Competitive Hybridization of a Microarray Identifies CMKLR1 as an Up-Regulated Gene in Human Bone Marrow-Derived Mesenchymal Stem Cells Compared to Human Embryonic Fibroblasts.

Mesenchymal stem cells (MSCs) have been widely applied to the regeneration of damaged tissue and the modulation of immune response. The purity of MSC preparation and the delivery of MSCs to a target region are critical factors for success in therapeutic application. In order to define the molecular identity of an MSC, the gene expression pattern of a human bone marrow-derived mesenchymal stem cell (hBMSC) was compared with that of a human embryonic fibroblast (hEF) by competitive hybridization of a microarray. A total of 270 and 173 genes were two-fold up- and down-regulated with FDR < 0.05 in the hBMSC compared to the hEF, respectively. The overexpressed genes in the hBMSC over the hEF, including transcription factors, were enriched for biological processes such as axial pattern formation, face morphogenesis and skeletal system development, which could be expected from the differentiation potential of MSCs. CD70 and CD339 were identified as additional CD markers that were up-regulated in the hBMSC over the hEF. The differential expression of CD70 and CD339 might be exploited to distinguish hEF and hBMSC. CMKLR1, a chemokine receptor, was up-regulated in the hBMSC compared to the hEF. RARRES2, a CMKLR1 ligand, stimulated specific migration of the hBMSC, but not of the hEF. RARRES2 manifested as ~two-fold less effective than SDF-1α in the directional migration of the hBMSC. The expression of CMKLR1 was decreased upon the osteoblastic differentiation of the hBMSC. However, the RARRES2-loaded 10% HA-silk scaffold did not recruit endogenous cells to the scaffold in vivo. The RARRES2−CMKLR1 axis could be employed in recruiting systemically delivered or endogenous MSCs to a specific target lesion.

4-Hexylresorcinol Treatment before Degumming Increases the ß-Sheet Structure of Silk Sericin and BMP-2 Expression in RAW264.7 Cells.

Silk sericin is a degumming product used by the silk industry. The degumming process can affect the protein structure and molecular weight of silk sericin. The present study examined how pretreatment with 4-hexylresorcinol (4HR) affects the biomedical properties of silk sericin. Before the degumming process, silkworm cocoons were treated with 4HR solution. The protein structure of the final degumming product was evaluated by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy. Untreated silk sericin (S) and silk sericin pretreated with 4HR (S+4HR) were added to RAW264.7 cells, and the expression of BMP-2 was determined. The bone-regenerating capacity of S+4HR was evaluated using the critical-sized rat calvarial defect model. Compared with S, S+4HR showed an increase in ß-sheet structures. Administration of S+4HR to RAW264.7 cells increased expression of BMP-2, mainly via the TLR-mediated signaling pathway. Bone volume, as measured by micro-computerized tomography, was significantly greater in the S+4HR group than in the S, gelatin alone, and unfilled control groups (p < 0.05 each). Expression of BMP-2 and runx2 in tissue specimens was significantly higher following treatment with S+4HR than with S (p < 0.05). Taken together, these findings show that 4HR pretreatment before the degumming process increased the ß-sheet structure of silk sericin, as well as inducing BMP-2 expression and bone regeneration ability.

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.

Oxya chinensis sinuosa (OC) Extracts Protects ARPE-19 Cells against Oxidative Stress via Activation of the Mitogen-Activated Protein Kinases (MAPKs)/ Nuclear Factor-κB (NF-κB) Pathway.

Oxya chinensis sinuosa (OC) is a well-known edible insect. Several researches on the health benefits of OC consumption have been performed to date; however, their effect on eye health remains largely unknown. This study aimed to assess the protective effects of OC extracts on the oxidative stress on the retinal pigment epithelium (RPE) cells. Oxidative damage has been identified as one of the key regulatory factors in age-related macular degeneration. H2O2-induced reactive oxygen species (ROS) production, a well-known oxidative stress factor, can cause cell death in retinal pigment epithelia cells. In this study, we found that three OC extracts effectively prevented H2O2-induced ROS production and subsequent death of ARPE-19 cells in a dose-dependent manner. In addition, the OC extracts inhibited the phosphorylation of mitogen-activated protein kinases including p38, JNK, and ERK. The OC extracts restored IκBα degradation induced by H2O2, indicating that OC extracts suppressed the activation of nuclear factor-κB. Furthermore, the three OC extracts were shown to have antioxidant effects by up-regulating the intracellular expression of key antioxidant proteins such as SOD, NQO, and HO-1. Here we demonstrated the antioxidant and anti-apoptotic effects of the OC extracts on ARPE-19, indicating their potential role in improving eye health. These results suggest that three OC extracts plays a critical role in oxidative stress-induced cell death protects in ARPE-19 cells.

Erratum to: Oxya chinensis sinuosa (OC) Extracts Protects ARPE-19 Cells against Oxidative Stress via Activation of the Mitogen-Activated Protein Kinases (MAPKs)/Nuclear Factor-κB (NF-κB) Pathway.

[This corrects the article DOI: 10.5851/kosfa.2024.e15.].

Cisplatin and 4-hexylresorcinol synergise to decrease metastasis and increase survival rate in an oral mucosal melanoma xenograft model: a preliminary study.

The present study was undertaken to examine the effects of cisplatin plus 4-hexylresorcinol (4-HR) combination therapy on oral mucosal melanoma (OMM) using cultured primary OMM cells in a tumour xenograft model. Cultured primary OMM cells were used for the MTT assay and DNA microarray. OMM cells were implanted into the submandibular glands of nude mice. The mice were then treated with cisplatin only or cisplatin plus 4-HR. Tumour size changes, survival rate and tumour metastasis were compared between the two groups by observation, micro-positron emission tomography (PET) and histological examination. In the MTT assay, the cisplatin plus 4-HR group showed significantly higher inhibition of OMM cell growth compared to the other groups (p<0.05). DNA microarray results showed significant inhibition of matrix metalloproteinase (MMP)-2 gene expression upon 4-HR application. The necropsy and micro-PET results showed that the mice from the cisplatin-only group had more distant metastases than the mice from the cisplatin plus 4-HR combination group (p=0.002). MMP-2 expression was lower in the primary tumours in the cisplatin plus 4-HR combination group than in the cisplatin-only group (p<0.001). Overall survival was longer in mice from the cisplatin plus 4-HR combination group than in the cisplatin-only group (p=0.049). In conclusion, the combined effect of cisplatin and 4-HR resulted in fewer metastases and longer survival than cisplatin-only treatment in the OMM xenograft model.

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.

The optimal scaffold for silk sericin-based bone graft: collagen versus gelatin.

BACKGROUND: Silk sericin is an active ingredient in bone grafts. However, the optimal scaffold for silk sericin has yet to be identified. METHOD: A critical-sized bone defect model in rat calvaria was used to evaluate bone regeneration. Silk sericin from Yeonnokjam, Bombyx mori, was incorporated into gelatin (group G, n = 6) and collagen (group C, n = 6). Bone regeneration was evaluated using micro-computed tomography (mCT) and histology. RESULTS: Group C showed a larger bone volume than group G in the mCT analysis (P = 0.001). Histological analysis showed a larger area of bony defects in group G than in group C. The bone regeneration area in group C was significantly larger than that in group G (P = 0.003). CONCLUSION: Compared with gelatin, collagen shows better bone regeneration in silk sericin-based bone grafts.

Protaetia brevitarsis seulensis larvae ethanol extract inhibits RANKL-stimulated osteoclastogenesis and ameliorates bone loss in ovariectomized mice.

Modulation of osteoclast formation could be a therapeutic target for inhibiting pathological bone destruction. The receptor activator of nuclear factor (NF)-κB ligand (RANKL) is known to be an essential factor in osteoclast differentiation and activation inducers. However, whether Protaetia brevitarsis seulensis (P. brevitarsis) larvae-a traditional animal-derived medicine used in many Asian countries-can inhibit RANKL-induced osteoclast formation and prevent ovariectomy (OVX)-induced bone loss has not been evaluated. Here, we aimed to investigate the anti-osteoporotic effects of P. brevitarsis larvae ethanol extract (PBE) in RANKL-stimulated RAW264.7 cells and OVX mice. In vitro, PBE (0.1, 0.5, 1, and 2 mg/mL) decreased RANKL­induced tartrate-resistant acid phosphatase (TRAP) activity and expression of osteoclastogenesis-associated genes and proteins. Furthermore, PBE (0.1, 0.5, 1, and 2 mg/mL) significantly inhibited the phosphorylation of p38 and NF-κB. Female C3H/HeN mice were divided into five groups (n = 5 per group), namely, sham-operated, OVX, OVX+PBEL (100 mg/kg, oral gavage), OVX+PBEH (200 mg/kg, oral gavage), and OVX+estradiol (0.03 µg/day, subcutaneous injection). High doses of PBE significantly increased femoral bone mineral density (BMD) and bone volume/tissue volume (BV/TV), whereas femoral bone surface/bone volume (BS/BV) and osteoclastogenesis-associated protein expression decreased compared to those in the OVX group. Moreover, PBE (200 mg/kg) significantly increased estradiol and procollagen type I N-terminal propeptide and decreased N-terminal telopeptide of type I collagen and C-terminal telopeptide of type I collagen compared to those in the OVX group. Our results suggest that PBE can be an effective therapeutic candidate for preventing or treating postmenopausal osteoporosis.

Effect of Extraction Ingredients on the Conformation and Stability of Silk Sericin (SS).

Silk sericin (SS) has different physicochemical properties depending on the extraction technique. In this study, SS was isolated in the presence of ingredients, including 5 to 10% ethanol (EtOH) and 5 to 10% glycine. Furthermore, temperature conditions of 80 °C, 100 °C, and 120 °C were used for 1, 3, and 5 h to evaluate the extraction rates. The extraction, gelation, structural, and cytotoxicity properties of SS extracted under different conditions were investigated. Extraction at 100 °C and 120 °C were found to have the highest SS yield, with 80 °C being the lowest. SS isolated at 100 °C and 120 °C for 1 and 3 h in water, and EtOH gelled at 4 °C in 2 to 3 days and 37 °C in 40 min. Glycine SS extracts were obtained at 100 °C and 120 °C for 1 h, gelled at 4 °C for 20 days and 37 °C for 16 h. SS was observed at 80 °C, with no gelation occurring. Glycine SS extracts obtained for 3, and 5 h at 120 °C showed no gelation. Circular dichroism (CD) results show glycine in SS induces α-helix and random coil structure. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC) were used to quantify the molecular weight distribution at 63 and 70 kDa, respectively. The MMT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) revealed no cytotoxicity in macrophage RAW 264.7 cells treated with this method SS; these findings present the significance and possibility of using selected extraction ingredients in SS that allow for the application of native SS at an initial extraction viscosity.

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.

Effects of Ultraviolet Light Irradiation on Silk Fibroin Films Prepared under Different Conditions.

Silk fibroin (SF)-based materials are exposed to both natural and artificial ultraviolet (UV) light during preparation or administration. However, the effects of UV irradiation on SF films prepared under different conditions have not yet been described in detail. In this study, four SF films with different molecular weight (MW) distribution were fabricated using SF solutions, which were prepared by dissolving degummed SF for 0.5-24 h. We observed UV (365 nm) irradiation on SF films induced the increase of yellowness and absorbance at 310 nm of SF films, indicating the formation of new photo-products and di-tyrosine bonds by photo-oxidation. Due to di-tyrosine cross-links between SF chains, UV-irradiated SF films were not fully dissociated in urea solution. In addition to formation of new products, UV reduced the crystallinity of SF films by breaking hydrogen bonds of ß-sheet conformation. Unlike the UV-induced decomposition of physical interactions, UV did not affect the covalent bonds (i.e., peptide bonds). Through these experiments, we could expect that SF with higher MW was more susceptible and SF with lower MW was more resistant to UV-induced photo-oxidation and photo-degradation. These results provide useful information about UV-induced aging of SF-based materials under natural sunlight and UV irradiating conditions.

Silk sericin application increases bone morphogenic protein-2/4 expression via a toll-like receptor-mediated pathway.

Bone morphogenic protein-2/4 (BMP-2/4) is an osteoinductive protein that accelerates osteogenesis when administered to bony defects. Sericin is produced by silkworms, and has a biological activity that differs depending on the degumming method used. Our results indicated that the high molecular weight fraction of silk sericin (MW > 30 kDa) obtained via sonication had a more abundant ß-sheet structure than the low molecular weight fraction. Administration of the ß-sheet structure silk sericin increased BMP-2/4 expression in a dose-dependent manner in RAW264.7 cells and human monocytes. This sericin increased the expression levels of toll-like receptor (TLR)-2, TLR-3, and TLR-4 in RAW264.7 cells. Application of a TLR-2 antibody or TLR pathway blocker decreased BMP-2/4 expression following sericin administration. In the animal model, the bone volume and BMP-2/4 expression were higher in rats treated with a sericin-incorporated gelatin sponge than in rats treated with a gelatin sponge alone or a sponge-incorporated with denatured sericin. In conclusion, sericin with a more abundant ß-sheet structure increased BMP-2/4 expression and bone formation better than sericin with a less abundant ß-sheet structure.

Comparison of methods for the repair of acute tympanic membrane perforations: Silk patch vs. paper patch.

We investigated the effects of repairing large tympanic membrane (TM) perforations in rats with a thin silk patch compared with the commonly used paper patch. We performed bilateral myringotomies of 1.8 mm in diameter on 50 adult Sprague-Dawley rats with intact TMs. The perforations in the right ears of 40 rats were treated with a silk patch, and the perforations in the left ears of the same rats were treated with a paper patch. Ten rats acted as controls. The mean healing times of the TM perforations on the silk-patch-treated ears and the paper-patch-treated ears were 7.2+/-1.48 and 9.1+/-1.11 days, respectively (control 10.38+/-1.70 days). The difference between silk-patch- and paper-patch-treated ears was statistically significant, with a mean difference of 1.9 days (0.6-4.5 days). The mean perforation closure times were significantly shorter in silk-patch- and paper-patch-treated ears than in the control animals. The endoscopic and histological findings of this study provide evidence that silk-patch treatment accelerates wound healing and shortens TM perforation closure time. We suggest that the silk patch may prove to be an effective material for repairing TM perforations in human patients in an outpatient clinical setting.

Effect of honey bee venom on microglial cells nitric oxide and tumor necrosis factor-alpha production stimulated by LPS.

Abnormal activation of microglial cells has been implicated in various neurodegenerative diseases. Results showed that venom (KBV) produced and purified in Korea regulated lipopolysaccharides (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in the murine microglia, BV-2 cell line. The production of proinflammatory cytokines, NO, and TNF-alpha was examined by LPS in BV-2 cell. The effect of KBV on the expression of inducible nitric oxide synthase (iNOS) and TNF-alpha was investigated by Western blot and RT-PCR in LPS-stimulated BV-2 cells. KBV suppressed the NO, iNOS, and TNF-alpha production, and decreased the levels of iNOS and TNF-alpha mRNA. These results suggest that KBV has anti-inflammatory properties that inhibit iNOS and TNF-alpha expression. KBV could be useful in inhibiting the production of inflammatory cytokine and NO production in neurodegenerative diseases. Further studies on the pharmacological aspects of the individual components of KBV are recommended.

Comparison of Bio-degradation for Ridge Preservation Using Silk Fibroin-based Grafts and a Collagen Plug.

A material for ridge preservation should have dimensional stability to resist bio-degradation. This study was designed to compare bio-degradation of ridge preservation materials. Collagen plug was used as a positive control. Untreated, ethanol-treated, and 4-hexylresorcinol (4HR)-treated silk plugs were used for the experimental group. Each material underwent a scanning electron microscopic exam and a Fourier transform infrared (FT-IR) spectroscopic exam. Bio-degradation was evaluated by analyzing cylindrical bony defects in rabbit tibias. There were no prominent differences in microstructure among the silk plug groups. FT-IR exam demonstrated that the ethanol- and 4HR-treated silk plug groups had enhanced ß-sheet structure. All silk plug groups exhibited significantly higher residual graft than the collagen plug group 4 weeks postoperative (p < 0.05). In conclusion, silk fibroin-based ridge preservation material was less bio-degradable than a collagen plug until at least 4 weeks after grafting.

Bone regeneration is associated with the concentration of tumour necrosis factor-α induced by sericin released from a silk mat.

To understand the osteogenic effect of the middle layer of the silk cocoon, sericin was examined for its cellular effects associated with tumor necrosis factor-α (TNF-α) signaling in this study. The fragmented sericin proteins in the silk mat were evaluated for the TNF-α expression level in murine macrophages. The concentration of protein released from silk mats was higher in the outermost and the innermost layers than in the middle layers, and the protein released from the silk mat was identified as sericin. The level of TNF-α in murine macrophages was dependent on the applied concentration of sericin, and the expression of genes associated with osteogenesis in osteoblast-like cells was dependent on the applied concentration of TNF-α. In animal experiments, silk mats from the middle layers led to a higher regenerated bone volume than silk mats from the innermost layer or the outermost layer. If TNF-α protein was incorporated into the silk mats from the middle layers, bone regeneration was suppressed compared with unloaded silk mats from the middle layers. Accordingly, silk mats from the silk cocoon can be considered to be a fragmented sericin-secreting carrier, and the level of sericin secretion is associated with TNF-α induction and bone regeneration.