Silk Fibroin Scaffolds Research Articles

Silk-Based Matrices and c-Kit-Positive Cardiac Progenitor Cells for a Cellularized Silk Fibroin Scaffold: Study of an in vivo Model

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-Kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the 3-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with 3 different geometries and tested their behavior in 3 different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocyte aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-Kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds which may preserve the survival of these cells in vivo also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

The stiffness variability of a silk fibroin scaffold during bone cell proliferation.

Complex assessment of gradual changes in scaffold morphology and stiffness is an essential step in bone filler development. Current approach, however, does not reflect long term cell proliferation effect as the mechanical tests are usually conducted on pristine materials without cells or cell influence on material stiffness is evaluated after one time period only. Here, biocompatible silk fibroin (SF) porous scaffolds envisioned for bone defect filling were prepared by dissolving of fibroin fibers, followed by dialysis, freeze-drying and final stabilization. Particular attention was devoted to the influence of bone cell proliferation up to 2 months on the stiffness of the material. The morphology of the material was studied in terms of its inner structure and the overall changes in the surface characteristics due to proliferation of MG 63 bone cell line. The SF scaffold stiffness significantly increased during first month followed by its decline during second month due to bone cell seeding. After 2 months, the SF scaffold was completely colonized, which resulted in a gradual decay of its structure. The length of degradation due to bone cell proliferation and mechanical behavior corresponded to the requirements set for reasonable filler material indicating that porous SF scaffolds comprise a promising biomaterial for bone regeneration.

Muscle-derived stem cells in silk fibroin hydrogels promotes muscle regeneration and angiogenesis in sheep models: an experimental study.

Silk fibroin (SF) hydrogels are of high interest in tissue engineering. However, angiogenesis is one of the major challenges in tissue regeneration and repair. In this study, we present a simple and effective method to develop a 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG)-SF hydrogel. The SF hydrogels had no immunogenicity and approached natural tissues. The SF scaffolds were first prepared from Bombyx mori silkworms and DMPG. The SF scaffold was seeded with muscle-derived stem cells derived from sheep embryo and implanted in the tibialis anterior muscle of mature sheep. Gelation time, H&E staining, and histochemistry were conducted and observed. The suitability of the hydrogels for 3D cell culture was assessed by living cell stain CM-Dil. The results showed that the SF hydrogels resembled the mechanical properties of natural soft tissues better. The results of H&E staining and histochemistry revealed that the degradation rate showed an S-type change, and muscle regeneration and angiogenesis were clearly visible. Adverse effects were not observed in the sheep models. DMPG-induced SF hydrogels can be successfully used for in situ cell encapsulation. It provides promising opportunities in biomedical applications, such as in tissue engineering and regeneration.

Fabrication of channeled scaffolds through polyelectrolyte complex (PEC) printed sacrificial templates for tissue formation.

One of the pivotal factors that limit the clinical translation of tissue engineering is the inability to create large volume and complex three-dimensional (3D) tissues, mainly due to the lack of long-range mass transport with many current scaffolds. Here we present a simple yet robust sacrificial strategy to create hierarchical and perfusable microchannel networks within versatile scaffolds via the combination of embedded 3D printing (EB3DP), tunable polyelectrolyte complexes (PEC), and casting methods. The sacrificial templates of PEC filaments (diameter from 120 to 500 μm) with arbitrary 3D configurations were fabricated by EB3DP and then incorporated into various castable matrices (e.g., hydrogels, organic solutions, meltable polymers, etc.). Rapid dissolution of PEC templates within a 2.00 M potassium bromide aqueous solution led to the high fidelity formation of interconnected channels for free mass exchange. The efficacy of such channeled scaffolds for in vitro tissue formation was demonstrated with mouse fibroblasts, showing continuous cell proliferation and ECM deposition. Subcutaneous implantation of channeled silk fibroin (SF) scaffolds with a porosity of 76% could lead to tissue ingrowth as high as 53% in contrast to 5% for those non-channeled controls after 4 weeks. Both histological and immunofluorescence analyses demonstrated that such channeled scaffolds promoted cellularization, vascularization, and host integration along with immunoregulation.

Platelet-Derived Growth Factor-Functionalized Scaffolds for the Recruitment of Synovial Mesenchymal Stem Cells for Osteochondral Repair.

Cartilage regeneration is still a challenge for clinicians because of avascularity, denervation, load-bearing, synovial movement, and the paucity of endogenous repair cells. We constructed a multilayered osteochondral bionic scaffold and examined its repair capacity using a rabbit osteochondral defect model. The cartilage phase and interface layer of the scaffold were prepared by freeze-drying, whereas the bone phase of the scaffold was prepared by high-temperature sintering. The three-phase osteochondral bionic scaffold was formed by joining the hydroxyapatite (HAp) and silk fibroin (SF) scaffolds using the repeated freeze-thaw method. Different groups of scaffolds were implanted into the rabbit osteochondral defect model, and their repair capacities were assessed using imaging and histological analyses. The cartilage phase and the interface layer of the scaffold had a pore size of 110.13 ± 29.38 and 96.53 ± 33.72 μm, respectively. All generated scaffolds exhibited a honeycomb porous structure. The polydopamine- (PDA-) modified scaffold released platelet-derived growth factor (PDGF) for 4 weeks continuously, reaching a cumulative release of 71.74 ± 5.38%. Synovial mesenchymal stem cells (SMSCs) adhered well to all scaffolds, but demonstrated the strongest proliferation ability in the HSPP (HAp-Silk-PDA-PDGF) group. Following scaffold-induced chondrogenic differentiation, SMSCs produced much chondrocyte extracellular matrix (ECM). In in vivo experiments, the HSPP group exhibited a significantly higher gross tissue morphology score and achieved cartilage regeneration at an earlier stage and a significantly better repair process compared with the other groups (P < 0.05). Histological analysis revealed that the new cartilage tissue in the experimental group had a better shape and almost filled the defect area, whereas the scaffold was nearly completely degraded. The new cartilage was effectively fused with the surrounding normal cartilage, and a substantial amount of chondrocyte ECM was formed. The SF/HAp three-layer osteochondral bionic scaffold exhibited favorable pore size, porosity, and drug sustained-release properties. It demonstrated good biocompatibility in vitro and encouraging repair effect at osteochondral defect site in vivo, thereby expected to enabling the repair and regeneration of osteochondral damage.

Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm−1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Silk Protein Composite Bioinks and Their 3D Scaffolds and In Vitro Characterization.

This paper describes the use of silk protein, including fibroin and sericin, from an alkaline solution of Ca(OH)2 for the clean degumming of silk, which is neutralized by sulfuric acid to create calcium salt precipitation. The whole sericin (WS) can not only be recycled, but completely degummed silk fibroin (SF) is also obtained in this process. The inner layers of sericin (ILS) were also prepared from the degummed silk in boiling water by 120 °C water treatment. When the three silk proteins (SPs) were individually grafted with glycidyl methacrylate (GMA), three grafted silk proteins (G-SF, G-WS, G-ILS) were obtained. After adding I2959 (a photoinitiator), the SP bioinks were prepared with phosphate buffer (PBS) and subsequently bioprinted into various SP scaffolds with a 3D network structure. The compressive strength of the SF/ILS (20%) scaffold added to G-ILS was 45% higher than that of the SF scaffold alone. The thermal decomposition temperatures of the SF/WS (10%) and SF/ILS (20%) scaffolds, mainly composed of a β-sheet structures, were 3 °C and 2 °C higher than that of the SF scaffold alone, respectively. The swelling properties and resistance to protease hydrolysis of the SP scaffolds containing sericin were improved. The bovine insulin release rates reached 61% and 56% after 5 days. The L929 cells adhered, stretched, and proliferated well on the SP composite scaffold. Thus, the SP bioinks obtained could be used to print different types of SP composite scaffolds adapted to a variety of applications, including cells, drugs, tissues, etc. The techniques described here provide potential new applications for the recycling and utilization of sericin, which is a waste product of silk processing.

Bionic tracheal tissue regeneration using a ring-shaped scaffold comprised of decellularized cartilaginous matrix and silk fibroin

Reconstruction of full-circumferential segmental tracheal defect remains an international dilemma and the key challenge is the lack of a bionic tracheal substitute, with a specific configuration of cartilaginous rings interspersed with vascularized fibrous tissue (CRVFT). We previously demonstrated the formation of a bionic tracheal substitute with CRVFT in-vivo. However, it is still desirable to develop a scaffold with proper mechanical strength and chondroinductive activity to promote in-vivo cartilage formation, which could circumvent the painstaking procedure of in-vitro cultivation. Herein, we prepared a ring-shaped porous silk fibroin (SF)-reinforced decellularized cartilaginous matrix (DCM) (DCM/SF) scaffold, which displayed suitable pore size (206.7 ± 12.5 μm) and porosity (92.7 ± 2.5%) and was biocompatible for cell colonization. The addition of SF considerable enhanced anti-contraction capacity and Young's modulus, while diminishing water absorption and degradation rate of the DCM/SF scaffold. Further, the DCM/SF scaffold obviously promoted chondrogenesis of the embedded bone marrow stem cells (BMSCs), compared to DCM or SF scaffold alone. In addition, a cartilaginous ring was formed using the DCM/SF scaffold, which was repopulated with BMSCs after subcutaneous implantation in nude mouse. Moreover, a bionic tracheal tissue with remarkable CRVFT was achieved via the interrupted stacking of BMSC-DCM/SF constructs on a stent, before subcutaneous implantation into a rabbit for 4 weeks. The bionic trachea was fully revascularized and displayed comparable biochemical compositions and mechanical strength resembling to those of normal trachea. This study introduces a reliable new approach for bionic tracheal tissue regeneration and significantly advances the ongoing repair of segmental tracheal defect.

3D Silk Fibroin-Gelatin/Hyaluronic Acid/Heparan Sulfate Scaffold Enhances Expression of Stemness and EMT Markers in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a stem cell-based cancer. The in vivo tumor microenvironment is not present in two-dimensional (2D) cultures, which is one of the limitations in cancer stem cell (CSC) research. Thus, we aimed to establish three-dimensional (3D) culture mimicking extracellular matrix (ECM) that could serve as a niche for CSC enrichment in CCA. Silk fibroin-gelatin/hyaluronic acid/heparan sulfate (SF-GHHs) scaffolds were fabricated by lyophilization in various ratios and compared to silk fibroin (SF) scaffold. The physical and biological characteristics of the scaffolds were investigated. The SF-GHHs 1:2 scaffold with pore size of 350±102 μm harbored optimal porosity, good water uptake, and stable beta-sheet that supported the increase in KKU-213A cell proliferation and aggregation. The CSC and the epithelial-mesenchymal transition (EMT) markers were significantly upregulated in this scaffold compared to 2D. Moreover, drug sensitivity against cisplatin and gemcitabine in 3D culture was significantly higher than that in 2D culture. The SF-GHHs 1:2 scaffold could simulate ECM that may serve as a CSC niche of CCA, and reinforce stemness and EMT properties, suggesting its suitability for 3D CCA model, which supports CSC and new targeting drug research in CCA.

Development of Silk Fibroin Scaffolds by Using Indirect 3D-Bioprinting Technology.

Due to the excellent biocompatibility of natural polymers, a variety of natural polymers have been widely used as biomaterials for manufacturing tissue engineered scaffolds. Despite the excellent biological activity of natural polymers, there have been obstacles in using them on their own to prepare 3D scaffolds with sufficient mechanical strength. Although multiple 3D-bioprinting technologies have recently emerged as effective manufacturing tools for scaffold preparation, scaffold preparation using only natural polymers with tunable mechanical properties is still difficult. Herein, we introduce novel scaffold fabrication methods using the natural polymer silk fibroin via indirect 3D-bioprinting technology. The developed silk fibroin scaffolds showed biocompatibility and tunable mechanical strength by changing the concentration of the silk fibroin. Furthermore, controlling the flexibility of the silk fibroin scaffolds was made possible by changing the solvent for the silk fibroin solution used to fabricate the scaffold. Consequently, silk fibroin scaffolds fabricated via our method can be considered for various applications in the bioengineering of either soft or musculoskeletal tissues.

Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.

Radially Aligned Porous Silk Fibroin Scaffolds as Functional Templates for Engineering Human Biomimetic Hepatic Lobules.

Bioengineering functional hepatic tissue constructs that physiologically replicate the human native liver tissue in vitro is sought for clinical research and drug discovery. However, the intricate architecture and specific biofunctionality possessed by the native liver tissue remain challenging to mimic in vitro. In the present study, a versatile strategy to fabricate lobular-like silk protein scaffolds with radially aligned lamellar sheets, interconnected channels, and a converging central cavity was designed and implemented. A proof-of-concept study to bioengineer biomimetic hepatic lobules was conducted through coculturing human hepatocytes and primary endothelial cells on these lobular-like scaffolds. Relatively long-term viability of hepatocyte/endothelial cells was found along with cell alignment and organization in vitro. The hepatocytes showed special epithelial polarity and bile duct formation, similar to the liver plate, while the aligned endothelial cells generated endothelial networks, similar to natural hepatic sinuses. This endowed the three-dimensional (3D) tissue constructs with the capability to recapitulate hepatic-like parenchymal-mesenchymal growth patterns in vitro. More importantly, the cocultured hepatocytes outperformed monocultures or monolayer cultures, displaying significantly enhanced hepatocyte functions, including functional gene expression, albumin (ALB) secretion, urea synthesis, and metabolic activity. Thus, this functional unit with a biomimetic phenotype provides a novel technology for bioengineering biomimetic hepatic lobules in vitro, with potential utility as a building block for bioartificial liver (BAL) engineering or as a robust tool for drug metabolism investigation.

Role of Hypoxia on Growth and Differentiation of Human Adipose Derived Stem Cells Grown on Silk Fibroin Scaffold Induced by Platelet Rich Plasma

Previous research has proven that 10% platelet-rich plasma (PRP) can enhance growth and differentiation of human adipose derived stem cells (hADSC) grown on silk fibroin scaffold into chondrocytes. A low oxygen concentration (hypoxia) condition is an important factor that potentially affects the ability of hADSC to grow and differentiate. The objective of this research was to analyze the difference in growth and differentiation capacity of hADSC grown on salt leached silk fibroin scaffold supplemented by 10% PRP under normoxic and hypoxic conditions. The growth capacity of the hADSC was determined by MTT assay and differentiation was tested using glycosaminoglycan (GAG) content analysis, while chondrocyte markers were visualized with the immunocytochemistry (ICC) method. This research observed hADSC proliferation under normoxic and hypoxic conditions for 21 days. Visualization of type 2 collagen showed that it was more abundant under hypoxia compared to normoxia. HIF-1α was only detected in the hADSC cultured in hypoxic conditions. In conclusion, culture under hypoxic conditions increases the capacity of hADSC to grow and differentiate into chondrocytes. This is the first study that has shown that hypoxia is able to enhance the proliferation and differentiation of hADSC grown on 3D salt leached silk fibroin scaffold supplemented by 10% PRP.

Fabrication of Silk Scaffold Containing Simvastatin-Loaded Silk Fibroin Nanoparticles for Regenerating Bone Defects

Background:In the present study, a tissue engineered SF scaffold containing simvastatin-loaded SFNPs were used to stimulate the regeneration of the defected bone.Methods:At first, the porous SF scaffold was prepared using freeze-drying. Then simvastatin-loaded SFNPs were made by dissolvation method and embedded in the SF scaffold. Afterwards, the scaffold and the NPs were characterized in terms of physicochemical properties and the ability to release the simvastatin small molecule.Results:The results exhibited that the SF scaffold had a porous structure suitable for releasing the small molecule and inducing the proliferation and attachment of osteoblast cells. SFNPs containing simvastatin had spherical morphology and were 174 ± 4 nm in size with -24.5 zeta potential. Simvastatin was also successfully encapsulated within the SFNPs with 68% encapsulation efficiency. Moreover, the small molecule revealed a sustained release profile from the NPs during 35 days. The results obtained from the in vitro cell-based studies indicated that simvastatin-loaded SFNPs embedded in the scaffold had acceptable capacity to promote the proliferation and ALP production of osteoblast cells while inducing osteogenic matrix precipitation. Conclusion:The SF scaffold containing simvastatin-loaded SFNPs could have a good potential to be used as a bone tissue-engineered construct.

Evaluation of Bi-Layer Silk Fibroin Grafts for Penile Tunica Albuginea Repair in a Rabbit Corporoplasty Model.

The use of autologous tissue grafts for tunica albuginea repair in Peyronie’s disease and congenital chordee is often restricted by limited tissue availability and donor site morbidity, therefore new biomaterial options are needed. In this study, bi-layer silk fibroin (BLSF) scaffolds were investigated to support functional tissue regeneration of tunica albuginea in a rabbit corporoplasty model. Eighteen adult male, New Zealand white rabbits were randomized to nonsurgical controls (NSC, N = 3), or subjected to corporoplasty with BLSF grafts (N = 5); decellularized small intestinal submucosa (SIS) matrices (N = 5); or autologous tunica vaginalis (TV) flaps (N = 5). End-point evaluations were cavernosography, cavernosometry, histological, immunohistochemical, and histomorphometric assessments. Maximum intracorporal pressures (ICP) following papaverine-induced erection were similar between all groups. Eighty percent of rabbits repaired with BLSF scaffolds or TV flaps achieved full rigid erections, compared to 40% of SIS reconstructed animals. Five-minute peak erections were maintained in 60% of BLSF rabbits, compared to 20% of SIS and TV flap reconstructed rabbits. Graft perforation occurred in 60% of TV group at maximum ICP compared to 20% of BLSF cohort. Neotissues supported by SIS and BLSF scaffolds were composed of collagen type I and elastin fibers similar to NSC. SIS and TV flaps showed significantly elevated levels of corporal fibrosis relative to NSC with a corresponding decrease in corporal smooth muscle cells expressing contractile proteins. BLSF biomaterials represent emerging platforms for corporoplasty and produce superior functional and histological outcomes in comparison to TV flaps and SIS matrices for tunica albuginea repair.

Silk Fibroin Scaffolds as Biomaterials for 3D Mesenchymal Stromal Cells Cultures

Silk fibroin (SF), a protein-based fiber extracted from Bombyx mori cocoons, has recently emerged with great potential for the biomedical field to be used as a biomaterial processable in a variety of formats and applications, due to its natural characteristics. The aims of the present study were to characterize the structural properties of the SF scaffolds, in the format of porous sponges, and to investigate their feasibility to support the adhesion of mesenchymal stromal/stem cells isolated from human Wharton’s jelly of the umbilical cord (WJ-MSC). Adhesion is a prerequisite for using the SF scaffold as biomaterial for supporting three-dimensional (3D) WJ-MSC cultures for several applications. The integration among micro-computed tomography, confocal analysis, and field emission scanning electron microscopy allowed carrying out a deep investigation based on quantitative morphological parameters and qualitative observations at high resolution. High levels of porosity, interconnection, and contact surface–volume ratio confirmed the appropriateness of the designed SF porous scaffolds as supports for cell cultures. WJ-MSC was demonstrated to be capable of adhering to and colonizing the SF scaffold applicable as a 3D cell culture system, of conducting in vitro experiments in a more controlled environment, and possibly of being used in tissue engineering, regenerative medicine, and applications in oncology.

A Naringin-loaded gelatin-microsphere/nano-hydroxyapatite/silk fibroin composite scaffold promoted healing of critical-size vertebral defects in ovariectomised rat

In this study, we investigated the effect of three-dimensional of naringin/gelatin microspheres/nano-hydroxyapatite/silk fibroin (NG/GMs/nHA/SF) scaffolds on repair of a critical-size bone defect of lumbar 6 in osteoporotic rats. In this work, a cell-free scaffold for bone-tissue engineering based on a silk fibroin (SF)/nano-hydroxyapatite (nHA) scaffold was developed. The scaffold was fabricated by lyophilization. Naringin (NG) was loaded into gelatin microspheres (GMs), which were encapsulated in the nHA/SF scaffolds. The materials were characterized using x ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis. Moreover, the biomechanics, degradation, and drug-release profile of the scaffold were also evaluated. In vitro, the effect of the scaffold on the adhesion, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) was evaluated. In vivo, at 3 months after ovariectomy, a critical-size lumbar defect was indued in the rats to evaluate scaffold therapeutic potential. A 3-mm defect in L6 developed in 60 SD rats, which were randomly divided into SF scaffold, nHA/SF scaffold, NG/nHA/SF scaffold, NG/GMs/nHA/SF scaffold, and blank groups (n = 12 each). At 4, 8, 12, and 16 weeks postoperatively, osteogenesis was evaluated by X-ray, micro-computed tomography, hematoxylin-eosin staining, and fast green staining, and by analysis of BMP-2, Runx2, and Ocn protein levels at 16 weeks. In our results, NG/GM/nHA/SF scaffolds exhibited good biocompatibility, biomechanical strength, and promoted BMSC adhesion, proliferation, and calcium nodule formation in vitro. Moreover, NG/GMs/nHA/SF scaffolds showed greater osteogenic differentiation potential than the other scaffolds in vitro. In vivo, gradual new bone formation was observed, and bone defects recovered by 16 weeks in the experimental group. In the blank group, limited bone formation was observed, and the bone defect was obvious. In conclusion, NG/GMs/nHA/SF scaffolds promoted repair of a lumbar 6 defect in osteoporotic rats. Therefore, the NG/GMs/nHA/SF biocomposite scaffold has potential as a bone-defect-filling biomaterial for bone regeneration.

Silk fibroin nanofibrous scaffolds incorporated with microRNA-222 loaded chitosan nanoparticles for enhanced neuronal differentiation of neural stem cells

Neural stem cells (NSCs) transplantation therapy is a promising method for neural tissue regeneration. How to enhance the neuronal differentiation of NSCs has been the most challenging aspect of NSCs application. Herein, the microRNA-222 loaded chitosan nanoparticles (miR-222/CS NPs) were incorporated with silk fibroin (SF) nanofibrous scaffolds to enhance neuronal differentiation of NSCs. The encapsulation efficiency of miR-222 in the miR-222/CS NPs was (96.4 ± 0.3) %. The results of the electrophoretic assay and cellular uptake assay confirmed that miR-222 was stable in the miR-222/CS NPs and can be effectively delivered into NSCs. The water contact angle decreased from (89 ± 3.05)° for the SF scaffolds to (14 ± 1.00)° for the composite scaffolds. The Western blot and RT-PCR results confirmed that the composite scaffolds could enhance neuronal differentiation of NSCs. In conclusion, the SF nanofibrous scaffolds in combination with miR-222/CS NPs are a promising approach for neural tissue regeneration.

A Comparative Analysis of the Structure and Biological Properties of Films and Microfibrous Scaffolds Based on Silk Fibroin.

A comparative analysis of the structure and biological properties of silk fibroin constructions was performed. Three groups of constructions were obtained: films obtained by casting an aqueous solution of silk fibroin and electrospun microfibrous scaffolds based on silk fibroin, with the addition of 30% gelatin per total protein weight. The internal structures of the films and single fibers of the microfibrous scaffolds consisted of densely packed globule structures; the surface area to volume ratios and volume porosities of the microfibrous scaffolds were calculated. All constructions were non-toxic for cells and provide high levels of adhesion and proliferation. The high regenerative potential of the constructions was demonstrated in a rat full-thickness skin wound healing model. The constructions accelerated healing by an average of 15 days and can be considered to be promising constructions for various tasks of tissue engineering and regenerative medicine.

Evaluation of Bi-Layer Silk Fibroin Grafts for Tubular Ureteroplasty in a Porcine Defect Model.

Ureteral reconstruction with autologous tissue grafts is often limited by tissue availability and donor site morbidity. This study investigates the performance of acellular, bi-layer silk fibroin (BLSF) scaffolds in a porcine model of ureteroplasty. Tubular ureteroplasty with BLSF grafts in combination with transient stenting for 8 weeks was performed in adult female, Yucatan, mini-swine (N = 5). Animals were maintained for 12 weeks post-op with imaging of neoconduits using ultrasonography and retrograde ureteropyelography carried out at 2 and 4 weeks intervals. End-point analyses of ureteral neotissues and unoperated controls included histological, immunohistochemical (IHC), histomorphometric evaluations as well as ex vivo functional assessments of contraction/relaxation. All animals survived until scheduled euthanasia and displayed mild hydronephrosis (Grades 1-2) in reconstructed collecting systems during the 8 weeks stenting period with one animal presenting with a persistent subcutaneous fistula at 2 weeks post-op. By 12 weeks of scaffold implantation, unstented neoconduits led to severe hydronephrosis (Grade 4) and stricture formation in the interior of graft sites in 80% of swine. Bulk scaffold extrusion into the distal ureter was also apparent in 60% of swine contributing to ureteral obstruction. However, histological and IHC analyses revealed the formation of innervated, vascularized neotissues with a-smooth muscle actin+ and SM22α+ smooth muscle bundles as well as uroplakin 3A+ and pan-cytokeratin + urothelium. Ex vivo contractility and relaxation responses of neotissues were similar to unoperated control segments. BLSF biomaterials represent emerging platforms for tubular ureteroplasty, however further optimization is needed to improve in vivo degradation kinetics and mitigate stricture formation.