Institute of Fundamental Technological Research

Results of the preparation of biodegradable porous scaffolds using an original modification of a wet phase inversion method were presented. Influence of gelatin non‐woven as a non‐classic pore precursor and polyvinylpyrrolidone, Pluronic as classic pore precursors on the structure of obtained scaffolds was analyzed. It was shown that the addition of gelatin non‐wovens enables the preparation of scaffolds, which allow for the growth of cells (size, distribution, and shape of pores). Mechanical properties of the obtained cell scaffolds were determined. The influence of pore precursors on mass absorption of scaffolds against isopropanol and plasma was investigated. Interaction of scaffolds with a T‐lymphocyte line (Jurkat) and with fibroblasts (L929) was investigated. Obtained scaffolds are not cytotoxic and can be used as implants, for example, the regeneration of cartilage tissue.

cell cultures, cytotoxic, fibroblasts, imprinted scaffolds

The main goal of tissue engineering (TE) is supporting the regeneration of damaged tissues that are difficult to regenerate. The experimental results of the preparation of semi-permeable membranes for cell cultures are presented. The effect of the PLA molecular weight and addition of pore precursors on the morphology of the membranes was studied. The pore precursor of choice was polyvinylpyrrolidone (PVP). It was found that semi-permeable membranes for application in tissue engineering can be prepared with polylactides of molecular weight more significant than 37,000 g/mol. Moreover, it was observed that the growth of the molecular weight of the polymer, the porosity, the size of the pores, the Young modulus and maximum tensile increased. Additionally, to obtain a better morphology of the membranes, PVP should be added to the polymeric solution. Positive growth of L929 fibroblast cells on the obtained scaffolds was shown.

biodegradable polymers, cell cultures, L929 fibroblasts, polylactide, scaffolds, tissue engineering

An innovative modification of the wet inversion phase method, consisting in the use of a polymer nano‐nonwoven as a nonclassic pore precursor. Mechanical properties of the obtained scaffolds were determined, their hydrophilic properties (serum absorbability) were tested, and the content of residues of materials used in the scaffold preparation was determined. Nontoxicity of the developed scaffolds toward T lymphocyte cells was proved. Cultures of primary chondrocytes were obtained successfully. It was proved that an addition of a polymer nano‐nonwoven changes the properties of the scaffolds favorably in respect of their subsequent application in tissue engineering.

cartilage regeneration, chondrocytes, nano-nonwoven, polyvinylpyrrolidone, T lymphocytes

The results of studying the effect of the vibration processing time on the size of calcium hydroxide particles are given. The physicochemical processes affecting the size and morphology of calcium hydroxide particles have been studied. A stage-by-stage mechanism of the process of the carbonation of lime, depending on its specific surface, is established. The results show that the optimal period for the vibration treatment of lime to obtain the most active material is 20 min. A longer period of vibration results in the merging of particles into larger agglomerates.

lime, portlandite, vibration treatment, carbonation, crystallization

Polyester 3D scaffolds were obtained by dry inversion phase method. The influence of a polymer and liquid pore precursor type on the 3D scaffolds morphology, porosity and mechanical properties was tested. Polymers and precursors forming a porous structure were identified. It was found that 3D scaffolds having the most preferable structure for cell cultures were obtained from polylactide with the addition of n‐butanol as the liquid pore precursor.

liquid pore precursors, mechanical properties, dry inversion phase method, 3D scaffolds

Bicomponent polycaprolactone/gelatin and polycaprolactone/collagen fibres were formed by electrospinning using two kinds of solvents: a representative of commonly used solvents with this polymer composition, highly toxic hexafluoroisopropanol (HFIP) and alternative, less harmful one, the mixture of acetic (AA) and formic (FA) acids. Both material types were subjected to investigations of structure and in-vitro cellular activity. Viscosity and Fourier transform infrared spectroscopy (FTIR) measurements shown that the type of solvent used influences the structure of solution and conformation of polymer molecules. In-vitro quantitative tests as well as cell culture morphology observations proved that materials electrospun with the use of 'green' solvents can yield similar results to those obtained by made with toxic ones. Slightly better cellular response to materials electrospun from HFIP can be explained by relatively well dispersed components within the fibre and more expanded conformation of molecules, resulting in better exposition of RGD (Arg-Gly-Asp) binding sites to cells' integrin receptors.

Cellular tests, Electrospinning, Biopolymers, Viscosity, Solvents

Zbadano wpływ dodatku ciekłych prekursorów porów na morfologię, porowatość i właściwości mechaniczne polilaktydowych rusztowań komórkowych. Rusztowania otrzymano metodą mokrej inwersji faz w wariancie freeze extraction. Oceniono cytotoksyczność wybranych rusztowań w stosunku do fibroblastów mysich oraz ich przydatność do hodowli komórkowych. Wykazano, że dodatek prekursora porów dopolilaktydu korzystnie zmienia morfologię wytworzonych rusztowań, jednocześnie pogarszając ich wytrzymałość mechaniczną. Stwierdzono, że polilaktydowe rusztowania komórkowe z powodzeniem mogą być wykorzystywane do hodowli komórkowych.

usztowania komórkowe, polilaktyd, hodowle komórkowe, fibroblasty

Bicomponent polycaprolactone/gelatin and polycaprolactone/collagen nanofibers formed by electrospinning using various solvents were subjected to biodegradation and compared. Hexafluoroisopropanol (HFIP) was used as a reference solvent, while the second, alternative solvent system was the mixture of acetic acid (AA) with formic acid (FA). Biodegradation of investigated materials was manifested mainly by the gelatin leaching, including collagen which is indeed denaturated to gelatin during electrospinning, leading to nanofibers erosion. There was no molecular degradation of PCL during 90 days of biodegradation procedure as deduced from no change in the elongation stress at break. The rate of biopolymer leaching was very fast from all materials during the first 24 h of biodegradation, being related to surface leaching, followed by a slower rate leaching from deeper material layers. Mass measurements showed much faster biopolymer leaching from nanofibers electrospun from AA/FA than from HFIP because of strongly emulsive nature of the solution in the former case. Irrespective of the solvent used, the leaching rate increased with initial content of gelatin. The analysis of Young modulus during biodegradation indicated complex mechanism of changes, including biopolymer mass loss, increase of PCL crystallinity and partial gelatin renaturation.

Bicomponent nanofibers, Biodegradation, Biopolymer

This study investigated the influence of the particle size on the adhesion force between ceramic particles and metal matrix in ceramic-reinforced metal matrix composites. The Cu-Al2O3 composites with 5 vol.% of ceramic phase were prepared by a powder metallurgy process. Alumina oxide powder as an electrocorundum (Al2O3) powder with different particle sizes, i.e., fine powder <3 µm and coarse powder of 180 µm was used as a reinforcement. Microstructural investigations included analyses using scanning electron microscopy with an integrated EDS microanalysis system and transmission microscopy. In order to measure the adhesion force (interface strength), we prepared the microwires made of the investigated materials and carried out the experiments with the use of the self-made tensile tester. We have observed that the interface strength is higher for the sample with coarse particles and is equal to 74 ± 4 MPa and it is equal to 68 ± 3 MPa for the sample with fine ceramic particles.

adhesion, interface strength, metal matrix composites, nanocomposites, tensile test

Bicomponent polycaprolactone/gelatin (PCL/Gt) nanofibers were successfully formed for the first time by electrospinning using a novel polymer–solvent system with solvents being alternative to the commonly used toxic solvents like fluorinated alcohols. The mixture of acetic acid (AA) with formic acid (FA; 90:10) was applied. Stable electrospinning was possible despite the fact the mixture of PCL and gelatin in AA/FA solvent showed emulsive structure. From the practical perspective, there is no doubt that it is possible to obtain PCL/Gt fibers using AA/FA mixture with morphology similar to that for fibers spun from hexafluoroisopropanol (HFIP) solutions.

Alternative solvents, electrospinning, gelatin, nanofibers, polycaprolactone, structure

The addition of natural fibers residue in cement based materials can be a sustainable technological alternative for traditional dispersed reinforcement, and can improve the performance of brittle matrix materials. The presence of a wool reinforcement can increase the fracture toughness and, at the same time, can reduce the environmental impact of cementitious mortars. The beneficial effects are similarly to those observed in presence of vegetal fibers (e.g., hemp), which have been largely investigated in the literature. However, there are some limits in the use of wool fibers due to their chemical compatibility with the cement matrix, as they can dissolve in alkaline environments. In the present paper, to investigate the compatibility between wool fibers and cementitious mortars, laboratory prototypes have been taken into consideration. Three series of wool-reinforced mortar beams have been cast and cured in water (20°C) or in dry conditions (temp. 20 °C, 50% R.H.) for some days. Portland-limestone cement CEM II has been used, whereas the content of fibers has been limited to about 1% in volume to maintain the workability of the mortars. To investigate the chemical compatibility, and the subsequent effects on the mechanical performances, prototypes have been tested in three point bending. After the mechanical test, the mortars microstructure was evaluated through SEM images and by thin section in transmitted light, in order to individuate a possible relationship between the dissolution of wool and curing conditions. The microstructure observation revealed the capability of wool fibers to bridge the cracks, and to reduce the brittleness of plain mortars. The differences in the mortars microstructure due to alternative curing conditions were also observed and described in the paper. Accordingly, wool could be effectively used to reduce the plastic shrinkage of cementbased composites, like the industrially manufactured polypropylene fibers.

Wool fibers, Plain cement-based mortar, Fiber-reinforced mortar, Polypropylene fibers, Three point bending tests, SEM analyses

Three types of membrane structures with wide pores were compared in this study. One of the membranes was obtained from polyethersulfone using cellulose fibers as the macropore precursors. Two of the fibers were obtained from poly(L-lactide). As the macropore precursors olyvinylpyrrolidone (1.2 MDa) and pork gelatin non-woven were used, the influence of non-woven fibers on the structure of membranes was shown. Necessity of specific membrane structure application was explained. The hoice of polymers and co-polymers with a range of biodegradation times can determine the scaffold type suitable for the age of a patient.

Polysulfone membrane, Polyester membranes, Membrane structures, Biodegradable membranes, 3D scaffold

Results of the preparation of semi-permeable membranes made of biodegradable polymers membranes were presented. Among known polyesters, polylactide was selected for research. The membranes were obtained using wet phase inversion method. The influence of polyvinylpyrrolidone and polymeric nano-non-wovens as pores precursors on the structure of obtained membranes was analysed. It was shown, that utilisation of polymeric nano-non-wovens enabled preparation of semi-permeable membranes, which could be used as wide-pore 3D-type cellular scaffolds.

Biodegradable polymers membranes, Biodegradable polyesters, Porous three-dimensional scaffolds, Inversion phase method

Electrospun nonwovens from PCL/gelatin and PCL/collagen structurally mimic native extracellular matrix and provide cells with chemical cues affecting them. Electrospinning of bicomponent nanofibres requires the use of a solvent which dissolves both of the polymers.
We have optimized the process of electrospinning of PCL/gelatin and PCL/collagen nanofibers based on the use of non-toxic, alternative solvents: acetic acid and formic acid (AA/FA) as previously described [1].
Bicomponent PCL/gelatin and PCL/collagen nanofibers were formed by electrospinning using the mixture of acetic acid and formic acid (9:1 w/w ratio), while hexafluoroisopropanol (HFIP) was used as a reference solvent. Nonwoven materials were subjected to cellular in vitro and biodegradation tests and compared.
All in vitro tests were performed using L929 mouse fibroblast cells. Cytotoxicity test was carried out on extracts and showed that all type of materials are not cytotoxic. Materials with 10% biopolymer content as well as made from PCL only underwent experiment in direct contact. Cells were cultured on materials for 3, 5 and 7 days and afterwards taken for SEM as well as fluorescent dying of nuclei and cytoskeleton. Obtained results proved that the addition of Arg-Gly-Asp (RGD) amino acid sequences from biopolymer, in comparison to pure PCL materials, facilitates cell adhesion and spreading on the surface of nonwovens regardless of solvent used in electrospinning.
PCL/gelatin and PCL/collagen nonwovens underwent biodegradation in PBS solution at 37°C. After different times, ranging from 1 to 90 days samples were subjected for comparative analysis via various methods.
Despite the fact that bicomponent nanofibers electrospun from alternative solvents have similar morphology to those electrospun from perfluorinated alcohols, they differ in the internal structure which seriously affects biodegradation process. Biodegradation of investigated materials is manifested mainly by the gelatin leaching, which leads to nanofibers erosion, particularly large for nanofibers spun from AA/FA.

electrospinning, bicomponent nanofibers, biodegradation, cellular studies

Bicomponent polycaprolactone/gelatin nanofibers were formed by electrospinning as previously described [1] using a novel polymer – solvent system with solvents being alternative to the commonly used toxic solvents like fluorinated alcohols. PCL/Gelatin nanofibres were electrospun from the mixture of acetic acid (AA) with formic acid (FA) (90:10) and from hexafluoroisopropanol (HFIP), that was used as reference solvent. PCL/Gelatin nanofibres with polymers w/w ratios 9:1, 8:2 and 7:3, underwent biodegradation in PBS solution at 37°C. After different times, ranging from 1 to 90 days, they were rinsed in demineralized water and dried. Weight loss and FTIR tests were performed to assess the kinetics of gelatin leaching, while SEM imaging and hydrophobicity tests to show its depletion from the surface. DSC measurements were carried out to examine changes in fibres’ internal structure and uniaxial tensile testing to compare their mechanical properties. Morphology of PCL/Gt fibers obtained from AA/FA is similar to that obtained from HFIP. Despite similar morphology, the internal structure of nanofibers formed from alternative solvents is different, reflecting the emulsive nature of PCL/gelati n mixture in AA/FA solvents contrary to clear, transparent solutions in HFIP. This apparent difference affects strongly the kinetics of leaching of gelatin from bicomponent fibres and thus how their mechanical and bioactive properties are changing in time after placing in living organism. There is substantial difference in kinetics of gelatin leaching depending on solvent used. Mass measurements show much faster gelatin degradation in nanofibres electrospun from AA/FA than from HFIP. For instance, for PCL/Gt 7:3 samples, gelatin content loss is 85% for AA/FA and 68% for HFIP after 90 days. Moreover, irrespective of the solvent used, the degradation rate increases with initial content of gelatin and is the highest in the first 24 hours: 27% for AA/FA 9:1 and 67% for 7:3 and 13% and 32% for HFIP respectively. The observed changes can be explained by nonuniform distribution of gelatin within fibres spun form AA/FA due to emulsive character of solution. Comparison of SEM images reveals linear groove-like sites remaining after gelatin leaching on a surface of fibres spun from AA/FA solvent. Contrary to this, fibres spun from HFIP remain smooth which can be attributed to molecular dispersion of both components.

nanofibers, biodegradation, polycaprolactone, gelatin

Few research groups have highlighted the unexpected degree of cell proliferation depending on the degree of crystallinity of the substrate. Commonly used methods of forming three-dimensional scaffolds do not take into account crystallinity optimisation.
The aim of proposed presentation is to investigate polycaprolactone (PCL) substrate supermolecular structure effect, mainly crystallinity, on cells spreading, activity and proliferation.

crystalllinity, PCL, solvent, molecular structure, L929

In this study bicomponent polycaprolactone/gelatin nanofibers were successfully formed by electrospinning using for the first time a novel polymer – solvent system consisting of acetic acid and formic acid. Such solvent system is alternative to the commonly used toxic solvents like fluorinated alcohols, mainly hexafluoroisopropanol. The effect of electrospinning conditions on morphology and structure of nanofibers were investigated.

nanofibers, electrospinning, polycaprolactone, gelatin, alternative solvents, structure

Bicomponent poly(caprolactone)/ chitosan (PCL/Chit) nanofibers are a promising alternative for cartilage tissue regeneration. Chitosan is characterized by high structural similarity to the glycosaminoglycans (GAG) which naturally occur in the extracellular matrix (ECM). Its hydrophilicity is beneficial for cells adhesion and proliferation [1]. The amino groups in chitosan are responsible for the formation of polycations, which subsequently form compounds with natural and synthetic anions (proteins, lipids, synthetic polymers which are negatively charged) [2, 3]. Electrospinning of polycations with positive charge on the needle, is difficult due to the instability of the stream resulting from large repulsion force in the polymer jet [3]. Introduction of synthetic polymer molecules to the solution decreases interactions between the chains of chitosan and reduces the viscosity of the solution, so they are easier to form by electrospinning, as well as with negative charge on the needle [4 ]. A synthetic polymer, which is poly(caprolactone), improves mechanical properties of the fibers and the time of the hydrolytic degradation of the scaffold [4]. Nanofibers are excellent material for cell scaffolds used in tissue engineering because of high similarity of their morphology to native extracellular matrix (ECM) [1, 2]. From the viewpoint of cartilage tissue regeneration scaffold in the form of nanofibers is particularly justified due to naturally occurring network of polymer fibers (proteins and glycosaminoglycans) called aggrecans, in ECM of cartilage. Chondrocytes are connected with aggrecans [4]. Both, the structure and composition of formed nanofibers may affect the time in which cells will reach their proper morphology and undertake its functions [4]. In order to study cell behavior on electrospun PCL/chitosan nonwoven, fibroblasts L929 were cultured. Actin Green staining was conducted in order to imagine actin cytoskeleton of fibroblasts. To characterize, both fibers structure and cell morphology, SEM imagining was done. AFM imaging was carried to describe fibers topography and phase distribution. Also conductivity and viscosity of the PCL/chitosan solution with various polymer ratio was measured.

electrospinninig, scaffolds, PCL, chitozan, cellular responce