Collagen-based scaffolds enriched with glycosaminoglycans isolated from skin of Salmo salar fish

In this study both collagen and glycosaminoglycans were isolated from biodegradable waste. Namely collagen was isolated from rat tail tendons and glycosaminoglycans (GAGs) from fish skin. Porous materials were then obtained based on the isolated collagen with 1 or 5% addition of GAGs by freeze-drying process. The scaffolds were studied by infrared spectroscopy, mechanical testing and examined for the porosity and density. The scaffolds structure was observed by scanning electron microscope. The adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on prepared scaffolds to assess their biocompability.The results showed that the addition of glycosaminoglycans improves the properties of collagen-based scaffolds. Mechanical strength was increased by GAGs addition as well as the porosity of studied materials. Each scaffold with and without GAGs displayed porous structure with interconnected regular shaped pores. The attachment of cells was better for pure collagen scaffold, however, GAGs additive promoted the cells proliferation on the scaffold.     

The comparison of physic-chemical properties of chitosan/collagen/hyaluronic acid composites with nano-hydroxyapatite cross-linked by dialdehyde starch and tannic acid

Scaffolds based on chitosan, collagen and hyaluronic acid, cross-linked by dialdehyde starch with hydroxyapatite were obtained with the use of the freeze-drying method. Scaffolds were cross-linked by tannic acid or dialdehyde starch addition. Composites were characterized by different analyses, e.g. SEM images, porosity, density, liquid uptake, and mechanical tests. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells were examined on the obtained scaffolds.The results showed that the properties of the scaffolds based on chitosan, collagen, and hyaluronic acid can be modified by cross-linkers addition. The compressive modulus for the scaffolds cross-linked by dialdehyde starch was higher than for those cross-linked by tannic acid. The porosity of scaffolds cross-linked by starch was higher than those in which tannic acid was applied. However, the former presented lower density. SEM images showed the homogeneous scaffold structure with interconnected pores. Scaffolds cross-linked by tannic acid exhibited higher biocompatibility than those cross-linked by dialdehyde starch. However, the results showed that both scaffolds, cross-linked by dialdehyde starch and by tannic acid can provide the support required in tissue engineering and regenerative medicine. The scaffolds presented here may be easily operated to fit such small bone defects without causing adverse reactions.     

Preparation and modification of collagen-based porous scaffold for tissue engineering

In the effort to generate cartilage tissues using mesenchymal stem cells, porous scaffolds with prescribed biomechanical properties were prepared. Scaffolds with interconnected pores were prepared via lyophilisation of frozen hydrogels made from collagen modified with chitosan nanofibres, hyaluronic acid, copolymers based on poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and itaconic acid (ITA), and hydroxyapatite nanoparticles. The modified collagen compositions were cross-linked using N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) combined with N-hydroxysuccinimide (NHS) in water solution. Basic physicochemical and mechanical properties were measured and an attempt to relate these properties to the molecular and supermolecular structure of the modified collagen compositions was carried out. Scaffolds containing hydrophilic chitosan nanofibres showed the highest swelling ratio (SR = 20–25) of all the materials investigated, while collagen modified with an amphiphilic PLGA-PEG-PLGA copolymer or functionalised with ITA exhibited the lowest swelling ratio (SR = 5–8). The best resistance to hydrolytic degradation was obtained for hydroxyapatite containing scaffolds. On the other hand, the fastest degradation rate was observed for synthetic copolymer-containing scaffolds. The results showed that the addition of hydroxyapatite or hyaluronic acid to the collagen matrix increases the rigidity in comparison to the collagen-chitosan scaffold. Collagen scaffold modified with hyaluronic acid presented reduced deformation at break while the presence of hydroxypatatite enhanced the scaffold deformation under tensile loading. The tensile elastic modulus of chitosan nanofibre collagen scaffold was the lowest but closest to the articular cartilage; however, the strength and deformation to failure increased up to 200 %. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August 2007.     

Microstructure and mechanical properties of bacterial cellulose/chitosan porous scaffold

A family of polysaccharide based scaffold materials, bacterial cellulose/chitosan (BC/CTS) porous scaffolds with various weight ratios (from 20/80 to 60/40 w/w%) were prepared by freezing (−30 and −80 °C) and lyophilization of a mixture of microfibrillated BC suspension and chitosan solution. The microfibrillated BC (MFC) was subjected to 2,2,6,6-tetramethylpyperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce surface carboxyl groups before mixing. The integration of MFC within chitosan matrix was performed by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated cross-linking. The covalent amide bond formation was determined by ATR-FTIR. Because of this covalent coupling, the scaffolds retain their original shapes during autoclave sterilization. The composite scaffolds are three-dimensional open pore microstructure with pore size ranging from 120 to 280 μm. The freezing temperature and mean pore size take less effect on scaffold mechanical properties. The compressive modulus and strength increased with increase in MFC content. The results show that the scaffolds of higher MFC content contribute to overall better mechanical properties.     

Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.     

化学修饰方法对聚乙二醇功能化碳纳米管的影响

通过酰氯化法与碳二亚胺缩合法(EDC/NHS)制备氨基化聚乙二醇(PEG1500N)修饰的多壁碳纳米管(MWNTs)并采用FTIR、Raman、TEM、原子力显微镜(AFM)、TGA-DTA-DSC、UV-Vis进行表征与分析。实验结果发现：两种方法PEG1500N都能很好地修饰MWNTs,但EDC/NHS缩合法采用更短的反应时间(反应1 d),达到了更好的接枝效果。EDC/NHS缩合法提高了碳管上羧基的利用率,接枝率大大提高。TGA-DTA分析表明缩合法接枝率为30%,而酰氯化法(反应4 d)为15%。UV-Vis分析表明EDC/NHS缩合法得到的产物溶解性也更好,溶解度由1.19 mg·mL^-1(酰氯化法得到的产物的溶解度)提高到2 mg·mL^-1以上。     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

An electrochemical immunosensor based on covalent immobilization of okadaic acid onto screen printed carbon electrode via diazotization-coupling reaction

In this work, an electrochemical method based on the diazonium-coupling reaction mechanism for the immobilization of okadaic acid (OA) on screen printed carbon electrode was developed. At first, 4-carboxyphenyl film was grafted by electrochemical reduction of 4-carboxyphenyl diazonium salt, followed by terminal carboxylic group activation by N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyle)-N′-ethyle-carbodiimide hydrochloride (EDC). Hexamethyldiamine was then covalently bound by one of its terminal amine group to the activated carboxylic group. The carboxyl group of okadaic acid was activated by EDC/NHS and then conjugated to the second terminal amine group on other side of the hexamethyldiamine through amide bond formation. After immobilization of OA, an indirect competitive immunoassay format was employed to detect OA. The immunosensor obtained using this novel approach allowed detection limit of 1.44 ng/L of OA, and was also validated with certified reference mussel samples.     

明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价

利用静电纺丝技术制备了明胶与聚乳酸的复合纤维膜, 研究了组分配比对复合膜的表面性能、孔隙结构和力学性能的影响, 并以复合膜为组织工程支架进行兔角膜上皮细胞的体外培养. 采用扫描电子显微镜、免疫荧光染色和噻唑蓝四氮唑溴化物(MTT)比色法综合评价了细胞在支架表面的黏附与增殖能力. 结果表明, 纺丝溶液的组分对纤维的直径分布和表面亲水性有显著影响, 不同组分配比的复合纤维膜均具有高孔隙率的通孔结构; 以明胶为基材可维持复合膜的细胞黏附性; 与聚乳酸复合可以明显提高复合膜的力学性能.     

多巴胺改性海藻酸多孔支架的制备与性能

生物大分子海藻酸(Alg)由于其安全、无毒、可生物降解等特性而被广泛应用于组织工程领域。 受海洋贻贝蛋白结构的启发,多巴胺(DA)具有优异的粘附性能,在碱性水溶液条件下可发生氧化自聚形成聚多巴胺(PDA)。 以Alg为基体,加入PDA纳米粒子复合,并通过冷冻干燥法制备得到Alg/PDA多孔支架材料。 结果表明,Alg/PDA多孔支架材料具有较为规整的内部结构。 改变Alg质量浓度,Alg/PDA支架材料的孔径可控制在60~120 μm之间,孔隙率可控于80%~88%。 所得的支架材料具有适宜大小的孔径和孔隙率,结果表明支架材料对细胞无毒副作用。     

Development of sodium alginate-xanthan gum based nanocomposite scaffolds reinforced with cellulose nanocrystals and halloysite nanotubes

Sodium alginate (Alg) and xanthan gum (XG) based nanocomposite scaffolds reinforced with various amounts of cellulose nanocrystals (CNCs) and/or halloysite nanotubes (HNTs) were prepared by freeze-casting/drying method. In this study, the structure-property-performance relationship was mainly focused and analysed. Morphological analysis showed high porosity and pore-interconnectivity (pore channels) in all obtained scaffolds. Structural analysis demonstrates the good interfacial interactions and uniform dispersion of the CNCs and HNTs, involving partial orientation within the polymeric network. The water uptake capacity (from 14.73.7 ± 0.46 g/g to 11.34 ± 0.32 g/g) and porosity (from 91.7 ± 0.81% to 88.5 ± 0.64%) were reduced. The compressive strengths (in dry state from 91.1 ± 1.2 kPa to 114.4 ± 0.6 kPa and in wet state from 9.0 ± 0.8 kPa to 10.6 ± 0.8 kPa), thermal stability, cytocompatibility (MC3T3-E1 osteoblastic cells) of the nanocomposite scaffolds improved as compared to Alg and AlgX scaffolds without CNCs and/or HNTs. The obtained scaffolds may be appropriate as scaffolding material in bone tissue engineering.     

Collagen and chitosan blends for 3D bioprinting: A rheological and printability approach

Collagen and chitosan are widely employed as biomaterials, including for 3D-bioprinting. However, the use of collagen and chitosan (col:chi) blends as bioinks is still scarce. In this work, the rheology of different hydrogel precursors (0.5–1.50% w/v chi: 0.18–0.54% w/v col) was analyzed through frequency and strain sweeps, as well as at different shear rates. Col:chi blends showed a shear-thinning behavior, with viscosity values at low shear rates between 0.35 and 2.80 Pa s. Considering the strain rate determined by the applied flow in a 3D-bioprinter, precursor viscosities during the extrusion were in the interval 0.5–0.8 Pa s. Printability (Pr) was measured comparing images of the printed meshes and the corresponding CAD grid design, using photograph analysis. Col:chi 0.36:1.00 was chosen to print mono-layered scaffolds for tissue engineering (TE) because of its suitable viscosity, printability and polymer ratio content. Hydrogels were obtained through NaHCO₃ nebulization and 37° incubation, and NHS/EDC were added to obtain scaffolds with improved mechanical behavior. They were stable after 44 h in PBS with collagenase at physiological level and showed no cytotoxic effect in NIH-3T3 fibroblasts.     

多肽在金纳米粒子表面偶联反应效率研究

通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)活化反应将多肽偶联到金纳米粒子表面,采用荧光光谱研究其形成的酰胺键的反应偶联效率.考察了实验条件,包括缓冲液的种类(HEPES、Tris-HCl、硼酸、PBS缓冲液)、pH值(pH 6.5~9.0)、缓冲液浓度(10, 25, 40和50 mmol/L)、NHS和EDC的浓度(NHS 0.2~1.0 mol/L,EDC 0.01~0.5 mol/L)及二者比例(0,0.5,1.0,2.0和2.5)、偶联反应时间(4, 8, 12, 24和36 h)等对偶联效率的影响,筛选出最佳实验条件.实验结果表明, 25 mmol/L 4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(pH 7.0), NHS/EDC浓度为0.4 mol/L/0.2 mol/L和24 h的反应时间为EDC-NHS活化反应将多肽偶联到金纳米粒子上的最佳实验条件.本研究结果可为相关研究提供技术参考.     

Modified silicon carbide NPs reinforced nanocomposite hydrogels based on alginate-gelatin by with high mechanical properties for tissue engineering

Hydrogels are encouraging for different clinical purposes because of their high water absorption and mechanical relation to native tissues. Injectable hydrogels can modify the invasiveness of utilization, which decreases recovery and surgical costs. Principal designs applied to create injectable hydrogels incorporate in situ formation owing to chemical or/and physical crosslinking. Here, we report nontoxic, thermosensitive, injectable hydrogels composed of gelatin (GEL) and oxidized alginate (OA) reinforced by silicon carbide nanoparticles (SiC NPs) and crosslinked with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The mechanical characteristics of the hydrogels were examined via rheological analysis. The outcomes reveal that extending the SiC NPs contents enhances the mechanical properties around five times. The cross-sectional microstructure of the scaffolds comprising 0.25, 1.0, and 1.5% SiC NPs was scrutinized by FESEM, verifying porous structure with interconnected pores. Because of the smaller pore sizes in the hydrogels, the swelling rate has reduced at the higher content of SiC, which diminishes the water uptake. Additionally, the biodegradation study unveils that the hydrogels with SiC are more long-lasting than the hydrogel without SiC. By adding SiC NPs, a decrease is observed in the biodegradation and swelling ratio. The scaffold with a higher SiC NPs content (1.5%) manifested better cell attachment and was less cytotoxic than hydrogel without SiC. OA/GEL composites embedded SiC NPs have manifested excellent physical properties for tissue engineering in comparison with hydrogel without nanoparticles.     

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.     

基于生物素-亲和素系统的压电免疫传感器检测相思子毒素研究

利用生物素-亲和素系统的放大作用和纳米金质量扩增效应,建立了压电免疫传感器检测相思子毒素的新方法.首先在石英晶体的金电极上依次组装二巯基丙酸、EDC和NHS进行表面修饰,然后通过亲和素固定生物素标记相思子毒素多抗来制备敏感膜,利用纳米金的质量扩增效应设计了一种"毒素-纳米金标记单抗"复合物,成功实现了对相思子毒素的检测,提高了传感器灵敏度和重现性.本传感器对相思子毒素响应的线性范围为0.05～5 mg/L; 回归方程为Δf＝50.81CAbrin+67.11(r=0.9903,n＝10,P<0.0001); 检测灵敏度为50.81 Hz · L/mg.     

Biopolymer/Glycopolypeptide‐Blended Scaffolds: Synthesis,Characterization and Cellular Interactions

Three‐dimensional (3D) scaffolds formed from natural biopolymers gelatin and chitosan that are chemically modified by galactose have shown improved hepatocyte adhesion, spheroid geometry and functions of the hepatocytes. Galactose specifically binds to the hepatocytes via the asialoglycoprotein receptor (ASGPR) and an increase in galactose density further improves the hepatocyte proliferation and functions. In this work, we aimed to increase the galactose density within the biopolymeric scaffold by physically blending the biopolymers chitosan and gelatin with an amphiphlic β‐galactose polypeptide (PPO‐GP). PPO‐GP, is a di‐block copolymer with PPO and β‐galactose polypeptide, exhibits lower critical solution temperature and is entrapped within the scaffold through hydrophobic interactions. The uniform distribution of PPO‐GP within the scaffold was confirmed by fluorescence microscopy. SEM and mechanical testing of the hybrid scaffolds indicated pore size, inter connectivity and compression modulus similar to the scaffolds made from 100 % biopolymer. The presence of the PPO‐GP on the surface of the scaffold was tested monitoring the interaction of an analogous mannose containing PPO‐GP scaffold and the mannose binding lectin Con‐A. In vitro cell culture experiments with HepG2 cells were performed on GLN‐GP and CTS‐GP and their cellular response was compared with GLN and CTS scaffolds for a period of seven days. Within three days of culture the Hep G2 cells formed multicellular spheroids on GLN‐GP and CTS‐GP more efficiently than on the GLN and CTS scaffolds. The multicellular spheroids were also found to infiltrate more in GLN‐GP and CTS‐GP scaffolds and able to maintain their round morphology as observed by live/dead and SEM imaging.     

Preparation and characterization of collagen/chitosan poly (ethylene glycol)/nanohydroxyapatite composite scaffolds

Porous three‐dimensional collagen/chitosan scaffolds combined with poly (ethylene glycol) (PEG) and hydroxyapatite were obtained through a freeze‐drying method. Physical cross‐linking was examined by dehydrothermal treatment. The prepared materials were characterized by different analyses, eg, scanning electron microscopy (SEM), measurements of porosity and swelling, mechanical properties, and resistance to enzymatic degradation. The porosity of scaffolds and their swelling ratio decreased with the addition of hydroxyapatite. Moreover, after exposure to collagenase, the collagen/chitosan matrices containing PEG showed much faster degradation rate than matrices with the addition of hydroxyapatite. The results indicated that the addition of hydroxyapatite led to improvement of stiffness. The highest degree of porosity and swelling were demonstrated by collagen/chitosan/PEG matrices without hydroxyapatite.     

Development of an amperometric immunosensor based on flow injection analysis for the detection of red blood cells

An amperometric immunosensor, based on a non-competitive sandwich assay and flow injection analysis (FIA), was developed for the detection of human red blood cells (RBCs). A dual working electrode, on which specific IgM and nonspecific IgM were chemically immobilised to form sensing and blank electrodes, respectively, was employed to determine the binding of specific blood cells and non-specific adsorption in one determination. Horseradish peroxidase (HRP)-labelled antiblood group A IgM was used in the assay. Sensor preparation involved chemical immobilisation of the IgMs on glassy carbon electrodes using l-ethyl-3(3-dimethyl aminopropyl)carbodiimide (EDC) as a coupling reagent in the presence of N-hydroxysuccinimide (NHS). The interference contributions, such as the non-specific adsorption of the enzyme conjugate and the blood cells, were determined and removed. A quantitative relationship between the cell binding response and its concentration was obtained in the region 1 − 30 × 10⁸ cells ml⁻¹.     

Functional and highly porous scaffolds for biomedical applications

Highly porous functional scaffolds were obtained from linear and cross-linked multifunctional poly(ε-caprolactone) and poly(L-lactide). The polymers were synthesized by ring-opening polymerization of ε-caprolactone and L-lactide using poly(but-2-ene-1,4-diyl malonate) (PBM) as macroinitiator and stannous 2-ethylhexanoate. The presence of a double bond in each repeating unit of PBM enabled cross-linking of both scaffolds and films. Soft and flexible scaffolds were created from cross-linked PBM. The mechanical properties of scaffolds and films were evaluated under cyclic conditions, with a focus on the compositions and molecular weights. It was obvious that PBM in the polymers and its cross-linking ability resulted in tunable material characteristics, including an increased ability to recover after repeated loading.