Academic literature on the topic 'Mineralisation in vitro'

Vascular pericytes can differentiate into osteoblast-like cells in vitro, suggesting that these cells may represent a potential source of osteoprogenitor cells in the adult. Pericyte differentiation is associated with a characteristic pattern of nodule formation and mineralisation. Nodules are formed in post-confluent cultures by the retraction of multilayered areas. Crystals of hydroxyapatite are deposited on the extracellular matrix of these nodules which then becomes mineralised. We now demonstrate that thrombospondin-1 (TSP-1) gene expression is modulated during pericyte differentiation in vitro. That is, the relative levels of TSP-1 (protein and mRNA) increased markedly during nodule formation and then decreased when mineralisation of the nodules had taken place. TSP-1 was localised throughout non-mineralised nodules but it was largely excluded from the inner mass of mineralised nodules. The production of a mineralised matrix by vascular pericytes was promoted by the presence of antibodies to TSP-1 in the culture medium and was inhibited by exogenous TSP-1. These effects did not appear to be mediated through the activation of latent TGF-beta, since neither exogenous TGF-beta nor neutralising antibodies to TGF-beta had any effect on the rate or extent of mineralisation seen in the pericyte cultures. Taken together these results suggest that high levels of TSP-1 inhibit pericyte mineralisation, supporting the view that this protein plays a role in pericyte differentiation and bone formation.

To understand more clearly the groundwater contamination potential of herbicides applied to New Zealand soils, experimental field plots were established on 2 different soil types: Himatangi, a sandy dune soil, and Kiripaka, a silty clay derived from basalt. A mix of triazine herbicides, containing atrazine, terbuthylazine, and hexazinone, was applied to the plots at 10 kg a.i./ha. At various times after application, soil was removed from the plots and analysed for residual levels of herbicides, in vitro rates of mineralisation of 14C-ring-labelled atrazine, and numbers of atrazine-degrading microbes. Atrazine and terbuthylazine were below detectable levels (<0.01 mg/kg) in Himatangi topsoil 18 months after pesticide application but still detectable in topsoil from the Kiripaka site. Hexazinone was detectable in topsoil from both soil plots 18 months after application. Atrazine adsorption isotherms were constructed for topsoil and subsoil from both plots, with estimated Kf values ranging from 0.53 to 4.69 μg1–n mLn/g. A single application of atrazine was sufficient to enhance the rate of 14C-atrazine mineralisation in vitro by topsoil from both plots, and subsoil from the Kiripaka site. Rates of mineralisation of atrazine in the soil from the plots increased 1–6 months after pesticide application and remained elevated for 18–24 months. The numbers of atrazine degraders detected did not correlate with atrazine mineralisation rates. An atrazine-degrading bacterium, identifed as Arthrobacter nicotinovorans, was isolated from Himatangi soil exhibiting enhanced rates of atrazine-mineralisation activity.

For the first time, the bioaccessibility of the mineral nutrients in ripe table olives and their contributions to the recommended daily intake (RDI), according to digestion methods (Miller’s vs. Crews’ protocols), digestion type (standard vs. modified, standard plus a post-digest re-extraction), and mineralisation system (wet vs. ashing) were studied. Overall, when the standard application was used, Miller’s protocol resulted in higher bioaccessibilities of Na, K, Ca, Mg, and Fe than the Crews’ method. The modified protocols improved most of these values, but the Crews’ results only approximated the Miller’s levels in the case of Na and K. The bioaccessibility of P was hardly affected by the factors studied, except that the modified Miller’s protocol led to higher levels when ashing. No significant effect of the mineralisation system was found. The modified Miller’s protocol, regardless of the mineralisation system, led to the overall highest bioaccessibility values in ripe olives, which were: Na (96%), K (95%), Ca (20%), Mg (73%), Fe (45%), and P (60%). Their potential contributions to the RDI, based on these bioaccessibilities and 100 g olive flesh service size, were then 29, 0.5, 4, 3, 33, and 1% respectively. The investigation has led to the development of a method for assessing the bioaccessibility of the mineral nutrients not only in ripe but also in the remaining table olive presentations and opens a new research line of great interest for producing healthier products.

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid–base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.

Currently, many polymeric biomaterials do not possess the most desirable surface properties for direct bone bonding due to the lack of suitable surface functionalities. The incorporation of negatively charged groups has been shown to enhance calcium phosphate formation in vitro and bone bonding ability in vivo. However, there are some conflicting literature reports that highlight the complicated nature of the mineralisation process as well as the sometimes apparent contradictory effect of the negatively charged groups. Surface modification using well-defined polymers offer a more precise control of the chain structures. The aims of this study were to synthesise well-defined polymers containing phosphate and carboxylic acid groups, and perform various surface modification techniques. The influence of the polymer structure on mineralisation was examined using a series of specially synthesised phosphate-containing polymers. The mineralisation ability of the fabricated surfaces was also tested. Soluble poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were synthesised using reversible addition fragmentation chain transfer (RAFT)-mediated polymerisation. The polymerisation conversions were monitored by in situ Raman spectroscopy. Subsequently 31P NMR investigation revealed the presence of large amounts of diene impurities as well as free orthophosphoric acids in both the MAEP and MOEP monomers. Elemental analyses of the polymers showed loss of phosphate groups due to hydrolysis during the polymerisation. Both gel and soluble PMAEP polymers were found to contain large amounts of carboxyl groups indicating hydrolysis at the C-O-C ester linkages. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy) ethyl methacrylate) PAAEMA were successfully prepared for the purpose of immobilisation of these polymers onto aminated slides. Well-defined fluorinated polymers, (poly(pentafluorostyrene) (PFS), poly(tetrafluoropropyl acrylate) (TFPA) and poly(tetrafluoropropyl methacrylate) (TFPMA)) were synthesised by RAFT-mediated polymerisation. It was found that the Mn values of PFS at higher conversions were significantly lower than those calculated from the theory, although the PDI's were low (<1.1). One possible explanation for this is that it may be a result of the self-initiation of FS which created more chains than the added RAFT agents. Both TFPA and TFPMA showed well-controlled RAFT polymerisations. Chain extension of the fluorinated polymers with tert-butyl acrylate (tBA) followed by hydrolysis of the tBA groups produced the amphiphilic block copolymers containing carboxylic acid groups. Block copolymers consisting of PAAEMA segments were further reacted with glycine and L-phenylalanyl glycine. Three types of surface modifications were carried out: Layer-by-Layer (LbL) assemblies of the soluble phosphate- and carboxylic acid-containing homopolymers, coupling reactions of block copolymers consisting of phosphate and keto groups onto aminated slides, and adsorption of fluorinated homo and block copolymers containing carboxylic acid groups onto PTFE. For LbL assemblies XPS analyses revealed that the thickness of the poly(acrylic acid) (PAA) layer was found to be strongly dependent on the pH at deposition. AFM images showed that the PMAEP LbL had a patchy morphology which was due to the carboxylate groups that were not deprotonated at low pH. Successful coupling of the block copolymers consisting of phosphate and keto groups onto aminated slides was evident in the XPS results. The conformation of attached P(MOEP-b-AAEMA) was investigated by ToF-SIMS. Adsorption of the fluorinated polymers onto the PTFE film was examined using different solvents. PFS showed the best adsorption onto PTFE. The block copolymers consisting of PFS and PtBA or PAA were successfully adsorbed onto PTFE. Contact angle measurements showed that the adsorbed block copolymers reorganised quickly to form a hydrophilic surface during the investigation. In vitro mineralisation of various phosphate-containing polymers and the fabricated surfaces were studied using the simulated body fluid (SBF) technique. The SEM/EDX investigation showed that either brushite or monetite, with a tile-like morphology, was formed on both soluble and gel PMAEP polymers after seven days in SBF. The PMOEP gel formed a similar layer as well as a secondary growth of hydroxyapatite (HAP) that exhibited a typical globular morphology. Fourier transform infrared (FTIR) spectroscopy of the PMOEP film prepared from soluble PMOEP showed large amounts of carbonated HAP formation after seven days in SBF. Carbonated HAP is the phase that most closely resembles that found in biological systems. Both the LbL surfaces and the block copolymer-attached aminated slides showed only patchy mineralisation even after 14 days in SBF. This indicates that ionic interactions of the negatively charged phosphates or carboxylates and protonated amines prevented chelation of calcium ions, which is believed to be the first step in mineralisation. The P(FS-b-AA) adsorbed PTFE film also showed only small amounts of mineral formation after 14 days in SBF. These results highlight the many features controlling the mineralisation outcomes.

Currently, many polymeric biomaterials do not possess the most desirable surface properties for direct bone bonding due to the lack of suitable surface functionalities. The incorporation of negatively charged groups has been shown to enhance calcium phosphate formation in vitro and bone bonding ability in vivo. However, there are some conflicting literature reports that highlight the complicated nature of the mineralisation process as well as the sometimes apparent contradictory effect of the negatively charged groups. Surface modification using well-defined polymers offer a more precise control of the chain structures. The aims of this study were to synthesise well-defined polymers containing phosphate and carboxylic acid groups, and perform various surface modification techniques. The influence of the polymer structure on mineralisation was examined using a series of specially synthesised phosphate-containing polymers. The mineralisation ability of the fabricated surfaces was also tested. Soluble poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were synthesised using reversible addition fragmentation chain transfer (RAFT)-mediated polymerisation. The polymerisation conversions were monitored by in situ Raman spectroscopy. Subsequently 31P NMR investigation revealed the presence of large amounts of diene impurities as well as free orthophosphoric acids in both the MAEP and MOEP monomers. Elemental analyses of the polymers showed loss of phosphate groups due to hydrolysis during the polymerisation. Both gel and soluble PMAEP polymers were found to contain large amounts of carboxyl groups indicating hydrolysis at the C-O-C ester linkages. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy) ethyl methacrylate) PAAEMA were successfully prepared for the purpose of immobilisation of these polymers onto aminated slides. Well-defined fluorinated polymers, (poly(pentafluorostyrene) (PFS), poly(tetrafluoropropyl acrylate) (TFPA) and poly(tetrafluoropropyl methacrylate) (TFPMA)) were synthesised by RAFT-mediated polymerisation. It was found that the Mn values of PFS at higher conversions were significantly lower than those calculated from the theory, although the PDI's were low (<1.1). One possible explanation for this is that it may be a result of the self-initiation of FS which created more chains than the added RAFT agents. Both TFPA and TFPMA showed well-controlled RAFT polymerisations. Chain extension of the fluorinated polymers with tert-butyl acrylate (tBA) followed by hydrolysis of the tBA groups produced the amphiphilic block copolymers containing carboxylic acid groups. Block copolymers consisting of PAAEMA segments were further reacted with glycine and L-phenylalanyl glycine. Three types of surface modifications were carried out: Layer-by-Layer (LbL) assemblies of the soluble phosphate- and carboxylic acid-containing homopolymers, coupling reactions of block copolymers consisting of phosphate and keto groups onto aminated slides, and adsorption of fluorinated homo and block copolymers containing carboxylic acid groups onto PTFE. For LbL assemblies XPS analyses revealed that the thickness of the poly(acrylic acid) (PAA) layer was found to be strongly dependent on the pH at deposition. AFM images showed that the PMAEP LbL had a patchy morphology which was due to the carboxylate groups that were not deprotonated at low pH. Successful coupling of the block copolymers consisting of phosphate and keto groups onto aminated slides was evident in the XPS results. The conformation of attached P(MOEP-b-AAEMA) was investigated by ToF-SIMS. Adsorption of the fluorinated polymers onto the PTFE film was examined using different solvents. PFS showed the best adsorption onto PTFE. The block copolymers consisting of PFS and PtBA or PAA were successfully adsorbed onto PTFE. Contact angle measurements showed that the adsorbed block copolymers reorganised quickly to form a hydrophilic surface during the investigation. In vitro mineralisation of various phosphate-containing polymers and the fabricated surfaces were studied using the simulated body fluid (SBF) technique. The SEM/EDX investigation showed that either brushite or monetite, with a tile-like morphology, was formed on both soluble and gel PMAEP polymers after seven days in SBF. The PMOEP gel formed a similar layer as well as a secondary growth of hydroxyapatite (HAP) that exhibited a typical globular morphology. Fourier transform infrared (FTIR) spectroscopy of the PMOEP film prepared from soluble PMOEP showed large amounts of carbonated HAP formation after seven days in SBF. Carbonated HAP is the phase that most closely resembles that found in biological systems. Both the LbL surfaces and the block copolymer-attached aminated slides showed only patchy mineralisation even after 14 days in SBF. This indicates that ionic interactions of the negatively charged phosphates or carboxylates and protonated amines prevented chelation of calcium ions, which is believed to be the first step in mineralisation. The P(FS-b-AA) adsorbed PTFE film also showed only small amounts of mineral formation after 14 days in SBF. These results highlight the many features controlling the mineralisation outcomes.

The steps involved in early bone mineralisation have been extensively studied. Studies have suggested various means by which bone mineralisation occurs: (1) a cell-independent (protein-assisted) process where non-collagenous proteins mediate soluble ions to form mineral on the collagen fibril; (2) a cell-controlled mechanism where mineral seeds formed within intracellular vesicles released from the plasma membrane would subsequently rupture and disperse their contents on the extracellular matrix; and (3) an acellular route in which amorphous calcium phosphate mineral precursors are produced and deposited in collagen fibrils where they transform into more crystalline apatite platelets. Despite extensive studies, there are still unanswered questions about how bone becomes bone. The first series of experiments in this thesis are aimed at studying the mineral characteristics of early in vitro osteoblast mineralisation at the extracellular matrix. These experiments seek to determine the sequence of possible mineralisation events that take place during mineral nucleation and growth on collagen. Transmission electron microscopy was used to provide high spatial resolution, which was compounded with chemical analysis of energy dispersive x-ray and electron energy-loss spectroscopies. We identified carbonate-rich calcium phosphate dense granules in the extracellular matrix that may act as seeds for growth into larger, submicron-sized, globular aggregates of apatite mineral with a different stoichiometry. These globules appear to mineralise the collagen fibrils forming crystalline textured crystals with higher calcium-to-phosphate ratio and lower carbonate content as the mineral phase of bone. We provide new evidence that the use of a carbonate rich, amorphous calcium phosphate spherical bioseed could be a process by which a soluble calcium phosphate phase is stabilised and delivered to the collagen for subsequent maturation and collagen mineralisation. We also examined the effect of strontium ion supplementation to bone mineralisation as a translational study. Previous studies showed the positive effects of in vivo strontium supplementation as an anti-osteoporotic drug. Strontium is able to: (1) stimulate bone formation; (2) increase osteoid surface, osteoblast surface, and bone forming surfaces; (3) decrease bone resorbing cells; (4) increase bone strength and mass; and (5) reduce the risk of fractures. In the in vitro system, studies have focused on how strontium ions increase bone mineral's a- and c-axis lattice parameters. The effect of strontium ions on the matrix component of bone is the aim for the second part of this thesis. Using Raman spectroscopy, TEM imaging, and biochemical quantification, we studied the effect of strontium ion supplementation on in vitro MC3T3 osteoblasts, with close focus on the osteoblast matrix. We observed that cultures treated with high strontium supplementation had impaired mineralisation, where nodules were formed but failed to mineralise. Periodic collagen banding was seen on TEM micrograph from all treatments. Collagen organisation was quantified using image analysis of TEM micrograph, and strontium supplementation seemed to affect fibril organisation. A slight addition of 0.1 mM strontium seemed to result in the less random organisation of collagen fibril, while 3 mM supplementation seemed to increase the random organisation. Only cultures treated with the highest amount of strontium supplementation showed an abundance of matrix vesicles around the collagen fibrils. Raman spectroscopy showed an increase in lipid detection on strontium supplemented groups, which may be due to the increased presence of matrix vesicles. Taken together, high strontium supplementation may decrease the rate of degradation of matrix vesicles and lead to altered mineralisation whereby nodules form but fail to mineralise. The relative amounts of collagen were also explored by Raman spectroscopy and hydroxyproline analysis; however, our findings were not statistically significant. Further experiments are needed to more completely elucidate the molecular mechanisms at play in strontium's effect on bone mineralisation.

L’objectif de ce travail était de mettre au point un modèle de culture cellulaire, afin d’étudier in vitro l’incidence sur la physiologie des cellules pulpaires et en particulier de l’odontoblaste, de biomatériaux utilisés pour traiter les effractions de pulpes dentaires. Ce modèle repose sur l’utilisation de culture sphéroïdes dont la conformation spatiale reproduit plus fidèlement l’environnement in vivo que les cultures bidimensionnelles. Après avoir élaboré le modèle sphéroïde à partir de lignées murines, des expérimentations visant à déterminer la cytotoxicité des matériaux ont été effectuées. Leurs capacités à induire la biominéralisation ont également été évaluées. La dernière partie de ce travail avait pour objectif d’immortaliser des primocultures de cellules pulpaires humaines en transfectant les gènes SV40 et hTERT, afin d’établir une lignée cellulaire
The aim of this study was to develop a cell culture model to assess in vitro the effects on the physiology of pulp cells, in particular the odontoblasts, of biomaterials used to treat dental pulp exposures. This model is based on the use of spheroid culture whose spatial configuration reproduces the in vivo environment more faithfully than do two-dimensional cultures. After developing the spheroid model from mouse lines, experiments to determine the cytotoxicity of the materials were conducted. Their ability to induce bio-mineralisation was also assessed. The last part of this work aimed to immortalise primo-cultures of human pulp cells by transfecting hTERT and SV40 genes, in order to establish a new cell line