Growth of fibroblast and vascular smooth muscle cells in fibroin/collagen scaffold

In our recent study, a novel fibroin/collagen scaffold with improved mechanical properties and controllable porous structure was prepared through freeze–drying method. In this research, the cyto-compatibility was further studied, using fibroblast and vascular smooth muscle cells (VSMC) as the model cells. MTT results indicated that the growth of fibroblast and VSMC both further improved in the fibroin/collagen scaffold than in pure fibroin scaffolds. The confocal and SEM results showed that fibroblast cells and VSMCs had better adhesion and spreading properties in the fibroin/collagen scaffolds. Although further studies, such as the extracellular matrix production and the functional gene expression, are necessary to clarify the biocompatibility of the fibroin/collagen scaffolds, the present results indicate that the fibroin/collagen scaffold is a new scaffold material suitable for tissue engineering. On the other hand, the mild and all-aqueous preparation processes also make it possible to embed different growth factors inside the scaffolds to maximize cell functions and the formation of specific tissues.     

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.     

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin,gelatin and hydroxyapatite

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as “non-irritant” to “slight-irritant”, compared to Gelfoam^® (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications.     

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.     

Controlling microstructure of three-dimensional scaffolds from regenerated silk fibroin by adjusting pH

For tissue engineering, it is very important to design and control the pore architecture of three-dimensional (3D) polymeric scaffolds, which plays an important role in directing tissue formation and function. In this study, 3D porous silk fibroin scaffolds produced using a freeze drying technique were prepared at pHs ranging from 5 to 9. The effects of pH on the pore microstructure of the silk fibroin scaffold were examined by rheometry, FESEM and FTIR. Different pore structures were formed according to the pH of silk fibroin because silk fibroin exhibits water-like behavior under basic conditions and gel-like behavior under acidic conditions.     

Facile fabrication of the porous three-dimensional regenerated silk fibroin scaffolds

In the present work, we report a new facile method to fabricate porous three-dimensional regenerated silk fibroin (RSF) scaffolds through n-butanol- and freezing-induced conformation transition and phase separation. The effects of RSF concentration, freezing temperature and n-butanol addition on the microstructure, the secondary structures of silk fibroin and apparent mechanical properties of the RSF scaffolds were investigated by SEM, ¹³C CP-MAS NMR spectra and mechanical testing, respectively. By adjusting the RSF concentration and n-butanol addition, the pore size of the scaffold could be controlled in the range from of 10 μm to 350 μm with 84%–98% of porosity. The tensile strength of the wet scaffold reached the maximum of 755.2 ± 33.6 kPa when the concentration of RSF solution was increased to 15% w/w. Moreover, post-treatment with ethanol further induced conformation transition of RSF from random coil or helix to β-sheet. The porous scaffolds prepared by this facile and energy-saving method with good biocompatibility will have great potential for application in tissue engineering.     

Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering

Plastic compression of hydrated collagen gels rapidly produces biomimetic scaffolds of improved mechanical properties. These scaffolds can potentially be utilised as cell seeded systems for bone tissue engineering. This work investigated the influence of multiple unconfined compression on the biocompatibility and mechanical properties of such systems. Single and double compressed dense collagen matrices were produced and characterised for protein dry weight, morphology and mechanical strength. Compression related maintenance of the seeded HOS TE85 cell line viability in relation to the extent of compression was evaluated up to 10 days in culture using the TUNEL assay. Fluorescence Live/Dead assay was conducted to examine overall cell survival and morphology. Cell induced structural changes in the dense collagenous scaffolds were assessed by routine histology. The mechanical properties of the cellular scaffolds were also evaluated as a function of time in culture. It is clear that a single plastic compression step produced dense collagenous scaffolds capable of maintaining considerable cell viability and function as signs of matrix remodeling, and maintenance of mechanical properties were evident. Such scaffolds should therefore be further developed as systems for bone tissue regeneration.     

β-CaSiO_3/丝素蛋白复合支架材料结构与性能的研究

利用冷冻干燥法制备了β-CaSiO_3/丝素蛋白复合支架材料,经XRD和FTIR分析表明复合支架中丝素的结构主要以β-折叠为主;SEM分析显示材料孔隙分布均匀,孔连通性较好,孔径尺寸约为100～300μm.对支架的孔隙率和机械强度等性能进行了表征,研究表明复合支架的孔隙率为83%～87%,机械强度有较大提高.应用模拟体液浸泡实验研究了复合支架的体外生物活性,并用XRD、FESEM和EDS对试样表面进行了表征;结果显示,样品经模拟体液浸泡3天后,表面都能沉积出类骨羟基磷灰石(HA)层,β-CaSiO_3的加入能加快复合支架表面沉积类骨HA的速度.研究结果显示β-CaSiO_3/丝素蛋白复合支架材料有望作为强度较好的生物活性硬组织修复材料.     

A novel method for the fabrication of homogeneous hydroxyapatite/collagen nanocomposite and nanocomposite scaffold with hierarchical porosity

Homogeneous nanocomposites composed of hydroxyapatite (HAp) and collagen were synthesized using a novel in situ precipitation method through dual template-driven. The morphological and componential properties of nanocomposites were investigated. The HAp particulates, in sizes of about 50–100 nm, were distributed homogeneously in the organic collagen hydrogel. Highly magnified TEM observation showed that HAp inorganic particles were composed of fine sub-particles (2–5 nm) without regular crystallographic orientation. Based on these homogeneous nanocomposites, a novel HAp/collagen nanocomposite scaffold with hierarchical porosity was prepared by multilevel freeze-drying technique. Compared to other conventional scaffolds for tissue engineering, this novel in situ method endows synthesized composite scaffolds with unique morphology—ultrafine HAp particles dispersed homogenously in collagen at nano level and the foam scaffold with hierarchical pore structures. The mechanical performance increased obviously compared with neat collagen. These results provided an efficient approach toward new biomimetic tissue scaffold for the biomedical applications with enhanced intensity/bioactivity and controlled resorption rates. This novel method, we expect, will lead to a wide application in many other hydrogel systems and may be useful for fabrication of various homogeneous inorganic/organic nanocomposites.     

Controlling the processing of collagen-hydroxyapatite scaffolds for bone tissue engineering

Scaffolds are an important aspect of the tissue engineering approach to tissue regeneration. This study shows that it is possible to manufacture scaffolds from type I collagen with or without hydroxyapatite (HA) by critical point drying. The mean pore sizes of the scaffolds can be altered from 44 to 135 μm depending on the precise processing conditions. Such pore sizes span the range that is likely to be required for specific cells. The mechanical properties of the scaffolds have been measured and behave as expected of foam structures. The degradation rate of the scaffolds by collagenase is independent of pore size. Dehydrothermal treatment (DHT), a common method of physically crosslinking collagen, was found to denature the collagen at a temperature of 120^∘C resulting in a decrease in the scaffold’s resistance to collagenase. Hybrid scaffold structures have also been manufactured, which have the potential to be used in the generation of multi-tissue interfaces. Microchannels are neatly incorporated via an indirect solid freeform fabrication (SFF) process, which could aid in reducing the different constraints commonly observed with other scaffolds.     

Crosslinked fibroin nanoparticles using EDC or PEI for drug delivery: physicochemical properties,crystallinity and structure

A new silk fibroin-based nanoparticles (FNPs) have been successfully developed utilizing crosslinker EDC (EDC-FNPs) or cationic polymer poly(ethyleneimine) (PEI-FNPs). All developed crosslinked FNPs show a similar spherical particle size of ~?300 nm. Depending on the amount of EDC or the addition of PEI, the zeta potential could be controlled favorably and range from ??15 mV to +?30 mV. Particle structures and crystallinity index are determined and compared using different techniques; X-ray diffraction (XRD), Fourier-transformed infrared spectroscopy (FT-IR), differential scanning calorimetry and nuclear magnetic resonance (NMR). The most reliable crystallinity calculation methods are based on FT-IR and NMR. The degree of fibroin crystallinity increases with increasing EDC content, whereas PEI reduces it. Exceptionally, XRD shows a reverse order of crystallinity due to the inability to detect short-range ordered structures. Proposed schematic particle structures match all experimental data. In conclusion, the crosslinked fibroin-based nanoparticle properties could be manipulated by using carbodiimide or PEI crosslinkers. In addition, both EDC-FNPs and PEI-FNPs showed high potential as a drug delivery system.     

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-polyurethane scaffolds for tissue engineering

Silk fibroin (SF) is a highly promising protein for its surface and structural properties, associated with a good bio- and hemo-compatibility. However, its mechanical properties and architecture cannot be easily tailored to meet the requirements of specific applications. In this work, SF was used to modify the surface properties of polyurethanes (PUs), thus obtaining 2D and 3D scaffolds for tissue regeneration. PUs were chosen for their well known advantageous properties and versatility; they can be obtained either as 2D (films) or 3D (foams) substrates. Films of a medical-grade poly-carbonate-urethane were prepared by solvent casting; PU foams were purposely designed and prepared with a morphology (porosity and cell size) adequate for cell growth. PU substrates were coated with fibroin by a dipping technique. To stabilize the coating layer, a conformational change of the protein from the alpha-form (water soluble) to the beta-form (not water soluble) was induced. Novel methodology in UV spectroscopy were developed for quantitatively analyzing the SF-concentration in dilute solutions. Pure fibroin was used as standard, as an alternative to the commonly used albumin, allowing real concentration values to be obtained. SF-coatings showed good stability in physiological-like conditions. A treatment with methanol further stabilized the coating. Preliminary results with human fibroblasts indicated that SF coating promote cell adhesion and growth, suggesting that SF-modified PUs appear to be suitable scaffolds for tissue engineering applications.     

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.     

Graphene Oxide/Polymer‐Based Biomaterials

Since its discovery in 2004, derivatives of graphene have been developed and heavily investigated in the field of tissue engineering. Among the most extensively studied forms of graphene, graphene oxide (GO), and GO/polymer‐based nanocomposites have attracted great attention in various forms such as films, 3D porous scaffolds, electrospun mats, hydrogels, and nacre‐like structures. In this review, the most actively investigated GO/polymer nanocomposites are presented and discussed, these nanocomposites are based on chitosan, cellulose, starch, alginate, gellan gum, poly(vinyl alcohol) (PVA), poly(acrylamide), poly(?‐caprolactone) (PCL), poly(lactic acid) (PLLA), poly(lactide‐co‐glycolide) (PLGA), gelatin, collagen, and silk fibroin (SF). The biological and mechanical performance of such nanocomposites are comprehensively scrutinized and ongoing research questions are addressed. The analysis of the literature reveals overall the great potential of GO/polymer nanocomposites in tissue engineering strategies and indicates also a series of challenges requiring further research efforts.

     

高强度力学性能丝素/羟基磷灰石多孔支架材料的初步制备

以丝素和羟基磷灰石为基材,磷酸盐缓冲液为溶剂,戊二醛为交联剂制备丝素/羟基磷灰石支架材料,对丝素生物材料在骨材料中的开发应用有积极意义。研究表明,丝素/羟基磷灰石复合材料具有结晶结构和β构象。当丝素与羟基磷灰石的比例为6/4时,支架材料的弹性模量和压缩强度达到最大,分别为48.087 MPa和1.427 MPa,孔隙率...     

Deposition behavior and properties of silk fibroin scaffolds soaked in simulated body fluid

Porous 3D silk fibroin (SF) scaffolds were prepared directly from the SF solution with the addition of methanol and glutaraldehyde by a freeze-drying method. The scaffolds were then soaked in the simulated body fluid (SBF) for the deposition of hydroxyapatite (HA) crystals. The XRD and FTIR results showed that the SF were in β-sheet structure, resulting in the high thermal stability and mechanical properties of scaffolds. The XRD and AAS data revealed that the SF scaffolds could induce the continuous growth and enrichment of HA crystals onto the scaffolds with the extension of soaking time. The mechanical properties of scaffolds increased first with the HA-deposition within 3 d of soaking, then it declined. During the full soaking period, no significant change was observed on the porosity and water-binding ability, which were kept at about 84% and 800%, respectively. The cell cultivation results showed that the scaffolds have the satisfied cell biocompatibility, which was promoted after the HA-deposition. This work suggests that the porous 3D SF scaffolds may be a potential candidate in the bone engineering.     

Effects of osteoblast-like cell seeding on the mechanical properties of porous composite scaffolds

In tissue engineering technology, polymer–ceramics or polymer–polymer composites have been considered as advanced scaffolds having mechanical stability, biocompatibility, cell proliferation, and easy processability. However, the relationship between the mechanical properties and the cell proliferation behavior of such composite scaffolds has not been clarified yet. In this study, two types of composite scaffolds, poly(ethylene terephthalate) (PET) fiber/collagen and β-tricalcium phosphate (β-TCP)/gelatin scaffolds, were investigated. MC3T3-E1 cells were cultured in these scaffolds under appropriate conditions. Compression tests were then periodically conducted to evaluate the compressive elastic modulus. It was found that the modulus of the scaffolds containing cells increased with the cell culture period. It is noted that the modulus of the β-TCP/gelatin with cells was approximately seven times larger than that of the PET fiber/collagen with cells.     

Nanoscale viscoelastic properties of an aligned collagen scaffold

Localised mechanical properties for aligned collagen scaffolds derived from Type 1 collagen were determined by application of nanoindentation based techniques. It was possible to measure the modulus and hardness with nanometre control over the depth of penetration and quasi-static testing under displacement control yielded average modulus values ranging from 1.71 GPa to 3.31 GPa; a narrower range of values than obtained by other methods. Hardness values of 222 MPa to 256 MPa were recorded and showed little scatter, highlighting the potential of nanoindentation hardness values as a reproducible and accurate measure of soft material properties. Open loop Load-displacement curves for the collagen exhibited the expected shapes for a viscoelastic material and it was thus possible to apply dynamic stiffness measurement at the nano scale. As well as determining the storage modulus (0.71 GPa) and the loss modulus (0.40 GPa) at the sub-micron length and nano depth resolution it was also possible to discriminate between surface and bulk readings allowing surface effects to be discarded if necessary. In addition to being a more accurate indentation method than atomic force microscopy, the localised dynamic mechanical properties of collagen were measured for the first time. These results demonstrate that this nanoindentation technique can serve as a powerful tool for the characterisation of collagen based biomaterials that are used as scaffolds for a variety of engineered tissues, such as artificial skin, skeletal muscle, heart valves and neuroregeneration guides.     

Manufacture and characterisation of EmDerm—novel hierarchically structured bio-active scaffolds for tissue regeneration

There are significant challenges for using emulsion templating as a method of manufacturing macro-porous protein scaffolds. Issues include protein denaturation by adsorption at hydrophobic interfaces, emulsion instability, oil droplet and surfactant removal after protein gelation, and compatible cross-linking methods. We investigated an oil-in-water macro-emulsion stabilised with a surfactant blend, as a template for manufacturing protein-based nano-structured bio-intelligent scaffolds (EmDerm) with tuneable micro-scale porosity for tissue regeneration. Prototype EmDerm scaffolds were made using either collagen, through thermal gelation, fibrin, through enzymatic coagulation or collagen-fibrin composite. Pore size was controlled via surfactant-to-oil phase ratio. Scaffolds were crosslink-stabilised with EDC/NHS for varying durations. Scaffold micro-architecture and porosity were characterised with SEM, and mechanical properties by tensiometry. Hydrolytic and proteolytic degradation profiles were quantified by mass decrease over time. Human dermal fibroblasts, endothelial cells and bone marrow derived mesenchymal stem cells were used to investigate cytotoxicity and cell proliferation within each scaffold. EmDerm scaffolds showed nano-scale based hierarchical structures, with mean pore diameters ranging from 40–100 microns. The Young’s modulus range was 1.1–2.9?MPa, and ultimate tensile strength was 4–16?MPa. Degradation rate was related to cross-linking duration. Each EmDerm scaffold supported excellent cell ingress and proliferation compared to the reference materials Integra? and Matriderm?. Emulsion templating is a novel rapid method of fabricating nano-structured fibrous protein scaffolds with micro-scale pore dimensions. These scaffolds hold promising clinical potential for regeneration of the dermis and other soft tissues, e.g., for burns or chronic wound therapies.