Substitut osseux injectable, antibactérien et résorbable : études physico-chimiques et biologiques d'un ciment composite à base d'apatite

Ce travail porte sur la recherche et le développement d'un matériau de substitution osseuse permettant une implantation par chirurgie mini invasive, limitant les infections post-opératoires et dont la résorbabilité serait adaptée à la cinétique de régénération osseuse. Nous nous sommes intéressés à un ciment à base de carbonate et de phosphate de calcium (CaCO3 – CaP) dont la réaction de prise conduit à la formation d'une apatite nanocristalline analogue au minéral osseux. Dans une première partie la cinétique de prise et le produit de réaction ont été caractérisés par différentes techniques, notamment la diffraction des RX et les spectroscopies FTIR et RMN du solide. Un sel d'argent – Ag3PO4 ou AgNO3, choisis pour leurs propriétés antibactériennes – a été ensuite introduit dans la formulation. Son effet sur la cinétique de la réaction chimique de prise a été mis en évidence par traitement des spectres FTIR et RMN et un mécanisme réactionnel original impliquant les ions argent et nitrate dans la formation de l'apatite a été proposé. L'ajout d'un polysaccharide, la carboxyméthylcellulose (CMC), dans la phase solide du ciment a montré une très nette amélioration de l'injectabilité de la pâte, avec la disparition du phénomène de séparation des phases qui limite généralement l'injectabilité des ciments minéraux. La résistance à la compression et le module élastique des ciments composites ont été par ailleurs augmentés, parallèlement à une diminution de leur porosité. Différentes études in vitro en présence de cellules ou de bactéries ont enfin été réalisées et ont mis en évidence respectivement la cytocompatibilité des différentes compositions de ciments étudiées et le caractère antibactérien de ces matériaux à partir d'une certaine concentration en argent. L'implantation in vivo de compositions choisies a présenté des résultats très prometteurs quant à la résorbabilité d'un ciment composite CaCO3 - CaP/CMC/Ag et à la néoformation osseuse. ABSTRACT : The present work concerns research and development of a material for bone substitution, enabling implantation through a mini-invasive surgery, limiting post-operative infections and whose resorbability is adapted to bone regeneration kinetics. This study focused on a calcium carbonate and phosphate based cement, whose setting reaction leads to the formation of a nanocrystalline apatite, similar to bone mineral. First, the setting kinetics and the reaction products were characterised using different techniques, especially X-ray diffraction and FTIR and solid-state NMR spectroscopies. A silver salt – Ag3PO4 or AgNO3, chosen for their antibacterial properties – was then introduced in the formulation. Its effect on the setting reaction kinetics was revealed by data processing of FTIR and NMR spectra and an original reaction mechanism which involves silver and nitrates in the formation of apatite was proposed. The addition of a polysaccharide, carboxymethylcellulose (CMC), in the solid phase of the cement showed a clear improvement of the injectability of the paste, preventing the occurrence of filter-pressing phenomenon, often limiting the injectability of mineral cements. The resistance to compressive strength and elastic modulus of the composite cement were also improved together with a decrease in their porosity. Different in vitro studies were carried out in the presence of cells or bacteria and demonstrated the cytocompatibility of different cement compositions and their antibacterial properties starting at a certain silver concentration, respectively. In vivo implantation of selected compositions showed promising results concerning resorbability of a composite CaCO3 - CaP/CMC/Ag cement and the associated bone neoformation

Mechanical properties of self-setting composites: influence of the carboxymethylcellulose content and hydration state

The impact of the carboxymethylcellulose (CMC) content on the mechanical properties of calcium phosphate–calcium carbonate–CMC composite cements for bone substitution was investigated. The relevance of the compressive test conditions (wet or dried composite cements) is discussed and models are proposed to better understand the mechanisms involved in the mechanical properties of the composite materials. Based on a modellisation using the Voigt model for dried composite cements, we show that a minimum of CMC content of around 10–20 % is needed to enhance the mechanical properties of the dried composite materials (up to 86 MPa for the composite including 50 wt% CMC) through the formation of a mineral–organic entangled network. The compressive strength of the wet samples is low (\3 MPa) but the gain observed in the dried composites is encouraging and might be extrapolated to wet conditions if we were to use a less hydrophilic polysaccharid

Optimization of spray-dried hyaluronic acid microspheres to formulate drug-loaded bone substitute materials

Wepresent here our first results concerning the evaluation of hyaluronic acid (HA) as a candidate to formulate an organic–mineral cement with sustained release properties. Incorporating drug-loaded microspheres in mineral bone cements is an alternative strategy to improve their ability as drug delivery materials. To synthesize microspheres according to a reproducible process and control at the same time their morphology and their encapsulation efficiency is one of the main challenges of the conception of such drug-loaded bone substitute. In this context, we investigated the potentialities of two HA, differing by theirmolecular weight, to form microspheres by a spray-drying technique. Erythrosin B (EB) was encapsulated as a model drug and spray-drying process conditionswere optimized. To performthis, the rheological behavior and viscosity of HA solutions have been related to their spray-drying ability, and then to the resulting microparticles morphological properties and size distribution. Reproducible microspheres, answering to the requirements in terms of size and encapsulation efficiency, have been obtained from both HA. However the HA exhibiting the lowest molecular weight, HA600, led to smaller microparticles, with a higher polydispersity index. Their swelling ability, related to their stability upon rehydration, also appeared reduced. In this context, HA850, with the highest molecular weight, was selected and the possibility to modulate drug release by HA850 microspheres incorporation into a mineral cement was explored. EB release kinetics from HA microspheres, HA microspheres loaded cement and reference cement were followed at 37 °C, in Tris buffer at pH 7.4, using European Pharmacopoeia flow-through cells. Results showed that HA microspheres incorporation into a mineral cement permitted to modify the cement drug release profile and led to a sustained release

Development of an injectable composite for bone regeneration

With the development of minimally invasive surgical techniques, there is a growing interest in the research and development of injectable biomaterials especially for orthopedic applications. In a view to enhance the overall surgery benefits for the patient, the BIOSINJECT project aims at preparing a new generation of mineral-organic composites for bone regeneration exhibiting bioactivity, therapeutic activity and easiness of use to broaden the application domains of the actual bone mineral cements and propose an alternative strategy with regard to their poor resorbability, injectability difficulties and risk of infection. First, a physical-chemical study demonstrated the feasibility of self-setting injectable composites associating calcium carbonate-calcium phosphate cement and polysaccharides (tailor-made or commercial polymer) in the presence or not of an antibacterial agent within the composite formulation. Then, bone cell response and antimicrobial activity of the composite have been evaluated in vitro. Finally, in order to evaluate resorption rate and bone tissue response an animal study has been performed and the histological analysis is still in progress. These multidisciplinary and complementary studies led to promising results in a view of the industrial development of such composite for dental and orthopaedic applications

Injectable, antibacterial and resorbable bone substitute : a physico-chemical and biological study of an apatite-based composite cement

Ce travail porte sur la recherche et le développement d'un matériau de substitution osseuse permettant une implantation par chirurgie mini invasive, limitant les infections post-opératoires et dont la résorbabilité serait adaptée à la cinétique de régénération osseuse. Nous nous sommes intéressés à un ciment à base de carbonate et de phosphate de calcium (CaCO3 – CaP) dont la réaction de prise conduit à la formation d'une apatite nanocristalline analogue au minéral osseux. Dans une première partie la cinétique de prise et le produit de réaction ont été caractérisés par différentes techniques, notamment la diffraction des RX et les spectroscopies FTIR et RMN du solide. Un sel d'argent – Ag3PO4 ou AgNO3, choisis pour leurs propriétés antibactériennes – a été ensuite introduit dans la formulation. Son effet sur la cinétique de la réaction chimique de prise a été mis en évidence par traitement des spectres FTIR et RMN et un mécanisme réactionnel original impliquant les ions argent et nitrate dans la formation de l'apatite a été proposé. L'ajout d'un polysaccharide, la carboxyméthylcellulose (CMC), dans la phase solide du ciment a montré une très nette amélioration de l'injectabilité de la pâte, avec la disparition du phénomène de séparation des phases qui limite généralement l'injectabilité des ciments minéraux. La résistance à la compression et le module élastique des ciments composites ont été par ailleurs augmentés, parallèlement à une diminution de leur porosité. Différentes études in vitro en présence de cellules ou de bactéries ont enfin été réalisées et ont mis en évidence respectivement la cytocompatibilité des différentes compositions de ciments étudiées et le caractère antibactérien de ces matériaux à partir d'une certaine concentration en argent. L'implantation in vivo de compositions choisies a présenté des résultats très prometteurs quant à la résorbabilité d'un ciment composite CaCO3 - CaP/CMC/Ag et à la néoformation osseuse.The present work concerns research and development of a material for bone substitution, enabling implantation through a mini-invasive surgery, limiting post-operative infections and whose resorbability is adapted to bone regeneration kinetics. This study focused on a calcium carbonate and phosphate based cement, whose setting reaction leads to the formation of a nanocrystalline apatite, similar to bone mineral. First, the setting kinetics and the reaction products were characterised using different techniques, especially X-ray diffraction and FTIR and solid-state NMR spectroscopies. A silver salt – Ag3PO4 or AgNO3, chosen for their antibacterial properties – was then introduced in the formulation. Its effect on the setting reaction kinetics was revealed by data processing of FTIR and NMR spectra and an original reaction mechanism which involves silver and nitrates in the formation of apatite was proposed. The addition of a polysaccharide, carboxymethylcellulose (CMC), in the solid phase of the cement showed a clear improvement of the injectability of the paste, preventing the occurrence of filter-pressing phenomenon, often limiting the injectability of mineral cements. The resistance to compressive strength and elastic modulus of the composite cement were also improved together with a decrease in their porosity. Different in vitro studies were carried out in the presence of cells or bacteria and demonstrated the cytocompatibility of different cement compositions and their antibacterial properties starting at a certain silver concentration, respectively. In vivo implantation of selected compositions showed promising results concerning resorbability of a composite CaCO3 - CaP/CMC/Ag cement and the associated bone neoformation

Substitut osseux injectable, antibactérien et résorbable (études physico-chimiques et biologiques d'un ciment composite à base d'apatite)

Ce travail porte sur la recherche et le développement d'un matériau de substitution osseuse permettant une implantation par chirurgie mini invasive, limitant les infections post-opératoires et dont la résorbabilité serait adaptée à la cinétique de régénération osseuse. Nous nous sommes intéressés à un ciment à base de carbonate et de phosphate de calcium (CaCO3 CaP) dont la réaction de prise conduit à la formation d'une apatite nanocristalline analogue au minéral osseux. Dans une première partie la cinétique de prise et le produit de réaction ont été caractérisés par différentes techniques, notamment la diffraction des RX et les spectroscopies FTIR et RMN du solide. Un sel d'argent Ag3PO4 ou AgNO3, choisis pour leurs propriétés antibactériennes a été ensuite introduit dans la formulation. Son effet sur la cinétique de la réaction chimique de prise a été mis en évidence par traitement des spectres FTIR et RMN et un mécanisme réactionnel original impliquant les ions argent et nitrate dans la formation de l'apatite a été proposé. L'ajout d'un polysaccharide, la carboxyméthylcellulose (CMC), dans la phase solide du ciment a montré une très nette amélioration de l'injectabilité de la pâte, avec la disparition du phénomène de séparation des phases qui limite généralement l'injectabilité des ciments minéraux. La résistance à la compression et le module élastique des ciments composites ont été par ailleurs augmentés, parallèlement à une diminution de leur porosité. Différentes études in vitro en présence de cellules ou de bactéries ont enfin été réalisées et ont mis en évidence respectivement la cytocompatibilité des différentes compositions de ciments étudiées et le caractère antibactérien de ces matériaux à partir d'une certaine concentration en argent. L'implantation in vivo de compositions choisies a présenté des résultats très prometteurs quant à la résorbabilité d'un ciment composite CaCO3 - CaP/CMC/Ag et à la néoformation osseuse.The present work concerns research and development of a material for bone substitution, enabling implantation through a mini-invasive surgery, limiting post-operative infections and whose resorbability is adapted to bone regeneration kinetics. This study focused on a calcium carbonate and phosphate based cement, whose setting reaction leads to the formation of a nanocrystalline apatite, similar to bone mineral. First, the setting kinetics and the reaction products were characterised using different techniques, especially X-ray diffraction and FTIR and solid-state NMR spectroscopies. A silver salt Ag3PO4 or AgNO3, chosen for their antibacterial properties was then introduced in the formulation. Its effect on the setting reaction kinetics was revealed by data processing of FTIR and NMR spectra and an original reaction mechanism which involves silver and nitrates in the formation of apatite was proposed. The addition of a polysaccharide, carboxymethylcellulose (CMC), in the solid phase of the cement showed a clear improvement of the injectability of the paste, preventing the occurrence of filter-pressing phenomenon, often limiting the injectability of mineral cements. The resistance to compressive strength and elastic modulus of the composite cement were also improved together with a decrease in their porosity. Different in vitro studies were carried out in the presence of cells or bacteria and demonstrated the cytocompatibility of different cement compositions and their antibacterial properties starting at a certain silver concentration, respectively. In vivo implantation of selected compositions showed promising results concerning resorbability of a composite CaCO3 - CaP/CMC/Ag cement and the associated bone neoformation.TOULOUSE-INP (315552154) / SudocSudocFranceF

ZnGa2O4:Cr3+: a new red long-lasting phosphor with high brightness

International audienc

Acta Biomaterialia

In the challenging quest for a solution to reduce the risk of implant-associated infections in bone substitution surgery, the use of silver ions is promising regarding its broad spectrum on planktonic, sessile as well as multiresistant bacteria. In view of controlling its delivery in situ at the desired dose, we investigated its encapsulation in carboxymethyl cellulose (CMC) microparticles by spray-drying and included the latter in the formulation of a self-setting calcium phosphate bone cement. We implemented an original step-by-step methodology starting from the in vitro study of the antibacterial properties and cytotoxicity of two silver salts of different solubility in aqueous medium and then in the cement to determine the range of silver loading able to confer anti-biofilm and non-cytotoxic properties to the biomaterial. A dose-dependent efficiency of silver was demonstrated on the main species involved in bone-implant infection (S. aureus and S. epidermidis). Loading silver in microspheres instead of loading it directly inside the cement permitted to avoid undesired silver-cement interactions during setting and led to a faster release of silver, i.e. to a higher dose released within the first days combining anti-biofilm activity and preserved cytocompatibility. In addition, a combined interest of the introduction of about 10% (w/w) silver-loaded CMC microspheres in the cement formulation was demonstrated leading to a fully injectable and highly porous (77%) cement, showing a compressive strength analogous to cancellous bone. This injectable silver-loaded biomimetic composite cement formulation constitutes a versatile bone substitute material with tunable drug delivery properties, able to fight against bone implant associated infection. Statement of significance This study is based on two innovative scientific aspects regarding the literature: i) Choice of silver ions as antibacterial agent combined with their way of incorporation: Carboxymethylcellulose has never been tested into bone cement to control its drug loading and release properties. ii) Methodology to formulate an antibacterial and injectable bone cement: original and multidisciplinary step-by-step methodology to first define, through (micro)biological tests on two silver salts with different solubilities, the targeted range of silver dose to include in carboxymethylcellulose microspheres and, then optimization of silver-loaded microparticles processing to fulfill requirements (encapsulation efficiency and size). The obtained fully injectable composite controls the early delivery of active dose of silver (from 3 h and over 2 weeks) able to fight against bone implant-associated infections

Composition and properties of silver-containing calcium carbonate–calcium phosphate bone cement

The introduction of silver, either in the liquid phase (as silver nitrate solution: Ag(L)) or in the solid phase (as silver phosphate salt: Ag(S)) of calcium carbonate–calcium phosphate (CaCO3–CaP) bone cement, its influence on the composition of the set cement (C-Ag(L)and C-Ag(S) cements with a Ca/Ag atomic ratio equal to 10.3) and its biological properties were investigated. The fine characterisation of the chemical setting of silver-doped and reference cements was performed using FTIR spectroscopy. We showed that the formation of apatite was enhanced from the first hours of maturation of C-Ag(L) cement in comparison with the reference cement, whereas a longer period of maturation (about 10 h) was required to observe this increase for C-Ag(S) cement, although in both cases, silver was present in the set cements mainly as silver phosphate. The role of silver nitrate on the setting chemical reaction is discussed and a chemical scheme is proposed. Antibacterial activity tests (S. aureus and S. epidermidis) and in vitro cytotoxicity tests (human bone marrow stromal cells (HBMSC)) showed that silver-loaded CaCO3–CaP cements had antibacterial properties (anti-adhesion and anti-biofilm formation) without a toxic effect on HBMSC cells, making C-Ag(S) cement a promising candidate for the prevention of bone implant-associated infections