Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo.

The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group (p = 0.044) and the AL group (p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering.

The effects of tetracycline-loaded silk fibroin membrane on proliferation and osteogenic potential of mesenchymal stem cells.

BACKGROUND: The main objective of this study was to investigate the effect of tetracycline-loaded silk fibroin membranes (TC-SFMs) on the proliferation and the osteogenic differentiation of human mesenchymal stem cells. MATERIALS AND METHODS: Four groups (0, 1, 5, and 10% concentration) of TC-SFMs were prepared for the experiments. We investigated cumulative tetracycline (TC) release profile for 7 d. Human gingiva-derived mesenchymal stem cells (GMSCs) were isolated from our previous study and seeded to the TC-SFMs. WST-8 assay (Cell Counting Kit-8; SigmaeAldrich Co, St. Louis, MO), staining of Phalloidin-FITC, and scanning electron microscope analyzed the cellular attachment and viability. Staining of Alizarin Red S (Sigma-Aldrich Co.) and osteogenic marker (osteocalcin) analyzed osteogenic differentiation. Additionally, quantitative polymerase chain reaction measured the expression of osteogenic lineage genes, including bone gamma-carboxyglutamic acid protein, bone sialoprotein, runt-related transcription factor 2, and collagen type I α1 according to TC concentration (0.05, 0.1, 0.25, and 0.5 mg/mL). RESULTS: The release of TC from TC-SFMs plateaued and neared completion in 24 h. Significantly higher viability was noted achieved in 1% and 5% TC-SFMs. The morphology of GMSCs on TC-SFMs at 0% and 1% concentration showed spindle shapes, but cells in 10% TC-SFMs appeared spheroid. During Alizarin Red S staining at 21 d of osteogenic differentiation, calcium and osteocalcin formation were significantly lower in the 10% TC-SFM group than in the 0, 1, and 5 groups. Compared with the control group, bone gamma-carboxyglutamic acid protein showed significantly low expression rate at TC concentration ≥0.05 mg/mL. Bone sialoprotein was low at TC concentration ≥0.1 mg/mL. Likewise, runt-related transcription factor 2 and collagen type I α1 were low at TC concentration of 0.5 mg/mL. CONCLUSIONS: Within the limits of this study, 1% and 5% TC-SFMs showed higher proliferation and osteogenic potential of GMSCs than 10% TC-SFM. Therefore, the use of 1% to 5% range of TC may be more suitable to silk fibroin membrane for stem cell tissue engineering.

Hydroxyapatite and silk combination-coated dental implants result in superior bone formation in the peri-implant area compared with hydroxyapatite and collagen combination-coated implants.

PURPOSE: The objective of this study was to compare bone formation after installation of uncoated (UC), hydroxyapatite-coated (HA), collagen plus HA-coated (CH), and silk plus HA-coated (SH) implants. MATERIALS AND METHODS: Implants in the UC group had acid-etched surfaces. Surface coating was applied using the aerosol deposition method. Cellular responses on the coated surfaces were examined with scanning electron microscopy. Cellular responses to the surfaces were studied with the corresponding coated discs and MG63 cells. Subsequently, 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphatase (ALP) assays were performed. Peri-implant bone formation was evaluated with the rabbit tibia model. Twenty-four implants from each group were installed. The animals were sacrificed 6 weeks after implant installation. Peri-implant bone formation and implant-to-bone contact were measured in histologic sections. Significance of differences across groups was evaluated using analysis of variance. RESULTS: Scanning electron microscopic images showed that the CH and SH groups exhibited cells that appeared more spread out than those in the other groups. The SH group exhibited the highest value in the MTT assay. The CH group exhibited the highest level of ALP activity. Comparisons of these modifications with the acid-etched surfaces showed that the CH and SH groups displayed significantly greater peri-implant bone formation (P < .001). CONCLUSION: The SH group displayed significantly greater new bone formation and bone-to-implant contact than did the other groups.

Comparison of silkworm-cocoon-derived silk membranes of two different thicknesses for guided bone regeneration.

The objective of this study was to compare the effectiveness of silk membranes (SMs) of different thicknesses for guided bone regeneration. Two kinds of SMs were prepared (SM1: 0.01 mm thickness, SM2: 0.5 mm thickness). Before use in animal experiments, scanning electron microscope images were taken to examine the gross morphology of each membrane. Ten New Zealand white rabbits were used for this study. Bilateral round-shaped defects were created in the parietal bone (diameter: 8.0 mm) and each defect was covered with SM1 or SM2. Animals were killed at 4 weeks and 8 weeks. Bone regeneration was analyzed in each specimen by micro-computed tomography (µ-CT) and histological analysis. In the µ-CT analysis, the average amount of newly formed bone in the SM2 group was greater than that in the SM1 group. There was a significant difference at 4 weeks after surgery (P = 0.004). In the histological analysis, the amount of formed lamellar bone was much greater in the SM2 group than in the SM1 group at 8 weeks after surgery (P = 0.021). In conclusion, the thick SM was much more effective for bone regeneration of bone defects than the thin SM.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

The objective of this study was to demonstrate that a silk fibroin (SF) and 4-hexylresorcinol (4-HR) incorporation membrane could be used for a guided bone regeneration technique. Fourier transform infrared measurements were obtained to determine change of physical property of SF membrane by 4-HR incorporation. Two peri-implant defects, 3.0 × 5.0 mm (width × length), were prepared on the lateral side of the implant hole in the tibia of New Zealand white rabbits (n = 8). The peri-implant defect was left unfilled in the control group. Silk fibroin + 4-HR membrane was applied to the peri-implant defect in the experimental group. The 8 animals were killed at 8 weeks after implantation. Subsequently, removal torque test and histomorphometric evaluation were done. Fourier transform infrared spectroscopy showed no specific chemical interaction between 4-HR and SF. In the histomorphometric analysis, the mean bone regeneration was 18.3 ± 1.9 mm(2) in the experimental group and 9.3 ± 0.9 mm(2) in the control group (P = 0.004). In conclusion, the SF and 4-HR incorporation membrane successfully regenerated bone in the rabbit tibia peri-implant bone defect model.

Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new bone substitute.

PURPOSE: This study involves a comparison between the bone regeneration of nano-hydroxyapatite (nHA), as derived from eggshells either with or without silk fibroin scaffolds, and the unfilled control in the rabbit calvarial bony defect model. MATERIALS AND METHODS: Sixteen 4-month-old New Zealand white rabbits, with a mean weight of 2.8 kg (range, 2.5-3.0 kg), were used in this experiment. After the formation of bilateral parietal bony defects (diameter, 8.0 mm), either an nHA or an nHA+silk fibroin combination (nHA+silk) was grafted. The control was unfilled defect. The bone regeneration was evaluated by micro-computed tomography (µCT) and histomorphometric analyses at 4 and 8 weeks. RESULTS: All measured variables of the µCT analysis were significantly higher in the grafted groups (nHA and nHA+silk) than in the unfilled control groups at both 4 and 8 weeks after operation (P < .05). On histomorphometric analysis, there was no significant difference between the groups at 4 weeks after operation. However, the nHA group exerted significantly higher bone regeneration (40.16% ± 8.27%) compared with the unfilled control group (25.66% ± 10.98%) or the nHA+silk group (16.62% ± 3.05%) (P < .05). CONCLUSION: The nHA from eggshells exerted better bone formation than the unfilled control group on both µCT and histomorphometric analyses. Considering the rapid healing in bony defect and easy availability, the nHA from the eggshells could prove to be a good new bone substitute.

Accelerated biodegradation of silk sutures through matrix metalloproteinase activation by incorporating 4-hexylresorcinol.

Silk suture material is primarily composed of silk fibroin and regarded as a non-resorbable material. It is slowly degraded by proteolysis when it is implanted into the body. 4-Hexylresorcinol (4HR) is a well-known antiseptic. In this study, the biodegradability of 4HR-incorporated silk sutures were compared to that of untreated silk sutures and polyglactin 910 sutures, a commercially available resorbable suture. 4HR-incorporated silk sutures exhibited anti-microbial properties. Matrix metalloproteinase (MMP) can digest a wide spectrum of proteins. 4HR increased MMP-2, -3, and -9 expression in RAW264.7 cells. MMP-2, -3, and -9 were able to digest not only silk fibroin but also silk sutures. Consequently, 59.5% of the 4HR-incorporated silk suture material remained at 11 weeks after grafting, which was similar to that of polyglactin 910 degradation (56.4% remained). The residual amount of bare silk suture material at 11 weeks after grafting was 91.5%. The expression levels of MMP-2, -3 and -9 were high in the 4HR-incorporated silk suture-implanted site 12 weeks after implantation. In conclusion, 4HR-treated silk sutures exhibited anti-microbial properties and a similar level of bio-degradation to polyglactin 910 sutures and induced higher expression of MMP-2, -3, and -9 in macrophages.

Uncommon presentation of potential medication-related osteonecrosis of the jaw.

BACKGROUND: This article presents a patient with potential atypical medication-related osteonecrosis of the jaw and reviews related literatures. CASE PRESENTATION: A 52-year-old male showed pain in the left buccal area and had numbness on the left lower lip area. He received medications having anti-angiogenic effect for 4 years. He did not receive irradiation of the jaw regions. In histological view, most of the adipocytes were destroyed and disappeared in the scanty vascular marrow tissue, resulting in the replacement of the fatty necrosis with variable sized vacuolated empty spaces. In the immunohistochemistry analysis, the infiltrated macrophages into the marrow stromal tissue were strongly positive for lysozymes. These findings demonstrate that the presented osteonecrosis underwent a chronic and persistent granulomatous inflammatory reaction. CONCLUSIONS: We conclude that the present case might have been caused by anti-angiogenic drug abuse, affecting the reduction of the mandibular marrow vascularity and subsequently inducing fatty necrosis and an extensive osteolytic change of the mandible.

Novel fabrication of fluorescent silk utilized in biotechnological and medical applications.

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine.

A novel degradable polycaprolactone networks for tissue engineering.

Polycaprolactone (PCL) macromer was obtained by the reaction of PCL diol with acryloyl chloride and confirmed using Fourier transform infrared and nuclear magnetic resonance spectrometer. Novel degradable PCL networks were prepared through photopolymerization of the PCL macromer. Thermal, mechanical, and morphological characteristics as well as degradability and biocompatibility of the PCL networks were investigated. Differential scanning calorimetry showed that the melting temperature and the calculated weight average crystallinity of PCL networks were decreased with a decrease of molecular weight of PCL diols due to the increased crosslinking density. Thermal stability of PCL networks was higher than that of PCL diols. PCL networks showed faster degradation, and higher compressive modulus and compressive recovery ratios than those of PCL itself because of their low crystallinity and the modification of terminal groups. The porosity of the PCL networks can be controlled by the amounts and size of porogen used. MG-63 osteoblast cell was attached and proliferated on PCL networks. PCL networks therefore may have considerable potential as scaffold for tissue engineering.

Biodegradation behavior of silk fibroin membranes in repairing tympanic membrane perforations.

Silk fibroin (SF) from silkworms has been widely studied as a biomaterial. The degradation behavior of silk biomaterials is important for medical applications, but few studies have examined long-term degradation behavior in vivo. In this study, we investigated the degradation behavior of SF membranes in vitro and in vivo. For the in vitro assay, we observed degradation of silk membranes in phosphate buffered saline, culture media, and an enzyme (proteinase K) solution. In the proteinase K solution, 80% of the silk membranes degraded within 10 days. Silk membranes exhibited no cytotoxicity toward L929 cells and rat tissues. To investigate the degradation of silk membranes in vivo, they were implanted subcutaneously in rats and harvested 19 months after surgery. Scanning electron microscopy imaging and histological analysis of silk membrane explants showed that they broke into several pieces after 16 months. Results show that silk membranes are biocompatible and display excellent long-term degradation behavior when used as biomaterials.

Accelerated healing with the use of a silk fibroin membrane for the guided bone regeneration technique.

OBJECTIVE: The purpose of this study was to evaluate the bone regeneration ability of silk fibroin (SF) membrane. STUDY DESIGN: Fourier-transform infrared (FT-IR) and solubility test against distilled water were performed with 3 different types of SF membrane (SM1, SM2, and SM3). Subsequently, microscopic computerized tomography (µ-CT) and histomorphometric analyses were performed in rabbit calvarial defect model after SF membrane application at 4 and 8 weeks after surgery. RESULTS: FT-IR showed that the conformation of the SF membrane was a random coil structure and that SM1 was the least soluble. When SM1 was used in the animal model, the groups with SM1 had significantly higher new bone formation than the uncovered control in both the µ-CT and the histomorphometric analyses (P < .05). CONCLUSIONS: The SF membrane had more new bone formation compared with the uncovered control.

Restoration of peri-implant defects in immediate implant installations by Choukroun platelet-rich fibrin and silk fibroin powder combination graft.

OBJECTIVE: The objective of this study was to determine the capability of silk fibroin powder as a biomaterial template for the restoration of peri-implant defects when mixed with Choukroun platelet-rich fibrin (PRF) in vivo. STUDY DESIGN: Ten New Zealand white rabbits were used for this study. Using a trephine bur (diameter 7.0 mm), 2 monocortical defects were prepared. Subsequently, 2 dental implants were installed into the tibia (diameter 3.0 mm, length 10.0 mm). In the experimental group, the peri-implant defect was filled with a combination graft of silk fibroin powder and Choukroun PRF. The control was left in an unfilled state. The animals were killed at 8 weeks. Subsequently, a removal torque test and a histomorphometric analysis were done. RESULTS: The removal torque for the experimental group was 30.34 +/- 5.06 N.cm, whereas it was 21.86 +/- 3.39 N.cm for the control. The difference between the 2 groups was statistically significant (P = .010). Mean new bone formation was 51.93 +/- 27.90% in the experimental group and 11.67 +/- 15.12% in the control (P = .003). Mean bone-to-implant contact was 43.07 +/- 21.96% in the experimental group and 15.37 +/- 23.84% in the control (P = .002). CONCLUSION: A peri-implant defect can be successfully repaired by the application of Choukroun PRF and silk fibroin powder.

Semi-interpenetrating polymer networks composed of silk fibroin and poly(ethylene glycol) for wound dressing.

Semi-interpenetrating polymer networks (SIPNs) composed of silk fibroin (SF) and poly(ethylene glycol) (PEG) were prepared by photopolymerization of a PEG macromer in the presence of SF to improve the mechanical properties of SF sponge as wound dressing. The morphological structure of the SF/PEG SIPNs was observed to be composed of an interconnected microporous surface and a cross-sectional area. SF/PEG SIPNs showed non-cytotoxicity evaluated by a cell proliferation method using L929 fibroblasts. Wound contraction treated with SF/PEG SIPNs sponges was faster than that of Vaseline gauze as a control. Histological observation confirmed that the deposition of collagen in the dermis was organized by covering the wound area with SF/PEG SIPNs. The above results indicated that SF/PEG SIPNs could be used as wound dressing.