Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment.

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Apatite/Chitosan Composites Formed by Cold Sintering for Drug Delivery and Bone Tissue Engineering Applications.

In the biomedical field, nanocrystalline hydroxyapatite is still one of the most attractive candidates as a bone substitute material due to its analogies with native bone mineral features regarding chemical composition, bioactivity and osteoconductivity. Ion substitution and low crystallinity are also fundamental characteristics of bone apatite, making it metastable, bioresorbable and reactive. In the present work, biomimetic apatite and apatite/chitosan composites were produced by dissolution-precipitation synthesis, using mussel shells as a calcium biogenic source. With an eye on possible bone reconstruction and drug delivery applications, apatite/chitosan composites were loaded with strontium ranelate, an antiosteoporotic drug. Due to the metastability and temperature sensitivity of the produced composites, sintering could be carried out by conventional methods, and therefore, cold sintering was selected for the densification of the materials. The composites were consolidated up to ~90% relative density by applying a uniaxial pressure up to 1.5 GPa at room temperature for 10 min. Both the synthesised powders and cold-sintered samples were characterised from a physical and chemical point of view to demonstrate the effective production of biomimetic apatite/chitosan composites from mussel shells and exclude possible structural changes after sintering. Preliminary in vitro tests were also performed, which revealed a sustained release of strontium ranelate for about 19 days and no cytotoxicity towards human osteoblastic-like cells (MG63) exposed up to 72 h to the drug-containing composite extract.

Consolidation of Spray-Dried Amorphous Calcium Phosphate by Ultrafast Compression: Chemical and Structural Overview.

A large amount of research in orthopedic and maxillofacial domains is dedicated to the development of bioactive 3D scaffolds. This includes the search for highly resorbable compounds, capable of triggering cell activity and favoring bone regeneration. Considering the phosphocalcic nature of bone mineral, these aims can be achieved by the choice of amorphous calcium phosphates (ACPs). Because of their metastable property, these compounds are however to-date seldom used in bulk form. In this work, we used a non-conventional "cold sintering" approach based on ultrafast low-pressure RT compaction to successfully consolidate ACP pellets while preserving their amorphous nature (XRD). Complementary spectroscopic analyses (FTIR, Raman, solid-state NMR) and thermal analyses showed that the starting powder underwent slight physicochemical modifications, with a partial loss of water and local change in the HPO42- ion environment. The creation of an open porous structure, which is especially adapted for non-load bearing bone defects, was also observed. Moreover, the pellets obtained exhibited sufficient mechanical resistance allowing for manipulation, surgical placement and eventual cutting/reshaping in the operation room. Three-dimensional porous scaffolds of cold-sintered reactive ACP, fabricated through this low-energy, ultrafast consolidation process, show promise toward the development of highly bioactive and tailorable biomaterials for bone regeneration, also permitting combinations with various thermosensitive drugs.

One-Pot Synthesis of Bioinspired Peptide-Decorated Apatite Nanoparticles for Nanomedicine.

Hybrid organic-inorganic bio-inspired apatite nanoparticles (NPs) are attractive for biomedical applications and especially in nanomedicine. Unfortunately, their applications in nanomedicine are limited by their broad particle size distributions and uncontrolled drug loading due to their multistep synthesis process.  Besides, very few attempts at exposing bioactive peptides on apatite NPs are made. In this work, an original one-pot synthesis of well-defined bioactive hybrid NPs composed of a mineral core of bioinspired apatite surrounded by an organic corona of bioactive peptides is reported. Dual stabilizing-bioactive agents, phosphonated polyethylene glycol-peptide conjugates, are prepared and directly used during apatite precipitation i) to form the organic corona during apatite precipitation, driving the size and shape of resulting hybrid NPs with colloidal stabilization and ii) to expose peptide moieties (RGD or YIGSR sequences) at the NPs periphery in view of conferring additional surface properties to enhance their interaction with cells. Here, the success of this approach is demonstrated, the functionalized NPs are fully characterized by Fourier-transform infrared, Raman, X-ray diffraction, solid and liquid state NMR, transmission electron microscopy, and dynamic light scattering, and their interaction with fibroblast cells is followed, unveiling a synergistic proliferative effect.

Long-Term Fate and Efficacy of a Biomimetic (Sr)-Apatite-Coated Carbon Patch Used for Bone Reconstruction.

Critical bone defect repair remains a major medical challenge. Developing biocompatible materials with bone-healing ability is a key field of research, and calcium-deficient apatites (CDA) are appealing bioactive candidates. We previously described a method to cover activated carbon cloths (ACC) with CDA or strontium-doped CDA coatings to generate bone patches. Our previous study in rats revealed that apposition of ACC or ACC/CDA patches on cortical bone defects accelerated bone repair in the short term. This study aimed to analyze in the medium term the reconstruction of cortical bone in the presence of ACC/CDA or ACC/10Sr-CDA patches corresponding to 6 at.% of strontium substitution. It also aimed to examine the behavior of these cloths in the medium and long term, in situ and at distance. Our results at day 26 confirm the particular efficacy of strontium-doped patches on bone reconstruction, leading to new thick bone with high bone quality as quantified by Raman microspectroscopy. At 6 months the biocompatibility and complete osteointegration of these carbon cloths and the absence of micrometric carbon debris, either out of the implantation site or within peripheral organs, was confirmed. These results demonstrate that these composite carbon patches are promising biomaterials to accelerate bone reconstruction.

Exploring the Formation Kinetics of Octacalcium Phosphate from Alpha-Tricalcium Phosphate: Synthesis Scale-Up, Determination of Transient Phases, Their Morphology and Biocompatibility.

Even with decades of research studies behind octacalcium phosphate (OCP), determination of OCP phase formation has proved to be a cumbersome challenge. Even though obtaining a large quantity of OCP is important for potential clinical uses, it still remains a hindrance to obtain high yields of pure OCP. Taking that into consideration, the purpose of this study was to scale-up OCP synthesis for the first time and to use a multi-technique approach to follow the phase transformation pathway at multiple time points. In the present study, OCP has been synthesized from α-tricalcium phosphate (α-TCP), and subsequently scaled-up tenfold and hundredfold (100 mg → 10 g). The hydrolysis mechanism has been followed and described by using XRD and FTIR spectroscopy, as well as Raman and SEM. Gradual transformation into the OCP phase transpired through dicalcium phosphate dihydrate (brushite, DCPD, up to ~36%) as an intermediary phase. Furthermore, the obtained transitional phases and final OCP phases (across all scale-up levels) were tested with human bone marrow-derived mesenchymal stem cells (hBMSCs), in order to see how different phase mixtures affect the cell viability, and also to corroborate the safety of the scaled-up product. Twelve out of seventeen specimens showed satisfactory percentages of cell viability and confirmed the prospective use of scaled-up OCP in further in vitro studies. The present study, therefore, provides the first scale-up process of OCP synthesis, an in depth understanding of the formation pathway, and investigation of the parameters able to contribute in the OCP phase formation.

Influence of Water on an Epoxy/Amine-Metal Interphase: A Combined DFT and Mixing Calorimetry Approach.

Epoxy-amine systems are ubiquitous in the field of industrial thermosetting polymers, often used in a moist atmosphere. In addition, previous studies showed amine-metal interactions through the formation of an interphase, with the formation of surface complexes that may involve the formation of water molecules. However, to date, the impact of water on an epoxy/amine-metal interphase has not been specifically addressed. In this work, we examined for the first time the role of this potential fourth component by way of a dual experimental/computational approach. The effect of water on the glass-transition temperature of the obtained polymers was quantified. The in situ formation of a DETA-Al-water interphase was followed by mixing calorimetry. The DETA-water interaction was highly exothermic, and the underlying mechanism was discussed on the basis of DETA hydration, which was confirmed by density functional theory (DFT) and Monte Carlo simulations. Taking into account the pre-existing interaction between diethylenetriamine (DETA) molecules allowed us to model all experimental data. Comparison of experimental and calculated IR spectra contributed to validate the simulation parameters used. Our findings indicate that the presence of water may noticeably affect epoxy-amine-based systems. Mixing calorimetry and computational modeling appear as particularly adapted tools for the comprehension of such complex systems.

Ozonized 2-hydroxypropyl-ß-cyclodextrins as novel materials with oxidative and bactericidal properties.

Ozonized (2-Hydroxypropyl)-ß-cyclodextrins (Oz-HPbCDs) were produced by direct gas/solid reaction between gaseous ozone (O3) and solid HPbCD. The solid materials obtained were first characterized using physical and chemical methods and compared to the initial HPbCD. The main process parameters of the synthesis were studied independently to assess their effect on the oxidizing power of Oz-HPbCDs. The ability of the Oz-HPbCDs to retain their oxidative properties over time was evaluated, at different storage temperatures, for a period of at least two months. Lastly, aqueous solutions of HPbCD and Oz-HPbCD at different concentrations were contacted with bacterial strains of Escherichia coli and Streptococcus uberis to see whether these materials might have bactericidal properties. Since normal bacterial growth was noted with HPbCD, the antimicrobial efficiency of Oz-HPbCDs was clearly demonstrated on these two types of bacteria.

Bio-inspired apatite particles limit skin penetration of drugs for dermatology applications.

Most treatments of skin pathologies involve local administration of active agents. One issue can however be the partial transcutaneous diffusion of the drug to blood circulation, leading to undesirable effects. In this work, the original use of submicron mineral particles based on bio-inspired calcium phosphate apatite was explored for the first time as drug carriers for favoring topical delivery. The permeation of a model drug across synthetic and biological membranes was investigated in both static and dynamic conditions. Our data show that adsorption of the drug on the apatite particles surface drastically limits its permeation, with lower effective diffusion coefficients (Peff) and smaller total released amounts. The retention of the apatite colloidal particles on porcine ear skin explants surface was demonstrated by combining histological observations and Raman confocal microscopy. All results converge to show that association of the drug to apatite particles favors skin surface effects. These findings point to the relevance of mineral-based particles as drug carriers for local delivery to the skin, and open the way to novel applications of bio-inspired apatites in dermatology. STATEMENT OF SIGNIFICANCE: Calcium phosphates (CaP) are major biomaterials in orthopedics and dentistry. Their resemblance to bone mineral allows new applications beyond bone repair, e.g. in nanomedicine. In 2018, a 14-page detailed review (M. Epple, Acta Biomaterialia 77 (2018) 1-14) provided clear facts in favor of the non-toxicity of nanosized CaP as an answer to discussions from EU and US study groups, thus clarifying the path to novel applications of nano CaP. In the present paper, bio-inspired apatite nanoparticles are used for the first time as drug carriers for dermatology for drastically limiting drug transcutaneous permeation and retaining a topical effect. We demonstrate this proof of concept via permeation cell tests, histology, Raman microscopy and photoluminescence after application on porcine ear skin.

A soft-chemistry approach to the synthesis of amorphous calcium ortho/pyrophosphate biomaterials of tunable composition.

The development of amorphous phosphate-based materials is of major interest in the field of biomaterials science, and especially for bone substitution applications. In this context, we herein report the synthesis of gel-derived hydrated amorphous calcium/sodium ortho/pyrophosphate materials at ambient temperature and in water. For the first time, such materials have been obtained in a large range of tunable orthophosphate/pyrophosphate molar ratios. Multi-scale characterization was carried out thanks to various techniques, including advanced multinuclear solid state NMR. It allowed the quantification of each ionic/molecular species leading to a general formula for these materials: [(Ca2+y Na+z H+3+x-2y-z)(PO43-)1-x(P2O74-)x](H2O)u. Beyond this formula, the analyses suggest that these amorphous solids are formed by the aggregation of colloids and that surface water and sodium could play a role in the cohesion of the whole material. Although the full comprehension of mechanisms of formation and structure is still to be investigated in detail, the straightforward synthesis of these new amorphous materials opens up many perspectives in the field of materials for bone substitution and regeneration. STATEMENT OF SIGNIFICANCE: The metastability of amorphous phosphate-based materials with various chain length often improves their (bio)chemical reactivity. However, the control of the ratio of the different phosphate entities has not been yet described especially for small ions (pyrophosphate/orthophosphate) and using soft chemistry, whereas it opens the way for the tuning of enzyme- and/or pH-driven degradation and biological properties. Our study focuses on elaboration of amorphous gel-derived hydrated calcium/sodium ortho/pyrophosphate solids at 70 °C with a large range of orthophosphate/pyrophosphate ratios. Multi-scale characterization was carried out using various techniques such as advanced multinuclear SSNMR (31P, 23Na, 1H, 43Ca). Analyses suggest that these solids are formed by colloids aggregation and that the location of mobile water and sodium could play a role in the material cohesion.

Multifunctionalization Modulates Hydroxyapatite Surface Interaction with Bisphosphonate: Antiosteoporotic and Antioxidative Stress Materials.

Multifunctionalized biomaterials with enhanced bone antiresorptive properties were obtained through adsorption of a bisphosphonate, risedronate, on hydroxyapatite (HA) nanocrystals functionalized with zinc ions and polyethylenimine (PEI). Zn incorporation into the HA structure amounts to about 8 atom %, whereas the PEI content of the bifunctionalized material ZnHAPEIBP is about 5.9 wt %. The mechanism of adsorption and release of the bisphosphonate on ZnHAPEI is compared with that on ZnHA: risedronate adsorption isotherm on ZnHA is a Langmuir type, whereas the isotherm of adsorption on ZnHAPEI is better fitted with a Freundlich model and involved a higher amount of adsorbed risedronate. In vitro cell tests were carried out with a coculture model of osteoblasts and osteoclasts using a model simulating oxidative stress and consequent cellular senescence and osteoporosis by the addition of H2O2. The conditions utilized in the coculture model strongly affect osteoblast behavior. The results show that the composite materials allow an increase in osteoblast viability and recover impairment, revealing a novel characteristic of risedronate that is able to counteract the negative effects of oxidative stress when associated with differently functionalized samples. Both PEI and the bisphosphonate reduce osteoclast viability. Moreover, PEI, and even more risedronate, exerts an inhibitory effect on osteoclast activity.

Interaction of Folic Acid with Nanocrystalline Apatites and Extension to Methotrexate (Antifolate) in View of Anticancer Applications.

Nanocrystalline apatites mimicking bone mineral represent a versatile platform for biomedical applications thanks to their similarity to bone apatite and the possibility to (multi)functionalize them so as to provide "à la carte" properties. One relevant domain is in particular oncology, where drug-loaded biomaterials and engineered nanosystems may be used for diagnosis, therapy, or both. In a previous contribution, we investigated the adsorption of doxorubicin onto two nanocrystalline apatite substrates, denoted HA and FeHA (superparamagnetic apatite doped with iron ions), and explored these drug-loaded systems against tumor cells. To widen their applicability in the oncology field, here we examine the interaction between the same two substrates and two other molecules: folic acid (FA), often used as cell targeting agent, and the anticancer drug methotrexate (MTX), an antifolate analogue. In a first stage, we investigated the adsorptive behavior of FA (or MTX) on both substrates, evidencing their specificities. At low concentration, typically under 100 mmol/L, adsorption onto HA was best described using the Sips isotherm model, while the formation of a calcium folate secondary salt was evidenced at high concentration by Raman spectroscopy. Adsorption onto FeHA was instead fitted to the Langmuir model. A larger adsorptive affinity was found for the FeHA substrate compared to HA; accordingly, a faster release was noticed from HA. In vitro tests carried out on human osteosarcoma cell line (SAOS-2) allowed us to evaluate the potential of these compounds in oncology. Finally, in vivo (subcutaneous) implantations in the mouse were run to ascertain the biocompatibility of the two substrates. These results should allow a better understanding of the interactions between FA/MTX and bioinspired nanocrystalline apatites in view of applications in the field of cancer.

Hydroxyapatite functionalization to trigger adsorption and release of risedronate.

Bisphosphonates are widely employed drugs for the treatment of pathologies characterized by excessive bone resorption, and display a great affinity for apatitic supports. In this work we investigate how hydroxyapatite functionalization can influence the processes of adsorption and release of a bisphosphonate, namely risedronate. To this aim, pure hydroxyapatite (HA), hydroxyapatite with a partial substitution of Zn to Ca (ZnHA) and poly-ethylenimine-functionalized hydroxyapatite (HAPEI) were submitted to interaction with risedronate solution. The results indicate that the mechanisms of adsorption and release are greatly influenced by the type of the apatitic support. All the apatitic supports display Langmuir isotherms for risedronate adsorption. However in the case of HAPEI the plateau is not reached even at high equilibrium concentrations in solution. The data suggest that risedronate adsorption on HAPEI mineral-organic support occurs not only through chemisorption on apatitic phase, as on HA and ZnHA, but also through physisorption involved by PEI coating, which modulates also bisphosphonate release. These properties of tailor-made hydroxyapatite supports could be exploited to develop delivery systems for antiresorptive agents directly on osteoporotic sites.

Development of a new family of monolithic calcium (pyro)phosphate glasses by soft chemistry.

UNLABELLED: The development of bioactive phosphate-based glasses is essential in biomaterials science, and especially for bone substitution applications. In this context, the preparation of amorphous calcium-phosphorus hydroxide/oxide monoliths at low temperature is a key challenge for being able to develop novel hybrid materials for these applications. We herein report for the first time the synthesis and physical chemical characterisation of a novel family of pyrophosphate-based glasses (with the formula: {[(Ca(2+))1-x(H(+)/K(+))2x]2[(P2O7(4-))1-y(PO4(3-))4y/3]} n(H2O)), which were prepared by soft chemistry using low temperatures (T<70°C) and water as a solvent. The effect of the initial Ca/Pyrophosphate ratio on the structure and morphology of these pyrophosphate glasses was investigated in detail. Depending on this ratio, a glass (mixed calcium pyro- and orthophosphate) or a glass-ceramic (Ca10K4(P2O7)6·9H2O crystals embedded in the amorphous phase) was obtained. The proportion of the crystalline phase increased with an increase in the Ca/Pyrophosphate ratio in the batch solution. As expected for a glass, the formation of the glassy material was demonstrated not to be thermodynamically but rather kinetically driven, and the washing step was found to be crucial to prevent crystallisation. The stability of the amorphous phase was discussed considering the structural degrees of freedom of pyrophosphate entities, ionic strength of the initial solution and the inhibitory effect of orthophosphate ions. Overall, this new strategy of preparation of monolithic calcium-(pyro)phosphate based glasses using soft chemistry in water is highly promising in view of preparing new functional organic-inorganic hybrids for bone substitution applications. STATEMENT OF SIGNIFICANCE: Phosphate-based glasses have gradually emerged as a potential alternative to silicate bioactive glasses in order to induce different biological mechanisms of degradation. The synthesis of such monolithic glasses at low temperature is a key step to allow new inorganic glass compositions to be reached and hybrid materials to be prepared. Although sol-gel and coacervate methods (respectively orthophosphate and metaphosphate precursors) have already been described to prepare such glasses, the use of toxic solvents and/or the final temperature treatment associated to these processes could limit the use of these materials for biomedical applications and/or the further development of hybrids. It is shown here that pyrophosphate precursors are an alternative strategy to obtain monolithic calcium (pyro)phosphate glasses under soft conditions (water solvent, 70°C).

Adsorption of tranexamic acid on hydroxyapatite: Toward the development of biomaterials with local hemostatic activity.

This work proposes to combine tranexamic acid (TAX), a clinically used antifibrinolytic agent, and hydroxyapatite (HA), widely used in bone replacement, to produce a novel bioactive apatitic biomaterial with intrinsic hemostatic properties. The aim of this study was to investigate adsorptive behavior of the TAX molecule onto HA and to point out its release in near physiological conditions. No other phase was observed by X-ray diffraction or transmission electron microscopy, and no apparent change in crystal size was detected. The presence of TAX on the powders was lightly detected on Raman spectra after adsorption. The adsorption data could be fitted with a Langmuir-Freundlich equation, suggesting a strong interaction between adsorbed molecules and the formation of multilayers. The concentration of calcium and phosphate ions in solution remained low and stable during the adsorption process, thus ion exchange during the adsorption process could be ruled out. The release of TAX was fast during the first hours and was governed by a complex process that likely involved both diffusion and dissolution of HA. Preliminary aPTT (activated partial thromboplastin time) hemostasis tests offered promising results for the development of osteoconductive apatitic biomaterials with intrinsic hemostatic properties, whether for dental or orthopedic applications.

Comparison of Physical-chemical and Mechanical Properties of Chlorapatite and Hydroxyapatite Plasma Sprayed Coatings.

Chlorapatite can be considered a potential biomaterial for orthopaedic applications. Its use as plasma-sprayed coating could be of interest considering its thermal properties and particularly its ability to melt without decomposition unlike hydroxyapatite. Chlorapatite (ClA) was synthesized by a high-temperature ion exchange reaction starting from commercial stoichiometric hydroxyapatites (HA). The ClA powder showed similar characteristics as the original industrial HA powder, and was obtained in the monoclinic form. The HA and ClA powders were plasma-sprayed using a low-energy plasma spraying system with identical processing parameters. The coatings were characterized by physical-chemical methods, i.e. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy, including distribution mapping of the main phases detected such as amorphous calcium phosphate (ACP), oxyapatite (OA), and HA or ClA. The unexpected formation of oxyapatite in ClA coatings was assigned to a side reaction with contaminating oxygenated species (O2, H2O). ClA coatings exhibited characteristics different from HA, showing a lower content of oxyapatite and amorphous phase. Although their adhesion strength was found to be lower than that of HA coatings, their application could be an interesting alternative, offering, in particular, a larger range of spraying conditions without formation of massive impurities.

Strontium-loaded mineral bone cements as sustained release systems: Compositions, release properties, and effects on human osteoprogenitor cells.

This study aims to evaluate in vitro the release properties and biological behavior of original compositions of strontium (Sr)-loaded bone mineral cements. Strontium was introduced into vaterite CaCO3 -dicalcium phosphate dihydrate cement via two routes: as SrCO3 in the solid phase (SrS cements), and as SrCl2 dissolved in the liquid phase (SrL cements), leading to different cement compositions after setting. Complementary analytical techniques implemented to thoroughly investigate the release/dissolution mechanism of Sr-loaded cements at pH 7.4 and 37°C during 3 weeks revealed a sustained release of Sr and a centripetal dissolution of the more soluble phase (vaterite) limited by a diffusion process. In all cases, the initial burst of the Ca and Sr release (highest for the SrL cements) that occurred over 48 h did not have a significant effect on the expression of bone markers (alkaline phosphatase, osteocalcin), the levels of which remained overexpressed after 15 days of culture with human osteoprogenitor (HOP) cells. At the same time, proliferation of HOP cells was significantly higher on SrS cements. Interestingly, this study shows that we can optimize the sustained release of Sr(2+) , the cement biodegradation and biological activity by controlling the route of introduction of strontium in the cement paste.