Effect of PEG-PLLA diblock copolymer on macroporous PLLA scaffolds by thermally induced phase separation

A regular and highly interconnected macroporous poly(L-lactic acid) (PLLA) scaffold was fabricated from a PLLA-dioxane-water ternary system with added polyethylene glycol (PEG)-PLLA diblock using thermally induced phase separation (TIPS). The morphology of the scaffold was investigated in detail by controlling the following TIPS parameters: quenching temperature, aging time, polymer concentration, molecular structure, and diblock concentration. The phase diagram was assessed visually on the basis of the turbidity. The cloud-point curve shifted to higher temperatures with increasing PEG content in the additives (PEG-PLLA diblocks), due to a stronger interaction between PEG and water in solution. The addition of diblock series (0.5 wt% in solution) stabilized interconnections of pores at a later stage without segregation or sedimentation. The pore size of the scaffold could be easily controlled in the range 50-300 microm. A macroporous PLLA scaffold was used to study an MC3T3-E1 cell (an osteoblast-like cell) culture. The cells successfully proliferated in the PLLA scaffold in the presence of added PEG-PLLA diblock for 4 weeks.     

热致相分离法制备聚羟基丁酸酯-羟基戊酸酯纳米纤维支架及其表征***

背景：由微生物合成的聚羟基丁酸酯-羟基戊酸酯(polyhydroxybutyrate-hydroxyvalerate,PHBV)是聚羟基脂肪酸酯的一种,具有良好的生物相容性和机械强度。目的：探讨热致相分离法制备PHBV纳米纤维支架的方法及结晶行为。方法：采用扫描电镜、广角X射线衍射、红外光谱和差示扫描量热分析分析基质的结构。结果与结论：凝胶温度对纳米纤维的结晶和热性质有很大的影响。当凝胶温度较高时,PHBV纳米纤维的结晶度和晶粒尺寸随着凝胶温度的降低而减小,而且随着凝胶温度的降低,其结晶的有序性增加。说明温度对PHBV支架形貌和结构的影响可能对PHBV支架的性质-包括生物降解性和对细胞活性的生物应答反应有一定的积极意义。关键词：聚羟基丁酸酯-羟基戊酸酯;纳米纤维;基质;支架;生物相容性doi:10.3969/j.issn.1673-8225.2012.03.002     

Polyurethane/poly(lactic-co-glycolic) acid composite scaffolds fabricated by thermally induced phase separation

In this study, we present a novel composite scaffold fabricated using a thermally induced phase separation (TIPS) process from poly(lactic-co-glycolic) (PLGA) and biomedical polyurethane (PU). This processing method has been tuned to allow intimate (molecular) mixing of these two very different polymers, giving rise to a unique morphology that can be manipulated by controlling the phase separation behaviour of an initially homogenous polymer solution. Pure PLGA scaffolds possessed a smooth, directional fibrous sheet-like structure with pore sizes of 0.1-200mum, a porous Young's modulus of 93.5kPa and were relatively brittle to touch. Pure PU scaffolds had an isotropic emulsion-like structure, a porous Young's modulus of 15.7kPa and were much more elastic than the PLGA scaffolds. The composite PLGA/PU scaffold exhibits advantageous morphological, mechanical and cell adhesion and growth supporting properties, when compared with scaffolds fabricated from PLGA or PU alone. This novel method provides a mechanism for the formation of tailored bioactive scaffolds from nominally incompatible polymers, representing a significant step forward in scaffold processing for tissue-engineering applications.     

Microporous collagen spheres produced via thermally induced phase separation for tissue regeneration

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.     

Fabrication and characterization of modified nanofibrous poly(L-lactic acid) scaffolds by thermally induced phase separation technique and aminolysis for promoting cyctocompatibility

Modified nanofibrous Poly(L-lactic acid) (PLLA) scaffolds were fabricated by aminolysis combined with thermally induced phase separation technique using PLLA/1,4-dioxane/urea-NaOH-H₂O system at ?40 °C freeze temperature. Aminolysis led to the modification of scaffold resulting in enhancement in the bioactivity. The surface of the modified nanofibrous scaffold provided a good environment for attachment and proliferation of MC3T3-E1 subclone 14 cells, exhibiting significant potential for bone tissue regeneration and for promoting cytocompatibility.     

Novel fabrication techniques to produce microspheres by thermally induced phase separation for tissue engineering and drug delivery

A novel application of thermally induced phase separation (TIPS) is described enabling the rapid formation of monodisperse porous microspheres. By taking advantage of TIPS processing parameters, the porosity, the pore morphology (bimodal/channel-like/radial towards the centre) and the presence of an open-pore or dense skin region can be tailored. Achievable sizes range from 10 to 2000microm in diameter. The technique facilitates the homogeneous inclusion of particulate fillers and drugs. Moreover, the combined TIPS/oil-in-water emulsion technique allows for the production of microspheres with isotropic pore morphology with interconnected spherical pores of 30-70microm and well-formed porous microspheres of 10-200microm in diameter with an open porous surface. This method is advantageous over existing techniques by avoiding the use of long-term exposure to an aqueous continuous phase as used in oil-in-water or water-in-oil-in-water processing and therefore drug encapsulation efficiencies will be higher.     

Polyurethane scaffold formation via a combination of salt leaching and thermally induced phase separation

Porous scaffolds have been made from two polyurethanes based on thermally induced phase separation of polymer dissolved in a DMSO/water mixture in combination with salt leaching. It is possible to obtain very porous foams with a very high interconnectivity. A major advantage of this method is that variables like porosity, pore size, and interconnectivity can be independently adjusted with the absence of toxic materials in the production process. The obtained compression moduli were between 200 kPa and 1 MPa with a variation in porosity between 76 and 84%. Currently the biological and medical aspects are under evaluation.     

Polymeric scaffolds prepared via thermally induced phase separation: tuning of structure and morphology

Scaffolds suitable for tissue engineering applications like dermal reconstruction were prepared by Thermally Induced Phase Separation (TIPS) starting from a ternary solution PLLA/dioxane/water. The experimental protocol consisted of three consecutive steps, a first quench from the homogeneous solution to an appropriate demixing temperature (within the metastable region), a holding stage for a given residence time, and a final quench from the demixing temperature to a low temperature (within the unstable region). A large variety of morphologies, in terms of average pore size and interconnection, were obtained upon modifying the demixing time and temperature, owing to the interplay of nucleation and growth processes during the residence in the metastable state. An interesting combination of micro and macroporosity was observed for long residence times in the metastable state (above 30 min at 35 degrees C). Preliminary degradation tests in a biological mimicking fluid (D-MEM with bovine serum) showed a significant weight loss during the initial stages (ca. 30% in 30 days) related to the degradation of the amorphous part, followed by a negligible weight loss in the next days (few percent from 30 to 60 days).     

Combining mechanical foaming and thermally induced phase separation to generate chitosan scaffolds for soft tissue engineering

In this paper, a novel foaming methodology consisting of turbulent mixing and thermally induced phase separation (TIPS) was used to generate scaffolds for tissue engineering. Air bubbles were mechanically introduced into a chitosan solution which forms the continuous polymer/liquid phase in the foam created. The air bubbles entrained in the foam act as a template for the macroporous architecture of the final scaffolds. Wet foams were crosslinked via glutaraldehyde and frozen at ?20 °C to induce TIPS in order to limit film drainage, bubble coalescence and Ostwald ripening. The effects of production parameters, including mixing speed, surfactant concentration and chitosan concentration, on foaming are explored. Using this method, hydrogel scaffolds were successfully produced with up to 80% porosity, average pore sizes of 120 μm and readily tuneable compressive modulus in the range of 2.6 to 25 kPa relevant to soft tissue engineering applications. These scaffolds supported 3T3 fibroblast cell proliferation and penetration and therefore show significant potential for application in soft tissue engineering.     

Enhanced attachment, growth and migration of smooth muscle cells on microcarriers produced using thermally induced phase separation

Microcarriers are widely used for the expansion of cells in vitro, but also offer an approach for combining cell transplantation and tissue bulking for regenerative medicine in a minimally invasive manner. This could be beneficial in conditions associated with muscle damage or atrophy, such as faecal incontinence, where the use of bulking materials or cell transplantation alone has proven to be ineffective. Microcarriers currently available have not been designed for this purpose and are likely to be suboptimal due to their physical and biochemical properties. The aim of this study was to investigate macroporous microspheres of polylactide-co-glycolide (PLGA), prepared using a thermally induced phase separation technique, for their suitability as cell microcarriers for the transplantation of smooth muscle cells. Cell attachment, growth and migration were studied and compared with commercially available porcine gelatin microcarriers (Cultispher-S) in suspension culture. Smooth muscle cells attached more rapidly to the PLGA microcarriers, which also significantly enhanced the rate of cell growth compared with Cultispher-S microcarriers. The majority of smooth muscle cells attached to the PLGA microcarriers in suspension culture were able to migrate away over a 15 day period of static culture, unlike Cultispher-S microcarriers which retained the majority of cells. The ability of PLGA microcarriers to enhance cell growth combined with their capacity to release cells at the sites of delivery are features that make them ideally suited for use as a cell transplantation delivery device in tissue engineering and regenerative medicine.     

Human nasoseptal chondrocytes maintain their differentiated phenotype on PLLA scaffolds produced by thermally induced phase separation and supplemented with bioactive glass 1393

Damage of hyaline cartilage such as nasoseptal cartilage requires proper reconstruction, which remains challenging due to its low intrinsic repair capacity. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. Despite so far mostly tested for bone tissue engineering, bioactive glass (BG) could exert stimulatory effects on chondrogenesis.

The aim of this work was to produce and characterize composite porous poly(L-lactide) (PLLA)/1393BG scaffolds via thermally induced phase separation (TIPS) technique and assess their effects on chondrogenesis of nasoseptal chondrocytes.

The PLLA scaffolds without or with 1, 2.5, 5% BG1393 were prepared via TIPS technique starting from a ternary solution (polymer/solvent/non-solvent) in a single step. Scaffolds were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetric analysis (DSC). Human nasoseptal chondrocytes were seeded on the scaffolds with 1 and 2.5% BG for 7 and 14 days and cell survival, attachment, morphology and expression of SOX9 and cartilage-specific extracellular cartilage matrix (ECM) components were monitored.

The majority of chondrocytes survived on all PLLA scaffolds functionalized with BG for the whole culture period. Also inner parts of the scaffold were colonized by chondrocytes synthesizing an ECM which contained glycosaminoglycans. Type II collagen and aggrecan gene expression increased significantly in 1% BG scaffolds during the culture. Chondrocyte protein expression for cartilage ECM proteins indicated that the chondrocytes maintained their differentiated phenotype in the scaffolds.

BG could serve as a cytocompatible basis for future scaffold composites for osteochondral cartilage defect repair.

Abbreviations: AB: alcian blue ACAN: gene coding for aggrecan; BG: Bioactive glass; 2D: two-dimensional; 3D: three-dimensional; COL2A1: gene coding for type II collagen; DAPI: 4?,6-diamidino-2-phenylindole; DMEM: Dulbecco’s Modified Eagle’s Medium; DMMB: dimethylmethylene blue; DSC: Differential scanning calorimetric analysis; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; EtBr: ethidium bromide; FCS: fetal calf serum; FDA: fluorescein diacetate; GAG: glycosaminoglycans; HDPE: high density polyethylene; HE: hematoxylin and eosin staining; HCA: hydoxylapatite; PBE: phosphate buffered EDTA100 mM Na₂HPO₄ and 5 mM EDTA, pH8; PBS: phosphate buffered saline; PFA: paraformaldehyde; PG: proteoglycans; PI: propidium iodide; PLLA: Poly-L-Lactic Acid Scaffold; RT: room temperature; SD: standard deviation; SEM: scanning electron microscopy; sGAG: sulfated glycosaminoglycans; SOX9/Sox9: SRY (sex-determining region Y)-box 9 protein; TBS: TRIS buffered saline; TIPS: Thermally Induced Phase Separation; XRD: X-ray diffraction analysis     

Preparation and properties of poly(lactide-co-glycolide) (PLGA)/ nano-hydroxyapatite (NHA) scaffolds by thermally induced phase separation and rabbit MSCs culture on scaffolds

Biodegradable polymer/bioceramic composites scaffold can overcome the limitation of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. To better mimic the mineral component and the microstructure of natural bone, novel nano-hydroxyapatite (NHA)/polymer composite scaffolds with high porosity and well-controlled pore architectures as well as high exposure of the bioactive ceramics to the scaffold surface is developed for efficient bone tissue engineering. In this article, regular and highly interconnected porous poly(lactide-co-glycolide) (PLGA)/NHA scaffolds are fabricated by thermally induced phase separation technique. The effects of solvent composition, polymer concentration, coarsening temperature, and coarsening time as well as NHA content on the micro-morphology, mechanical properties of the PLGA/NHA scaffolds are investigated. The results show that pore size of the PLGA/NHA scaffolds decrease with the increase of PLGA concentration and NHA content. The introduction of NHA greatly increase the mechanical properties and water absorption ability which greatly increase with the increase of NHA content. Mesenchymal stem cells are seeded and cultured in three-dimensional (3D) PLGA/NHA scaffolds to fabricate in vitro tissue engineering bone, which is investigated by adhesion rate, cell morphology, cell numbers, and alkaline phosphatase assay. The results display that the PLGA/NHA scaffolds exhibit significantly higher cell growth, alkaline phosphatase activity than PLGA scaffolds, especially the PLGA/NHA scaffolds with 10 wt.% NHA. The results suggest that the newly developed PLGA/NHA composite scaffolds may serve as an excellent 3D substrate for cell attachment and migration in bone tissue engineering.     

Bordetella bronchiseptica phase variation induced by crystal violet.

A method for effective induction of phase variation in Bordetella bronchiseptica by treatment with crystal violet (CV) is presented. When grown in CV-broth, phase I cells dissociated into three serial phases. Appearance of variant cells was observed simultaneously with the beginning of cell multiplication. The maximum effect of CV was obtained at a concentration of 8 micrograms/ml, when the proportion of variants in the population reached 100%. The main factors which affected phase variation were concentration of CV, culture age, and temperature of treatment. The phase variants obtained were phenotypically stable upon serial passages on Bordet-Gengou agar plates. By this treatment, no reversion of phase descendants to former phases was observed.     

Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development

During tissue formation, skeletal muscle precursor cells fuse together to form multinucleated myotubes. To understand this mechanism, in vitro systems promoting cell alignment need to be developed; for this purpose, micrometer-scale features obtained on substrate surfaces by photolithography can be used to control and affect cell behaviour. This work was aimed at investigating how differently microgrooved polymeric surfaces can affect myoblast alignment, fusion and myotube formation in vitro. Microgrooved polymeric films were obtained by solvent casting of a biodegradable poly-l-lactide/trimethylene carbonate copolymer (PLLA–TMC) onto microgrooved silicon wafers with different groove widths (5, 10, 25, 50, 100 μm) and depths (0.5, 1, 2.5, 5 μm), obtained by a standard photolithographic technique. The surface topography of wafers and films was evaluated by scanning electron microscopy. Cell assays were performed using C2C12 cells and myotube formation was analysed by immunofluorescence assays. Cell alignment and circularity were also evaluated using ImageJ software. The obtained results confirm the ability of microgrooved surfaces to influence myotube formation and alignment; in addition, they represent a novel further improvement to the comprehension of best features to be used. The most encouraging results were observed in the case of microstructured PLLA–TMC films with grooves of 2.5 and 1 μm depth, presenting, in particular, a groove width of 50 and 25 μm.     

B cell tolerance induced by polymeric antigens. IV. Antigen-mediated inhibition of antibody-forming cells.

The role of antibody-forming cell (AFC) blockade in the genesis of hapten-specific tolerance induced by hapten (2,4-dinitrophenyl)-coupled pneumococcal polysaccharide (DNP-lys-S3) was investigated. Tolerizing doses of DNP-lys-S3 given to mice making antibodies to DNP-hemocyanin a day before assay markedly reduced numbers of detectable anti-DNP AFC. Furthermore, anti-DNP AFC were specifically inhibited by brief in vitro incubation with DNP-lys-S3, followed by extensive washing. This inhibition requires multivalent binding of tolerogen to immunoglobulin receptors on AFC, and appears to result from a decrease in the rate of antibody secretion per cell. A variety of data indicate that IgM AFC are more susceptible to blockade than IgG AFC, and the susceptibility of both cell types decreases with time after immunization. This probably reflects differences in the density and in the rate of loss of surface immunoglobulin receptors on IgM and IgG AFC and their immediate precursors. However, we conclude that under “conventional” tolerizing conditions, i.e. when tolerogen is given prior to immunogen, DNP-lys-S3 suppresses antibody formation primarily by inactivating DNP-specific precursor cells. AFC blockade is therefore probably only relevant in the suppression of (primary?) IgM responses by large doses of tolerogen. The relative importance of precursor inactivation and AFC blockade in tolerogenesis of IgM responses to T cell-independent antigens (such as 53) remains to be elucidated.     

Studies on the phase separation of a polyetherimide‐modified epoxy resin, 4. Kinetic effect on the phase separation mechanism of a blend at different cure rates

Keeping the same thermodynamics of the blend, the kinetic effect on the phase separation mechanism of polyetherimide modified epoxy resin systems was studied. With the changes of cure temperature and amount of cure agent, at different cure rates phase separation in the reacted blends takes place. The process of phase separation is traced by time‐resolved light scattering (TRLS) and the final morphology is observed by scanning electron microscopy (SEM). The early stage of phase separation is analyzed using Cahn's linearized theory, while the scaling postulate is tested in the late stage. The effect of the competition between thermodynamics and kinetics on phase separation mechanism is discussed.     

Serogroup identification of meningococci by a modified antiserum agar method.

Modifications in the antiserum agar method for serogroup identification of meningococci, which reduce the amount of group-specific antisera required and increase long-term storage of prepoured antiserum agar plates, are described.     

B cell tolerance induced by polymeric antigens. VI. Kinetics and reversibility of the inhibition of antibody-forming cells by antigen.

Injection of mice already making antibodies to 2,4-dinitrophenylated (DNP) Ficoll with tolerizing doses of DNP-pneumococcal polysaccharide (DNP-lys-S3) markedly inhibits the secretion of anti-DNP antibodies by IgM antibody-forming cells. The present study shows that the degree of inhibition depends not only on the dose of DNP-lys-S3 but also on the duration of exposure to antigen. DNP-lys-S3 was detectable on the surface of antibody-forming cells at a time when their rate of secretion was unimpaired, thus suggesting that the inhibition involves intracellular events subsequent to the binding of antigen to the cell membrane. The inhibition was reversible if antibody-forming cells were exposed to antigen for 24 h, and then cultured for 18 h in its absence, but became irreversible if the treatment period was extended to 48 h. The relevance of this model of inhibition of lymphocyte function by antigen to possible mechanisms of B cell tolerance is discussed.     

Silicone rubber-hydrogel composites as polymeric biomaterials III. An investigation of phase distribution by scanning electron microscopy

The structure of silicone rubber-hydrogel composite materials was investigated by scanning electron microscopy (SEM) and light microscopy. The polymer phases in these materials composed of the polysiloxane matrix and very small particles of lightly cross-linked poly(2-hydroxyethylmethacrylate) or poly(2-hydroxyethylmethacrylate-co-methacrylic acid) were visualized using both methods. The distribution of polymer phases was studied by SEM of fracture surfaces of the materials. The results are discussed in relation to the transport properties of the materials.     

An acute model for IgA-mediated glomerular inflammation in rats induced by monoclonal polymeric rat IgA antibodies.

An acute model for IgA-mediated glomerular inflammation in rats was induced by the in situ deposition of IgA directly into the glomerular mesangium. F(ab')2 anti-Thy1 MoAb was used to anchor an antigen, DNP (2,4-dinitrophenol), in the glomeruli of rats. Subsequent infusion of rat polymeric (p-) or monomeric (m-) IgA MoAb with specificity for DNP resulted in mesangial deposition of IgA in both groups of rats. However, acute proteinuria was observed only in p-IgA-treated rats and not in PBS- or m-IgA-treated rats. Immunofluorescence analysis revealed deposition of C3 in an identical pattern to that of IgA in the glomeruli of p-IgA-treated rats. No mesangial deposits of C4 or C1q were seen in these animals. Rats receiving m-IgA or PBS displayed no detectable C3, C4 or C1q deposition. The amount of proteinuria in p-IgA-treated rats was related to the amount of deposited C3. The presence of intraglomerular monocytes was only observed 2 days after p-IgA injection. By light microscopy, aneurysm formation, mesangial hypercellularity and matrix expansion were seen only in p-IgA-treated rats. However, by 37 days post-injection complete resolution of the lesions was observed. No histological renal changes were observed in PBS- or m-IgA-treated rats. In conclusion, an acute form of IgA-mediated nephritis in rats was induced by p-IgA but not by m-IgA. This reproducible model provides a basis for further study into the mechanisms of IgA-mediated glomerular inflammation.