Fibroin Scaffolds Research Articles

Silver-Treated Silk Fibroin Scaffolds for Prevention of Critical Wound Infections

The risk of infections in chronic wounds represents a serious issue, particularly in aged people and in patients affected by diseases such as diabetes and obesity. Moreover, the growing resistance demonstrated by many bacterial strains has significantly reduced the therapeutic options for clinicians and has become a great challenge for the researchers in the definition of novel approaches that promote the wound healing process and reduce the healing time. Tissue engineering approaches based on biomaterials and three-dimensional scaffolds have demonstrated huge potential in supporting cell proliferation; among them, Bombyx mori-derived silk fibroin is a very appealing possibility for the development of devices with regenerative properties for wound healing applications. However, due to the high risk of infections in chronic wounds, an antibacterial treatment is also strongly encouraged for preventing bacterial proliferation at the wound site. In this work, to develop a device with regenerative and antibacterial properties, antibacterial silver coatings were deposited onto silk fibroin scaffolds, and the effect of the treatment in terms of chemical–physical and microbiological properties was investigated. The results demonstrated that the silver treatment improved the mechanical properties of the protein scaffold and provided good antibacterial efficacy against representative bacterial strains in wound infection, namely Escherichia coli and antibiotic-resistant Pseudomonas aeruginosa.

Ultralight 3D silk nanofiber scaffolds with enhanced hemostatic properties and biocompatibility for rapid hemostasis

Developing an effective hemostatic material that combines excellent biocompatibility with high blood absorption capacity remains a critical clinical challenge. In this study, ultralight three-dimensional (3D) silk fibroin (SF) scaffolds, composed of continuously interconnected nanofibers, are introduced as a novel hemostatic material. The distinctive fluffy nanofiber architecture endows the scaffolds with a remarkable porosity of 78.9 % and exceptional liquid absorption capacity. The integration of montmorillonite (MMT) further enhances the hemostatic properties of the scaffolds, while SF naturally promotes the intrinsic coagulation pathway. The scaffolds exhibit an impressive blood absorption capacity of 1687.7 %. In vitro evaluations indicate that the SF nanofiber scaffolds exhibit a blood clotting index rate of 24.6 %, a hemolysis ratio of less than 4 %, and outstanding biocompatibility. The blood coagulation time is significantly reduced from 449 s to 230 s. Additionally, the scaffolds show adhesion rates to erythrocytes and platelets of 52 % and 45.9 %, respectively. The nanofiber scaffolds aggregate and activate platelets extensively, accelerating the formation of platelet emboli, which collectively contribute to their superior hemostatic performance. Consequently, these nanofiber scaffolds hold substantial promise as absorbable hemostatic agents for achieving rapid hemostasis in clinical applications.

Assessment of the advantages and limitations of an innovative silk fibroin scaffold for the reconstruction of the anterior cruciate ligament with preclinical in vitro and in vivo evaluations

The gold standard treatment in anterior cruciate ligament (ACL) reconstruction involves autologous tissue transplantation, but this can have complications. Artificial grafts are an alternative, but the best option is debated. This study aimed to assess the biocompatibility and integration of a silk fibroin textile prosthesis (SF-TP) with peri-implant bone tissue and the native ACL. Twenty-six sheep underwent ACL reconstruction with SF-TP or autologous femoral fascia lata (FFL). Sheep were divided into two groups (3 and 6 months) and retrieved joints processed for histological, morphometrical and mechanical analysis. In vitro, SF-TP showed no cytotoxicity and good cell interaction up to 14 days. Histology revealed fibro-vascular tissue around SF-TP, with a progressive attempt of ligamentous-like tissue formation at 6 months. However, SF-TP group had higher joint damage scores. Micro-CT showed tunnel enlargement in SF-TP group, while FFL group had a decrease. SF-TP reconstructions had lower stiffness and strength (44 % and 64 % decrease) than those of autologous FFL reconstruction and often failed by pull-out from the bone tunnel due to tunnel enlargement. These results indicate poor osteointegration and graft motion with SF-TP, leading to joint damage/bone resorption and reduced mechanical competence. These results do not support the use of SF-TP for ACL reconstruction.

Mg doped ZnO containing silk nanocomposite scaffolds for biofilm prevention during alveolar bone regeneration

Biofilms are created when micro-colonies adhere to a solid surface and produce a strong extracellular matrix which increases the risk of infection and impedes the healing process of bone defects. Although post-operative antibiotics are commonly prescribed to prevent infections, they carry the risk of side effects and antibiotic resistance. Current research explored the potential of magnesium doped zinc oxide incorporated silk fibroin scaffolds to prevent the biofilms formation for alveolar bone regeneration applications. Novel silk fibroin (SF)/hydroxyapatite (HA) scaffolds incorporated with magnesium-doped zinc oxide nanoparticles (Mg–ZnO NPs with 0.5, 1 and 2 % Mg) were fabricated by freeze gelation method. The data suggested that new materials exhibit remarkable antibacterial activity against Staphylococcus aureus. Moreover, these materials are also found to be both biodegradable and biocompatible. The study revealed that scaffolds displayed excellent antibacterial activity that was increased with increasing concentration of Mg in ZnO, with a maximum of 49.39 % was observed for 2 % of Mg doped ZnO. Additionally, biodegradation of the material also increases with the increase in concentration of Mg doping in ZnO. The adequate porosity in the range of 67 %–74.3 % was recorded of new materials substituted with Mg–ZnO, which led to improved cell viability (99.6 %) and uniform deposition of apatite over the surface of scaffolds, making it an excellent candidate for biomedical applications that require antibacterials properties with high cell viability.

Enhanced effects of slowly co-released TGF-β3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects

Bioactive agents have demonstrated regenerative potential for cell-free bone tissue engineering. Nevertheless, certain challenges persist, including ineffective delivery methods and confined therapeutic potency. Here, we demonstrated that the biomimetic calcium phosphate coating system (BioCaP) could effectively uptake and slowly release the incorporated bioactive agents compared to the surface absorption system via osteoclast-mediated degradation of BioCaP coatings. The release kinetics were determined as a function of time. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Then, we developed the bi-functional BioCaP-coated silk fibroin scaffolds enabling the effective co-delivery of TGF-β3 and BMP-2 (SFI-T/SFI-B) and the corresponding slow release of TGF-β3 and BMP-2 exhibited superior potential in promoting chondrogenesis and osteogenesis without impairing cell vitality in vitro. The SFI-T/SFI-B scaffolds could improve cartilage and bone regeneration in 5 × 4 mm rabbit osteochondral (OC) defect. These findings indicate that the biomimetic calcium-phosphate coated silk fibroin scaffolds with slowly co-released TGF-β3 and BMP-2 effectively promote the repair of OC defects, hence facilitating the future clinical translation of controlled drug delivery in tissue engineering.Graphic

Gold nanoclusters on a silk fibroin scaffold accelerate fibroblast proliferation and improve burn recovery in a mouse burn model

Delayed wound healing in skin burn injuries is common owing to inhibition of proliferation caused by excessive inflammation, necrosis, autophagy, and oxidative stress. Gold nanoclusters modified with lipoic acid (AuNCs) exhibited antioxidant and anti-inflammatory effects in a murine burn healing model. However, the impact of the AuNCs on dermal cell proliferation and granulation tissue formation remains unclear. We therefore examined their effects on dermal cell proliferation, the cell cycle, and the signaling pathways involved. AuNCs were combined with a silk fibroin scaffold and tested in a mouse burn model and human dermal fibroblasts in vitro. Combined with the silk fibroin scaffold, 50 nmol/L of AuNCs activated fibroblast differentiation, collagen deposition, and angiogenesis, increasing granulation-tissue formation, reducing tissue necrosis and systemic inflammatory response, and improving wound healing in the model. The AuNCs increased PI3K/Akt signaling pathway phosphorylation in human dermal fibroblasts, upregulating protein expression of cyclin A, B, and CDK1, which participate in the G2/M cell cycle phase, ultimately promoting fibroblast proliferation. These findings support the beneficial effects of AuNCs on burn wound healing. We suggested that AuNCs on a silk fibroin scaffold or other vector is a potential clinical strategy for burn wound treatment.

Improved biocompatibility and osteogenesis of porous graphene oxide/silk fibroin scaffold for potential applications in bone tissue engineering

To better mimic the inherent porous architecture of the natural bone, we fabricated graphene oxide/silk fibroin (GO/SF) scaffolds using a salting leaching method, followed by in situ reduction and reinforcement with ascorbic acid. SEM images revealed that the scaffolds exhibited an interconnectivity porous structure both on the surface and throughout the interior. We found that the pore size could be readily adjusted by varying the concentration of GO in the scaffolds. FTIR results demonstrated that the addition of GO didn’t form chemical interaction between GO and SF, but it significantly increased the content of β-sheet structure from 46.36 % to 51.12 % and improved the thermal stability of the scaffolds. Moreover, the reduced graphene oxide/silk fibroin (rGO/SF) scaffolds exhibited a superior capacity to promote the adhesion, proliferation, and mineral deposition of mouse mesenchymal stem cell (MSCs) when compared to pure SF scaffolds. Notably, the equilibrium Young's modulus of the scaffolds gradually increased from 5.5±0.5 KPa to 14.6±2.1 KPa with the increment of rGO content. Osteogenic differentiation with higher BSP (2. 84-fold) and RUNX2 (2.91-fold) expression level was also observed in 3 % rGO scaffold, suggesting a strong inclination towards bone formation. Based on these findings, the prepared rGO/SF scaffold in this study hold great potential as a viable option for bone tissue engineering.

Precisely Printable Silk Fibroin/Carboxymethyl Cellulose/Alginate Bioink for 3D Printing.

Three-dimensional (3D) bioprinting opens up many possibilities for tissue engineering, thanks to its ability to create a three-dimensional environment for cells like an extracellular matrix. However, the use of natural polymers such as silk fibroin in 3D bioprinting faces obstacles such as having a limited printability due to the low viscosity of such solutions. This study addresses these gaps by developing highly viscous, stable, and biocompatible silk fibroin-based inks. The addition of 2% carboxymethyl cellulose sodium and 1% sodium alginate to an aqueous solution containing 2.5 to 5% silk fibroin significantly improves the printability, stability, and mechanical properties of the printed scaffolds. It has been demonstrated that the more silk fibroin there is in bioinks, the higher their printability. To stabilize silk fibroin scaffolds in an aqueous environment, the printed structures must be treated with methanol or ethanol, ensuring the transition from the silk fibroin's amorphous phase to beta sheets. The developed bioinks that are based on silk fibroin, alginate, and carboxymethyl cellulose demonstrate an ease of printing and a high printing quality, and have a sufficiently good biocompatibility with respect to mesenchymal stromal cells. The printed scaffolds have satisfactory mechanical characteristics. The resulting 3D-printing bioink composition can be used to create tissue-like structures.

3D-printed nanohydroxyapatite/methylacrylylated silk fibroin scaffold for repairing rat skull defects

The repair of bone defects remains a major challenge in the clinic, and treatment requires bone grafts or bone replacement materials. Existing biomaterials have many limitations and cannot meet the various needs of clinical applications. To treat bone defects, we constructed a nanohydroxyapatite (nHA)/methylacrylylated silk fibroin (MASF) composite biological scaffold using photocurable 3D printing technology. In this study, scanning electron microscopy (SEM) was used to detect the changes in the morphological structure of the composite scaffold with different contents of nanohydroxyapatite, and FTIR was used to detect the functional groups and chemical bonds in the composite scaffold to determine the specific components of the scaffold. In in vitro experiments, bone marrow mesenchymal stem cells from SD rats were cocultured with scaffolds soaking solution, and the cytotoxicity, cell proliferation, Western blot analysis, Quantitative real-time PCR analysis, bone alkaline phosphatase activity and alizarin red staining of scaffolds were detected to determine the biocompatibility of scaffolds and the effect of promoting proliferation and osteogenesis of bone marrow mesenchymal stem cells in vitro. In the in vivo experiment, the skull defect was constructed by adult SD rats, and the scaffold was implanted into the skull defect site. After 4 weeks and 8 weeks of culture, the specific osteogenic effect of the scaffold in the skull defect site was detected by animal micro-CT, hematoxylin and eosin (HE) staining and Masson's staining. Through the analysis of the morphological structure of the scaffold, we found that the frame supported good retention of the lamellar structure of silk fibroin, when mixed with nHA, the surface of the stent was rougher, the cell contact area increased, and cell adhesion and lamellar microstructure for cell migration and proliferation of the microenvironment provided a better space. FTIR results showed that the scaffold completely retained the β -folded structure of silk fibroin, and the scaffold composite was present without obvious impurities. The staining results of live/dead cells showed that the constructed scaffolds had no significant cytotoxicity, and thw CCK-8 assay also showed that the constructed scaffolds had good biocompatibility. The results of osteogenic induction showed that the scaffold had good osteogenic induction ability. Moreover, the results also showed that the scaffold with a MASF: nHA ratio of 1: 0.5 (SFH) showed better osteogenic ability. The micro-CT and bone histometric results were consistent with the in vitro results after stent implantation, and there was more bone formation at the bone defect site in the SFH group.This research used photocurable 3D printing technology to successfully build an osteogenesis bracket. The results show that the constructed nHA/MASF biological composite material, has good biocompatibility and good osteogenesis function. At the same time, in the microenvironment, the material can also promote bone defect repair and can potentially be used as a bone defect filling material for bone regeneration applications.

Numerical analysis of scaffold degradation in cryogenic environment: impact of cell migration and cell apoptosis

The analysis of degradation in the presence of cell death and migration is a critical aspect of research in various biological fields, such as tissue engineering, regenerative medicine, and disease pathology. In present study, numerical study of degradation of scaffold were performed in present of cells, cell apoptosis and cell migration. A poly electrolyte complex (PEC) silk fibroin scaffold was used for degradation study. Degradation study in the presence of cells and migration were performed at fixed pH concentration 7.2. Similarly, degradation study of scaffold were performed at different pH cell apoptosis. A transient analysis of scaffold was evaluated in COMSOL 5.5 in presence of cryogenic temperature at different temperature gradient. The parameters; temperature, stress, strain tensor and deformation gradient associated with the degradation of polyelectrolyte complex scaffold were evaluated. Result shows that in both geometries minimum temperature had been achieved as 230.051 K at point P4 in series view and parallel view and at a point P3 for cell migration study for −5 k min−1 and −1 k min−1, respectively. The maximum stress had been generated for 5.57 × 107 N m−2 for the temperature gradient of −2 K min−1 at T cycle in the case of cell migration study. In contrast in series view the maximum stress 2.9 × 107 N m−2 were observed at P4 which was higher as compare to P3. Similarly, for a parallel view, maximum stress (3.93 × 107 N m−2) was obtained for point P3. It had been observed that the maximum strain tensor 5.21 × 10−3, 5.15 × 10−3 and 5.26 × 10−3 was generated in series view at 230 k on a point P3 for − 1, −2 and −5 K min−1, respectively. Similarly, the maximum strain tensor 8.16 × 10−3, 8.09 × 10−3 and 8.09 × 10−3 was generated in parallel view at 230 k on a point P3 for −1, −2 and −5 K min−1, respectively. In the presence of cells, at a point P4 for temperature gradient of −1 and −2 K min−1, it had been closed to the scaffold wall, which had a different temperature profile than the point P3 and scaffold comes to the contact with the cells. The analysis of PEC scaffold degradation in the presence of cells, including cell apoptosis and migration, offers significant insights into the relationship between scaffold properties, cell behaviour, and tissue regeneration.

Impact of crystalline domains on long-term stability and mechanical performance of anisotropic silk fibroin sponges.

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.

Evaluation of rabbit adipose derived stem cells fate in perfused multilayered silk fibroin composite scaffold for Osteochondral repair.

Development of osteochondral tissue engineering approaches using scaffolds seeded with stem cells in association with mechanical stimulations has been recently considered as a promising technique for the repair of this tissue. In this study, an integrated and biomimetic trilayered silk fibroin (SF) scaffold containing SF nanofibers in each layer was fabricated. The osteogenesis and chondrogenesis of stem cells seeded on the fabricated scaffolds were investigated under a perfusion flow. 3-Dimethylthiazol-2,5-diphenyltetrazolium bromide assay showed that the perfusion flow significantly enhanced cell viability and proliferation. Analysis of gene expression by stem cells revealed that perfusion flow had significantly upregulated the expression of osteogenic and chondrogenic genes in the bone and cartilage layers and downregulated the hypertrophic gene expression in the intermediate layer of the scaffold. In conclusion, applying flow perfusion on the prepared integrated trilayered SF-based scaffold can support osteogenic and chondrogenic differentiation for repairing osteochondral defects.

The naringin/carboxymethyl chitosan/sodium hyaluronate/silk fibroin scaffold facilitates the healing of diabetic wounds by restoring the ROS-related dysfunction of vascularization and macrophage polarization

Chronic diabetic wounds remain a globally recognized clinical challenge, which occurs mainly due to the disturbances of wound microenvironmental induced by high concentrations of reactive oxygen species (ROS). Impairments in angiogenesis and inflammation in the wound microenvironment ultimately impede the normal healing process. Therefore, targeting macrophage and vascular endothelial cell dysfunction is a promising therapeutic strategy. In our study, we fabricated artificial composite scaffolds composed of naringin/carboxymethyl chitosan/sodium hyaluronate/silk fibroin (NG/CMCS/HA/SF) to promote wound healing. The NG/CMCS/HA/SF scaffold demonstrated favorable anti-inflammatory, anti-oxidative, and pro-angiogenic properties in both in vitro and in vivo experiments, effectively promoting the healing of diabetic wounds. The positive therapeutic effects observed indicate that the composite scaffolds have great potential in clinical wound healing applications.

Anti-Inflammatory Peptide-Conjugated Silk Fibroin/Cryogel Hybrid Dual Fiber Scaffold with Hierarchical Structure Promotes Healing of Chronic Wounds.

Chronic wounds resulting from diabetes, pressure, radiation therapy, and other factors continue to pose significant challenges in wound healing. To address this, this study introduces a novel hybrid fibroin fibrous scaffold (FFS) comprising randomly arranged fibroin fibers and vertically aligned cryogel fibers (CFs). The fibroin scaffold is efficiently degummed at room temperature and simultaneously formed a porous structure. The aligned CFs are produced via directional freeze-drying, achieved by controlling solution concentration and freezing polymerization temperature. The incorporation of aligned CFs into the expanded fibroin fiber scaffold leads to enhanced cell infiltration both in vitro and in vivo, further elevating the hybrid scaffold's tissue compatibility. The anti-inflammatory peptide 1 (AP-1) is also conjugated to the hybrid fibrous scaffold, effectively transforming the inflammatory status of chronic wounds from pro-inflammatory to pro-reparative. Consequently, the FFS-AP1+CF group demonstrates superior granulation tissue formation, angiogenesis, collagen deposition, and re-epithelialization during the proliferative phase compared to the commercial product PELNAC. Moreover, the FFS-AP1+CF group displays epidermis thickness, number of regenerated hair follicles, and collagen density closer to normal skin tissue. These findings highlight the potential of random fibroin fibers/aligned CFs hybrid fibrous scaffold as a promising approach for skin tissue filling and tissue regeneration.

Rationally designed ultra-fast laser surface texturation of biocompatible temporal scaffolds

The aim of the current work is to create 2D silk fibroin and 3D calcium phosphate temporal cellular scaffolds with specifically enhanced porous topographical design by means of femtosecond laser-induced nano- and micro-scale hybrid surface structuring for application in muscle and bone tissue engineering. The created cellular matrices were ablated in a multistep manner using a Solstice Ace system, delivering laser pulses with pulse duration of 70 fs, at λ=800 nm and a focus spot of 25μm. The samples were positioned on a motorized XY translation stage perpendicular or in a variable angle in respect to the laser beam. Based on the previously performed comparative experimental study, optimal laser structuring conditions and raster patterns were chosen for further enhancement of muscular and Schwann cells environment. The most valuable contribution of the study presented is related to the optimization of the bioactivity properties of the studied cell matrices and their subsequent practical application in tissue engineering.

A bilayer bioengineered patch with sequential dual-growth factor release to promote vascularization in bladder reconstruction.

Bladder tissue engineering holds promise for addressing bladder defects resulting from congenital or acquired bladder diseases. However, inadequate vascularization significantly impacts the survival and function of engineered tissues after transplantation. Herein, a novel bilayer silk fibroin (BSF) scaffold was fabricated with the capability of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB) sequential release. The outer layer of the scaffold was composed of compact SF film with waterproofness to mimic the serosa of the bladder. The inner layer was constructed of porous SF matrix incorporated with SF microspheres (MS) loaded with VEGF and PDGF-BB. We found that the 5% (w/v) MS-incorporated scaffold exhibited a rapid release of VEGF, whereas the 0.2% (w/v) MS-incorporated scaffold demonstrated a slow and sustained release of PDGF-BB. The BSF scaffold exhibited good biocompatibility and promoted endothelial cell migration, tube formation and enhanced endothelial differentiation of adipose derived stem cells (ADSCs) in vitro. The BSF patch was constructed by seeding ADSCs on the BSF scaffold. After in vivo transplantation, not only could the BSF patch facilitate the regeneration of urothelium and smooth muscle, but more importantly, stimulate the regeneration of blood vessels. This study demonstrated that the BSF patch exhibited excellent vascularization capability in bladder reconstruction and offered a viable functional bioengineered patch for future clinical studies.

IN VITRO MODELS MIMICKING THE BONE REMODELLING CYCLE

Metabolic bone diseases, such as osteoporosis and osteopetrosis, result from an imbalanced bone remodeling process. In vitro bone models are often used to investigate either bone formation or resorption independently, while in vivo, these processes are coupled. Combining these processes in a co-culture is challenging as it requires finding the right medium components to stimulate each cell type involved without interfering with the other cell type's differentiation. Furthermore, differentiation stimulating factors often comprise growth factors in supraphysiological concentrations, which can overshadow the cell-mediated crosstalk and coupling.To address these challenges, we aimed to recreate the physiological bone remodeling process, which follows a specific sequence of events starting with cell activation and bone resorption by osteoclasts, reversal, followed by bone formation by osteoblasts. We used a mineralized silk fibroin scaffold as a bone-mimetic template, inspired by bone's extracellular matrix composition and organization. Our model supported osteoclastic resorption and osteoblastic mineralization in the specific sequence that represents physiological bone remodeling.We also demonstrated how culture variables, such as different cell ratios, base media, and the use of osteogenic/osteoclast supplements, and the application of mechanical load, can be adjusted to represent either a high bone turnover system or a self-regulating system. The latter system did not require the addition of osteoclastic and osteogenic differentiation factors for remodeling, therefore avoiding growth factor use.Our in vitro model for bone remodeling has the potential to reduce animal experiments and advance in vitro drug development for bone remodeling pathologies like osteoporosis. By recreating the physiological bone remodeling cycle, we can investigate cell-cell and cell-matrix interactions, which are essential for understanding bone physiology and pathology. Furthermore, by tuning the culture variables, we can investigate bone remodeling under various conditions, potentially providing insights into the mechanisms underlying different bone disorders.

Electrical stimulation with polypyrrole-coated polycaprolactone/silk fibroin scaffold promotes sacral nerve regeneration by modulating macrophage polarisation.

Peripheral nerve injury poses a great threat to neurosurgery and limits the regenerative potential of sacral nerves in the neurogenic bladder. It remains unknown whether electrical stimulation can facilitate sacral nerve regeneration in addition to modulate bladder function. The objective of this study was to utilise electrical stimulation in sacra nerve crush injury with newly constructed electroconductive scaffold and explore the role of macrophages in electrical stimulation with crushed nerves. As a result, we generated a polypyrrole-coated polycaprolactone/silk fibroin scaffold through which we applied electrical stimulation. The electrical stimulation boosted nerve regeneration and polarised the macrophages towards the M2 phenotype. An in vitro test using bone marrow derived macrophages revealed that the pro-regenerative polarisation of M2 were significantly enhanced by electrical stimulation. Bioinformatics analysis showed that the expression of signal transducer and activator of transcriptions (STATs) was differentially regulated in a way that promoted M2-related genes expression. Our work indicated the feasibility of electricals stimulation used for sacral nerve regeneration and provided a firm demonstration of a pivotal role which macrophages played in electrical stimulation.

Microstructured silk fiber scaffolds with enhanced stretchability.

Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of β-sheet nanocrystals that severely embrittle SF, as well as hierarchical fiber organization at both micro- and macroscale. Here, we introduce a fabrication process based on electrowriting of aqueous SF solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH2PO4). This approach enables gelation of SF chains via controlled β-sheet formation and partial conservation of compliant random coil structures. Moreover, this process allows for precise architecture control in microfiber scaffolds, enabling the creation of 3D flat and tubular macro-geometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 μm and ∼97% open porosity. Remarkably, the crosslinked printed structures demonstrated a balanced coexistence of β-sheet and random coil conformations, which is uncommon for organic solvent-based crosslinking methods. This synergy of printing and post-processing yielded stable scaffolds with high compliance (modulus = 0.5-15 MPa) and the ability to support elastic cyclic loading up to 20% deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human renal epithelial and endothelial cells with viability above 95%. These cells formed homogeneous monolayers that aligned with the fiber direction and deposited type-IV collagen as a specific marker of healthy extracellular matrix, indicating that both cell types attach, proliferate, and organize their own microenvironment within the SF scaffolds. These findings represent a significant development in fabricating organized stable SF scaffolds with unique microfiber structures and mechanical and biological properties that make them highly promising for tissue engineering applications.

Biomimetic triphasic silk fibroin scaffolds seeded with tendon-derived stem cells for tendon-bone junction regeneration.

The regeneration of tendon and bone junctions (TBJs), a fibrocartilage transition zone between tendons and bones, is a challenge due to the special triphasic structure. In our study, a silk fibroin (SF)-based triphasic scaffold consisting of aligned type I collagen (Col I), transforming growth factor β (TGF-β), and hydroxyapatite (HA) was fabricated to mimic the compositional gradient feature of the native tendon-bone architecture. Rat tendon-derived stem cells (rTDSCs) were loaded on the triphasic SF scaffold, and the high cell viability suggested that the scaffold presents good biocompatibility. Meanwhile, increased expressions of tenogenic-, chondrogenic-, and osteogenic-related genes in the TBJs were observed. The in vivo studies of the rTDSC-seeded scaffold in a rat TBJ rupture model showed tendon tissue regeneration with a clear transition zone within 8 weeks of implantation. These results indicated that the biomimetic triphasic SF scaffolds seeded with rTDSCs have great potential to be applied in TBJ regeneration.