Preparation of three-dimensional fibroin/collagen scaffolds in various pH conditions

Three dimensional (3-D) fibroin/collagen scaffolds are the novel fibroin based scaffolds derived from aqueous solution. In this article, we investigated the effect of pH on the formation of fibroin/collagen scaffolds. In the range of pH from 4 to 8.5, the fibroin/collagen scaffolds with good porous structures can be prepared using freeze-drying method, which would facilitate the adding of other biopolymers. The structures of different fibroin-based scaffolds were investigated with FTIR and DSC, which indicated that the interaction of fibroin and collagen affected the methanol-induced transformation of fibroin from random-coil to β-sheet conformation. The mechanical properties were also studied. The results mean that all the fibroin-based scaffolds prepared in various pH values had better mechanical properties than other reported fibroin scaffolds. Since the fibroin/collagen scaffolds can be prepared in the range of pH from 4 to 8.5, it is very easy to prepare different multifunctional scaffolds such as fibroin/collagen/chitosan scaffolds and fibroin/collagen/heparin scaffolds in acidic or neutral conditions. These new fibroin-based blend materials extend the range of biomaterial properties that can promote the use in biomedical applications such as drug release and tissue engineering.     

Fabrication and characterization of bioactive silk fibroin/wollastonite composite scaffolds

Composite scaffolds of silk fibroin (SF) with bioactive wollastonite were prepared by freeze-drying. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis showed that random coil and β-sheet structure co-existed in the SF scaffold. The mechanical performance, surface hydrophilicity and water-uptake capacity of the composite scaffolds were improved compared with those of pure SF scaffold. The bioactivity of the composite scaffold was evaluated by soaking in a simulated body fluid (SBF), and formation of a hydroxycarbonate apatite (HCA) layer was determined by FT-IR and XRD. The results showed that the SF/wollastonite composite scaffold was bioactive as it induced the formation of HCA on the surface of the composite scaffold after soaking in SBF for 5 days. In vitro cell attachment and proliferation tests showed that the composite scaffold was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, the incorporation of wollastonite into the SF scaffold can enhance both the mechanical strength and bioactivity of the scaffold, which suggests that the SF/wollastonite composite scaffold may be a potential biomaterial for tissue engineering.     

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 μm to 150 μm, 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 of 3-D regenerated fibroin scaffolds with freeze drying method and freeze drying/foaming technique

Although three-dimensional fibroin scaffolds have been prepared with freeze drying method, the porosity and pore sizes still can not satisfy the requirement of tissue engineering. In this article, fibroin porous scaffold with high porosity and > 100μm diameter interconnected pores was firstly prepared with freeze drying method through adjusting fibroin concentration. The morphology of different scaffolds lyophilized from different fibroin concentration was observed by SEM. A novel freeze drying improved method, freeze drying/foaming technique, was also devised to prepare fibroin scaffolds at different fibroin concentrations. Using the said method, the porosity and pore size of fibroin scaffolds prepared from 12% concentration were 85.8 ± 4% and 109 ± 20 μm respectively with yield strength up to 450 ± 6 KPa while the porosity and pore size of fibroin scaffolds prepared from 8% concentration were 96.9 ± 3.6% and 120 ± 30 μm respectively with yield strength up to 30 ± 1 KPa. The freeze drying/foaming technique produced scaffolds with a useful combination of high yield strength, interconnected pores, and pore sizes greater than 100 μm in diameter. Through adjusting fibroin concentration and thawing time, the porosity, pore sizes and mechanical properties could be controlled to satisfy the different requirements of tissue engineering. The results suggested that fibroin scaffolds prepared with the above methods could be formed for utility in biomaterial application.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.     

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.     

Attachment of stem cells on porous chitosan scaffold crosslinked by Na5P3O10

In this study, porous chitosan scaffolds were prepared by freeze-dried method using Na₅P₃O₁₀ as a crosslinking agent. The three-dimensional pore structure of the scaffold was interconnected with a mean pore size about 40 to 100 μm. The remained weight of crosslinked scaffold was about 76% after being exposed to PBS for 30 days. Mouse embryonic stem (E. S.) cells could grow on these crosslinked scaffolds. E. S. cells differentiated to other cells after 21 days of culturing on the scaffolds. The growth rate of E. S. cells was improved by post surface treatment of the scaffolds with collagen. However, there was no significant increase in growth rate of E. S. cells when scaffolds were surface treated with argon plasma. These porous chitosan scaffolds present a promising approach for tissue engineering applications.     

The comparison of the Wnt signaling pathway inhibitor delivered electrospun nanoyarn fabricated with two methods for the application of urethroplasty

Urethral strictures were common disease caused by over-expression of extracellular matrix from fibroblast. In this study, we compare two nanoyarn scaffolds for improving fibroblasts infiltration without inhibition the over-expression of extracellular matrix. Collagen/poly(L-lactide-co-caprolactone) (Col/P(LLA-CL)) nanoyarn scaffolds were prepared by conjugated electrospinning and dynamic liquid electrospinning, respectively. In addition, co-axial electrospinning technique was combined with the nanoyarn fabrication process to produce nanoyarn scaffolds loading Wnt signaling pathway inhibitor. The mechanical properties of the scaffolds were examined and morphology was observed by SEM. Cell morphology, proliferation and infiltration on the scaffolds were investigated by SEM, MTT assay and H&E staining, respectively. The release profiles of different scaffolds were determined using HPLC. The results indicated that cells showed an organized morphology along the nanoyarns and considerable infiltration into the nanoyarn scaffolds prepared by dynamic liquid electrospinning (DLY). It was also observed that the DLY significantly facilitate cell proliferation. The D-DLY could facilitate the infiltration of the fibroblasts and could be a promising scaffold for the treatment of urethra stricture while it may inhibit the collagen production.     

Optimization of macroporous 3-D silk fibroin scaffolds by salt-leaching procedure in organic solvent-free conditions

A novel all-aqueous process is described to form three-dimensional porous silk fibroin (SF) scaffolds, which not only avoided the use of organic solvents or harsh chemicals, but also can form scaffolds with various sizes and in large quantities. The scaffolds show a rough surface on the pores and the pores are highly interconnected. The porosity of the scaffolds, which varied between a large range (67.6~99.3%), can be controlled by the SF concentrations and the salt/fibroin ratio. The results of measurements indicated that this novel process can improve and enforce the transformation in SF structure from a random coil to a β-sheet. Swelling studies showed that the scaffold has excellent properties of hydrophilicity. The cell culture experiments demonstrated that the scaffolds facilitated the human osteosarcoma cells attachment and proliferation in vitro.     

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.     

Fabrication of a composite vascular scaffold using electrospinning technology

Intermolecular forces and morphology demonstrated that there was an excellent compatibility between silk fibroin and gelatin. The silk fibroin/gelatin composite vascular scaffold (inner diameter 4.5 mm) was prepared successfully by electrospinning. The scaffold was treated with ethanol to enhance the water-resistant ability and biomechanical properties. After ethanol treatment, the scaffold could hardly dissolve in the water, and FTIR showed that the conformation of the treated silk fibroin/gelatin composite vascular scaffold was mainly β-sheets. The electrospun silk fibroin/gelatin vascular scaffold possessed outstanding biomechanical properties. In vitro cell culture and in vivo subcutaneous implantation demonstrated that the electrospun silk fibroin/gelatin vascular scaffold had an appropriate biocompatibility. The results indicated that the electrospun silk fibroin/gelatin composite vascular scaffold could be considered as an ideal candidate for tissue-engineered blood vessel.     

Preparation of porous biodegradable poly(lactide-co-glycolide)/ hyaluronic acid blend scaffolds: Characterization, in vitro cells culture and degradation behaviors

A series of poly(lactide-co-glycolide) (PLGA)/ hyaluronic acid (HA) blend with different HA composition were used to fabricate scaffolds successfully. The pores of the three dimensional scaffold were prepared by particle leaching and freeze drying. The pore size was about 50–200 μ m and the porosity was about 85%. The characterizations of the scaffold, such as mechanical properties, hydrophilicity and surface morphologies were determined. Mouse 3T3 fibroblast was directly seeded on the scaffolds. The cell adhesion efficiency, cell morphology observed by scanning electron microscopy (SEM) and the degradation behavior of the blend scaffold were evaluated. In summary, the results show that the adhesion efficiency of cells on the PLGA/HA blend scaffold is higher than that on the PLGA scaffold. Moreover, the incorporation of HA in PLGA not only helps to increase the cell affinity but also tends to lead the water and nutrient into the scaffold easily.     

Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.     

Heparin-functionalized collagen matrices with controlled release of basic fibroblast growth factor

Tissue engineering scaffolds with controlled long-term release of growth factors are constructed in an attempt to mimic the intelligent ability of the extracellular matrix (ECM) to release endogenous growth factors. In this study, collagen sponges (Collagen group) were modified by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) crosslinking (EDC/NHS group) and heparin immobilization (EDC/NHS-H group), and subsequently seeded with human umbilical vein endothelial cells (HUVECs). Native and modified sponges were pre-adsorbed with basic fibroblast growth factor (bFGF) to evaluate the sustained release and bioactive maintenance of bFGF from the sponges. We found that modified collagen matrices permitted HUVECs to proliferate and migrate well and to distribute uniformly. The EDC/NHS-H group exhibited an excellent sustained-release profile and bioactive maintenance of the pre-adsorbed bFGF as compared with the Collagen and EDC/NHS groups. These results suggest that heparin-functionalized collagen matrices can support a controlled release of bFGF and thus, have potential as a tissue engineering scaffold.     

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.     

Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.     

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

Abstract

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (?20 and ?80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types.     

Systematic studies of a self-assembling peptide nanofiber scaffold with other scaffolds

A designer self-assembling peptide nanofiber scaffold has been systematically studied with 10 commonly used scaffolds in a several week study using neural stem cells (NSC), a potential therapeutic source for cellular transplantations in nervous system injuries. These cells not only provide a good in vitro model for the development and regeneration of the nervous system, but may also be helpful in testing for cytotoxicity, cellular adhesion, and differentiation properties of biological and synthetic scaffolds used in medical practices. We tested the self-assembling peptide nanofiber scaffold with the most commonly used scaffolds for tissue engineering and regenerative medicine including PLLA, PLGA, PCLA, collagen I, collagen IV, and Matrigel. Additionally, each scaffold was coated with laminin in order to evaluate the utility of this surface treatment. Each scaffold was evaluated by measuring cell viability, differentiation and maturation of the differentiated stem cell progeny (i.e. progenitor cells, astrocytes, oligodendrocytes, and neurons) over 4 weeks. The optimal scaffold should show high numbers of living and differentiated cells. In addition, it was demonstrated that the laminin surface treatment is capable of improving the overall scaffold performance. The designer self-assembling peptide RADA16 nanofiber scaffold represents a new class of biologically inspired material. The well-defined molecular structure with considerable potential for further functionalization and slow drug delivery makes the designer peptide scaffolds a very attractive class of biological material for a number of applications. The peptide nanofiber scaffold is comparable with the clinically approved synthetic scaffolds. The peptide scaffolds are not only pure, but also have the potential to be further designed at the molecular level, thus they promise to be useful for cell adhesion and differentiation studies as well as for future biomedical and clinical studies.     

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young’s modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.     

Silk fibroin modified porous poly(ε-caprolactone) scaffold for human fibroblast culture in vitro

In order to develop scaffolds with improved biocompatibility for cell culture, hybrid scaffolds were fabricated by modifying poly(epsilon-caprolactone) (PCL) with silk fibroin (SF) in a porous structure. Scanning electronic microscopy revealed that the morphology of the PCL-SF hybrid scaffold was affected by the concentration of the SF solution. Availability of SF on the surface and the conformational transition induced by methanol treatment were proved by attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR), and wettability of the hybrid scaffold was greatly improved. To evaluate scaffold biocompatibility, human fibroblasts were cultured on the hybrid scaffold with the unmodified PCL scaffold as control. An MTT assay indicated that although fewer cells were initially held on the hybrid scaffold after one day of culture, comparable cell numbers were achieved after four days and significantly more cells proliferated on the hybrid after seven days. The cell morphology also indicated that the PCL-SF hybrid scaffold was favorable for cell culture. This study suggests that surface modification with SF would be an effective way to improve the biocompatibility of PCL, facilitating its application in practical tissue engineering.