Advanced carriers as ultrafine fibers and laser-treated films
Advances in science and technology have led to the increasing use of various high-molecular-weight polymer compounds in different fields of medicine. Polymers play a big part in manufacture of high-tech materials and biomedical devices, and in improvement of novel reconstruction technologies. The great diversity of polymers, whose composition, stereoconfiguration, and molecular weight can be widely varied, and the possibility of producing polymer composites with different substances provide a basis for fabricating a very wide range of materials with novel valuable properties.
The most extensively used polyesters for biomedical purposes are polylactides (PLA) and polyglycolides (PGA); in 1970, they were approved by the United States Food and Drug Administration (USFDA) for medical use in the USA. The second most commonly used and comprehensively studied polymers are hydroxy derivatives of alkanoic acids, polyhydroxyalkanoates (PHAs). PHAs are biodegradable, biocompatible, and thermoplastic polymers; PHA-based products are mechanically strong. Poly(3-hydroxybutyrate [P(3HB)] – the most comprehensively studied and commonly used PHA – is not soluble in aqueous media, and, thus, it is biodegraded at a slower rate, producing butyric acid, which does not cause dramatic acidification of the tissues [1–4]. PHAs are promising for constructing biomedical devices, including nonwoven and disposable products, sutures, and wound dressings, controlled drug delivery systems, scaffolds for cellular and tissue engineering, and elements for surgical reconstruction and transplantation [4–9].
Processing properties of P(3HB) can be improved by employing a number of approaches. These are construction of recombinant strains capable of producing PHA copolymers, which incorporate combination of the key genes responsible for the synthesis of monomers of PHA cellular cycle from various microorganisms; creation of special conditions of cultivation and carbon nutrition for wild-type strains to produce polymers with various compositions and improved processing properties – a decreased degree of crystallinity and increased ductility; and construction of composite materials and blends of P(3HB), and other natural and synthetic materials.
A new approach to the modification of polymer products is to treat them by physical methods or chemical reagents in order to enhance adhesive properties of the surface and facilitate attachment of the cultured cells, to improve gas-dynamic properties of the products, and to increase their permeability for substrates and metabolic products of cells and tissues. Laser treatment is a relatively new approach in the modification of polymer products. Its main advantage over other treatments is that it modifies the surface selectively, without destroying the material or producing toxic substances.
Electrospinning (electrostatic spinning) is a promising technique that can be used for fabricating micro and ultrafine fibers and fibrous scaffolds (mats) and membranes. This technique was introduced in the 20th century to fabricate synthetic fibers. Electrospinning studies using PHAs have not been conducted until quite recently.
The purpose of this study were: to produce electrospun ultrafine fibers differing in their physicochemical properties using PHAs with dissimilar chemical structures and to investigate the influence of electrospinning parameters and PHA chemical composition on the morphology of ultrafine fibers and physical-mechanical and biological properties of fibrous scaffolds; to investigate the effects of different types of laser processing on the structure and physical, mechanical, and biological properties of P(3HB) films.
PHA samples were extracted from bacterial biomass with chloroform and precipitated in hexane. The optimized extraction procedure enabled the production of medically pure specimens that contained no organic impurities (proteins, carbohydrates or lipids, including fatty acids) and were suitable for use in contact with blood .
Ultrafine fibers were prepared by electrospinning from high-purity PHA specimens with different chemical structure containing different monomer fractions (3-hydroxybutyric acid, 3-hydroxybutyric and 3-hydroxyvaleric, 3-hydroxybutyric and 3-hydrohexanoic, 3-hydroxybutyric and 4-hydroxybutyric acids).
Ultrafine fibers were electrospun from PHA solutions using a Nanon 01A automatic setup (MECC Inc., Japan). Chloroform solutions with polymer concentration varied from 1 to 10 wt.% were prepared from all types of PHAs. The polymer solution was poured into a plastic syringe (13 mm inside diameter). The syringe was fixed horizontally in the setup, the solution feeding rate varied from 4 to 8 ml/h, the applied voltage from 15 to 30 kV, and the working distance from 11 to 15 cm. Randomly oriented or aligned ultrafine fibers were collected on a flat steel plate or a rotating drum (at 1000 rpm), respectively; both collectors were covered with aluminum foil to collect ultrafine fibers more effectively.
Laser treatment of the surface of flexible transparent polymer films was performed by moderate uniform irradiation of the surface, using CO2 lasers. In the first series of experiments, polymer films were treated using a LaserPro Explorer II system (Coherent, U.S.), with power varying between 1.5 and 16.5 W and the speed between 0.8 and 2 m/s. Under these conditions, the film surface was uniformly irradiated, and no considerable damage or perforation occurred. The treatment was performed in the focused and defocused modes. In the second series of experiments, films were treated using a LaserPro Spirit system (Sunrad, U.S.) at its highest power, 25 W.
The microstructures of ultrafine fibers and laser-treated films were analyzed using scanning electron microscopy (S-5500 (Hitachi, Japan), and TM 3000 (Hitachi, Japan). Prior to microscopy, the samples were sputter-coated with platinum (at 10 mA, for 40 s), with an Emitech K575X sputter coater.
The effect of the density of polymer solutions on fiber properties was studied using the homopolymer of 3-hydroxybutyric acid, in order to avoid the influence of chemical composition of the PHA on the electrospinning process and properties of the products. P(3HB) chloroform solutions with polymer concentration varied from 1 to 10 wt.% were used. The process parameters were as follows: needle diameter – 1 mm, applied voltage – 30 kV, solution feeding rate – 5 ml/h, and working distance – 15 cm, a flat steel collection plate.
Stable electrospinning of ultrafine fibers in the Nanon 01A setup was attained from P(3HB) solutions with polymer concentrations from 2 to 8 wt.% (solution viscosity 200–800 cP). Polymer concentration significantly influenced the diameter of the ultrafine fibers, which varied from 0.45 to 3.14 μm. Within the study range of polymer concentration, the diameter of the ultrafine fibers is linearly related to the solution density. The viscosity of the solutions with polymer concentrations above 8 wt.% was too high (about 1000 cP) to allow successful formation of ultrafine fibers.
We prepared ultrafine fibers using PHAs with different chemical structures and studied the effect of polymer composition on the surface structure and physical-mechanical properties of the fibers. First, electrospun aligned fibrous scaffolds differed from randomly oriented ones in that they had much higher mechanical strength. Second, the effects of the second monomers of the copolymers used to prepare the fibers on the properties of the aligned fibrous mats were different from their effects on the properties of the randomly oriented fibrous mats. The aligned fibrous mats prepared from copolymers containing 3HV and 3HHx had similar values of tensile strength and Young’s modulus, and they were not significantly lower than those of P(3HB) fibers, but their elasticity values differed by a factor of two. In P(3HB-co-10 mol.%-4HB) fibers, both parameters characterizing mechanical strength were lower than in P(3HB) ones, but this difference was not as significant as in randomly oriented fibers, while elasticity was more than four times higher.
The most important difference between randomly oriented and aligned copolymer fibrous scaffolds was that in the latter, increased molar fractions of the second monomers had pronounced effect on the properties of the scaffolds.
Biological properties of fibrous scaffolds were studied in the culture of NIH 3T3 fibroblast cells. Investigation of cell attachment and proliferation on scaffolds prepared from solutions of the P(3HB) homopolymer of different densities (2, 4, 6, and 8 wt.%) did not reveal any significant differences at p ≤ 0.05, showing that all fibrous mats were suitable for use as cell culture scaffolds. At 24 h after seeding, the number of cells on scaffolds was 1.52–1.95 × 103 cells/cm2 irrespective of fiber diameter; at Day 3, the number of cells reached 6.55–8.02 × 103 cells/cm2. No statistically significant differences were found in the counts of proliferating and viable cells between scaffolds of different types. Hence, the diameter of electrospun P(3HB) fibers did not influence the attachment and growth of NIH 3T3 mouse fibroblast cells.
Thus, MTT assay showed that all PHA fibrous scaffolds facilitated fibroblast cell growth better than the reference scaffolds. Thus, results of MTT assay suggest that all types of fibrous scaffolds facilitate proliferation of fibroblast cells more effectively than the reference scaffolds and that randomly oriented scaffolds are more advantageous for growth and development of this kind of cells than aligned ones.
Results of processing of films using a LaserPro Explorer II system, with the power varied between 1.5 and 12.0 W, corresponding to power density (Q) 0.6 × 104 and 5.3 × 104 W/cm2, and the speed between 0.8 and 2 m/s are given in Table 1. These treatments did neither cause any considerable damage to films nor generated perforations. The use of a defocused laser beam, with lower radiative flux density, decreased surface deformation.
Measurements of water contact angles on the film surface showed a decrease in this parameter on laser-treated films. In the focused mode, the decrease was more pronounced, and the angle was reduced to 67.4°, while in the defocused mode, it decreased to 79.4°, at the processing speed of 0.8 m/s and 1.8 m/s and power 9 W (or 4 × 104 W/cm2) and 12 W (0.12 × 104 W/cm2), respectively. The use of the majority of irradiation modes caused a slight (10–16%) increase in the surface free energy of the film surfaces and a considerable increase (by a factor of 3–5) in its polar component, especially at higher power values. This may suggest that under high-energy impact, new polar functional groups may be generated on the surface and increase the water affinity of the polymer surface.
Changes in surface morphology influence the adhesive properties of the surface and the number of the viable cells attached to it. Fluorescent and electron microscopy of the films with NIH 3T3 mouse fibroblast cells attached to the film surface showed a great number of adherent viable cells. The most highly populated scaffolds were the ones that had been treated at the power and speed of 1.5 W and 1.8 m/s, 3 W and 0.8 m/s, and 9 W and 2 m/s, respectively, in the focused mode, and at 6 W and 2 m/s, respectively, in the defocused mode.
Comparative counts of physiologically active and viable cells cultivated on laser-treated films in MTT assay showed that at day 1, cell counts were similar on all films (about 1.2–1.46 × 105 cells/cm2). At day 4, the number of cells on laser-treated films was greater than in the control. The largest number of viable cells was observed on the films processed by laser radiation at 2 m/s (3.84–5.07 × 105 cells/cm2). The other scaffolds, including the untreated film (control), had similar amounts of adherent cells: 3.2–3.8 × 105 cells/cm2. At day 7, the populations of cells on scaffolds treated with medium intensity of energy, i.e. 1.5 W/1.8 m/s; 3 W/0.8 m/s; 9 W/2 m/s of focused irradiation and 6 W/2 m/s of defocused irradiation were similar to each other (8.1–8.8 × 105 cells/cm2). These films showed a moderate decrease in the water contact angles (hydrophilicity increase). The number of cells on the films treated with more intensive laser radiation (9 W/0.8 m/s of focused irradiation and 12 W/1.8 m/s of defocused irradiation) was lower – 6.45 and 7.1 × 105 cells/cm2. These results are consistent with the data obtained by microscopy of samples.
Thus, experiments showed that laser treatment influenced the surface structure and properties of the poly(3-hydroxybutyrate) films, increasing surface porosity, and, hence, improving cell adhesion and proliferation.
This study investigated the main parameters of electrospinning of fibers from solutions of PHAs with different compositions that influenced fiber diameter and properties; also polymer products were processed with laser radiation in different modes and tested to find their most promising biomedical uses. The most biocompatible films were produced in experiments with continuous laser treatment, in both focused and defocused modes. Thus, targeted laser modification of films from poly(3-hydroxybutyrate) improves their biomedically important properties. The study revealed electrospinning parameters for the production of high-quality fibers from different types of PHA and determined which parameters should be varied to tailor the properties of the products (fiber diameter, surface morphology, and physical-mechanical properties). None of the fibrous scaffolds produced from PHAs by electrospinning had any adverse effects on attachment, growth, and viability of NIH 3T3 mouse fibroblast cells, and all of them were found to be suitable for tissue engineering applications.
 Sudesh K, Abe H. Prog. Polym. Sci. 2000; 25:1503–1555.
 Hasirci V. Biodegradable biomedical polymers. In: Wase DL, editor. Biomaterials and bio- engineering handbook. New York (NY): Marcel Dekker; 2000. p. 141–155.
 Chanprateep S. J. Biosci. Bioeng. 2010;110:621–632.
 Steinbüchel A, Chen GQ. Berlin: Springer-Verlag; 2010. p. 1862–5576.
 Volova TG. Polyhydroxyalkanoates – plastic materials of the 21st century: production, properties, application. New York (NY): Nova Science; 2004.
 Sudesh K, Abe H. Shrewsbury: Smithes Rapra Technology; 2010. 151 p.
 Volova TG, Shishatskaya EI, Sinskey AJ. Degradable polymers: production, properties and applications. New York (NY): Nova Science; 2013. 380 p.
 Laycock B, Halley P, Pratt S, et al. Prog. Polym. Sci. 2013;38:536–583.
 Wu LP. Polyhydroxyalkanoates (PHA): biosynthesis, industrial production and applications in medicine. New York (NY): Nova Science; 2014.
 Sevastianov VI, Perova NV, Shishatskaya EI, et al. J. Biomater. Sci. Polym. 2003;14:1029–1042.