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.     

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.     

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.     

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.     

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.     

Fabrication and characterization of curcumin-loaded silk fibroin/P（LLA-CL） nanofibrous scaffold

Curcumin exhibited excellent properties including antioxidant, anti- inflammatory, antiviral, antibacterial, antifungal, anticancer, and anticoagulant activities. In this study, curcumin was incorporated into silk fibroin （SF）/poly（L-lactic acid-co-e- caprolactone） （P（LLA-CL）） nanofibrous scaffolds via electrospinning, and changes brought about by raising the curcumin content were observed： SEM images showed that the average nanofibrous diameter decreased at the beginning and then increased, and the nanofibers became uniform; FTIR showed that the conformation of SF transforming from random coil form to β-sheet structure had not been induced, while SF conformation converted to β-sheet after being treated with 75% ethanol vapor; XRD results confirmed that the crystal structure of （P（LLA-CL）） had been destroyed; The mechanical test illustrated that nanofibrous scaffolds still maintained good mechanical properties. Further, curcumin-loaded nanofibrous scaffolds were evaluated for drug release, antioxidant and antimicrobial activities in vitro. The results showed that curcumin presented a sustained release behavior from nanofibrous scaffolds and maintained its free radical scavenging ability, and such scaffolds could effectively inhibit S. aureus growth （〉 95%）. Thus, curcumin-loaded SF/P（LLA-CL） nanofibrous scaffolds might be potential candidates for wound dressing and tissue engineering scaffolds.     

Silk RGD融合蛋白修饰羟基磷灰石/丝素蛋白支架对成骨细胞生长的影响

通过浸渍吸附的方法, 用桑蚕丝素-RGD融合蛋白(简称Silk-RGD)对多孔状磷灰石/丝素蛋白(HA/SF)复合支架材料进行表面修饰, 研究了复合支架材料在不同浓度Silk-RGD蛋白溶液中浸渍后对两种不同成骨细胞MG-63和MC3T3-E1黏附、增殖和分化的影响。结果表明, Silk-RGD融合蛋白修饰的复合支架材料的细胞黏附性能显著高于未经修饰的对照组, 且其促黏附性能具有Silk-RGD浓度依赖性; 体外培养7天时, 细胞增殖能力较对照组更显著,当Silk-RGD的吸附量为11 μg/mg时, MG-63的增殖率较对照样提高了21%, MC3T3-E1提高了50%; 而碱性磷酸酶活性检测结果显示, 复合支架经Silk-RGD表面修饰后对MC3T3-E1细胞的分化有一定的促进作用, 但对MG-63细胞的影响不明显。      

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.     

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.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Preparation,characterization and biological test of 3D-scaffolds based on chitosan,fibroin and hydroxyapatite for bone tissue engineering

This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2 ± 0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7 ± 2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7 days and 21 days and the level of alkaline phosphatase at 14 and 21 days was statistically superior compared to other tested materials.     

造孔剂和发泡剂结合法制备氟化羟基磷灰石多孔支架研究

本研究采用添加造孔剂和发泡剂相结合法制备多孔氟化羟基磷灰石, 合成具有大孔与小孔套连、3D方向上分布的多孔支架。通过化学沉淀法制备了氟化羟基磷灰石(FHA)粉体, 以碳酸氢铵为发泡剂、PMMA为造孔剂, 通过烘干和烧结工艺制备孔洞均匀且相互贯通、坯体致密的多孔支架。经X射线衍射分析, 支架的相组成是FHA和β-TCP。SEM观察结果显示支架孔洞形貌规整、大孔尺寸100~400 µm, 小孔尺寸10~50 µm。以HA和β-TCP为原料对造孔方法的普适性进行验证, 并对支架孔的形成及其影响因素进行了分析。     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Modification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potential

The modification of human cancellous bone (hBONE) with silk fibroin/gelatin (SF/G) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccini-mide (NHS) crosslinking was established. The SF/G solutions at a weight ratio of 50/50 and the solution concentrations of 1, 2, and 4 wt % were studied. SF/G sub-matrix was formed on the surface and inside pore structure of hBONE. All hBONE scaffolds modified with SF/G showed smaller pore sizes, less porosity, and slightly lower compressive modulus than unmodified hBONE. SF/G sub-matrix was gradually biodegraded in collagenase solution along 4 days. The hBONE scaffolds modified with SF/G, particularly at 2 and 4 wt % solution concentrations, promoted attachment, proliferation, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSC), comparing to the original hBONE. The highest cell number, ALP activity and calcium production were observed for MSC cultured on the hBONE scaffolds modified with 4 wt % SF/G. The mineralization was also remarkably induced in the cases of modified hBONE scaffolds as observed from the deposited calcium phosphate by EDS. The modification of hBONE with SF/G was, therefore, the promising method to enhance the osteoconductive potential of human bone graft for bone tissue engineering.     

静电纺丝天然-人工合成聚合物复合纳米纤维及其对细胞黏附、增殖和迁移的影响

生物材料组成成分对细胞生物功能有不同的影响。利用静电纺丝技术制备了基于聚己内酯(PCL,polycaprolactone)的不同天然蛋白、多糖(丝素蛋白(SF,silk fibroin)、透明质酸(HA,hyaluronicacid))的混合组分纳米纤维,采用了扫描电镜和接触角对纳米纤维进行基础表征。同时,进一步考察了纳米纤维作为组织工程支架的可行性。研究结果表明SF组分能增加材料的可纺性,有利于细胞的前期黏附,并能够促进细胞增殖。HA组分可以改善材料的亲水性,增加细胞伪足并促进细胞迁移。重要的是,PCL/SF/HA纳米纤维能同时结合SF和HA的优点,有望在组织工程领域得到应用。     

A novel electrospun silk fibroin/hydroxyapatite hybrid nanofibers

A novel electrospinning of silk fibroin/hydroxyapatite hybrid nanofibers with different composition ratios was performed with methanoic acid as a spinning solvent. The silk fibroin/hydroxyapatite hybrids containing up to 30% hydroxyapatite nanoparticles could be electrospun into the continuous fibrous structure. The electrospun silk fibroin/hydroxyapatite hybrid nanofibers showed bigger diameter and wider diameter distribution than pure silk fibroin nanofibers, and the average diameter gradually increased from 95 to 582 nm. At the same time, the secondary structure of silk fibroin/hydroxyapatite nanofibers was characterized by X-ray diffraction, Fourier transform infrared analysis, and DSC measurement. Comparing with the pure silk fibroin nanofibers, the crystal structure of silk fibroin was mainly amorphous structure in the hybrid nanofibers. X-ray diffraction results demonstrated the hydroxyapatite crystalline nature remained as evidenced from the diffraction planes (002), (211), (300), and (202) of the hydroxyapatite crystallites, which was also confirmed by Fourier transform infrared analysis. The thermal behavior of hybrid nanofibers exhibited the endothermic peak of moisture evaporation ranging from 86 to 113 °C, and the degradation peak at 286 °C appeared. The SF/HAp nanofibers mats containing 30% HAp nanoparticles showed higher breaking tenacity and extension at break for 1.1688 ± 0.0398 MPa and 6.55 ± 1.95%, respectively. Therefore, the electrospun silk fibroin/hydroxyapatite hybrid nanofibers should be provided potentially useful options for the fabrication of biomaterial scaffolds for bone tissue engineering.     

静电纺SF/PLA共混纳米纤维的形态结构及热性能

以六氟异丙醇(HFIP)为溶剂,通过静电纺丝方法纺制了不同比例的丝素蛋白/聚乳酸(SF/PLA)共混纳米纤维,并通过SEM、FT-IR、X射线衍射和TG-DTA研究了该纳米纤维非织造网的形态特征、分子结构和热性能。结果表明,纤维直径在246nm～2000nm之间,混纺纤维直径随着SF含量的增大而变粗;FT-IR和X射线衍射谱图都表明混纺纤维有着较高的结晶度,并有相分离发生;TG-DTA曲线中明显的SF和PLA分解峰,进一步证明了在本实验条件下SF与PLA的共混不能形成共溶体系,共混SF/PLA纳米纤维存在两个分离的结晶区,即SF晶区与PLA晶区。     

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.     

Silk fibroin-based scaffolds for tissue engineering

Silk fibroin (SF) from the Bombyx mori silkworm exhibits attractive potential applications as biomechanical materials, due to its unique mechanical and biological properties. This review outlines the structure and properties of SF, including of its biocompatibility and biodegradability. It highlights recent researches on the fabrication of various SF-based composites scaffolds that are promising for tissue engineering applications, and discusses synthetic methods of various SF-based composites scaffolds and valuable approaches for controlling cell behaviors to promote the tissue repair. The function of extracellular matrices and their interaction with cells are also reviewed here.     

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.