Actively separated microneedle patch for sustained-release of growth hormone to treat growth hormone deficiency

Growth hormone deficiency (GHD) has become a serious healthcare burden, and presents a huge impact on the physical and mental health of patients. Here, we developed an actively separated microneedle patch (PAA/NaHCO3-Silk MN) based on silk protein for sustained release of recombinant human growth hormone (rhGH). Silk protein, as a friendly carrier material for proteins, could be constructed in mild full-water conditions and ensure the activity of rhGH. After manually pressing PAA/NaHCO3-Silk MN patch to skin for 1 min, active separation is achieved by absorbing the interstitial fluid (ISF) to trigger HCO3 ‒ in the active backing layer to produce carbon dioxide gas (CO2). In rats, the MN patch could maintain the sustained release of rhGH for more than 7 days, and produce similar effects as daily subcutaneous (S.C.) injections of rhGH in promoting height and weight with well tolerated. Moreover, the PAA/NaHCO3-Silk MN patch with the potential of painless self-administration, does not require cold chain transportation and storage possess great economic benefits. Overall, the PAA/NaHCO3-Silk MN patch can significantly improve patient compliance and increase the availability of drugs, meet current unmet clinical needs, improve clinical treatment effects of GHD patients.

Good cell compatibility and permeability of silk fibroin/chitosan composite scaffolds / 中国组织工程研究

BACKGROUND: Silk fibroin and chitosan are commonly used as scaffolds In tissue engineering, but there are some shortcomings In their separate application. When they are mixed, they can be modified each other. They give full play to each other’s advantages and become Ideal composite scaffolds. OBJECTIVE: To prepare Silk fibroin/chitosan composite scaffold and determineits properties. METHODS: The silk fibroin/chitosan composite scaffolds were prepared by freeze-drying method. The morphology and structure of the composite scaffolds were examined by scanning electron microscopy. The properties of the composite scaffolds were tested by thermogravlmetric analysis, mechanical properties test, and cytotoxicity test. The quaternion chitosan was prepared. The nuclear magnetic resonance spectrum was detected by nuclear magnetic resonance instrument. The potential and particle size distribution were detected by Zeta potentiometer. The protection of DNA was detected by gel electrophoresis. The binding with DNA was observed by transmission electron microscope. RESULTS AND CONCLUSION: (1 ) The results of scanning electron microscopy showed that the silk fibroin/chitosan composite scaffolds had a good three-dimensional pore structure, with a pore size of 50-100 urn. (2) The results of thermogravimetric analysis showed that when the temperature was less than 200 °C, the mass loss rate of silk fibroin/chitosan composite scaffold was lower. When the temperature Increased to 200-500 °C, the mass loss rate of the scaffold began to accelerate, and the loss amount increased. At 800 °C, the residual mass of the composite scaffold was 38%. (3) The maximum strain of silk fibroin/chitosan composite scaffold reached 94.94%, and the maximum stress was 7.01 MPa. (4) The results of CCK-8 experiment showed that silk fibroin/chitosan composite scaffolds had no cytotoxicity to rabbit bone marrow mesenchymal stem cells and had good cell compatibility. (5) The results of nuclear magnetic resonance spectra showed that the quaternion degree of quaternary ammonium chitosan was about 20%. (6) The particle size distribution of quaternized chitosan was (588.56±52.39) nm, and the surface of quaternized chitosan was positively charged with a potential of (16.3±3.92) mV, which was beneficial to the combination with DNA. (7) The results of gel electrophoresis experiments showed that the higher the proportion of quaternion chitosan, the better the encapsulation of DNA. When the ratio of chitosan/DNA was 1 : 3, the encapsulation effect was achieved. (8) The results of transmission electron microscopy showed that most of the particles of quaternized chitosan/DNA were solid and round; the particle size difference was small; and the average particle size was about 200 nm. (9) The results showed that silk fibroin/chitosan composite scaffolds had a good biocompatibility and cell permeability, which was conducive to the growth of cells between scaffolds.

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression / 中国天然药物

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

Functionalized genetic engineered silk-based biomaterials and their applications / 生物工程学报

Silk-based biomaterials are featured with excellent mechanical properties, good biocompatibility and biodegradability, which contribute to their potential applications in biomedical field. The current recognition of silk protein materials in structure and function provides a basic theory for the transformation of silk protein into new types of biomaterials. In addition, exogenous sequences encoding new peptide or structural domain can be inserted into the maternal gene sequences encoding silk proteins through genetic engineering technology to synthesize novel silk-based biomaterials with unique functions. This review summarizes the current trend and development perspective of genetically engineered functional silk-based materials for biomedical applications.

Construction of spider silk functional platform via intein trans-splicing / 生物工程学报

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.

Preparation of bilayer spider silk protein vascular scaffold and its biomechanical properties and cell compatibility / 医用生物力学

Objective To prepare a bilayer spider silk protein vascular scaffold using electrospinning, observe microstructure of the vascular scaffold and study its biomechanical properties and cell compatibility. Methods Spinning solution was electrospun to prepare (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) bilayer spider silk protein vascular scaffold using rotating receiving rod as the collection device. The effects of mass fraction and wall thickness on the porosity, bursting strength, tensile properties, suture retention strength and water permeability of the vascular scaffold were investigated, and cytotoxicity and cell adhesion property of the vascular scaffold were tested. Results The vascular scaffold presented three-dimensional porous microstructure with randomly distributed fibers. The bursting strength, tensile strength and suture retention strength were directly proportional to mass fraction and wall thickness, but the porosity, water permeability and elongation at break were inversely proportional to mass fraction and wall thickness. The bursting strength range of vascular scaffold was 43~183 kPa, which was higher than the physiological blood pressure; the suture strength was above 0.19 N, which was consistent with the transplantation requirement in vivo; the tensile strength was higher than that of human radial artery, which met the transplantation requirement in vivo; the range of water permeability was 0.3~0.6 mL•min-1•cm-2. The vascular scaffold had no cytotoxicity and facilitated the adhesion and proliferation of endothelial cells. Conclusions It is feasible to prepare the bilayer spider silk protein vascular scaffold through electrospinning. The superior biomechanical properties and biocompatibility properties show that the bilayer spider silk protein vascular can be used for construction of the tissue engineered blood vessels in vitro, with prospect for further vascular graft study, which lays a foundation for its clinical application.

Dietary effect of silk protein on epidermal levels of free sphingoid bases and phosphate metabolites in NC/Nga mice

In our previous studies, dietary supplements of silk protein, sericin, and fibroin, were beneficial for improving epidermal levels of ceramides, which are the major lipids for maintaining the epidermal barrier. In this study, we investigated the dietary effects of silk protein on epidermal levels of free sphingoid bases and their phosphates such as C18 sphingosine (So), C18 sphinganine (Sa), C18 sphingosine-1-phosphate (S1P), and C18 sphinganine-1-phosphate (Sa1P), which are either synthetic substrate or degradative metabolites of ceramides. Forty-five male NC/Nga mice, an animal model of atopic dermatitis (AD), were divided into three groups: group CA was an atopic control and fed a control diet, group S was fed a 1% sericin diet, and group F was fed a 1% fibroin diet. Fifteen male BALB/c mice served as group C (control group) and were fed the control diet. All mice were fed with diets and water ad libitum for 10 weeks. Sa in group CA was lower than that in group C, but So in group CA was similar to that in group C. So and Sa were higher in groups S and F than those in group CA; So level was even higher than that in group C, and Sa level was similar to that of group C. The So/Sa ratio in group CA, which is reported to increase in AD, was significantly higher than that of group C. The So/Sa ratio was lower in groups S and F than that in group CA, and decreased further in group F. However, S1P and Sa1P in groups S and F were similar to those in group CA. Taken together, we demonstrated that silk protein, sericin and fibroin dietary supplements, increased So and Sa levels, and decreased the So/Sa ratio.

Resistance to Toxoplasma gondii Infection in Mice Treated with Silk Protein by Enhanced Immune Responses

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4+ and CD8+ T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-gamma, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4+ and CD8+ T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.

Optimization of Fermentation Medium with Lactose as an Inducer for High Molecular Weight Recombinant Spider Silk Protein / 中国生物工程杂志

Based on M9 culture medium,the concentration of ingredients of culture medium was optimized for the fermentation of pNSR32/BL21(DE3),the engineering bacterial with spider silk protein,and lactose as an inducer.The condition of optimum culture medium was obtained for the expression of the high molecular weight recombinant spider silk protein by using orthogonal and individual factor experimental design.The result was showed that the optimum culture medium was consisted of 0.3% glycerol,3% yeast,0.75% tryptone,0.05%(NH_4)_2SO_4 and a little inorganic salt_.It was confirmed that the optimum culture medium will benefit the growth of bacterial and expression of recombinant spider silk protein.The production level of propose protein will attain at 20% of the total proteins in the fermentation.