Silk Fibroin-chitosan Scaffolds Research Articles

Protective effect of brain-derived neurotrophic factor and neurotrophin-3 overexpression by adipose-derived stem cells combined with silk fibroin/chitosan scaffold in spinal cord injury

ABSTRACT Objectives: Spinal cord injury (SCI) is a most debilitating traumatic injury, and cytotherapy is a promising alternative treatment strategy. Here we investigated the effect and mechanism of adipose-derived stem/stromal cells (ASCs) with overexpressing brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) (BDNF-NT3) in combination with silk fibroin/chitosan scaffold (SFCS) in SCI. Methods: Female Sprague-Dawley rats were used as an SCI model. SFCS,SFCS and ASCs, or ASCs overexpressing NT3, BDNF, and BDNF-NT3 were implanted into SCI rats. Basso, Beattie, and Bresnahan score, pathological changes, and spinal cord tissue and nerve fiber morphology were observed and assayed. GAP-43, GFAP, and caspase-3 expression was determined using immunohistochemistry and western blotting. Results: Smoother spinal cords, less scar tissue, and lower inflammatory activity were found in the SFCS, SFCS and ASCs, ASCs with NT3, BDNF, and BDNF-NT3 overexpression treatment than in the untreated SCI rat groups. Increasing formation of nerve fibers was observed in the above groups in order. GAP-43 expression significantly increased, while GFAP and caspase-3 expression significantly decreased. These results indicated obvious alleviation in pathological changes and BDNF-NT3 overexpression in ASCs combined with SFCS treatment in SCI rats. Conclusion: Thus, BDNF-NT3 overexpression from ASCs with SFCS had synergistic neuroprotective effects on SCI and may be a treatment option for SCI.

Synthesis of and in vitro and in vivo evaluation of a novel TGF-β1-SF-CS three-dimensional scaffold for bone tissue engineering.

The role of transforming growth factor-β1 (TGF-β1) in normal human fracture healing has been previously demonstrated. The objective of the present study was to examine the biocompatibility of TGF-β1-silk fibroin-chitosan (TGF-β1-SF-CS) three-dimensional (3D) scaffolds in order to construct an ideal scaffold for bone tissue engineering. We added TGF-β1 directly to the SF-CS scaffold to construct a 3D scaffold for the first time, to the best of our knowledge, and performed evaluations to determine whether it may have potential applications as a growth factor delivery device. Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded on the TGF-β1-SF-CS scaffolds and the silk fibroin-chitosan (SF-CS) scaffolds. On the TGF-β1-SF-CS and the SF-CS scaffolds, the cell adhesion rate increased in a time-dependent manner. Using a Cell Counting Kit-8 (CCK-8) assay and analyzing the alkaline phosphatase (ALP) expression proved that TGF-β1 significantly enhanced the growth and proliferation of BMSCs on the SF-CS scaffolds in a time-dependent manner. To examine the in vivo biocompatibility and osteogenesis of the TGF-β1-SF-CS scaffolds, the TGF-β1-SF-CS scaffolds and the SF-CS scaffolds were implanted in rabbit mandibles and studied histologically and microradiographically. The 3D computed tomography (CT) scan and histological examinations of the samples showed that the TGF-β1-SF-CS scaffolds exhibited good biocompatibility and extensive osteoconductivity with the host bone after 8 weeks. Moreover, the introduction of TGF-β1 to the SF-CS scaffolds markedly enhanced the efficiency of new bone formation, and this was confirmed using bone mineral density (BMD) and biomechanical evaluation, particularly at 8 weeks after implantation. We demonstrated that the TGF-β1-SF-CS scaffolds possessed as good biocompatibility and osteogenesis as the hybrid ones. Taken together, these findings indicate that the TGF-β1-SF-CS scaffolds fulfilled the basic requirements of bone tissue engineering, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery. Thus, TGF-β1-SF-CS composite scaffolds represent a promising, novel type of scaffold for use in bone tissue engineering.

Synthesis of the New-Type Vascular Endothelial Growth Factor-Silk Fibroin-Chitosan Three-Dimensional Scaffolds for Bone Tissue Engineering and In Vitro Evaluation.

The objective of the study was to discuss the biocompatibility of the vascular endothelial growth factor-silk fibroin-chitosan (VEGF-SF-CS) scaffolds. To offer an ideal scaffold for bone tissue engineering, the author added vascular endothelial growth factor (VEGF) into silk fibroin-chitosan (SF-CS) scaffold directly to reconstruct a three-dimensional scaffold for the first time, SF-CS scaffold was loaded with VEGF and evaluated as a growth factor-delivery device. Human fetal osteoblast cell was seeded on the VEGF-SF-CS scaffolds and SF-CS scaffolds. On VEGF-SF-CS and SF-CS scaffolds, the cell adhesion rate was increased as time went on. Scanning electron microscopy: the cells grew actively and had normal multiple fissions, granular and filamentous substrates could be seen around the cells, and cell microfilaments were closely connected with the scaffolds. The cells could not only show the attached growth on surfaces of the scaffolds, but also extend into the scaffolds. Cell Counting Kit-8 and alkaline phosphatase analysis proved that the VEGF could significantly promote human fetal osteoblast1.19 cells growth and proliferation in the SF-CS scaffolds, but the enhancement of osteoblasts cell proliferation and activity by VEGF was dependent on time.

Construction and in vitro characterization of three-dimensional silk fibroinchitosan scaffolds.

The objective of this study was to discuss the construction method, characterization, and biocompatibility of three-dimensional silk fibroin-chitosan (SF-CS) scaffolds which met the requirements of bone tissue engineering scaffolds. Silk fibroin (SF) and chitosan (CS) were mixed at different ratios -3 to 7, 5 to 5, and 7 to 3- to fabricate the composite materials. To find out the optimum mixing ratio of SF and CS, parameters such as pore size, porosity, water absorption, and the mechanical properties were evaluated. Osteoblast cells hFOB1.19 were seeded on SF-CS scaffolds and pure CS scaffolds for the first time. Cell adhesion rate, cell proliferation, and cell activity were evaluated, and cell growth and formation of mineralized nodules were observed. Results showed that SF-CS scaffolds are a suitable candidate for bone tissue engineering.

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin–chitosan scaffold and its interactions with endothelial cells for tissue engineering applications

Aligned three-dimensional nanofibrous silk fibroin–chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100kHz to 10MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10MHz in the presence of 5mM NaCl had a fibril size of 77.96±4.69nm and an apparent elastic modulus of 39.9±22.4kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36±2.37kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.

Use of a Silk Fibroin-Chitosan Scaffold to Construct a Tissue-Engineered Corneal Stroma

Purpose: To construct a scaffold using silk fibroin (SF) and chitosan (CS) that could replace the corneal stroma with the biological characteristics of the scaffold materials still intact. Methods: To develop an organotypic corneal stroma, SF and CS were chosen to synthesise the tissue-engineered bioscaffold. We cultured primary rabbit corneal epithelial cells and corneal stromal cells in vitro. Keratocytes were used to assess cytotoxicity on SF-CS (SFCS) blends, which was determined by a Cell Counting Kit-8 assay. The corneal lamellar scaffolds were developed with sequential culture techniques to form cell-scaffold constructs. Implantation was tested in 15 New Zealand White rabbits. The corneal substitutes were analysed by light and electron microscopy. Results: The reconstructed lamellar cornea was comparable to native tissue, with high levels of K3/12 expression in the corneal epithelial cells and vimentin in the stromal cells; moreover, the morphology and the position of the cells could be distinguished by histological methods. There was no sign of any immune reaction in or around the transplanted discs 12 weeks after implantation. Conclusion: A SFCS scaffold might be a suitable blend for corneal tissue engineering.

Biocompatibility study of a silk fibroin-chitosan scaffold with adipose tissue-derived stem cells in vitro

The use of tissue engineering technology in the repair of spinal cord injury (SCI) is a topic of current interest. The success of the repair of the SCI is directly affected by the selection of suitable seed cells and scaffold materials with an acceptable biocompatibility. In this study, adipose tissue-derived stem cells (ADSCs) were incorporated into a silk fibroin-chitosan scaffold (SFCS), which was constructed using a freeze-drying method, in order to assess the biocompatibility of the ADSCs and the SFCS and to provide a foundation for the use of tissue engineering technology in the repair of SCI. Following the seeding of the cells onto the scaffold, the adhesion characteristics of the ADSCs and the SFCS were assessed. A significant difference was observed between the experimental group (a composite of the ADSCs with the SFCS) and the control group (ADSCs without the scaffold) following a culture period of 6 h (P<0.05). The differences in the results at the following time-points were statistically insignificant (P>0.05). The use of an MTT assay to assess the proliferation of the cells on the scaffold revealed that there were significant differences between the experimental and control groups following culture periods of 2 and 4 days (P<0.05). However, the results at the subsequent time-points were not statistically significantly different (P>0.05). Scanning electron microscopy (SEM), using hematoxylin and eosin (H&E) staining, was used to observe the cellular morphology following seeding, and this revealed that the cells displayed the desired morphology. The results indicate that ADSCs have a good biocompatibility with a SFCS and thus provide a foundation for further studies using tissue engineering methods for the repair of SCI.

Ice-template-induced silk fibroin–chitosan scaffolds with predefined microfluidic channels and fully porous structures

Scaffold-based tissue engineering has made great progress in fabricating relatively simple tissues. One of the major challenges in creating thick complex organs is to achieve sufficient nutrient supply as well as uniform cell distribution in a three-dimensional (3D) scaffold. Here we employed microstructured ice templates to fabricate silk fibroin–chitosan (SF–CS) scaffolds with predefined microfluidic channels, open-pore surface and oriented porous structures. The effects of these structural organizations in ice-template-induced (ITI) scaffolds on nutrient delivery, cell seeding as well as cell growth were well investigated in comparison with that of polydimethylsiloxane-template-induced scaffolds. The ITI scaffolds exhibited better structural properties in promoting mass transport, facilitating uniform cell distribution and growth. The ITI scaffolds uniformly seeded with living cells could be further rolled up to form a thick tissue-engineered construct with predefined microfluidic channels. We envision that our ITI scaffolds can be potentially used to engineer thick prevascularized organs when the oriented porous structures are uniformly seeded with primary cells and the predefined microfluidic channels are incorporated with endothelial cells.

Adhesion, migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix

Silk fibroin–chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 ± 4.8% at 15 min, increasing to 92.8 ± 1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47 ± 2.7 μm h −1 and a mean persistence time of 41.4 ± 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC–ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher ( p < 0.0001) for ASCs seeded on SFCS (69.0 ± 9.0 kPa) than on glass (6.1 ± 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher ( p < 0.05) for ASC seeded on SFCS (0.42 ± 0.02 fibers μm −1) than on glass-seeded controls (0.24 ± 0.03 fibers μm −1). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell–cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.

Silk fibroin/chitosan/heparin scaffold: preparation, antithrombogenicity and culture with hepatocytes

AbstractAntithrombogenicity is very important for tissue engineering scaffolds used in situations involving contact with blood. A silk fibroin/chitosan (SFCS) scaffold has been developed for liver tissue engineering with porous structure, suitable mechanical properties and biocompatibility. Because the interaction between silk fibroin and blood coagulation factors can lead to blood coagulation, the anticoagulant property of the SFCS scaffold should be improved. Heparin was added into SFCS scaffold under mild conditions. The effects of heparin on the morphology, swelling properties, structure, porosity, mechanical properties, antithrombogenicity and cytocompatibility of the SFCS scaffold were studied. SFCS scaffold containing heparin maintains the porous structure and good mechanical properties of the fibroin‐based scaffold; moreover, it is not cytotoxic. Addition of heparin leads to the SFCS scaffold being blood‐compatible and an effective heparin‐delivering system. The anticoagulant property of the SFCS scaffold can be improved by the addition of heparin, which may be helpful for scaffolds used in situations involving contact with blood. Copyright © 2009 Society of Chemical Industry

Self-assembly model, hepatocytes attachment and inflammatory response for silk fibroin/chitosan scaffolds

Silk fibroin is an attractive natural fibrous protein for biomedical application due to its good biocompatibility and high tensile strength. Silk fibroin is apt to form a sheet-like structure during the freeze-drying process, which is not suitable for the scaffold of tissue engineering. In our former study, the adding of chitosan promoted the self-assembly of silk fibroin/chitosan (SFCS) into a three-dimensional (3D) homogeneous porous structure. In this study, a model of the self-assembly is proposed; furthermore, hepatocytes attachment and inflammatory response for the SFCS scaffold were examined. The rigid chain of chitosan may be used as a template for β-sheet formation of silk fibroin, and this may break the sheet structure of the silk fibroin scaffold and promote the formation of a 3D porous structure of the SFCS scaffold. Compared with the polylactic glycolic acid scaffold, the SFCS scaffold further facilitates the attachment of hepatocytes. To investigate the inflammatory response, SFCS scaffolds were implanted into the greater omentum of rats. From the results of implantation, we could demonstrate in vivo that the implantation of SFCS scaffolds resulted in only slight inflammation. Keeping the good histocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold could be a prominent candidate for soft tissue engineering, for example, in the liver.

Preparation and cytocompatibility of silk fibroin/chitosan scaffolds

One challenge in soft tissue engineering is to find an applicable scaffold, not only having suitable mechanical properties, porous structures, and biodegradable properties, but also being abundant in active groups and having good biocompatibility. In this study, a three-dimensional silk fibroin/chitosan (SFCS) scaffold was successfully prepared with interconnected porous structure, excellent hydrophilicity, and proper mechanical properties. Compared with polylactic glycolic acid (PLGA) scaffold, the SFCS scaffold further facilitated the growth of HepG2 cells (human hepatoma cell line). Keeping the good cytocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold should be a prominent candidate for soft tissue engineering, for example, liver.

IFATS Collection: Human Adipose-Derived Stem Cells Seeded on a Silk Fibroin-Chitosan Scaffold Enhance Wound Repair in a Murine Soft Tissue Injury Model

Soft tissue loss presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a silk fibroin-chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose-derived stem cells (ASCs) in a murine soft tissue injury model. Green fluorescent protein (GFP)-labeled ASCs were seeded on SFCS scaffolds at a density of 1 x 10(5) ASCs per cm(2) for 48 hours and then suture-inlaid to a 6-mm, full-thickness skin defect in 6-week-old male athymic mice. Wound healing was tracked for 2 weeks by planimetry. Histology was evaluated at 2 and 4 weeks. Our data show that the extent of wound closure was significantly enhanced in the ASC-SFCS group versus SFCS and no-graft controls at postoperative day 8 (90% +/- 3% closure vs. 75% +/- 11% and 55% +/- 17%, respectively). Microvessel density at wound bed biopsy sites from 2 weeks postoperative was significantly higher in the ASC-SFCS group versus SFCS alone (7.5 +/- 1.1 vs. 5.1 +/- 1.0 vessels per high-power field). Engrafted stem cells were positive for the fibroblastic marker heat shock protein 47, smooth muscle actin, and von Willebrand factor at both 2 and 4 weeks. GFP-positive stem cells were also found to differentiate into epidermal epithelial cells at 4 weeks postoperative. In conclusion, human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound healing and show differentiation into fibrovascular, endothelial, and epithelial components of restored tissue.

Silk fibroin/chitosan scaffold: preparation, characterization, and culture with HepG2 cell

Tissue engineering requires the development of three-dimensional water-stable scaffolds. In this study, silk fibroin/chitosan (SFCS) scaffold was successfully prepared by freeze-drying method. The scaffold is water-stable, only swelling to a limited extent depending on its composition. Fourier Transform Infrared (FTIR) spectra and X-Ray diffraction curves confirmed the different structure of SFCS scaffolds from both chitosan and silk fibroin. The homogeneous porous structure, together with nano-scale compatibility of the two naturally derived polymers, gives rise to the controllable mechanical properties of SFCS scaffolds. By varying the composition, both the compressive modulus and compressive strength of SFCS scaffolds can be controlled. The porosity of SFCS scaffolds is above 95% when the total concentration of silk fibroin and chitosan is below 6 wt%. The pore sizes of the SFCS scaffolds range from 100 microm to 150 microm, which can be regulated by changing the total concentration. MTT assay showed that SFCS scaffolds can promote the proliferation of HepG2 cells (human hepatoma cell line) significantly. All these results make SFCS scaffold a suitable candidate for tissue engineering.

Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold

Degradation behaviors of porous scaffolds play an important role in the engineering process of a new tissue. In this study, three-dimensional porous silk fibroin/chitosan (SFCS) scaffolds were successfully prepared by freeze-drying method. In vitro degradation behaviors of SFCS scaffolds have been systematically investigated up to 8 weeks in phosphate buffer saline (PBS) solution at 37 °C. The following properties of the scaffolds were measured as a function of degradation time: pore morphology, structure, weight loss, and wet/dry weight value. The pH value of the PBS solution during degradation was also detected. SFCS scaffolds maintained its porous structure till 6 weeks of degradation. During the first 2 weeks, the pH value fluctuated in a narrow range from 6.53 to 6.93. SFCS scaffolds degraded much more quickly during the first 2 weeks, and the weight loss reached 19.28 wt% after 8 weeks of degradation. The degradation process affects little SFCS scaffolds' swelling properties.