Sustained release of proteins from electrospun biodegradable fibers

Electrospinning is a simple and versatile technique of producing polymeric fibers ranging from submicron to micron in diameter. Incorporation of bioactive agents into the fibers could make a biofunctional tissue engineering scaffold. In this study, we investigated the feasibility of encapsulating human beta-nerve growth factor (NGF), which was stabilized in a carrier protein, bovine serum albumin (BSA) in a copolymer of epsilon-caprolactone and ethyl ethylene phosphate (PCLEEP) by electrospinning. Partially aligned protein encapsulated fibers were obtained and the protein was found to be randomly dispersed throughout the electrospun fibrous mesh in aggregate form. A sustained release of NGF via diffusion process was obtained for at least 3 months. PC12 neurite outgrowth assay confirmed that the bioactivity of electrospun NGF was retained, at least partially, throughout the period of sustained release, thus clearly demonstrating the feasibility of encapsulating proteins via electrospinning to produce biofunctional tissue scaffolds.     

Surface functionalized electrospun biodegradable nanofibers for immobilization of bioactive molecules

A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.     

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.     

Biomimetic sensing layer based on electrospun conductive polymer webs

The aim of the present study is to combine a bio-inspired nanofibrous artificial epithelium to the electronic nose (e-nose) principles. The sensing device set up was an electronic nose consisting of an array of 9 micro-chemoresistors (Cr-Au, 3×3) coated with electrospun nanofibrous structures. These were comprised of doped polyemeraldine base blended with 3 different polymers: polyethylene oxide, polyvinilpyrrolidone and polystyrene, which acted as carriers for the conducting polymer and were the major responsible of the features of each fibrous overlay (electrical parameters, selectivity and sensitivity ranges). The two sensing strategies here adopted and compared consisted in the use of 2 different textural coatings: a single- and a double-overlay, where the double-overlay resulting from overdeposition of 2 different polymer blends. Such e-nose included a plurality of nanofibres whose electrical parameters were at the same time depending on each polymer exposure to analytes (NO(2), NH(3)) and on the spatial distribution of the interlacing fibres. The morphology of the coating arrangements of this novel e-nose was investigated by scanning electron microscopy (SEM) and its sensor responses were processed by multicomponent data analyses (PCA and PLS) reporting encouraging results for detection and recognition of analytes at ppb levels.     

Functionalized synthetic biodegradable polymer scaffolds for tissue engineering

Scaffolds (artificial ECMs) play a pivotal role in the process of regenerating tissues in 3D. Biodegradable synthetic polymers are the most widely used scaffolding materials. However, synthetic polymers usually lack the biological cues found in the natural extracellular matrix. Significant efforts have been made to synthesize biodegradable polymers with functional groups that are used to couple bioactive agents. Presenting bioactive agents on scaffolding surfaces is the most efficient way to elicit desired cell/material interactions. This paper reviews recent advancements in the development of functionalized biodegradable polymer scaffolds for tissue engineering, emphasizing the syntheses of functional biodegradable polymers, and surface modification of polymeric scaffolds.     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.     

Biocompatibility and long-term toxicity of InnoPol implant, a biodegradable polymer scaffold.

InnoPol, a poly((D,L)-lactic-co-glycolic acid) [PLGA] 65/35 scaffold manufactured by special gas foaming methods in Korea, was subjected to tests to evaluate the degradation and tissue compatibility characteristics and long-term systemic toxicity in mice and rats. C57BL/6 mice and SD rats were implanted subcutaneously with 3-mm- and 1-mm-thick InnoPol circular discs, 10 mm in diameter, respectively, and sacrificed 8, 12, and 24 weeks after implantation. No test material-related effects were observed in mortality, clinical signs, body weight gain, food and water consumption, ophthalmologic signs, urinalysis, hematology, serum biochemistry parameters and organ weights of all animals implanted with InnoPol. Also, there were no systemic symptoms including metabolic alterations and inflammatory reactions in either mice or rats. In addition, no gross pathological findings, except skin lesions around the implantation sites, were found in the major organs. Although mild inflammation at the site of InnoPol implantation was confirmed from hematoxylin and eosin or Masson's trichrome staining at 8-12 weeks, the reactions had disappeared at 24 weeks following complete degradation of the scaffold, leaving granulomatous tissues that were similar to surgical wounds in sham operation controls without implants. These results suggest that InnoPol possesses good mechanical properties and tissue compatibility and does not cause any systemic toxicity other than transient local inflammatory reactions at the implantation site, and that it might be useful in applications as a medical device for implantation.     

Nanostructured biodegradable polymer networks using lyotropic liquid crystalline templates

The physical properties, porosity, and physiological behavior of synthetic biodegradable hydrogels have been identified as highly critical design parameters in most tissue engineering materials applications. Nanotechnology may provide the means to manipulate these parameters by accessing control over the network structure of the biomaterial, providing unique property relationships that often result from nanostructured materials. In this study, a lyotropic liquid crystal (LLC) was used as a polymerization template in the formation of a photopolymerizable biodegradable PLA-b-PEG-b-PLA (PEG = poly(ethylene glycol); PLA = poly(lactic acid)) material with nanoscale lamellar morphology. Through ordering of the biodegradable monomer within the liquid crystal assembly, a 2-fold increase in maximum polymerization rate and a 30% increase in double bond conversion were realized over isotropic monomer formulations. The resulting network structure of the templated PLA-b-PEG-b-PLA material has a dramatic affect on the physical properties of the hydrogel including an 80% increase in network swelling and an approximately 230% increase in diffusivity. This increase in permeability and solvent uptake leads to rapid degradation of the lamellar templated samples, further demonstrating the influence of the LLC directed network structure on the porosity and physical properties of the biodegradable material. The ability to control the porosity, physical properties, and behavior of a biodegradable hydrogel simply by imparting LLC network structure, without changing the chemistry or biocompatibility of the polymer, could prove highly advantageous in the design of synthetic biomaterials for potential medical applications.     

Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.     

Starlike branched and hyperbranched biodegradable polymer systems as DNA carriers

The information on the synthesis of new biologically compatible and biologically degradable DNA carriers based on starlike polymer conjugates of proteins (lysine dendrimers and their derivatives) and hyperbranched polyamino acids is reviewed. Their capacity to bind and compact DNA and to provide for transfection is discussed based on results obtained with model systems. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Starlike branched and hyperbranched biodegradable polymer systems as DNA carriers

The information on the synthesis of new biologically compatible and biologically degradable DNA carriers based on starlike polymer conjugates of proteins (lysine dendrimers and their derivatives) and hyperbranched polyamino acids is reviewed. Their capacity to bind and compact DNA and to provide for transfection is discussed based on results obtained with model systems.     

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.     

Synthesis of novel biodegradable cationic polymer: N,N-diethylethylenediamine polyurethane as a gene carrier

A new cationic polymer, N,N-diethylethylenediamine-polyurethane (DEDA-PU), bearing tertiary amines in the backbone and side chains, was synthesized and used as a nonviral vector for gene delivery. The DEDA-PU readily self-assembled with the plasmid DNA (pCMV-betagal) in water and buffer at physiological pH, as determined by agarose gel retardation, dynamic light scattering, zeta potential, atomic force microscopy (AFM), and restriction endonuclease protection assays. The results revealed that DEDA-PU was able to bind with plasmid DNA, yielding positively charged complexes with a size around 100 nm at a DEDA-PU/DNA ratio of 50/1 (w/w). The DEDA-PU/DNA complexes were able to transfect HEK 293 cells in vitro with an efficiency comparable to a well-known gene carrier [poly(2-dimethylaminoethyl methacrylate), PDMAEMA]. The cytotoxicity of DEDA-PU was substantially lower than PDMAEMA. The degradation studies indicated that DEDA-PU degrades hydrolytically in 20 mM HEPES buffer at pH 7.4 with a half-life of approximately 60 h. This study shows that DEDA-PU holds promise as biodegradable polycations for gene delivery and is interesting candidate for further study.     

A human antibody fragment with high affinity for biodegradable polymer film

Antibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 A, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein-ligand interaction.     

Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine

Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.     

Elaboration of biodegradable polymer substrate for cultivation of human dermal fibroblasts

The influence of polylactic acid (PLA) surface films on the pattern of cell behavior was studied. The human dermal fibroblasts were cultivated on PLA covered glasses. The hydrophobic nature of PLA films depends on the availability of polymer solvent in the film preparation. PLA films obtained from a more polar solvent--aceton--appeared to be more hydrophilic than those obtained from methylene chloride. More hydrophilic polymer films also appeared to be more preferable for cell cultivation, and human dermal fibroblasts demonstrated a better adhesion and proliferation on hydrophilic rather than on hydrophobic PLA films.     

Poly(sebacic acid-co-ricinoleic acid) biodegradable injectable in situ gelling polymer

The investigated polymers, poly(sebacic acid-co-ricinoleic acid) containing > or =70% ricinoleic acid, may be injected via a 22 gauge needle and become gel upon contact with aqueous medium, both in vitro and in vivo. Various properties of the polymers including viscosity, thermal analysis, and in vivo behavior, before and after exposure to aqueous medium, were determined. These polymers were observed using scanning electron microscopy (SEM) at dry and wet states. It was found that the viscosity and melting temperature of P(SA:RA) increased after exposure to buffer. The viscosity at 37 degrees C of P(SA:RA)3:7 had the highest increase: from 4200 cP before to 8940 cP after exposure to buffer; in the case of P(SA:RA)25:75 before exposure to buffer the viscosity was 1150 cP while after it raised to 3200 cP. The viscosity of P(SA:RA)2:8 also increased from 400 cP before exposure to buffer to 1000 cP after. On the other hand polymer without sebacic acid, (poly(ricinoleic acid)), did not show gelation properties. Thermal analysis also showed an increase in the melting point of the polymers exposed to the aqueous medium during the first 24 h of incubation. Images obtained by SEM showed formation of a three-dimensional network in polymers exposed to buffers. When injected into animals, P(SA:RA) forms a solid implant in the injection site already at 8 h postinjection.     

A biodegradable polymer as a cytokine delivery system for inducing bone formation

Bone morphogenetic proteins (BMPs) that have the potential to elicit new bone in vivo have been used in a tissue-engineering approach for the repair of bone injuries and bone defects. Although it is now possible to generate large amounts of recombinant human (rh) BMPs for medical use, the major challenge remains in the development of optimal local delivery systems for these proteins. Here we describe the development of a synthetic biodegradable polymer, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG). This polymer exhibits promising degradation characteristics for BMP delivery systems and good biocompatibility under test conditions. PLA-DX-PEG/rhBMP-2 composite implants induced ectopic new bone formation effectively when tested in vivo, and can repair large bone defects orthotopically. This polymeric delivery system represents an advance in the technology for the enhancement of bone repair.