Combined therapy (Rho-A-kinase inhibitor and chitosan/collagen porous scaffold) provides a supportive environment for endogenous regenerative processes after spinal cord trauma.

Due to the complexity of pathological processes in spinal cord injury (SCI), it is increasingly recognized that combined strategies are more effective than single treatments. The aim of the present study was to enhance neural tissue regeneration and axon regrowth using Rho-A-kinase inhibitor (Y-27632) in a rat SCI model (Th9 compression) and to bridge the lesion with a chitosan/collagen porous scaffold (ChC-PS) applied two weeks after SCI. In addition, to see the synergic effect of Y-27632 and ChC-PS, we combined these single therapeutic strategies to enhance the regenerative capacity of injured spinal cord tissue. The animals survived eight weeks. Application of Y-27632 modulated the inhibitory milieu by specifically targeting gray and white matter integrity, glial fibrillary acidic protein (GFAP)-immunoreactivity, and the outgrowth of neurofilaments and growth-associated protein-43 (GAP-43) immunoreactive axons across the lesion sites, leading to significant positive functional outcome from day 20 to 56. Compared to single treatments, combined Y-27632/ChC-PS therapy was more effective in neurofilaments and GAP-43 expression and GFAP immunoreactivity in the perilesional area of dorsal, lateral and ventral columns, and in enhancing the gray and white matter integrity throughout the cranio-caudal extent. The findings indicate that combined therapy provides a supportive environment for endogenous regenerative processes.

Microstructure and properties of polyhydroxybutyrate-chitosan-nanohydroxyapatite composite scaffolds.

Polyhydroxybutyrate-chitosan-hydroxyapatite (PHB-CHT-HAP) composite scaffolds were prepared by the precipitation of biopolymer-nanohydroxyapatite suspensions and following lyophilisation. The propylene carbonate and acetic acid were used as the polyhydroxybutyrate and chitosan solvents, respectively. The high porous microstructure was observed in composites and the macroporosity of scaffolds (pore sizes up to 100 µm) rose with the chitosan content. It was found the reduction in both the PHB melting (70°C) and thermal degradation temperatures of polyhydroxybutyrate and chitosan biopolymers in composites, which confirms the mutual ineraction between polymers and the decrease of PHB lamellar thickness. No preferential preconcentration of individual biopolymers was verified in composites, and the compressive strengths of macroporous PHB-CHT-HAP scaffolds were approximately 2.5 MPa. The high toxic fluorinated cosolvents were avoided from the preparation process.

Physical, mechanical and in vitro evaluation of a novel cement based on akermantite and dicalcium phosphate dihydrate phase.

Magnesium containing calcium silicates have recently shown that they are promising materials for various biomedical application with potential use in the form of bulk ceramic, composite scaffold or coatings on metallic substrates. A novel akermanite (AK; Ca2MgSi2O7)/dicalcium phosphate dihydrate (DCPD, CaHPO4. H2O) cement mixture was tested in this work in order to produce an alternative AK/DCPD biocement for orthopedic applications. For comparison, we have prepared two cements mixed with 2.5 wt% NaH2PO4 solution (labeled as NaH2PO4 cement) and with the solution composed of organic 2.5 wt% citric acid a 2.5 wt% trisodium citrate (citrate cement) respectively. The results demonstrated only a partial dissolution of AK, regardless of the type of liquid used. On the other hand, the DCPD was completely hydrolyzed much faster in the citrate cement. The final hydration product was an amorhous quarternary phase of CaO-MgO-SiO2-P2O5 composition with the remaining unreacted akermanite embeded in the cement matrix. The highest early compressive strength was observed in the citrate cement (33 MPa), but much lower value was measured in NaH2PO4 cement (7 MPa) after 1 d setting. Different cell responses have been observed when the cells were cultured on the surfaces of cement substrates. While the NaH2PO4 cement demonstrated high proliferation activity of osteoblast, the citrate cement showed strong cytotoxic cell response, probably as a result of higher concentration of citrates on the cement surface, which can negatively affect the attachment and proliferation of osteoblastic cells.

Effect of tetracalcium phosphate/monetite toothpaste on dentin remineralization and tubule occlusion in vitro.

OBJECTIVES: To investigate the tubule occlusion and remineralization potential of a novel toothpaste with active tetracalcium phosphate/monetite mixtures under de/remineralization cycling. METHODS: Dentin de/remineralization cycling protocol consisted of demineralization in 1% citric acid at pH 4.6 with following remineralization with toothpastes and soaking in artificial saliva. Effectiveness of toothpastes to promote remineralization was evaluated by measurement of microhardness recovery, analysis of surface roughness, thickness of coating and scanning electron microscopy. RESULTS: The novel tetracalcium phosphate/monetite dentifrice had comparable remineralization potential as commercial calcium silicate/phosphate (SENSODYNE®) and magnesium aluminum silicate (Colgate®) toothpastes and significantly higher than control saliva (p<0.02). Surface roughness was significantly lower after treatment with prepared and SENSODYNE® dentifirice (p<0.05). The coatings on dentin surfaces was significantly thicker after applying toothpastes as compared to negative control (p<0.001). CONCLUSIONS: The new fluoride toothpaste formulation with bioactive tetracalcium phosphate/monetite calcium phosphate mixture effectively occluded dentin tubules and showed good dentin remineralization potential under de/remineralization cycling. It could replace professional powder preparation based on this mixture. It was demonstrated that prepared dentifrice had comparable properties with commercial fluoride calcium silicate/phosphate or magnesium aluminum silicate dentifrices.

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.

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 ß-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.

A study of cytocompatibility and degradation of iron-based biodegradable materials.

Biodegradable metallic implants are of significant importance in the replacement of bones or the repair of bone defects. Iron-phosphate-coated carbonyl iron powder (Fe/P) was prepared by the phosphating method. Moreover, Fe/P-Mn alloy was produced by sintering the Fe/P powder mixed with manganese powder. Bare carbonyl iron samples and the Fe/P and Fe/P-Mn sintered samples were evaluated for their microstructure, cytotoxicity, and hemocompatibility. The microstructure of the sintered samples was examined using an optical microscope and scanning electron microscopic analysis. Corrosion behavior was evaluated by potentiodynamic polarization in Hank's solution. The in vitro biocompatibilities were investigated by cytotoxicity and hemolysis tests. The results obtained demonstrate that the addition of Mn resulted in higher surface inhomogeneity, porosity and roughness as well as in increased cytotoxicity. The phosphate coating has a moderately negative effect on the cytotoxicity. The corrosion rates determined from Tafel diagrams were ordered in the following sequence: Fe/P-Mn, Fe, Fe/P from high to low. The hemocompatibility of experimental samples was ordered in the following sequence: Fe/P, Fe/P-Mn, Fe from high to low. All samples were found to be hemocompatible.

Carbonate release from carbonated hydroxyapatite in the wide temperature rage.

Synthetic carbonated apatite ceramics are considered as promising alternative to auto- and allograft materials for bone substitute. The aim of this study was to investigate the thermal stability of an AB-type carbonated apatite in the wide temperature range. The data on the thermal stability have to allow the conditions of the sintering of the ceramics to be controlled. Initial carbonated apatite powders were prepared by interaction between calcium oxide and ammonium hydrogen phosphate with addition of ammonium carbonate. Decomposition process was monitored by infra red spectroscopy, weight loss and X-ray diffraction of solid, and by infra red analysis of condensed gas phase resulted from the thermal decomposition of the sample in equilibrium conditions. Features of carbon monoxide and carbon dioxide release were revealed. The synthesized AB-type carbonated apatite is started to decompose at about 400 degrees Celsius releasing mainly carbon dioxide, but retained some carbonate groups and apatite structure at the temperature 1100 degrees Celsius useful to prepare porous carbonate-apatite ceramics intended for bone tissue engineering scaffolds.