In vivo transmission electron microscopy of the alternating structure of the protrusions between adjacent macrophage-like cells on micropatterns.

OBJECTIVES: We investigated the behavior of macrophages in the defined microtopography of materials. METHODS: Patterned cyclo-olefin polymer films were implanted into the femurs of seven-week-old rats. After 1 and 4 weeks, the rats were fixed with glutaraldehyde and OsO4, and their bones were observed with transmission electron microscopy (TEM). RESULTS: TEM and segmentation revealed an alternating structure in which multiple protrusions of adjacent macrophage-like cells overlapped. They were approximately 2 µm long and almost uniform in width, and were induced by the limited topography. CONCLUSION: New structures appeared between the macrophage-like cells as a result of microtopography.

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.

Development of Autopolymerizing Resin Material with Antimicrobial Properties Using Montmorillonite and Nanoporous Silica.

Although autopolymerizing resin offers numerous applications in orthodontic treatment, plaque tends to accumulate between the appliance and the mucosa, which increases the number of microorganisms present. In this study, we added cetylpyridinium chloride (CPC) loaded montmorillonite (Mont) and nanoporous silica (NPS) to autopolymerizing resin (resin-Mont, resin-NPS) and evaluated their drug release capacity, antimicrobial capacity, drug reuptake capacity, mechanical strength, and color tone for the devolvement of autopolymerizing resin with antimicrobial properties. As observed, resin-Mont and resin-NPS were capable of the sustained release of CPC for 14 d, and a higher amount of CPC was released compared to that of resin-CPC. Additionally, resin-Mont and resin-NPS could reuptake CPC. Moreover, the antimicrobial studies demonstrated that resin-Mont and resin-NPS could release effective amounts of CPC against Streptococcus mutans for 14 d and 7 d after reuptake, respectively. Compared to resin-CPC, resin-Mont exhibited a higher sustained release of CPC in all periods, both in the initial sustained release and after reuptake. However, the mechanical strength decreased with the addition of Mont and NPS, with a 36% reduction observed in flexural strength for resin-Mont and 25% for resin-NPS. The application of these results to the resin portion of the orthodontic appliances can prevent bacterial growth on the surface, as well as on the interior, of the appliances and mitigate the inflammation of the mucosa.

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.

Extracted tissue-specific atelocollagens have distinctive textural properties.

Food texture is a very important factor for elderly persons, children, and patients who have difficulty swallowing. Collagen and its hydrolysis product, gelatin, are used as ingredients in foods, dietary supplements, and medical materials. In this study, we extracted atelocollagen from nonedible porcine tissues, including ear, nose, and skin, and analyzed the biophysical properties of each tissue. Extracted whole auricle collagen (AEC) showed superior springiness, while only the skin region of auricle collagen (ASC) showed superior hardness, springiness, and brittleness. Body skin collagen showed high hardness but low springiness. In a shear stress test, ASC gels showed high shear strength, and their strains coincided with hardness in a textural examination, while nose and AEC showed low maximum strains. In viscosity, the auricular collagens showed higher viscosity regardless of the region of the ear. Fibril formation in collagen from each tissue and organ varied a great deal in width and morphology. We found that the same type of collagen had a unique texture and viscosity under physiological conditions depending on the tissue or organ of extraction. The results show that the collagen extracted from each organ has a unique texture and unique possibilities to serve as an ingredient in food or supplements.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces.

OBJECTIVES: Osteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). METHODS: Various cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. RESULTS: Osteoclast formation was promoted on pillars (diameter, 1 µm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). CONCLUSIONS: We demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 µm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

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.

Ion Capture and Release Ability of Glass Ionomer Cement Containing Nanoporous Silica Particles with Different Pore and Particle Size.

This study prepared glass ionomer cement (GIC) containing nanoporous silica (NPS) (GIC-NPS) at 5 wt% concentrations using 3 types of NPS with different pore and particle sizes and evaluated the differences in their cationic ion capture/release abilities and mechanical properties. The cationic water-soluble dye was used as cationic ion. The test GIC-NPS complexes captured dyes by immersion in 1 wt% dye solutions. All the GIC-NPS complexes released dyes for 28 d, and the amount of dye released from the complexes increased with decreasing pore size; however, the particle size of NPS did not affect the amount of dye released. Additionally, GIC-NPS was able to recharge the dye, and the amount of released the dye by the complexes after recharge was almost identical to the amount released on the first charge. Although not significantly different, the compressive strength of GIC-NPS was slightly greater than that of GIC without NPS regardless of the type of NPS. These results suggest that the degree of capture and release of cationic molecules, such as drugs, can be controlled by optimizing the pore size of NPS without sacrificing its mechanical strength when its content is 5 wt%.

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.

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.

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.

Proliferation of Saos-2 and Ca9-22 cells on grooved and pillared titanium surfaces.

BACKGROUND: Surface nanostructures in titanium (Ti) oral implants are critical for rapid osseointegration. OBJECTIVE: The purpose of this study was to evaluate the growth of osteoblast-like (Saos-2) and epithelial-like (Ca9-22) cells on nanopatterned Ti films. METHODS: Ti films with 500 nm grooves and pillars were fabricated by nanoimprinting, and seeded with Saos-2 and Ca9-22 cells. Cell viability and morphology were assessed by cell proliferation assay and scanning electron microscopy, respectively. RESULTS: As assessed after 1 hour, proliferation of Saos-2 cells was most robust on grooved films than on pillared and smooth films, in this order. These cells approximately doubled on grooved and pillared substrates in 24 hours and after 5 days, but not on smooth surfaces. In contrast, Ca9-22 cells favored smooth surfaces, followed by grooved and pillared films. Indeed, cells sparsely adhered to pillared films over 5 days of incubation (p < 0.05). CONCLUSIONS: The data show that Saos-2 and Ca9-22 cells respond differently to different nanostructures, and highlight the potential use of nanopatterns to promote bone regeneration or to prevent epithelial downgrowth at the implant-bone interface.

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.

Comparative biological assessments of endodontic root canal sealer containing surface pre-reacted glass-ionomer (S-PRG) filler or silica filler.

Surface pre-reacted glass-ionomer (S-PRG) filler releases several ions, such as fluoride, borate and strontium ions, to exert bioactive effects. We fabricated an endodontic root canal sealer containing S-PRG fillers (S-PRG sealer) and then evaluated the antibacterial and anti-inflammatory properties of S-PRG sealer compared with sealer containing conventional silica fillers (silica sealer). Antibacterial tests showed that S-PRG sealer significantly reduced the turbidity of Enterococcus faecalis compared with silica sealer. Implantation of S-PRG or silica sealer blocks in rat subcutaneous tissue showed that S-PRG sealer decreased the proinflammatory response compared with silica sealer at 10 days post-implantation. In addition, immunostaining revealed that infiltration of CD68- and peroxidase-positive cells around the S-PRG sealer was significantly lower than that in silica sealer. Therefore, it was suggested that S-PRG sealer exhibits antibacterial and anti-inflammatory effects.

Size- and Morphology-Controlled Preparation of Surface-Modified Water-Dispersible Fullerene Nanoparticles for Bioapplications.

In this study, we investigated water-dispersible surface modification for size- and shape-controlled fullerene nanoparticles (C60P) based on a condensation reaction with di-amino alkane. This modification provided for water dispersibility of C60P and the capability for secondary modification as well. The resultant C60P particles have several useful physical properties: water-dispersibility for ease of injection; fluorescence for detection and quantification; and a characteristic morphology to assist identification. These properties will widely extend the applications of these particles, especially into the biological fields of bioimaging and drug delivery.

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.

Antimicrobial photodynamic activity and cytocompatibility of Au25(Capt)18 clusters photoexcited by blue LED light irradiation.

Antimicrobial photodynamic therapy (aPDT) has beneficial effects in dental treatment. We applied captopril-protected gold (Au25(Capt)18) clusters as a novel photosensitizer for aPDT. Photoexcited Au clusters under light irradiation generated singlet oxygen (1O2). Accordingly, the antimicrobial and cytotoxic effects of Au25(Capt)18 clusters under dental blue light-emitting diode (LED) irradiation were evaluated. 1O2 generation of Au25(Capt)18 clusters under blue LED irradiation (420-460 nm) was detected by a methotrexate (MTX) probe. The antimicrobial effects of photoexcited Au clusters (0, 5, 50, and 500 µg/mL) on oral bacterial cells, such as Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis, were assessed by morphological observations and bacterial growth experiments. Cytotoxicity testing of Au clusters and blue LED irradiation was then performed against NIH3T3 and MC3T3-E1 cells. In addition, the biological performance of Au clusters (500 µg/mL) was compared to an organic dye photosensitizer, methylene blue (MB; 10 and 100 µg/mL). We confirmed the 1O2 generation ability of Au25(Capt)18 clusters through the fluorescence spectra of oxidized MTX. Successful application of photoexcited Au clusters to aPDT was demonstrated by dose-dependent decreases in the turbidity of oral bacterial cells. Morphological observation revealed that application of Au clusters stimulated destruction of bacterial cell walls and inhibited biofilm formation. Aggregation of Au clusters around bacterial cells was fluorescently observed. However, photoexcited Au clusters did not negatively affect the adhesion, spreading, and proliferation of mammalian cells, particularly at lower doses. In addition, application of Au clusters demonstrated significantly better cytocompatibility compared to MB. We found that a combination of Au25(Capt)18 clusters and blue LED irradiation exhibited good antimicrobial effects through 1O2 generation and biosafe characteristics, which is desirable for aPDT in dentistry.

Dose effects of beta-tricalcium phosphate nanoparticles on biocompatibility and bone conductive ability of three-dimensional collagen scaffolds.

Three-dimensional collagen scaffolds coated with beta-tricalcium phosphate (ß-TCP) nanoparticles reportedly exhibit good bioactivity and biodegradability. Dose effects of ß-TCP nanoparticles on biocompatibility and bone forming ability were then examined. Collagen scaffold was applied with 1, 5, 10, and 25 wt% ß-TCP nanoparticle dispersion and designated TCP1, TCP5, TCP10, and TCP25, respectively. Compressive strength, calcium ion release and enzyme resistance of scaffolds with ß-TCP nanoparticles applied increased with ß-TCP dose. TCP5 showed excellent cell-ingrowth behavior in rat subcutaneous tissue. When TCP10 was applied, osteoblastic cell proliferation and rat cranial bone augmentation were greater than for any other scaffold. The bone area of TCP10 was 7.7-fold greater than that of non-treated scaffold. In contrast, TCP25 consistently exhibited adverse biological effects. These results suggest that the application dose of ß-TCP nanoparticles affects the scaffold bioproperties; consequently, the bone conductive ability of TCP10 was remarkable.