Enhanced bone regeneration by silicon-substituted hydroxyapatite derived from cuttlefish bone.

OBJECTIVE: There is growing interest in the use of cuttlefish bone (CB) as a bone graft material. Silicon (Si) plays an important role in bone formation and calcification. This study aimed to prepare Si-substituted CB-derived hydroxyapatite (Si-CB-HAp) using a natural CB to improve the bioactivity for bone formation. MATERIALS AND METHODS: We prepared Si-HAp from CB (Si-CB-HAp) using a hydrothermal and solvothermal method. The microstructure and chemical composition were characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectrometer (EDS). The bioactivity of the Si-CB-HAp was evaluated using human mesenchymal stem cells. Furthermore, the in vivo bone regeneration efficiency was evaluated using a rabbit calvarial defect model. RESULTS: Our results show that the Si content was 0.77 wt% in Si-CB-HAp, and its original microstructure was conserved. The presence of Si was shown to enhance cell proliferation and early cellular attachment of human mesenchymal stem cells. Additionally, results of alkaline phosphatase activity and real-time PCR for osteoblast marker genes show that Si substitution into CB-HAp enhanced osteoblast differentiation. In addition, in vivo bone defect healing experiments show that the formation of bone with Si-CB-HAp is higher than that with CB-HAp. CONCLUSION: These results indicate that Si-CB-HAp may potentially be used as a bone graft material to enhance bone healing.

The effect of alendronate soaking and ultraviolet treatment on bone-implant interface.

OBJECTIVE: Rapid and stable fixation of dental implants is crucial for successful treatment. Herein, we examined whether the simultaneous treatment of titanium implants with ultraviolet (UV) and alendronate (ALN) synergistically improved the bone-to-implant contact. MATERIALS AND METHODS: We assessed the in vitro effects of UV radiation-treated (UV+/ALN-), ALN-soaked (UV-/ALN+), and UV radiation/ALN-treated (UV+/ALN+) titanium implants on cell proliferation, cytotoxicity, cell adhesion, and osteoblast differentiation using MG-63 osteoblast-like cells by the assays of MTS, live/dead, scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity, and alizarin red S (AR-S) staining, respectively. Furthermore, in vivo bone formation at the bone-implant interface efficiency determined using a rabbit tibia implantation. Implants were divided into 3 experimental groups (UV+/ALN-, UV-/ALN+, UV+/ALN+) and the non-treated control (UV-/ALN-) group and transplanted into the proximal tibia of rabbits. At 1, 2, 4, and 8 weeks post-operation, bone formation at the bone-implant interface was evaluated by micro-computed tomography and histological analysis. RESULTS: MG-63 cells cultured on UV+/ALN+ implants showed significantly higher cell proliferation, ALP activity, and calcium mineralization than those cultured on other implants (P < 0.05). Furthermore, SEM observation showed the highest increase in cell attachment and growth on the UV+/ALN+ implants. In vivo, experimental groups at all time points showed greater peri-implant bone formation than the control group. At 8 weeks post-implantation, in the UV+/ALN+ group, significantly higher bone formation was observed than the UV+/ALN- or UV-/ALN+ group, respectively (P < 0.05). CONCLUSIONS: Treatment of titanium surfaces with UV and ALN may synergistically enhance osteoblastic differentiation and mineralization in vitro and enhance bone formation at the bone-implant interface in vivo. These data suggest that UV and ALN treatment may improve the osseointegration of titanium implants.

Methyl Red Decolorization Efficiency of a Korea Strain of Aspergillus sp. Immobilized into Different Polymeric Matrices.

Intensive research studies have revealed that fungal decolorization of dye wastewater is a promising replacement for the current process of dye wastewater decolorization. The authors isolated an Aspergillus sp. from the effluent of a textile industry area in Korea and assessed the effects of a variety of operational parameters on the decolorization of methyl red (MR) by this strain of Aspergillus sp. This Aspergillus sp. was then immobilized by entrapment in several polymeric matrices and the effects of operational conditions on MR decolorization were investigated again. The optimal decolorization activity of this Aspergillus sp. was observed in 1% glucose at a temperature of 37 °C and pH of 6.0. Furthermore, stable decolorization efficiency was observed when fungal biomass was immobilized into alginate gel during repeated batch experiment. These results suggest that the Aspergillus sp. isolated in Korea could be used to treat industrial wastewaters containing MR dye.

Enhanced bone healing by improved fibrin-clot formation via fibrinogen adsorption on biphasic calcium phosphate granules.

OBJECTIVES: Fibrin clots play an important role in bone tissue regeneration. This study aimed at improving the fibrin-clotting rate by coating the surface of biphasic calcium phosphate (BCP) granules with fibrinogen (FNG). METHODS AND MATERIALS: FNG was coated on the BCP surface using an adsorption and freeze-drying method. The surface morphology of FNG-adsorbed BCP (FNG-BCP) was characterized using scanning electron microscopy (SEM), and the stability of the adsorbed FNG evaluated by gel electrophoresis and circular dichroism (CD) analysis. The biocompatibility of FNG-BCP was evaluated in vitro using human mesenchymal stem cells, and in vivo bone-healing efficiency determined using a rabbit calvarial bone defect model. RESULTS: SEM studies showed numerous irregularly distributed FNG fractions adsorbed onto the surface of BCP granules. Gel electrophoresis, CD analysis, and in vitro coagulation results showed that the adsorbed FGN maintained its native protein structure and clotting properties. Biocompatibility experiments showed that cell proliferation and adhesion were improved in cells cultivated on the FNG-BCP granules. After surgical implantation into the bone defects, the FNG-BCP granules coagulated at the defect site by reacting with the blood discharged from the surgical site tissue. In addition, at 8 weeks, the volume of FNG40-BCP (P = 0.012) was significantly higher than that of BCP alone in the newly formed bone. CONCLUSIONS: These results indicate that self-coagulating FNG-CBP granules may have the potential to be used as a bone substitute for enabling effective bone repair through rapid fibrin-clot formation.

The effects of fibrinogen concentration on fibrin/atelocollagen composite gel: an in vitro and in vivo study in rabbit calvarial bone defect.

OBJECTIVE: This study aimed to optimize the fibrinogen concentration in fibrin and atelocollagen (AT-COL) (fibrin/AT-COL) composite gel for improving bone regeneration. METHODS AND MATERIALS: The fibrin/AT-COL composite gels were fabricated using various fibrinogen concentrations, and the microstructure and mechanical properties of the resulting composite gels analyzed. The cytocompatibility of the composite gels was examined using human mesenchymal stem cell (hMSCs). Furthermore, in nine rabbits, the in vivo bone regeneration efficiency was evaluated using a rabbit calvarial defects model at 2 weeks (n = 3), 4 weeks (n = 3), and 8 weeks (n = 3). RESULTS: Scanning electron microscopy analysis revealed the formation of a fibrin layer matrix and collagen fibril networks. The composite gel containing 40 mg/ml fibrinogen showed a densely packed fibrin matrix and displayed superior mechanical properties. Cells cultured in the composite gels prepared with 5-20 mg/ml fibrinogen appeared elongated, with a spindle-like morphology. At a higher fibrinogen concentration (40 mg/ml), many cells were rounded and showed limited viability. In an in vivo study, at 8 weeks, the volume of fibrin/AT-COL gel (P = 0.02) was significantly higher than that of fibrin gel alone in the newly formed bone. Histological analysis revealed more islands of newly formed bone filling the central area of the defect in the fibrin/AT-COL gel-implanted animals. CONCLUSION: Our results demonstrate that optimization of the fibrinogen content of fibrin/AT-COL composites should be beneficial for bone tissue engineering.

NIR-Triggered Hyperthermal Effect of Polythiophene Nanoparticles Synthesized by Surfactant-Free Oxidative Polymerization Method on Colorectal Carcinoma Cells.

In this work, polythiophene nanoparticles (PTh-NPs) were synthesized by a surfactant-free oxidative chemical polymerization method at 60 °C, using ammonium persulphate as an oxidant. Various physicochemical properties were studied in terms of field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), Fourier transform infra-red (FT-IR) spectroscopy, and differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA). Photothermal performance of the as-synthesized PTh-NPs was studied by irradiating near infra-red of 808 nm under different concentration of the substrate and power supply. The photothermal stability of PTh-NPs was also studied. Photothermal effects of the as-synthesized PTh-NPs on colorectal cancer cells (CT-26) were studied at 100 µg/mL concentration and 808 nm NIR irradiation of 2.0 W/cm2 power. Our in vitro results showed remarkable NIR laser-triggered photothermal apoptotic cell death by PTh-NPs. Based on the experimental findings, it is revealed that PTh-NPs can act as a heat mediator and can be an alternative material for photothermal therapy in cancer treatment.

Synthesis of polypyrrole nanorods via sacrificial removal of aluminum oxide nanopore template: A study on cell viability, electrical stimulation and neuronal differentiation of PC12 cells.

Synthesis of nanomaterials having uniform shape and size is a challenging task. Properties exhibited by such substrates would be compatible and homogeneous compared to the average properties displayed by those substrates with heterogeneous size. Herein, we report the synthesis of polypyrrole nanorods (PPy-NRs) of almost uniform size via oxidative chemical polymerization of pyrrole within anodized aluminum oxide nanopores followed by sacrificial removal of the template. Field emission scanning electron microscopy (FE-SEM), fourier transformed infra-red (FT-IR) spectra, X-ray diffraction (XRD), and ultra-violet-visible-near infra-red (UV-Vis-NIR) spectra of the substrate were used to analyze the physicochemical properties of as-synthesized PPy-NRs. PPy-NRs treated MC3T3-E1 and PC12 cells exhibited good biocompatibility in CCK-8 and live/dead assays. The assay showed more cell viability on PC12 cell lines. Electrical stimulation through PPy-NRs treated PC12 cells accelerated neuronal differentiation compared to those without electrical stimulation during in vitro cell culture.

A conducting neural interface of polyurethane/silk-functionalized multiwall carbon nanotubes with enhanced mechanical strength for neuroregeneration.

A fibrous scaffold, fully assimilating polyurethane (PU) and silk fibroin associated with functionalized multi-walled carbon nanotubes (fMWCNTs) was developed by electrospinning technique. Herein, we engineered the PU/Silk fibroin-fMWCNTs-based biomaterial that shows great promise as electrospun scaffolds for neuronal growth and differentiation, because of its unique mechanical properties, hydrophilicity, and biodegradability, with outstanding biocompatibility in nerve tissue engineering. The morphology and structural properties of the scaffolds were studied using various techniques. In particular, the presence of fMWCNTs enhances the electrical conductivity and plausible absorption of sufficient extracellular matrix (ECM). The in vitro tests revealed that the aligned scaffolds (PU/Silk-fMWCNTs) significantly stimulated the growth and proliferation of Schwann cells (S42), together with the differentiation and spontaneous neurite outgrowth of rat pheochromocytoma (PC12) cells that were particularly guided along the axis of fiber alignment. The conductive PU/Silk-fMWCNTs scaffold significantly improves neural expression in vitro with successful axonal regrowth, which was confirmed by immunocytochemistry and qRT-PCR analysis. Inspired by the comprehensive experimental results, the fMWCNTs-based scaffold affords new insight into nerve-guided conduit design from both conductive and protein rich standpoints, and opens a new perspective on peripheral nerve restoration in preclinical applications.

The intracellular uptake ability of chitosan-coated Poly (D,L-lactide-co-glycolide) nanoparticles.

In this study, we prepared chitosan-coated Poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles. Specifically, we utilized a double emulsion-solvent evaporation technique to formulate nanoparticles containing paclitaxel as a model macromolecule and 6-coumarin as a fluorescent marker. SEM images verified that all nanoparticles were spherical in shape with smooth surfaces. Chitosan coating slightly increased the size distribution of the PLGA/PVA nanoparticles, from 202.2+/-3.2 nm to 212.2+/-2.9 nm, but the encapsulation efficiency was not significantly different. In contrast, coating with chitosan slowed the in vitro drug release rate and significantly changed the zeta potential from negative (-30.1+/-0.6 mV) to positive (26+/-1.2 mV). At the initial burst time, the drug release rate from chitosancoated nanoparticles was slightly slower than that of the uncoated nanoparticles. Chitosan-coated nanoparticles were also taken up much more efficiently than uncoated nanoparticles. This study demonstrated the efficacy of chitosancoated PLGA nanoparticles as an efficient delivery system.

Incorporation of BMP-2 nanoparticles on the surface of a 3D-printed hydroxyapatite scaffold using an ε-polycaprolactone polymer emulsion coating method for bone tissue engineering.

Hydroxyapatite (HAp)-based three-dimensional (3D) scaffolding is an excellent method for the fabrication of complex-shaped scaffolds to reconstruct bone defects. This study aimed at improving the osteoinductivity and compressive strength of the HAp-based 3D scaffold for bone regeneration. Bone morphogenetic protein-2-loaded nanoparticles (BMP-2/NPs) were prepared by a double emulsion-solvent evaporation method and incorporated onto the surface of 3D scaffolds using ε-polycaprolactone (PCL) and NPs emulsion solution. The surface morphology of the scaffold was characterized using scanning electron microscopy and its biocompatibility and osteogenic effects evaluated in vitro using human mesenchymal stem cells. The in vivo bone regeneration efficiency was determined using a rabbit calvarial bone defect model. We obtained 3D HAp scaffolds with NPs using PCL coating process. BMP-2/NPs were uniformly distributed on the scaffold surface and BMP-2 was gradually released. Furthermore, PCL coating improved the compressive strength of the scaffold. The cell proliferation, adhesion, and osteogenic differentiation properties were improved with PCL_BMP-2/NPs coated scaffold. In vivo experiments showed that the formation of new bone was significantly higher in the PCL_BMP-2/NPs group than in the uncoated scaffold-implanted group. The coating method using PCL and NPs emulsion solutions was useful not only to incorporate BMP-2/NPs onto the surface of the scaffold, but also to improve the compressive strength, which enhanced bone regeneration.

Injectable methylcellulose hydrogel containing calcium phosphate nanoparticles for bone regeneration.

A novel injectable methylcellulose (MC) hydrogel containing calcium phosphate nanoparticles (CaP NPs) was prepared by an in situ formation process, in which the precursor salts induced a salt-out effect in the MC solution. The thermo-sensitive properties of MC-CaP NPs composite hydrogels with different crystalline phases were characterized by rheometry, infrared spectroscopy and injectability test. The as-prepared MC hydrogels with bioactive CaP NPs had a suitable injectability at the body temperature, irrespective of the crystalline phases of CaP NPs. At the physiological pH condition, the structure of the MC hydrogel containing CaP NPs was analyzed by scanning electron microscopy, X-ray diffraction (XRD). The XRD results indicate that the in situ synthesized CaP NPs had a crystalline phase of hydroxyapatite (HAP). The in vitro study using mesenchymal stem cells showed that MC-HAP NPs composite hydrogel was biocompatible. The in vivo study indicated that the regeneration rate of new mature bone was also higher in the MC-HAP NPs composite hydrogel than in the pure MC hydrogel. The results of this study indicate that injectable MC-HAP NPs composite hydrogel has a great potential for bone tissue regeneration.

pH/NIR-Responsive Polypyrrole-Functionalized Fibrous Localized Drug-Delivery Platform for Synergistic Cancer Therapy.

Localized drug-delivery systems (LDDSs) are a promising approach for cancer treatment because they decrease systematic toxicity and enhance the therapeutic effect of the drugs via site-specific delivery of active compounds and possible gradual release. However, the development of LDDS with rationally controlled drug release and intelligent functionality holds great challenge. To this end, we have developed a tailorable fibrous site-specific drug-delivery platform functionalized with pH- and near-infrared (NIR)-responsive polypyrrole (PPy), with the aim of cancer treatment via a combination of photothermal ablation and chemotherapy. First, a paclitaxel (PTX)-loaded polycaprolactone (PCL) (PCL-PTX) mat was prepared by electrospinning and subsequently in situ membrane surface-functionalized with different concentrations of PPy. The obtained PPy-functionalized mats exhibited excellent photostability and heating property in response to NIR exposure. PPy-coated mats exhibited enhanced PTX release in a pH 5.5 environment compared to pH 7.4. Release was further accelerated in response to NIR under both conditions; however, superior release was observed at pH 5.5 compared to pH 7.4, indicating a dual stimuli-responsive (pH and NIR) drug-delivery platform. More importantly, the 808 nm NIR irradiation enabled markedly accelerated PTX release from PPy-coated PCL-PTX mats and slowed and sustained release following termination of laser irradiation, confirming representative stepwise drug-release properties. PPy-coated PCL-PTX mats presented significantly enhanced in vitro and in vivo anticancer efficacy under NIR irradiation compared to PPy-coated PCL-PTX mats not exposed to NIR or uncoated mats (PCL-PTX). This study has thus developed a promising fibrous site-specific drug-delivery platform with NIR- and pH-triggering that notably utilizes PPy as a dopant for synergistic photothermal chemotherapy.

Precoating of biphasic calcium phosphate bone substitute with atelocollagen enhances bone regeneration through stimulation of osteoclast activation and angiogenesis.

Type I collagen (Col) is a naturally polymerizing protein and important extracellular matrix bone component. The aim of this study was to improve bone regeneration capacity by precoating the surface of biphasic calcium phosphate (BCP) granules with AT-Col, and evaluating its biological effects. BCP granules were precoated with AT-Col using adsorption and lyophilization method. Morphology of AT-Col precoated surfaces was observed using scanning electron microscopy (SEM). Biocompatibility and osteogenic activity of AT-Col were determined in vitro with human mesenchymal stem cell (hMSCs). In vivo bone healing efficiency and related biological effects were determined using a rabbit calvarial defect model. SEM results revealed numerous irregularly distributed AT-Col polymer clusters on BCP granule surface. Biocompatibility experiments demonstrated that AT-Col was non-cytotoxic, and that cell proliferation, adhesion, and osteogenic activity were improved by AT-Col precoating. After in vivo surgical implantation into bone defects, new bone formation was improved by AT-Col granule precoating. Specifically, 8 weeks post-surgery, percentage bone volume was significantly higher in AT-Col/BCP animals (35.02 ± 1.89%) compared with BCP-treated animals (8.94 ± 1.47%) (p < 0.05). Furthermore, tartrate-resistant acid phosphatase staining and CD31 immunohistochemical staining revealed that osteoclast activation and new blood vessel formation in vivo were also induced by AT-Col precoating. Collectively, these data indicate that AT-Col/BCP may be potentially used as a bone substitute to enable effective bone regeneration through enhanced new blood vessel formation and osteoclast activation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1446-1456, 2017.

Improvement of mechanical strength and osteogenic potential of calcium sulfate-based hydroxyapatite 3-dimensional printed scaffolds by ε-polycarbonate coating.

Powder-based three-dimensional (3D) printing is an excellent method to fabricate complex-shaped scaffolds for tissue engineering. However, their lower mechanical strength restricts their application in bone tissue engineering. Here, we created a 3D-printed scaffold coated with a ε-polycaprolactone (PCL) polymer solution (5 and 10 w/v %) to improve the mechanical strength of the scaffold. The 3D scaffold was fabricated from calcium sulfate hemihydrate powder (CaSO4-1/2 H2O), transformed into hydroxyapatite (HAp) by treatment with a hydrothermal reaction in an NH4H2PO4 solution. The surface properties and composition of the scaffold were evaluated using scanning electron microscopy and X-ray diffraction analysis. We demonstrated that the 3D scaffold coated with PCL had an improved mechanical modulus. Coating with 5 and 10% PCL increased the compressive strength significantly, by about 2-fold and 4-fold, respectively, compared with that of uncoated scaffolds. However, the porosity was reduced significantly by coating with 10% PCL. In vitro biological evaluation demonstrated that MG-63 cells adhered well and proliferated on the 3D scaffold coated with PCL, and the scaffold was not cytotoxic. In addition, alkaline phosphatase activity and real time polymerase chain reaction demonstrated that osteoblast differentiation also improved in the PCL-coated 3D scaffolds. These results indicated that PCL polymer coating could improve the compressive strength and biocompatibility of 3D HAp scaffolds for bone tissue engineering applications.

Evaluation of bone regeneration with biphasic calcium phosphate substitute implanted with bone morphogenetic protein 2 and mesenchymal stem cells in a rabbit calvarial defect model.

OBJECTIVE: The aim of this study was to evaluate the in vivo osteogenic potential of biphasic calcium phosphate (BCP), bone morphogenetic protein 2 (BMP-2), and/or mesenchymal stem cell (MSC) composites by using a rabbit calvarial defect model. STUDY DESIGN: Bone formation was assessed by using three different kinds of implants in rabbit calvarial defects, BCP alone, BCP/recombinant human (rh) BMP-2, and BCP/rhBMP-2/MSCs composite. The implants were harvested after 2 or 8 weeks, and the area of new bone formation was quantified by micro-computed tomography (micro-CT) and histologic studies. RESULTS: The highest bone formation was achieved with the BCP/rhBMP-2/MSCs treatment, and it was significantly higher than that achieved with the empty or BCP-alone treatment. The quantity of new bone at 8 weeks was greater than at 4 weeks in each group. The relative density of osteocalcin immunoreactivity also increased during this interval. CONCLUSIONS: These results indicate that the combination of BCP, rhBMP-2, and MSCs synergistically enhances osteogenic potential during the early healing period and could be used as a bone graft substitute.

Effect of nanofiber content on bone regeneration of silk fibroin/poly(ε-caprolactone) nano/microfibrous composite scaffolds.

The broad application of electrospun nanofibrous scaffolds in tissue engineering is limited by their small pore size, which has a negative influence on cell migration. This disadvantage could be significantly improved through the combination of nano- and microfibrous structure. To accomplish this, different nano/microfibrous scaffolds were produced by hybrid electrospinning, combining solution electrospinning with melt electrospinning, while varying the content of the nanofiber. The morphology of the silk fibroin (SF)/poly(ε-caprolactone) (PCL) nano/microfibrous composite scaffolds was investigated with field-emission scanning electron microscopy, while the mechanical and pore properties were assessed by measurement of tensile strength and mercury porosimetry. To assay cell proliferation, cell viability, and infiltration ability, human mesenchymal stem cells were seeded on the SF/PCL nano/microfibrous composite scaffolds. From in vivo tests, it was found that the bone-regenerating ability of SF/PCL nano/microfibrous composite scaffolds was closely associated with the nanofiber content in the composite scaffolds. In conclusion, this approach of controlling the nanofiber content in SF/PCL nano/microfibrous composite scaffolds could be useful in the design of novel scaffolds for tissue engineering.

A polycaprolactone/cuttlefish bone-derived hydroxyapatite composite porous scaffold for bone tissue engineering.

Cuttlefish bone (CB) is an attractive natural biomaterial source to obtain hydroxyapatite (HAp). In this study, a porous polycaprolactone (PCL) scaffold incorporating CB-derived HAp (CB-HAp) powder was fabricated using the solvent casting and particulate leaching method. The presence of CB-HAp in PCL/CB-HAp scaffold was confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) and porosity analysis showed that the average pore dimension of the fabricated scaffold was approximately 200-300 µm, with ∼85% porosity, and that the compressive modulus increased after addition of CB-HAp powders. In vitro tests such as cell proliferation assay, cytotoxicity analysis, cell attachment observations, and alkaline phosphatase activity assays showed that the PCL/CB-HAp scaffold could improve the proliferation, viability, adherence, and osteoblast differentiation rate of MG-63 cells. When surgically implanted into rabbit calvarial bone defects, consistent with the in vitro results, PCL/CB-HAp scaffold implantation resulted in significantly higher new bone formation than did implantation of PCL alone. These findings suggest that addition of CB-HAp powder to the PCL scaffold can improve cellular response and that the PCL/CB-HAp composite scaffold has great potential for use in bone tissue engineering.

Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration.

Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering.

Comparison of in vitro and in vivo bioactivity: cuttlefish-bone-derived hydroxyapatite and synthetic hydroxyapatite granules as a bone graft substitute.

Bone reconstruction in clinical settings often requires bone substitutes. Hydroxyapatite (HAp) is a widely used bone substitute due to its osteoconductive properties and bone bonding ability. The aim of this study was to evaluate HAp granules derived from cuttlefish bone (CB-HAp) as a substitute biomaterial for bone grafts. In this study, HAp granules were prepared from raw CB by using a hydrothermal reaction. The formation of HAp from CB was confirmed by scanning electron microscopy and x-ray diffraction analysis. The bioactivity of the CB-HAp granules was evaluated both in vitro and in vivo. Our results show that CB-HAp is non-toxic and that CB-HAp granules supported improved cell adhesion, proliferation and differentiation compared to stoichiometric synthetic HAp granules. Furthermore, in vivo bone defect healing experiments show that the formation of bone with CB-HAp is higher than that with pure HAp. These results show that CB-HAp granules have excellent potential for use as a bone graft material.

A simplified technique for tumor localization using preoperative endoscopic clipping and radio-opaque markers during totally laparoscopic gastrectomy.

Tumor localization during intracorporeal anastomosis after totally laparoscopic distal gastrectomy (TLDG) is challenging. The aim of this study was to assess the simplicity and feasibility of locating tumors in the stomach using radio-opaque markers and preoperative endoscopic clipping. The intra- and postoperative findings of 29 patients who underwent TLDG with intracorporeal anastomosis between January 2012 and March 2013 were reviewed. Preoperative endoscopic clips were applied just proximal to the tumor by specialized endoscopists, and surgical gauze with an attached radio-opaque marker (3 mm × 60 mm) was prepared. The marker was fixed to either the anterior or posterior of the stomach, above the predicted site of the tumor, using suture ties. Portable abdominal radiography was used during the laparoscopic surgery, and the stomach was resected using guidance by the radiomarker. The radio-opaque marker and the endoscopic clips were clearly visible by intraoperative abdominal radiography. All patients received curative resection. No complications or deaths were encountered. The mean distance between the endoscopic clips and the radiomarker by portable intraoperative radiography was 21.3 ± 18.3 mm, whereas the actual in situ mean distance was 20.7 ± 17.6 mm. This difference was not statistically significant (P > 0.05). It is imperative that preoperative endoscopic clips are applied just proximal to the tumor by specialized endoscopists. The use of a radio-opaque marker is a simple and feasible way to locate tumors during totally laparoscopic gastrectomy.