Electrospinning of aniline pentamer-graft-gelatin/PLLA nanofibers for bone tissue engineering

Blends of aniline pentamer-graft-gelatin (AP-g-GA) and poly(l-lactide) (PLLA) were electrospun to prepare uniform nanofibers as biomimetic scaffolds. The nanofibers exhibited good electroactivity, thermal stability and biodegradability. The biocompatibility of the nanofibers in vitro was evaluated by the adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. The cellular elongation was significantly greater on electroactive AP-g-GA/PLLA nanofibers than on PLLA nanofibers. Moreover, the AP-g-GA/PLLA nanofibers stimulated by an electrical pulsed signal could promote the differentiation of MC3T3-E1 cells compared with pure PLLA nanofibers. Our results demonstrated that the biodegradable and electroactive AP-g-GA/PLLA nanofibers had potential application in vivo as bone repair scaffold materials in tissue engineering.     

Electrospinning of collagen and elastin for tissue engineering applications

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (M(w)=8 x 10(6)) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (T(d)=79 degrees C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.     

Electrospinning: applications in drug delivery and tissue engineering

Despite its long history and some preliminary work in tissue engineering nearly 30 years ago, electrospinning has not gained widespread interest as a potential polymer processing technique for applications in tissue engineering and drug delivery until the last 5-10 years. This renewed interest can be attributed to electrospinning's relative ease of use, adaptability, and the ability to fabricate fibers with diameters on the nanometer size scale. Furthermore, the electrospinning process affords the opportunity to engineer scaffolds with micro to nanoscale topography and high porosity similar to the natural extracellular matrix (ECM). The inherently high surface to volume ratio of electrospun scaffolds can enhance cell attachment, drug loading, and mass transfer properties. Various materials can be electrospun including: biodegradable, non-degradable, and natural materials. Electrospun fibers can be oriented or arranged randomly, giving control over both the bulk mechanical properties and the biological response to the scaffold. Drugs ranging from antibiotics and anticancer agents to proteins, DNA, and RNA can be incorporated into electrospun scaffolds. Suspensions containing living cells have even been electrospun successfully. The applications of electrospinning in tissue engineering and drug delivery are nearly limitless. This review summarizes the most recent and state of the art work in electrospinning and its uses in tissue engineering and drug delivery.     

Electrospinning of polyvinyl alcohol/gelatin nanofiber composites and cross-linking for bone tissue engineering application

A three-dimensional polymer composite system consisting of polyvinyl alcohol/gelatin (PVA/GE) was fabricated via the electrospinning method and physically cross linked by methanol treatment. The effects of cross-linking between PVA/GE blend on physical, mechanical, and biological properties were investigated. After treating with methanol, PVA/GE mats become dense, hard, and aggregative with increased resistance to water dissolution. Osteoblasts like MG-63 cells were seeded on the surfaces of the cross linked PVA/GE mats and were found to attach firmly by expressing philopodial extensions. In addition, MTT assay and Western Blot analysis confirmed that the cells readily proliferated on the cross linked PVA/GE scaffolds. The osteoblast cell-matrix interaction demonstrated that the active biocompatibility of the mats was facilitated by using GE and cross-linking. In conclusion, our results suggest that cross-linked PVA/GE scaffolds hold promise for tissue engineering applications, especially in the field of artificial bone implant.     

Polymeric nanofibers in tissue engineering

Polymeric nanofibers can be produced using methods such as electrospinning, phase separation, and self-assembly, and the fiber composition, diameter, alignment, degradation, and mechanical properties can be tailored to the intended application. Nanofibers possess unique advantages for tissue engineering. The small diameter closely matches that of extracellular matrix fibers, and the relatively large surface area is beneficial for cell attachment and bioactive factor loading. This review will update the reader on the aspects of nanofiber fabrication and characterization important to tissue engineering, including control of porous structure, cell infiltration, and fiber degradation. Bioactive factor loading will be discussed with specific relevance to tissue engineering. Finally, applications of polymeric nanofibers in the fields of bone, cartilage, ligament and tendon, cardiovascular, and neural tissue engineering will be reviewed.     

Novel porous gelatin scaffolds by overrun/particle leaching process for tissue engineering applications

Porous gelatin scaffolds were prepared using a modified overrun process, which is a novel method for preparing a porous matrix by injecting air and mixing polymer solution at low temperature. The pores in the scaffolds formed by the overrun process exhibited a dual-pore structure due to the injection of air bubbles and ice recrystallization. However, the morphology of the overrun-processed gelatin scaffolds had closed pore structures. The closed pore structure was reformed into a uniformly distributed and interconnected open structure by the combination of the overrun process and a particle-leaching technique (NaCl and sucrose). The mechanical strength and biodegradation rate of gelatin scaffolds were controlled by the matrix porosity and concentration of gelatin solution. Despite higher porosity, overrun processed gelatin scaffolds showed similar mechanical strength to freeze-dried scaffolds. After 1 week of in vitro culturing, the fibroblasts in overrun-processed scaffolds were widely distributed on the surface of the scaffold pores, whereas cells seeded in freeze-dried scaffolds were mainly placed on the top and bottom of the scaffolds. Therefore, the overrun process combined with a particle-leaching technique can be applied to fabricate porous scaffolds with a desirable cellular structure for tissue engineering applications.     

Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications

Electrospinning is a promising approach to create nanofiber structures that are capable of supporting adhesion and guiding extension of neurons for nerve regeneration. Concurrently, electrical stimulation of neurons in the absence of topographical features also has been shown to guide axonal extension. Therefore, the goal of this study was to form electrically conductive nanofiber structures and to examine the combined effect of nanofiber structures and electrical stimulation. Conductive meshes were produced by growing polypyrrole (PPy) on random and aligned electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, as confirmed by scanning electron micrographs and X-ray photon spectroscopy. PPy–PLGA electrospun meshes supported the growth and differentiation of rat pheochromocytoma 12 (PC12) cells and hippocampal neurons comparable to non-coated PLGA control meshes, suggesting that PPy–PLGA may be suitable as conductive nanofibers for neuronal tissue scaffolds. Electrical stimulation studies showed that PC12 cells, stimulated with a potential of 10 mV/cm on PPy–PLGA scaffolds, exhibited 40–50% longer neurites and 40–90% more neurite formation compared to unstimulated cells on the same scaffolds. In addition, stimulation of the cells on aligned PPy–PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy–PLGA fibers, suggesting a combined effect of electrical stimulation and topographical guidance and the potential use of these scaffolds for neural tissue applications.     

Fabrication of porous gelatin scaffolds for tissue engineering.

A novel method which employs water present in swollen hydrogels as a porogen for shape template was suggested for preparing porous materials. Biodegradable hydrogels were prepared through crosslinking of gelatin with glutaraldehyde in aqueous solution, followed by rinsing and washing. After freezing the swollen hydrogels, the ice formed within the hydrogel network was sublimated by freeze-drying. This simple method produced porous hydrogels. Irrespective of any rinsing and washing processes, water was homogeneously distributed into the hydrogel network, allowing the hydrogel network to uniformly enlarge and the ice to act as a porogen during the freezing process. Different porous structures were obtained by varying the freezing temperature. Hydrogels frozen in liquid nitrogen, had a two-dimensionally ordered structure, while the hydrogels prepared at freezing temperatures near -20 degrees C, showed a three-dimensional structure with interconnected pores. As the freezing temperature was lowered, the hydrogel structure gradually became more two-dimensionally ordered. These results suggest that the porosity of dried hydrogels can be controlled by the size of ice crystals formed during freezing. It was concluded that the present freeze-drying procedure is a bio-clean method for formulating biodegradable sponges of different pore structures without use of any additives and organic solvents.     

Advancing tissue engineering by using electrospun nanofibers

Electrospinning is a versatile technique that enables the development of nanofiber-based scaffolds, from a variety of polymers that may have drug-release properties. Using nanofibers, it is now possible to produce biomimetic scaffolds that can mimic the extracellular matrix for tissue engineering. Interestingly, nanofibers can guide cell growth along their direction. Combining factors like fiber diameter, alignment and chemicals offers new ways to control tissue engineering. In vivo evaluation of nanomats included their degradation, tissue reactions and engineering of specific tissues. New advances made in electrospinning, especially in drug delivery, support the massive potential of these nanobiomaterials. Nevertheless, there is already at least one product based on electrospun nanofibers with drug-release properties in a Phase III clinical trial, for wound dressing. Hopefully, clinical applications in tissue engineering will follow to enhance the success of regenerative therapies.     

Photopolymerizable hydrogels for tissue engineering applications

Photopolymerized hydrogels are being investigated for a number of tissue engineering applications because of the ability to form these materials in situ in a minimally invasive manner such as by injection. In addition, hydrogels, three-dimensional networks of hydrophilic polymers that are able to swell large amounts of water, can be made to resemble the physical characteristics of soft tissues. Hydrogel materials also generally exhibit high permeability and good biocompatibility making, these materials attractive for use in cell encapsulation and tissue engineering applications. A number of hydrogel materials can be formed via photopolymerization processes mild enough to be carried out in the presence of living cells. This allows one to homogeneously seed cells throughout the scaffold material and to form hydrogels in situ. This review presents advantages of photopolymerization of hydrogels and describes the photoinitiators and materials in current use. Applications of photopolymerized hydrogels in tissue engineering that have been investigated are summarized.     

Carbon nanotube applications for tissue engineering

As the field of tissue engineering advances, new tools for better monitoring and evaluating of engineered tissues along with new biomaterials to direct tissue growth are needed. Carbon nanotubes may be an important tissue engineering material for improved tracking of cells, sensing of microenvironments, delivering of transfection agents, and scaffolding for incorporating with the host's body. Using carbon nanotubes for optical, magnetic resonance and radiotracer contrast agents would provide better means of evaluating tissue formation. In addition, monitoring and altering intra and intercellular processes would be useful for design of better engineered tissues. Carbon nanotubes can also be incorporated into scaffolds providing structural reinforcement as well as imparting novel properties such as electrical conductivity into the scaffolds may aid in directing cell growth. Potential cytotoxic effects associated with carbon nanotubes may be mitigated by chemically functionalizing the surface. Overall, carbon nanotubes may play an integral role as unique biomaterial for creating and monitoring engineered tissue.     

Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering

To develop a single-unit osteochondral tissue with demineralized bone matrix gelatin (BMG), rabbit chondrocytes were cultured on demineralized bone matrix gelatin for 6 weeks. The engineered osteochondral tissue was characterized with histology, immunolocalization, TEM, SEM, biochemical assay, and gene expression analysis. About 1.3mm viable neo-cartilage was produced on demineralized BMG. RT-PCR, immunohistochemistry, TEM, biochemical assay, and histology revealed hyaline-like cartilage with zonal layers, intense type II collagen expression, and abundant proteoglycan content formed upon BMG compared with normal cartilage. But hydroxyproline content and type I collagen gene and protein expressions were significantly lower. We consider engineering cartilage tissue with chondrocytes cultured on allogenic demineralized BMG is a good approach for osteochondral tissue engineering.     

Amido-modified polylactide for potential tissue engineering applications

Poly(ester amide) copolymers based on L-lactide (2) and a new depsipeptide (1) were prepared by ring opening polymerization in the presence of Sn(Oct)2 as the catalyst. Variable monomer feed ratios up to 2.3 mol% 1 afforded copolymers containing ester and amido functional groups in the backbone. Lower glass transition temperatures and reduced crystallization kinetics and crystallinity compared to homo-polylactide (PLA) was achieved with low levels of amido incorporation. A reactivity comparison between enchainment of 2 and 1 was determined using in situ infrared spectroscopy. An increase in shear viscosity was observed with the increase of 1 content as determined by rheology studies. Cellular compatibility of the co-polymers was investigated by seeding D1 mouse stem cells onto films and characterizing cell morphology by optical microscopy. Preliminary results indicate that these novel materials exhibit reduced cell attachment compared to PLA and, pending further exploration, may have potential use in biomedical applications.     

Amido-modified polylactide for potential tissue engineering applications

Poly(ester amide) copolymers based on _L-lactide (2) and a new depsipeptide (1) were prepared by ring opening polymerization in the presence of Sn(Oct)₂ as the catalyst. Variable monomer feed ratios up to 2.3 mol% 1 afforded copolymers containing ester and amido functional groups in the backbone. Lower glass transition temperatures and reduced crystallization kinetics and crystallinity compared to homo-polylactide (PLA) was achieved with low levels of amido incorporation. A reactivity comparison between enchainment of 2 and 1 was determined using in situ infrared spectroscopy. An increase in shear viscosity was observed with the increase of 1 content as determined by rheology studies. Cellular compatibility of the co-polymers was investigated by seeding D1 mouse stem cells onto films and characterizing cell morphology by optical microscopy. Preliminary results indicate that these novel materials exhibit reduced cell attachment compared to PLA and, pending further exploration, may have potential use in biomedical applications.     

Transglutaminase crosslinked gelatin as a tissue engineering scaffold

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.     

Electrospinning polydioxanone for biomedical applications

Polydioxanone (PDS) is a colorless, crystalline, bioabsorbable polymer that was first developed specifically for wound closure sutures. The compatibility, degradation rate, and mechanical properties (including shape memory) of PDS are of interest when considering the design of tissue engineering scaffolds. This research presents the electrospinning of PDS to fabricate unique nanofibrous structures for a variety of biomedical applications. Electrospinning is a polymer processing technique that utilizes an electric field to form fibers from a polymer solution or melt and allows the fabrication of nanofibrous non-woven structures. Results demonstrate the ability to control the fiber diameter of PDS as a function of solution concentrations and the fiber orientation with our prototype electrospinning apparatus. The results also show dependence between the fiber orientation and the elastic modulus, peak stress, and strain to failure of PDS in a uniaxial model.     

Combining electrospun nanofibers with cell-encapsulating hydrogel fibers for neural tissue engineering

A promising component of biomaterial constructs for neural tissue engineering are electrospun fibers, which differentiate stem cells and neurons as well as direct neurite growth. However, means of protecting neurons, glia, and stem cells seeded on electrospun fibers between lab and surgical suite have yet to be developed. Here we report an effort to accomplish this using cell-encapsulating hydrogel fibers made by interfacial polyelectrolyte complexation (IPC). IPC-hydrogel fibers were created by interfacing acid-soluble chitosan (AsC) and cell-containing alginate and spinning them on bundles of aligned electrospun fibers. Primary spinal astrocytes, cortical neurons, or L929 fibroblasts were mixed into alginate hydrogels prior to IPC-fiber spinning. The viability of each cell type was assessed at 30 min, 4 h, 1 d, and 7 d after encapsulation in IPC hydrogels. Some neurons were encapsulated in IPC-hydrogel fibers made from water-soluble chitosan (WsC). Neurons were also stained with Tuj1 and assessed for neurite extension. Neuron survival in AsC-fibers was worse than astrocytes in AsC-fibers (p < 0.05) and neurons in WsC-fibers (p < 0.05). As expected, neuron and glia survival was worse than L929 fibroblasts (p < 0.05). Neurons in IPC-hydrogel fibers fabricated with WsC extended neurites robustly, while none in AsC fibers did. Neurons remaining inside IPC-hydrogel fibers extended neurites inside them, while others de-encapsulated, extending neurites on electrospun fibers, which did not fully integrate with IPC-hydrogel fibers. This study demonstrates that primary neurons and astrocytes can be encapsulated in IPC-hydrogel fibers at good percentages of survival. IPC hydrogel technology may be a useful tool for encapsulating neural and other cells on electrospun fiber scaffolds.     

Biomimetic nanofibrous gelatin/apatite composite scaffolds for bone tissue engineering

Mimicking certain features (e.g. nanoscale topography and biological cues) of natural extracellular matrix (ECM) is advantageous for the successful regeneration of damaged tissue. In this study, nanofibrous gelatin/apatite (NF-gelatin/apatite) composite scaffolds have been fabricated to mimic both the physical architecture and chemical composition of natural bone ECM. A thermally induced phase separation (TIPS) technique was developed to prepare nanofibrous gelatin (NF-gelatin) matrix. The NF-gelatin matrix mimicked natural collagen fibers and had an average fiber diameter of about 150 nm. By integrating the TIPS method with porogen leaching, three-dimensional NF-gelatin scaffolds with well-defined macropores were fabricated. In comparison to Gelfoam^® (a commercial gelatin foam) with similar pore size and porosity, the NF-gelatin scaffolds exhibited a much higher surface area and mechanical strength. The surface area and compressive modulus of NF-gelatin scaffolds were more than 700 times and 10 times higher than that of Gelfoam^®, respectively. The NF-gelatin scaffolds also showed excellent biocompatibility and mechanical stability. To further enhance pre-osteoblast cell differentiation as well as improving mechanical strength, bone-like apatite particles (<2 μm) were incorporated onto the surface of NF-gelatin scaffolds via a simulated body fluid (SBF) incubation process. The NF-gelatin/apatite scaffolds 5 days after SBF treatment showed significantly higher mechanical strength than NF-gelatin scaffolds 5 days after SBF treatment. Furthermore, the incorporated apatite in the NF-gelatin/apatite composite scaffold enhanced the osteogenic differentiation. The expression of BSP and OCN in the osteoblast–(NF-gelatin/apatite composite) constructs was about 5 times and 2 times higher than in the osteoblast–(NF-gelatin) constructs 4 weeks after cell culture. The biomimetic NF-gelatin/apatite scaffolds are, therefore, excellent for bone tissue engineering.