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.     

Fabrication of bioactive composite by developing PLLA onto the framework of sintered HA scaffold

Composite porous scaffolds of hydroxyapatite (HA)/poly-l-lactide (PLLA) were fabricated by a two-step immersing replication method. Structure and mechanical properties of both the single HA scaffold and the composite HA/PLLA scaffold were determined. The bioactivity of the scaffolds was evaluated by soaking in a simulated body fluid (SBF), and the formation of the apatite layer was determined by X-ray diffraction (XRD), Scanning Electron Microscope (SEM) and Energy-Dispersive Spectrometer (EDS). The results showed that without changing the highly interconnected porous structure, the HA/PLLA composite scaffold was mechanically enhanced to a great deal of extent compared with single HA scaffold. On the other hand, it is also suggested that the HA/PLLA scaffold was bioactive as it induced the formation of apatite on the surface of the composite scaffolds after soaking in SBF for 7 days.     

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 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。      

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.     

聚乳酸/聚乙二醇/羟基磷灰石多孔骨支架的3D打印制备及其生物相容性

聚乳酸(PLA)是一种应用广泛的生物高分子材料,但在应用过程中存在韧性、亲水性、生物活性差等缺点。用聚乙二醇(PEG)和羟基磷灰石(HA)对PLA进行改性。通过熔融共混制备不同质量比的PLA/PEG/HA复合3D打印线材,并通过分析PLA/PEG/HA线材的力学性能、结晶性能、热性能、流变性能等,筛选更适合熔融沉积成型(FDM)的3D打印成型线材,进而利用3D打印制备精度高的力学性能试样及生物相容性好、细胞可增殖和分化的生物多孔支架。结果表明:PEG的添加提高了PLA的韧性,降低了PLA的熔点。HA的添加则提高PLA/PEG/HA复合材料的弹性模量和冷结晶温度,同时HA也可以改善复合材料的加工性能。SEM与荧光标记结果表明多孔支架与细胞具有良好的生物相容性。生物支架对体外细胞的成功培养,为进一步发掘生物多孔支架在动物体内、生物医学及定制化应用方面提供了潜在可能。     

Hydrothermal synthesis of zirconia-based nanocomposite powder reinforced by graphene and its application for bone scaffold with 3D printing

Hydrothermal method is a cheap and green approach for the synthesis of composite powders. In this study, the zirconia (ZrO₂)-based nanocomposite powder was reinforced with reduced graphene oxide (ZrO₂/RGO) and was synthesized in a one-pot as a precursor for bone scaffold applications. Moreover, for the stimulation of osseointegration in bone scaffolds, Hydroxyapatite (HA) was used in 10 wt%. In this regard, the two types of ZrO₂/RGO and ZrO₂/RGO/HA precursors were applied for the fabrication of bone scaffolds via 3D printing and finally, the mechanical and biological properties of scaffolds were evaluated. For characterization, the X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), compress strength, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide as MTT assay protocol were performed. The results demonstrated that the ZrO₂/RGO scaffolds with a tolerance of compressive stress of 240.11 MPa depicted better mechanical properties compared with ZrO₂/RGO/HA with the compress strength of 141.66 MPa. Moreover, after 7 days of bone scaffolds immersion in simulated body fluid (SBF) the growth of compressive strength began while after 28 days reached 260.15 MPa for ZrO₂/RGO and 192.31 for ZrO₂/RGO/HA. Finally, the cellular response of the scaffolds indicated the lack of cellular toxicity of the scaffolds during MTT assay.     

Interaction of cell culture with composition effects on the mechanical properties of polycaprolactone-hydroxyapatite scaffolds fabricated via selective laser sintering (SLS)

In the current study PCL/HA composites were fabricated using SLS as two- and three-dimensional lattice structures and exposed to a cellular component (MC 3T3 osteoblast-like cells). The main aims were to determine the mechanical differences due to powder composition and to observe the physical and mechanical changes pertaining to cell presence. These structures were characterized by compressive mechanical testing, and the effects of cell culturing and degradation on mechanical properties of the scaffolds with different PCL/HA compositions were determined. Moreover, changes in the scaffold morphology due to the cell culture conditions were determined by μ-CT analysis.Cells steadily grew on the scaffolds for 21 days with preferential distribution around the macropores and initially PCL/HA(15%) composites had higher cell numbers. Removal of loosely sintered parts was observable during the culturing period. Cell culture conditions did not change the compressive moduli significantly but had a distinct effect on compressive strength. For PCL/HA(15%) composites, an initial loss in strength caused by cell culture was reversed by longer cell exposure, with compressive strength of the structures restored to the initial properties (p ≤ 0.05). μ-CT measurements showed widespread morphological changes in the scaffolds, such as a decrease in the roughness of the struts. In general, in the initial period composites with lower HA content (15 wt.%) showed better metabolic activity compared to the higher HA content, however by day 14 the performance of the two compositions was equal. These results suggest that changes in sintering due to the differences in powder composition can have profound effects on the short and long term mechanical properties of the scaffold particularly under cell culture conditions, and this should be closely considered for SLS processing of scaffolds.     

Novel silk fibroin/hydroxyapatite composite films: Structure and properties

Novel composite films of Bombyx mori silk fibroin (SF) and hydroxyapatite (HA) composite films, with glycerin as an additive, were fabricated by means of co-precipitation, where the theoretical HA content was varied from 2 (w/w)% to 31 (w/w)%. The structure and properties of the composite films were investigated by SEM, XRD, AFM, TGA and tensile testing. The results showed that the composite films were smooth and transparent with the uniform distribution of HA into the composites when the final HA content was lower than 21 (w/w)%. XRD and TGA data showed that the silk fibroin in the composites was predominantly in a β-sheet crystalline structure, which was induced not only by the addition of glycerin, also by the HA crystal growth during the composite fabrication, leading to the thermal stable composite films. On the other hand, the HA crystals had the anisotropic growth with high extent of lattice imperfection and the preferential orientation along c-axis, probably promoted by the silk fibroin. The mechanical testing results showed that both break strain and stress were declined with the increase of HA content in the composites, presumably due to the original brittleness of HA compound.     

Enhanced cell affinity of the silk fibroin- modified PHBHHx material

Cell affinity is one of the important issues required for developing tissue engineering materials. Although the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has been attractive for its controllable mechanical properties recent years, its cell affinity is still necessary to be improved for the requirements. For this purpose, the regenerated silk fibroin (SF) was coated on the PHBHHx films and its porous scaffolds. The mechanical test showed that SF-modified PHBHHx (SF/PHBHHx) film has a maximum tensile strength of 11.5 ± 0.5 MPa and elongation at break of 175 ± 5%. ATR-FTIR spectroscopy demonstrated that SF firmly attached on the scaffold by the hydrogen bonding interaction between SF and PHBHHx even flushed for 21 days in the phosphate-buffer saline (PBS) solution (pH = 7.4). In order to characterize the cell affinity of the SF-modified material, endothelial-like cell line ECV304 cells were seeded on the SF/PHBHHx films and its porous scaffolds. The histochemical analyses of cells stained by the hematoxylin and eosin (HE) as well as cell nuclei stained by the 4′,6-diamindine-2′-phenylindole (DAPI) demonstrated that cell attached and reached nearly 100% confluence on the SF/PHBHHx films when cultured for 4 days, which was much faster than that on the pure PHBHHx film. Moreover, the assay of cell activity by the 3-(4, 5-dimethyl thiazol -2-yl)-2, 5-diphenyl terazolium bromide (MTT) showed quantitatively that the number of cells on the SF/PHBHHx porous scaffolds was significant more than that on the unmodified ones after 4, 8, and 14 days culture, respectively. Scanning electron microscopy (SEM) revealed the similar results. Therefore, the SF-modified PHBHHx material is maybe a potential material applicable in the cardiovascular tissue engineering.     

Nanoindentation on porous bioceramic scaffolds for bone tissue engineering

We report nanoindentation mechanical properties measurements on porous ceramic scaffolds made for tissue engineering applications. The scaffolds have been made from tricalcium phosphate (TCP), hydroxyapatite (HA) nanopowder and mixed powders of HA (50 wt%) and TCP (50 wt%) using the polyurethane sponge method, which produces open porous ceramic scaffolds through replication of a porous polymer template. The scaffolds prepared by this method have a controllable pore size and interconnected pore structure. The crystal structures and morphology of porous scaffolds were determined by X-ray diffraction (XRD) and atomic force microscopy (AFM) respectively. Nanoindentation measurements to a depth of 600 nm showed a Young's modulus value of 10.3 GPa for HA+TCP composite scaffolds and 1.5 GPa for TCP scaffolds. The hardness values were 240 MPa for HA+TCP composites and 21 MPa for TCP sample respectively. The results showed that the mechanical properties of the biodegradable scaffolds can be considerably enhanced with the addition of HA while maintaining the interconnected open pores and pore geometry desirable for bone tissue engineering.     

Fabrication and characterization of novel hydroxyapatite/porous carbon composite scaffolds

Novel hydroxyapatite (HA)/porous carbon composite scaffolds were prepared by applying sonoelectrodeposition and a subsequent hydrothermal treatment to previous carbonized phenolic resin coated polyurethane sponges. The interconnected pore network and morphology of HA/porous carbon composite scaffolds were determined by scanning electron microscope (SEM), and the whole surface of porous carbons were evenly coated with the deposited HA layer which was confirmed by EDS and XRD. The porosity (83.5 ± 0.3%) and the bulk density (0.297 ± 0.009 g·cm⁻³) of HA/porous carbon scaffolds were detected by the Archimedes method. The compressive and flexural strength of the scaffolds is 1.187 ± 0.064 MPa and 0.607 ± 0.268 MPa, respectively. Compared with the polymeric surface of 24-well cell culture plates, these novel scaffolds significantly promote the proliferation of human osteoblast-like MG-63 cells, indicating that this novel HA/porous carbon composite scaffold could be used for in vitro 3D culture of osteoblasts.     

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.     

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.     

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。     

木垛型壳聚糖多孔支架的磷酸化改性和仿生矿化

为提高壳聚糖支架材料的孔隙率及矿化程度, 通过磷酸化表面改性和仿生矿化制备了磷酸化(PCSW)和生物矿化(BMCW)木垛型壳聚糖多孔支架。FTIR结果显示, 壳聚糖分子中有磷酸根的引入。XRD结果表明, 矿化24 h后支架上形成结晶度较高的磷酸钙盐晶体, 矿化48 h后结晶度明显增加并形成单纯的羟基磷灰石(HA)结晶。SEM观察发现, 在支架的内外表面均致密地沉积了HA晶体层。压缩强度测试结果表明, 复合支架BMCW矿化48 h的压缩强度为(0.54±0.005) MPa, 压缩模量为(5.47±0.65) MPa, BMCW可用作非承重骨组织修复材料。     

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.     

In vitro study of hydroxyapatite/polycaprolactone (HA/PCL) nanocomposite synthesized by an in situ sol–gel process

Hydroxyapatite (HA) is the most substantial mineral constituent of a bone which has been extensively used in medicine as implantable materials, owing to its good biocompatibility, bioactivity high osteoconductive, and/or osteoinductive properties. Nevertheless, its mechanical property is not utmost appropriate for a bone substitution. Therefore, a composite consist of HA and a biodegradable polymer is usually prepared to generate an apt bone scaffold. In the present work polycaprolactone (PCL), a newly remarkable biocompatible and biodegradable polymer, was employed as a matrix and hydroxyapatite nanoparticles were used as a reinforcement element of the composite. HA/PCL nanocomposites were synthesized by a new in situ sol–gel process using calcium hydroxide and phosphoric acid precursors in the presence of Tetrahydrofuran (THF) as a solvent. Chemical and physical characteristics of the nanocomposite were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) analyses. The results indicated that pure HA nanoparticles were well-incorporated and homogenously dispersed in the PCL matrix. It was found that the mechanical property of PCL was improved by addition of 20 wt.% HA nanoparticles. Furthermore, the biological property of nanocomposites was investigated under in vitro condition. For this purpose, HA/PCL scaffolds were prepared through a salt leaching process and immersed in a saturated simulated body fluid (SBF) after 3 and 7 days. It was found that a uniform layer of biomimetic HA could be deposited on the surface of HA/PCL scaffolds. Therefore, the prepared HA/PCL scaffolds showed good potential for bone tissue engineering and could be used for many clinical applications in orthopedic and maxillofacial surgery.     

Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures

Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique ‘elastic–plastic’ mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65–70%) but different pore architectures, described as ‘lamellar’ (pore width = 25 ± 5 μm) and ‘cellular’ (pore diameter = 100 ± 10 μm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications.     

3D打印HA微球支架的制备与表征

生物材料表面微结构对于成骨具有重要的影响,该研究以不同粒径(<60μm)的羟基磷灰石(HA)微球状粉体为原料,通过3D打印技术制备了一系列(HA0、HA10、HA30、HA50)生物陶瓷支架。不同支架具有相似的理化性能,由于微球粒径不同形成了不同的微结构,对其生物学性能造成不同的影响。相比传统非微球颗粒打印的支架(HA0), HA微球构成的支架能够提供更多细胞粘附和生长位点, 24 h的粘附实验显示HA30支架能显著促进骨髓间充质干细胞的伪足伸长;培养5 d的细胞增殖实验显示,微球支架上的细胞数量与HA0支架出现显著性差异,表面微球结构与细胞尺度相当的HA30支架具有最好的促增殖效果。因此,3D打印技术在可控制备HA支架宏观结构的同时,还可以通过控制生物陶瓷粉体的颗粒形貌,调控3D打印支架的表面微结构,从而优化其生物学效应,在骨组织工程领域具有良好的应用前景。