Porous poly(alpha-hydroxyacid)/Bioglass composite scaffolds for bone tissue engineering. I: Preparation and in vitro characterisation.

Highly porous composites scaffolds of poly-D,L-lactide (PDLLA) and poly(lactide-co-glycolide) (PLGA) containing different amounts (10, 25 and 50 wt%) of bioactive glass (45S5 Bioglass)were prepared by thermally induced solid-liquid phase separation (TIPS) and subsequent solvent sublimation. The addition of increasing amounts of Bioglass into the polymer foams decreased the pore volume. Conversely, the mechanical properties of the polymer materials were improved. The composites were incubated in phosphate buffer saline at 37 degrees C to study the in vitro degradation of the polymer by measurement of water absorption, weight loss as well as changes in the average molecular weight of the polymer and in the pH of the incubation medium as a function of the incubation time. The addition of Bioglass to polymer foams increased the water absorption and weight loss compared to neat polymer foams. However, the polymer molecular weight, determined by size exclusion chromatography, was found to decrease more rapidly and to a larger extent in absence of Bioglass. The presence of the bioactive filler was therefore found to delay the degradation rate of the polymer as compared to the neat polymer foams. Formation of hydroxyapatite on the surface of composites, as an indication of their bioactivity, was recorded by EDXA, X-ray diffractometry and confirmed by Raman spectroscopy.

Osteoblast attachment and mineralized nodule formation on rough and smooth 45S5 bioactive glass monoliths.

Human primary osteoblast responses to smooth and roughened bioactive glass of 45S5 (Bioglass trade mark ) composition (46.1% SiO(2), 26.9% CaO, 2.6% P(2)O(5), 24.4% Na(2)O) were analysed in vitro. The smooth and rough surfaces had R(a) values and peak to valley distances of 0.04, 4.397, 2.027, and 21.328 microm, respectively. Cell attachment and morphology was observed using phalloidin staining of the actin cytoskeleton and revealed significant differences between smooth and rough surfaces. Cells that were spiky in appearance on the rough compared to the smooth surface formed an organized actin matrix much later on the rough surface. Scanning electron microscopy revealed many cell filipodia extending from more rounded cell bodies on the rough surface. A significantly greater number of nodules on the rough surface was observed, and these were shown to mineralize when supplemented with beta-glycerophosphate and dexamethasone. Raman spectroscopy confirmed the presence of hydroxyapatite in the mineralized cultures showing a definite peak at 964 cm(-1). FTIR analysis showed hydroxyapatite formation occurred more rapidly on the rough surface. This study demonstrates that although initial cell morphology was less advanced on the roughened surface, the cells were able to form mineralized nodules in greater numbers. This may have implications to bone tissue engineering using bioactive glasses.

Preparation, characterization, and in vitro degradation of bioresorbable and bioactive composites based on Bioglass-filled polylactide foams.

Highly porous poly(D,L-lactide)/Bioglass composites scaffolds were prepared by thermally induced phase separation process of polymer solutions and subsequent solvent sublimation. A series of composite foams with different polymer/Bioglass weight ratios was prepared to study the influence of Bioglass content on the foam characteristics such as porous structure, density, and pore volume. The pore volume was decreased from 9.5 to 5.7 cm(3)/g when the Bioglass content was increased up to 40 wt %, but the overall pore morphology was not affected very much by changing the polymer/glass composition ratio. The composites foams were then incubated in phosphate-buffered saline at 37 degrees C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation as an indication of their bioactivity. The addition of Bioglass to polymer foams increased the water absorption and weight loss as compared with pure polymer foams. However, the polymer molecular weight, determined by size exclusion chromatography, was found to decrease more rapidly and to a larger extent in absence of Bioglass. This delayed degradation rate in the composite foams was probably caused by the dissolution of alkaline ions from the Bioglass, resulting in a buffering effect of the incubation medium. After incubation for 7 days, HA was detected by X-ray diffractometry and Raman spectroscopy and confirmed by environmental scanning electron microscopy and energy-dispersive X-ray analysis. The porous composites developed here are promising materials for bone regeneration applications because the formation of HA on the surface of the pore walls should provide good environment for the adhesion and proliferation of osteoblasts and osteoprogenitor cells.

In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds.

Highly porous poly(DL-lactic acid) (PDLLA) foams and Bioglass-filled PDLLA composite foams were characterized and evaluated in vitro as bone tissue engineering scaffolds. The hypothesis was that the combination of PDLLA with Bioglass in a porous structure would result in a bioresorbable and bioactive composite, capable of supporting osteoblast adhesion, spreading and viability. Composite and unfilled foams were incubated in simulated body fluid (SBF) at 37 degrees C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation, which is a measure of the materials' in vitro bioactivity. HA was detected on all the composite samples after incubation in SBF for just 3 days. After 28 days immersion the foams filled with 40 wt % Bioglass developed a continuous layer of HA. The formation of HA for the 5 wt % Bioglass-filled foams was localized to the Bioglass particles. Cell culture studies using a commercially available (ECACC) human osteosarcoma cell line (MG-63) were conducted to assess the biocompatibility of the foams and cell attachment to the porous substrates. The osteoblast cell infiltration study showed that the cells were able to migrate through the porous network and colonize the deeper regions within the foam, indicating that the composition of the foams and the pore structures are able to support osteoblast attachment, spreading, and viability. Rapid formation of HA on the composites and the attachment of MG-63 cells within the porous network of the composite foams confirms the high in vitro bioactivity and biocompatibility of these materials and their potential to be used as scaffolds in bone tissue engineering and repair.

Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy.

A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 microM and 500 microM, and 100% for 1,000 microM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.

Bioactive glass-induced osteoblast differentiation: a noninvasive spectroscopic study.

Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect real-time biochemical changes in foetal osteoblasts (FOBs) following exposure to 45S5 Bioglass (BG)-conditioned media. Bio-Raman spectroscopy, combined with multivariate statistical analysis techniques (principal component analysis and least squares analysis), was able to noninvasively identify biochemical differences in FOBs cultured for different time periods and between FOBs exposed/or not to BG-conditioned media. Gene and protein expression studies were also performed for known markers of osteoblastic differentiation, namely, alkaline phosphatase, bone sialoprotein, and collagen type I. Quantitative RT-PCR confirmed upregulation of genes associated with osteoblast differentiation after exposure to BG-conditioned media. These results suggest that Raman spectroscopy can noninvasively detect biochemical changes in FOBs associated with differentiation. This technique could have important applications in the field of regenerative medicine by enabling rapid characterization of cell or organoid behavior on novel bioactive scaffolds without damage to either cell or biomaterial.

Fingering instabilities in dewetting nanofluids.

The growth of fingering patterns in dewetting nanofluids (colloidal solutions of thiol-passivated gold nanoparticles) has been followed in real time using contrast-enhanced video microscopy. The fingering instability on which we focus here arises from evaporatively driven nucleation and growth in a nanoscopically thin precursor solvent film behind the macroscopic contact line. We find that well-developed isotropic fingering structures only form for a narrow range of experimental parameters. Numerical simulations, based on a modification of the Monte Carlo approach introduced by Rabani et al. [Nature (London) 426, 271 (2003)10.1038/nature02087], reproduce the patterns we observe experimentally.

Bioresorbable and bioactive composite materials based on polylactide foams filled with and coated by Bioglass particles for tissue engineering applications.

Poly(DL-lactide) (PDLLA) foams and bioactive glass (Bioglass) particles were used to form bioresorbable and bioactive composite scaffolds for applications in bone tissue engineering. A thermally induced phase separation process was applied to prepare highly porous PDLLA foams filled with 10 wt % Bioglass particles. Stable and homogeneous layers of Bioglass particles on the surface of the PDLLA/Bioglass composite foams as well as infiltration of Bioglass particles throughout the porous network were achieved using a slurry-dipping technique. The quality of the bioactive glass coatings was reproducible in terms of thickness and microstructure. In vitro studies in simulated body fluid (SBF) were performed to study the formation of hydroxyapatite (HA) on the surface of the PDLLA/Bioglass composites, as an indication of the bioactivity of the materials. Formation of the HA layer after immersion in SBF was confirmed by X-ray diffraction and Raman spectroscopy measurements. The rate of HA formation in Bioglass-coated samples was higher than that observed in non-coated samples. SEM analysis showed that the HA layer thickness rapidly increased with increasing time in SBF in the Bioglass-coated samples. The high bioactivity of the developed composites suggests that the materials are attractive for use as bioactive, resorbable scaffolds in bone tissue engineering.

In situ monitoring of cell death using Raman microspectroscopy.

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells.

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.