New fabrication method of silk fibroin plate and screw based on a centrifugal casting technique.

Recently, a newer generation of absorbable biomaterials has been developed from silk. Silk is approved by the US Food and Drug Administration, has robust mechanical features, and is biocompatible. Moreover, it offers the ability to be functionalized with bioactive compounds, making it ideal for use in new medical devices. Thus, many researchers have considered that absorbable devices made from silk may be able to overcome current limitations and could be used to meet a broader range of fixation needs. Here, we describe a novel method for the fabrication of silk fibroin (SF)-based bioabsorbable fixation systems using a centrifugal casting technique and incorporating a 3D printer. This approach allows us to create the desired geometric design for the fixation system easily. Moreover, our products demonstrated smoother surface profiles and more homogenous and dense cross-sectional architectures. Furthermore, our plates exhibited very similar mechanical properties compared with commercially used one, and our screws showed more than 70% of their initial mass after 7 weeks on the enzymatic degradation test. On in vivo analysis, we found that our devices were well-maintained in the location of initial fixation, and new bone formation was also observed around this. By these results, we suggest that the SF-based plate/screw prepared by our novel method might be used for the internal fixation of fracture sites.

Three dimensional poly(ε-caprolactone) and silk fibroin nanocomposite fibrous matrix for artificial dermis.

Ideal dermal substitutes should have comparable physicochemical and biological properties to the natural skin tissue. In this study, we report a novel strategy to "engineer" controlled 3D nanocomposite fibrous matrix of poly(ε-caprolactone) (PCL) and silk fibroin (SF) for an artificial dermis application. Using a custom-designed cold-plate electrospinning and automatic magnet agitation system, up to 6mm of the thickness was achieved resulting from the accumulation of ice crystal layers on the PCL nanofibers surface-modified with the SF particles. The sacrificed ice crystals induced interconnected macro-pores ranging from tens to hundreds µm. The agitation system introduced uniform distribution of the SF protein within/on the nanofibers, preventing the particles from precipitation and agglomeration. NIH 3T3 fibroblasts proliferated in vitro on the PCL and PCL/SF scaffolds for 7days, but there was no statistical difference between the groups. Conversely, In vivo rat model studies revealed that the wound healing rate and collagen deposition increased with the SF content within the nanocomposites. The unique 3D construct with the PCL/SF nanocomposite fibers provided desirable spatial cues, surface topography, and surface chemistry for the native cells to infiltrate into the scaffolds. The wound healing potential of the nanocomposites was comparable to the commercial Matriderm® artificial dermis.

Osteoinductive silk fibroin/titanium dioxide/hydroxyapatite hybrid scaffold for bone tissue engineering.

The present study demonstrated the fabrication that incorporation of titanium isopropoxide (TiO2) and hydroxyapatite (HA) nanoparticles into the silk fibroin (SF) scaffolds. In this process, we prepared TiO2 nanoparticles using sol-gel synthesis and the porous structure was developed by salt-leaching process. Homogeneous distribution of TiO2 and HA nanoparticles were confirmed by images of VP-FE-SEM and those equipped with energy dispersive X-ray spectrometer. Structural characteristics of the porous SF/TiO2/HA hybrid scaffold were also determined using FTIR analysis and X-ray diffractometer. In this study, the porous SF/TiO2/HA hybrid scaffold showed similar porosity, enhanced mechanical property, but decreased water binding abilities, compared with the porous SF scaffold. For evaluation of the osteogenic differentiation of rat bone marrow mesenchymal stem cells, alkaline phosphatase activity and osteogenic gene expression were employed. Our results revealed that the porous SF/TiO2/HA hybrid scaffold had improved osteoinductivity compared with the porous SF scaffold. These results suggest that the osteogenic property as well as mechanical property of the porous SF/TiO2/HA hybrid scaffold could be better than the porous SF scaffold. Therefore, the porous SF/TiO2/HA hybrid scaffold may be a good promising biomaterial for bone tissue engineering application.

Hybrid scaffolds based on PLGA and silk for bone tissue engineering.

Porous silk scaffolds, which are considered to be natural polymers, cannot be used alone because they have a long degradation rate, which makes it difficult for them to be replaced by the surrounding tissue. Scaffolds composed of synthetic polymers, such as PLGA, have a short degradation rate, lack hydrophilicity and their release of toxic by-products makes them difficult to use. The present investigations aimed to study hybrid scaffolds fabricated from PLGA, silk and hydroxyapatite nanoparticles (Hap NPs) for optimized bone tissue engineering. The results from variable-pressure field emission scanning electron microscopy (VP-FE-SEM), equipped with EDS, confirmed that the fabricated scaffolds had a porous architecture, and the location of each component present in the scaffolds was examined. Contact angle measurements confirmed that the introduction of silk and HAp NPs helped to change the hydrophobic nature of PLGA to hydrophilic, which is the main constraint for PLGA used as a biomaterial. Thermo-gravimetric analysis (TGA) and FT-IR spectroscopy confirmed thermal decomposition and different vibrations caused in functional groups of compounds used to fabricate the scaffolds, which reflected improvement in their mechanical properties. After culturing osteoblasts for 1, 7 and 14 days in the presence of scaffolds, their viability was checked by MTT assay. The fluorescent microscopy results revealed that the introduction of silk and HAp NPs had a favourable impact on the infiltration of osteoblasts. In vivo experiments were conducted by implanting scaffolds in rat calvariae for 4 weeks. Histological examinations and micro-CT scans from these experiments revealed beneficial attributes offered by silk fibroin and HAp NPs to PLGA-based scaffolds for bone induction.

Fabrication of duck's feet collagen-silk hybrid biomaterial for tissue engineering.

Collagen constituting the extracellular matrix has been widely used as biocompatible material for human use. In this study, we have selected duck's feet for extracting collagen. A simple method not utilizing harsh chemical had been employed to extract collagen from duck's feet. We fabricated duck's feet collagen/silk hybrid scaffold for the purpose of modifying the degradation rate of duck's feet collagen. This study suggests that extracted collagen from duck's feet is biocompatible and resembles collagen extracted from porcine which is commercially used. Duck's feet collagen is also economically feasible and it could therefore be a good candidate as a tissue engineering material. Further, addition of silk to fabricate a duck's feet collagen/silk hybrid scaffold could enhance the biostability of duck's feet collagen scaffold. Duck's feet collagen/silk scaffold increased the cell viability compared to silk alone. Animal studies also showed that duck's feet collagen/silk scaffold was more biocompatible than silk alone and more biostable than duck's feet or porcine collagen alone. Additionally, the results revealed that duck's feet collagen/silk hybrid scaffold had high porosity, cell infiltration and proliferation. We suggest that duck's feet collagen/silk hybrid scaffold could be used as a dermal substitution for full thickness skin defects.

Wound healing effect of electrospun silk fibroin nanomatrix in burn-model.

Silk fibroin has recently become an important biomaterial for tissue engineering application. In this study, silk fibroin nanomatrix was fabricated by electrospinning and evaluated as wound dressing material in a burn rat model. The wound size reduction, histological examination, and the quantification of transforming growth factor TGF-ß1 and interleukin IL-1α, 6, and 10 were measured to evaluate the healing effects. The silk fibroin nanomatrix treatment exhibited effective performance in decreasing the wound size and epithelialization. Histological finding also revealed that the deposition of collagen in the dermis was organized by covering the wound area in the silk fibroin nanomatrix treated group. The expression level of pro-inflammatory cytokine (IL-1α) was significantly reduced in the injured skin following the silk fibroin nanomatrix treatment compared to the medical gauze (control) at 7 days after burn. Also, the expression level of TGF-ß1 in the wound treated with silk fibroin nanomatrix peaked 21-days post-treatment whereas expression level of TGF-ß1 was highest at day 7 in the gauze treated group. In conclusion, this data demonstrates that silk fibroin nanomatrix enhances the burn wound healing, suggesting it is a good candidate for burn wound treatment.

Fabrication of 3D porous SF/ß-TCP hybrid scaffolds for bone tissue reconstruction.

Bio-ceramic is a biomaterial actively studied in the field of bone tissue engineering. But, only certain ceramic materials can resolve the corrosion problem and possess the biological affinity of conventional metal biomaterials. Therefore, the recent development of composites of hybrid composites and polymers has been widely studied. In this study, we aimed to select the best scaffold of silk fibroin and ß-TCP hybrid for bone tissue engineering. We fabricated three groups of scaffold such as SF (silk fibroin scaffold), GS (silk fibroin/small granule size of ß-TCP scaffold) and GM (silk fibroin/medium granule size of ß-TCP scaffold), and we compared the characteristics of each group. During characterization of the scaffold, we used scanning electron microscopy (SEM) and a Fourier transform infrared spectroscopy (FTIR) for structural analysis. We compared the physiological properties of the scaffold regarding the swelling ratio, water uptake and porosity. To evaluate the mechanical properties, we examined the compressive strength of the scaffold. During in vitro testing, we evaluated cell attachment and cell proliferation (CCK-8). Finally, we confirmed in vivo new bone regeneration from the implanted scaffolds using histological staining and micro-CT. From these evaluations, the fabricated scaffold demonstrated high porosity with good inter-pore connectivity, showed good biocompatibility and high compressive strength and modulus. In particular, the present study indicates that the GM scaffold using ß-TCP accelerates new bone regeneration of implanted scaffolds. Accordingly, our scaffold is expected to act a useful application in the field of bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1779-1787, 2016.

Effect of simulated pulpal pressure on dentin bond strength of self-etching bonding systems.

PURPOSE: To evaluate in vitro the influence of dentin perfusion on dentin bond strengths of etch and rinse and self-etch adhesives. METHODS: 64 extracted human molars were ground flat and resin composite (Brilliant) was bonded using the following bonding systems (each n=16): OptiBond FL including phosphoric acid etching (OFL+), OptiBond FL without phosphoric acid etching (OFL-), Clearfil SE-Bond (CSE) and Prompt L-Pop (LP). In each bonding system, half of the specimens (n=8) were bonded without (PP-) and half with (PP+) simulated intrapulpal pressure. After water storage (37 degrees C, 24 hours), a 1 mm thick slice was cut vertically from the middle of each sample. Microtensile bond strength (microTBS) was determined and the debonded surfaces were investigated in SEM for the mode of failure. Statistical significance was determined by ANOVA, Tukey-test, and t-test (P= 0.05). RESULTS: Mean microTBS measured were (MPa +/- SD): OFL+/PP- 58.6 (5.8), OFL+/PP+ 38.1 (3.8), OFL-/PP- 37.9 (3.7), OFL-/PP+ 33.3 (3.5), CSE/PP- 41.1 (4.1), CSE/PP+ 39.0 (4.4), LP/PP- 35.8 (6.4), LP/PP+ 29.0 (4.8). The presence of PP resulted in a decrease of microTBS in all bonding systems, which reached significance in OFL+ (P< 0.0001), OFL- (P= 0.023) and LP (P= 0.032). Both, with and without PP, the bonding systems showed significant differences in bond strength (ANOVA: P< 0.0001).

Three-layered scaffolds for artificial esophagus using poly(ɛ-caprolactone) nanofibers and silk fibroin: An experimental study in a rat model.

The purpose of this study was to determine the feasibility of an artificial esophagus using a three-layered poly(ε-caprolactone) (PCL)-silk fibroin (SF) scaffold in a rat model. The artificial esophagus was a three-layered, hybrid-type prosthesis composed of an outer and inner layer of PCL with a middle layer of SF. After depositing the inner layer of the PCL scaffold by electrospinning, the lyophilized middle SF layer was created. The outer layer of PCL was produced following the same procedure used to make the inner PCL layer. Eleven rats were anesthetized using inhaled anesthesia. Circumferential defects of the cervical esophagus (n=11) were created and reconstructed. Groups of rats were sacrificed after the 1st and 2nd weeks. Three rats died of an esophageal fistula and wound infection. No gross evidence of a fistula, perforation, abscess formation, seroma accumulation, or surrounding soft-tissue necrosis was observed in the other rats sacrificed after the 1st and 2nd weeks. The artificial esophagus constructs produced complete healing of the circumferential defects by the 2nd week. The composition of the three-layered artificial esophagus was confirmed histologically to have an outer and inner layer of PCL and a middle layer of SF. The fusion of the PCL-SF scaffold and the regenerative tissue remained intact. Our study proposes a more practical experimental model for studying a three-layered PCL-SF scaffold in the esophagus. However, further studies on circumferential defect reconstruction in a rat model are still required.

Fabrication of 3D porous silk scaffolds by particulate (salt/sucrose) leaching for bone tissue reconstruction.

Silk fibroin is a biomaterial being actively studied in the field of bone tissue engineering. In this study, we aimed to select the best strategy for bone reconstruction on scaffolds by changing various conditions. We compared the characteristics of each scaffold via structural analysis using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), the swelling ratio, water uptake, porosity, compressive strength, cell infiltration and cell viability (CCK-8). The scaffolds had high porosity with good inter pore connectivity and showed high compressive strength and modulus. In addition, to confirm bone reconstruction, animal studies were conducted in which samples were implanted in rat calvaria and investigated by micro-CT scans. In conclusion, the presented study indicates that using sucrose produces scaffolds showing better pore interconnectivity and cell infiltration than scaffolds made by using a salt process. In addition, in vivo experiments showed that hydroxyapatite accelerates bone reconstruction on implanted scaffolds. Accordingly, our scaffold will be expected to have a useful application in bone reconstruction.

Modified oligosaccharides as potential dental plaque control materials.

Metabolic acids produced by oral pathogens demineralize tooth surfaces, leading to dental caries. Glucosyltransferases are the key factor in this process. We synthesized various modified oligosaccharides and tested them for their inhibitory effects on glucosyltransferase activity. Oligosaccharides were produced using a mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides and then further modified as iron- and sulfate-oligosaccharides. Iron- and sulfate-oligosaccharides reduced glucosyltransferase activity of Streptococci from 17% to 43% and prevented the formation of insoluble biomass on the surface of glass vials or stainless steel wires in the presence of sucrose. They also reduced the growth and acid productions of oral pathogens including S. mutans, S. sobrinus, Eikenella corrodens, Prevotella intermedia, and Actinobacillus actinomycetemcmitans.

Bond strength of adhesive/composite combinations to dentin involving total- and self-etch adhesives.

PURPOSE: To compare the bonding potential to human dentin of adhesive/composite combinations including five 2-step and two 3-step total-etch (TE) bonding systems, two systems with self-conditioning (SC) primers, and one SC all-in-one adhesive by use of the microtensile bond test. METHODS: Hybrid resin composites were bonded to the occlusal dentin of 50 extracted human molars. After water storage (37 degrees C, 24 h), 31-mm-thick slabs were cut from the middle of the teeth perpendicular to their long axis. Microtensile bond strength was determined and debonded surfaces were examined under the SEM for mode of failure. STATISTICS: GLM multivariate procedure for repeated measurements, Student-Newman-Keuls test (SPSS version 10.0; p = 0.05). RESULTS: Mean bond strengths of the simplified (2-step) TE systems (OptiBond Solo, Gluma One Bond, Solobond M, Prime&Bond NT, One Coat Bond; 19.9 MPa to 39.9 MPa) were not significantly lower than that of the traditional 3-step TE systems (EBS Multi: 26.0 MPa; OptiBond FL: 32.7 MPa), and not related to phosphoric acid concentration. Dentin treatment with SC primers (Clearfil Liner Bond 2: 22.0 MPa; Clearfil Liner Bond 2V: 22.4 MPa) was as effective as etching with phosphoric acid. The SC all-in-one adhesive (Etch&Prime 3.0: 10.1 MPa) produced significantly lower bond strength than all other systems evaluated. CONCLUSION: The use of adhesive/composite combinations including simplified bonding systems does not necessarily result in reduced bond strength to dentin. SC primers offer a promising alternative to phosphoric acid etching as far as bonding to dentin is concerned. In contrast, the SC all-in-one adhesive evaluated needs to be improved.

Fabrication of poly(lactic-co-glycolic acid) scaffolds containing silk fibroin scaffolds for tissue engineering applications.

The present study deals with the fabrication of poly(lactic-co-glycolic acid) (PLGA) scaffolds modified with silk fibroin for biomedical application. The PLGA solutions were added with salt particles and pressed with high pressures; which were further subjected to salt leaching resulting in the creation of large sized pores in the PLGA scaffolds. To fill up these pores, 2%, 4%, and 8% of silk solutions were added, however, the addition created extra small sized pores. The scaffolds were characterized by various state of techniques; the scanning electronic microscopy revealed the large sized pores in the pristine scaffold can be tailored into smaller architecture by the addition of silk fibroin. The contact angle measurements confirmed the introduction of silk helped to change the hydrophobic nature of PLGA into hydrophilic, which is the main constrain for PLGA. The mechanical properties of scaffold can be easily improved by applying the higher amounts of silk into the scaffolds. The thermal gravimetric analyses and fourier transform infrared spectroscopy confirmed the presence of silk fibroin in scaffolds. The cell viability and cell attachment was checked by culturing the scaffolds with NIH 3T3 fibroblasts and chondrocytes. Furthermore, these results revealed that the introduction of silk had significant impact on the viability of fibroblast also had a good affinity for cell attachment and infiltration of human chondrocytes in scaffolds after culturing the cells for 2 and 5 weeks of time.

Hypoxia inducible factor-1α directly induces the expression of receptor activator of nuclear factor-κB ligand in periodontal ligament fibroblasts.

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1α (HIF-1α) protein in PDLFs. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL expression. To investigate further whether HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1α significantly increased RANKL promoter activity, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL promoter activity. Furthermore, mutations of putative HIF-1α binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1α increased the DNA binding of HIF-1α to RANKL promoter. These results suggest that HIF-1α mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs.

The effect of antioxidants on the production of pro-inflammatory cytokines and orthodontic tooth movement.

Orthodontic force causes gradual compression of the periodontal ligament tissues, which leads to local hypoxia in the compression side of the tissues. In this study, we investigated whether antioxidants exert a regulatory effect on two factors: the expression of pro-inflammatory cytokines in human periodontal ligament fibroblasts (PDLFs) that were exposed to mechanical compression and hypoxia and the rate of orthodontic tooth movement in rats. Exposure of PDLFs to mechanical compression (0.5-3.0 g/cm(2)) or hypoxic conditions increased the production of intracellular reactive oxygen species. Hypoxic treatment for 24 h increased the mRNA levels of IL-1ß, IL-6 and IL-8 as well as vascular endothelial growth factor (VEGF) in PDLFs. Resveratrol (10 nM) or N-acetylcysteine (NAC, 20 mM) diminished the transcriptional activity of hypoxiainducible factor-1 and hypoxia-induced expression of VEGF. Combined treatment with mechanical compression and hypoxia significantly increased the expression levels of IL-1ß, IL-6, IL-8, TNF-α and VEGF in PDLFs. These levels were suppressed by NAC and resveratrol. The maxillary first molars of rats were moved mesially for seven days using an orthodontic appliance. NAC decreased the amount of orthodontic tooth movement compared to the vehicle-treated group. The results from immunohistochemical staining demonstrated that NAC suppressed the expression of IL-1ß and TNF-α in the periodontal ligament tissues compared to the vehicle-treated group. These results suggest that antioxidants have the potential to negatively regulate the rate of orthodontic tooth movement through the down-regulation of pro-inflammatory cytokines in the compression sides of periodontal ligament tissues.

Synthetic peptide-coated bone mineral for enhanced osteoblastic activation in vitro and in vivo.

A 15-mer synthetic peptide, designated P1, was derived from the bone morphogenetic protein (BMP) receptor I and BMP receptor II binding domains of BMP-2 for the purpose of enhancing bone regeneration capacity of inorganic bovine bone mineral. A second peptide, denoted P2, was designed by adding seven glutamic acid residues to the N-terminal of P1 to increase the surface coating efficiency onto bone mineral. The coating efficiency of P1 increased with the concentration of peptide. P2 peptide, in contrast, had a higher coating efficiency at lower peptide concentrations. The peptides properly transduced intracellular signals properly via the Smad and ERK pathways, thereby increasing mineralization in vitro, implying that the peptides alone can induce osteoblastic differentiation. Adhesion of cells to bone mineral was greater when peptides were present than in bone mineral alone. P1- and P2-coated bone mineral increased osteoblastic differentiation, as demonstrated by ALPase activity. P1-coated bone mineral stimulated more new bone regeneration in bone defect sites after 2 weeks than the peptide-free control. These peptides, in combination with bone grafts or implants, have the potential to enhance osteoblastic differentiation and bone regeneration.