Silk Fibroin Scaffolds Research Articles

Mechanically enhanced biodegradable scaffold based on SF microfibers for repairing bone defects in the distal femur of rats

Silk-based biodegradable materials play an important role in tissue engineering, especially in the field of bone regeneration. However, while optimizing mechanical properties and bone regeneration characteristics, modified silk fibroin (SF)-based materials also increase the complexity of scaffold systems, which is not conducive to clinical translation. In this study, we first added synthetic biomimetic mineralized collagen (MC) particles to SF-based materials to improve the bone regeneration properties of the scaffolds and simultaneously regulated the degradation rate of the scaffolds to match the bone regeneration rate. Second, SF microfibers were prepared by hydrolysis with alkaline heating and added to SFMC scaffolds with excellent osteogenic stimulation ability to prepare SF microfiber (mf)-modified SFMC-mf scaffolds with excellent mechanical properties, whose compression modulus increased from 4.58±0.23 MPa to 14.63±0.88 MPa. Finally, the SFMC-mf scaffold was implanted into the weight-bearing bone defect area of the distal femur of rats, and the results showed that the SFMC-mf scaffold significantly promoted functional recovery of the affected limb and increased the amount of new bone in the defect area compared with those in the SFC-mf group and the blank control group. In addition, the RNA-seq results suggested that the genes with upregulated expression in the SFMC-mf scaffold group were mainly enriched in vascular regeneration. In conclusion, this SF microfiber modification method effectively improved the mechanical properties of SFMC scaffolds without moving the SF scaffold system in the direction of compositional complexity, providing new insights for the subsequent development of more effective bionic repair materials for bone defects and assisting in their clinical translation.

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.

Piezoelectric silk fibroin nanofibers: Structural optimization to enhance piezoelectricity and biostability for neural tissue engineering

Silk fibroin (SF) has garnered significant interest in tissue engineering due to its excellent biocompatibility and bioactivity. Notably, SF exhibits piezoelectric properties that can be harnessed to electrically stimulate cells, enhancing tissue morphogenesis. However, a systematic approach to control SF's piezoelectricity and biodegradation, and its application in neural morphogenesis, has not been fully explored. In this study, SF was electrospun into nanofibers of various sizes and subjected to a post-electrospinning chemical treatment to examine the effects of phase composition, especially the β-sheet formation, on the piezoelectricity and biostability of SF. The optimized SF scaffolds having a nanofiber diameter of 750 nm and a scaffold thickness of 250 µm allowed for the maintenance of nanofibrous structure for at least 6 weeks in aqueous environments while maintaining piezoelectric potential outputs of at least 200 mVp-p. This enabled cell membrane depolarization over an extended cell culture duration, facilitating the functional maturation of human neural stem cells (hNSCs). Specifically, acoustic piezo-activation of the SF scaffolds, resulting in mechano-electrical stimulation (MES) of the cells, accelerated their differentiation into neurons, astrocytes, and especially oligodendrocytes, leading to robust axon myelination within two weeks. Furthermore, MES enhanced neural connectivity and functional maturity, as evidenced by significant increases in excitatory and inhibitory synapse formation (46 % and 58 %, respectively), action potential amplitude (252 %), and velocity (210 %) compared to static control conditions. These findings demonstrate the potential of biodegradable electrospun SF nanofibers with balanced piezoelectricity and biostability for advancing neural tissue engineering.

Silk fibroin/chitosan thiourea hydrogel scaffold with vancomycin and quercetin-loaded PLGA nanoparticles for treating chronic MRSA osteomyelitis in rats

Chronic osteomyelitis presents significant treatment challenges, necessitating an efficient system for infection elimination and bone repair. This study developed a natural hydrogel scaffold using silk fibroin (SF) and chitosan thiourea (CST), incorporating vancomycin (VC) and quercetin (QC) loaded PLGA nanoparticles (NPs) for dual-purpose treatment. SF/CST hydrogel scaffolds exhibited homogeneous porosity and smaller interconnected pore size than pure SF and pure CST hydrogel scaffolds. Optimal PLGA/QC NPs measured 206 nm in size, displayed spherical morphology, had uniform distribution, and achieved 87 % QC loading. The release study showed sustained long-term release of VC and QC from the hydrogel scaffolds for over 20 days. Biocompatibility tests indicated that hydrogel scaffolds promoted osteoblast adhesion without cytotoxicity, with QC-containing scaffolds enhancing osteoblast growth. Antibacterial tests confirmed retained VC activity against methicillin-resistant Staphylococcus aureus (MRSA) in SF/CST. An experimental study assessed the efficacy of the hydrogel scaffolds in a MRSA-infected rat osteomyelitis model. Radiographic scores demonstrated a significant reduction for SF/CST-VC-PLGA/QC NPs compared to control, indicating reduced osteomyelitis effects. Macroscopic evaluations showed notable reductions in gross pathological effects for VC-containing groups. Histopathological assessments revealed significantly lower osteomyelitis scores and higher healing scores in the SF/CST-VC-PLGA/QC NPs, with reduced inflammatory cell infiltration and more organized connective tissue formation. In conclusion, SF/CST-VC-PLGA/QC NPs is an effective dual drug delivery system for osteomyelitis treatment, demonstrating significant antibacterial activity, enhanced bone regeneration, and reduced infection rate.

An In Vitro Macrophage Response Study of Silk Fibroin and Silk Fibroin/Nano-Hydroxyapatite Scaffolds for Tissue Regeneration Application.

In recent years, silk fibroin (SF) has been incorporated with low crystallinity nanohydroxyapatite (nHA) as a scaffold for various tissue regeneration applications due to the mechanical strength of SF and osteoconductive properties of nHA. However, currently, there is a lack of understanding of the immune response toward the degradation products of SF with nHA composite after implantation. It is known that particulate fragments from the degradation of a biomaterial can trigger an immune response. As the scaffold is made of degradable materials, the degradation products may contribute to the inflammation. Therefore, in this study, the effects of the enzymatic degradation of the SF/nHA scaffold on macrophage response were investigated in comparison to the control SF scaffold. Since the degradation products of a scaffold can influence macrophage polarization, it can be hypothesized that as the SF and SF/nHA scaffolds were degraded in vitro using protease XIV solution, the degradation products can contribute to the polarization of THP-1-derived macrophages from pro-inflammatory M1 to anti-inflammatory M2 phenotype. The results demonstrated that the initial (day 1) degradation products of the SF/nHA scaffold elicited a pro-inflammatory response, while the latter (day 24) degradation products of the SF/nHA scaffold elicited an anti-inflammatory response. Moreover, the degradation products from the SF scaffold elicited a higher anti-inflammatory response due to the faster degradation of the SF scaffold and a higher amino acid concentration in the degradation solution. Hence, this paper can help elucidate the contributory effects of the degradation products of SF and SF/nHA scaffolds on macrophage response and provide greater insights into designing silk-based biomaterials with tunable degradation rates that can modulate macrophage response for future tissue regeneration applications.

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.

A facile nanopattern modification of silk fibroin electrospun scaffold and corresponding impact on cell proliferation and osteogenesis

Abstract As a well-known natural protein biomaterial, silk fibroin (SF) has shown broad application prospects in typical biomedical fields. However, the mostly used SF from Bombyx mori silkworm lacks specific cell adhesion sites and other bioactive peptide sequences, and there is still significant room for further improvement of their biological functions. Therefore, it is crucial to develop a facile and effective modification strategy for this widely researched biomaterial. In this study, the SF electrospun scaffold has been chosen as a typical SF biomaterial, and air plasma etching has been adopted as a facile nanopattern modification strategy to promote its biological functions. Results demonstrated that the plasma etching could feasibly and effectively create nano-island-like patterns on the complex surface of SF scaffolds, and the detailed nanopattern features could be easily regulated by adjusting the etching time. In addition, the mesenchymal stem cell responses have illustrated that the nanopattern modification could significantly regulate corresponding cell behaviors. Compared with the non-etched scaffold, the 10 min-etched scaffold (10E scaffold) significantly promoted stem cell proliferation and osteogenic differentiation. Moreover, 10E scaffold has also been confirmed to effectively accelerate vascularization and ectopic osteogenesis in vivo using a rat subcutaneous implantation model. However, the mentioned promoting effects would be weakened or even counteracted with the increase of etching time. In conclusion, this facile modification strategy demonstrated great application potential for promoting cell proliferation and differentiation. Thus, it provided useful guidance to develop excellent SF-based scaffolds suitable for bone and other tissue engineering.

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.

Tissue scaffolds mimicking hierarchical bone morphology as biomaterials for oral maxillofacial surgery with augmentation: structure, properties, and performance evaluation for in vitro testing

In this study, tissue scaffolds mimicking hierarchical morphology are constructed and proposed for bone augmentation. The scaffolds are fabricated using lyophilization, before coating them with collagen (Col). Subsequently, the Col-coated scaffolds undergo a second lyophilization, followed by silk fibroin (SF) coating, and a third lyophilization. Thereafter, the scaffolds are divided into six groups with varying ratios of Col to SF: Col/SF = 7:3, 5:5, 3:7, 10:0, and 0:10, with an SF scaffold serving as the control group. The scaffold morphology is examined using a scanning electron microscope, while molecular and structural formations are characterized by Fourier transform infrared spectrometer and differential scanning calorimeter, respectively. Physical and mechanical properties including swelling and compression are tested. Biological functions are assessed throughin vitroosteoblast cell culturing. Biomarkers indicative of bone formation-cell viability and proliferation, alkaline phosphatase activity, and calcium content-are analyzed. Results demonstrate that scaffolds coated with Col and SF exhibit sub-porous formations within the main pore. The molecular formation reveals interactions between the hydrophilic groups of Col and SF. The scaffold structure contains bound water and SF formation gets disrupted by Col. Physical and mechanical properties are influenced by the Col/SF ratio and morphology due to coating. The biological functions of scaffolds with Col and SF coating show enhanced potential for promoting bone tissue formation, particularly the Col/SF (7:3) ratio, which is most suitable for bone augmentation in small defect areas.

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.

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

Streamlining Skin Regeneration: A Ready-To-Use Silk Bilayer Wound Dressing.

Silk proteins have been highlighted in the past decade for tissue engineering (TE) and skin regeneration due to their biocompatibility, biodegradability, and exceptional mechanical properties. While silk fibroin (SF) has high structural and mechanical stability with high potential as an external protective layer, traditionally discarded sericin (SS) has shown great potential as a natural-based hydrogel, promoting cell-cell interactions, making it an ideal material for direct wound contact. In this context, the present study proposes a new wound dressing approach by developing an SS/SF bilayer construct for full-thickness exudative wounds. The processing methodology implemented included an innovation element and the cryopreservation of the SS intrinsic secondary structure, followed by rehydration to produce a hydrogel layer, which was integrated with a salt-leached SF scaffold to produce a bilayer structure. In addition, a sterilization protocol was developed using supercritical technology (sCO2) to allow an industrial scale-up. The resulting bilayer material presented high porosity (>85%) and interconnectivity while promoting cell adhesion, proliferation, and infiltration of human dermal fibroblasts (HDFs). SS and SF exhibit distinct secondary structures, pore sizes, and swelling properties, opening new possibilities for dual-phased systems that accommodate the different needs of a wound during the healing process. The innovative SS hydrogel layer highlights the transformative potential of the proposed bilayer system for biomedical therapeutics and TE, offering insights into novel wound dressing fabrication.

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.

An In Situ-Gelling Conductive Hydrogel for Potential Use in Neural Tissue Engineering.

Cerebral cavitation is usual following acute brain injuries, such as stroke and traumatic brain injuries, as well as after tumor resection. Minimally invasive implantation of an injectable scaffold in the cavity is a promising approach for potential regeneration of tissue loss. This study aimed at designing an in situ-gelling conductive hydrogel containing silk fibroin (SF), brain decellularized extracellular matrix (dECM), and carbon nanotubes (CNT) for potential use in brain tissue regeneration. Two percent w/v SF hydrogels with different concentrations of dECM (0.1%, 0.2%, or 0.3% w/v) and CNTs (0.05%, 0.1%, or 0.25% w/v) were fabricated and characterized. It was observed that with the addition of dECM, the porosity decreased, whereas swelling and electrical conductivity tended to increase. The addition of dECM also led to a faster resorption rate, but no significant change in compressive modulus. Addition of CNTs, on the other hand, led to a denser, stronger, and more regular porous structure, higher swelling ratio, faster gelation time, slower degradation rate, and a significant increase in electrical conductivity. dECM and CNTs combined together resulted in superior porosity, swelling, resorption rate, mechanical properties, and electrical conductivity compared with SF scaffolds containing only dECM or CNTs. Hydrogel samples containing 2% SF, 0.3% dECM, and 0.1% CNTs had a high porosity (58.9%), low swelling ratio (15.9%), high conductivity (2.35 × 10-4 S/m), and moderate degradation rate (37.3% after 21 days), appropriate for neural tissue engineering applications. Cell evaluation studies also showed that the hydrogel systems support the cell adhesion and growth, with no sign of significant cytotoxicity. Impact statement Tissue loss and formation of a fluid-filled cavity following stroke, traumatic brain injury, or brain tumor resection lead to sensorimotor and/or cognitive deficits. The lack of a healthy extracellular matrix in the cavity avoids the endogenous cell migration and axonal sprouting and may also worsen the secondary injuries to peri-lesional tissue. Due to the brain anatomy, simple implantation of tissue engineering scaffolds to the injured site is not possible in many cases. Therefore, the development of injectable scaffolds that support neural growth and differentiation is crucial for tissue repair or limiting the expansion of damage region.

Dual therapeutic approach: Biodegradable nanofiber scaffolds of silk fibroin and collagen combined with silver and gold nanoparticles for enhanced bacterial infections treatment and accelerated wound healing

The development of novel multifunctional wound healing materials based on natural bioactives has garnered significant interest in recent years due to their ability to stimulate the healing process and alleviate problems associated with standard wound dressings. In the presented work, a bimetallic combination of silver (Ag) and gold (Au) nanoparticles incorporated into silk fibroin (SF) and collagen (CL) composite nanofiber (CNF) using the electrospinning technique. The physicochemical investigations confirmed the successful integration of Ag and Au nanoparticles sizes of approximately 6.76 nm and 9.75 nm, with an average nanofiber diameter of 189.46 ± 108.73 nm. The evaluation of composite nanofiber treated in the in vitro antibacterial tests zone of inhibition value S. aureus (17 mm) and E. coli (18 mm). Additionally, we analyzed the cell proliferation study using NIH3T3 fibroblast cell line treated with the composite nanofiber material. The SF/CL/Ag–Au composite nanofiber exhibited the highest level of cell proliferation compared to other matrices. This increased cell proliferation offers significant advantages in wound healing applications. Furthermore, in vivo study on wound healing activity in Sprague-Dawley rats demonstrated a remarkable healing percentage of 98.85%. These findings collectively highlight the promising potential of the SF/CL/Ag–Au composite nanofiber in promoting accelerated wound healing. The composite nanofibers demonstrated remarkable hemocompatibility of erythrocyte lysis percentages varying between 1.5% and 3.9%. Besides, swelling tests revealed that the material can absorb exudates from wound sites, facilitating tissue regeneration and cell adhesion. Finally, this research highlights the potential of the SF/CL/Ag–Au composite nanofiber excellent antibacterial activity, highest biocompatibility, enhanced wound healing, and highly suitable for various biomedical applications.

Incorporating bioactive glass nanoparticles in silk fibroin/bacterial nanocellulose composite scaffolds improves their biological and osteogenic properties for bone tissue engineering applications

Blend polymers composed of natural polymers are a ubiquitous biomaterial class due to their suitable mechanical and biological characterization. In the present study, composite scaffolds based on bacterial cellulose (BC)/silk fibroin (SF) with bioactive glass nanoparticles (BGNPs) were developed to enhance osteogenesis in human adipose derived stem cells (hASCs). The scanning electron microscopy (SEM) results of BGNPs indicated a spherical morphology and size ranging from 15 to 30 nm. The presence of BC and BGNPs reduced the pore diameter of SF scaffolds to about 210 ± 10 μm and 205 ± 10 μm, respectively, while increasing their compressive strength and compressive modulus. FTIR analyses proved the presence of BGNPs, BC and SF in the scaffolds. Flow cytometry data confirmed the surface markers for hASCs. The results also showed that BC and BGNPs addition to BC/SF scaffolds decreased degradation and swelling rate. The gene expression (Runx2, alkaline phosphatase and osteocalcin) studies signified the osteogenic potential of BGNPs in BC/SF scaffolds on hASCs. Eventually, the increased cell adhesion, viability and differentiation in the BC/SF and BC/SF/BGNPs composite scaffolds drawn from MTT, SEM, Alizarin red staining and alkaline phosphatase activity confirmed that these scaffolds promise to serve as a therapeutic candidate for bone defects.

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.