Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

Roadmap on thermoelectricity.

The increasing energy demand and the ever more pressing need for clean technologies of energy conversion pose one of the most urgent and complicated issues of our age. Thermoelectricity, namely the direct conversion of waste heat into electricity, is a promising technique based on a long-standing physical phenomenon, which still has not fully developed its potential, mainly due to the low efficiency of the process. In order to improve the thermoelectric performance, a huge effort is being made by physicists, materials scientists and engineers, with the primary aims of better understanding the fundamental issues ruling the improvement of the thermoelectric figure of merit, and finally building the most efficient thermoelectric devices. In this Roadmap an overview is given about the most recent experimental and computational results obtained within the Italian research community on the optimization of composition and morphology of some thermoelectric materials, as well as on the design of thermoelectric and hybrid thermoelectric/photovoltaic devices.

Genipin-crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization.

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td  = 57-63°C) and a significant amount of Coll and GAG deposition during the 3 weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral-like matrix deposition by hMSCs if compared to the results obtained from the Gp "two-step" functionalization procedure.

Selenium-doped hydroxyapatite nanoparticles for potential application in bone tumor therapy.

In the present study we have studied the incorporation and release of selenite ions (SeO32-) in hydroxyapatite nanoparticles for the treatment of bone tumors. Two types of selenium-doped hydroxyapatite (HASe) nanoparticles (NPs) with a nominal Se/(P + Se) molar ratio ranging from 0.01 up to 0.40 have been synthesized by a new and mild wet method. The two series of samples were thoroughly characterized and resulted to be slightly different in chemical composition, but they had similar properties in terms of morphology and degree of crystallinity. Selenium release from HASe was investigated under neutral and acidic conditions to simulate both healthy tissues and the low-pH environment surrounding a tumor mass, respectively. The comparison of the release profiles at two pH values clearly showed the possibility of modulating the Se release by simply changing the amount of Se in the HASe particles. The correlation between the physicochemical properties of HASe and their dissolution as a function of pH has been also investigated to facilitate future application of the NPs as chemotherapeutic adjuvant agents. Finally, the cytotoxic activity of HASe was evaluated using prostate (PC3) and breast (MDA-MB-231) cancer cells as well as healthy human bone marrow stem cells (hBMSc). HASe NPs exerted a good cytocompatibility at low concentration of Se but, with high Se doping concentration, they displayed strong cytotoxicity.

Evidence of Modular Responsiveness of Osteoblast-Like Cells Exposed to Hydroxyapatite-Containing Magnetic Nanostructures.

The development of nanocomposites with tailored physical-chemical properties, such as nanoparticles containing magnetic iron oxides for manipulating cellular events at distance, implies exciting prospects in biomedical applications for bone tissue regeneration. In this context, this study aims to emphasize the occurrence of differential responsiveness in osteoblast-like cells to different nanocomposites with diverse features: dextran-grafted iron oxide (DM) nanoparticles and their hybrid nano-hydroxyapatite (DM/n-HA) counterpart. Here, responsiveness of cells in the presence of DMs or DM/n-HAs was evaluated in terms of cytoskeletal features. We observed that effects triggered by the DM are no more retained when DM is embedded onto the DM/n-HA nanocomposites. Also, analysis of mRNA level variations of the focal adhesion kinase (FAK), P53 and SLC11A2/DMT1 human genes showed that the DM/n-HA-treated cells retain tracts of physiological responsiveness compared to the DM-treated cells. Overall, a shielding effect by the n-HA component can be assumed, masking the DM's cytotoxic potential, also hinting a modular biomimicry of the nanocomposites respect to the physiological responses of osteoblast-like cells. In this view, the biocompatibility of n-HA together with the magnetic responsiveness of DMs represent an optimized combination of structural with functional features of the DM/n-HA nano-tools for bone tissue engineering, for finely acting within physiological ranges.

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.

Platinum-loaded, selenium-doped hydroxyapatite nanoparticles selectively reduce proliferation of prostate and breast cancer cells co-cultured in the presence of stem cells.

Chemotherapeutic treatment of patients with bone tumors or bone metastases often leads to severe side effects such as high drug toxicity, lack of tumor specificity and induced drug resistance. A novel strategy to treat early stages of bone metastases involves local co-delivery of multiple chemotherapeutic agents to synergistically improve the curative effect and overcome shortcomings of traditional chemotherapy. Herein we show that selenite-doped hydroxyapatite nanoparticles loaded with a hydroxyapatite-binding anti-tumor platinum complex (PtPP-HASe) selectively reduce proliferation of cancer cells without reducing proliferation of bone marrow stem cells. These PtPP-HASe particles were nanocrystalline with selenium (Se) and platinum (Pt) contents ranging between 0-10 and 1.5-3 wt%, respectively. Release kinetics of Se and Pt from PtPP-HASe nanoparticles resulted in a cumulative release of ∼10 and ∼66 wt% after 7 days, respectively. At a Pt/Se ratio of 8, released Pt and Se species selectively reduced cell number of human prostate (PC3) and human breast cancer cells (MDA-MB-231) by a factor of >10 with limited effects on co-cultured human bone marrow stem cells (hBMSc). These novel nanoparticles demonstrate high anti-cancer selectivity, which offers ample opportunities for the design of novel biomaterials with potent and selective chemotherapeutic efficacy against cancer cells.

Sintering of magnesium-strontium doped hydroxyapatite nanocrystals: Towards the production of 3D biomimetic bone scaffolds.

The development of biomimetic scaffolds is a challenging aim in the field of bone repair for fabrication of osteoconductive and osteoinductive scaffolds. Biogenic hydroxyapatite (HA) is not stoichiometric but is substituted by several ions. An approach to improve synthetic scaffolds biomimetism can be the doping with osteoinductive ions. To this aim, herein thermally stable magnesium-strontium hydroxyapatite (HAMgSr) nanocrystals were synthesized and used for the fabrication of sintered highly porous scaffolds. The chemical and physical properties of the obtained scaffolds were analyzed by X-ray diffraction, scanning electron microscopy, mechanical testing. Three different substituting ions percentage were analyzed and among these, the copresence of Mg and Sr at 0.5 wt% has shown the best results in terms of thermal stability and mechanical properties. The potential utilization of these materials for bone regeneration purposes was preliminarily evaluated in vitro, by assaying proliferation of viable osteoblast-like cells; the experimental evidences suggest that the scaffolds can be exploited as bone-mimicking substrates suitable to support cell growth and proliferation. These observations underline the importance of the presence of Mg and Sr in scaffolds for bone remodeling as well as the good potential of the newly developed scaffolds for clinical use in tissue engineering.

Evaluation of in Vivo Response of Three Biphasic Scaffolds for Osteochondral Tissue Regeneration in a Sheep Model.

Osteochondral defects are a common problem in both human medicine and veterinary practice although with important limits concerning the cartilaginous tissue regeneration. Interest in the subchondral bone has grown, as it is now considered a key element in the osteochondral defect healing. The aim of this work was to generate and to evaluate the architecture of three cell-free scaffolds made of collagen, magnesium/hydroxyapatite and collagen hydroxyapatite/wollastonite to be implanted in a sheep animal model. Scaffolds were designed in a bilayer configuration and a novel "Honey" configuration, where columns of hydroxyapatite were inserted within the collagen matrix. The use of different types of scaffolds allowed us to identify the best scaffold in terms of integration and tissue regeneration. The animals included were divided into four groups: three were treated using different types of scaffold while one was left untreated and represented the control group. Evaluations were made at 3 months through CT analysis. The novel "Honey" configuration of the scaffold with hydroxyapatite seems to allow for a better reparative process, although we are still far from obtaining a complete restoration of the defect at this time point of follow-up.

Bioactive chitosan-based scaffolds with improved properties induced by dextran-grafted nano-maghemite and l-arginine amino acid.

Over the past years, fundamentals of magnetism opened a wide research area of interest, in the field of tissue engineering and regenerative medicine. The integration of magnetic nanoarchitectures into synthetic/natural scaffold formulations allowed obtaining "on demand" responsive structures able to guide the regeneration process. The aim of this work was the design and characterization of three-dimensional (3D) chitosan-based scaffolds containing dextran-grafted maghemite nanoarchitectures (DM) and functionalized with l-arginine (l-Arg) amino acid as bioactive agent. A homogeneous pore distribution and a high degree of interconnection were obtained for all the structures with DMs, which resulted well distributed inside the polymer matrix. All the results suggest that the simultaneous presence of DMs and l-Arg conferred interesting mechano-structural and bioactive properties toward osteoblast-like and human mesenchymal stem cells, differentially stimulating their proliferation both in the absence and in the presence of a time-dependent magnetic field. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1244-1252, 2019.

Influence of Nanofiber Orientation on Morphological and Mechanical Properties of Electrospun Chitosan Mats.

This work explored the use of chitosan (Cs) and poly(ethylene oxide) (PEO) blends for the fabrication of electrospun fiber-orientated meshes potentially suitable for engineering fiber-reinforced soft tissues such as tendons, ligaments, or meniscus. To mimic the fiber alignment present in native tissue, the CS/PEO blend solution was electrospun using a traditional static plate, a rotating drum collector, and a rotating disk collector to get, respectively, random, parallel, circumferential-oriented fibers. The effects of the different orientations (parallel or circumferential) and high-speed rotating collector influenced fiber morphology, leading to a reduction in nanofiber diameters and an improvement in mechanical properties.

Biomechanical evaluation of hMSCs-based engineered cartilage for chondral tissue regeneration.

Articular cartilage regeneration is still an open challenge in the field of tissue engineering. Although autologous chondrocytes seeded on collagen scaffolds (CSs) have already showed interesting results in the long-term repair of chondral lesions, they are not exempt from disadvantages that could be overcome using mesenchymal stem cells (MSCs). The ability of polymeric scaffolds to support MSCs proliferation and differentiation has been widely documented. However, few studies assessed their mechanical performances and additionally performing a single mechanical test, i.e. stress-strain or stress-relaxation in compression. Articular cartilage, though, possesses unique and multifaceted mechanical properties that can be exhaustively described only implementing a complete set of mechanical tests. Hence, the final aim of this study was to in depth assess the mechanical properties of human MSCs-cultured collagen scaffolds applying unconfined stress-strain, stress-relaxation and dynamic compression tests and identify key mechanical parameters. Firstly, plain CSs were fabricated and cultured under chondrogenic conditions with human MSCs (hMSCs). CSs displayed a high-interconnected porosity permitting uniform hMSCs distribution along the scaffold depth. Within CSs, hMSCs differentiated in chondroblasts, characterized by the presence of the lacunae and by a pericellular matrix positive for GAGs and for type 2 collagen deposition. The deep implemented mechanical characterization highlighted that the engineered constructs display (i) higher resistance to compression, (ii) more marked viscoelastic behavior over time and (iii) increased dynamic properties compared to naked CSs. In particular, stress-strain testes showed significant increase in the engineered constructs' stiffness that can be related to the proteoglycan deposition, observed by histology at the end of culture. Stress-relaxation and dynamic tests pointed out a substantial increase of peak and equilibrium stresses, relaxation time and dynamic modulus in the engineered constructs compared to empty CSs, suggesting a considerable decrease in scaffold permeability due to a strong chondral matrix deposition. Overall, the obtained results indicate a significant improvement of cell/CS mechanical performance toward a cartilage-like mechanical behavior.

Simplified preparation and characterization of magnetic hydroxyapatite-based nanocomposites.

Authors aimed to provide a magnetic responsiveness to bone-mimicking nano-hydroxyapatite (n-HA). For this purpose, dextran-grafted iron oxide nanoarchitectures (DM) were synthesized by a green-friendly and scalable alkaline co-precipitation method at room temperature and used to functionalize n-HA crystals. Different amounts of DM hybrid structures were added into the nanocomposites (DM/n-HA 1:1, 2:1 and 3:1weight ratio) which were investigated through extensive physicochemical (XRD, ICP, TGA and Zeta-potential), microstructural (TEM and DLS), magnetic (VSM) and biological analyses (MTT proliferation assay). X-ray diffraction patterns have confirmed the n-HA formation in the presence of DM as a co-reagent. Furthermore, the addition of DM during the synthesis does not affect the primary crystallite domains of DM/n-HA nanocomposites. DM/n-HAs have shown a rising of the magnetic moment values by increasing DM content up to 2:1 ratio. However, the magnetic moment value recorded in the DM/n-HA 3:1 do not further increase showing a saturation behaviour. The cytocompatibility of the DM/n-HA was evaluated with respect to the MG63 osteoblast-like cell line. Proliferation assays revealed that viability, carried out in the absence of external magnetic field, was not affected by the amount of DM employed. Interestingly, assays also suggested that the DM/n-HA nanocomposites exhibit a possible shielding effect with respect to the anti-proliferative activity induced by the DM particles alone.

In vitro efficacy of a novel potassium oxalate hydrogel for dentin hypersensitivity.

A novel potassium oxalate-based hydrogel is proposed for treating dentin hypersensitivity and this study evaluates its in vitro performance as a remineralizing, desensitizing agent. Etched disks of human dentin were treated for 10 or 20 min using the test hydrogel, to mimic a professional application with dental mouth guards. Dentin disks were evaluated in terms of permeability indexes in a fluid-filled system, the surface morphology was assessed by scanning electron microscopy, and the structural properties were studied using X-ray diffraction analysis. The potassium oxalate hydrogel significantly reduced dentin permeability, in a time-dependent manner, and occluded most of the patent dentinal tubules via crystal precipitation, forming a remineralized layer. After hydrogel treatments, an acid solution (pH 4.2) was applied to the disks for 30 s, or 1, 2, or 5 min, in order to reproduce a plaque-like oral acidity, and further analysis showed a good resistance of the remineralized layer to the acid challenge. The potassium oxalate-based hydrogel showed a better performance over commercially available products and artificial saliva, appearing a promising candidate for the treatment of dentin hypersensitivity.

Effect of Poly-L-Lysine coating on titanium osseointegration: from characterization to in vivo studies.

Dental implant prostheses cannot preclude a correct and stable implant osseointegration, which is still a challenge and greatly depends on biomaterial-cell interface. Titanium (Ti) coating using polyelectrolyte poly-L-lysine (PLL) may represent an interesting and simple approach, to provide a charged surface net able to improve cell adherence. However, in vitro and in vivo effects of Ti coated with PLL have been poorly investigated. The aims of the present study are (1) to obtain and characterize, chemically and physically, Ti disks coated with PLL (TiPLL); (2) to perform in vitro studies on osteoblast cell lines' cytocompatibility and functionality (alkaline phosphatase [ALP] activity, calcium deposition, proinflammatory interleukin 6 production); (3) to obtain in vivo evidence of osseointegration, using a sheep animal model. XPS, AFM, and contact-angle analyses demonstrated that the Ti disk was successfully covered with PLL, providing higher hydrophilicity to the Ti disk. No cellular toxicity, enhanced calcium deposition, and a decreased tendency toward interleukin-6 production were observed in the osteoblast seeded onto TiPLL. In vivo experiments showed cortical bone microhardness at 3 months significantly improved in the presence of the PLL coating. PLL coating on Ti implants seemed to safely enhance calcium deposition and implant early osseointegration in animals, suggesting promising evidence to optimize the surface properties of dental implants.

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.

Agarose gel as biomaterial or scaffold for implantation surgery: characterization, histological and histomorphometric study on soft tissue response.

Maxillofacial, orthopedic, oral, and plastic surgery require materials for tissue augmentation, guided regeneration, and tissue engineering approaches. In this study, the aim was to develop and characterize a new extrudable hydrogel, based on agarose gel (AG; 1.5% wt) and to evaluate the local effects after subcutaneous implantation in comparison with collagen and hyaluronic acid. AG chemical-physical properties were ascertained through Fourier transform infrared (FT-IR) spectroscopy and rheological analysis. In vivo subcutaneous implants were performed, and histological and histomorphometric evaluations were done at 1, 4, 12, and 16 weeks. FT-IR confirmed that spectroscopic properties were the same for the baseline agarose and rheological characterization established that AG is a weak hydrogel. Subcutaneous AG implants induced new vessels and fibrous tissue formation rich in neutrophils; the capsule thickness around AG increased until the 12th week but remained thinner than those around hyaluronic acid and collagen. At 16 weeks, the thickness of the capsule significantly decreased around all materials. This study confirmed that 1.5% wt AG possesses some of the most important features of the ideal biocompatible material: safety, effectiveness, costless, and easily obtained with specific chemical and geometrical characters; the AG can represent a finely controllable and biodegradable polymeric system for cells and drug delivery applications.

Nanocrystalline carbonate-apatites: role of Ca/P ratio on the upload and release of anticancer platinum bisphosphonates.

In the present study two nanocrystalline apatites have been investigated as bone-specific drug delivery devices to be used for treatment of bone tumors either by local implantation or by injection. In order to assess how the Ca/P ratio can influence the adsorption and release of anticancer platinum-bisphosphonate complexes, two kinds of apatite nanocrystals having different Ca/P ratios but similar morphologies, degree of crystallinity, and surface areas have been synthesized and characterized. The two platinum-bisphosphonate complexes considered were the bis-{ethylenediamineplatinum(ii)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and the bis-{ethylenediamineplatinum(ii)}medronate. The Ca/P ratio plays an important role in the adsorption as well as in the release of the two drugs. In fact, the apatite with a higher Ca/P ratio showed greater affinity for both platinum complexes. Also the chemical structure of the two Pt complexes appreciably affects their affinity towards as well as their release from the two kinds of apatites. In particular, the platinum complex whose bisphosphonate contains a free aminic group showed greater upload and smaller release. The cytotoxicity of the Pt complexes released from the apatite was tested against human cervical, colon, and lung cancer cells as well as against osteosarcoma cells. In agreement with previous work, the Pt complexes released were found to be more cytotoxic than the unmodified complexes.

Degradable polymers may improve dental practice.

The use of biomaterials in dentistry is more widespread than in any other medical field in terms of both amount and variety. Most of them were not originally designed for dental applications but for other medical applications or, sometimes, for no medical purposes. Among these materials, biodegradable materials play an important role, especially in bone regeneration and in periodontal surgery. This paper briefly reviews some degradable polymers developed as tools for the treatment of periodontal and bone diseases. We discuss materials previously applied in other industrials contexts, such as polyesters, methylcellulose, and chitosan and we provide perspectives for their use in periodontal regeneration.

Amino acid synergetic effect on structure, morphology and surface properties of biomimetic apatite nanocrystals.

Alanine, arginine and aspartic acid have been used as co-reagents during the synthesis of a biomimetic calcium-deficient hydroxyapatite (CDHA) for the synergistic coupling of synthesis and functionalization. The surface and bulk characterizations are consistent with a binding model in which the amino acid is preferentially bound to the CDHA nanocrystal surface by its lateral chain group. The zeta-potential measurements show that the amino acid-functionalized CDHA surface charge shifts towards neutral compared to CDHA synthesized in the absence of amino acids. Amino acids bound to the crystal induce crystal growth inhibition predominantly at the Ca-rich surfaces during the initial stages of crystallization. Moreover, high-resolution transmission electron microscopy measurements suggest a model for needle-shaped CDHA nanocrystals formation in the presence of either arginine or aspartic acid based on the oriented aggregation of primary crystallite domains specifically along the c-axis direction and the self-assembly of preformed nanoparticles. The results have significant importance for the control of the shape, morphology and aggregation of the CDHA nanocrystals, while the observed surface modifications are of marked importance for the nature, stability and reactivity of the functionalized surfaces produced.