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.

Enhanced differentiation and mineralization of human fetal osteoblasts on PDLLA containing Bioglass composite films in the absence of osteogenic supplements.

This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue culture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.

Gene therapy progress and prospects: in tissue engineering.

Tissue engineering (TE) has existed for several years as an area spanning many disciplines, including medicine and engineering. The use of stem cells as a biological basis for TE coupled with advances in materials science has opened up an entirely new chapter in medicine and holds the promise of major contributions to the repair, replacement and regeneration of damaged tissues and organs. In this article, we review the spectrum of stem cells and scaffolds being investigated for their potential applications in medicine.

Preparation of bioactive glass-polyvinyl alcohol hybrid foams by the sol-gel method.

A new class of materials based on inorganic and organic species combined at a nanoscale level has received large attention recently. In this work the idea of producing hybrid materials with controllable properties is applied to obtain foams to be used as scaffolds for tissue engineering. Hybrids were synthesized by reacting poly(vinyl alcohol) in acidic solution with tetraethylorthosilicate. The inorganic phase was also modified by incorporating a calcium compound. Hydrated calcium chloride was used as precursor. A surfactant was added and a foam was produced by vigorous agitation, which was cast just before the gel point. Hydrofluoric acid solution was added in order to catalyze the gelation. The foamed hybrids were aged at 40 degrees C and vacuum dried at 40 degrees C. The hybrid foams were analyzed by Scanning Electron Microscopy, Mercury Porosimetry, Nitrogen Adsorption, X-ray Diffraction and Infra-red Spectroscopy. The mechanical behavior was evaluated by compression tests. The foams obtained had a high porosity varying from 60 to 90% and the macropore diameter ranged from 30 to 500 microm. The modal macropore diameter varied with the inorganic phase composition and with the polymer content in the hybrid. The surface area and mesopore volume decreased as polymer concentration increased in the hybrids. The strain at fracture of the hybrid foams was substantially greater than pure gel-glass foams.

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.

Osteoblast responses to tape-cast and sintered bioactive glass ceramics.

The advantage of tape-cast bioactive glasses lies in the manufacturing procedure, which allows the build-up of layers and, therefore, the production of complex shapes. This, therefore, has applications to tissue engineering, where specific shapes are required such as repair of craniofacial defects. The bioactivity of tape-cast discs sintered at temperatures ranging from 800 degrees C to 1000 degrees C and for 3 or 6 h was analyzed by FTIR. Tape-cast discs were used to culture primary human osteoblasts, and cell attachment, cell death, collagen production, nodule formation, and mineralization were studied. These responses were dependent upon Si and Na release profiles of the tape-cast discs, and development of the hydroxyapatite layer.

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.

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.

In vitro release kinetics of proteins from bioactive foams.

This study describes an approach to obtaining 3-D scaffolds for tissue engineering that allows the incorporation and release of biologically active proteins to stimulate cell function. Laminin was adsorbed on the textured surfaces of binary 70S30C (70 mol % SiO(2), 30 mol % CaO) and ternary 58S (60 mol % SiO(2), 36 mol % CaO, 4 mol % P(2)O(5)) foams. The covalent bonds between the binding sites of the proteins and the ligands on the scaffolds' surfaces did not denaturate the proteins. In vitro studies show that the foams modified with chemical groups and coated with laminin were bioactive, as demonstrated by the formation of a crystalline hydroxy carbonate apatite (HCA) layer formed on the surfaces of the foams upon exposure to simulated body fluid (SBF). The release of proteins from the foams also was investigated. Sustained and controlled release from the scaffolds over a 30-day period was achieved. Laminin release from the bioactive foams followed the dissolution rate of the material network. These results suggest that bioactive foams have the potential to act as scaffolds for soft-tissue engineering with a controlled release of proteins that can induce tissue formation or regeneration.

In vitro bioactivity of S520 glass fibers and initial assessment of osteoblast attachment.

Bioactive glass fibers are attractive materials for use as tissue-engineering scaffolds and as the reinforcing phase for resorbable bioactive composites. The bioactivity of S520 glass fibers (52.0 mol % SiO(2), 20.9 Na(2)O, 7.1 K(2)O, 18.0 CaO, and 2.0 P(2)O(5)) was evaluated in two media, simulated body fluid (SBF) and Dulbecco's modified Eagle's medium (DMEM), for up to 20 days at 37 degrees C. Hydroxyapatite formation was observed on S520 fiber surfaces after 5 h in SBF. After a 20-day immersion, a continuous hydroxyapatite layer was present on the surface of samples immersed in SBF as well as on those samples immersed in DMEM [fiber surface area to solution volume ratio (SA:V) of 0.10 cm(2)/mL]. Backscattered electron imaging and EDS analysis revealed that the hydroxyapatite layer formation was more extensive for samples immersed in SBF. Decreasing the SA:V ratio to 0.05 cm(2)/mL decreased the time required to form a continuous hydroxyapatite surface layer. ICP was used to reveal Si, Ca, and P release profiles in DMEM after the 1st h (15.1, 83.8, and 29.7 ppm, respectively) were similar to those concentrations previously determined to stimulate gene expression in osteoblasts in vitro (16.5, 83.3, and 30.4 ppm, respectively). The tensile strength of the 20-microm diameter fibers was 925 +/- 424 MPa. Primary human osteoblast attachment to the fiber surface was studied by using SEM, and mineralization was studied by using alizarin red staining. Osteoblast dorsal ruffles, cell projections, and lamellipodia were observed, and by 7 days, cells had proliferated to form monolayer areas as shown by SEM. At 14 days, nodule formation was observed, and these nodules stained positive for alizarin red, demonstrating Ca deposition and, therefore mineralization.

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.

Development and in vitro characterisation of novel bioresorbable and bioactive composite materials based on polylactide foams and Bioglass for tissue engineering applications.

Bioactive and bioresorbable composite materials were fabricated using macroporous poly(DL-lactide) (PDLLA) foams coated with and impregnated by bioactive glass (Bioglass) particles. Stable and homogeneous Bioglass coatings on the surface of PDLLA foams as well as infiltration of Bioglass particles throughout the porous network were achieved using a slurry-dipping technique in conjunction with pre-treatment of the foams in ethanol. The quality of the bioactive glass coatings was reproducible in terms of thickness and microstructure. Additionally, electrophoretic deposition was investigated as an alternative method for the fabrication of PDLLA foam/Bioglass composite materials. In vitro studies in simulated body fluid (SBF) were performed to study the formation of hydroxyapatite (HA) on the surface of PDLLA/Bioglass composites. SEM analysis showed that the HA layer thickness rapidly increased with increasing time in SBF. The high bioactivity of the PDLLA foam/Bioglass composites indicates the potential of the materials for use as bioactive, resorbable scaffolds in bone tissue engineering.

In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses.

Effects of powder type, particle size (5-20 microm; 90-300 microm; 90-710 microm), and type of dissolution medium on the dissolution behavior of bioactive glasses were investigated in vitro using melt-derived 45S5 and sol-gel derived 58S bioactive glass powders. Dissolution studies were performed in simulated body fluid and in alpha-MEM based cell culture medium at 37 degrees C under dynamic conditions (1 Hz) for periods of 30 min, 1, 2, 4, 8, 17, and 22 h. The concentrations of elements dissolved from the glasses were evaluated using inductively coupled plasma analysis. The reacted powders were analyzed for bioactivity using Fourier transform infrared spectrometry to observe the formation of a calcium phosphate layer on the surface. The non-porous surfaces of melt-derived 45S5 glass powders exhibited lower dissolution rates and rate of surface layer formation than 58S gel-glass powders. The rates of dissolution for both types of powders were lower in culture medium, compared to simulated body fluid, and increased as the particle size decreased. Thus, particle size range, glass type, and powder volume fraction can be used as a means to control the release rate of active ions that stimulate the gene expression and cellular response for tissue proliferation and repair.

Evaluation of Bioglass/dextran composite as a bone graft substitute.

Allogenic and alloplastic bone graft substitutes serve either as bioinert or bioactive osteoconductors. Bioglass is a bioactive osteoconductor and also shows osteoproductive effects due to its high level of bioactivity. However, the material lacks some cohesiveness when used in augmenting certain bony surfaces, i.e. large or pleomorphic defects. The addition of medium molecular weight dextran modifies the particulate to a putty consistency and improves the handling characteristics. The objective of this study is to evaluate the influence of dextran upon the bioactive properties of Bioglass. Standardized bony defects in the lateral femoral condyles in adult New Zealand white rabbits were filled with one of five material groups: (1) autogenous bone; (2) Bioglass particulate; (3) Bioglass particulate mixed with dextran to a putty-like consistency; (4) a mixture of Bioglass and autogenous bone; (5) a mixture of Bioglass putty with autogenous bone. Postoperative healing was observed after periods of 2 days, 1, 2, 3, 6 and 12 weeks. Results showed no evidence of toxicity in the dextran-containing materials, and defects in all test groups showed 100% bony ingrowth within 6 weeks. The addition of medium molecular weight dextran did not appear to alter the bioactive properties of Bioglass and had no adverse influence upon the ingrowth of bone into the defect sites.

Interaction of bioactive glasses with peritoneal macrophages and monocytes in vitro.

Macrophage activation was analyzed following exposure to pure, crystalline alpha-quartz powders, two bioactive gel-glass powders of different compositions, and a melt-derived glass, 45S5 Bioglass. The release of reactive oxygen metabolites (chemiluminescence test), modifications of cell morphology, the amount of tumor necrosis factor alpha (TNFalpha) secreted, and the amount of TNFalpha mRNA expression were evaluated. The 45S5 Bioglass powders elicited the highest chemiluminescence response while the two solgel glasses had a lower response with less of an oxidative burst difference between them. Particulate bioactive glasses are actively ingested by mouse peritoneal macrophages, and only the 58S solgel glass had a moderate toxic effect on the macrophages. Macrophage cell morphology showed increased size and cell spreading, consistent with the high level of cytokine secretion induced by 45S5 Bioglass. The 45S5 Bioglass powders led to an increased release of TNFalpha and expression of TNFalpha mRNA relative to unstimulated and control treated monocytes. Bioactive glasses (and particularly 45S5 Bioglass) that in vivo induce rapid bone growth appear to activate an autocrine-like process in which the response evoked by the material (for example monocyte and macrophage activation with cytokine production) enhances subsequent interactions with cells in contact with the material.

Surface-modified 3D scaffolds for tissue engineering.

The aim of this work was to use sol-gel processing to develop bioactive materials to serve as scaffolds for tissue engineering that will allow the incorporation and release of proteins to stimulate cell function and tissue growth. We obtained organofunctionalized silica with large content of amine and mercaptan groups (up to 25%). The developed method can allow the incorporation and delivery of proteins at a controlled rate. We also produced bioactive foams with binary SiO(2)-CaO and ternary SiO(2)-CaO-P(2)O(5) compositions. In order to enhance peptide-material surface properties, the bioactive foams were modified with amine and mercaptan groups. These materials exhibit a highly interconnected macroporous network and high surface area. These textural features together with the incorporation of organic functionally groups may enable them to be used as scaffolds for the engineering of soft tissue.

Mechanical properties of biodegradable polymer sutures coated with bioactive glass.

Combining commercially available Polyglactin 910 (Vicryl) sutures with bioactive glass powder offers new possibilities for application of composite materials in tissue engineering. Commercial bioactive glass (45S5 Bioglass) powder was used to coat Vicryl sutures and the tensile strength of the sutures was tested before and after immersion in simulated body fluid (SBF) as a means to assess the effect of the bioactive glass coating on suture degradation. Different gauge lengths (126.6 and 111.6 mm) and strain rates (2.54, 11.4 and 25.4 mm/min) were tested. The tensile strength of composite sutures was slightly lower than that of as-received Vicryl sutures (404 MPa versus 463 MPa). However after 28 days immersion in SBF the residual tensile strength of the coated sutures was significantly higher, indicating a protective function of the Bioglass coating. The tensile strength results were similar for the different gauge lengths and strain rates investigated. A qualitative explanation for the effect of bioactive glass coating on polymer degradation is offered.

Novel bioresorbable and bioactive composites based on bioactive glass and polylactide foams for bone tissue engineering.

Bioresorbable and bioactive tissue engineering scaffolds based on bioactive glass (45S5 Bioglass(R)) particles and macroporous poly(DL-lactide) (PDLLA) foams were fabricated. A slurry dipping technique in conjunction with pretreatment in ethanol was used to achieve reproducible and well adhering bioactive glass coatings of uniform thickness on the internal and external surfaces of the foams. In vitro studies in simulated body fluid (SBF) demonstrated rapid hydroxyapatite (HA) formation on the surface of the composites, indicating their bioactivity. For comparison, composite foams containing Bioglass(R) particles as filler for the polymer matrix (in concentration of up to 40 wt %) were prepared by freeze-drying, enabling homogenous glass particle distribution in the polymer matrix. The formation of HA on the composite surfaces after immersion in phosphate buffer saline (PBS) was investigated to confirm the bioactivity of the composites. Human osteoblasts (HOBs) were seeded onto as-fabricated PDLLA foams and onto PDLLA foams coated with Bioglass(R) particles to determine early cell attachment and spreading. Cells were observed to attach and spread on all surfaces after the first 90 min in culture. The results of this study indicate that the fabricated composite materials have potential as scaffolds for guided bone regeneration.

Dose-dependent behavior of bioactive glass dissolution.

The effect of glass dosage (0.001 g ml(-1) to 0.015 g ml(-1)) on the in vitro dynamic dissolution behavior of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses, in simulated body fluid (SBF) at 37 degrees C, was evaluated. These glasses differ significantly in texture, especially the specific surface area and porosity, as a result of differences in manufacturing route. The concentrations of elements (Si, Ca, P, and Na) leached from the glasses into the dissolution medium, from 1 to 22 h, were evaluated with the use of induced coupled plasma analysis (ICP). The reacted powders were analyzed with the use of FTIR to observe the formation of a hydroxycarbonate apatite layer on the surface. The results show that the rate of HCA formation on both gel- and melt-derived bioactive glass powders in vitro depends on the concentration of the powders in solution. This result must be taken into account when carrying out in vitro cell-culture studies to simulate conditions in vivo and in experiments using extracts of the bioactive glass powders.

Characterization of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses.

The ability of bioactive glasses to form a bond to living bone and also to stimulate bone-cell proliferation depends on the chemical composition and on the surface texture of the glasses. In this work, the differences in physical properties between the melt-derived 45S5 and sol-gel-derived 58S Bioglass powders of various particle-size ranges were studied. The powders were characterized for particle-size distribution by laser spectrometry, for specific surface area and porosity by nitrogen sorption analysis, and for morphological features by scanning electron microscopy. Melt-derived 45S5 powders exhibited a low-porosity texture with surface area in the range 0.15-2.7 m(2)/g. In contrast, the sol-gel-derived powders exhibited a highly mesoporous texture, with surface area in the range of 126.5-164.7 m(2)/g and a large fraction of 6-9 nm pore sizes. These differences in texture, as well as variations in chemical composition, account for significant changes in the resorption and in vivo responses.