Lubomir Medvecky

Properties and in vitro characterization of polyhydroxybutyrate–chitosan scaffolds prepared by modified precipitation method

Porous polyhydroxybutyrate (PHB)–chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends.

Phase transformations, microstructure formation and in vitro osteoblast response in calcium silicate/brushite cement composites

Self-setting simple calcium silicate/brushite (B) biocements with various Ca/P ratios were prepared by mutual mixing of both monocalcium silicate hydrate (CSH) or β-wollastonite (woll) powders with B and the addition of 2 wt% NaH2PO4 solution as a hardening liquid. The phase composition of the final composites and the texture of the surface calcium phosphate/silica layer were controlled by the starting Ca/P ratio in composites and the pH during setting. It was verified that the presence of continuous bone-like calcium phosphate coating on the surface of the samples was not essential for in vitro osteoblast proliferation. The nanocrystalline calcium deficient hydroxyapatite and amorphous silica were found as the main setting products in composite mixtures with a Ca/P ratio close to the region of the formation of deficient hydroxyapatite-like calcium phosphates. No CSH phase with a lower Ca/Si ratio was identified after transformation. The results confirmed a small effect of the monocalcium silicate addition on the compressive strength (CS) of cements up to 30 wt% (around 20-25 MPa) and a significant rise of the value in 50 woll/B cement (65 MPa). The final setting times of the cement composites varied between 5 and 43 min depending on the P/L ratio and the type of monocalcium silicate phase in the cement mixture. 10CSH/B and 50 woll/B cements with different textures but free of both the needle-like and perpendicularly-oriented hydroxyapatite particles on the surface of the samples had low cytotoxicity.

Brain Cortex Microglia Derived Exosomes: novel nanoparticles for glioma therapy

The function and integrity of the nervous system require interactive exchanges among neurons and glial cells. Exosomes and other extracellular vesicles (EVs) are emerging as a key mediator of intercellular communication, capable of transferring nucleic acids, proteins and lipids influencing numerous functional and pathological aspects of both donor and recipient cells. The immune response mediated by microglia-derived exosomes is most prominently involved in the spread of neuroinflammation, neurodegenerative disorders, and brain cancer. Therefore, in the present study we describe a reproducible and highly efficient method for yielding purified primary microglia cells, followed by exosome isolation and their characterization. An in vitro biological assay demonstrates that microglia-derived exosomes tested on a 3D spheroid glioma culture were able to inhibit tumor invasion in time course. These results evidence that brain microglia-derived exosomes could be used as nanotherapeutic agents against glioma cells.

Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response

Polyhydroxybutyrate/chitosan/calcium phosphate composites are interesting biomaterials for utilization in regenerative medicine and they may by applied in reconstruction of deeper subchondral defects. Insufficient informations were found in recent papers about the influence of lysozyme degradation of chitosan in calcium phosphate/chitosan based composites on in vitro cytotoxicity and proliferation activity of osteoblasts. The effect of enzymatic chitosan degradation on osteoblasts proliferation was studied on composite films in which the porosity of origin 3D scaffolds was eliminated and the surface texture was modified. The significantly enhanced proliferation activity with faster population growth of osteoblasts were found on enzymatically degraded biopolymer composite films with α-tricalcium phosphate and nanohydroxyapatite. No cytotoxicity of composite films prepared from lysozyme degraded scaffolds containing a large fraction of low molecular weight chitosans (LMWC), was revealed after 10 days of cultivation. Contrary to above in the higher cytotoxicity origin untreated nanohydroxyapatite films and porous composite scaffolds. The results showed that the synergistic effect of surface distribution, morphology of nanohydroxyapatite particles, microtopography and the presence of LMWC due to chitosan degradation in composite films were responsible for compensation of the cytotoxicity of nanohydroxyapatite composite films or porous composite scaffolds.

Novel approach to obtain composite poly-L-lactide based films blended with starch and calcium phosphates and their bioactive properties

A novel approach for preparation of composite materials based on the poly-L-lactide (PL), starch and calcium phosphates is provided, applying the polymer crazing process in liquid absorption active medium. For composite films preparation, PL and blends of PL and starch have been chosen as polymeric matrixes. Pores formation in the polymer films occurred during the process of uniaxial stretching in the presence of ethanol or water-ethanol mixtures via the solvent-crazing mechanism. The diameter of pores and fibrils in crazed PL was 20–30 nm. Porous polymer matrixes have been loaded with starch, using crazing mechanism, and filled with calcium phosphates (CP) by the countercurrent diffusion method, the content of starch and CP being up to 11.0 and 14.5 wt%, respectively. The obtained composites were investigated by XRD, SEM-EDS and TEM methods. It was found out that the starch filled the porous crazed structure of polymer, while the CP synthesis in the PL pores resulted in the formation of amorphous particles with the diameter of about 20 nm. These CP nanoparticles aggregated into the submicron particles of 100–300 nm in diameter. The bioactivity of the initial and porous poly-L-lactide films, and composites, based on PL with starch and calcium phosphates, was investigated. It was shown that the bioactivity depends on the chemical composition and surface morphology of the prepared materials. After 10 days of cultivation of the pre-osteoblastic MC3T3E1 cells, an intense, 6 times, growth of osteoblast population was achieved for the composite containing calcium phosphates. This result is perceptibly higher than those obtained for other samples (4 times growth) and for the control sample (5 times growth). Based on the results of in vitro tests, it can be concluded that the prepared materials are promising for biomedical applications.

Effect of microstructure characteristics on tetracalcium phosphate-nanomonetite cement in vitro cytotoxicity.

MC3T3E1 murine pre-osteoblastic cells were used to evaluate the cytotoxicity of tetracalcium phosphate (TTCP)-nanomonetite (DCPA) cement. The starting cement powder mixture was prepared by the in situ reaction between TTCP and a diluted solution of orthophosphoric acid in a planetary ball mill. The cements in the form of pressed cement powder mixture discs differ from each other by the method of pre-treatment and degree of the transformation of cement components in phosphate-buffered saline (PBS). For the evaluation of TTCP-DCPA cement to be non-cytotoxic, it was sufficient to apply the short-time soaking in PBS solution, regardless of whether the cement components were completely transformed or not. If the texture motif and hydroxyapatite particle morphology were properly developed during the initial stage of hardening, the cement cytotoxicity or osteoblast proliferation were insignificantly influenced by the soaking time or the texture stability during cell cultivation, but the lattice ordering enhanced cell proliferation. Results showed that the surface texture and the hydroxyapatite particle morphology are crucial for in vitro cement cytotoxicity evaluation.

Hydrolysis, setting properties and in vitro characterization of wollastonite/newberyite bone cement mixtures

Bone cements based on magnesium phosphates such as newberyite (N; MgHPO4.3H2O) have been shown as potential bone substitutes due to their biocompatibility, biodegradability and ability to support osteoblast differentiation and proliferation. Newberyite can hydrolyze to hydrated magnesium phosphate compounds (e.g. bobierite (Mg3(PO4)2.8H2O)) at alkaline conditions. In this study, 25 and 50 wt% of crystalline β -wollastonite (woll; CaSiO3) was admixed to newberyite powder in order to both enhance the acid-base hydrolysis of newberyite and to produce a functional bone cement. The setting process of wollastonite/newberyite cement mixtures started with the hydrolysis of the wollastonite with further transformation of newberyite into bobierite and the formation of magnesium silicate phase. The results demonstrated that 25 wollastonite/newberyite and 50 wollastonite/newberyite cement pastes at optimal powder/liquid ratios had final setting times of ∼34 and 25 min and compressive strength values of 18 and 32 MPa after seven days setting, respectively. The tests of cytotoxicity of cement extracts on osteoblastic cells and contact cytotoxicity of the cement substrates showed different results. The osteoblasts cultured in cement extracts readily proliferated which confirmed the non-cytotoxic concentration of ions released from both cements. On the other hand, a strong cytotoxic character of 25 wollastonite/newberyite sample surface in contrary to high (∼80%) proliferation activity of cells on the 50 wollastonite/newberyite cement substrate was observed. The differences in cell proliferation activity was attributed to different surface topographies of cement substrates, where needle-like precipitated microcrystals of magnesium phosphate phase (in 25 wollastonite/newberyite cement) prevented the adhesion and proliferation of osteoblasts contrary to the smoother surface covered by extremely fine nanoparticles in the 50 wollastonite/newberyite cement.

Properties of CaO–SiO2–P2O5 reinforced calcium phosphate cements and in vitro osteoblast response

Non-cytotoxic and bioactive tetracalcium phosphate/nanomonetite/calcium silicate-phosphate cements were prepared by simple mechanical mixing of starting powder precursors based on acid or basic tetracalcium phosphate/nanomonetite mixtures with 1 or 5 wt% addition of precititated amorphous or crystalline calcium silicate phosphate phases. The small additions (1-2 wt%) of crystalline CaSiP phase caused about a two-fold rise in the compressive strength of cements (up to 70 MPa) with simultaneous preservation of short setting time (around 5 min) and refinement of nanohydroxyapatite particles in microstructure. The results verified a close pH to body fluids and enhanced steady state concentrations of Ca2+, silicate and phosphate ions during the soaking of acid than the basic composite mixtures in physiological solution. No cytotoxicity or suppressing in proliferation activity of osteoblasts were revealed after the addition of CaSiP phases to cement powder mixtures. The ALP activity of osteoblasts during the first two days of culture on all composite systems was significantly higher than on pure tetracalcium phosphate/nanomonetite substrates. The superior enhancing in ALP osteoblast activity was found on cements with amorphous CaSiP glass component (even at low contents), which confirms excellent in vitro osteoblast activity on composites and their possible utilization as bone cements in reconstruction medicine.