Multinuclear solid state NMR of novel bioactive glass and nanocomposite tissue scaffolds

Sol-gel derived bioactive glasses are promising candidates for bone regeneration, where bone is a natural nanocomposite of collagen (organic polymer) and hydroxyapatite (inorganic mineral) with a complex hierarchical structure and excellent mechanical properties. Solid-state NMR is a sensitive probe and offers atomic-level information on the structure of sol-gel derived bioactive glasses. In this thesis, a multinuclear solid state NMR approach, as part of an extensive study, has been applied to a key range of sol-gel derived materials related to novel nanocomposites to act as tissue scaffolds. The nanostructure evolution of sol-gel derived bioactive glasses 70S30C (70 mol% SiO2 and 30 mol% CaO) was characterised by 29Si, 1H and 13C CP MAS NMR. Calcium was found to be incorporated into the silica network during the stabilisation stage and to increases its disorder. The inhomogeneity found within 70S30C bioactive glass monoliths showed that the calcium concentration was higher in the outer region of the monolith caused by the way calcium only enters into the structure after breakbown of the nitrate. Trimethylsilylation reaction mechanisms used to tailor the nanoporosity of sol-gel derived 70S30C bioactive glass was also studied. The 29Si NMR results showed that the modification processes affected the atomic scale structure of the glass, such as Qn structure and network connectivity. 1H and 13C NMR was used to follow the loss of hydroxyls and organic groups directly. The study was extended to 58S (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) systems and compared for two synthesis routes: inorganic and alkoxide. Via the inorganic route high temperatures were needed for calcium incorporation, while via alkoxide route calcium was found to be incorporated at low temperatures. Reactive surface Ca ions were involved in the formation of different types of carbonates for the two routes. The addition of P2O5 to the silica-calcium oxide system results in a scavenging of calcium ions by phosphate groups to give orthophosphate and pyrophosphate units. Solid-state NMR of new organic-inorganic hybrid scaffolds, class II, in the silicagelatin and silica-calcium oxide-poly(γ-glutamic acid) (γ-PGA) systems indicates that 3- glycidoxypropyltrimethoxysilane (GPTMS) provides a covalent link between the organic and inorganic networks and increased the inorganic condensation. 1H-1H intra- and intermolecular proximities have been identified using 1H DQ (double-quantum) CRAMPS (combined rotation and multiple pulse spectroscopy) techniques. 13C NMR results indicate that an efficient promotion of epoxide ring opening of GPTMS was reached by either gelatin or γ-PGA. 43Ca NMR identified different calcium environments in the hybrid systems. The last part of this thesis is focused on the comparison studies in the mechanism of apatite growth on both melt-derived (Bioglass®) and sol-gel derived (TheraGlass®) bioactive glass surfaces. By using a combination of 1H, 13C, 31P, 29Si and 23Na, using one and two dimensional NMR spectroscopy, the inhibitive effects of serum proteins in the mechanism of the apatite growth was revealed. The solid-state NMR experimental data support the hydroxycarbonate apatite formation mechanism proposed by Hench. Apatite formation takes place from the largely amorphous phosphate ions initially deposited on the glass surface. Serum proteins adsorbed on the glass surface have been found to significantly inhibit the apatite formation. Multiple sodium sites have been identified in Bioglass® composition with the formation of a more ordered local structure on increasing immersion time

Multinuclear solid state NMR of novel bioactive glass and nanocomposite tissue scaffolds

Sol-gel derived bioactive glasses are promising candidates for bone regeneration, where bone is a natural nanocomposite of collagen (organic polymer) and hydroxyapatite (inorganic mineral) with a complex hierarchical structure and excellent mechanical properties. Solid-state NMR is a sensitive probe and offers atomic-level information on the structure of sol-gel derived bioactive glasses. In this thesis, a multinuclear solid state NMR approach, as part of an extensive study, has been applied to a key range of sol-gel derived materials related to novel nanocomposites to act as tissue scaffolds. The nanostructure evolution of sol-gel derived bioactive glasses 70S30C (70 mol% SiO2 and 30 mol% CaO) was characterised by 29Si, 1H and 13C CP MAS NMR. Calcium was found to be incorporated into the silica network during the stabilisation stage and to increases its disorder. The inhomogeneity found within 70S30C bioactive glass monoliths showed that the calcium concentration was higher in the outer region of the monolith caused by the way calcium only enters into the structure after breakbown of the nitrate. Trimethylsilylation reaction mechanisms used to tailor the nanoporosity of sol-gel derived 70S30C bioactive glass was also studied. The 29Si NMR results showed that the modification processes affected the atomic scale structure of the glass, such as Qn structure and network connectivity. 1H and 13C NMR was used to follow the loss of hydroxyls and organic groups directly. The study was extended to 58S (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) systems and compared for two synthesis routes: inorganic and alkoxide. Via the inorganic route high temperatures were needed for calcium incorporation, while via alkoxide route calcium was found to be incorporated at low temperatures. Reactive surface Ca ions were involved in the formation of different types of carbonates for the two routes. The addition of P2O5 to the silica-calcium oxide system results in a scavenging of calcium ions by phosphate groups to give orthophosphate and pyrophosphate units. Solid-state NMR of new organic-inorganic hybrid scaffolds, class II, in the silicagelatin and silica-calcium oxide-poly(γ-glutamic acid) (γ-PGA) systems indicates that 3- glycidoxypropyltrimethoxysilane (GPTMS) provides a covalent link between the organic and inorganic networks and increased the inorganic condensation. 1H-1H intra- and intermolecular proximities have been identified using 1H DQ (double-quantum) CRAMPS (combined rotation and multiple pulse spectroscopy) techniques. 13C NMR results indicate that an efficient promotion of epoxide ring opening of GPTMS was reached by either gelatin or γ-PGA. 43Ca NMR identified different calcium environments in the hybrid systems. The last part of this thesis is focused on the comparison studies in the mechanism of apatite growth on both melt-derived (Bioglass®) and sol-gel derived (TheraGlass®) bioactive glass surfaces. By using a combination of 1H, 13C, 31P, 29Si and 23Na, using one and two dimensional NMR spectroscopy, the inhibitive effects of serum proteins in the mechanism of the apatite growth was revealed. The solid-state NMR experimental data support the hydroxycarbonate apatite formation mechanism proposed by Hench. Apatite formation takes place from the largely amorphous phosphate ions initially deposited on the glass surface. Serum proteins adsorbed on the glass surface have been found to significantly inhibit the apatite formation. Multiple sodium sites have been identified in Bioglass® composition with the formation of a more ordered local structure on increasing immersion time.EThOS - Electronic Theses Online ServiceUniversity of WarwickEngineering and Physical Sciences Research Council (EPSRC)GBUnited Kingdo

Composition-dependent in vitro apatite formation at mesoporous bioactive glass-surfaces quantified by solid-state NMR and powder XRD

Silicate-based bioactive glasses exhibit bone-bonding properties due to the formation of a hydroxycarbonate apatite (HCA) layer at the glass surface on its contact with living tissues. This bone-healing process is triggered by ionic exchange between the glass and the surrounding fluids and thereby depends on the glass composition. In this work, the HCA formation from three mesoporous bioactive glasses (MBGs) of different compositions immersed in a simulated body fluid (SBF) was monitored for variable time intervals between 15 minutes to 30 days. By utilizing two independent assessment techniques, solid-state P-31 NMR spectroscopy and powder X-ray diffraction (PXRD), we report the first quantitative assessment of the HCA growth (i.e., "in vitro bioactivity") from a bioactive glass: both techniques allow for monitoring the crystallization of the amorphous calcium phosphate (ACP) precursor into HCA, i.e., a profile of the relative ACP/HCA fractions of the biomimetic phosphate layer formed at each MBG surface and SBF-exposure period. The amount of HCA present in each solid specimen after the SBF treatment, as well as the composition of the remaining cation-depleted MBG phase, was determined from PXRD data in conjunction with measured concentrations of Ca, Si, and P in the solution. In contrast with previous findings from in vitro bioactivity assessments of the same MBG compositions, the HCA formation is herein observed to increase concurrently with the Ca and P contents of the MBG; these apparently different composition-bioactivity observations stem from a significantly lower MBG-loading in the SBF solution utilized herein. The results are discussed in relation to the general task of performing bioactivity testing in SBF, where we highlight the importance of adapting the concentration of the biomaterial to its composition to avoid perturbing the HCA crystallization and thereby altering the outcome of the test

Proton Environments in Biomimetic Calcium Phosphates Formed from Mesoporous Bioactive CaO-SiO2- P2O5 Glasses in vitro: Insights from Solid-State NMR

When exposed to body fluids, mesoporous bioactive glasses (MBGs) of the CaO{SiO2{P2O5 system develop a bone-bonding surface layer that initially consists of amorphous calcium phosphate(ACP), which transforms into hydroxy-carbonate apatite (HCA) with a very similar composition as bone/dentin mineral. Information from various 1H-based solid-state nuclear magnetic resonance (NMR) experiments were combined to elucidate the evolution of the proton speciations both at the MBG surface and within each ACP/HCA constituent of the biomimetic phosphate layer formed when each of three MBGs with distinct Ca, Si, and P contents was immersed in a simulated body fluid (SBF) for variable periods between 15 min and 30 days. Directly excited magic-angle-spinning (MAS) 1H NMR spectra mainly reflect the MBG component, whose surface is rich in water and silanol (SiOH) moieties. Double-quantum{single-quantum correlation 1H NMR experimentation at fast MAS revealed their interatomic proximities. The comparatively minor H species of each ACP and HCA component were probed selectively by heteronuclear 1H{31P NMR experimentation. The initially prevailing ACP phase comprises H2O and "non-apatitic" HPO2

Silica–gelatin hybrids for tissue regeneration : inter-relationships between the process variables

Owing to their diverse range of highly tailorable material properties, inorganic/organic hybrids have the potential to meet the needs of biodegradable porous scaffolds across a range of tissue engineering applications. One such hybrid platform, the silica–gelatin sol–gel system, was examined and developed in this study. These hybrid scaffolds exhibit covalently linked interpenetrating networks of organic and inorganic components, which allows for independent control over their mechanical and degradation properties. A combination of the sol–gel foaming process and freeze drying was used to create an interconnected pore network. The synthesis and processing of the scaffolds has many variables that affect their structure and properties. The focus of this study was to develop a matrix tool that shows the inter-relationship between process variables by correlating the key hybrid material properties with the synthesis parameters that govern them. This was achieved by investigating the effect of the organic (gelatin) molecular weight and collating previously reported data. Control of molecular weight of the polymer is as an avenue that allows the modification of hybrid material properties without changing the surface chemistry of the material, which is a factor that governs the cell and tissue interaction with the scaffold. This presents a significant step forward in understanding the complete potential of the silica–gelatin hybrid system as a medical device

Role of pH and temperature on silica network formation and calcium incorporation into sol–gel derived bioactive glasses

Bioactive glasses and inorganic/organic hybrids have great potential as implant materials. Bioactive glasses can bond to bone through the formation of a bone-like hydroxycarbonate apatite (HCA) layer and stimulate bone growth via their dissolution products. The brittle nature of these glasses can be combined with the toughness of a biodegradable polymer by forming a hybrid through the sol–gel process. However, for polymer incorporation, lower temperatures and milder pH conditions are required rather than the current method which uses pH 2 were mesoporous. This indicates a difference in gel formation about the isoelectric point of silicic acid, which was confirmed by 29Si solid state NMR. When immersed in simulated body fluid (SBF), the glasses stabilised at 600 °C were more porous, yet had a slower ion release rate than the gels dried at 40 °C. All gels and glasses formed an HCA layer in SBF; however, calcium was only incorporated into the silica network after stabilisation at 600 °C and thus a new way of incorporating calcium at low temperatures must still be found. This work is an important foundation for hybrid synthesis as raising the pH of the sol–gel process from pH < 1 to pH 5.5 was found to have no adverse effects on silica network formation and thus polymer can be incorporated into the sol–gel process at milder pH conditions without the concern of acid catalysed polymer degradation by chain scission

Silicon oxycarbonitrides synthesized by ammonia-assisted thermolysis route from polymers: A total X-ray scattering, solid-state NMR, and TEM structural study

Solid-state nuclear magnetic resonance (NMR) spectroscopy, total X-ray scattering with a pair distribution function (PDF) analysis, and transmission electron microscopy (TEM) were employed to explore the structures of microporous and non-porous ceramics synthesized by an NH3-assisted thermolysis from polymers. Polysiloxane (SPR-212a, Starfire® Systems) and polysilazane (HTT-1800, KiON Speciality Polymers) polymers form microporous silicon oxycarbonitride ceramics with accessible and tailored micropores. 29Si magic-angle-spinning NMR showed that the introduction of nitrogen leads to structures incorporating considerable amounts of SiN4 and SiO2N2 building blocks. The samples derived from a polycarbosilane (SMP-10, Starfire® Systems) remained non-porous: for such a C-rich and N-bearing phase, the NMR, TEM, and PDF results suggested a Si network exhibiting domains dominated by either Sisingle bondN or Sisingle bondC bonds. 13C NMR revealed primarily “carbidic” CSi4 environments in the C-rich phases, as well as the formation of an amorphous sp2-hybridized carbon phase; both are believed to be detrimental for the micropore formation

Direct Probing of the Phosphate-Ion Distribution in Bioactive Silicate Glasses by Solid-State NMR: Evidence for Transitions between Random/Clustered Scenarios

By employing ³¹P multiple-quantum coherence-based solid-state nuclear magnetic resonance spectroscopy, we present the first comprehensive experimental assessment of the nature of the orthophosphate-ion distributions in silicate-based bioactive glasses (BGs). Results are provided both from melt-prepared BG and evaporation-induced self-assembly-derived mesoporous bioactive glass (MBG) structures of distinct compositions. The phosphate species are randomly dispersed in melt-derived BGs (comprising 44–55 mol % SiO₂) of the Na₂O–CaO–SiO₂–P₂O₅ system, whereas a Si-rich (86 mol % SiO₂) and Ca-poor ordered MBG structure exhibits nanometer-sized amorphous calcium phosphate clusters, conservatively estimated to comprise at least nine orthophosphate groups. A Ca-richer MBG (58 mol % SiO₂) reveals a less pronounced phosphate clustering. We rationalize the variable structural role of P in these amorphous biomaterials

Effect of calcium source on structure and properties of sol–gel derived bioactive glasses

The aim was to determine the most effective calcium precursor for synthesis of sol–gel hybrids and for improving homogeneity of sol–gel bioactive glasses. Sol–gel derived bioactive calcium silicate glasses are one of the most promising materials for bone regeneration. Inorganic/organic hybrid materials, which are synthesized by incorporating a polymer into the sol–gel process, have also recently been produced to improve toughness. Calcium nitrate is conventionally used as the calcium source, but it has several disadvantages. Calcium nitrate causes inhomogeneity by forming calcium-rich regions, and it requires high temperature treatment (>400 °C) for calcium to be incorporated into the silicate network. Nitrates are also toxic and need to be burnt off. Calcium nitrate therefore cannot be used in the synthesis of hybrids as the highest temperature used in the process is typically 40–60 °C. Therefore, a different precursor is needed that can incorporate calcium into the silica network and enhance the homogeneity of the glasses at low (room) temperature. In this work, calcium methoxyethoxide (CME) was used to synthesize sol–gel bioactive glasses with a range of final processing temperatures from 60 to 800 °C. Comparison is made between the use of CME and calcium chloride and calcium nitrate. Using advanced probe techniques, the temperature at which Ca is incorporated into the network was identified for 70S30C (70 mol % SiO2, 30 mol % CaO) for each of the calcium precursors. When CaCl2 was used, the Ca did not seem to enter the network at any of the temperatures used. In contrast, Ca from CME entered the silica network at room temperature, as confirmed by X-ray diffraction, 29Si magic angle spinning nuclear magnetic resonance spectroscopy, and dissolution studies. CME should be used in preference to calcium salts for hybrid synthesis and may improve homogeneity of sol–gel glasses