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.

Growth on poly(L-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells.

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Natural, biphasic calcium phosphate from fish bones for enamel remineralization and dentin tubules occlusion.

OBJECTIVES: A calcium phosphate extracted from fish bones (CaP-N) was evaluated for enamel remineralization and dentinal tubules occlusion. METHODS: CaP-N was characterized by assessing morphology by SEM, crystallinity by PXRD, and composition by ICP-OES. CaP-N morphology, crystallinity, ion release, and pH changes over time in neutral and acidic solutions were studied. CaP-N was then tested to assess remineralization and dentinal tubules occlusion on demineralized human enamel and dentin specimens (n = 6). Synthetic calcium phosphate in form of stoichiometric hydroxyapatite nanoparticles (CaP-S) and tap water were positive and negative controls, respectively. After treatment (brush every 12 h for 5d and storage in Dulbecco's modified PBS), specimens' morphology and surface composition were assessed (by SEM-EDS), while the viscoelastic behavior was evaluated with microindentation and DMA. RESULTS: CaP-N consisted of rounded microparticles (200 nm - 1 µm) composed of 33 wt% hydroxyapatite and 67 wt% ß-tricalcium phosphate. In acidic solution, CaP-N released calcium and phosphate ions thanks to the preferential ß-tricalcium phosphate phase dissolution. Enamel remineralization was induced by CaP-N comparably to CaP-S, while CaP-N exhibited a superior dentinal tubule occlusion than CaP-S, forming mineral plugs and depositing new nanoparticles onto demineralized collagen. This behavior was attributed to its bigger particle size and increased solubility. DMA depth profiling and SEM showed an excellent interaction between the newly formed mineralized structures and the pristine tissue, particularly at the exposed collagen fibrils. SIGNIFICANCE: CaP-N demonstrated very good remineralizing and occlusive activity in vitro, comparable to CaP-S, thus could be a promising circular economy alternative therapeutic agent for dentistry.

Preparation of core-shell poly(L-lactic) acid-nanocrystalline apatite hollow microspheres for bone repairing applications.

In this paper, hybrid inorganic-organic core-shell hollow microspheres, made of poly(L-lactic acid) (PLLA) and biomimetic nano apatites (HA), were prepared from biodegradable and biocompatible substances, suitable for bone tissue applications. Preparation is started from Pickering emulsification, i.e., solid particle-stabilized emulsions in the absence of any molecular surfactant, where solid particles adsorbed to an oil-water interface. Stable oil-in-water emulsions were produced using biomimetic 20 nm sized HA nanocrystals as particulate emulsifier and a dichloromethane (CH(2)Cl(2)) solution of PLLA as oil phase. Hybrid hollow PLLA microspheres at three different HA nanocrystals surface coverage, ranging from 10 to 50 µm, were produced. The resulting materials were completely characterized with spectroscopic, calorimetric and microscopic techniques and the cytocompatibility was established by indirect contact tests with both fibroblasts and osteoblasts and direct contact with these latter. They displayed a high level of cytocompatibility and thus represent promising materials for drug delivery systems, cell carriers and scaffolds for regeneration of bone useful in the treatment of orthopaedic, maxillofacial and dental fields.

Amorphous calcium phosphate, the lack of order is an abundance of possibilities.

For almost three decades from its discovery, amorphous calcium phosphate (ACP) was not considered a suitable biomaterial due to its structural instability. Thanks to its unique properties in respect to crystalline calcium phosphate phases, nowadays ACP is used in promising devices for hard tissue regeneration. Here we have highlighted the features of ACP that were harnessed to create excellent biomaterials for dental remineralization, self-setting bone cements, drug delivery, and coatings of prostheses. Its current limitations as well as future perspectives of development were concisely described. Despite more research works are needed, we envisage that the future of ACP is bright.

Dental tissue remineralization by bioactive calcium phosphate nanoparticles formulations.

Recent health care products are based on formulations claimed to provide enamel remineralization and dentinal tubules occlusion through calcium-phosphate bioactive nanocompounds (ion-doped hydroxyapatite and precursor, amorphous calcium phosphate nanoparticles). This study aimed to characterize, test, and compare for the first time the structure and performance of a representative, market-available sample of remineralizing toothpastes and topical mousses. Formulations were characterized to determine their composition and investigate the presence of bioactive compounds and doping elements. A conventional fluoride-containing toothpaste was used as reference. The enamel remineralization and efficacy of dentinal tubules occlusion by tested formulations were investigated ex vivo on human hard tissues. All formulations containing Ca-P bioactive nanocompounds showed remineralizing ability by epitaxial growth of a layer showing the morphology and composition of human hydroxyapatite. Such layers also embedded nanosilica clusters. The presence of doping elements or casein phosphopeptide seemed essential to allow such performances, especially when hydroxyapatite and amorphous calcium phosphate compounds were doped with small amounts of CO32-, F-, Mg2+, and Sr2+. Topical mousse formulations showed a higher tubules occlusion capability than toothpastes, independently from their composition. Therefore, all tested formulations could be useful in restoring tooth structures in a biomimetic way, contrasting dental demineralization processes leading to caries.

Thermal crystallization of amorphous calcium phosphate combined with citrate and fluoride doping: a novel route to produce hydroxyapatite bioceramics.

Amorphous calcium phosphate (ACP) is a material of high interest for dentistry, orthopedics, and other biomedical sectors. Being intrinsically metastable, the process of transformation of ACP into a crystalline phase upon heating is of high relevance for the development of innovative bioceramics. Here we have first studied the thermal behavior of a citrate-stabilized ACP (Cit-ACP) also doped with fluoride ions (Cit-FACP) prepared at three different nominal Cit/Ca ratios (i.e. 4, 2, 1) by differential thermal analysis. Next, the physico-chemical features of the crystalline products as well as the in vitro cell response to the materials were investigated. A citrate and fluoride free ACP sample was also tested as the blank. We have found that the activation energy of crystallization of Cit-(F)ACP samples is lower in comparison to the blank ACP and this is influenced by the nominal Cit/Ca molar ratio. Interestingly, we have discovered that the thermal treatment of Cit-(F)ACP at 800 °C yields hydroxyapatite (HA) or fluorapatite (FHA) as the main products differently from blank ACP that, like most of the ACPs reported in the literature, yields ß-tricalcium phosphate. This was attributed to the Ca/P ratio of Cit-(F)ACP, which is similar to HA. A study of the crystalline products has revealed that all the (F)HA samples were non-cytotoxic, and retained carbonate ions in the crystal structure despite the heat treatment that should have induced decarbonation. The morphology of the products is influenced by the nominal Cit/Ca ratio and the presence of fluoride, ranging from spherical nanoparticles to micrometric hexagonal rods. Overall, our results prove that the thermal crystallization of Cit-(F)ACP is markedly different from classic ACP based materials and the thermal treatment of Cit-(F)ACP represents an attractive route for producing pure bioactive HA ceramics.

Characterization of a Toothpaste Containing Bioactive Hydroxyapatites and In Vitro Evaluation of Its Efficacy to Remineralize Enamel and to Occlude Dentinal Tubules.

Demineralization of dental hard tissues is a well-known health issue and the primary mechanism responsible for caries and dentinal hypersensitivity. Remineralizing toothpastes are nowadays available to improve conventional oral care formulations regarding the prevention and repair of demineralization. In this paper, we analyzed the chemical-physical features of a commercial toothpaste (Biosmalto Caries Abrasion and Erosion, Curasept S.p.A., Saronno, Italy), with particular attention paid to the water-insoluble fraction which contains the remineralizing bioactive ingredients. Moreover, the efficacy of the toothpaste to induce enamel remineralization and to occlude dentinal tubules has been qualitatively and semiquantitatively tested in vitro on human dental tissues using scanning electron microscopy and X-ray microanalysis. Our results demonstrated that the water-insoluble fraction contained silica as well as chitosan and poorly crystalline biomimetic hydroxyapatite doped with carbonate, magnesium, strontium, and fluoride ions. The formulation showed excellent ability to restore demineralized enamel into its native structure by epitaxial deposition of a new crystalline phase in continuity with the native one. It was also able to occlude the dentinal tubules exposed completely by acid-etching. Overall, this study demonstrated that the tested toothpaste contained a biomimetic ionic-substituted hydroxyapatite-based active principle and that, within the in vitro conditions analyzed in this study, it was effective in dental hard tissue remineralization.

Superparamagnetic hybrid microspheres affecting osteoblasts behaviour.

The present work describes biomimetic hybrid microspheres made of collagen type I-like peptide matrix (RCP) mineralised with Fe2+/Fe3+ doping hydroxyapatite (RCPFeHA) by a bio-inspired process. Superparamagnetic RCPFeHA microspheres are obtained by emulsification of the hybrid slurries in the presence of citrate ions, to achieve a biomimetic surface functionalisation improving the bioactivity and the dispersion ability in cell culture medium. A biological in vitro study correlates the osteoblast cells behaviour to calcium and iron ions released by the hybrid microspheres in culture media mimicking physiological or inflammatory environment, evidencing a clear triggering of cell activity and bio-resorption ability. In presence of the microspheres, the osteoblast cells maintain their typical morphology and no cell damage were detected, whereas also showing up-regulation of osteogenic markers. The ability of the hybrid microspheres to undergo bio-resorption and release bioactive ions in response to different environmental stimuli without harmful effects opens new perspectives in bone regeneration, as magnetically active bone substitute with potential ability of drug carrier and smart response in the presence of inflammatory states.

Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remineralization.

Demineralization of dental hard tissue is a widespread problem and the main responsible for dental caries and dentin hypersensitivity. The most promising strategies to induce the precipitation of new mineral phase are the application of materials releasing gradually Ca2+ and PO43- ions or mimicking the mineral phase of the host tissue. However, the design of formulations covering both processes is so far a challenge in preventive dentistry. In this work, we have synthesized innovative biomimetic amorphous calcium phosphate (ACP), which has been, for the first time, doped with fluoride ions (FACP) to obtain materials with enhanced anti-caries and remineralizing properties. Significantly, the doping with fluoride (F) did not vary the physico-chemical features of ACP but resulted in a faster conversion to the crystalline apatite phase in water, as observed by in-situ time-dependent Raman experiments. The efficacy of the as synthesized ACP and FACP samples to occlude dentinal tubules and induce enamel remineralization has been tested in vitro in human molar teeth. The samples showed good ability to partially occlude the tubules of acid-etched dentin and to restore demineralized enamel into its native structure. Results demonstrate that ACP and FACP are promising biomimetic materials in preventive dentistry to hinder demineralization of dental hard tissues.

Polyester fibers can be rendered calcium phosphate-binding by surface functionalization with bisphosphonate groups.

Fibers are often used as structural elements to improve the mechanical properties of materials such as brittle ceramic matrices by facilitating the dissipation of energy. However, this energy dissipation is mainly controlled by the interface between the two components, and a poorly designed fiber-matrix interface strongly reduces the efficacy of fiber reinforcement. Here, we present a versatile approach to control the affinity of biocompatible fibers to calcium-containing matrices to maximize the efficacy of reinforcement of calcium phosphates-based bioceramics by means of polymeric fibers. To this end, polyester fibers of tunable length were produced by electrospinning and aminolysis, followed by covalent attachment of alendronate, a bisphosphonate molecule with strong calcium-binding affinity, to the surface of the fibers. The proposed method allowed for selective control over the amount of alendronate conjugation, thereby improving the affinity of polyester fibers toward calcium phosphate bioceramics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2335-2342, 2017.

Bioinspired negatively charged calcium phosphate nanocarriers for cardiac delivery of MicroRNAs.

AIM: To develop biocompatible and bioresorbable negatively charged calcium phosphate nanoparticles (CaP-NPs) as an innovative therapeutic system for the delivery of bioactive molecules to the heart. MATERIALS & METHODS: CaP-NPs were synthesized via a straightforward one-pot biomineralization-inspired protocol employing citrate as a stabilizing agent and regulator of crystal growth. CaP-NPs were administered to cardiac cells in vitro and effects of treatments were assessed. CaP-NPs were administered in vivo and delivery of microRNAs was evaluated. RESULTS: CaP-NPs efficiently internalized into cardiomyocytes without promoting toxicity or interfering with any functional properties. CaP-NPs successfully encapsulated synthetic microRNAs, which were efficiently delivered into cardiac cells in vitro and in vivo. CONCLUSION: CaP-NPs are a safe and efficient drug-delivery system for potential therapeutic treatments of polarized cells such as cardiomyocytes.

Controlled Release of Chemotherapeutic Platinum-Bisphosphonate Complexes from Injectable Calcium Phosphate Cements.

Herein, we present a method to release chemotherapeutic platinum-bisphosphonate (Pt-BP) complexes from apatitic calcium phosphate cements (CPCs). Pt-BP-loaded hydroxyapatite nanoparticles (HA NPs) were added at different ratios to the powder phase of the cements, which contained poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres as porogens to accelerate their degradation. In vitro release kinetics of Pt-BP complexes revealed that the release rate of Pt species can be tuned by varying the amount of drug-loaded HA NPs as well as modifying the chemical structure of the Pt-BP complex to tailor its affinity with HA NPs. In addition, the incorporation of PLGA microspheres into the CPCs increased the degradation rate of the materials without affecting the release rate of Pt species. Finally, the antiproliferative activity of the free Pt-BP complexes and Pt-BP-loaded CPCs was evaluated using both human osteosarcoma cancer cells (MG-63) and human bone marrow-derived mesenchymal stromal cells (h-BMMSCs). This study demonstrated that both free Pt-BP complexes and the releasates from the CPCs were antiproliferative in a dose-dependent manner. Moreover, their antiproliferative activity was higher on MG-63 cells compared to h-BMMSC primary cells. In summary, it was shown that injectable CPCs can be rendered chemotherapeutically active by incorporation of HA NPs loaded with HA-binding Pt-BP complexes.

Magnetic bioinspired hybrid nanostructured collagen-hydroxyapatite scaffolds supporting cell proliferation and tuning regenerative process.

A bioinspired mineralization process was applied to develop biomimetic hybrid scaffolds made of (Fe(2+)/Fe(3+))-doped hydroxyapatite nanocrystals nucleated on self-assembling collagen fibers and endowed with super-paramagnetic properties, minimizing the formation of potentially cytotoxic magnetic phases such as magnetite or other iron oxide phases. Magnetic composites were prepared at different temperatures, and the effect of this parameter on the reaction yield in terms of mineralization degree, morphology, degradation, and magnetization was investigated. The influence of scaffold properties on cells was evaluated by seeding human osteoblast-like cells on magnetic and nonmagnetic materials, and differences in terms of viability, adhesion, and proliferation were studied. The synthesis temperature affects mainly the chemical-physical features of the mineral phase of the composites influencing the degradation, the microstructure, and the magnetization values of the entire scaffold and its biological performance. In vitro investigations indicated the biocompatibility of the materials and that the magnetization of the super-paramagnetic scaffolds, induced applying an external static magnetic field, improved cell proliferation in comparison to the nonmagnetic scaffold.

Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats.

The prevalence of osteoporosis will increase within the next decades due to the aging world population, which can affect the bone healing response to dental and orthopedic implants. Consequently, local drug targeting of peri-implant bone has been proposed as a strategy for the enhancement of bone-implant integration in osteoporotic conditions. In the present study, an established in-vivo femoral condyle implantation model in osteoporotic and healthy bone is used to analyze the osteogenic capacity of titanium implants coated with bisphosphonate (BP)-loaded calcium phosphate nanoparticles (nCaP) under compromised medical conditions. After 4 weeks of implantation, peri-implant bone volume (%BV; by µCT) and bone area (%BA; by histomorphometry) were significantly increased within a distance of 500 µm from implant surfaces functionalized with BP compared to control implants in osteoporotic and healthy conditions. Interestingly, the deposition of nCaP/BP coatings onto implant surfaces increased both peri-implant bone contact (%BIC) and volume (%BV) compared to the deposition of nCaP or BP coatings individually, in osteoporotic and healthy conditions. The results of real-time PCR revealed similar osteogenic gene expression levels to all implant surfaces at 4-weeks post-implantation. In conclusion, simultaneous targeting of bone formation (by nCaP) and bone resorption (by BP) using nCaP/BP surface coatings represents an effective strategy for synergistically improvement of bone-implant integration, especially in osteoporotic conditions.

Bio-inspired citrate-functionalized apatite thin films crystallized on Ti-6Al-4V implants pre-coated with corrosion resistant layers.

In this paper the crystallization of a bioinspired citrate-functionalized apatite (cit-Ap) thin film (thickness about 2µm) on Ti-6Al-4V supports pre-coated with bioactive and corrosion resistant buffer layer of silicon nitride (Si3N4), silicon carbide (SiC) or titanium nitride (TiN) is reported. The apatitic coatings were produced by a new coating technique based on the induction heating of the implants immersed in a flowing calcium-citrate-phosphate solution at pH11. The influence of the buffer layers and the surface roughness of the substrate on the chemical-physical features and adhesion of the cit-Ap films were investigated. The best plasticity, compactness and adherence properties have been found in the Ap layer grown on Si3N4, followed by the Ap grown on SiC and TiN, respectively. The adhesion property was likely related to the roughness of the buffered substrates, whereas the compactness and plasticity were closely related to the operating conditions during the Ap crystallization (flow rate of the solution and increase of temperature) rather than to the nature of the buffer layer.

Silica xerogels and hydroxyapatite nanocrystals for the local delivery of platinum-bisphosphonate complexes in the treatment of bone tumors: a mini-review.

The present review focuses on the "drug targeting and delivery" approach of the selective transportation of cisplatin to bone tumors and bone metastases. This aim is realized by binding cisplatin to (bis)phosphonate ligands or their derivatives. Geminal bisphosphonates are in clinical use in the treatment of several bone-related diseases because of their high affinity for calcium ions and hence for bones. Platinum-bisphosphonate complexes may be easily loaded onto calcium-containing inorganic matrices, such as calcium-doped sol-gel derived silica xerogels and hydroxyapatite nanocrystals, for local administration at the site of the bone malignancy. The composites may be used as bone-filler materials that, in addition to their action as bone substitutes, can also act as controlled platinum-drug releasing agents. The release kinetics of the drug can be tailored for specific therapeutic applications modulating the physico-chemical features of the inorganic matrices. Moreover, apatite nanocrystals loaded with platinum-bisphosphonate prodrugs can be used as injectable material for nanomedical applications (e.g. intracellular drug delivery).

The binding of CNA35 contrast agents to collagen fibrils.

CryoTEM demonstrates that a CNA35-bearing liposomal MRI contrast agent selectively binds to poorly assembled collagen type I as opposed to well-assembled collagen fibrils, whereas monomeric CNA35 binds to all forms of collagen. It is shown that upon conjugation to liposomes and micelles CNA35 loses its ability to dissociate ordered collagen fibrils and thereby to create its own binding sites.

Evolving application of biomimetic nanostructured hydroxyapatite.

By mimicking Nature, we can design and synthesize inorganic smart materials that are reactive to biological tissues. These smart materials can be utilized to design innovative third-generation biomaterials, which are able to not only optimize their interaction with biological tissues and environment, but also mimic biogenic materials in their functionalities. The biomedical applications involve increasing the biomimetic levels from chemical composition, structural organization, morphology, mechanical behavior, nanostructure, and bulk and surface chemical-physical properties until the surface becomes bioreactive and stimulates cellular materials. The chemical-physical characteristics of biogenic hydroxyapatites from bone and tooth have been described, in order to point out the elective sides, which are important to reproduce the design of a new biomimetic synthetic hydroxyapatite. This review outlines the evolving applications of biomimetic synthetic calcium phosphates, details the main characteristics of bone and tooth, where the calcium phosphates are present, and discusses the chemical-physical characteristics of biomimetic calcium phosphates, methods of synthesizing them, and some of their biomedical applications.

Controlled release of vancomycin from cross-linked gelatine.

This paper explores the possibility of using biodegradable cross-linked gelatines as antibiotic devices for a long-term elution (80 days). Capillary electrophoresis (CE) has been utilized to evaluate the mass percentage of vancomycin and gelatine contemporary released from differently cross-linked vancomycin loaded gelatine samples in an elution time ranging from 24 to 1920 h. While the solubilization kinetic of gelatine samples differently cross-linked can be very close described by the simplified Higuchi model, the vancomycin release kinetic is contemporary governed by both the Fickian diffusion process trough the gelatine matrix network and the dissolution process of the matrix due to its degradation. Comparing the antibiotic eluting kinetics from gelatine at diverse cross-linking degree we observed that the degradation of the proteic matrix appears to have a minor influence in the drug release control. Vancomycin released from all the gelatine partially cross-linked samples results active against Staphylococcus aureus and Streptococcus faecalis which represent the most pathogens commonly isolated in orthopaedic infections. Vancomycin overcomes the minimum inhibitory concentration for both the bacteria in the whole range of elution time. Cross-linked gelatine devices appear to represent a useful biodegradable delivery system for local anti-infective therapy in arthoplasty.