Chemical interactions in composites of gellan gum and bioactive glass: self-crosslinking and in vitro dissolution.

We investigated the interactions between the organic-inorganic phases in composites and the impact on in vitro dissolution. The composite consists of a hydrogel-forming polysaccharide gellan gum (GG, organic phase) and a borosilicate bioactive glass (BAG, inorganic phase). The BAG loading in the gellan gum matrix varied from 10 to 50 wt%. While mixing GG and BAG, the ions released from BAG microparticles crosslinked with the carboxylate anions of GG. The nature of the crosslinking was assessed, and its impact on mechanical properties, swelling ratio, and enzymatic degradation profile upon immersion for up to 2 weeks was studied. Loading up to 30 wt% of BAG in GG caused an increase in mechanical properties associated with an increasing crosslinking density. At higher BAG loading, excess divalent ions and percolation of particles led to a decrease in the fracture strength and compressive modulus. Upon immersion, a decrease in the composite mechanical properties was attributed to the dissolution of the BAG and the loosening of the glass/matrix interface. The enzymatic degradation of the composites was inhibited at higher BAG loadings (40 and 50 wt%) even when the specimen was immersed for 48 h in PBS buffer with lysozyme. During in vitro dissolution in both SBF and PBS, the ions released from the glass led to the precipitation of hydroxyapatite already at day 7. In conclusion, we thoroughly discussed the in vitro stability of the GG/BAG composite and established the maximum BAG loading to enhance the GG crosslinking and mechanical properties. Based on this study, 30, 40, and 50 wt% of BAG in GG will be further investigated in an in vitro cell culture study.

Polymer-Based Honeycomb Films on Bioactive Glass: Toward a Biphasic Material for Bone Tissue Engineering Applications.

The development of innovative materials for bone tissue engineering to promote bone regeneration while avoiding fibrous tissue infiltration is of paramount importance. Here, we combined the known osteopromotive properties of bioactive glasses (BaGs) with the biodegradability, biocompatibility, and ease to shape/handle of poly-l-co-d,l-lactic acid (PLDLA) into a single biphasic material. The aim of this work was to unravel the role of the surface chemistry and topography of BaG surfaces on the stability of a PLDLA honeycomb membrane, in dry and wet conditions. The PLDLA honeycomb membrane was deposited using the breath figure method (BFM) on the surface of untreated BaG discs (S53P4 and 13-93B20), silanized with 3-aminopropyltriethoxysilane (APTES) or conditioned (immersed for 24 h in TRIS buffer solution). The PLDLA membranes deposited onto the BaG discs, regardless of their composition or surface treatments, exhibited a honeycomb-like structure with pore diameter ranging from 1 to 5 µm. The presence of positively charged amine groups (APTES grafting) or the precipitation of a CaP layer (conditioned) significantly improved the membrane resistance to shear as well as its stability upon immersion in the TRIS buffer solution. The obtained results demonstrated that the careful control of the substrate surface chemistry enabled the deposition of a stable honeycomb membrane at their surface. This constitutes a first step toward the development of new biphasic materials enabling osteostimulation (BaG) while preventing migration of fibrous tissue inside the bone defect (honeycomb polymer membrane).

Phosphate/oxyfluorophosphate glass crystallization and its impact on dissolution and cytotoxicity.

The role of fluorine in bioactive glasses is of interest due to the potential of precipitating fluorapatite, a phase with higher chemical resistance than the typical hydroxyapatite precipitated from oxide bioactive glasses. However, the introduction of fluorine in silicate bioactive glasses was found deleterious to the bioactivity of the glass. Here, phosphate glasses with the composition 75NaPO3-(25-x) CaO-xCaF2 (in mol%), with x = 0-20 and glass-ceramics were investigated to evaluate their potential as substitutes to the traditional silicate bioactive glass. An increase in CaF2 substitution for CaO led to an increase in the glass solubility, due to an increase in highly soluble F(M)n species (where M is a cation) and to an increased polymerization of the phosphate network. Structural analysis reveals the formation of FP bonds, in addition to the F(M)n species, in the glass with the higher CaF2 content. Furthermore, with heat treatment, CaF2 crystals precipitate within the bulk in the newly developed glass, when x = 20. This bulk crystallization reduces the glass dissolution without compromising the precipitation of a reactive layer at the glass surface. Finally, in vitro cell tests were performed using MC3T3 pre-osteoblastic cells. While the substitution of CaF2 for CaO led to an increased cytotoxicity, the controlled crystallization of the fluorine containing glasses decreased such cytotoxicity to similar values than traditional bioactive phosphate glass (x0). This study reports on new oxyfluorophosphate glass and glass-ceramics able, not only, to precipitate a Ca-P reactive layer but also to be processed into glass-ceramics with controlled crystal size, density and cellular activity. STATEMENT OF SIGNIFICANCE: Uncontrolled crystallization of bioactive glasses has negative effect on the materials' bioactivity. While in silicate glass the bioactivity is solely reduced, in phosphate glasses it is often completely suppressed. Furthermore, the need for fluorine containing bioactive glasses, not only for use in bone reconstruction but also in toothpaste as emerged. The addition of F in both silicate and phosphate has led to challenges due the lack of Si-F or P-F bonds, generally leading to a decrease in bioactivity. Here, we developed a bioactive invert phosphate glass where up to 20 mol% of CaO was replaced with CaF2. In the new developed glasses, NMR demonstrated formation of P-F bonds. The content of fluorine was tailored to induce CaF2 bulk crystallization. Overall an increase in F was associated with an increase network connectivity. In turns it led to an increased dissolution rate which was linked to a higher cytotoxicity. Upon (partial to full) surface crystallization of the F-free glass, the bioactivity (ability to form a reactive layer) was loss and the cytotoxicity again increased due to the rapid dissolution of one crystal phase and of the remaining amorphous phase. On another hand, the controlled bulk precipitation of CaF2 crystals, in the F-containing glass, was associated with a reduced cytotoxicity. The new oxyfluorophosphate glass-ceramic developed is promising for application in the biomedical field.

Dissolution, bioactivity and osteogenic properties of composites based on polymer and silicate or borosilicate bioactive glass.

Bioactive glass (BAG)/Poly (Lactic Acid) (PLA) composites have great potential for bone tissue engineering. The interest in these materials is to obtain a scaffold with tailorable properties bringing together the advantages of the composites' constituents such as the biodegradability, bioactivity and osteoinduction. The materials studied are PLA/13-93 and PLA/13-93B20 (20% of SiO2 is replaced with B2O3 in the 13-93 composition). To characterize them, they were dissolved in TRIS buffer and Simulated Body Fluid (SBF) in vitro. Over the 10 weeks of immersion in TRIS, the ion release from the composites was constant. Following immersion in SBF for 2 weeks, the hydroxyapatite (HA) layer was found to precipitate at the composites surface. By adding Boron, both these reactions were accelerated, as the borosilicate glass dissolves faster than pure silicate glass alone. Polymer degradation was studied and showed that during immersion, the pure PLA rods maintained their molecular weight whereby the composites decreased with time, but despite this the mechanical properties remained stable for at least 10 weeks. Their ability to induce osteogenic differentiation of myoblastic cells was also demonstrated with cell experiments showing that C2C12 cells were able to proliferate and spread on the composites. The Myosin Heavy Chain and Osteopontin were tracked by immunostaining the cells and showed a suppression of the myosin signal and the presence of osteopontin, when seeded onto the composites. This proves osteoinduction occurred. In studying the mineralization of the cells, it was found that BAG presence conditions the synthesizing of mineral matter in the cells. The results show that these composites have a potential for bone tissue engineering.

Nucleation and growth behavior of Er3+ doped oxyfluorophosphate glasses.

The nucleation and growth behavior of glasses with the composition (75 NaPO3-25 CaF2)100-x -(TiO2/ZnO/MgO) x , with x = 0 and x = 1.5 (in mol%) is investigated. The glasses possess similar activation energy for crystallization and Johnson-Mehl-Avrami exponent, with value 2 confirming bulk crystallization of crystals with needle like shape. The Ti and Mg glasses exhibit broader nucleation curve and higher T n max than the x = 0 and Zn glasses due to their stronger field strength. The crystal growth rates were determined and validated using SEM. Finally, we showed that the nucleation and growth of glasses can be controlled due to the large difference between onset of crystallization and maximum nucleation temperature which is crucial when preparing novel transparent glass-ceramics.

Core-clad phosphate glass fibers for biosensing.

Recently, a phosphate glass with composition 20 CaO-20 SrO-10 Na2O-50 P2O5 (mol%) was found to have good potential as a biomaterial and to possess thermal properties suitable for fiber drawing. This study opened the path towards the development of fully bioresorbable fibers promising for biosensing. In the past, this phosphate glass with CeO2 was found to increase the refractive index and the glass stability. Therefore, a new SrO-containing glass was prepared with 1 mol% of CeO2 and core fibers were drawn from it. A core-clad fiber was also processed, where the core was a Ce-doped glass and the clad undoped, to allow for total internal reflection. The mechanical properties of the core and core-clad fibers are discussed as a function of immersion time in TRIS-buffer solution. Finally, a sensing region was created, in the core-clad fiber, by etching the cladding using phosphoric acid. Then, the change in light transmission, upon immersion in TRIS-buffer solution, was quantified to assess the potential use of the novel core-clad fiber as a biosensor. Upon immersion in TRIS, the core-clad fiber was found to guide light effectively and to maintain a tensile strength of ~150-200 MPa up to 6 weeks in TRIS, clearly showing that this fiber has potential as a biosensing device.

In vitro Evaluation of Porous borosilicate, borophosphate and phosphate Bioactive Glasses Scaffolds fabricated using Foaming Agent for Bone Regeneration.

In this work, glasses within the borosilicate borophosphate and phosphate family were sintered into 3D porous scaffolds using 60 and 70 vol. % NH4(HCO3) as a foaming agent. All scaffolds produced remained amorphous; apart from one third of the glasses which crystallized. All produced scaffolds had porosity >50% and interconnected pores in the range of 250-570 µm; as evidenced by µCT. The in-vitro dissolution of the scaffolds in SBF and changes in compression were assessed as a function of immersion time. The pH of the solution containing the borosilicate scaffolds increased due to the typical non-congruent dissolution of this glass family. Borophosphate and phosphate scaffolds induced a decrease in pH upon dissolution attributed to the congruent dissolution of those materials and the large release of phosphate within the media. As prepared, scaffolds showed compressive strength of 1.29 ± 0.21, 1.56 ± 0.63, 3.63 ± 0.69 MPa for the borosilicate, borophosphate and phosphate samples sintered with 60 vol. % NH4 (HCO3), respectively. Evidence of hydroxyapatite precipitation on the borosilicate glass scaffolds was shown by SEM/EDS, XRD and ICP-OES analysis. The borophosphate scaffolds remained stable upon dissolution. The phosphate scaffolds were fully crystallized, leading to very large release of phosphate in the media.

Effects of Sintering Temperature on Crystallization and Fabrication of Porous Bioactive Glass Scaffolds for Bone Regeneration.

In this work the sintering ability of borosilicate (S53B50), borophosphate (P40B10) and phosphate (Sr) bioactive glasses was investigated. The glass powders were crushed and sintered in air at a heating rate of 10 °C/min for 2 hours at sintering temperatures between 480 °C-600 °C. The aim was to define the optimum sintering temperature prior to glass crystallization. The density of the samples was found to decrease when the temperature was increased up to 580 °C; probably due to the inhibition of the viscous flow of the particles during sintering thereby reducing the densification of the material. Such low porosity is not suitable in tissue engineering. To process highly porous scaffolds with porosity required for scaffold applicable to tissue engineering, the powders were further mixed with 60 vol.% and 70 vol.% of NH4(HCO3) foaming agent. Meanwhile, the density of the samples sintered with NH4(HCO3) was found to decrease with an increase in NH4(HCO3) content. This indicates an increase in porosity of the samples. The glass compositions reached an open porosity of more than 60% at the addition of 70 vol.% NH4(HCO3). In addition, SEM micrograph revealed large pores with good interconnection between the pores.

Crystallization and sintering of borosilicate bioactive glasses for application in tissue engineering.

Typical silicate bioactive glasses are known to crystallize readily during the processing of porous scaffolds. While such crystallization does not fully suppress the bioactivity, the presence of significantly large amounts of crystals leads to a decrease in the rate of reaction of the glass and an uncontrolled release of ions. Furthermore, due to the non-congruent dissolution of silicate glasses, these materials have been shown to remain within the surgical site even 14 years post-operation. Therefore, bioactive materials that can dissolve more effectively and have higher conversion rates are required. Within this work, boron was introduced, in the FDA approved S53P4 glass, at the expense of SiO2. The crystallization and sintering-ability of the newly developed glasses were investigated by differential thermal analysis. All the glasses were found to crystallize primarily from the surface, and the crystal phase precipitation was dependent on the quantity of B2O3 incorporated. The rate of crystallization was found to be lower for the glasses when 25, 50 and 75% of SiO2 was replaced with B2O3. These glasses were further sintered into porous scaffolds using simple heat sintering. The impact of glass particle size and heat treatment temperature on the scaffold porosity and average pore size was investigated. Scaffolds with porosity ranging from 10 to 60% and compressive strength ranging from 1 to 35 MPa were produced. The scaffolds remained amorphous during processing and their ability to rapidly precipitate hydroxycarbonate apatite was maintained. This is of particular interest in the field of tissue engineering as scaffold degradation and reaction is generally faster and offers higher controllability as opposed to the current partially/fully crystallized scaffolds obtained from the FDA approved bioactive glasses.

The influence of SrO and CaO in silicate and phosphate bioactive glasses on human gingival fibroblasts.

In this paper, we investigate the effect of substituting SrO for CaO in silicate and phosphate bioactive glasses on the human gingival fibroblast activity. In both materials the presence of SrO led to the formation of a CaP layer with partial Sr substitution for Ca. The layer at the surface of the silicate glass consisted of HAP whereas at the phosphate glasses it was close to the DCPD composition. In silicate glasses, SrO gave a faster initial dissolution and a thinner reaction layer probably allowing for a continuous ion release into the solution. In phosphate glasses, SrO decreased the dissolution process and gave a more strongly bonded reaction layer. Overall, the SrO-containing silicate glass led to a slight enhancement in the activity of the gingival fibroblasts cells when compared to the SrO-free reference glass, S53P4. The cell activity decreased up to 3 days of culturing for all phosphate glasses containing SrO. Whereas culturing together with the SrO-free phosphate glass led to complete cell death at 7 days. The glasses containing SrO showed rapid cell proliferation and growth between 7 and 14 days, reaching similar activity than glass S53P4. The addition of SrO in both silicate and phosphate glasses was assumed beneficial for proliferation and growth of human gingival fibroblasts due to Sr incorporation in the reaction layer at the glass surface and released in the cell culture medium.

Phosphate-based glass fiber vs. bulk glass: Change in fiber optical response to probe in vitro glass reactivity.

This paper investigates the effect of fiber drawing on the thermal and structural properties as well as on the glass reactivity of a phosphate glass in tris(hydroxymethyl)aminomethane-buffered (TRIS) solution and simulated body fluid (SBF). The changes induced in the thermal properties suggest that the fiber drawing process leads to a weakening and probable re-orientation of the POP bonds. Whereas the fiber drawing did not significantly impact the release of P and Ca, an increase in the release of Na into the solution was noticed. This was probably due to small structural reorientations occurring during the fiber drawing process and to a slight diffusion of Na to the fiber surface. Both the powders from the bulk and the glass fibers formed a Ca-P surface layer when immersed in SBF and TRIS. The layer thickness was higher in the calcium and phosphate supersaturated SBF than in TRIS. This paper for the first time presents the in vitro reactivity and optical response of a phosphate-based bioactive glass (PBG) fiber when immersed in SBF. The light intensity remained constant for the first 48h after which a decrease with three distinct slopes was observed: the first decrease between 48 and 200h of immersion could be correlated to the formation of the Ca-P layer at the fiber surface. After this a faster decrease in light transmission was observed from 200 to ~425h in SBF. SEM analysis suggested that after 200h, the surface of the fiber was fully covered by a thin Ca-P layer which is likely to scatter light. For immersion times longer than ~425h, the thickness of the Ca-P layer increased and thus acted as a barrier to the dissolution process limiting further reduction in light transmission. The tracking of light transmission through the PBG fiber allowed monitoring of the fiber dissolution in vitro. These results are essential in developing new bioactive fiber sensors that can be used to monitor bioresponse in situ.

Thermal properties and surface reactivity in simulated body fluid of new strontium ion-containing phosphate glasses.

In this paper, we investigate the effect of SrO substitution for CaO in 50P2O5-10Na2-(40-x)CaO-xSrO glass system (x from 0 to 40) on the thermal and structural properties and also on the glass reactivity in simulated body fluid (SBF) in order to find new glass candidates for biomedical glass fibers. The addition of SrO at the expense of CaO seems to restrain the leaching of phosphate ions in the solution limiting the reduction of the solution pH. We observed the formation of an apatite layer at the surface of the glasses when in contact with SBF. SrO and MgO were found in the apatite layer of the strontium ion-containing glasses, the concentration of which increases with an increase of SrO content. We think that it is the presence of MgO and SrO in the layer which limits the leaching of phosphate in the solution and thus the glass dissolution in SBF.

Phase composition and in vitro bioactivity of porous implants made of bioactive glass S53P4.

This work studied the influence of sintering temperature on the phase composition, compression strength and in vitro properties of implants made of bioactive glass S53P4. The implants were sintered within the temperature range 600-1000°C. Over the whole temperature range studied, consolidation took place mainly via viscous flow sintering, even though there was partial surface crystallization. The mechanical strength of the implants was low but increased with the sintering temperature, from 0.7 MPa at 635°C to 10 MPa at 1000°C. Changes in the composition of simulated body fluid (SBF), the immersion solution, were evaluated by pH measurements and ion analysis using inductively coupled plasma optical emission spectrometry. The development of a calcium phosphate layer on the implant surfaces was verified using scanning electron microscopy-electron-dispersive X-ray analysis. When immersed in SBF, a calcium phosphate layer formed on all the samples, but the structure of this layer was affected by the surface crystalline phases. Hydroxyapatite formed more readily on amorphous and partially crystalline implants containing both primary Na(2)O·CaO·2SiO(2) and secondary Na(2)Ca(4)(PO(4))(2)SiO(4) crystals than on implants containing only primary crystals.