An in situ study of thermal crystallization of amorphous calcium phosphates.

The heat-induced crystallization of amorphous calcium phosphate (ACP) is an intriguing process not yet well comprehended. This is because most of the works on this topic are based on ex situ studies where the materials are characterized after the heat and cooldown cycles, thus missing transient structural changes. Here, we used time-resolved energy dispersive X-ray diffraction and infrared spectroscopy to study, for the first time, the thermal crystallization of ACP in situ. The thermal crystallization of two kinds of citrate-stabilized carbonated ACP was studied, as they are promising materials for the preparation of advanced bioceramics. The behavior of these samples was compared to that of two citrate-free ACPs, either doped or non-doped with carbonate ions. Our results evinced that several phenomena occur during ACP thermal annealing. Before crystallization, all ACP samples undergo a decrease in the short-range order process, followed by several internal reorganizations. We have assessed that differently from carbonate-free ACP, carbonated ACPs with and without citrate directly crystallize into a biomimetic poorly crystalline carbonated hydroxyapatite. Citrate-stabilized ACPs in comparison to citrate-free ACPs have a faster hydroxyapatite formation kinetics, which is due to their higher specific surface area. This work reveals the necessity and the potentialities of using in situ techniques to effectively probe complex processes such as the heat-induced crystallization of ACPs.

Nanotechnological approach and bio-inspired materials to face degenerative diseases in aging.

The aging of the world population is increasingly claimed as an alarming situation, since an ever-raising number of persons in advanced age but still physically active is expected to suffer from invalidating and degenerative diseases. The impairment of the endogenous healing potential provoked by the aging requires the development of more effective and personalized therapies, based on new biomaterials and devices able to direct the cell fate to stimulate and sustain the regrowth of damaged or diseased tissues. To obtain satisfactory results, also in cases where the cell senescence, typical of the elderly, makes the regeneration process harder and longer, the new solutions have to possess excellent ability to mimic the physiological extracellular environment and thus exert biomimetic stimuli on stem cells. To this purpose, the "biomimetic concept" is today recognized as elective to fabricate bioactive and bioresorbable devices such as hybrid osteochondral scaffolds and bioactive bone cements closely resembling the natural hard tissues and with enhanced regenerative ability. The review will illustrate some recent results related to these new biomimetic materials developed for application in different districts of the musculoskeletal system, namely bony, osteochondral and periodontal regions, and the spine. Further, it will be shown how new bioactive and superparamagnetic calcium phosphate nanoparticles can give enhanced results in cardiac regeneration and cancer therapy. Since tissue regeneration will be a major demand in the incoming decades, the high potential of biomimetic materials and devices is promising to significantly increase the healing rate and improve the clinical outcomes even in aged patients.

Mussel Shell-Derived Macroporous 3D Scaffold: Characterization and Optimization Study of a Bioceramic from the Circular Economy.

Fish industry by-products constitute an interesting platform for the extraction and recovery of valuable compounds in a circular economy approach. Among them, mussel shells could provide a calcium-rich source for the synthesis of hydroxyapatite (HA) bioceramics. In this work, HA nanoparticles have been successfully synthesized starting from mussel shells (Mytilus edulis) with a two steps process based on thermal treatment to convert CaCO3 in CaO and subsequent wet precipitation with a phosphorus source. Several parameters were studied, such as the temperature and gaseous atmosphere of the thermal treatment as well as the use of two different phosphorus-containing reagents in the wet precipitation. Data have revealed that the characteristics of the powders can be tailored, changing the conditions of the process. In particular, the use of (NH4)2HPO4 as the phosphorus source led to HA nanoparticles with a high crystallinity degree, while smaller nanoparticles with a higher surface area were obtained when H3PO4 was employed. Further, a selected HA sample was synthesized at the pilot scale; then, it was employed to fabricate porous 3D scaffolds using the direct foaming method. A highly porous scaffold with open and interconnected porosity associated with good mechanical properties (i.e., porosity in the range 87-89%, pore size in the range 50-300 µm, and a compressive strength σ = 0.51 ± 0.14 MPa) suitable for bone replacement was achieved. These results suggest that mussel shell by-products are effectively usable for the development of compounds of high added value in the biomedical field.

Calcium phosphate-based nanosystems for advanced targeted nanomedicine.

Synthetic calcium phosphates (CaPs) are the most widely accepted bioceramics for the repair and reconstruction of bone tissue defects. The recent advancements in materials science have prompted a rapid progress in the preparation of CaPs with nanometric dimensions, tailored surface characteristics, and colloidal stability opening new perspectives in their use for applications not strictly related to bone. In particular, the employment of CaPs nanoparticles as carriers of therapeutic and imaging agents has recently raised great interest in nanomedicine. CaPs nanoparticles, as well as other kinds of nanoparticles, can be engineered to specifically target the site of the disease (cells or organs), thus minimizing their dispersion in the body and undesired organism-nanoparticles interactions. The most promising and efficient approach to improve their specificity is the 'active targeting', where nanoparticles are conjugated with a targeting moiety able to recognize and bind with high efficacy and selectivity to receptors that are highly expressed only in the therapeutic site. The aim of this review is to give an overview on advanced targeted nanomedicine with a focus on the most recent reports on CaP nanoparticles-based systems, specifically designed for the active targeting. The distinctive characteristics of CaP nanoparticles with respect to the other kinds of nanomaterials used in nanomedicine are also discussed.

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.

Bioinspired oriented calcium phosphate nanocrystal arrays with bactericidal and osteogenic properties.

The global diffusion of antibiotic resistance poses a severe threat to public health. Addressing antibiotic-resistant infections requires innovative approaches, such as antibacterial nanostructured surfaces (ANSs). These surfaces, featuring ordered arrays of nanostructures, exhibit the ability to kill bacteria upon contact. However, most currently developed ANSs utilize bioinert materials, lacking bioactivity crucial for promoting tissue regeneration, particularly in the context of bone infections. This study introduces ANSs composed of bioactive calcium phosphate nanocrystals. Two distinct ANSs were created through a biomineralization-inspired growth of amorphous calcium phosphate (ACP) precursors. The ANSs demonstrated efficient antibacterial properties against both Gram-negative (P. aeruginosa) and Gram-positive (S. aureus) antibiotic resistant bacteria, with up to 75 % mortality in adhered bacteria after only 4 h of contact. Notably, the ANS featuring thinner and less oriented nano-needles exhibited superior efficacy attributed to simultaneous membrane rupturing and oxidative stress induction. Moreover, the ANSs facilitate the proliferation of mammalian cells, enhancing adhesion, spreading, and reducing oxidative stress. The ANSs displayed also significant bioactivity towards human mesenchymal stem cells, promoting colonization and inducing osteogenic differentiation. Specifically, the ANS with thicker and more ordered nano-needles demonstrated heightened effects. In conclusion, ANSs introduced in this work have the potential to serve as foundation for developing bone graft materials capable of eradicate site infections while concurrently stimulating bone regeneration. STATEMENT OF SIGNIFICANCE: Nanostructured surfaces with antibacterial properties through a mechano-bactericidal mechanism have shown significant potential in fighting antibiotic resistance. However, these surfaces have not been fabricated with bioactive materials necessary for developing devices that are both antibacterial and able to stimulate tissue regeneration. This study demonstrates the feasibility of creating nanostructured surfaces of ordered calcium phosphate nano-needles through a biomineralization-inspired growth. These surfaces exhibit dual functionality, serving as effective bactericidal agents against Gram-negative and Gram-positive antibiotic-resistant bacteria while also promoting the proliferation of mammalian cells and inducing osteogenic differentiation of human mesenchymal stem cells. Consequently, this approach holds promise in the context of bone infections, introducing innovative nanostructured surfaces that could be utilized in the development of antimicrobial and osteogenic grafts.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure.

BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

Fluorescent Carbon Dots from Food Industry By-Products for Cell Imaging.

Herein, following a circular economy approach, we present the synthesis of luminescent carbon dots via the thermal treatment of chestnut and peanut shells, which are abundant carbon-rich food industry by-products. As-synthesized carbon dots have excellent water dispersibility thanks to their negative surface groups, good luminescence, and photo-stability. The excitation-emission behaviour as well as the surface functionalization of these carbon dots can be tuned by changing the carbon source (chestnuts or peanuts) and the dispersing medium (water or ammonium hydroxide solution). Preliminary in vitro biological data proved that the samples are not cytotoxic to fibroblasts and can act as luminescent probes for cellular imaging. In addition, these carbon dots have a pH-dependent luminescence and may, therefore, serve as cellular pH sensors. This work paves the way towards the development of more sustainable carbon dot production for biomedical applications.

Inhalable Microparticles Embedding Biocompatible Magnetic Iron-Doped Hydroxyapatite Nanoparticles.

Recently, there has been increasing interest in developing biocompatible inhalable nanoparticle formulations, as they have enormous potential for treating and diagnosing lung disease. In this respect, here, we have studied superparamagnetic iron-doped calcium phosphate (in the form of hydroxyapatite) nanoparticles (FeCaP NPs) which were previously proved to be excellent materials for magnetic resonance imaging, drug delivery and hyperthermia-related applications. We have established that FeCaP NPs are not cytotoxic towards human lung alveolar epithelial type 1 (AT1) cells even at high doses, thus proving their safety for inhalation administration. Then, D-mannitol spray-dried microparticles embedding FeCaP NPs have been formulated, obtaining respirable dry powders. These microparticles were designed to achieve the best aerodynamic particle size distribution which is a critical condition for successful inhalation and deposition. The nanoparticle-in-microparticle approach resulted in the protection of FeCaP NPs, allowing their release upon microparticle dissolution, with dimensions and surface charge close to the original values. This work demonstrates the use of spray drying to provide an inhalable dry powder platform for the lung delivery of safe FeCaP NPs for magnetically driven applications.

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.