompatibility and osteogenic ability of RCP granules combined with ß-tricalcium phosphate (TCP) submicron particles (ß-TCP/RCP) were recently demonstrated. In the present study, ß-TCP/RCP was implanted into experimental periodontal tissue defects created in beagles to investigate its regenerative effects. METHODS: An RCP solution was lyophilized, granulated, and thermally cross-linked into particles approximately 1 mm in diameter. ß-TCP dispersion (1 wt%; 500 µL) was added to 100 mg of RCP granules to form ß-TCP/RCP. A three-walled intrabony defect (5 mm × 3 mm × 4 mm) was created on the mesial side of the mandibular first molar and filled with ß-TCP/RCP. RESULTS: A micro-computed tomography image analysis performed at 8 weeks postoperative showed a significantly greater amount of new bone after ß-TCP/RCP grafting (2.2-fold, P < 0.05) than after no grafting. Histological findings showed that the transplanted ß-TCP/RCP induced active bone-like tissue formation including tartaric acid-resistant acid phosphatase- and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements performed at 8 weeks postoperative revealed that ß-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control condition and significantly suppressed gingival recession and epithelial downgrowth (P < 0.05). CONCLUSIONS: ß-TCP/RCP implantation promoted bone-like and periodontal ligament-like tissue formation, suggesting its efficacy as a periodontal tissue regenerative material.

Sustained antibacterial coating with graphene oxide ultrathin film combined with cationic surface-active agents in a wet environment.

Antimicrobial surfactants contained in mouthrinse have excellent efficacy, but are not retained on the tooth surface (are rinsed away) due to their low water resistance and thus do not exhibit sustained antibacterial activity. We have developed a new coating method using graphene oxide (GO) that retains the surfactant on the tooth surface even after rinsing with water, thus providing a sustained antibacterial effect. Ultra-thin films of GO and an antimicrobial agent were prepared by (1) applying GO to the substrate surface, drying, and thoroughly rinsing with water to remove excess GO to form an ultrathin film (almost a monolayer, transparent) on the substrate surface, then (2) applying antimicrobial cationic surface active agents (CSAAs) on the GO film to form a composite coating film (GO/CSAA). GO/CSAA formation was verified by scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and ζ-potential and contact angle measurements. GO/CSAA was effective at inhibiting the growth of oral pathogens for up to 7 days of storage in water, and antibacterial activity was recovered by reapplication of the CSAA. Antibacterial GO/CSAA films were also formed on a tooth substrate. The results suggest that GO/CSAA coatings are effective in preventing oral infections.

Ultrasonic irrigation of periodontal pocket with surface pre-reacted glass-ionomer (S-PRG) nanofiller dispersion improves periodontal parameters in beagle dogs.

OBJECTIVES: Surface pre-reacted glass-ionomer (S-PRG) nanofiller, an antibacterial ion-releasing bioactive glass, has been shown to adhere to tooth surfaces and reported to improve inflammatory parameters in experimental periodontitis. In this study, cementum substrate was irrigated ultrasonically with dispersion to examine in-vitro nanofiller adhesion and antibacterial activity. Moreover, periodontal pockets in a beagle dog were ultrasonically irrigated with dispersion to assess periodontal healing. METHODS: The morphology of human cementum irrigated with S-PRG nanofiller dispersion was examined by scanning electron microscopy and energy dispersive X-ray spectrometry. The antibacterial activity of the treated cementum was tested using Actinomyces naeslundii. In addition, experimentally formed periodontal pockets in beagle dog were ultrasonically irrigated with S-PRG nanofiller dispersion. Periodontal parameters (gingival index, bleeding on probing, probing pocket depth, and clinical attachment level) were measured from baseline (0 weeks) through 12 weeks. Moreover, the effects of irrigation with S-PRG nanofiller on changes in periodontal microflora and bone healing were analyzed. RESULTS: After ultrasonic irrigation, S-PRG nanofiller adhered to the cementum and exhibited antibacterial activity. The periodontal parameters were shown to improve following ultrasonic irrigation with S-PRG nanofiller dispersion. Analysis by next-generation sequencing revealed that the ratio of red-complex species decreased in the pockets irrigated with S-PRG nanofiller dispersion. In addition, the S-PRG nanofiller showed the potential to promote bone healing. CONCLUSIONS: Ultrasonic irrigation with S-PRG nanofiller dispersion using an ultrasonic scaler system permitted delivery of the S-PRG nanofiller to the root surface, providing improved parameters in experimental periodontitis and modifying the composition of subgingival periodontal microflora.

Periodontal tissue engineering using an apatite/collagen scaffold obtained by a plasma- and precursor-assisted biomimetic process.

BACKGROUND AND OBJECTIVES: In the treatment of severe periodontal destruction, there is a strong demand for advanced scaffolds that can regenerate periodontal tissues with adequate quality and quantity. Recently, we developed a plasma- and precursor-assisted biomimetic process by which a porous collagen scaffold (CS) could be coated with low-crystalline apatite. The apatite-coated collagen scaffold (Ap-CS) promotes cellular ingrowth within the scaffold compared to CS in rat subcutaneous tissue. In the present study, the osteogenic activity of Ap-CS was characterized by cell culture and rat skull augmentation tests. In addition, the periodontal tissue reconstruction with Ap-CS in a beagle dog was compared to that with CS. METHODS: The plasma- and precursor-assisted biomimetic process was applied to CS to obtain Ap-CS with a low-crystalline apatite coating. The effects of apatite coating on the scaffold characteristics (i.e., surface morphology, water absorption, Ca release, protein adsorption, and enzymatic degradation resistance) were assessed. Cyto-compatibility and the osteogenic properties of Ap-CS and CS were assessed in vitro using preosteoblastic MC3T3-E1 cells. In addition, we performed in vivo studies to evaluate bone augmentation and periodontal tissue reconstruction with Ap-CS and CS in a rat skull and canine furcation lesion, respectively. RESULTS: As previously reported, the plasma- and precursor-assisted biomimetic process generated a low-crystalline apatite layer with a nanoporous structure that uniformly covered the Ap-CS surface. Ap-CS showed significantly higher water absorption, Ca release, lysozyme adsorption, and collagenase resistance than CS. Cell culture experiments revealed that Ap-CS was superior to CS in promoting the osteoblastic differentiation of MC3T3-E1 cells while suppressing their proliferation. Additionally, Ap-CS significantly promoted (compared to CS) the augmentation of the rat skull bone and showed the potential to regenerate alveolar bone in a dog furcation defect. CONCLUSION: Ap-CS fabricated by the plasma- and precursor-assisted biomimetic process provided superior promotion of osteogenic differentiation and bone neoformation compared to CS.

Prospective Longitudinal Changes in the Periodontal Inflamed Surface Area Following Active Periodontal Treatment for Chronic Periodontitis.

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/-173, 161+/-276, 184+/-320, 175+/-417, and 209+/-469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

Antibacterial and Antibiofilm Photodynamic Activities of Lysozyme-Au Nanoclusters/Rose Bengal Conjugates.

Antibacterial photodynamic therapy (aPDT) utilizes reactive oxygen species such as singlet oxygen (1O2) and free radicals via photosensitizers, which are light and light-sensitive agents, to reduce bacterial infections. It has been utilized as a treatment for dental diseases in place of antibiotic therapies. However, aPDT does not always cause the desired therapeutic effect due to the instability of organic photosensitizers and the formation of bacterial biofilms. To promote the antibacterial and antibiofilm effects of aPDT, we have proposed a lysozyme (Lys)-gold nanoclusters (Au NCs)/rose bengal (Lys-Au NCs/RB) conjugate as a novel photosensitizer. This conjugate was found to effectively impede the growth of both gram-positive and gram-negative bacteria when exposed to white light-emitting diode (LED) irradiation. The photoexcited Lys-Au NCs/RB showed significantly higher antibacterial activity than photoexcited Lys-Au NCs or RB alone. The synergistic effect is a result of the combination of Lys (an antibacterial protein) and enhanced 1O2 generation related to resonance energy transfer (RET) in the Au NCs/RB conjugate. Photoexcited Lys-Au NCs/RB increased the effects of aPDT in a dose- and time-dependent manner. Furthermore, the photoexcited Lys-Au NCs/RB successfully decreased Streptococcus mutans biofilm formation. However, in contrast, it did not have a negative effect on the proliferation, adhesion, or spread of mammalian cells, indicating low cytotoxicity. Lys-Au NCs/RB is a novel photosensitizer with low cytotoxicity that is capable of bacterial inactivation and the suppression of biofilm formation, and could help to improve dental treatments in the future.

Antibacterial coating of tooth surface with ion-releasing pre-reacted glass-ionomer (S-PRG) nanofillers.

OBJECTIVES: Surface pre-reacted glass-ionomer (S-PRG) fillers release antibacterial borate and fluoride ions. We fabricated nanoscale S-PRG fillers (S-PRG nanofillers) for antibacterial coating of tooth surfaces and assessed the antibacterial effects of this coating in vitro. In addition, we creating a canine model of periodontitis to evaluate the effectiveness of S-PRG nanofiller application on tooth roots and improvement of periodontal parameters. METHODS: Human dentin blocks were coated with S-PRG nanofiller (average particle size: 0.48 µm) and then characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectrometer (EDX), and ion-releasing test. Antibacterial effects of dentin blocks coated with S-PRG nanofiller were examined using bacterial strains, Streptococcus mutans and Actinomyces naeslundii. Next, we created an experimental model of periodontitis in furcation of premolars of beagle dogs. Then, S-PRG nanofiller coating was applied onto exposed tooth root surfaces. Periodontal parameters, gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL), were measured from baseline until 4 weeks. In addition, bone healing was radiographically and histologically examined. RESULTS: SEM and EDX revealed that S-PRG nanofillers uniformly covered the dentin surface after coating. Dentin blocks coated with S-PRG nanofiller showed ion-releasing property, bacterial growth inhibition, and sterilization effects. In the experimental periodontitis model, S-PRG nanofiller coating significantly reduced clinical inflammatory parameters, such as GI (P < 0.01) and BOP (P < 0.05), compared to uncoated samples. In addition, PPD and CAL significantly decreased by S-PRG nanofiller coating (2 weeks: P < 0.05; 3 and 4 weeks: P < 0.01), suggesting the improvement of periodontitis. Micro-CT and histology revealed that bone healing of furcation defects was enhanced by S-PRG nanofiller coating. CONCLUSION: S-PRG nanofiller coating provides antibacterial effects to tooth surfaces and improves clinical parameters of periodontitis.

Estimation of the Periodontal Inflamed Surface Area by Simple Oral Examination.

The periodontal inflamed surface area (PISA) is a useful index for clinical and epidemiological assessments, since it can represent the inflammation status of patients in one contentious variable. However, calculation of the PISA is difficult, requiring six point probing depth measurements with or without bleeding on probing on 28 teeth, followed by data input in a calculation program. More simple methods are essential for screening periodontal disease or in epidemiological studies. In this study, we tried to establish a convenient partial examination method to estimate PISA. Cross-sectional data of 254 subjects who completed active periodontal therapy were analyzed. Teeth that represent the PISA value were selected by an item response theory approach. The maxillary second molar, first premolar, and lateral incisor and the mandibular second molar and lateral incisor were selected. The sum of the PISAs of these teeth was significantly correlated with the patient's PISA (R2 = 0.938). More simply, the sum of the maximum values of probing pocket depth with bleeding for these teeth were also significantly correlated with the patient's PISA (R2 = 0.6457). The simple model presented in this study may be useful to estimate PISA.

Evaluation of antibacterial and cytocompatible properties of multiple-ion releasing zinc-fluoride glass nanoparticles.

Zinc-fluoride glass nanoparticles (Zinc-F) release several ions, such as fluoride, zinc and calcium ions, through acid-base reactions. The aim of this study was to evaluate the antibacterial and cytotoxic properties of Zinc-F. Antibacterial tests showed that a Zinc-F eluting solution significantly reduced the turbidity and colony-forming units of Streptococcus mutans and Actinomyces naeslundii, compared to that of calcium-fluoroaluminosilicate glass nanoparticles without zinc ions. In live/dead staining, Zinc-F eluate significantly decreased green-stained bacterial cells, indicating live cells, compared with the control (no application). Human dentin coated with Zinc-F showed suppressed S. mutans and A. naeslundii biofilm formation. Additionally, Zinc-F eluate showed low cytotoxic effects in osteoblastic and fibroblastic cells. Therefore, our findings suggested that Zinc-F exhibits antibacterial and biocompatible properties through multiple-ion release.

Optimal Examination Sites for Periodontal Disease Evaluation: Applying the Item Response Theory Graded Response Model.

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites-maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)-were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

Bone forming ability of recombinant human collagen peptide granules applied with ß-tricalcium phosphate fine particles.

Recombinant human collagen peptide, developed based on human collagen type I, contains an arginyl-glycyl-aspartic acid (RGD)-rich motif to enhance cell behavior and is anticipated as a xeno-free polymer material for use in tissue engineering. We fabricated granules containing recombinant human collagen peptide (RCP) applied with beta-tricalcium phosphate fine particles (RCP/ß-TCP) as bone filling scaffold material and assessed the bone forming ability of RCP/ß-TCP. Recombinant peptide was thermal crosslinked and freeze-dried to prepare RCP. An aqueous dispersion of ß-TCP fine particles was added to RCP to obtain RCP/ß-TCP. Subsequently, RCP/ß-TCP were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX), and cell culture assessments. Furthermore, RCP/ß-TCP were implanted into rat cranial bone defects for radiographic and histological evaluations. In SEM and EDX analyses of RCP/ß-TCP, ß-TCP particles dose-dependently covered the surface of RCP. Cell culture tests showed that RCP/ß-TCP remarkably promoted proliferation and mRNA expression of various genes, such as integrin ß1 and osteogenic markers, of osteoblastic MC3T3-E1 cells. Histomorphometric assessment at 4 weeks showed that RCP/ß-TCP significantly promoted new skull bone formation compared to RCP (p < 0.05) and control (no application) (p < 0.01). Accordingly, these findings suggest RCP/ß-TCP possess bone forming capability and would be beneficial for bone tissue engineering therapy.

Photodynamic inactivation of oral bacteria with silver nanoclusters/rose bengal nanocomposite.

Antimicrobial photodynamic therapy (a-PDT) is a promising anti-infective technique for generation of singlet oxygen (1O2) to target dental disease. However, conventional organic photosensitizers have problems for clinical use in terms of cytotoxicity, quenching of a-PDT activity by self-dimerization, and the lack of long-term antibacterial effect. We herein propose silver nanoclusters/rose bengal nanocomposite (AgNCs/RB) as a novel photosensitizer with two primary antibacterial effects: (1) 1O2 generation by irradiated RB and (2) Ag+ ion release from AgNCs. AgNCs/RB irradiated with white light-emitting diode (LED) for a short irradiation time of 1 min significantly decreased the bacterial turbidity of Streptococcus mutans, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans (P < 0.05). In SEM, TEM and LIVE/DEAD staining images, photoexcited AgNCs/RB reduced S. mutans colonization, destroyed the cell membrane, and increased the number of dead cells. The antibacterial efficiency of photoexcited AgNCs/RB was greater than that of AgNCs or RB alone (P < 0.05), suggesting a synergistic effect of 1O2 and Ag+ ions from photoexcited AgNCs/RB. By contrast, photoexcited AgNCs/RB did not affect WST-8 and LDH activities and morphology of NIH3T3 mammalian cells, indicating low cytotoxicity. Interestingly, the antibacterial activity of AgNCs/RB on S. mutans was maintained even after the cessation of LED irradiation, indicating a long-term antibacterial effect due to released Ag+ ions. The present AgNCs/RB photosensitizers provide effective synergistic antibacterial effects for dental a-PDT via 1O2 and Ag+ ions coupled with low cytotoxicity.

Prolonged release of bone morphogenetic protein-2 in vivo by gene transfection with DNA-functionalized calcium phosphate nanoparticle-loaded collagen scaffolds.

In the combination of scaffolds immersed in growth factor solutions, the release of growth factors mainly depends on scaffold degradation. However, the release of bone morphogenetic protein (BMP)-2 at an appropriate concentration during the stage of tissue regeneration would enhance bone regeneration. To achieve this condition, the present study was performed to investigate the effects of scaffolds combined with gene transfection using non-viral vectors. Nanohydroxyapatite-collagen (nHAC) scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or ascorbic acid/copper chloride, and a collagen scaffold (Terdermis®) were prepared, loaded with BMP-2-encoding plasmid DNA-functionalized calcium phosphate nanoparticles (CaP), naked plasmid DNA, or BMP-2 solution, and implanted in rats. The yield of released BMP-2 and its releasing period, respectively, were larger and longer from the scaffolds loaded with CaP than from those incubated with BMP-2 solution. In addition, the alkaline phosphatase activity induced by the CaP-loaded scaffolds was higher. Histological analysis showed that released BMP-2 could be observed on the macrophages or multinuclear giant cells surrounding the nHAC fragments or collagen fibres. TRAP-positive or OCN-positive sites were observed in all groups and a mineralization area was observed in the Terdermis®/CaP sample. The present study demonstrates that gene transfection by scaffold loaded with CaP gene transfer vectors induces a larger yield of BMP-2 for a longer period than by scaffolds loaded with BMP-2 solution or naked plasmid.

Preparation of micro/nanopatterned gelatins crosslinked with genipin for biocompatible dental implants.

Background: Collagen is a basic component of the periodontium and plays an important role in the function of the periodontal unit. Therefore, coating with collagen/gelatin has been applied to enable dental implants to positively interact with peri-implant tissues. Although the micro/nanoscale topography is an important property of the surface of dental implants, smaller collagen/gelatin surface patterns have not been sufficiently developed. Furthermore, only few reports on the behavior of cells on gelatin surfaces with different patterns and sizes exist. In this study, we developed micro/nanometer-scaled gelatin surfaces using genipin crosslinking, with the aim of understanding the use of patterning in surface modification of dental implants. Results: Grooves, holes, and pillars, with widths or diameters of 2 µm, 1 µm, or 500 nm were fabricated using a combination of molding and genipin crosslinking of gelatin. The stability of the different gelatin patterns could be controlled by the degree of genipin crosslinking. The gelatin patterns at 20 mM concentration of genipin and 41% crosslinking maintained a stable, patterned shape for at least 14 days in a cell culture medium. A cell morphology study showed that the cells on groves were aligned along the direction of the grooves. In contrast, the cells on pillars and holes exhibited randomly elongated filopodia. The vinculin spots of the cells were observed on the top of ridges and pillars or the upper surface of holes. The results of a cell attachment assay showed that the number of surface-attached cells increased with increasing patterning of the gelatin surface. Unlike the cell attachment assay, the results of a cell proliferation assay showed that Saos-2 cells prefer grooves with diameters of approximately 2 µm and 1 µm and pillars with diameters of 1 µm and heights of 500 nm. The number of cells on pillars with heights of 2 µm was larger than those of the other gelatin surface patterns tested. Conclusion: These data support that a detailed design of the gelatin surface pattern can control both cell attachment and proliferation of Saos-2 cells. Thus, gelatin surfaces patterned using genipin crosslinking are now an available option for biocompatible material patterning.