Preparation, physicochemical characterization and cytocompatibility study of self-assembled silk fibroin nanoparticles / 药学学报

This study aimed to prepare silk fibroin nanoparticles (SF-NPs) and assess the physicochemical properties and biocompatibility of the formulation. An optimized and simplified solvent displacement method was employed to obtain SF-NPs. Single-factor prescription screening, such as silk fibroin (SF) solution concentration, the ratio of SF solution to organic solvent, ultrasonication power and time, and different types of organic phases, was used to optimize the formulation. The characterization of the optimal formulation included particle size, polydispersity index (PDI), zeta potential, morphology, and stability. The in vitro cell compatibility of the nanoparticles was evaluated using CCK-8 and Calcein-AM/PI cell viability staining. The results showed that when SF concentration was 20 mg·mL-1, volume ratio of aqueous phase to acetone was 1∶6, ultrasonic power was 80 W and ultrasonic time was 3 min, the best SF-NPs was obtained. The nanoparticles prepared in this study exhibit a near-spherical shape, with a uniform size distribution, having an average size of 144.8 nm, a PDI of 0.174, and a zeta potential of -27.35 mV. Results from in vitro cell experiments demonstrate excellent cell compatibility of SF-NPs, showing the ability to promote cell proliferation. The SF-NPs which were successfully prepared in this study exhibit uniform particle size and excellent biocompatibility.

Preparation and Performance of Three-Dimensional Silk FibroinScaffolds with Different Matrix Stiffness / 医用生物力学

Objective After hydrogen bonding between collagen ( COL) and silk fibroin ( SF ) at different concentrations, a composite scaffold with adjustable stiffness was prepared by combining with gel system, and its physical and chemical properties were characterized. Methods SF with different qualities was dissolved in sodium alginate (SA) solution, then COL solution at different concentration and calcium carbonate ( CaCO3 ) powder were added. The hydrogels of SC1, SC2, and SC3 groups were obtained by taking out the mixed solution and adding some gluconic acid lactone ( GDL) powder, and different SF scaffolds were obtained after freeze drying. Results The SF scaffolds with adjustable stiffness were successfully prepared. The compression moduli of SC1, SC2, and SC3 groups were (17. 31±2. 73), (24. 12±1. 81), (32. 54±1. 81) kPa, respectively. The innerstructure of the scaffolds was observed. From SC1 group to SC3 group, pores of the scaffolds were smaller and fewer, and hydrophilicity of the materials become better and better. Conclusions Three-dimensional ( 3D) porous scaffolds with different matrix stiffness can be prepared by changing the concentration of SF and COL solution. The concentration of SF and COL is proportional to the compression modulus, water absorption, water retention and swelling rate of SF scaffolds, while inversely proportional to porosity. The findings of this study are expected to provide theoretical guidance for construction of scaffolds with appropriate matrix stiffness for inducing osteogenic differentiation of mesenchymal stem cells

Preparation of silk fibroin/hyaluronic acid composite hydrogel based on thiol-ene click chemistry / 浙江大学学报·医学版

OBJECTIVES@#To design and prepare silk fibroin/hyaluronic acid composite hydrogel.@*METHODS@#The thiol modified silk fibroin and the double-bond modified hyaluronic acid were rapidly cured into gels through thiol-ene click polymerization under ultraviolet light condition. The grafting rate of modified silk fibroin and hyaluronic acid was characterized by 1H NMR spectroscopy; the gel point and the internal microstructure of hydrogels were characterized by rheological test and scanning electron microscopy; the mechanical properties were characterized by compression test; the swelling rate and degradation rate were determined by mass method. The hydrogel was co-cultured with the cells, the cytotoxicity was measured by the lactate dehydrogenase method, the cell adhesion was measured by the float count method, and the cell growth and differentiation on the surface of the gel were observed by scanning electron microscope and fluorescence microscope.@*RESULTS@#The functional group substitution degrees of modified silk fibroin and hyaluronic acid were 17.99% and 48.03%, respectively. The prepared silk fibroin/hyaluronic acid composite hydrogel had a gel point of 40-60 s and had a porous structure inside the gel. The compressive strength was as high as 450 kPa and it would not break after ten cycles. The water absorption capacity of the composite hydrogel was 4-10 times of its own weight. Degradation experiments showed that the hydrogel was biodegradable, and the degradation rate reached 28%-42% after 35 d. The cell biology experiments showed that the cytotoxicity of the composite gel was low, the cell adhesion was good, and the growth and differentiation of the cells on the surface of the gel were good.@*CONCLUSIONS@#The photocurable silk fibroin/hyaluronic acid composite hydrogel can form a gel quickly, and has excellent mechanical properties, adjustable swelling rate and degradation degree, good biocompatibility, so it has promising application prospects in biomedicine.

Pro-angiogenic photo-crosslinked silk fibroin hydrogel: a potential candidate for repairing alveolar bone defects

Abstract Objective: This study aimed to develop a pro-angiogenic hydrogel with in situ gelation ability for alveolar bone defects repair. Methodology: Silk fibroin was chemically modified by Glycidyl Methacrylate (GMA), which was evaluated by proton nuclear magnetic resonance (1H-NMR). Then, the photo-crosslinking ability of the modified silk fibroin was assessed. Scratch and transwell-based migration assays were conducted to investigate the effect of the photo-crosslinked silk fibroin hydrogel on the migration of human umbilical vein endothelial cells (HUVECs). In vitro angiogenesis was conducted to examine whether the photo-crosslinked silk fibroin hydrogel would affect the tube formation ability of HUVECs. Finally, subcutaneous implantation experiments were conducted to further examine the pro-angiogenic ability of the photo-crosslinked silk fibroin hydrogel, in which the CD31 and α-smooth muscle actin (α-SMA) were stained to assess neovascularization. The tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also stained to evaluate inflammatory responses after implantation. Results: GMA successfully modified the silk fibroin, which we verified by our 1H-NMR and in vitro photo-crosslinking experiment. Scratch and transwell-based migration assays proved that the photo-crosslinked silk fibroin hydrogel promoted HUVEC migration. The hydrogel also enhanced the tube formation of HUVECs in similar rates to Matrigel®. After subcutaneous implantation in rats for one week, the hydrogel enhanced neovascularization without triggering inflammatory responses. Conclusion: This study found that photo-crosslinked silk fibroin hydrogel showed pro-angiogenic and inflammation inhibitory abilities. Its photo-crosslinking ability makes it suitable for matching irregular alveolar bone defects. Thus, the photo-crosslinkable silk fibroin-derived hydrogel is a potential candidate for constructing scaffolds for alveolar bone regeneration.

The study on the tissue engineered bladder patch constructed with the double-layer silk scaffold and adipose-derived stem cells for bladder repair and reconstruction / 中华泌尿外科杂志

Objective:To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells (ADSCs) in the repair and reconstruction of bladder.Methods:This study was conducted from May 2020 to March 2021. The silk fibroin (SF) aqueous solution was obtained from silkworm cocoons, and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared. The rat ADSCs were isolated, cultured, and the ADSCs surface markers (CD29, CD90, CD45, CD106) were identified by flow cytometry. The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch. Thirty-six male SD rats were randomly divided into three groups: tissue engineered bladder patch group (SF-ADSCs group, n=15), double-layer silk scaffold group (SF group, n=15), control group ( n=6). The tissue engineered bladder patch (SF-ADSCs group) and double-layer silk scaffold (SF group) were wrapped on the omentum to promote vascularization. The vascularization was evaluated by HE and immunofluorescence staining. The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder. In the control group (six rats), the incision was closed immediately after the bladder tissue fully exposed. At 4 weeks and 12 weeks after operation, the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology. After the graft was harvested, HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue. Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation. Results:The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90, and there was no significant expression of CD45 and CD106. Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting, but also had good toughness and was facilitated to surgical suture. The number (43.50±2.66) and area (0.73±0.03)% of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group [(24.50±3.51), (0.55±0.05)%], and the difference was statistically significant ( P<0.05). At 4 weeks after bladder repair, the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold. At 12 weeks, the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology, which was similar to that of normal bladder tissue. Immunofluorescence staining showed that the continuous urothelial layer, abundant smooth muscle tissue, vascular structure and regenerated neurons could be observed in the SF-ADSCs group. Urodynamic test showed that the bladder maximum volume (0.74±0.03)ml and compliance (16.68±0.44)μl/cm H 2O in the SF-ADSCs group, which were better than that in the SF group [(0.47±0.05)ml, (14.89±0.37)μl/cm H 2O], but lower than that in the control group [(1.12±0.08)ml, (19.34±0.45)μl/cm H 2O], and the difference was statistically significant ( P<0.05). Conclusions:The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue, the regeneration of bladder wall structure and the recovery of bladder physiological function.

Study on preparation ，safety and anti-tumor activity of apigenin silk fibroin nanoparticles / 中国药房

OBJECTIVE To prepare apigenin silk fibroin（API@SF）nanoparticles and to evaluate their safety and anti-tumor activity. METHODS API@SF nanoparticles were prepared by nanoprecipitation method ，and their morphology ，particle size ，Zeta potential，drug loading amount and in vitro release were characterized. The safety of nanoparticles was evaluated by hemolysis test and HE staining. MTT assay was adopted to evaluate inhibitory effects of API@SF nanoparticles on breast cancer 4T1 cells in mice. RESULTS The prepared API@SF nanoparticles were spherical with uniform distribution. The average particle size was 406.61 nm， the polydispersity index was 0.154，the Zeta potential was －18.4 mV，and the average drug-loading amount was 5.20％. The in vitro release results showed that the release rate of the nanoparticles was relatively fast in the release medium of pH 5.0 and relatively slow in the release medium of pH 7.4. Results of hemolysis test and HE staining showed that the nanoparticles had good biocompatibility. Results of MTT assay showed that the inhibitory effect of API@SF nanoparticles on 4T1 cells was significantly higher than that of API raw materials （P＜0.05），and its mechanism may be related to increasing the level of reactive oxygen species in cells. CONCLUSIONS API@SF nanoparticles are prepared successfully ，which possess good safety and anti-tumor activity.

Research progress of silk fibroin-based nanoparticulate drug delivery systems / 药学学报

Silk fibroin is a natural polymer with certain water solubility, structural modification, good biocompatibility and biodegradability, which can be used as a drug delivery carrier material. As a promising drug delivery system, drug-loaded silk fibroin nanoparticles can control drug release, reduce toxicity and improve therapeutic effects. In this paper, the basic characteristics of silk fibroin, the preparation methods of drug-loaded silk fibroin nanoparticles and the application of silk fibroin in nanoparticulate drug delivery systems are reviewed, and on this basis, the further development of drug-loaded silk fibroin nanoparticles is prospected.

Recovery of motor function in rats with complete spinal cord injury following implantation of collagen/silk fibroin scaffold combined with human umbilical cord-mesenchymal stem cells

SUMMARY OBJECTIVE: This study aimed to assess the effect of the collagen/silk fibroin scaffolds seeded with human umbilical cord-mesenchymal stem cells on functional recovery after acute complete spinal cord injury. METHODS: The fibroin and collagen were mixed (mass ratio, 3:7), and the composite scaffolds were produced. Forty rats were randomly divided into the Sham group (without spinal cord injury), spinal cord injury group (spinal cord transection without any implantation), collagen/silk fibroin scaffolds group (spinal cord transection with implantation of the collagen/silk fibroin scaffolds), and collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (spinal cord transection with the implantation of the collagen/silk fibroin scaffolds co-cultured with human umbilical cord-mesenchymal stem cells). Motor evoked potential, Basso-Beattie-Bresnahan scale, modified Bielschowsky's silver staining, and immunofluorescence staining were performed. RESULTS: The BBB scores in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group were significantly higher than those in the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). The amplitude and latency were markedly improved in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). Meanwhile, compared to the spinal cord injury and collagen/silk fibroin scaffolds groups, more neurofilament positive nerve fiber ensheathed by myelin basic protein positive structure at the injury site were observed in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (p<0.01, p<0.05). The results of Bielschowsky's silver staining indicated more nerve fibers was observed at the lesion site in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.01, p< 0.05). CONCLUSION: The results demonstrated that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen/silk fibroin scaffolds could promote nerve regeneration, and recovery of neurological function after acute spinal cord injury.

Preparation Method of Silk Fibroin and Its Application in Field of Biomedical Materials / 中国医疗器械杂志

Silk fibroin has the characteristics of good biocompatibility, mechanical properties, degradation performance and easy shaping, which makes silk fibroin become the focus of biomedical material preparation and research, and has received extensive attention. This article reviews the prior art methods of silk fibroin degumming, dissolution and regeneration processing. The specific applications of silk fibroin materials in the field of biomedical materials are reviewed, and the application prospects of silk fibroin in the field of biomedical materials are prospected.

Evaluation of silk fibroin/curcumin composite film for promoting wound healing / 中国组织工程研究

BACKGROUND: In the previous study, we have successfully prepared a silk fibroin/curcumin composite film, which possesses good physicochemical properties, sustained-release properties, antibacterial activity and biocompatibility. OBJECTIVE: To further observe the effect of the prepared silk fibroin/curcumin composite film, as the wound dressing, in the field of wound healing. METHODS: The silk fibroin/curcumin composite film was prepared by referring to the previously established methods. The mechanical properties and light transmittance of the prepared composite film were then investigated. Escherichia coli and Staphylococcus aureus were used as strains. The plate-counting technique was applied to evaluate the antibacterial activity of the light illuminated silk fibroin/curcumin composite film, silk fibroin/curcumin composite film without light exposure, and silk fibroin film without light exposure. The full-thickness excisional lesions were created on the dorsum of 20 Kunming mice. The wounds of 10 mice were covered with silk fibroin/curcumin composite film, and the wounds of other 10 mice were dressed with sterile gauze. The healing status and histological morphology of the wound sites were observed at 3, 7, and 14 days post operation. The animal experimental design and the protocols were approved by the Laboratory Animal Ethics Committee of Southwest University (approval number: IACUC-2019011). RESULTS AND CONCLUSION: (1) Compared to silk fibroin film, the fracture strength of silk fibroin/curcumin composite film increased significantly (P < 0.05), while the Young’s modulus of silk fibroin/curcumin composite film decreased significantly (P < 0.01). (2) Both silk fibroin film and the silk fibroin/curcumin composite film exhibited excellent transparency, but the silk fibroin/curcumin composite film showed a stronger absorption in visible light region at a wavelength of 400-450 nm. (3) Without light illumination, the silk fibroin/curcumin composite film exhibited stronger antibacterial activity than that of silk fibroin film. While, the silk fibroin/curcumin composite film with light illumination demonstrated stronger antibacterial activity than that of silk fibroin/curcumin composite film without light illumination. (4) The wound healing rate of the experimental group was higher than that of the control group at each time point (P <0.05). Analysis on histological morphology reveals that the length of regenerated epithelia of dermal tissue and the thickness of granulation tissue in the experimental group were greater than those in the control group at 3 and 7 days post operation (P < 0.01). While, the thickness of granulation tissue was smaller than that in the control group at 14 days post operation (P < 0.01). (5) The results indicated that the silk fibroin/curcumin composite film can facilitate skin wound healing.

Effect of cell sheet combined with 3D printing an antler powder/silk fibroin/polyvinyl alcohol scaffold on the repair of mandibular defects in sheep / 口腔疾病防治

Objective @#To explore the feasibility of antler powder/silk fibroin/polyvinyl alcohol scaffolds as tissue engineering bone scaffolds and the relationship between their degradation performance and the healing speed of bone defects.@*Methods @# Antler powder/silk fibroin/polyvinyl alcohol scaffolds and nano hydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds were prepared by 3D printing. The whole bone marrow culture method was used to prepare blood cell sheets of Altay big tail sheep’s iliac bone marrow. With observation times of 1, 2 and 3 months, the mandibular defects of 4 sheep were established. The experimental group was coated with antler powder/silk fibroin/polyvinyl alcohol scaffolds. The control group was coated with nanohydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds. The negative control group was coated with gel-free sponges. According to the self-control method of the bilateral mandible defect area, scaffolds wrapped with cell membranes or gel sponges wrapped with cell membranes were implanted. At the ends of the first, second and third months after implantation, the experimental animals were killed, cone beam CT was performed, and paraffin sections were taken for HE staining to evaluate the effect of different scaffold materials on bone regeneration and scaffold degradation.@* Results@# Scanning electron microscopy showed that both groups had regular pores and good continuity, and there was no difference in pore size and porosity between the two groups (P > 0.05). The results of CBCT imaging showed that in 3 months after operation, the experimental group had significantly better repair effects on bone defects than the control group, and the degradation rate matched the bone repair rate. The bone mineral density in the center of the defect was higher than that of the control group, which was close to that of normal bone tissue. The central bone mineral density of the experimental group at each time point was higher than those of the control group and the negative control group, and the difference was statistically significant (P < 0.05). HE staining results showed that the bone cells in the experimental group were more active, with more new capillaries and bone trabeculae formed, and the scaffold material absorbed more than the control group. @*Conclusion @#The antler powder/silk fibroin/polyvinyl alcohol scaffold can promote the repair of critical bone defects. Its degradability matches its bone tissue healing rate. It is expected to become a promising scaffold material for bone tissue engineering.

Glyoxal modification mediates conformational alterations in silk fibroin: Induction of fibrillation with amyloidal features

Various animal models, especially rodents, are used to study pain, due to the difficulty of studying it inhumans. Many drugs that produce analgesia have been studied and there is evidence among whichNSAIDs deserve to be highlighted. Dexketoprofen (DEX) provides a broad antinociceptive profile indifferent types of pain; therefore, this study was designed to evaluate the profile of antinociceptivepotency in mice. Analgesic activity was evaluated using the acetic acid abdominal constriction test(writhing test), a chemical model of visceral pain. Dose-response curves for i.p. DEX administration (1,3, 10, 30 and 100 mg/kg), using at least six mice in each of at least five doses, was obtained before and30 min after pre-treatment with different pharmacological agents. Pretreatment of the mice with opioidreceptor antagonists was not effective; however, the serotonin receptor antagonist and nitric oxidesynthase inhibitor produce a significant increase in DEX-induced antinociception. The data from thepresent study shows that DEX produces antinociception in the chemical twisting test of mice, which isexplained with difficulty by the simple inhibition of COX. This effect appears to be mediated by othermechanisms in which the contribution of the NO and 5-HT pathways has an important effect on DEXinduced antinociception.

Biocompatibility of tissue engineered cartilage constructedin vivoby silk fibroin-chitosan scaffold carrying bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Our previous studies have found that silk fibroin-chitosan scaffold carrying bone marrow mesenchymal stem cells can repair cartilage defect in rabbits, but further exploration on the biocompatibility of tissue engineered cartilage is yet to be done. OBJECTIVE: To explore the biocompatibility of tissue engineered cartilage that is constructed in vitro by silk fibroin-chitosan scaffold with bone marrow mesenchymal stem cells. METHODS: Three-dimensional silk fibroin-chitosan scaffolds were prepared in a ratio of 1:1. Rabbit bone marrow mesenchymal stem cells were extracted, induced and seeded onto the silk fibroin-chitosan scaffold to construct the cell-scaffold composite. The composite was then implanted into a rabbit joint defect model for cartilage repair. There were three groups in the present study: Experiment group with implantation of induced bone marrow mesenchymal stem cells+silk fibroin-chitosan scaffold into the cartilage defect model, control group with implantation of silk fibroin-chitosan scaffold into the cartilage defect model, and blank group without implantation. RESULTS AND CONCLUSION: The three-dimensional silk fibroin-chitosan scaffolds were successfully prepared and combined with bone marrow mesenchymal stem cells (BMSCs) to construct the tissue engineered cartilage for repair cartilage defects in rabbits. Blood routine parameters, procalcitonin levels, erythrocyte sedimentation rates and C-reactive protein levels detected at 2, 4, 8, and 12 weeks post-implantation indicated no obvious signs of systemic infection, and there was no damage to liver and kidney functions in the three groups. There were also no significant differences between the three groups in terms of blood routines and liver and kidney functions (P > 0.05). As shown by gross observation, hematoxylin-eosin staining and scanning electron microscope, in the experimental group, cartilage defects were repaired, with scaffold degradation, no presence of inflammatory cells, and good integration with surrounding tissues. Therefore, tissue engineered cartilage constructed in vitro by silk fibroin-chitosan scaffolds carrying bone marrow mesenchymal stem cells has good biocompatibility, which provides an experimental basis for tissue engineering approaches to cartilage repair.

Safety and antibacterial properties of gentamicin-loaded silk fibroin in meniscus repair / 中国组织工程研究

BACKGROUND: Silk fibroin material has good biocompatibility, excellent mechanical properties, biodegradation and other advantages. However, it has not been used as a kind of suture fixation material in the repair of meniscus tear. OBJECTIVE: To prepare gentamicin loaded silk fibroin material and analyze its biocompatibility and antibacterial properties. METHODS: After dissolving the degummed natural silkworm cocoon with ternary solution, the silk fibroin material was prepared by dialysis, freeze-drying, redissolution and molding, and gentamicin was dissolved in silk fibroin solution to prepare gentamicin loaded silk fibroin material. Silk fibroin material and gentamicin loaded silk fibroin material were co-cultured with mouse pre chondroblasts respectively. After 1, 3, and 5 days, cell proliferation was detected by MTT assay. After 24 hours of co-culture, the cytotoxic effect of the material was detected by calcein staining. The early apoptosis of cells was detected by flow cytometry. The adhesion of cells on the surface of the material was observed by scanning electron microscope. Silk fibroin material and gentamicin loaded silk fibroin material were mixed with anticoagulant separately. The hemolysis rate of the material was detected by hemolysis experiment. The silk fibroin material and gentamicin loaded silk fibroin material were co-cultured with S.aureus and E.coli respectively. The bacterial count, scanning electron microscope observation and bacteriostatic circle experiment were carried out. RESULTS AND CONCLUSION: (1) The two materials had no significant effect on cell proliferation and early apoptosis. (2) There was no obvious cytotoxicity between the two materials, and the cells adhered on the surfaces of the two materials grew well, arranged tightly, and the morphology of the cells expanded. (3) The two materials had good blood compatibility and did not cause hemolytic reaction. (4) Gentamicin loaded silk fibroin material had obvious inhibition on S.aureus and E.coli, and the inhibition on S.aureus was more obvious. (5) The results showed that the gentamicin loaded silk fibroin material has good biocompatibility and antibacterial property.

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.

Diffusion tensor imaging predicting locomotor function recovery with 3D printing scaffold after spinal cord injury / 中国组织工程研究

BACKGROUND: Diffusion tensor imaging, as a relatively new method based on MRI, has become an important means of examination and diagnosis in the field of neuroimaging. OBJECTIVE: To investigate the role of using diffusion tensor tensor imaging data to predict 3D-bioprinted collagen/silk fibroin scaffolds in the locomotor function recovery after spinal cord injury. METHODS: Ordinary and 3D-bioprinted collagen/silk fibroin scaffold were prepared. Forty adult female SD rats provided by the Laboratory Animal Center of the Academy of Military Medical Sciences of the People’s Liberation Army were randomly divided into four groups with 10 rats in each group. In the sham operation group, only T10 vertebral plate was removed. In the model group, spinal cord injury was induced by total transection of spinal cord at T10 segment. In the ordinary collagen scaffold and 3D-printed scaffold groups, after induction of T10 spinal cord injury, ordinary collagen scaffold and 3D-printed scaffold were implanted, respectively. At 1, 2, 3, 4, 6 and 8 weeks after surgery, Basso, Beattie and Bresnahan (BBB) locomotor function scoring and oblique plate test of the hind limbs were carried out. At 8 weeks after surgery, electrophysiological test of the hind limbs was performed to evaluate locomotor function. At 8 weeks after surgery, diffusion tensor imaging of the lumbar spine was performed and the correlation between diffusion tensor imaging parameter and rat locomotor function was analyzed. Animal experiments were approved by the Animal Ethics Committee of Characteristic Medical Center of the Chinese people’s Armed Police Force (approval No. 27653/58). RESULTS AND CONCLUSION: (1) From 3 weeks after surgery, BBB score in the 3D-printed group was significantly higher than that in the model and ordinary collagen scaffold groups (P < 0.05 or P < 0.01). From 2 weeks after surgery, the slope angle in the 3D-printed scaffold group was significantly higher than that in the model and ordinary scaffold groups (P < 0.05 or P < 0.01). (2) The amplitude of motor evoked potential in the 3D-printed scaffold group was significantly greater than that in the model and ordinary collagen scaffold groups (P < 0.05 or P < 0.01). The latency of motor evoked potential in the 3D-printed scaffold group was significantly shorter than that in the model and ordinary collagen scaffold groups (P < 0.05 or P < 0.01). (3) Diffusion tensor imaging showed that the nerve fiber trajectories in the three groups were irregular and lacked the continuity of nerve fibers, but the number of regenerated nerve fiber bundles in the 3D-printed collagen scaffold group was greater than that in the model and ordinary collagen scaffold groups (P < 0.01). The fractional anisotropy at 9, 7.5, 4.5, -3, -6, -7.5, -9 mm from the center of spinal cord injury in 3D-printed collagen scaffold group was significantly higher than that in model and ordinary collagen scaffold groups (P < 0.05 or P < 0.01). (4) The BBB score, slope angle, amplitude of motor evoked potential, latency of motor evoked potential were positively correlated with the fractional anisotropy value of diffusion tensor imaging from head to tail of rats. (5) These results suggest that diffusion tensor imaging can be used as an effective predictor to evaluate the recovery of neurological function after spinal cord injury in experimental animals and clinical cases.

Analysis of protective mechanism of silk protein based cryoprotectants / 生物医学工程学杂志

Dimethyl sulfoxide (Me SO) supplemented with fetal bovine serum (FBS) is a widely used cryoprotectant combination. However, high concentration of Me SO is toxic to cells, and FBS presents problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a kind of natural macromolecule fiber protein with good biocompatibility and hydrophilicity. The aim of this paper is to analyze the cryoprotective mechanism of silk protein as cryoprotectant. Firstly, differential scanning calorimetry (DSC) was used to measure the thermal hysteresis activity (THA) of silk protein. The THA of 10 mg/mL sericin protein was 0.96°C, and the THA of 10% (V/V) fibroin protein was 1.15°C. Then the ice recrystallization inhibition (IRI) of silk protein-PBS solution was observed with cryomicroscope. The cold stage was set at - 7°C, after 40 minutes' incubation, the mean grain size rate (MGSR) of sericin protein and fibroin protein were 28.99% and 3.18%, respectively, which were calculated relative to phosphate buffer saline (PBS) control. It is indicated that sericin and silk fibroin have certain effects of inhibiting recrystallization of ice crystals. Finally, the structure and physicochemical properties of silk protein were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the content of the random coil was 75.62% and the β-sheet structure was 24.38% in the secondary of sericin protein. The content of the β-sheet structure was 56.68%, followed by random coil structure 22.38%, and α-helix 16.84% in the secondary of fibroin protein. The above analysis demonstrates the feasibility of silk fibroin as a cryoprotectant, and provides a new idea for the selection of cryoprotectants in the future.

Lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells combined with silk fibroin scaffold for osteoblast transformation / 中国骨伤

OBJECTIVE@#To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation.@*METHODS@#The lentivirus BMP-2 overexpression vector was constructed, bone marrow mesenchymal stem cells were cultured, and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits, weighing 3.2 to 4.5 kg(averaging 3.9 kg), aged (2.89±0.45) years old, were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length, 2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex, while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex.@*RESULTS@#Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells), the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group, the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group, which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group, and a small amount of calcium nodules could be seen under the microscope. In the experimental group, obvious red area staining was observed by naked eye, and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group, and no obvious change in the microscopic observation. In the experimental group, purple area staining was observed by naked eyes, and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group, inflammatory cells were significantly reduced, scaffolds disappeared and angiogenesis was observed.@*CONCLUSIONS@#Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²⁺ to promote bone defect repair.

Carbon-Fiber Based Microelectrode Array Embedded with a Biodegradable Silk Support for In Vivo Neural Recording

BACKGROUND: Recently, carbon fibers have been utilized to develop a depth-type microelectrode array for chronic neural recording. Since the diameter of carbon fibers is smaller than the conventional electrodes made of metal wires or microfabricated silicon, the carbon fiber electrodes showed an improved capability for chronic neural recording with less tissue damages. However, the carbon fiber based microelectrodes have a limitation of short insertion depth due to a low stiffness. METHODS: We proposed a carbon fiber based microelectrode array embedded with a mechanical support structure to facilitate the penetration into the deeper brain. The support is made of biodegradable silk fibroin to reduce the reactive tissue responses. The 4-channel carbon fiber based microelectrode arrays were fabricated and accessed in terms of electrochemical impedance, recording capability for 1-month implantation in rat hippocampi. The electrodes with tungsten supports were fabricated and tested as a control group. Immunohistochemical analysis was performed to identify the reactive glial responses. RESULTS: The carbon fiber based electrode arrays with silk supports showed about 2-fold impedance increase 2 weeks after implantation while the number of active electrodes decreased simultaneously. However, after 1 month, the electrode impedance decreased back to its initial value and the percentage of active electrodes also increased above 70%. Immunohistochemical staining clearly showed that the electrodes with silk supports induced less reactive glial responses than that with tungsten supports. CONCLUSION: The proposed carbon fiber based microelectrode array is expected to be used for long-term in vivo neural recording from deep brain regions with the minimized reactive tissue response.

Application of composite 3D scaffolds in repairing skull defects / 中国综合临床

Bone graft is an important basic condition for repairing skull defect caused by various reasons,but whether autologous bone graft or allograft bone transplantation,the clinical limitations was increasingly significant.With the development of bone tissue engineering,the introduction of bone progenitor cells and growth factors into composite scaffolds made from natural or synthetic materials,and as a substitute for bone graft has become a hot spot of research.It will review the research progress on bone graft substitute and bone biomimetic scaffold material,so as to provides reference on the further investigations.