Silk fibroin reinforced decellularized porcine pericardial patch with superior strength, biocompatibility, and non-toxicity as a regenerative replacement for cardiovascular applications

Highlights Silk fibroin is resistant to early degradation, focal calcification, adsorbs albumin, and does not cause pronounced local and systemic inflammation during subcutaneous implantation in rats, which is comparable and even superior in properties to bovine pericardium used in vascular surgery. Aim. To study the hydrolytic degradation of silk fibroin (SF), to evaluate in a comparative aspect the surface adsorption of proteins by matrices from SF and bovine pericardium (BP), calcification of biomaterials, as well as their biocompatibility, the dynamics of biodegradation and systemic inflammation after subcutaneous implantation in rats.Methods. Matrices from SF were made by electrospinning. The BP flap was used as a comparison material. The loss of SF mass and the change in pH of the incubation solution during its hydrolytic degradation in vitro were studied at periods of 1, 3, 6, and 12 months. The surface adsorption of albumin and fibrinogen by matrices was assessed in vitro. Samples of SF and BP were implanted subcutaneously in rats for periods of 7, 14, 30, and 60 days. After explantation of the matrices, a histological examination of the samples was performed and their calcification was assessed. The levels of interleukins 6 and 8, tumor necrosis factor-α, and monocyte chemotactic protein-1 were studied in the blood serum of rats using the enzyme immunoassay method.Results. Hydrolytic degradation of SF in vitro resulted in a 6% loss of the matrix mass and was accompanied by a decrease in the pH of the incubation solution to 6.56. Twice as much albumin and six times as much fibrinogen were adsorbed on the SF surface than on the BP surface. Signs of SF biodegradation and a thin connective tissue capsule around the matrix appeared after 30 days of subcutaneous implantation in rats. No pronounced inflammation or calcification of SF were detected. Subcutaneous implantation of BP was accompanied by the formation of a pronounced connective tissue capsule around the sample after 60 days, fraying, and degradation of the material. Localized foci of calcification were not detected at any time point of subcutaneous implantation. The content of proinflammatory cytokines in the blood serum of rats after subcutaneous implantation of SF and BP decreased by day 60 of the study without statistically significant intergroup differences.Conclusion. Due to its resistance to premature degradation and calcification, increased surface adsorption of albumin, optimal biocompatibility and the absence of pronounced peri-implantation and systemic inflammation, SF is comparable and also has a number of advantages compared to BP used in vascular surgery.

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.

To evaluate the effects of combined treatment with tannic acid and ferric ions on the biomechanical and anti-calcification properties of glutaraldehyde-fixed bovine jugular veins after xenografting. Two-point bending test and uniaxial tensile test were used to evaluate the flexural and biomechanical properties; Subcutaneous implantation in rat and right ventricular outflow tract reconstruction of sheep were used to evaluate the anti-calcification effects; The performance of the graft in sheep models was evaluated every month after the surgery with echocardiography examination. Markers of macrophages, T lymphocytes, smooth muscle cell osteogenic differentiation and matrix metalloproteinases in sheep explants were detected by immunohistochemistry. The flexibility of the bovine jugular veins cotreated with ferric ions-tannic acid was improved while maintaining biomechanical properties and excellent anti-calcification effects. Echocardiography results showed that the grafts functioned well in the animals without stenosis or reflux of the valve. Immunohistochemical studies showed that the osteogenic differentiation marker (Runx2) was detected in calcified regions and colocalised with the SMC marker (α-SMA). Compared to the glutaraldehyde-treated samples, T-cell marker (CD3), matrix metalloproteinase-2 and 9 expressions were reduced in the ferric ions-tannic acid treated group. Ferric ions-tannic acid treatment can give the conduits better flexibility with excellent biomechanical properties and anti-calcification effects, making it a promising bovine jugular veins processing method.

Silk fibroin/sodium alginate composite porous materials with controllable degradation

Guided bone regeneration (GBR) technology is a commonly used surgical procedure for the repair of damaged periodontal tissues. Poor mechanical property and rapid degradation rate are the major reasons for GBR membrane failure in clinical applications. Herein, we applied a green papermaking method to fabricate silk fibroin (SF) membranes blended with collagen and tested their performance. The results showed that the blended SF75 (SF and collagen in a weight ratio of 75:25) membranes are biocompatible with good mechanical properties in the wet condition and appropriate biodegradation rate. MC3T3-E1 osteoblast cell adhesion and proliferation on the membranes were improved by the hybrid biological functions of SF and collagen. Subcutaneous implantation in rats for 9 weeks demonstrated that the membranes induced a less severe inflammatory response. The biodegradation time of the SF75 membranes was appropriate for tissue regeneration. This research, for the first time, reports a blended membrane prepared from silk fibroin and collagen with an ecofriendly method, which shows promise for application in guided bone regeneration.

We report the effects of distinct concentrations of genipin and silk fibroin (SF):chitosan (CS) ratios on the formation of SF–CS composite microspheres. We selected microspheres featuring an SF:CS ratio of 1:1, encapsulated various concentrations of bovine serum albumin (BSA), and then compared their encapsulation efficiency and sustained-release rate with those of pure CS microspheres. We determined that the following five groups of microspheres were highly spherical and featured particle sizes ranging from 70 μm to 147 μm: mass ratio of CS:SF =1:0.5, 0.1 g or 0.5 g genipin; CS:SF =1:1, 0.05 g or 1 g genipin; and CS:SF =1:2, 0.5 g genipin. The microspheres prepared using 1:1 CS:SF ratio and 0.05 g genipin in the presence of 10 mg, 20 mg, and 50 mg of BSA exhibited encapsulation efficiencies of 50.16%±4.32%, 56.58%±3.58%, and 42.19%±7.47%, respectively. Fourier-transform infrared spectroscopy (FTIR) results showed that SF and CS were cross-linked and that the α-helices and random coils of SF were converted into β-sheets. BSA did not chemically react with CS or SF. Moreover, thermal gravimetric analysis (TGA) results showed that the melting point of BSA did not change, which confirmed the FTIR results, and X-ray diffraction results showed that BSA was entrapped in microspheres in a noncrystalline form, which further verified the TGA and FTIR data. The sustained-release microspheres prepared in the presence of 10 mg, 20 mg, and 50 mg of BSA burst release 30.79%±3.43%, 34.41%±4.46%, and 41.75%±0.96% of the entrapped BSA on the 1st day and cumulatively released 75.20%±2.52%, 79.16%±4.31%, and 89.04%±4.68% in 21 days, respectively. The pure CS microspheres prepared in the presence of 10 mg of BSA burst release 39.53%±1.76% of BSA on the 1st day and cumulatively released 83.57%±2.33% of the total encapsulated BSA in 21 days. The SF–CS composite microspheres exhibited higher sustained release than did the pure CS microspheres, and thus these composite microspheres might function as a superior drug carrier.

The development of flexible, biocompatible, and environment-friendly sensors has attracted a significant amount of scientific interest for the past few decades. Among all the natural materials, silk fibroin (SF), due to its tunable biodegradability, biocompatibility, ease of processing, presence of functional groups, and controllable dimensions, has opened up opportunities for immobilizing multitudinous biomolecules and conformability to the skin, among other attractive opportunities. The silk fibroins also offer good physical properties, such as superior toughness and tensile strength. The sensors made of SF as an immobilization matrix have demonstrated excellent analytical performance, sensing even at low concentrations. The significant advantage of silk fibroins is the presence of functional groups along with a controllable conformation transition that enables immobilization of receptor molecules using silk fibroins as an immobilization matrix enables us to entrap the receptor molecules without using any chemical reagents. This review encompasses a detailed discussion on sensors, the advantages of using silk fibroins as an immobilization matrix for various receptors, their applications, and the future research scope in this state-of-the-art technology based upon the explorable applications for silk fibroin-based sensors.

The present work describes the development of a drug-loaded ureteral stent with antistricture function based on a trilayer design in which the middle layer was braided from biodegradable poly(p-dioxanone) (PDO) monofilament. Antistenosis drugs rapamycin and paclitaxel were loaded into a silk fibroin (SF) solution and coated on the inner and outer layers of the braided PDO stent. The cumulative release of rapamycin and paclitaxel was sustained over 30 days, with a total release above 80%. The drug-loaded ureteral stents inhibited the proliferation of fibroblasts and smooth muscle cells in vitro. Subcutaneous implantation in rats showed that the drug-loaded ureteral stents were biocompatible with durable mechanical properties in vivo, revealing the inhibition of an excessive growth of fibroblasts and excessive deposition of collagen fibers. In conclusion, the dual-drug-loaded biodegradable ureteral stents show the possibility for treatment of ureteral strictures and avoid the occurrence of complications such as inflammation and restricture.

Promotion of bone defect repairs using multiscale 3D printed silk porous hydrogel scaffolds.

Biomineralized silk fibroin hydrogel with ultra-high strength for supporting bodyweight-bearing bone defects.

Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to β-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become β-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent β-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.

The skin, acting as the outer protection of the human body, is most vulnerable to injury. Wound healing can often be impaired, leading to chronic, hard-to-heal wounds. For this reason, searching for the most effective dressings that can significantly enhance the wound healing process is necessary. In this regard, silk fibroin, a protein derived from silk fibres that has excellent properties, is noteworthy. Silk fibroin is highly biocompatible and biodegradable. It can easily make various dressings, which can be loaded with additional substances to improve healing. Dressings based on silk fibroin have anti-inflammatory, pro-angiogenic properties and significantly accelerate skin wound healing, even compared to commercially available wound dressings. Animal studies confirm the beneficial influence of silk fibroin in wound healing. Clinical research focusing on fibroin dressings is also promising. These properties make silk fibroin a remarkable natural material for creating innovative, simple, and effective dressings for skin wound healing. In this review, we summarise the application of silk fibroin biomaterials as wound dressings in full-thickness, burn, and diabetic wounds in preclinical and clinical settings.

Sponge-like materials made from regenerated silk fibroin biopolymers are a tunable and advantageous platform for in vitro engineered tissue culture and in vivo tissue regeneration. Anisotropic, three-dimensional (3D) silk fibroin sponge-like scaffolds can mimic the architecture of contractile muscle. Herein, we use silk fibroin solution isolated from the cocoons of Bombyx mori silkworms to form aligned sponges via directional ice templating in a custom mold with a slurry of dry ice and ethanol. Hydrated tensile mechanical properties of these aligned sponges were evaluated as a function of silk polymer concentration (3% or 5%), freezing time (50% or 100% ethanol), and post-lyophilization method for inducing crystallinity (autoclaving, water annealing). Hydrated static tensile tests were used to determine Young's modulus and ultimate tensile strength across sponge formulations at two strain rates to evaluate rate dependence in the calculated parameters. Results aligned with previous reports in the literature for isotropic silk fibroin sponge-like scaffolds, where the method by which beta-sheets were formed and level of beta-sheet content (crystallinity) had the greatest impact on static parameters, while polymer concentration and freezing rate did not significantly impact static mechanical properties. We estimated the crystalline organization using molecular dynamics simulations to show that larger crystalline regions may be responsible for strength at low strain amplitudes and brittleness at high strain amplitudes in the autoclaved sponges. Within the parameters evaluated, extensional Young's modulus is tunable in the range of 600-2800 kPa. Dynamic tensile testing revealed the linear viscoelastic region to be between 0% and 10% strain amplitude and 0.2-2 Hz frequencies. Long-term stability was evaluated by hysteresis and fatigue tests. Fatigue tests showed minimal change in the storage and loss modulus of 5% silk fibroin sponges for more than 6000 min of continuous mechanical stimulation in the linear regime at 10% strain amplitude and 1 Hz frequency. Furthermore, we confirmed that these mechanical properties hold when decellularized extracellular matrix is added to the sponges and when the mechanical property assessments were performed in cell culture media. We also used nano-computed tomography (nano-CT) and simulations to explore pore interconnectivity and tortuosity. Overall, these results highlight the potential of anisotropic, sponge-like silk fibroin scaffolds for long-term (>6 weeks) contractile muscle culture with an in vitro bioreactor system that provides routine mechanical stimulation.

IntroductionDespite the rapid development of small diameter vascular graft (SDVG), the ability of synthetic grafts to facilitate tissue remodeling and regeneration remains an important challenge within regenerative medicine.MethodsBased on our previous research work, silk fibroin (SF)/fibrin vascular grafts were successfully fabricated using electrospinning technology, and it was demonstrated that the grafts had superior mechanical strength, good cytocompatibility and histocompatibility. This indicated that the vascular graft was an ideal SDVG. We developed SF/fibrin vascular grafts with circumferentially aligned nanofibers to explore some of its properties in vivo.ResultsThe graft exhibited randomly arranged microstructure, excellent mechanical properties and compliance properties. These vascular grafts were transplanted into the abdominal aorta of rats, maintaining normal blood flow, vascular patency, and functionality. The M2/M1 ratio value in SF/fibrin grafts increased over time after implantation. Whereas the expression level of inflammatory cytokines initially increased and then eventually reached the normal levels. Moreover, the circumferentially aligned vascular grafts could guide the regeneration of neoarteries, endothelialization formation, enhanced functionality, rapid cellular infiltration and improved extracellular matrix (ECM) deposition, as well as generated more microvessels and fewer calcification.DiscussionOur research focused on the long-term performance in vivo of SF/fibrin tubular grafts, which could become a new type of SDVG and help guide the development of next-generation vascular grafts.

Biomimetic silk fibroin and xanthan gum blended hydrogels for connective tissue regeneration