可降解海藻酸钠-壳聚糖支架用于细胞黏附生长的研究

通过调节海藻酸钠与壳聚糖的比例制备了具有不同降解速度的组织工程支架,并以HepG2为细胞模型考察可降解支架对细胞黏附生长的影响.研究表明:壳聚糖含量越高,支架被溶菌酶降解的速度越快,但支架在培养液中的稳定性越高;MTT结果显示细胞在壳聚糖含量为100%和67%的支架中培养时活性较高,但活死染色显示细胞多以分散状态黏附在支架上,降低壳聚糖含量时细胞活性较低且多以聚集形式存在于支架空隙内部.通过调节海藻酸钠与壳聚糖的比例制备出可降解的组织工程支架,可以控制细胞的生长速度及黏附状态,有望为细胞的生长及功能发挥提供更适宜的环境.     

Biocompatibility of highly macroporous ceramic scaffolds: cell adhesion and morphology studies

Hydroxyapatite porous scaffolds can be used for tissue engineering applications since they can serve as templates for cell adhesion, proliferation and ultimately for tissue repair. One way to address this issue is to evaluate the cell adhesion using several characterization techniques namely, cytotoxicity assays and cell visualization. On the other hand, when using highly macroporous scaffolds some techniques may not be adequate for evaluation, such as MTT. In this work, cytotoxicity assay (MTS), scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM) were used to evaluate cell adhesion in highly macroporous hydroxyapatite scaffolds. It was possible to observe that some techniques were not suitable to evaluate cell adhesion. In addition, it was shown that for this kind of scaffolds, confocal laser scanning microscopy is a powerful tool for cell adhesion and proliferation evaluation.     

Oxygen-generating nanofiber cell scaffolds with antimicrobial properties

Many next-generation biomaterials will need the ability to not only promote healthy tissue integration but to simultaneously resist bacterial colonization and resulting biomaterials-associated infection. For this purpose, antimicrobial nanofibers of polycaprolactone (PCL) were fabricated by incorporating calcium peroxide. PCL nanofibers containing different ratios of calcium peroxide (1%, 5% and 10% (w/w)) with or without ascorbic acid were fabricated using an electrospinning technique. Antimicrobial evaluations confirmed the inhibitory properties of the nanofibers on the growth of E. coli and S. epidemidis because of a significant burst release of calcium peroxide from the nanofibers. Analysis of tissue cell response showed that despite an initial toxic effect over the first 24 h, after 4 days of culture, osteoblast viability and morphology were both healthy. These results demonstrate that oxygen-generating nanofibers can be designed and developed to provide a short-term peroxide-based antimicrobial response while still maintaining attractive tissue-integration properties.     

Microstructuring ceramic scaffolds for hepatocyte cell culture

Both extracorporeal liver support devices and tissue engineering of liver for transplantation require the maintenance of functionality of liver cells (hepatocytes) in cell culture for a long time. One approach to achieve this is to optimize hepatocyte in vitro environment by using a scaffold with topographic structure at sub-millimeter scale which controls cell distribution. Therefore, a set of new type of titania ceramic scaffolds, containing cavities of several sizes, has been produced for deducing the best choice of cavity dimensions for culturing hepatocytes. The aim of this paper is to describe in detail the production methods and characterization of such ceramic scaffolds. Experimental production of the scaffolds consists of microfabrication of silicon templates as well as preparation and molding of titania ceramics. The templates, containing arrays of conical protrusions arranged in close-packed hexagonal order, have been achieved using microfabrication methods of photolithography and anisotropic etching in KOH at 50 °C. Protrusion dimensions and overall quality of the templates has been evaluated by scanning electron microscopy. The microfabricated templates have resulted in well-defined and reproducible cavities of corresponding dimensions on the titania ceramic surface after injection-molding. Alternatively, simple embossing of the plastified green ceramics with the silicon templates attached to a metal plate also creates cavities on the ceramic surface. While both methods yield good results, they have different advantages: the injection-molding provides a higher quality of imprints while embossing is quicker and less complicated, and is not limited by dimensions of specific molding equipment. © 2001 Kluwer Academic Publishers     

Osteoblast-like cell ingrowth,adhesion and proliferation on porous Ti-6Al-4V with particulate and fiber scaffolds

This paper presents the results of an experimental study of osteoblast-like cell ingrowth into porous Ti-6Al-4V structures with well-controlled geometries. The effects of pore size and strut geometry are elucidated in in-vitro cell ingrowth experiments on porous Ti-6Al-4V structures with particulate and fiber geometries. The initial stages of cell spreading and proliferation are examined using cell culture experiments. Scanning electron microscope (SEM) and a methylthiazol tetrazolium (MTT) assay are used to reveal the initial stages of cell spreading and attachment. Enzymatic detachment tests are also used to examine cell adhesion after 48 h of cell culture. The results show a strong effect of pore size on the rate of cell bridging over gaps. The extent of cell ingrowth, initial cell adhesion and cell proliferation also increase with decreasing pore size. A lower incidence of cell bridging (over gaps) is observed on the fiber porous structures. However, fiber geometries enable contact guidance during cell spreading along the fiber directions. This enhances the extent of cell ingrowth into the fiber porous structures. No significant differences are observed in cell adhesion and proliferation on porous structures with similar pore sizes.     

A novel method for fabrication of biodegradable scaffolds with high compression moduli

It has been previously shown that, when used for meniscal reconstruction, porous copoly(L-lactide/-caprolactone) implants enhanced healing of meniscal lesions owing to their excellent adhesive properties. However, it appeared that the materials had an insufficient compression modulus to accomplish 100% fibrocartilage formation. In addition, to be used for meniscal prosthesis, the compression modulus of the porous materials should be larger than 150 kPa in order to protect the articular cartilage. A technique was developed to prepare stiff porous materials of a high molecular weight 50/50 copoly(L-lactide/-caprolactone) suitable for fibrocartilage regeneration in meniscal implants and meniscal prosthesis. Porous microspheres (50–250 m) were agglutinated in the presence of NaCl crystals (250–300 m). The microspheres were mixed with solid solvent in order to obtain a homogeneous distribution of solvent over the spheres. By changing the amount of solvent and crystals, the density and the compression modulus could be varied over a range of 0.07 g ml-1 to 0.5 g dl-1 and 40–1100 kPa, respectively.     

Influence of porosity and fibre diameter on the degradation of chitosan fibre-mesh scaffolds and cell adhesion

The state of the art approaches for tailoring the degradation of chitosan scaffolds are based on altering the chemical structure of the polymer. Nevertheless, such alterations may lead to changes in other properties of scaffolds, such as the ability to promote cell adhesion. The aim of this study was to investigate the influence of physical parameters such as porosity and fibre diameter on the degradation of chitosan fibre-mesh scaffolds, as a possible way of tailoring the degradation of such scaffolds. Four sets of scaffolds with distinct fibre diameter and porosity were produced and their response to degradation and cell adhesion was studied. The degradation study was carried out at 37^∘C in a lysozyme solution for five weeks. The extent of degradation was expressed as percentage of weight loss of the dried scaffolds after lysozyme treatment. Cell adhesion was assessed by Confocal Microscopy. The results have shown that the scaffolds with higher porosity degrade faster and that, within the same range of porosity, the fibres with smaller diameter degrade slightly faster. Furthermore, the morphological differences between the scaffolds did not affect the degree of cell adhesion, and the cells were observed throughout the thickness of all four types of scaffolds.     

Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

Piezoelectric materials that generate electrical signals in response to mechanical strain can be used in tissue engineering to stimulate cell proliferation.Poly (vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)),a piezoelectric polymer,is widely used in biomaterial applications.We hypothesized that incorporation of zinc oxide (ZnO) nanoparticles into the P(VDF-TrFE) matrix could promote adhesion,migration,and proliferation of cells,as well as blood vessel formation (angiogenesis).In this study,we fabricated and comprehensively characterized a novel electrospun P(VDF-TrFE)/ZnO nanocomposite tissue engineering scaffold.We analyzed the morphological features of the polymeric matrix by scanning electron microscopy,and utilized Fourier transform infrared spectroscopy,X-ray diffraction,and differential scanning calorimetry to examine changes in the crystalline phases of the copolymer due to addition of the nanoparticles.We detected no or minimal adverse effects of the biomaterials with regard to blood compatibility in vitro,biocompatibility,and cytotoxicity,indicating that P(VDF-TrFE)/ZnO nanocomposite scaffolds are suitable for tissue engineering applications.Interestingly,human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells cultured on the nanocomposite scaffolds exhibited higher cell viability,adhesion,and proliferation compared to cells cultured on tissue culture plates or neat P(VDF-TrFE) scaffolds.Nanocomposite scaffolds implanted into rats with or without hMSCs did not elicit immunological responses,as assessed by macroscopic analysis and histology.Importantly,nanocomposite scaffolds promoted angiogenesis,which was increased in scaffolds pre-seeded with hMSCs.Overall,our results highlight the potential of these novel P(VDF-TrFE)/ZnO nanocomposites for use in tissue engineering,due to their biocompatibility and ability to promote cell adhesion and angiogenesis.     

Genipin-crosslinked gelatin scaffolds for articular cartilage tissue engineering with a novel crosslinking method

A novel crosslinking method with directly crosslinking the gelatin gel, being cut to a disc of chosen size beforehand, for the fabrication of porous gelatin scaffold was proposed. This novel method of gel-crosslinking was compared with the traditional methods of mixing-crosslinking and scaffold-crosslinking. The structure of the scaffold fabricated by the gel-crosslinking method shows uniformly distributed and interconnected pores which can be much smaller than those made by the other two methods. All three methods have the last step as freeze-drying; nevertheless, freeze-drying once more will increase the uniformity of the structure and the interconnecting pores. Crosslinking of gelatin was carried out at room temperature with glutaraldehyde (GTA) or genipin (GP). In vitro cell culture of Wistar rat's joint chondrocytes demonstrates that the GTA-crosslinked scaffold is much worse than the GP-crosslinked one; a tissue containing collagen and glycosaminoglycan was produced in the GP-crosslinked scaffold in just 9 days after cell seeding, and a tissue with a cell distribution resembling that of the native cartilage was developed after 30 day cell culture. It was concluded that the novel method is feasible for application in articular cartilage tissue engineering.     

Controlling stem cell behavior with decellularized extracellular matrix scaffolds

Decellularized tissues have become a common regenerative medicine platform with multiple materials being researched in academic laboratories, tested in animal studies, and used clinically. Ideally, when a tissue is decellularized the native cell niche is maintained with many of the structural and biochemical cues that naturally interact with the cells of that particular tissue. This makes decellularized tissue materials an excellent platform for providing cells with the signals needed to initiate and maintain differentiation into tissue-specific lineages. The extracellular matrix (ECM) that remains after the decellularization process contains the components of a tissue specific microenvironment that is not possible to create synthetically. The ECM of each tissue has a different composition and structure and therefore has unique properties and potential for affecting cell behavior. This review describes the common methods for preparing decellularized tissue materials and the effects that decellularized materials from different tissues have on cell phenotype.     

Mesenchymal stem cellular adhesion and cytotoxicity study of random biopolyester scaffolds for tissue engineering

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rabbits and inoculated respectively on 3D scaffolds of poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV), poly(butylenes succinate) (PBS) and different blends (100/0, 80/20, 50/50, 20/80, 0/100) (Wt%) in vitro. It was found that the (50/50) blends possessed the best performance on adhesion and cytotoxicity of MSCs. The scanning electronic microscopy (SEM) results showed that the (50/50) blends had the appropriate roughness for MSCs to attach and grow, which may be used as a suitable biomaterial to create small caliber vascular grafts.     

Effect of collagen II coating on mesenchymal stem cell adhesion on chitosan and on reacetylated chitosan fibrous scaffolds

The biocompatibility and biomimetic properties of chitosan make it attractive for tissue engineering but its use is limited by its cell adhesion properties. Our objectives were to produce and characterize chitosan and reacetylated-chitosan fibrous scaffolds coated with type II collagen and to evaluate the effect of these chemical modifications on mesenchymal stem cell (MSC) adhesion. Chitosan and reacetylated-chitosan scaffolds obtained by a wet spinning method were coated with type II collagen. Scaffolds were characterized prior to seeding with MSCs. The constructs were analyzed for cell binding kinetics, numbers, distribution and viability. Cell attachment and distribution were improved on chitosan coated with type II collagen. MSCs adhered less to reacetylated-chitosan and collagen coating did not improve MSCs attachment on those scaffolds. These findings are promising and encourage the evaluation of the differentiation of MSCs in collagen-coated chitosan scaffolds. However, the decreased cell adhesion on reacetylated chitosan scaffold seems difficult to overcome and will limit its use for tissue engineering.     

Universal method for determining electrolyte temperatures in capillary electrophoresis

Temperature increase in capillary electrophoresis (CE) due to Joule heating is an inherent limitation of this powerful separation technique. Active cooling systems can decrease the temperature of a large part of the capillary but they leave "hot spots" at the capillary ends which can completely ruin some CE analyses despite their short lengths. Here, we introduce a "universal method for determining electrolyte temperatures" (UMET) that can determine temperatures in both efficiently- and inefficiently-cooled parts of the capillary. UMET can be applied to all electrolytes, as it does not involve any probe; it requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. To demonstrate the universality of UMET, we measured temperatures for electrolytes of different ionic strengths as well as for different capillary diameters. We further propose a "simplified universal method for predicting electrolyte temperatures" (SUMET) which only requires one measurement of current and voltage (that can be completed in 1 min) and uses two empirical equations to predict temperatures in the efficiently- and inefficiently-cooled parts of the capillary. The equations include several instrument-specific empirical parameters that are determined using a large set of current-voltage data obtained with UMET for a range of electrolytes and different capillaries. To demonstrate the utility of SUMET, we obtained the required data set for a Beckman MDQ CE instrument and produced all required empirical parameters that enable a user of this instrument to predict the temperature for every new experimental set in a matter of minutes. We confirmed the accuracy of SUMET by measuring the temperature-sensitive dissociation rate constant of a protein-DNA complex. We foresee that UMET will be used to produce instrument-specific empirical parameters for all CE instruments and then SUMET will be routinely used for temperature prediction in CE.     

Degradable polyester scaffolds with controlled surface chemistry combining minimal protein adsorption with specific bioactivation

Advanced biomaterials and scaffolds for tissue engineering place high demands on materials and exceed the passive biocompatibility requirements previously considered acceptable for biomedical implants. Together with degradability, the activation of specific cell–material interactions and a three-dimensional environment that mimics the extracellular matrix are core challenges and prerequisites for the organization of living cells to functional tissue. Moreover, although bioactive signalling combined with minimization of non-specific protein adsorption is an advanced modification technique for flat surfaces, it is usually not accomplished for three-dimensional fibrous scaffolds used in tissue engineering. Here, we present a one-step preparation of fully synthetic, bioactive and degradable extracellular matrix-mimetic scaffolds by electrospinning, using poly(D,L-lactide-co-glycolide) as the matrix polymer. Addition of a functional, amphiphilic macromolecule based on star-shaped poly(ethylene oxide) transforms current biomedically used degradable polyesters into hydrophilic fibres, which causes the suppression of non-specific protein adsorption on the fibres’ surface. The subsequent covalent attachment of cell-adhesion-mediating peptides to the hydrophilic fibres promotes specific bioactivation and enables adhesion of cells through exclusive recognition of the immobilized binding motifs. This approach permits synthetic materials to directly control cell behaviour, for example, resembling the binding of cells to fibronectin immobilized on collagen fibres in the extracellular matrix of connective tissue.     

Ultrafine fibrous gelatin scaffolds with deep cell infiltration mimicking 3D ECMs for soft tissue repair

In this research, ultrafine fibrous scaffolds with deep cell infiltration and sufficient water stability have been developed from gelatin, aiming to mimic the extracellular matrices (ECMs) as three dimensional (3D) stromas for soft tissue repair. The ultrafine fibrous scaffolds produced from the current technologies of electrospinning and phase separation are either lack of 3D oriented fibrous structure or too compact to be penetrated by cells. Whilst electrospun scaffolds are able to emulate two dimensional (2D) ECMs, they cannot mimic the 3D ECM stroma. In this work, ultralow concentration phase separation (ULCPS) has been developed to fabricate gelatin scaffolds with 3D randomly oriented ultrafine fibers and loose structures. Besides, a non-toxic citric acid crosslinking system has been established for the ULCPS method. This system could endow the scaffolds with sufficient water stability, while maintain the fibrous structures of scaffolds. Comparing with electrospun scaffolds, the ULCPS scaffolds showed improved cytocompatibility and more importantly, cell infiltration. This research has proved the possibility of using gelatin ULCPS scaffolds as the substitutes of 3D ECMs.     

Evaluation method for the adhesion strength of vitreous enamel

A new quantitative evaluation method and the formula for vitreous enamel were proposed. The three-point bending strength was measured by loading on the superposition of test piece which sandwiched glass layer partly between two steel sheets. The adhesion strength () was calculated using the following equation, = 3PLh/2b·Eg/[Egh3 + Es(h3 – h3)]w here, P; bending strength, L; length of span, b; width of glass layer, Eg; Young's modulus of glass, Es; Young's modulus of steel, h; thickness of glass layer, h'; thickness of superposition of test piece. The evaluation method for vitreous enamel of this experiment agreed with the empirical evaluation and may be applied to an actual situation.     

Instrumental method for quantitative evaluation of cell or particle adhesion, based on transport measurements in capillary flow

An instrumental method is proposed for the quantitative evaluation of cell/particle adhesion at solid/liquid interfaces. As a measure of adhesion, the retardation of convective transport in capillary flow tubes is determined. The flow tubes, for such purposes, have been internally coated with the substance under investigation. AC-operated electronic gating is applied, preferentially, for the determination of the elution rate. This technique, to some extent, is similar to that of high-pressure liquid chromatography (HPLC). However, the transport mechanism is different as in this case, where transport mainly takes place about halfway between the capillary axis and the wall (Segre-Silberberg effect). At specific flow rates of the carrier liquid, cells/particles repeatedly contact the wall, only to be released again as a consequence of increasing lift and drag. This phenomenon depends on the chemical nature of the capillary wall coating and causes measurable retardation of transport. A transport model is proposed that predicts flow conditions under which transport retardation and adhesive interaction are to be expected. Experiments prove that the Segre-Silberberg effect, i.e., the occurrence of a preferential pathway of transport, is fully established already at capillary lengths of a few decimeters, and that the occurring retardation of transport, if any, is governed by the chemical nature of the capillary wall coatings. The area enclosed between the cumulative elution curves of sample and reference materials offers a reproducible measure of adhesion. The technique is fast, sensitive, and reliable.     

Microfluidic shear devices for quantitative analysis of cell adhesion

We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytic modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In agreement with established literature, we show that fibroblasts cultured in the combined device reduced their adhesion strength to the substrate in response to epidermal growth factor stimulation.     

Novel,simple and reproducible method for preparation of composite hierarchal porous structure scaffolds

Demand to develop a simple and adaptable method for preparation the hierarchical porous scaffolds for bone tissue regeneration is ever increasing. This study presents a novel and reproducible method for preparing the scaffolds with pores structure spanning from nano, micro to macro scale. A macroporous Sr-Hardystonite (Sr–Ca₂ZnSi₂O₇, Sr–HT) scaffold with the average pore size of ~ 1200 μm and porosity of ~ 95% was prepared using polymer sponge method. The struts of the scaffold were coated with a viscous paste consisted of salt (NaCl) particles and polycaprolactone (PCL) to provide a layer with thickness of ~ 300–800 μm. A hierarchical porous scaffold was obtained with macro, micro and nanopores in the range of 400–900 μm, 1–120 μm and 40–290 nm, after salt leaching process. These scales could be easily adjusted based on the starting foam physical characteristics, salt particle size, viscosity of the paste and salt/PCL weight ratio.