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.

Skin sensitization study of bee venom (Apis mellifera L.) in guinea pigs and rats.

BACKGROUND: Bee venom (Apis mellifera L., BV) possessing a rich source of pharmacologically active substances has the potential to be used as a cosmetic ingredient for antiaging, antiinflammatory and antibacterial functions. The aim of this study was to assess the skin sensitization of BV on experimental animals using the Buehler test. MATERIALS AND METHODS: Guinea pigs were randomly allocated into three groups of BV-sensitization, positive control-sensitization, and ethyl alcohol-sensitization group for induction and challenge. On the other hand, two groups of rats were administered with BV at doses of 0 and 1500 mg/kg. Clinical signs, mortality and body weight changes were continually monitored during the study period. RESULTS: No treatment-related clinical signs or body weight changes were observed in both animal models. The average skin reaction evaluated by erythema and edema on the challenge sites, and sensitization rate in the BV-sensitization group of guinea pigs were substantially low compared with those in positive control group, representing a negligible sensitizing potential of BV. CONCLUSION: It was concluded that BV was well tolerated and exhibited no dermal irritation potential in guinea pigs and rats. Our findings may provide a developmental basis of BV for a cosmetic ingredient or external application for topical uses.

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.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

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 bee venom on transforming growth factor-beta1-treated hepatocytes.

Bee venom (BV) has been used as treatment against a wide variety of ailments, including inflammatory diseases. Various studies have demonstrated anti-inflammatory and anticancer effects of BV. Transforming growth factor (TGF)-beta1 induces hepatocyte apoptosis via the mitochondrial permeability transition. However, there is no evidence or information regarding the antiapoptotic effect of BV on hepatocytes. The authors investigated the antiapoptotic effect of BV on TGF-beta1-treated hepatocytes. The results showed significant protection from DNA damage by BV treatment compared to corresponding TGF-beta1-treated hepatocytes without BV. BV suppressed TGF-beta1-induced activation of the bcl-2 family and caspase family of proteins, which resulted in inhibition of poly ADP-ribose polymerase (PARP) cleavage. Furthermore, BV is not cytotoxic in the low concentrations used in this study. Low concentrations of BV potently suppress the apoptotic response in TGF-beta1-treated hepatocytes; therefore, BV may have therapeutic potential for the treatment of liver diseases.

Erratum to: Milk Containing BF-7 Enhances the Learning and Memory, Attention, and Mathematical Ability of Normal Persons.

[This corrects the article DOI: 10.5851/kosfa.2009.29.2.278.].

Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression.

Cancer cells, characterized by local invasion and distant metastasis, are very much dependent on the extracellular matrix. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we reported the effects of disulfiram, a clinically used anti-alcoholism drug, on tumor invasion suppression, as well as its effects on the activity of MMP-2 and MMP-9 in human osteosarcoma cells (U2OS). Disulfiram has been used for alcohol aversion therapy. However, recent reports have shown that disulfiram may have potential in the treatment of human cancers. Herewith, we showed that the anti-tumor effects of disulfiram, in an invasion assay using U2OS cells and that disulfiram has a type IV collagenase inhibitory activity that inhibits expression of genes and proteins responsible for both cell and non-cell mediated invasion on pathways. In conclusion, disulfiram inhibited expression of MMP-2 and MMP-9 and it regulated the invasion of human osteosarcoma cells. These observations raise the possibility of disulfiram being used clinical for the inhibition of cancer invasion.

Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat.

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 microgram/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.

Comparison of unprocessed silk cocoon and silk cocoon middle layer membranes for guided bone regeneration.

BACKGROUND: Silk cocoon is composed of multiple layers. The natural silk cocoon containing all layers was cut as a rectangular shape as defined as total group. The inner and outermost layers were removed from the total group and the remained mat was defined as the middle group. The objectives of this study was to compare the total group with the middle group as a barrier membrane for the guided bone regeneration. METHODS: The effects of these materials on the cellular proliferation and alkaline phosphatase (ALP) expression of MG63 cells were explored. For comparing bone regeneration ability, bilateral bone defects were created in calvarial areas in ten adult New Zealand white rabbits. The defects were covered with silk membranes of the middle group, with silk membrane of the total group used as the control on the contralateral side. The defects were allowed to heal for 4 and 8 weeks. Micro-computerized tomography (µCT) and histological examination were performed. RESULTS: The middle group exhibited a higher MTT value 48 and 72 h after treatment compared to the total group. ALP expression was also higher in the middle group. The results of µCT and histologic examination showed that new bone formation was significantly higher in the middle group compared to the total group 8 weeks postoperatively (P < 0.05). CONCLUSIONS: In conclusion, the middle layer of the silk cocoon supports guided bone regeneration better than unprocessed silk cocoon.

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats.

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10-mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p<0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p<0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p<0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury.

Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway.

The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca(2+)-activated K(+) channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21(Cip1) in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.

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.

The role of formic acid in solution stability and crystallization of silk protein polymer.

In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1-2 days of storage times. The beta-sheet crystallization of SF molecules was occurred by the elimination of formic acid upon drying. The SF molecules in formic acid solution are stable and have low hydrodynamic radius values. This may be closely related to the fact that formic acid has two opposite functions of dissolution and crystallization simultaneously. The turbidity, dynamic light scattering and FTIR measurements elucidate that the solution stability and crystallization of SF are attributed to compact molecular shape of SF in formic acid, resulted from the molecular interactions between formic acid and polar groups in SF molecules.

Preparation of semi-interpenetrating polymer networks composed of silk fibroin and poloxamer macromer.

A system was designed to utilize silk fibroin (SF) as a matrix for wound dressing. For this system, we prepared a sponge type of porous semi-interpenetrating networks (SIPNs) hydrogel composed of SF and poloxamer 407 macromer to enhance the mechanical and functional properties of SF. The thermal and mechanical properties of the hydrogels as well as their swelling behaviors were studied by means of differential scanning calorimetry, compressive modulus measurement, and gravimetric method, respectively. The morphology and crystalline structure of these SIPN hydrogels were also investigated by scanning electron microscopy (SEM) and wide-angle diffractometry, respectively. Conformational change of SF from random coil to beta-sheet structure was accelerated by formation of SIPNs with poloxamer. The melting temperature of poloxamer in the SIPNs decreased due to the prevention of crystallization by the incorporation of SF. The mechanical strength of SIPNs hydrogel was much higher than those of SF itself or SF/poloxamer blend and increased with the poloxamer content. The equilibrium water content of SF was remarkably increased by formation of SIPNs with poloxamer due to the hydrophilicity of poloxamer. The crystallinity and morphology of SIPNs hydrogel were affected by SIPNs hydrogel composition.

Preparation of self-assembled silk sericin nanoparticles.

Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the conformational change from random coil to beta-sheet. DSC thermogram of sericin-PEG conjugate suggests that mutual miscibility between PEG and SS chains was poor. Nanoparticles of sericin-PEG conjugate with sizes measured by dynamic light scattering ranging about 200-400 nm in diameter, were prepared by the diafiltration method. Shape of sericin-PEG conjugate nanoparticles observed by scanning and transmission electron microscopes was spherical. The results suggest that sericin-PEG conjugates are self-associated to form spherical nanoparticles through hydrophobic interaction.

Wet spinning of silk polymer. I. Effect of coagulation conditions on the morphological feature of filament.

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The effect of coagulation conditions, such as coagulant type and coagulation temperature, was investigated on the morphological feature of SF filaments and a theoretical approach was also performed to understand the coagulation phenomena. SEM observation revealed that as the R group size of alcoholic coagulant (ROH) increased, the cross-sectional shape deviated more from a circular form. This is attributed to the fact that as the R group size increased, the mass transfer rate difference increased, but the coagulation rate decreased due to a reduced diffusion rate. Most non-alcoholic coagulants exhibited this circular cross-sectional shape, except dioxane, which showed a clover shape. As the coagulation bath temperature increased, the cross-section deviated less from a circular form, with the reduction of fiber diameter. When methanol/water mixture was used as a coagulant, an ellipse or a dog-bone shape was obtained with higher water content in methanol, which was attributed to the decrease of coagulation rate. Although methanol exhibited a positive value of mass transfer rate difference, a circular shape of cross-section was obtained due to the density difference of the coagulated and uncoagulated parts in the coagulating SF filament.

Wet spinning of silk polymer. II. Effect of drawing on the structural characteristics and properties of filament.

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The rheological behavior of the SF dope solution prepared with formic acid was examined and the drawing effect on the structural characteristics and mechanical properties of SF filament was comparatively studied with those of natural silk fiber. SF dope exhibited shear thinning, but, as the dope concentration increased, the effect of shear thinning decreased, an indication that a higher concentration of dope solution will result in good spinnability. Wet-spun SF filaments exhibited a uniform and circular cross-sectional shape and dense morphology under SEM observation. X-ray diffraction (XRD) results revealed that the crystallinity of wet-spun regenerated filaments was hardly affected by the draw ratio, whereas the crystalline and amorphous orientation of regenerated SF filament showed different features depending on the drawing. The crystalline orientation of regenerated filaments increased with an increase of draw ratio and was lower than that of natural silk fiber. On the contrary, the amorphous orientation was constant throughout 1X-5X draw ratios, after an abrupt increase at 1X, and was higher than that of natural silk fiber. These differences in the orientation behaviors are attributed to the different spinning mechanisms involved. The tensile property was strongly dependent on the draw ratio. The breaking strength and elongation of the regenerated filament at 5X draw ratio were 2.2 g/day and 17%, respectively.

Powdered Wound Dressing Materials Made from wild Silkworm Antheraea pernyi Silk Fibroin on Full-skin Thickness Burn Wounds on Rats.

PURPOSE: This study evaluated powdered burn wound dressing materials from wild silkworm fibroin in an animal model. METHODS: Fifteen rats were used in this experiment. Full-thickness 2×2 cm burn wounds were created on the back of rats under anesthesia. In the two experimental groups, the wounds were treated with two different dressing materials made from silkworm fibroin. In the Control Group, natural healing without any dressing material was set as control. The wound surface area was measured at five days, seven days and 14 days. Wound healing was evaluated by histologic analysis. RESULTS: By gross observation, there were no infections or severe inflammations through 14 days post-injury. The differences among groups were statistically significant at seven days and 14 days, postoperatively (P <0.037 and 0.001, respectively). By post hoc test, the defect size was significantly smaller in experimental Group 1 compared with the Control Group and experimental Group 2 at seven days postoperatively (P =0.022 and 0.029, respectively). The difference between Group 1 and Group 2 was statistically significant at 14 days postoperatively (P <0.001). Group 1 and control also differed significantly (P =0.002). Group 1 showed a smaller residual scar than the Control Group and Group 2 at 14 days post-injury. Histologic analysis showed more re-epithelization in Groups 1 and 2 than in the Control Groups. CONCLUSION: Burn wound healing was accelerated with silk fibroin spun by wild silkworm Antheraea pernyi. There was no atypical inflammation with silk dressing materials. In conclusion, silk dressing materials can be used for treatment of burn wound.

Inhibitory effects of bee venom on Propionibacterium acnes-induced inflammatory skin disease in an animal model.

Propionibacterium acnes (P. acnes) is a major contributing factor to the inflammatory component of acne. The many prescription medications for acne allow for a large number of potential combination treatments. However, several antibiotics, apart from their antibacterial effects, exert side­effects, such as the suppression of host inflammatory responses. Purified bee venom (BV) is a natural toxin produced by honeybees (Apis mellifera L.). BV has been widely used as a traditional medicine for various diseases. In the present study, to investigate the therapeutic effects of BV against P. acnes-induced inflammatory skin disease, P. acnes was intradermally injected into the ears of mice. After the injection, BV was applied to the skin surface of the right ear. Histological observation revealed that P. acnes induced a considerable increase in the number of infiltrated inflammatory cells. However, treatment with BV markedly reduced these reactions compared with the P. acnes-injected mice not treated with BV. Moreover, the expression levels of tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß were significantly reduced in the BV-treated mice compared with the untreated P. acnes-injected mice. In addition, treatment with BV significantly inhibited Toll-like receptor (TLR)2 and CD14 expression in P. acnes-injected tissue. The binding activity of nuclear factor-κB (NF-κB) and activator protein (AP)-1 was markedly suppressed following treatment with BV. The results from our study, using an animal model, indicate that BV exerts an inhibitory effect on inflammatory skin diseases. In conclusion, our data indicate that BV has potential for use as an anti-acne agent and may be useful in the pharmaceutical and cosmetics industries.