A novel wet extrusion technique to fabricate self-assembled microfiber scaffolds for controlled drug delivery

We have developed a novel wet extrusion process to fabricate nonwoven self-assembled microfiber scaffolds with uniform diameters less than 5 μm and without any postmanipulation. In this method, a poly(L-lactic acid) solution flows dropwise into a stirring nonsolvent bath, deforming into liquid polymer streams that self-assemble into a nonwoven microfiber scaffold. The ability to tune fiber diameter was achieved by decreasing polymer spin dope concentration and increasing the silicon oil to petroleum ether ratio of the nonsolvent spin bath. To demonstrate the drug delivery capabilities of scaffolds, heparin was encapsulated using a conventional water-in-oil (W/O) emulsion technique and a cryogenic emulsion technique developed in our laboratory. Spin dope preparation was found to significantly effect the release kinetics of self-assembled scaffolds by altering the interconnectivity of pores within the precipitating filaments. After 35 days, scaffolds prepared from W/O emulsions released up to 45% encapsulated heparin, whereas nearly 80% release of heparin was observed from cryogenic emulsion formulations. The versatility of our system, combined with the prolonged release of small molecules and the ability to control the homogeneity of self-assembling scaffolds, could be beneficial for many tissue regeneration and engineering applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2793-2802, 2012.     

Polymer particle-based micromolding to fabricate novel microstructures

Conventional micromolding provides rapid and low-cost methods to fabricate polymer microstructures, but has limitations when producing sophisticated designs. To provide more versatile micromolding techniques, we developed methods based on filling micromolds with polymer microparticles, as opposed to polymer melts, to produce microstructures composed of multiple materials, having complex geometries, and made using mild processing conditions. Polymer microparticles of 1 to 30 μm in size were made from PLA, PGA and PLGA using established spray drying and emulsion techniques either with or without encapsulating model drug compounds. These polymer microparticles were filled into PDMS micromolds at room temperature and melted or bonded together to form microstructures according to different protocols. Porous microstructures were fabricated by ultrasonically welding microparticles together in the mold while maintaining the voids inherent in their packing structure. Multi-layered microstructures were fabricated to have different compositions of polymers and encapsulated compounds located in different regions of the microstructures. More complex arrowhead microstructures were fabricated in a two-step process using a single mold. To assess possible applications, microstructures were designed as microneedles for minimally invasive drug delivery. Multi-layer microneedles were shown to insert into cadaver tissue and, according to design, detach from their base substrate and remain embedded in the tissue for controlled release drug delivery over time. We conclude that polymer particle-based micromolding can encapsulate compounds within microstructures composed of multiple materials, having complex geometries, and made using mild processing conditions.     

Bicomponent electrospinning to fabricate three-dimensional hydrogel-hybrid nanofibrous scaffolds with spatial fiber tortuosity

Electrospun fibrous mats have emerged as powerful tissue engineering scaffolds capable of providing highly effective and versatile physical guidance, mimicking the extracellular environment. However, electrospinning typically produces a sheet-like structure, which is a major limitation associated with current electrospinning technologies. To address this challenge, highly porous, volumetric hydrogel-hybrid fibrous scaffolds were fabricated by one Taylor cone-based side-by-side dual electrospinning of poly (ε-caprolactone) (PCL) and poly (vinyl pyrrolidone) (PVP), which possess distinct properties (i.e., hydrophobic and hydrogel properties, respectively). Immersion of the resulting scaffolds in water induced spatial tortuosity of the hydrogel PVP fibers while maintaining their aligned fibrous structures in parallel with the PCL fibers. The resulting conformational changes in the entire bicomponent fibers upon immersion in water led to volumetric expansion of the fibrous scaffolds. The spatial fiber tortuosity significantly increased the pore volumes of electrospun fibrous mats and dramatically promoted cellular infiltration into the scaffold interior both in vitro and in vivo. Harmonizing the flexible PCL fibers with the soft PVP-hydrogel layers produced highly ductile fibrous structures that could mechanically resist cellular contractile forces upon in vivo implantation. This facile dual electrospinning followed by the spatial fiber tortuosity for fabricating three-dimensional hydrogel-hybrid fibrous scaffolds will extend the use of electrospun fibers toward various tissue engineering applications.     

Novel approach to fabricate keratin sponge scaffolds with controlled pore size and porosity

A compression-molding/particulate-leaching (CM/PL) method was developed to fabricate S-sulfo keratin sponges with the controlled pore size and porosity. The S-sulfo keratin was extracted from wool and was then spray-dried to give S-sulfo keratin powder. The S-sulfo keratin powder mixed with urea in advance was compression-molded together with the sieved NaCl particulates above the melting temperature of urea. The following removal of the salts and urea in water created the sponges composed of interconnected pores and the continuous S-sulfo keratin matrix. The S-sulfo keratin sponges were strong enough to handle and water-insoluble. By contrast, the sponges prepared without urea were very fragile and readily collapsed, because most of S-sulfo keratin matrix remained powdery. The pore size was in good accordance with the size of the salts, indicating that the pores were formed by leaching-out the salts. The S-sulfo keratin sponges with the regulated sizes of pores (<100, 100-300 and 300-500 microm) were fabricated, all of which had more than 90% of the porosity. Thus, CM/PL method is able to give the S-sulfo keratin sponge with the desired pore size and porosity, which might be a good scaffold for the cells in tissue engineering.     

制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为“Cartilage tissue engineering;scaffolds;fabrication”,中文检索词为“软骨组织工程;制备方法;支架材料;多孔支架”。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

A one-step method to fabricate PLLA scaffolds with deposition of bioactive hydroxyapatite and collagen using ice-based microporogens

Porous poly(l-lactic acid) (PLLA) scaffolds with bioactive coatings were prepared by a novel one-step method. In this process, ice-based microporogens containing bioactive molecules, such as hydroxyapatite (HA) and collagen, served as both porogens to form the porous structure and vehicles to transfer the bioactive molecules to the inside of PLLA scaffolds in a single step. Based on scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy analysis, the bioactive components were found to be transferred successfully from the porogens to PLLA scaffolds evenly. Osteoblast cells were used to evaluate the cellular behaviors of the composite scaffolds. After culturing for 8 days, MTT assay and alkaline phosphatase activity results suggested that HA/collagen could improve the interactions between osteoblast cells and the polymeric scaffold.     

Paraffin spheres as porogen to fabricate poly(L-lactic acid) scaffolds with improved cytocompatibility for cartilage tissue engineering

Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and pore size ranging from 150 to 700 microm were conveniently prepared with paraffin spheres used as porogen. PLLA/1,4-dioxane solution containing a given amount of paraffin spheres was frozen at -25 degrees C to obtain a solidified mixture, followed with freeze drying and subsequent leaching with hexane to remove the 1,4-dioxane and paraffin spheres, respectively. The fabricated PLLA scaffolds were highly porous with evenly distributed and interconnected pores. The microstructures of the PLLA scaffolds as a function of paraffin-sphere size, paraffin-sphere dosage, and PLLA concentration were characterized by confocal laser scanning microscopy (CLSM) and scanning-electronic microscopy (SEM). To improve the cytocompatibility of the bioinert material, a hybrid PLLA scaffold containing Type I collagen was prepared by pressing the collagen solution into the scaffold under reduced pressure. The amounts of the collagen introduced in the scaffolds were detected by ninhydrin method. The distribution of the collagen in the scaffolds was studied with CLSM. Finally, in vitro cell culture was performed by injecting a chondrocyte suspension into the scaffolds. The results showed that the chondrocytes were more evenly distributed and more spread out in the collagen-modified PLLA scaffolds than in the unmodified ones.     

A novel method to fabricate thermoresponsive microstructures with improved cell attachment/detachment properties

A novel, simple, and rapid method to fabricate thermoresponsive micropatterned substrate for cell adhesion, growth, and thermally induced detachment was developed. Thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), was grafted onto the surface of polystyrene (PS) film with microstructure by plasma-induced graft polymerization technique. The thermoresponsive micropatterned films were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, hydrogen nuclear magnetic resonance ((1) H NMR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). These results indicated that the grafting ratio of PNIPAAm increased with increasing roughness of PS film. However, the microstructures on the substrate were not affected by grafted PNIPAAm. The optimal grafting conditions, such as plasma treatment time, monomer concentration, graft polymerization time, and graft medium were investigated. The thermoresponsive micropatterned films were investigated with the fibroblast cell (L929) adhesion, proliferation, and thermally induced detachment assay. The microstructure on the thermoresponsive micropatterned substrate facilitated cell adhesion above the lower critical solution temperature (LCST) of PNIPAAm and cell detachment below the LCST. Moreover, it can be used to regulate cell organization and tissue growth.     

Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds

2-Hydroxyethyl methacrylate-L-lactate (HEMA-LLA) and HEMA-L-lactate-dextran (HEMA-LLA-D) were synthesized. 1H-NMR confirmed the formation of these oligomers and macromers. Cryogels with different pore structures were prepared using different amounts of HEMA, HEMA-LLA and HEMA-LLA-D by a cryogelation technique. SEM micrographs exhibited pore morphologies. Cryogels were highly porous with interconnected pore structures, opaque, spongy and highly elastic. It was possible to compress them to remove the water in the pores and to return to their original form just by immersing them in water in few minutes, which was quite reproducible. Their swelling abilities, compressive strengths and degradation in buffer solutions were found to be related with their structural properties which was controlled by changing the cryogelation recipe.     

Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds

2-Hydroxyethyl methacrylate-L-lactate (HEMA-LLA) and HEMA-L-lactate-dextran (HEMA-LLA-D) were synthesized. ¹H-NMR confirmed the formation of these oligomers and macromers. Cryogels with different pore structures were prepared using different amounts of HEMA, HEMA-LLA and HEMA-LLA-D by a cryogelation technique. SEM micrographs exhibited pore morphologies. Cryogels were highly porous with interconnected pore structures, opaque, spongy and highly elastic. It was possible to compress them to remove the water in the pores and to return to their original form just by immersing them in water in few minutes, which was quite reproducible. Their swelling abilities, compressive strengths and degradation in buffer solutions were found to be related with their structural properties which was controlled by changing the cryogelation recipe.     

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies. The cryogel scaffolds integrated with the surrounding tissue and the formation of a new tissue were accompanied with significant ingrowth of connective tissue cells and new blood vessels into the cryogel. The tissue responses were significantly lower in auricular and calvarial implantations when compared with the subcutanous and the submuscular implantations. The degradation of the scaffold was slower in bone comparing to soft tissues. The biodegradable cryogels are highly biocompatible and combine extraordinary properties including having soft and elastic nature, open porous structure, and very rapid and controllable swelling. Therefore, the cryogels could be promising candidates for further clinical applications in tissue regeneration.     

A novel rotating electrochemically anodizing process to fabricate titanium oxide surface nanostructures enhancing the bioactivity of osteoblastic cells

Titanium oxide (TiO(2) ) surface layers with various surface nanostructures (nanotubes and nanowires) have been developed using an anodizing technique. The pore size and length of TiO(2) nanotubes can be tailored by changing the anodizing time and applied voltage. We developed a novel method to transform the upper part of the formed TiO(2) nanotubes into a nanowire-like structure by rotating the titanium anode during anodizing process. The transformation of nanotubes contributed to the preferential chemical dissolution of TiO(2) on the areas with intense interface tension stress. Furthermore, we further compared the effect of various TiO(2) surface nanostructures including flat, nanotubes, and nanowires on bioactive applications. The MG-63 osteoblastic cells cultured on the TiO(2) nanowires exhibited a polygonal shape with extending filopodia and showed highest levels of cell viability and alkaline phosphatase activity (ALP). The TiO(2) nanowire structure formed by our novel method can provide beneficial effects for MG-63 osteoblastic cells in attachment, proliferation, and secretion of ALP on the TiO(2) surface layer.     

Engineering bone regeneration with bioabsorbable scaffolds with novel microarchitecture

Critical-sized defects (CSDs) were introduced into rat calvaria to test the hypothesis that absorption of surrounding blood, marrow, and fluid from the osseous wound into a bioabsorbable polymer matrix with unique microarchitecture can induce bone formation via hematoma stabilization. Scaffolds with 90% porosity, specific surface areas of approximately 10 m2/g, and median pore sizes of 16 and 32 microm, respectively, were fabricated using an emulsion freeze-drying process. Contact radiography and radiomorphometry revealed the size of the initial defects (50 mm2) were reduced to 27 +/- 11 mm2 and 34 +/- 17 mm2 for CSDs treated with poly(D,L-lactide-co-glycolide). Histology and histomorphometry revealed scaffolds filled with significantly more de novo bone than negative controls (p < 0. 007), more osteoid than both the negative and autograft controls (p < 0.002), and small masses of mineralized tissue (< 15 mm in diameter) observed within the scaffolds. Based on these findings, we propose a change in the current paradigm regarding the microarchitecture of scaffolds for in vivo bone regeneration to include mechanisms based on hematoma stabilization.     

Bone tissue engineering with novel rhBMP2-PLLA composite scaffolds

The aims of the present study were to fabricate a novel porous polylactic acid (PLLA) composite scaffold and evaluate the capacity of the scaffold in carrying recombinant bone morphogenetic protein 2 (rhBMP2) for engineering bone formation. The structures of the PLLA scaffolds were evaluated by SEM and the controlled release of rhBMP2 from the composite scaffolds was assayed by ELISA. Bone induction by the scaffolds loaded with or without rhBMP2 was performed in the calf muscle of twenty Wistar rats for 3, 7, 10, 14, and 28 days. Tissue specimens were examined by Masson's trichrome and von Kossa stainings, and immunohistochemistry of bone proteins. Our results indicated that a moderate foreign body reaction was found in control scaffolds, which lasted for 4 weeks. The addition of rhBMP2 to this novel scaffold dramatically alleviated the adverse responses to PLLA. Enhanced deposition of collagen matrix and endochondral formation were observed in rhBMP2-PLLA scaffolds at 7-10 days, compatible with an early release of rhBMP2 in the composite scaffolds. Bone sialoprotein and osteopontin were demonstrated simultaneously. Von Kossa staining was observed in the test group at 10-14 days. In conclusion, the PLLA scaffolds exhibited the capability of carrying rhBMP2 for inducing bone formation within 2 weeks. These results suggest that rhBMP2-PLLA scaffold may be applicable in tissue engineering.     

Development of novel biodegradable polymer scaffolds for vascular tissue engineering

Functional connective tissues have been developed using tissue engineering approach by seeding cells on biodegradable scaffolds such as polyglycolic acid (PGA). However, a major drawback of tissue engineering approaches that utilize synthetic polymers is the persistence of polymer remnants in engineered tissues at the end of culture. Such polymer fragments may significantly degrade tissue mechanics and stimulate local inflammatory responses in vivo. In this study, several polymeric materials with a range of degradation profiles were developed and evaluated for their potential applications in construction of collagen matrix-rich tissues, particularly tissue-engineered blood vessels. The degradation characteristics of these polymers were compared as were their characteristics vis-à-vis cell adhesion and proliferation, collagen synthesis, and ability to support growth of engineered vessels. Under aqueous conditions at 37°C, Polymer I (comprising 84% glycolide and 16% trimethylene carbonate [TMC]) had a similar degradation profile to PGA, Polymer II (comprising 84% glycolide, 14% TMC, and 2% polyethylene succinate) degradedly more slowly, but Polymer III (comprising 87% glycolide, 7% TMC, and 6% polyethylene glycol) had a more extensive degradation as compared to PGA. All polymers supported cell proliferation, but Polymer III improved collagen production and engineered vessel mechanics as compared with PGA. In addition, more slowly degrading polymers were associated with poorer final vessel mechanics. These results suggest that polymers that degrade more quickly during tissue culture actually result in improved engineered tissue mechanics, by virtue of decreased disruption of collagenous extracellular matrix.     

Solvent/non-solvent sintering: a novel route to create porous microsphere scaffolds for tissue regeneration

Solvent/non-solvent sintering creates porous polymeric microsphere scaffolds suitable for tissue engineering purposes with control over the resulting porosity, average pore diameter, and mechanical properties. Five different biodegradable biocompatible polyphosphazenes exhibiting glass transition temperatures from -8 to 41 degrees C and poly (lactide-co-glycolide), (PLAGA) a degradable polymer used in a number of biomedical settings, were examined to study the versatility of the process and benchmark the process to heat sintering. Parameters such as: solvent/non-solvent sintering solution composition and submersion time effect the sintering process. PLAGA microsphere scaffolds fabricated with solvent/non-solvent sintering exhibited an interconnected porosity and pore size of 31.9% and 179.1 mum, respectively which was analogous to that of conventional heat sintered PLAGA microsphere scaffolds. Biodegradable polyphosphazene microsphere scaffolds exhibited a maximum interconnected porosity of 37.6% and a maximum compressive modulus of 94.3 MPa. Solvent/non-solvent sintering is an effective strategy for sintering polymeric microspheres, with a broad spectrum of glass transition temperatures, under ambient conditions making it an excellent fabrication route for developing tissue engineering scaffolds and drug delivery vehicles.     

Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds

Dynamic processes such as diffusion and binding/unbinding of macromolecules (e.g. growth factors or nutrients) are crucial parameters for the design and application of effective artificial tissue materials. Here, dynamics of selected macromolecules were studied in two different composite tissue engineering scaffolds containing an electrospun nanofiber mesh (polycaprolactone or hydrophobically plasma modified polyvinylalcohol-chitosan) encapsulated in agarose hydrogels by a conventional approach fluorescence recovery after photobleaching (FRAP) and a novel technique, raster image correlation spectroscopy (RICS). The two approaches are compared, and it is shown that FRAP is unable to determine processes occurring at low molecular concentrations, especially accurately separating binding/unbinding from diffusion, and its results depend on the concentration of the studied molecules. RICS measures processes of single molecules and, because of its multiple adjustable timescales, can distinguish whether diffusion or binding controls molecular movement and separates fast diffusion from slow transient binding. In addition, RICS provides a robust read-out parameter quantifying binding affinity. Finally, the combination of FRAP and RICS helps to characterize diffusion and binding of macromolecules in tested artificial tissues better, and therefore predicts the behavior of biologically active molecules in these materials for medical applications.     

X-ray diffraction enhanced imaging as a novel method to visualize low-density scaffolds in soft tissue engineering

Scaffold visualization is challenging yet essential to the success of various tissue engineering applications. The aim of this study was to explore the potential of X-ray diffraction enhanced imaging (DEI) as a novel method for the visualization of low density engineered scaffolds in soft tissue. Imaging of the scaffolds made from poly(L-lactide) (PLLA) and chitosan was conducted using synchrotron radiation-based radiography, in-line phase-contrast imaging (in-line PCI), and DEI techniques as well as laboratory-based radiography. Scaffolds were visualized in air, water, and rat muscle tissue. Compared with the images from X-ray radiography and in-line PCI techniques, DEI images more clearly show the structure of the low density scaffold in air and have enhanced image contrast. DEI was the only technique able to visualize scaffolds embedded in unstained muscle tissue; this method could also define the microstructure of muscle tissue in the boundary areas. At a photon energy of 20?KeV, DEI had the capacity to image PLLA/chitosan scaffolds in soft tissue with a sample thickness of up to 4?cm. The DEI technique can be applied at high X-ray energies, thus facilitating lower in vivo radiation doses to tissues during imaging as compared to conventional radiography.     

A novel route in bone tissue engineering: Magnetic biomimetic scaffolds

In recent years, interest in tissue engineering and its solutions has increased considerably. In particular, scaffolds have become fundamental tools in bone graft substitution and are used in combination with a variety of bio-agents. However, a long-standing problem in the use of these conventional scaffolds lies in the impossibility of re-loading the scaffold with the bio-agents after implantation. This work introduces the magnetic scaffold as a conceptually new solution. The magnetic scaffold is able, via magnetic driving, to attract and take up in vivo growth factors, stem cells or other bio-agents bound to magnetic particles. The authors succeeded in developing a simple and inexpensive technique able to transform standard commercial scaffolds made of hydroxyapatite and collagen in magnetic scaffolds. This innovative process involves dip-coating of the scaffolds in aqueous ferrofluids containing iron oxide nanoparticles coated with various biopolymers. After dip-coating, the nanoparticles are integrated into the structure of the scaffolds, providing the latter with magnetization values as high as 15 emu g^?1 at 10 kOe. These values are suitable for generating magnetic gradients, enabling magnetic guiding in the vicinity and inside the scaffold. The magnetic scaffolds do not suffer from any structural damage during the process, maintaining their specific porosity and shape. Moreover, they do not release magnetic particles under a constant flow of simulated body fluids over a period of 8 days. Finally, preliminary studies indicate the ability of the magnetic scaffolds to support adhesion and proliferation of human bone marrow stem cells in vitro. Hence, this new type of scaffold is a valuable candidate for tissue engineering applications, featuring a novel magnetic guiding option.