丝素蛋白/羟基磷灰石复合材料的制备及性能表征

为了改善羟基磷灰石( HAP) 的脆性和新骨诱导性, 采用共沉淀法合成HAP , 盐溶法制备丝素蛋白(SF) , 在胶体状态下将HAP 和SF 复合得到了SF/ HAP 复合材料。采用扫描电镜(SEM) 、X 射线衍射(XRD) ,傅立叶红外光谱( FIR) 对复合材料结构和化学组成进行了分析, 在模拟体液中检验了复合材料的生物活性, 并对其抗压强度进行了测定。结果表明: HAP 与SF 在纳米尺度进行了复合, 复合材料中SF 主要以β-折叠构象存在,酰胺Ⅴ红外特征峰消失,β-折叠构象的其他峰发生了移动, 表明HAP 与SF 间存在化学结合; 模拟体液中浸泡18 天后, 复合材料表面形成了片层状的HAP ; 与纯的HAP 晶体比较, 复合材料结构稳定, 具有较好生物活性和骨诱导性, 其抗压强度可达63 MPa , 可望成为理想的骨组织替换和工程支架材料。      

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.     

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.     

Viscoelastic properties of composites of calcium alginate and hydroxyapatite

Calcium alginate was reinforced with hydroxyapatite (HA) particles, whose dimensions were a few micrometres, with mass fraction, m _f, values in the range 0–0.8. Cylindrical samples of these composite materials were subjected to cyclic compression in the frequency range f = 0.001–20 Hz; in these tests a sinusoidal load of amplitude 1 N was applied either side of a static compression of 2 N. Storage and loss moduli, E′ and E′′, respectively, were found to be independent of particle size; however, E′ increased with frequency consistent with the materials undergoing a glass transition. Above frequencies of about 0.05 Hz, for all materials. For each frequency, the dependence of the moduli on log₁₀ f could be represented by a third order polynomial; these equations can be used to calculate E′ and E′′ for a range of compositions. Approximate values of are predicted by a Reuss model.     

"一步法"制备纳米相丝素蛋白/羟基磷灰石生物复合材料

系统分析了羟基磷灰石(HA)的制备方法和丝素蛋白纤维(SF)的溶解方法,提出一种制备纳米丝素蛋白/羟基磷灰石生物复合材料(SF/HA)的新型反应复合方法--"一步法".并对由"一步法"制得的SF/HA分别进行了钙磷比测定、红外光谱测试、透射电镜观察和x射线衍射测试.结果表明:SF/HA中的钙磷比是1.6692,与标准HA中的钙磷比1.67一致;SF/HA中同时含有SF和HA中各自的官能团;SF/HA的晶粒横向尺度小于100nm,SF/HA呈针状或柱状晶粒,SF和HA能够形成复合;SF/HA的晶型属于六方晶系,当SF在SF/HA中所占质量分数为10%时,晶胞参数a=b=9.0319(A),c=7.0148(A),沿c轴方向平均品粒尺寸是230.7645(A)."一步法"制备SF/HA具有合理性和可行性.     

Nano-scaled hydroxyapatite/silk fibroin sheets support osteogenic differentiation of rat bone marrow mesenchymal cells

A novel biomaterial that was composed of nano-scaled sintered hydroxyapatite (HAp) and silk fibroin (SF) was fabricated. We cultured rat marrow mesenchymal cells (MMCs) on this biomaterial (nano-HAp/SF sheet), on bare SF sheets, and on tissue culture polystyrene (TCPS) dishes as controls, then evaluated cell adhesion, proliferation, and differentiation of the MMCs. After 1 h of culture, a large number of viable cells were observed on the nano-HAp/SF sheets in comparison to the controls. In addition, after 3 h of culture, the morphology of the cells on the nano-HAp/SF sheets was quite different from that on the SF sheets. MMCs extrude their cytoplasmic processes to nano-HAp particles and are well attached to the sheets. After 14 days of culture, under osteogenic conditions, the alkaline phosphatase (ALP) activity and bone-specific osteocalcin secretion of the cells on nano-HAp/SF sheets were higher than were those on the controls. These results indicated that the surface of the nano-HAp/SF sheets is covered with appropriate HAp crystal for MMC adhesion/proliferation and that the sheets effectively support the osteogenic differentiation of MMCs. Therefore, the nano-HAp/SF sheet is an effective biomaterial that is applicable in bone reconstruction surgery.     

“一步法”制备纳米相丝素蛋白/羟基磷灰石生物复合材料

系统分析了羟基磷灰石（HA）的制备方法和丝素蛋白纤维（SF）的溶解方法,提出一种制备纳米丝素蛋白/羟基磷灰石生物复合材料（SF/HA）的新型反应复合方法——“一步法”。并对由“一步法”制得的SF/HA分别进行了钙磷比测定、红外光谱测试、透射电镜观察和X射线衍射测试。结果表明：SF/HA中的钙磷比是1.6692,与标准HA中的钙磷比1.67一致;SF/HA中同时含有SF和HA中各自的官能团;SF/HA的晶粒横向尺度小于100 nm,SF/HA呈针状或柱状晶粒, SF和HA能够形成复合; SF/HA的晶型属于六方晶系, 当SF在SF/HA中所占质量分数为10%时,晶胞参数a=b=9.0319 A,c=7.0148 A,沿c轴方向平均晶粒尺寸是230.7645 A。“一步法”制备SF/HA具有合理性和可行性。     

Effect of silicon on the formation of silk fibroin/calcium phosphate composite

The silk fibroin/calcium phosphate composites were prepared by adding the different amount of Na₂SiO₃ to assess the effect of silicon on the HA (hydroxyapatite) formation in the composites. FTIR and XRD results suggested that the inorganic phase was constituted mainly by the amorphous DCPD (dicalcium phosphate dehydrate), a precursor of HA in the bone mineral, when the composites were prepared at the final Na₂SiO₃ concentration lower than 0.008%. Otherwise, HA was formed as the predominant one in the as-prepared composite, accompanied with a conformational transition in the organic phase of silk fibroin protein from silk I (α-helix and/or polyglycine II (3₁–helix) conformations) to silk II (antiparallel β-sheet conformation). SEM images showed the different morphologies with the samples, i.e., sheet-like crystals in the composites prepared at a low Na₂SiO₃ concentration and rod-like bundles in other composites. The rod-like bundles were connected together to form the porous network, due to the fact that the HA crystals grew with the aggregation of silk fibroin, and further accreted onto the silk fibroin fibrils. TG curves indicated that the composites prepared with a certain amount of additional SiO₃²⁻ had the higher thermal stability because of its high molecular orientation and crystallinity, and high water-holding capacity due to the porous microstructure.     

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。      

Micro-patterned hydroxyapatite/silk fibroin coatings on Mg--Zn--Y--Nd--Zr alloys for better corrosion resistance and cell behavior guidance

In this study, a micro-patterned hydroxyapatite/silk fibroin (HA-SF) coating was firstly fabricated on the surface of Mg–Zn–Y–Nd–Zr alloy by template-assisted electrospraying technique coupling with spin coating technique. Two types of micro-patterns were achieved with high contour accuracy, namely HA-SF(line-pattern) and HA-SF(dot-pattern). The microstructure, composition, surface wettability and corrosion behaviors of the coatings were investigated by SEM, EDS, FTIR, XRD, water contact angle and potentiodynamic polarization test. The results revealed the hydrophilic nature of coatings and two orders of magnitude reduction of corrosion density (i_corr) as compared with that of the substrate. All the micro-patterned surfaces promoted the attachment of MC3T3-E1 cells with visible filopodia after 1 d incubation. In addition, coatings with line pattern exhibited the superior guidance to cell migration as compared to dot pattern, and the preference of cell attachment in the convex zone was observed. In summary, the obtained micro-patterned HA-SF coatings possessed the remarkably improvement of anticorrosion ability and good efficacy in guidance of cell attachment and alignment, which can serve as a promising strategy for cellular response modulation at the interface of magnesium-based implants and bone.     

Electrophoretic-deposited novel ternary silk fibroin/graphene oxide/hydroxyapatite nanocomposite coatings on titanium substrate for orthopedic applications

The combination of graphene oxide (GO) with robust mechanical property, silk fibroin (SF) with fascinating biological effects and hydroxyapatite (HA) with superior osteogenic activity is a competitive approach to make novel coatings for orthopedic applications. Herein, the feasibility of depositing ternary SF/GO/HA nanocomposite coatings on Ti substrate was firstly verified by exploiting electrophoretic nanotechnology, with SF being used as both a charging additive and a dispersion agent. The surface morphology, microstructure and composition, in vitro hemocompatibility and in vitro cytocompatibility of the resulting coatings were investigated by SEM, Raman, FTIR spectra and biocompatibility tests. Results demonstrated that GO, HA and SF could be co-deposited with a uniform, smooth thin-film morphology. The hemolysis rate analysis and the platelet adhesion test indicated good blood compatibility of the coatings. The human osteosarcoma MG63 cells displayed well adhesion and proliferation behaviors on the prepared coatings, with enhanced ALP activities. The present study suggested that SF/GO/HA nanocomposite coatings could be a promising candidate for the surface functionalization of biomaterials, especially as orthopedic implant coating.     

Genipin交联丝素蛋白纳米纤维膜的制备与性能

利用Genipin对再生丝素蛋白进行交联改性,并通过静电纺丝法制备交联的丝素蛋白纳米纤维膜.利用场发射扫描电镜、红外光谱仪、X射线衍射仪、热重分析仪以及拉力机等对其结构与性能进行表征与测试.结果表明,随着交联剂Genipin质量比的增加,交联度增加,静电纺丝素蛋白纳米纤维平均直径增大,标准偏差增大;Genipin交联对丝素蛋白纳米纤维结晶结构影响不大,但热性能提高;常温条件下,随着Genipin质量比从2％提高至15％,丝素蛋白纳米纤维膜的力学性能逐渐增强,质量比为10％时,其力学性能较好,拉伸强度和断裂应变分别为19.6 MPa和5.9％;随着试验温度从40℃升高到200℃,丝素蛋白纳米纤维膜的拉伸强度和断裂应变先增大然后减小,当试验温度为80℃时,其力学性能较好,拉伸强度和断裂应变分别为41.6 MPa和8.6％.     

Genipin交联丝素蛋白纳米纤维膜的制备与性能

利用Genipin对再生丝素蛋白进行交联改性, 并通过静电纺丝法制备交联的丝素蛋白纳米纤维膜。利用场发射扫描电镜、 红外光谱仪、 X射线衍射仪、 热重分析仪以及拉力机等对其结构与性能进行表征与测试。结果表明, 随着交联剂Genipin质量比的增加, 交联度增加, 静电纺丝素蛋白纳米纤维平均直径增大, 标准偏差增大;Genipin交联对丝素蛋白纳米纤维结晶结构影响不大, 但热性能提高;常温条件下, 随着Genipin质量比从2%提高至15%, 丝素蛋白纳米纤维膜的力学性能逐渐增强, 质量比为10%时, 其力学性能较好, 拉伸强度和断裂应变分别为19.6 MPa和5.9%;随着试验温度从40 ℃升高到200 ℃, 丝素蛋白纳米纤维膜的拉伸强度和断裂应变先增大然后减小, 当试验温度为80 ℃时, 其力学性能较好, 拉伸强度和断裂应变分别为41.6 MPa和8.6%。     

甲醇、戊二醛交联剂对丝素蛋白/明胶多孔支架的性能影响

为了对比甲醇、戊二醛两种交联剂对丝素蛋白/明胶复合多孔支架的性能影响,采用冷冻干燥法等比例制备该支架,分两组分别用甲醇和戊二醛进行交联。通过观察支架的微观形貌,测量支架的孔隙率、吸水率、溶胀率,测试热稳定性及力学性能,比较经两种交联剂处理后支架结构和性能的变化。结果表明,经戊二醛交联后的支架孔隙分布更加规则、均匀,孔隙率、吸水率、溶胀率更高,力学性能更强。采用戊二醛交联丝素蛋白/明胶复合多孔支架,能够使支架性能更加优良。     

Preparation and properties of calcium sulfate bone cement incorporated with silk fibroin and Sema3A-loaded chitosan microspheres

To search for new bioactive materials which can be used as the substitute of bone repairing and drug carriers, Sema3A-loaded chitosan microspheres (SLCM) and silk fibroin (SF) were mixed with calcium sulfate cement (CSC). SEM, particle size analysis and swelling rate determination were performed to study properties of the microspheres. The drug loading, encapsulation efficiency and drug release rate were determined by ELISA. Microspheres with different SLCM weight contents (0.5%, 1% and 5%) were prepared to determine which one has the strongest mechanical properties and the appropriate setting time. It was revealed that CSC/SF/0.5SLCM has satisfactory mechanical properties, and its in vitro biocompatibility was assessed by MTS. Chitosan microspheres (5--18 μm) were globular, the surface was smooth, and the swelling rate is (77.02±5.57)%. With this formula, the setting time was increased with the addition of SLCM in CSC/SF, and the cumulative drug release rate is 44.62% in 28 d. XRD results demonstrate that the main component is calcium sulfate. Also it was found that CSC/SF/0.5SLCM supports the growth of MC3T3 cells. Thus the preparation of CSC/SF/0.5SLCM was reliable, and the products had good structures, physical properties and biocompati-bility, appearing to be a promising bone substitute material.     

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 an alternative regenerated silk fibroin nanofiber and carbonated hydroxyapatite multilayered composite via layer-by-layer

A novel multilayered composite consisting of regenerated silk fibroin (RSF) nanofiber and carbonated hydroxyapatite (CHA) was fabricated with the combination of electrospinning of RSF aqueous solution and soaking in CaCl₂ and Na₂HPO₄ solutions alternately. The chemical composition and morphologies of RSF/CHA composite were characterized by FT-IR, XRD, TGA, EDX, and SEM. The results showed that such an organic/inorganic composite had an alternate layered structure, while the CHA mineral partly penetrated into the porous RSF mats, which was similar to the structure of natural nacre. By tuning the CHA deposition procedure and RSF electrospinning condition independently, the thickness of each layer of CHA and RSF, as well as the layer numbers of composite, could be easily regulated. For example, the average thickness of CHA layers with 5 and 10 mineralization cycles were 1.63 and 3.19 μm, while 9.03 and 30.12 μm of porous RSF nanofiber layers could be formed with 7 and 24 h electrospinning process, respectively. Thus, it may provide an efficient and general approach to produce a series of inorganic/organic multilayered biomaterials for biomedical 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.     

过氧化物预硫化天然橡胶/丝素蛋白抗菌复合材料的制备与性能研究

采用三元混合体系溶解丝素蛋白,纯化后与过氧化物预硫化天然橡胶复合,得到了具有抗菌性能的天然橡胶/丝素蛋白(PVNR/SF)复合材料,并对胶乳和胶膜的相关性能分别进行测试分析。结果表明,SF的加入保持了天然胶乳的稳定性。在干燥制成胶膜后,与基体材料有很好的相容性,并且可以在一定程度上起到补强的作用,SF使天然橡胶获得良好的抗菌性能的同时,对基体的热稳定、粘弹等性能没有很大的影响。另外,通过红外光谱我们可以推测SF在胶膜中的抗菌性能与酰胺I结构有关。     

Electrospun composites of PHBV,silk fibroin and nano-hydroxyapatite for bone tissue engineering

Electrospinning of fibrous scaffolds containing nano-hydroxyapatite (nHAp) embedded in a matrix of functional biomacromolecules offers an attractive route to mimicking the natural bone tissue architecture. Functional fibrous substrates will support cell attachment, proliferation and differentiation, while the role of HAp is to induce cells to secrete extracellular matrix (ECM) for mineralization to form bone. Electrospinning of biomaterials composed of polyhydroxybutyrate-co-(3-hydroxyvalerate) with 2% valerate fraction (PHBV), nano-hydroxyapatite (nHAp), and Bombyx mori silk fibroin essence (SF), Mw = 90KDa, has been achieved for nHAp and SF solution concentrations of 2 (w/vol) % each and 5 (w/vol) % each. The structure and properties of the nanocomposite fibrous membranes were investigated by means of Scanning Electron Microscopy in combination with Energy Dispersive X-Ray Analysis (SEM/EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), uniaxial tensile and compressive mechanical testing, degradation tests and in vitro bioactivity tests. SEM images showed smooth, uniform and continuous fibre deposition with no bead formation, and fibre diameters of between 10 and 15 μm. EDX and FT-IR confirmed the presence of nHAp and SF. After one month in deionised water, tests showed less than 2% weight loss with the samples retaining their fibrous morphology, confirming that this material biodegrades slowly. After 28 days of immersion in Simulated Body Fluid (SBF) an apatite layer was visible on the surface of the fibres, proving their bioactivity. Preliminary in vitro biological assessment showed that after 1 and 3 days in culture, cells were attached to the fibres, retaining their morphology while presenting a flattened appearance and elongated shape on the surface of fibres. Young's modulus was found to increase from 0.7 kPa (± 0.33 kPa) for electrospun samples of PHBV only to 1.4 kPa (± 0.54 kPa) for samples with 2 (w/vol) % each of nHAp and SF. Samples prepared with 5 (w/vol) % each of nHAp and SF did not show a similar improvement.