Silk Fibroin Scaffolds Research Articles

The osteogenic effects of sponges synthesized with biomaterials and nano-hydroxyapatite

Artificial bone substitutes have been developed using various biomaterials for use in medicine. Silk fibroin (SF) displays excellent mechanical properties and cell compatibility. Nonetheless, the mechanical properties of silk fibroin scaffolds used in artificial bone substitutes are weaker than those of natural bone, and silk fibroin is deficient as an osteogenic agent. This limits their effectiveness in bone tissue engineering. We added nano-hydroxyapatite (nHAp) particles to an existing cell-based artificial bone substitute with a silk fibroin scaffold, which will improve its mechanical properties and osteogenic efficacy, leading to significant bone regeneration. The mechanical characters of silk fibroin modifying with nHAp were measured by Atomic Force Microscopy Analysis, dispersive x-ray spectroscopy, Porosity measurement, and Microcomputed Tomography. The proliferation and toxicity of a fibroin/dextran/collagen sponge (FDS) containing nHAp were evaluated in vitro, and its osteogenic efficacy was evaluated using nude mouse and rabbit radius defect models. The defect area was repaired and showed callus formation of new bone in the rabbit radius defect models of the nHAp-FDS-treated group, whereas the defect area was unchanged in the FDS-treated group. The nHAp-FDS manufactured in this study showed significant bone regeneration owing to the synergistic effects of the components, such as those due to the broad range of pore sizes in the sponge and protein adsorbability of the nHAp, which could be suggested as a better supportive material for bone tissue engineering.

Effect of silk fibroin scaffold loaded with 17-β estradiol on the proliferation and differentiation of BMSCs.

In this study, different concentrations of 17-β estradiol silk fibroin (SF)porous scaffolds (SFPS) were prepared using freeze-drying technique, with a hope for optimal concentration and apply it locally to the bone defect area. In this study, the porous scaffold morphology structure was characterized by SEM, FTIR and universal capacity testing machines, and the invitro cytocompatibility and biological activity of scaffold materials were studied by cell adhesion, viability and proliferation experiments. The results showed that SFPS boasts better physicochemical properties, while 17-β estradiol SF scaffolds with low concentrations of 10-10mol/L and 10-12mol/L had more growth and proliferation of SF scaffolds with higher concentrations, and 10-10mol/L was the optimal concentration of 17-β estradiol SFPS, which was more conducive to cell adhesion and proliferation. On the other hand, after osteogenesis induction of BMSCs inoculated on 17-β estradiol SFPS at different concentrations, it was found that the expression of alkaline phosphatase in BMSCs on different concentrations of 17-β estradiol porous scaffolds was not large. No conflict of interest exits in the submission of this manuscript.

Resistance to Degradation of Silk Fibroin Hydrogels Exposed to Neuroinflammatory Environments.

Central nervous system (CNS) diseases represent an extreme burden with significant social and economic costs. A common link in most brain pathologies is the appearance of inflammatory components that can jeopardize the stability of the implanted biomaterials and the effectiveness of therapies. Different silk fibroin scaffolds have been used in applications related to CNS disorders. Although some studies have analyzed the degradability of silk fibroin in non-cerebral tissues (almost exclusively upon non-inflammatory conditions), the stability of silk hydrogel scaffolds in the inflammatory nervous system has not been studied in depth. In this study, the stability of silk fibroin hydrogels exposed to different neuroinflammatory contexts has been explored using an in vitro microglial cell culture and two in vivo pathological models of cerebral stroke and Alzheimer's disease. This biomaterial was relatively stable and did not show signs of extensive degradation across time after implantation and during two weeks of in vivo analysis. This finding contrasted with the rapid degradation observed under the same in vivo conditions for other natural materials such as collagen. Our results support the suitability of silk fibroin hydrogels for intracerebral applications and highlight the potentiality of this vehicle for the release of molecules and cells for acute and chronic treatments in cerebral pathologies.

Mechanically Robust Shape‐Memory Organohydrogels Based on Silk Fibroin with Organogel Microinclusions of Various Sizes

AbstractOrganohydrogels (OHGs) are soft materials with antagonistic hydrophilic and hydrophobic domains that have great interest for many different applications. This study presents the preparation of mechanically strong OHGs with shape‐memory function by incorporating semicrystalline organo‐microgels within the pores of silk fibroin (SF) scaffolds. In the first step, SF cryogels with various pore diameters between 26 ± 8 and 17 ± 4 µm are synthesized by cryogelation of aqueous SF solutions at concentrations between 5 and 20 w/v%. In the second step, the pores of SF scaffolds are filled with an organogel precursor solution containing n‐octadecyl acrylate (C18A), acrylic acid, N,N'‐methylene(bis)acrylamide, and an initiator. Once the free‐radical polymerization took place inside the pores, OHGs containing organo‐microgels of various sizes are obtained. The incorporation of the organogel component in the cryogels generates crystalline areas due to the side‐by‐side packed C18 side chains. OHGs' melting temperature and crystallinity level can be varied from 42 to 54 °C and from 2 to 16%, respectively. The stiffness of OHGs increases from 5.9 ± 0.5 to 18 ± 1 MPa with increasing SF concentration from 5 to 20 w/v%, which is attributed to the decreasing pore size of the cryogels and increasing thickness of the pore walls.

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation.

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na2 CO3 -degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.

Sudan Black B Pretreatment to Suppress Autofluorescence in Silk Fibroin Scaffolds.

Natural polymers are extensively utilized as scaffold materials in tissue engineering and 3D disease modeling due to their general features of cytocompatibility, biodegradability, and ability to mimic the architecture and mechanical properties of the native tissue. A major limitation of many polymeric scaffolds is their autofluorescence under common imaging methods. This autofluorescence, a particular challenge with silk fibroin materials, can interfere with the visualization of fluorescently labeled cells and proteins grown on or in these scaffolds, limiting the assessment of outcomes. Here, Sudan Black B (SBB) was successfully used prefixation prior to cell seeding, in various silk matrices and 3D model systems to quench silk autofluorescence for live cell imaging. SBB was also trialed postfixation in silk hydrogels. We validated that multiple silk scaffolds pretreated with SBB (hexafluoro-2-propanol-silk scaffolds, salt-leached sponges, gel-spun catheters, and sponge-gel composite scaffolds) cultured with fibroblasts, adipose tissue, neural cells, and myoblasts demonstrated improved image resolution when compared to the nonpretreated scaffolds, while also maintaining normal cell behavior (attachment, growth, proliferation, differentiation). SBB pretreatment of silk scaffolds is an option for scaffold systems that require autofluorescence suppression.

Characterization of three-dimensional bone-like tissue growth and organization under influence of directional fluid flow.

The transition in the field of bone tissue engineering from bone regeneration to in vitro models has come with the challenge of recreating a dense and anisotropic bone-like extracellular matrix (ECM). Although the mechanism by which bone ECM gains its structure is not fully understood, mechanical loading and curvature have been identified as potential contributors. Here, guided by computational simulations, we evaluated cell and bone-like tissue growth and organization in a concave channel with and without directional fluid flow stimulation. Human mesenchymal stromal cells were seeded on donut-shaped silk fibroin scaffolds and osteogenically stimulated for 42 days statically or in a flow perfusion bioreactor. After 14, 28, and 42 days, constructs were investigated for cell and tissue growth and organization. As a result, directional fluid flow was able to improve organic tissue growth but not organization. Cells tended to orient in the tangential direction of the channel, possibly attributed to its curvature. Based on our results, we suggest that organic ECM production but not anisotropy can be stimulated through the application of fluid flow. With this study, an initial attempt in three-dimensions was made to improve the resemblance of in vitro produced bone-like ECM to the physiological bone ECM.

A Novel Immobilization Method of Urease in Silk Fibroin Scaffolds by Annealing at Constant Temperature and Humidity

AbstractTemperature and humidity (TH) post‐treatment is a relatively mild way to change the silk fibroin (SF) secondary structure. Here, urease is immobilized in SF scaffold (SFS) by TH post‐treatment, and the effects of annealing on the immobilized urease activity are examined. Fourier transform infrared indicates that the SFS secondary structure started to transform when the scaffold is annealed at 35 °C with 100% relative humidity for 20 min. The higher the temperature, the lower the treatment time is required to change SFS structure. Compared to methanol annealing, TH post‐treatment maintains the original form of the sample, and the scaffold shows better water‐binding abilities. The maximum recovery ratio of immobilized urease is 98.4 ± 7.0%, which is obtained after the scaffold is postprocessed at 35 °C for 60 min, and the relative recovery ratio of the urease activity is still as high as 80.6 ± 12.1% after repeatedly measuring 10 times. In addition, the relative recovery ratio of urease is not significantly decrease within 4 weeks of storage. Therefore, TH annealing is a relatively mild and effective way to immobilize urease in SFS. The urease‐immobilized SFS with hydrophilic properties and prolonged enzyme activity may be used for peritoneal dialysate regeneration systems.

Development of a More Environmentally Friendly Silk Fibroin Scaffold for Soft Tissue Applications.

A push for environmentally friendly approaches to biomaterials fabrication has emerged from growing conservational concerns in recent years. Different stages in silk fibroin scaffold production, including sodium carbonate (Na2CO3)-based degumming and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-based fabrication, have drawn attention for their associated environmental concerns. Environmentally friendly alternatives have been proposed for each processing stage; however, an integrated green fibroin scaffold approach has not been characterized or used for soft tissue applications. Here, we show that the combination of sodium hydroxide (NaOH) as a substitute degumming agent with the popular "aqueous-based" alternative silk fibroin gelation method yields fibroin scaffolds with comparable properties to traditional Na2CO3-degummed aqueous-based scaffolds. The more environmentally friendly scaffolds were found to have comparable protein structure, morphology, compressive modulus, and degradation kinetics, with increased porosity and cell seeding density relative to traditional scaffolds. Human adipose-derived stem cells showed high viability after three days of culture while seeded in each scaffold type, with uniform cell attachment to pore walls. Adipocytes from human whole adipose tissue seeded into scaffolds were found to have similar levels of lipolytic and metabolic function between conditions, in addition to a healthy unilocular morphology. Results indicate that our more environmentally friendly methodology for silk scaffold production is a viable alternative and well suited to soft tissue applications.

STUDY OF SILK STRAIN AND ITS PREPARATION FOR MENISCUS TISSUE ENGINEERING SCAFFOLD

Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study.Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I, collagen II, and aggrecan at day 7 and 14, was studied. All experiment were done in triplicated samples.Generally, method 1 SF extraction showed higher cell viability in all strains. Cell viability from Nanglai Saraburi, Laung Saraburi and Nangtui strains were higher than those without SF in every time point while Wanasawan and J108 had higher viability at day 1 and decreased by time. Expression in collagen 1, collagen 2 and aggrecan in method 1 are higher at day 7 and day 14. Collagen 1 expression was highest in Nangnoi Srisaket, followed by Laung Saraburi and Nanglai Saraburi in day 7. Nangnoi Srisaket also had highest expression at day 14, followed by Nanglai Saraburi and Laung Saraburi respectively. Nangseaw had highest collagen 2 expression, follow by Laung Saraburi and Nangnoi Srisaket respectively. Higher aggrecan gene expression of Tubtimsiam, Wanasawan, UB 1 and Nangnoi Srisaket was observed at day 7 and increased expression of all strains at day 14.SF extraction using CaCl2/Ethanol/H2O offered better cell viability and chondrogenic expression. Nangseaw, Laung Saraburi and Nangnoi Srisaket strains expressed more chondrogenic phenotype.

Facile construction of calcium titanate-loaded silk fibroin scaffolds hybrid frameworks for accelerating neuronal cell growth in peripheral nerve regeneration

Different concentrations of calcium titanate (CaTiO3) nanoparticles were loaded into the Silk fibroin (SF) solution to construct porous SF@CaTiO3 hybrid scaffolds, which were shown to have enhanced properties for stimulating peripheral nerve regeneration. Surface charges, crystallization intensity, wettability, porosity, and morphology were measured and analyzed. We analyzed the hybrid porous SF@CaTiO3 scaffolds that affected the expansion of Schwann cells. The results demonstrated a concentration-dependent influence on the dispersion of nanoparticles in the CaTiO3 hybridized SF scaffolds. Incorporating CaTiO3-NPs into the porous SF@CaTiO3 hybrid scaffolds can boost hydrophobicity while decreasing surface charge density and porosity. The hybridized scaffolds mostly had an orthorhombic calcium titanate crystal structure with amorphous Silk fibroin mixed. Schwann cell cultures revealed that SF@CaTiO3 hybrid scaffolds containing an optimal CaTiO3-NPs concentration could stimulate the proliferation, attachment, and protection of Schwann cell biological functions, suggesting the scaffolds' potential for use in peripheral nerve regeneration.

Three-dimensional silk fibroin-gelatin/chondroitin sulfate/hyaluronic acid-aloe vera scaffold supports in vitro chondrogenesis of bone marrow mesenchymal stem cells and reduces inflammatory effect.

A limited self-healing ability of injured articular cartilage results in osteoarthritis and a joint dysfunction afterward. Cartilage tissue engineering is a promising approach to increase the treatment efficiency. Moreover, host response to implanted biomaterial has been increasingly concerned. Thus, this study aimed to establish three-dimensional (3D) scaffold that could support cartilage tissue engineering and reduce inflammatory. The various ratios of silk fibroin (SF), gelatin (G), chondroitin sulfate (C), hyaluronic acid (H), and aloe vera (A) were used to fabricate 3D scaffolds by lyophilization, designated as SF, SF-A, SF-gelatin/chondroitin sulfate/hyaluronic acid (GCH)-A-411, and SF-GCH-A-111. The physical and biological characteristics of the scaffolds were investigated. All scaffolds possessed interconnected porous structures, which the highest pore size of 209 μm was found in SF and SF-GCH-A-411 scaffolds. Moreover, high porosity, high water uptake, and good mechanical strength were observed in the SF-GCH-A-411 scaffold. The SF, SF-A, and SF-GCH-A-411 scaffolds could retain their structures up to 21 days, while SF-GCH-A-111 was rapidly degraded. The proliferation of human bone marrow mesenchymal stem cells (BM-MSCs) was significantly higher in SF-A and SF-GCH-A-411 than in the SF scaffold. Besides, the SF-A and SF-GCH-A-411 revealed significantly lower expression of pro-inflammatory cytokine, interleukin-1 beta than the SF scaffold, suggesting the beneficial role of aloe vera in anti-inflammatory effect. Furthermore, the SF-GCH-A-411 scaffold could support chondrogenic differentiation of BM-MSCs. In conclusion, based on its superior physical and biological characteristics that support chondrogenesis of BM-MSCs, the SF-GCH-A-411 scaffold is recommended for cartilage tissue engineering.

Skin precursor-derived Schwann cells accelerate in vivo prevascularization of tissue-engineered nerves to promote peripheral nerve regeneration.

Prevascularization strategies have become a hot spot in tissue engineering. As one of the potential candidates for seed cells, skin precursor-derived Schwann cells (SKP-SCs) were endowed with a new role to more efficiently construct prevascularized tissue-engineered peripheral nerves. The silk fibroin scaffolds seeded with SKP-SCs were prevascularized through subcutaneously implantation, which was further assembled with the SKP-SC-containing chitosan conduit. SKP-SCs expressed pro-angiogenic factors in vitro and in vivo. SKP-SCs significantly accelerated the satisfied prevascularization in vivo of silk fibroin scaffolds compared with VEGF. Moreover, the NGF expression revealed that pregenerated blood vessels adapted to the nerve regeneration microenvironment through reeducation. The short-term nerve regeneration of SKP-SCs-prevascularization was obviously superior to that of non-prevascularization. At 12 weeks postinjury, both SKP-SCs-prevascularization and VEGF-prevascularization significantly improved nerve regeneration with a comparable degree. Our figures provide a new enlightenment for the optimization of prevascularization strategies and how to further utilize tissue engineering for better repair.

Quantitative T2 mapping monitoring the maturation of engineered elastic cartilage in a rabbit model

BackgroundCartilage tissue engineering provides a promising approach to reconstruct craniofacial defects, and a noninvasive method is needed to assess its effectiveness. Although magnetic resonance imaging (MRI) has been used to evaluate articular cartilage in vivo, few studies focused on its feasibility in monitoring engineered elastic cartilage (EC).MethodsAuricular cartilage, silk fibroin (SF) scaffold, and EC consisting of rabbit auricular chondrocytes and SF scaffold were transplanted subcutaneously into the rabbit back. In eight weeks after transplantation, grafts were imaged by MRI using PROSET, PDW VISTA SPAIR, 3D T2 VISTA, 2D MIXED T2 Multislice, and SAG TE multiecho sequences, followed by histological examination and biochemical analysis. Statistical analyses were performed to identify the association between T2 values and biochemical indicator values of EC.ResultsIn vivo imaging shows that 2D MIXED T2 Multislice sequence (T2 mapping) clearly distinguished the native cartilage, engineered cartilage and fibrous tissue. T2 values showed high correlations with cartilage-specific biochemical parameters at different time points, especially the elastic cartilage specific protein elastin (ELN, r= -0.939, P < 0.001).ConclusionQuantitative T2 mapping can effectively detect the in vivo maturity of engineered elastic cartilage after subcutaneously transplantation. This study would promote the clinical application of MRI T2 mapping in monitoring engineered elastic cartilage in the repair of craniofacial defects.

A review of recent advances on osteogenic applications of Silk fibroin as a potential bio-scaffold in bone tissue engineering

Silk fibroin (SF) have been increasingly used as bone substitutes in tissue engineering (TE) due to their sufficient mechanical strength, good biocompatibility, osteoconductive, and controllable degradation rates. The SF scaffold as a 3D-matrix allows and stimulates the proliferation, attachment and differentiation of stem cells toward osteogenic cells. Herein, we have summarized the applications of SF for regeneration of bone defects in a comprehensive manner. Additionally, we have discussed three critical factors for osteogenesis regulation, including biological (cells and growth factors), physical (mechanics and morphology), and chemical (composition) aspects with their current drawbacks and future perspective of SF in bone TE.

Biodegradable silk fibroin scaffold doped with mineralized collagen induces bone regeneration in rat cranial defects

Compared with most nondegradable or slowly degradable bone repair materials, bioactive biodegradable porous scaffolds with certain mechanical strengths can promote the regeneration of both new bone and vasculature while the cavity created by their degradation can be replaced by the infiltration of new bone tissue. Mineralized collagen (MC) is the basic structural unit of bone tissue, and silk fibroin (SF) is a natural polymer with adjustable degradation rates and superior mechanical properties. In this study, a three-dimensional porous biomimetic composite scaffold with a two-component SF-MC system was constructed based on the advantages of both materials. The spherical mineral agglomerates of the MC were uniformly distributed on the surface and inside the SF skeleton, which ensured good mechanical properties while regulating the degradation rate of the scaffold. Second, the SF-MC scaffold had good osteogenic induction of bone marrow mesenchymal stem cells (BMSCs) and preosteoblasts (MC3T3-E1) and also promoted the proliferation of MC3T3-E1 cells. Finally, in vivo 5 mm cranial defect repair experiments confirmed that the SF-MC scaffold stimulated vascular regeneration and promoted new bone regeneration in vivo by means of in situ regeneration. Overall, we believe that this low-cost biomimetic biodegradable SF-MC scaffold with many advantages has some clinical translation prospects.

Elastic Fiber-Reinforced Silk Fibroin Scaffold with A Double-Crosslinking Network for Human Ear-Shaped Cartilage Regeneration

Elastic Fiber-Reinforced Silk Fibroin Scaffold with A Double-Crosslinking Network for Human Ear-Shaped Cartilage Regeneration

Plasmid containing VEGF-165 and ANG-1 dual genes packaged with fibroin-modified PEI to promote the regeneration of vascular network and dermal tissue

Reducing the cytotoxicity of cationic polymers is the major issue to their use as a gene delivery carrier. In this study, plasmids containing encoding vascular endothelial cell growth factor 165 and angiopoietin-1 were packaged with the conjugates of cationic fibroin (CSF) and polyethylenimine (PEI), instead of packaging pDNA with PEI alone, to prepare nanocomplexes (CSF+PEI)/pDNA. The complexes were loaded into a silk fibroin scaffold to enhance its function to induce microvascular network generation and dermal tissue regeneration. The results of transfecting EA.hy926 cells with the complexes in vitro showed that (CSF+PEI)/pDNA had a stronger transfection ability than PEI/pDNA. Importantly, compared with PEI as the gene carrier alone, the cell viability was significantly increased and the cytotoxicity was effectively reduced after the conjugate of CSF and PEI was used as the gene carrier. The results of angiogenesis in chick embryo chorioallantoic membranes showed that compared with scaffolds loaded with PEI/pDNA, the neovascularization ratio in scaffolds loaded with (CSF+PEI)/pDNA was significantly increased. In vivo experimental results of scaffolds implantation for full-thickness skin defects in SD rats showed that, compared with loading PEI/pDNA complex, loading (CSF+PEI)/pDNA complex in the scaffold more effectively promoted the formation of vascular network in the scaffold and accelerated the regeneration of dermal tissue. The gene delivery system established in this study has application potential not only in the regeneration of vascular-containing tissues, but also in tumor gene therapy.

MXene-modified 3D printed scaffold for photothermal therapy and facilitation of oral mucosal wound reconstruction

The most prevalent oral and maxillofacial cancer is oral squamous cell carcinoma (OSCC). Patient survival is compromised by relapse due to post-operative tumor remnants and significant mucosal defects. Photothermal therapy (PTT) is used to ablate local malignancies, but the capacity of tumor cells to resist it is correlated with the overexpression of heat shock protein 70 (HSP70). In this work, PTT and tissue engineering scaffold were rationally integrated to construct a Ti3C2 MXene, collagen, silk fibroin and quercetin composite scaffold (M-CSQ scaffold), a 3D printed biomaterial that simultaneously kill OSCC cells and promote the regeneration of the mucosal defects. The M-CSQ scaffolds were prepared by cryogenic 3D printing and freeze-drying techniques with sufficient pores that allow an abundant cell migration and provide a proliferation space. Ti3C2 MXene has excellent photothermal conversion ability and stability. Quercetin targeted HSP70 to decrease its expression in OSCC cells, consequently weakening their resistance to high temperature and enhancing the effect of PTT. The M-CSQ scaffold effectively killed OSCC cells in vitro and inhibited tumor growth in vivo. In addition, the M-CSQ scaffold provided adhesion sites for Sprague-Dawley rat (SD rat) buccal mucosal fibroblasts and promoted the repair of buccal mucosal wounds.

Well-aligned three-dimensional silk fibroin protein scaffold for orientation regulation of cells

The similar characteristics of biomaterials to the extracellular matrix are essential for efficient tissue repair through dictating cell behaviors. But the scaffold fabrication with complex shapes and controlled alignment have proven to be a difficult task. Herein, a well-designed three-dimensional silk fibroin scaffold is fabricated through ice template technology. The effect of the silk fibroin protein concentration and the freezing temperature on the microstructure and mechanical properties of scaffolds are investigated systematically. Cells behavior mediated by the obtained silk fibroin scaffolds is detected. The results show that the protein concentration plays a vital role in microstructure and scaffold strength. A well-aligned scaffold can be obtained when silk fibroin solution is kept at 12 wt%, which holds the highest mechanical properties. The pore size can be further adjusted in the range of 5–80 µm by changing the freezing temperature from −60 to −196 °C. The well-oriented scaffold with the appropriate pore size of 10–20 µm has the best ability to guide cell alignment. The resulting scaffolds provide an excellent matrix to guide cells behaviors and have a potential application in tissue engineering.