Hydroxyapatite Nanocrystals Research Articles

Electrospun ELR-like nano-fibrous scaffold with intra-fibrous mineralization for dentin-pulp regeneration

Event Abstract Back to Event Electrospun ELR-like nano-fibrous scaffold with intra-fibrous mineralization for dentin-pulp regeneration Caixia Lan1, Yuping Li1 and Conrado Aparicio1 1 University of Minnesota, Restorative Sciences, United States Introduction: With the high incidence of pulpitis and obstacles of current endodontic treatments, tissue engineering has attracted increasing interest in dental tissue regeneration. Although great effort has been made on using synthetic and natural biomaterials in dentin regeneration, most materials fail to mimic the complex properties of collagen in dentin matrix, especially in inducing the biomimetic mineralization, a key factor for normal cell activity. Our study aimed to produce an elastin-like statherin peptide-containing recombinamer (st-ELR) scaffold with nanofibrous structure to promote dentin regeneration. Based on the ability of ELR fibers to undergo thermal-sensitive phase transformation, we used the biomimetic polymer induced liquid precursor (PILP) process to intra-fibrously mineralize them. We further investigated the role of mineralized ELR scaffolds in the proliferation and differentiation of human dental pulp stem cells (hDPSCs). Materials and Methods: St-ELR, and ELR without statherin-derived peptide (Ref-ELR) were electrospun to form nano-fibrous scaffolds and then subjected to supersaturated solution of Ca and PO4 supplemented with poly-aspartic acid as polymeric precursor to induce intra-fibrous mineralization. Mineralization was characterized by SEM, TEM, EDS, XRD and nano-indentation after 1, 3, and 7days. hDPSCs were cultured on dentin, mineralized and un-mineralized ELR surfaces. Cell proliferation was evaluated using immunofluorescence microscopy. ALP activity and expression of DMP1, DSP were analysed using ELISA assay, RT-PCR and western blot. Results and Discussion: SEM analysis revealed both St-ELR and Ref-ELR were highly intra- and extra-fibrous mineralized. A marked formation of needle-like apatitic mineral was aligned homogeneously along ELR fibers(Fig. 1C), which is similar to those seen in natural dentin. Mineral embedded within fibers were observed under SEM when looking at the cross-section surfaces (Fig.1D), indicating the successful intra-fibrous mineralization. TEM images revealed that St-ELR exhibited parallel orientation of hydroxyapatite crystals to the longitudinal axis of the fibrils while Ref-ELR showed random orientated growth of minerals (Fig.2). EDS and XRD confirmed that we obtained hydroxyapatite nano-crystals. The high degree of extra- and intra-fibrous mineralization resulted in significant increase of elastic modulus and hardness to 0.99GPa and 0.058 GPa in dry state after 7 days. Cell proliferation, ALP activity, expression of DMP1 and DSP of hDPSCs were significantly enhanced on mineralized groups with respect to non-mineralized controls. This data suggests fibrous ELR scaffolds with mineralization promote stem cell activities. Conclusion: Electrospun St-ELR recombinamers along with Ref-ELR can template dentin matrix to obtain intra-fibrous mineralization using PILP process and thus, further promote dental pulp stem cell proliferation and differentiation. The combination of physical and biological properties demonstrates ELR is a potential novel scaffold for dental tissue regeneration. G. Heo; A. Aksan Keywords: Biomimetic, Scaffold, Tissue Regeneration, Calcium phosphate Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in dental applications Citation: Lan C, Li Y and Aparicio C (2016). Electrospun ELR-like nano-fibrous scaffold with intra-fibrous mineralization for dentin-pulp regeneration. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01783 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Caixia Lan Yuping Li Conrado Aparicio Google Caixia Lan Yuping Li Conrado Aparicio Google Scholar Caixia Lan Yuping Li Conrado Aparicio PubMed Caixia Lan Yuping Li Conrado Aparicio Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Self-assembled peptide-polymer hydrogels as a 3D nanofiber substrate for biomimetic hydroxyapatite mineralization

Event Abstract Back to Event Self-assembled peptide-polymer hydrogels as a 3D nanofiber substrate for biomimetic hydroxyapatite mineralization Elham Radvar1, C Remzi Becer1 and Helena S. Azevedo1 1 Institute of Bioengineering, Queen Mary University of London, School of Engineering and Materials Science, United Kingdom Introduction: Hard tissues in vertebrates, such as bones, are produced by controlled biomineralization of an organic matrix that regulates the growth of the inorganic phase. In bone, the fundamental subunit is a collagen fibril, formed by self-assembly of single tropocollagen molecules (triple helix), where hydroxyapatite nanocrystals grow with their crystallographic c-axes aligned with the fibril long axes. Molecular self-assembly offers the possibility to recreate the same degree of nanoscale order in synthetic biomaterials and promote epitaxial mineralization mediated by an organic matrix. The self-assembly between a negatively charged polymer and a peptide amphiphile (PA) of opposite charge was shown to result in the formation of sacs and membranes[1]. Inspired by this seminal work, here we investigate the use of the negatively charged synthetic polymer polystyrene sulfonate (PSS) with a positively charged PA to form self-assembling matrices and their ability to direct hydroxyapatite mineralization. Materials and Methods: PSS (1 MDa, Fig 1-A) was commercially available from Sigma. C15H31CONH-V3A3K3-CONH2 PA (Fig 1-A) was synthesized by solid phase and purified as described elsewhere[2]. PSS/PA hydrogels were obtained by self-assembly combining aqueous solutions of PSS (2% w/v) and PA (2% w/v). Mineralization studies were performed by incubating the PSS/PA hydrogels in a simulated body fluid (SBF[3]) solution at 37°C for several days. Then, the gels were washed with distilled water and prepared for surface analysis by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Results and Discussion: Conversely to previous work reporting the self-assembly between PAs and oppositely charged polysaccharides, combining an aqueous PSS solution with a positively charged PA resulted in the formation of a self-supporting gel (Fig 1-B) exhibiting a random nanofibrilar network (Fig 1-C). PSS has been shown to direct the nucleation and growth of calcium carbonate mineral by binding to calcium ions[4]. Thus, we hypothesize that these nanofiber PSS/PA gels could be used as a 3D matrix to drive the nucleation of hydroxyapatite and create a mineralized matrix resembling the structure of natural bone. The formation of hydroxyapatite mineral on the surface of hydrogels after 14 days of incubation in SBF was confirmed by SEM (Fig 1-D, E) and EDS (Fig 1-F) analysis which show a typical morphology of hydroxyapatite crystals and the presence of calcium and phosphorus, respectively. Conclusions: While further studies are necessary to characterize the formed mineral, these self-assembled PSS/PA gels present an interesting approach to develop new biomaterials for bone regeneration considering the potential for further functionalization of the peptide segment with bioactive sequences. I would like to acknowledge the help of Dr. Daniela Ferreira and Sofia Ribeiro in peptide synthesis and SBF preparation, respectively. This work is supported by School of Engineering and Materials Science (SEMS)

Development and in vitro evaluation of bioinspired, chitosan based, 3D hybrid nanohydroxyapatite scaffolds.

Event Abstract Back to Event Development and in vitro evaluation of bioinspired, chitosan based, 3D hybrid nanohydroxyapatite scaffolds. Despoina Brasinika1, Athena Tsetsekou1, Kyriaki Kyriakidou2, Ioannis K. Karoussis2 and Triantafyllos Papadopoulos2 1 National Technical University of Athens, School of Mining and Metallurgical Engineering, Greece 2 National and Kapodistrian University of Athens, Faculty of Dentistry, School of Health Sciences, Greece Introduction: In the present work the synthesis of 3D hybrid porous scaffolds was performed through a freeze drying method, aiming at the development of implantable materials with enhanced biocompatibility and bioactivity. These hybrid materials constitute an innovative alternative for implant development, in order to successfully replace damaged human tissue and provide adequate mechanical stability till the natural regeneration of the desired tissue. The hybrid scaffolds consist of hydroxyapatite nanocrystals, synthesized biomimetically in the presence of natural biomolecules, in an effort to tailor the nucleation process and control the hydroxyapatite’s crystal growth. Since these scaffolds serve as a template to support and guide formation of new tissue, the material is required to present a porous network with high interconnectivity and mechanical stability[1],[2]. However, the porous structure of the scaffolds, inevitably affects the material’s mechanical properties. For this purpose, chemical cross-linking was performed with organic crosslinkers, through bonding with the biomolecules’ amino groups[3],[4]. Materials and Methods: Aqueous suspensions of hybrid nanohydroxyapatite were prepared through a bioinspired approach[5],[6] and then they were undertaken lyophilization to develop porous 3D scaffolds. To improve scaffolds’ mechanical properties, cross-linking of the biomolecules was performed prior to lyophilization. Finally, the physicochemical and biological properties of the as-obtained scaffolds were examined. Results: Evaluation of scaffolds’ internal porosity with Scanning Electron Microscopy (SEM) and X-ray computed microtomography (micro-CT) indicates the development of a highly interconnected porous network with bimodal pore size distribution (Fig. 1). Fig. 1: SEM images of the internal structure of hybrid scaffolds differing in the biomolecules employed for increasing mechanical stability. Optimization work provided scaffolds with pore size ranging from 50 to 100 μm for the large pores and around 5 to 10 μm for the smaller ones. Cell viability and cytotoxicity tests (MTT assay and fluorescein diacetate/propidium iodide staining (FDA/PI)) proved that after 7 days of cultivation a well-populated biomaterial was obtained with viable cells distributed through the scaffold (Fig. 2). Fig. 2: SEM image of osteoblasts adhered into the porous structure of a 3D scaffold after 7 days of cultivation. Discussion: The adopted bioinspired approach led to the fabrication of 3D hybrid porous scaffolds with a complex architecture and high interconnectivity among the pores. Their chemical composition and nanostructure - resembling that of natural bone - along with their complex porous network provide a cell friendly environment which proved beneficial for cell distribution, attachment and proliferation. Conclusions: The as-produced hybrid scaffolds comprise biocompatible biomaterials with promising properties for tissue engineering applications. The research leading to these results has received funding from National Strategic Reference Framework 2007–2013, Cooperation programs, Project BIOMINY – 09SYN-41-757

Highly selective aerobic oxidation of alkyl arenes and alcohols: cobalt supported on natural hydroxyapatite nanocrystals

Cobalt was successfully immobilized on natural hydroxyapatite nanocrystals which was obtained from cow bones (Co–NHAp). NHAp as the support enhanced both catalytic activity and selectivity in aerobic oxidation of alkyl arenes and alcohols.

Fabrication of porous hydroxyapatite-carbon nanotubes composite

Porous hydroxyapatite (HA)/carbon nanotubes (CNTs) composites were produced by using polymeric sponge route through double firing method (500°C for 0.5h and 1000°C for 1h). HA nano crystals were synthesized by the hydrothermal method at 200°C for 2h and mixed with aqueous CNTs suspension. For the functionalization of CNTs, acidic treatment was used. Polyvinyl Alcohol (PVA) was used as a binder for the formation process of HA/CNTs composite ceramics. Addition of CNTs can enhance its mechanical, biological and filtration performance of the porous HA/CNTs composite ceramics. The novelty of this paper relies on the fabrication of the HA/CNTs composites have been accomplished with tailored pore structure. The research shows that developed new technique is able to design and control pore structure of ceramic/CNTs composite for potential use in biomedical and environmental applications.

Hierarchical porous hydroxyapatite microspheres: synthesis and application in water treatment

Hydroxyapatite (HA) microspheres with hierarchical porous structure were synthesized using a facile gas diffusion method. The morphology and structure of the as-synthesized products were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, and Brunauer–Emmett–Teller gas sorptometry analyses. Results showed that the change in pH resulting from ammonia diffusion provided the driving force for the formation of HA crystals. Under the cooperative influences of reaction time and reaction temperature, the nucleation and growth processes of HA nanocrystals were effectively controlled. Therefore, porous HA microspheres with large surface areas were produced at 60 °C for 24 h. On the basis of the time-dependent experiments, a possible formation mechanism of hierarchical porous HA microsphere was proposed. Moreover, hierarchical porous HA microspheres were investigated as adsorbent, and they exhibited a high adsorptive capacity for heavy metals Pb2+ and Cu2+ of approximately 213.58 ± 2.6375 and 59.68 ± 3.4436 mg/g, respectively. Therefore, hierarchical porous HA microspheres may be applied to heavy metal-contaminated water treatment.

Biomimetic Mineralization of Recombinamer-Based Hydrogels toward Controlled Morphologies and High Mineral Density.

The use of insoluble organic matrices as a structural template for the bottom-up fabrication of organic-inorganic nanocomposites is a powerful way to build a variety of advanced materials with defined and controlled morphologies and superior mechanical properties. Calcium phosphate mineralization in polymeric hydrogels is receiving significant attention in terms of obtaining biomimetic hierarchical structures with unique mechanical properties and understanding the mechanisms of the biomineralization process. However, integration of organic matrices with hydroxyapatite nanocrystals, different in morphology and composition, has not been well-achieved yet at nanoscale. In this study, we synthesized thermoresponsive hydrogels, composed of elastin-like recombinamers (ELRs), to template mineralization of hydroxyapatite nanocrystals using a biomimetic polymer-induced liquid-precursor (PILP) mineralization process. Different from conventional mineralization where minerals were deposited on the surface of organic matrices, they were infiltrated into the frameworks of ELR matrices, preserving their microporous structure. After 14 days of mineralization, an average of 78 μm mineralization depth was achieved. Mineral density up to 1.9 g/cm(3) was found after 28 days of mineralization, which is comparable to natural bone and dentin. In the dry state, the elastic modulus and hardness of the mineralized hydrogels were 20.3 ± 1.7 and 0.93 ± 0.07 GPa, respectively. After hydration, they were reduced to 4.50 ± 0.55 and 0.10 ± 0.03 GPa, respectively. These values were lower but still on the same order of magnitude as those of natural hard tissues. The results indicated that inorganic-organic hybrid biomaterials with controlled morphologies can be achieved using organic templates of ELRs. Notably, the chemical and physical properties of ELRs can be tuned, which might help elucidate the mechanisms by which living organisms regulate the mineralization process.

High-performance ultrafiltration mixed-matrix membranes based on cellulose acetate and nanohydroxyapatite

The asymmetric cellulose acetate mixed-matrix membrane (MMM) filled with nanohydroxyapatite was prepared by a modified phase inversion method. To prevent the agglomeration of the hydroxyapatite nanocrystals and to obtain ultrafiltration composite membranes, a surfactant (12-hydroxystearic acid) was used. The hydroxyapatite nanopowder was dispersed in acetone-dissolved 12-hydroxystearic acid and then mixed homogeneously with acetone-dissolved cellulose acetate (CA). The process of addition of hydroxyapatite nanosuspension to CA solution resulted in asymmetric composite membranes with higher pure water flux and superior NaCl rejection in comparison to the CA membrane. The best performing composite membrane, the CHM-30 sample, shows a retention of 99.1% and a high water permeability of 34.96 L/m2 h bar. The results showed that the nanohydroxyapatite—cellulose acetate MMMs can be used in ultrafiltration applications.

The effects of strontium on bone mineral: A review on current knowledge and microanalytical approaches

The interest in effects of strontium (Sr) on bone has greatly increased in the last decade due to the development of the promising drug strontium ranelate. This drug is used for treating osteoporosis, a major bone disease affecting hundreds of millions of people worldwide, especially postmenopausal women. The novelty of strontium ranelate compared to other treatments for osteoporosis is its unique effect on bone: it simultaneously promotes bone formation by osteoblasts and inhibits bone resorption by osteoclasts. Besides affecting bone cells, treatment with strontium ranelate also has a direct effect on the mineralized bone matrix. Due to the chemical similarities between Sr and Ca, a topic that has long been of particular interest is the incorporation of Sr into bones replacing Ca from the mineral phase, which is composed by carbonated hydroxyapatite nanocrystals. Several groups have analyzed the mineral produced during treatment; however, most analysis were done with relatively large samples containing numerous nanocrystals, resulting thus on data that represents an average of many crystalline domains. The nanoscale analysis of the bone apatite crystals containing Sr has only been described in a few studies. In this study, we review the current knowledge on the effects of Sr on bone mineral and discuss the methodological approaches that have been used in the field. In particular, we focus on the great potential that advanced microscopy and microanalytical techniques may have on the detailed analysis of the nanostructure and composition of bone apatite nanocrystals produced during treatment with strontium ranelate.

Fabrication of Cell–Hydroxyapatite Nanocrystal Composites Assisted with Layer-by-layer Nanometer-sized Extracellular Matrix Films on Individual Stem Cells

Abstract The ability to control cell function is one of the challenges for cell-based therapy in regenerative medicine. In this study, we describe a novel method to fabricate cell–hydroxyapatite (HAp) nanocrystal composites to modulate cellular microenvironment. By preparation of layer-by-layer nanometer-sized extracellular matrix films onto cell surfaces, HAp nanocrystals were fabricated onto cell surfaces without cytotoxicity. This technology can modulate cellular functions such as adhesion, growth, and differentiation.

Transmission electron microscopy analysis of hydroxyapatite nanocrystals from cattle bones

In this present study, hydroxyapatite which was obtained from cattle bones has been heat treated at temperature 400°C and 600°C. The microstructure after the treatment has been studied in detail using Transmission electron microscopy (TEM) and X-ray diffraction techniques. The TEM results indicate that natural bone consists of collagen and hydroxyapatite nano-crystals which are needle shaped. The heat treatment influences the crystallinity and growth of these hydroxyapatite nano-crystals known as ‘crystal maturation’ or ‘crystal ageing’.

Biomimetic synthesis of ribbon-like hydroxyapatite employing poly(l-arginine)

Binding of a polypeptide on colloidal particles can affect the dissolution of mineral, initiate crystal nucleation and change the growth kinetics of the precipitated crystal. In this study the synthesis of hydroxyapatite nanocrystals was performed in the presence of poly(l-arginine), PA. Aqueous solutions of Ca2+, phosphate, and PA were employed at calcium:PA molar ratios ranging from 2:1 to 1:2 and the resulting suspensions were hydrothermally treated at 80°C for 16h, or at 130°C for 6h. The resulting nanomaterials were characterized by XRD, FTIR, TEM, SEM, and TGA. It was found that the presence of PA promotes HAP formation and affects its crystal size and morphology possibly through a rather specific interaction between the homopeptide that is positively charged and also that adopts a β-sheet conformation and the negatively charged c-plane of the growing HAP crystal. In all cases, hexagonal HAP crystals with thin ribbon-like morphology were obtained. Increase of the PA ratio and of the hydrothermal temperature leads to more homogeneous and narrower size distributions with crystallites having widths ranging between 5 to 50nm and lengths ranging from 50 to 450nm.

In situ Biomimetic Mineralization and Characterization of Carbon Nanotubes/chitosan Composite Microspheres†

Carbon nanotubes / chitosan( CNTs / CS) composite microspheres were prepared by emulsification and cross-linking method. Nano-hydroxyapatite crystals were biomimetically formed on the surface of the composite microspheres. The influence of CNTs on the mineralization of the microspheres was investigated. The morphologies,structurs and stability properties of the microspheres before and after mineralization were studied through scanning electronic microscopy( SEM),X-ray diffraction( XRD),swelling ratio and water content tests. The results show that the formation ability of the nano-hydroxyapatite on the CNTs / CS microspheres is significantly higher than that on pure CS microspheres. And the mineralized CNTs / CS microspheres exhibit better structure stability. After culturing with MG63 cells for 7 d,the proliferation of cells cultured with mineralized CNTs / CS microspheres shows significant difference from that of the cells cultured with mineralized CS microspheres.

Synthesis of Poly(ϵ-caprolactone) Grafted Poly(2-hydroxyethyl methacrylate) Functionalized Hydroxyapatite by RAFT and ROP

Poly(ϵ-caprolactone) grafted poly(2-hydroxyethyl methacrylate) functionalized hydroxyapatite (HAP@PHEMA-g-PCL) nanocomposites were synthesized by the combination of reversible addition fragmentation chain transfer (RAFT) polymerization and ring-opening polymerization (ROP). The RAFT agent was anchored on the surface of hydroxyapatite nanocrystals (HAPs) through the silane condensation process of 3-chloropropyltrimethoxysilane followed by reaction with potassium xanthogenate. Poly(2-hydroxyethyl methacrylate) (PHEMA) was covalently functionalized on the surface of HAPs by RAFT polymerization. Then, poly(ϵ-caprolactone) (PCL) was grafted on HAPs by ROP based on the hydroxyl groups of PHEMA to afford HAP@PHEMA-g-PCL. The structure and composition of HAP@PHEMA-g-PCL nanocomposites were characterized by FT-IR, XRD, and TGA analyses. The morphology and formation of the polymer encapsulating HAPs were demonstrated from SEM and TEM images, while the 1H MNR analysis of the cleaved PHEMA-g-PCL confirmed the grafting.

TEM characterization of hydroxyapatite nanocrystals grown on the bacterial cellulose fibrils

TEM characterization of hydroxyapatite nanocrystals grown on the bacterial cellulose fibrils

Ultrasound-induced albumin gelation method for the preparation of nanostructured hydroxyapatite

An ultrasound-induced albumin gelation method was utilized to prepare nanostructured hydroxyapatite (HAP). After ultrasound irradiation, hybrid colloids of bovine serum albumin (BSA) and amorphous calcium phosphate (ACaP) were formed, where the nanosized ACaP was homogenously distributed in the fibril-like BSA matrix. The freeze-dried BSA/ACaP sponge showed an oriented pore structure and the ACaP nanoparticles were transformed into nanostructured HAP after calcinations at 400–600°C. With the aid of BSA/ACaP sponge, HAP nanocrystals were aggregated into rod-like nanostructures, which were merged into large rod-like HAP crystals at 700°C. This study demonstrates that ultrasound-induced albumin gelation method can be successfully used to fabricate nanostructured HAP.

Combination of Surface Initiated Reversible Addition Fragmentation Chain Transfer Polymerization, Thiol-Ene Click Chemistry and Coordination Chemistry for the Fabrication of a Novel Photoluminescent Hydroxyapatite Nanohybrids.

Hydroxyapatite nanocrystals (HAPs) were encapsulated by poly(allyl methacrylate) (PAMA) employing controlled surface-initiated reversible addition-fragmentation chain transfer polymerization of allyl methacrylate to give HAP-PAMA nanohybrids. The subsequent thiol-ene coupling of nanohybrids with 2-mercaptosuccinic acid resulted in HAP-PAMA-COOH possessing multicarboxyl functionality. The coordination of the nanohybrids with Tb3+ ions in the presence of 1,10-phenanthroline (Phen) afforded photoluminescent Tb3+ tagged HAPs-PAMA nanohybrid complexes (HAP-PAMA-Tb3+-Phen). The structure, morphology, and fluorescence properties of the Tb3+ coordinated nanohybrid complexes were investigated by respective physical and spectral studies.

Synthesis, characterization and luminescence properties of Eu3+-doped hydroxyapatite nanocrystal and the thermal treatment effects

In this work, we present the synthesis, characterization and the luminescence properties of Ca10(PO4)6(OH)2 (hydroxyapatite/HAp) nanocrystals doped with europium trivalent ions. The most important processes that lead to europium emissions in the visible region were identified. Eu:HAp nanopowder excited at 394nm (or 460nm) exhibits several emissions: (i) weak emissions at 579nm, 592nm and 616nm due to the 5D0→7F0, 5D0→7F1 and 5D0→7F2 transitions, respectively, with europium ion occupying site I in hydroxyapatite structure and (ii) strong emissions due to the 5D0→7F0 (574nm), 5D0→7F1 (602nm) and 5D0→7F2 (610–630nm) transitions, when Eu3+ is occupying site II. The emission spectrum and the time-resolved luminescence analysis showed that the HAp nanocrystals (nanopowder) thermally treated at temperature (T) between 500 and 800°C have a change in the initial Eu3+ site distribution of 100 % of Eu3+ at site I to a more stable one where the majority of europium ions are at site II: 30% remains at site I and 70% migrates to site II. In addition, an enhancement of the Eu3+ emission intensity is observed due to the increasing crystallite size. A time-resolved luminescence investigation using a short pulse laser excitation at 460nm was employed to measure the luminescence decays and to determine the most important mechanisms involved in the deexcitation process of 5D0 excited state of Eu3+, where it is seen a fast (2.9μs) energy transfer from Eu3+- site I (donor) to Eu3+- site II (acceptor) in the thermally treated nanopowders with T>500°C. The initial presence of 100% of Eu3+ at site I in the synthesized nanocrystals is gradually modified by the thermal treatments with temperatures above 500°C by thermal activation of Ca2+ vacancy (the charge compensator) diffusion through the HAp lattice, which propitiates the Ca2+- vacancies and Eu3+ ions to exchange positions in the lattice. By this thermal activated mechanism, Eu3+ ion migrates through the lattice until get the final distribution of 30% at site I and 70% at site II. As a result, the complete description of the Eu3+ (5D0) decay and the energy transfer process from Eu3+ (site I)→Eu3+ (site II) were proposed.

Coupling Hydroxyapatite Nanocrystals with Lactoferrin as a Promising Strategy to Fine Regulate Bone Homeostasis.

Lactoferrin (LF) is an interesting glycoprotein in the field of bone biology for its regulatory effect on cells involved in bone remodeling, that results compromised in several pathological conditions, as osteoporosis. In a previous study we observed that the coupling of LF and biomimetic hydroxyapatite nanocrystals (HA), a material well-known for its bioactivity and osteoconductive properties, leads to a combined effect in the induction of osteogenic differentiation of mesenchymal stem cells. On the basis of this evidence, the present study is an extension of our previous work aiming to investigate the synergistic effect of the coupling of HA and LF on bone homeostasis. Biomimetic HA nanocrystals were synthesized and functionalized with LF (HA-LF) and then pre-osteoblasts (MC3T3-E1) and monocyte/macrophage cells lines (RAW 264.7), using as osteoclastogenesis in vitro model, were cultured separately or in co-culture in presence of HA-LF. The results clearly revealed that HA and LF act in synergism in the regulation of the bone homeostasis, working as anabolic factor for osteoblasts differentiation and bone matrix deposition, and as inhibitor of the osteoclast formation and activity.

Multi-Layered Scaffolds for Osteochondral Tissue Engineering: In Vitro Response of Co-Cultured Human Mesenchymal Stem Cells.

A promising strategy for osteochondral interface regeneration consists in the development of hybrid scaffolds, composed of distinct but integrated layers able to mimic the different regions of cartilage and bone. We developed multi-layered scaffolds by assembling a gelatin layer with layers containing different amounts of gelatin and hydroxyapatite nanocrystals, and using a gelatin solution (as a glue) to stick layers together. The scaffolds exhibit a high, interconnected porosity and mechanical properties varying with composition along the thickness of the scaffolds up to values of compressive stress and modulus of about 1 and 14 MPa, respectively. In vitro tests demonstrate that the different layers of the scaffolds promote chondrogenic and osteogenic differentiation of Human Mesenchimal Stem Cells (hMSC).