Monica Montesi

Hydroxyapatite-magnetite-MWCNT nanocomposite as a biocompatible multifunctional drug delivery system for bone tissue engineering

New magnetic hydroxyapatite-based nanomaterials as bone-specific systems for controlled drug delivery have been synthesized. The synthesized hydroxyapatite, HA, decorated with magnetite nanoparticles by a deposition method (HA/Fe3O4) and the nanocomposite system obtained using magnetic multi-walled carbon nanotubes (HA/MWCNT/Fe3O4) as a filler for HA have been characterized by chemical and morphological analyses, and their biological behavior was investigated. The systems have also been doped with clodronate in order to combine the effect of bone biomineralization induced by hydroxyapatite-based composites with the decrease of osteoclast formation induced by the drug. An analysis of the preosteoclastic RAW264.7 cell proliferation by MTT assay confirmed the high biocompatibility of the three systems. TRAP staining of RAW 264.7 conditioned with sRAKL to induce osteoclastogenesis, cultured in the presence of the systems doped and undoped with clodronate, showed the inhibitory effect of clodronate after we counted the MNC TRAP(+)cells but only in the osteoclast formation; in particular, the system HA/Fe3O4-Clo exerted a high inhibitory effect compared to the drug alone. These results demonstrate that the synthesized nanocomposites are a biocompatible magnetic drug delivery system and can represent a useful multimodal platform for applications in bone tissue engineering.

Superparamagnetic iron-doped nanocrystalline apatite as a delivery system for doxorubicin

The development of non-toxic and biodegradable magnetic nanoparticles (NPs) that can be easily functionalized with drugs or biomolecules and employed, under magnetic fields, as targeted nano-carriers or components of scaffolds with on-demand functionalities, is a big challenge in the biomaterials research. In the present work, the feasibility of previously synthesized iron-doped superparamagnetic apatite (FeHA) NPs to bind and then release the anticancer drug doxorubicin (DOX) under an applied low-frequency pulsed electromagnetic field (PEMF) was investigated. The behavior of FeHA towards DOX has been compared to that of synthetic biomimetic apatite (HA) NPs prepared ad hoc with characteristics close to those of bone mineral. The DOX adsorption kinetics and isotherms on FeHA and HA were explored and fitted according to different mathematical models (Elovich, Sips and Freundlich) revealing enhanced uptake of DOX on FeHA than HA, due to the better interaction of the drug with the surface iron cations and formation of multi-molecular DOX assemblies. In the absence of the PEMF, the quantity of DOX released from HA was higher than that released from FeHA, in agreement with the lower affinity of DOX for HA than FeHA. Interestingly, in the presence of the PEMF, the extent of DOX released from FeHA after 3 and 6 days increased significantly. The higher DOX release from FeHA under PEMF can be explained by the mechanical shacking of superparamagnetic FeHA NPs breaking the bonding with the drug and allowing detachment of DOX assemblies from the NP surface. In vitro assays demonstrated that DOX loaded on HA and FeHA displayed cytotoxicity against the human osteosarcoma cell line (SAOS-2) at the same level as free DOX, for all the concentrations and time points tested. Confocal microscopy analyses showed that drug-loaded NPs were rapidly internalized within cells and released DOX, which accumulated in the nuclei where it exerted the desired cytotoxic activity.

Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration

Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration.

Investigation of different cross-linking approaches on 3D gelatin scaffolds for tissue engineering application: A comparative analysis

The present study aims to investigate the physical-chemical and biological features exhibited by porous scaffolds for regeneration of cartilaginous tissues obtained through stabilization of 3D gelatin hydrogels by physical (DHT), chemical (BDDGE) and natural (Genipin) cross-linking approaches. The study aimed at comparatively assessing the porous microstructure and the long-term resistance of the scaffolds upon degradation in wet physiological conditions (37°C, pH=7.4). The degree of cross-linking increases as function of incorporation of cross-linkers which was maximum up to 73% for BDDGE. The infrared spectroscopy and thermal analysis confirmed the gelatin structure was preserved during the cross-linking treatments. Mechanical properties of the scaffolds were analysed by static and dynamic compression test, which showed different viscoelastic behaviour upon various cross-linking strategies. The biological performance of the scaffolds investigated using human chondrocytes showed good cell adhesion, viability and proliferation, as well as extensive 3D scaffold colonization. Besides, the analysis of gene expression related to the formation of new chondral tissue reported increasing ability with time in the formation of new extra-cellular matrix. In conclusion, out of three different cross-linking methods, the gelatin scaffolds subjected to dehydrothermal treatment (DHT) represented to be the most favourable 3D scaffold for cartilage regeneration.

Effect of hydroxyapatite nanocrystals functionalized with lactoferrin in osteogenic differentiation of mesenchymal stem cells.

Lactoferrin (LF) is a bioactive glycoprotein that became recently interesting in the field of bone regeneration for its modulatory effect on bone cells. On the basis of this evidence this work aims to functionalize biomimetic hydroxyapatite (HA) nanocrystals with LF to study their effect on osteogenic differentiation of mesenchymal stem cells (MSCs). The orientation of LF on the HA surface was analyzed by spectroscopic and thermal techniques. Three samples with different amounts of LF attached to HA nanocrystals were tested in vitro. The combined effect of HA and LF on MSC proliferation and morphology, alkaline phosphatase (ALP) activity, and gene expression were evaluated at different time points. The sample with the lowest LF amount showed the best bioactivity probably due to the formation of a single layer of protein with a better molecular orientation. Coupling of HA-LF did not affect cell proliferation and morphology, while analysis of HA-LF on ALP activity and messenger RNA expression of the selected genes, demonstrated the role of HA-LF in the induction of osteogenic markers. HA-LF represents a promising system to be used to manufacture bioactive functional materials in tissue engineering (as scaffolds, injectable cements, or coatings for metallic implants) with enhanced anabolic activity to treat bone diseases.

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering.

Fe-Doping-Induced Magnetism in Nano-Hydroxyapatites

Doping of biocompatible nanomaterials with magnetic phases is currently one of the most promising strategies for the development of advanced magnetic biomaterials. However, especially in the case of iron-doped magnetic hydroxyapatites, it is not clear if the magnetic features come merely from the magnetic phases/ions used as dopants or from complex mechanisms involving interactions at the nanoscale. Here, we report an extensive chemical-physical and magnetic investigation of three hydroxyapatite nanocrystals doped with different iron species and containing small or no amounts of maghemite as a secondary phase. The association of several investigation techniques such as X-ray absorption spectroscopy, Mössbauer, magnetometry, and TEM allowed us to determine that the unusual magnetic properties of Fe2+/3+-doped hydroxyapatites (FeHA) occur by a synergy of two different phenomena: i.e., (i) interacting superparamagnetism due to the interplay between iron-doped apatite and iron oxide nanoparticles as well as to the occurrence of dipolar interactions and (ii) interacting paramagnetism due to Fe3+ ions present in the superficial hydrated layer of the apatite nanophase and, to a lesser extent, paramagnetism due to isolated Fe3+ ions in the apatite lattice. We also show that a major player in the activation of the above phenomena is the oxidation of Fe2+ into Fe3+, as induced by the synthesis process, and their consequent specific positioning in the FeHA structure.

Surface morphology, tribological properties and in vitro biocompatibility of nanostructured zirconia thin films.

Deposition of nanostructured and low-wear zirconia (ZrO2) thin films on the metallic component of a total joint implant is envisaged to reduce wear of the soft ultra-high molecular weight polyethylene (UHMWPE) counterpart. In this work, morphological surface features, wear resistance and in vitro-biocompatibility of zirconia thin films deposited by the novel Pulsed Plasma Deposition (PPD) method have been investigated. Film thickness, roughness and wettability were found to be strongly dependent on deposition gas pressure. Interestingly, wear rate of UHMWPE disks coupled to zirconia-coated titanium spheres was only poorly correlated to the contact angle values, while film roughness and thickness seemed not to affect it. Furthermore, wear of UHMWPE, when coupled with zirconia coated-titanium spheres, significantly decreased with respect to uncoated spheres under dry or NaCl-lubricated conditions; besides, when using bovine serum, similar results were obtained for coated and uncoated spheres. Finally, suitable mesenchymal stem and osteoblast cells adhesion, proliferation and viability were observed, suggesting good biocompatibility of the nanostructured zirconia films. Taken together, the results shown in this work indicate that zirconia thin films deposited by the PPD method deserve further investigations as low-wear materials for biomedical applications such as total joint replacement.

Biomimetic Scaffold with Aligned Microporosity Designed for Dentin Regeneration

Tooth loss is a common result of a variety of oral diseases due to physiological causes, trauma, genetic disorders, and aging and can lead to physical and mental suffering that markedly lowers the individual’s quality of life. Tooth is a complex organ that is composed of mineralized tissues and soft connective tissues. Dentin is the most voluminous tissue of the tooth and its formation (dentinogenesis) is a highly regulated process displaying several similarities with osteogenesis. In this study, gelatin, thermally denatured collagen, was used as a promising low-cost material to develop scaffolds for hard tissue engineering. We synthetized dentin-like scaffolds using gelatin biomineralized with magnesium-doped hydroxyapatite and blended it with alginate. With a controlled freeze-drying process and alginate cross-linking, it is possible to obtain scaffolds with microscopic aligned channels suitable for tissue engineering. 3D cell culture with mesenchymal stem cells showed the promising properties of the new scaffolds for tooth regeneration. In detail, the chemical–physical features of the scaffolds, mimicking those of natural tissue, facilitate the cell adhesion, and the porosity is suitable for long-term cell colonization and fine cell–material interactions.

Evaluation of different crosslinking agents on hybrid biomimetic collagen-hydroxyapatite composites for regenerative medicine.

This study focuses on the development of novel bone-like scaffolds by bio-inspired, pH-driven, mineralization of type I collagen matrix with magnesium-doped hydroxyapatite nanophase (MgHA/Coll). To this aim, this study evaluates the altered modifications in the obtained composite due to different crosslinkers such as dehydrothermal treatment (DHT), 1,4-butanediol diglycidyl ether (BDDGE) and ribose in terms of morphological, physical-chemical and biological properties. The physical-chemical properties of the composites evaluated by XRD, FTIR, ICP and TGA demonstrated that the chemical mimesis of bone was effectively achieved using the in-lab biomineralization process. Furthermore, the presence of various crosslinkers greatly promoted beneficial enzymatic resistivity and swelling ability. The morphological results revealed highly porous and fibrous micro-architecture with total porosity above 85% with anisotropic pore size within the range of 50-200μm in all the analysed composites. The mechanical behaviour in response to compressive forces demonstrated enhanced compressive modulus in all crosslinked composites, suggesting that mechanical behaviour is largely dependent on the type of crosslinker used. The biomimetic compositional and morphological features of the composites elicited strong cell-material interaction. Therefore, the results showed that by activating specific crosslinking mechanisms, hybrid composites can be designed and tailored to develop tissue-specific biomimetic biomaterials for hard tissue engineering.