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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.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

Injectable calcium phosphate cements (CPCs) represent promising candidates for the regeneration of complex-shape bone defects, thanks to self-hardening ability, bioactive composition and nanostructure offering high specific surface area for cell attachment and conduction. Such features make CPCs also interesting for functionalization with various biomolecules, towards the generation of multifunctional devices with enhanced therapeutic ability. In particular, strontium-doped CPCs have been studied in the last years due to the intrinsic antiosteoporotic character of strontium. In this work, a SrCPC previously reported as osteointegrative and capable to modulate the fate of bone cells was enriched with hydroxyapatite nanoparticles (HA-NPs) functionalized with tetracycline (TC) to provide antibacterial activity. We found that HA-NPs functionalized with TC (NP-TC) can act as modulator of the drug release profile when embedded in SrCPCs, thus providing a sustained and tunable TC release. In vitro microbiological tests on Escherichia coli and Staphylococcus aureus strains proved effective bacteriostatic and bactericidal properties, especially for the NP-TC loaded SrCPC formulations. Overall, our results indicate that the addition of NP-TC on CPC acted as effective modulator towards a tunable drug release control in the treatment of bone infections or cancers.

The development of new materials based on abundant elements, reduced toxicity is today crucial for the next generation of energy device. Titanium-doped hydroxyapatite (TiHA) was tested for the first time as photoanode material for Dye – Sensitized Solar Cells (DSSCs). The chemical composition and energy structure of TiHA powders with increasing titanium content (5 wt%, 10 wt% and 15 wt%) were extensively characterized by surface electron spectroscopies, XPS and UPS. Their compatibility with conventional ruthenium-based dyes molecules was also assessed, producing considerable uptake. TiHA films were produced by screen-printing technique and XRD analyses confirm the presence of apatitic structure. The film properties were completely determined by optical, morphological (FE–SEM) and functional characterizations. Finally, TiHA-based DSSCs were assembled and their photovoltaic performance were assessed. The best efficiency, equal to 0.14%, was obtained for the TiHA containing 15 wt% of titanium. These results open the path for the possible application of doped hydroxyapatite as novel materials for energy conversion systems.

Bone is a complex biologic tissue, which is extremely relevant for various physiological functions, in addition to movement, organ protection, and weight bearing. The repair of critical size bone defects is a still unmet clinical need, and over the past decades, material scientists have been expending efforts to find effective technological solutions, based on the use of scaffolds. In this context, biomimetics which is intended as the ability of a scaffold to reproduce compositional and structural features of the host tissues, is increasingly considered as a guide for this purpose. However, the achievement of implants that mimic the very complex bone composition, multi-scale structure, and mechanics is still an open challenge. Indeed, despite the fact that calcium phosphates are widely recognized as elective biomaterials to fabricate regenerative bone scaffolds, their processing into 3D devices with suitable cell-instructing features is still prevented by insurmountable drawbacks. With respect to biomaterials science, new approaches maybe conceived to gain ground and promise for a substantial leap forward in this field. The present review provides an overview of physicochemical and structural features of bone tissue that are responsible for its biologic behavior. Moreover, relevant and recent technological approaches, also inspired by natural processes and structures, are described, which can be considered as a leverage for future development of next generation bioactive medical devices.

This work describes the development of electroconductive hydrogels as injectable matrices for neural tissue regeneration by exploiting a biocompatible conductive polymer - poly(3,4-ethylenedioxythiophene)- poly(styrenesulfonate) (PEDOT:PSS) - combined with a biomimetic polymer network made of gelatin. Our approach involved also genipin - a natural cross-linking agent - to promote gelation of gelatin networks embedding PEDOT:PSS. The achieved results suggest that physical-chemical properties of the resulting hydrogels, like impedance, gelation time, mechanical properties, swelling and degradation in physiological conditions, can be finely tuned by the amount of PEDOT:PSS and genipin used in the formulation. Furthermore, the presence of PEDOT:PSS (i) enhances the electrical conductivity, (ii) improves the shear modulus of the resulting hydrogels though (iii) partially impairing their resistance to shear deformation, (iv) reduces gelation time and (v) reduces their swelling ability in physiological medium. Additionally, the resulting electroconductive hydrogels demonstrate enhanced adhesion and growth of primary rat cortical astrocytes. Given the permissive interaction of hydrogels with primary astrocytes, the presented biomimetic, electroconductive and injectable hydrogels display potential applications as minimally invasive systems for neurological therapies and damaged brain tissue repair.

The degeneration of osteochondral tissue represents one of the major causes of disability in modern society and it is expected to fuel the demand for new solutions to repair and regenerate the damaged articular joints. In particular, osteoarthritis (OA) is the most common complication in articular diseases and a leading cause of chronic disability affecting a steady increasing number of people. The regeneration of osteochondral (OC) defects is one of the most challenging tasks in orthopedics since this anatomical region is composed of different tissues, characterized by antithetic features and functionalities, in tight connection to work together as a joint. The altered structural and mechanical joint environment impairs the natural tissue metabolism, thus making OC regeneration even more challenging. In this scenario, marine-derived ingredients elicit ever-increased interest for biomedical applications as a result of their outstanding mechanical and multiple biologic properties. The review highlights the possibility to exploit such unique features using a combination of bio-inspired synthesis process and 3D manufacturing technologies, relevant to generate compositionally and structurally graded hybrid constructs reproducing the smart architecture and biomechanical functions of natural OC regions.

Gelatine is a well-known and extensively studied biopolymer, widely used in recent decades to create biomaterials in many different ways, exploiting its molecular resemblance with collagen, the main constituent of the extra-cellular matrix, from which it is derived. Many have employed this biopolymer in tissue engineering and chemically modified (e.g., gelatin methacryloyl) or blended it with other polymers (e.g., alginate) to modulate or increase its performances and printability. Nevertheless, little is reported about its use as a stand-alone material. Moreover, despite the fact that multiple works have been reported on the realization of mould-casted and three-dimensional printed scaffolds in tissue engineering, a clear comparison among these two shaping processes, towards a comparable workflow starting from the same material, has never been published. Herein, we report the use of gelatine as stand-alone material, not modified, blended, or admixed to be processed or crosslinked, for the realization of suitable scaffolds for tissue engineering, towards the two previously mentioned shaping processes. To make the comparison reliable, the same pre-process (e.g., the gelatin solution preparation) and post-process (e.g., freeze-drying and crosslinking) steps were applied. In this study, gelatine solution was firstly rheologically characterized to find a formulation suitable for being processed with both the shaping processes selected. The realized scaffolds were then morphologically, phisico-chemically, mechanically, and biologically characterized to determine and compare their performances. Despite the fact that the same starting material was employed, as well as the same pre- and post-process steps, the two groups resulted, for most aspects, in diametrically opposed characteristics. The mould-casted scaffolds that resulted were characterized by small, little-interconnected, and random porosity, high resistance to compression and slow cell colonization, while the three-dimensional printed scaffolds displayed big, well-interconnected, and geometrically defined porosity, high elasticity and recover ability after compression, as well as fast and deep cell colonization.

This work describes the preparation, characterization and functionalization with magnetic nanoparticles of a bone tissue-mimetic scaffold composed of collagen and hydroxyapatite obtained through a biomineralization process. Bone remodeling takes place over several weeks and the possibility to follow it

The regeneration of load-bearing segmental bone defects remains a significant clinical problem in orthopedics, mainly due to the lack of scaffolds with composition and 3D porous structure effective in guiding and sustaining new bone formation and vascularization in large bone defects. In the present study, biomorphic calcium phosphate bone scaffolds (GreenBone™) featuring osteon-mimicking, hierarchically organized, 3D porous structure and lamellar nano-architecture were implanted in a critical cortical defect in sheep and compared with allograft. Two different types of scaffolds were tested: one made of ion-doped hydroxyapatite/β-tricalcium-phosphate (GB-1) and other made of undoped hydroxyapatite only (GB-2). X-ray diffraction patterns of GB-1 and GB-2 confirmed that both scaffolds were made of hydroxyapatite, with a minor amount of β-TCP in GB-1. The chemical composition analysis, obtained by ICP-OES spectrometer, highlighted the carbonation extent and the presence of small amounts of Mg and Sr as doping ions in GB-1. SEM micrographs showed the channel-like wide open porosity of the biomorphic scaffolds and the typical architecture of internal channel walls, characterized by a cell structure mimicking the natural parenchyma of the rattan wood used as a template for the scaffold fabrication. Both GB-1 and GB-2 scaffolds show very similar porosity extent and 3D organization, as also revealed by mercury intrusion porosimetry. Comparing the two scaffolds, GB-1 showed slightly higher fracture strength, as well as improved stability at the stress plateau. In comparison to allograft, at the follow-up time of 6 months, both GB-1 and GB-2 scaffolds showed higher new bone formation and quality of regenerated bone (trabecular thickness, number, and separation). In addition, higher osteoid surface (OS/BS), osteoid thickness (OS.Th), osteoblast surface (Ob.S/BS), vessels/microvessels numbers, as well as substantial osteoclast-mediated implant resorption were observed. The highest values in OS.Th and Ob. S/BS parameters were found in GB-1 scaffold. Finally, Bone Mineralization Index of new bone within scaffolds, as determined by micro-indentation, showed a significantly higher microhardness for GB-1 scaffold in comparison to GB-2. These findings suggested that the biomorphic calcium phosphate scaffolds were able to promote regeneration of load-bearing segmental bone defects in a clinically relevant scenario, which still represents one of the greatest challenges in orthopedics nowadays.

A three dimensional magnetic patterning of two cell types was realised in vitro inside an additive manufactured magnetic scaffold, as a conceptual precursor for the vascularised tissue. The realisation of separate arrangements of vascular and osteoprogenitor cells, labelled with biocompatible magnetic nanoparticles, was established on the opposite sides of the scaffold fibres under the effect of non-homogeneous magnetic gradients and loading magnetic configuration. The magnetisation of the scaffold amplified the guiding effects by an additional trapping of cells due to short range magnetic forces. The mathematical modelling confirmed the strong enhancement of the magnetic gradients and their particular geometrical distribution near the fibres, defining the preferential cell positioning on the micro-scale. The manipulation of cells inside suitably designed magnetic scaffolds represents a unique solution for the assembling of cellular constructs organised in biologically adequate arrangements.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122

Long bone defects still represent a major clinical challenge in orthopedics, with the inherent loss of function considerably impairing the quality of life of the affected patients. Thus, the purpose of this study was to assess the safety and potential of bone regeneration offered by a load-bearing scaffold characterized by unique hierarchical architecture and high strength, with active surface facilitating new bone penetration and osseointegration in critical size bone defects. The results of this study showed the potential of bio-ceramization processes applied to vegetable hierarchical structures for the production of new wood-derived bone scaffolds, further improved by surface functionalization, with good biological and mechanical properties leading to successful treatment of critical size bone defects in the sheep model. Future studies are needed to evaluate if these scaffolds prototypes, as either biomaterial alone or in combination with augmentation strategies, may represent an optimal solution to enhance bone regeneration in humans.

Osteomyelitis (OM) is an infectious disease of the bone primarily caused by the opportunistic pathogen Staphylococcus aureus (SA). This Gram-positive bacterium has evolved a number of strategies to evade the immune response and subvert bone homeostasis, yet the underlying mechanisms remain poorly understood. OM has been modeled in vitro to challenge pathogenetic hypotheses in controlled conditions, thus providing guidance and support to animal experimentation. In this regard, traditional 2D models of OM inherently lack the spatial complexity of bone architecture. Three-dimensional models of the disease overcome this limitation, however, they poorly reproduce composition and texture of the natural bone. Here, we developed a new 3D model of OM based on cocultures of SA and murine osteoblastic MC3T3-E1 cells on magnesium-doped hydroxyapatite/collagen I (MgHA/Col) scaffolds that closely recapitulate the bone extracellular matrix. In this model, matrix-dependent effects were observed in proliferation, gene transcription, protein expression, and cell–matrix interactions both of the osteoblastic cell line and of bacterium. Additionally, these had distinct metabolic and gene expression profiles, compared to conventional 2D settings, when grown on MgHA/Col scaffolds in separate monocultures. Our study points to MgHA/Col scaffolds as biocompatible and bioactive matrices and provides a novel and close-to-physiology tool to address the pathogenetic mechanisms of OM at the host–pathogen interface.

Microbial infections occurring during bone surgical treatment, the cause of osteomyelitis and implant failures, are still an open challenge in orthopedics. Conventional therapies are often ineffective and associated with serious side effects due to the amount of drugs administered by systemic routes. In this study, a medicated osteoinductive and bioresorbable bone graft was designed and investigated for its ability to control antibiotic drug release in situ. This represents an ideal solution for the eradication or prevention of infection, while simultaneously repairing bone defects. Vancomycin hydrochloride and gentamicin sulfate, here considered for testing, were loaded into a previously developed and largely investigated hybrid bone-mimetic scaffold made of collagen fibers biomineralized with magnesium doped-hydroxyapatite (MgHA/Coll), which in the last ten years has widely demonstrated its effective potential in bone tissue regeneration. Here, we have explored whether it can be used as a controlled local delivery system for antibiotic drugs. An easy loading method was selected in order to be reproducible, quickly, in the operating room. The maintenance of the antibacterial efficiency of the released drugs and the biosafety of medicated scaffolds were assessed with microbiological and in vitro tests, which demonstrated that the MgHA/Coll scaffolds were safe and effective as a local delivery system for an extended duration therapy—promising results for the prevention of bone defect-related infections in orthopedic surgeries.

A 3-D porous apatite scaffold (B-HA), recently obtained through biomorphic transformation of a natural wood, is investigated on its multi-scale porous structure determining superior mechanical properties and biological behaviour. B-HA shows hierarchical pore architecture with wide aligned channels interconnected with smaller tubules, thus recapitulating in detail the lymphatic network of the original wood template. As induced by its biomimetic architecture, B-HA displays values of compression and tensile strength and stiffness, higher than the values usually measured in sintered ceramics with isotropic porosity. Furthermore, B-HA shows a ductility not common for a pure ceramic body and a tensile strength higher than its compression strength, thus occupying a zone in the Ashby chart where ceramics are usually not present. Cell co-culture tests in bioreactor report encouraging results in enhancing the complex tissue regeneration process, thus making B-HA very promising as a scaffold able to promote bone regeneration, particularly for large bone defects., 16 pages, 12 figures

Obtaining 3-D inorganic devices with designed chemical composition, complex geometry, hierarchic structure and effective mechanical performance is a major scientific goal, still prevented by insurmountable technological limitations. With particular respect to the biomedical field, there is a lack in solutions ensuring the regeneration of long, load-bearing bone segments such as the ones of limbs, due to the still unmet goal of converging, in a unique device, bioactive chemical composition, multi-scale cell-conducive porosity and a hierarchically organized architecture capable of bearing and managing complex mechanical loads in a unique 3D implant. An emerging, but still very poorly explored approach in this respect, is given by biomorphic transformation processes, aimed at converting natural structures into functional 3D inorganic constructs with smart mechanical performance. Recent studies highlighted the use of heterogeneous gas-solid reactions as a valuable approach to obtain effective transformation of natural woods into hierarchically structured apatitic bone scaffolds. In this light, the present review illustrates critical aspects related to the application of such heterogeneous reactions when occurring in the 3D state, showing the relevance of a thorough kinetic control to achieve controlled phase transformations while maintaining the multi-scale architecture and the outstanding mechanical performance of the starting natural structure. These first results encourage the further investigation towards the biologic structures optimized by nature along the ages and then the development of biomorphic transformations as a radically new approach to enable a technological breakthrough in various research fields and opening to still unexplored industrial applications.

Over the past decades, age-related pathologies have increased abreast the aging population worldwide. The increased age of the population indicates that new tools, such as biomaterials/scaffolds for damaged tissues, which display high efficiency, effectively and in a limited period of time, for the regeneration of the body's tissue are needed. Indeed, scaffolds can be used as templates for three-dimensional tissue growth in order to promote the tissue healing stimulating the body's own regenerative mechanisms. In tissue engineering, several types of biomaterials are employed, such as bioceramics including calcium phosphates, bioactive glasses, and glass–ceramics. These scaffolds seem to have a high potential as biomaterials in regenerative medicine. In addition, in conjunction with other materials, such as polymers, ceramic scaffolds may be used to manufacture composite scaffolds characterized by high biocompatibility, mechanical efficiency and load-bearing capabilities that render these biomaterials suitable for regenerative medicine applications. Usually, bioceramics have been used to repair hard tissues, such as bone and dental defects. More recently, in the field of soft tissue engineering, this form of scaffold has also shown promising applications. Indeed, soft tissues are continuously exposed to damages, such as burns or mechanical traumas, tumors and degenerative pathology, and, thereby, thousands of people need remedial interventions such as biomaterials-based therapies. It is known that scaffolds can affect the ability to bind, proliferate and differentiate cells similar to those of autologous tissues. Therefore, it is important to investigate the interaction between bioceramics and somatic/stem cells derived from soft tissues in order to promote tissue healing. Biomimetic scaffolds are frequently employed as drug-delivery system using several therapeutic molecules to increase their biological performance, leading to ultimate products with innovative functionalities. This review provides an overview of essential requirements for soft tissue engineering biomaterials. Data on recent progresses of porous bioceramics and composites for tissue repair are also presented.

Hybrid superparamagnetic microspheres with bone-like composition, previously developed by a bio-inspired assembling/mineralization process, are evaluated for their ability to uptake and deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) in therapeutically-relevant doses along with prolonged release pro- files. The comparison with hybrid non-magnetic and with non-mineralized microspheres highlights the role of nanocrystalline, nanosize mineral phases when they exhibit surface charged groups enabling the chemical linking with the growth factor and thus moderating the release kinetics. All the microspheres show excellent osteogenic ability with human mesenchymal stem cells whereas the hybrid mineralized ones show a slow and sustained release of rhBMP-2 along 14 days of soaking into cell culture medium with substantially bioactive effect, as reported by assay with C2C12 BRE-Luc cell line. It is also shown that the release extent can be modulated by the application of pulsed electromagnetic field, thus showing the potential of remote controlling the bioactivity of the new micro-devices which is promising for future application of hybrid biomimetic mi- crospheres in precisely designed and personalized therapies. info:eu-repo/semantics/publishedVersion

In bone tissue engineering, the design of 3D systems capable of recreating composition, architecture and micromechanical environment of the native extracellular matrix (ECM) is still a challenge. While perfusion bioreactors have been proposed as potential tool to apply biomechanical stimuli, its use has been limited to a low number of biomaterials. In this work, we propose the culture of human mesenchymal stem cells (hMSC) in biomimetic mineralized recombinant collagen scaffolds with a perfusion bioreactor to simultaneously provide biochemical and biophysical cues guiding stem cell fate. The scaffolds were fabricated by mineralization of recombinant collagen in the presence of magnesium (RCP.MgAp). The organic matrix was homogeneously mineralized with apatite nanocrystals, similar in composition to those found in bone. X-Ray microtomography images revealed isotropic porous structure with optimum porosity for cell ingrowth. In fact, an optimal cell repopulation through the entire scaffolds was obtained after 1 day of dynamic seeding in the bioreactor. Remarkably, RCP.MgAp scaffolds exhibited higher cell viability and a clear trend of up-regulation of osteogenic genes than control (non-mineralized) scaffolds. Results demonstrate the potential of the combination of biomimetic mineralization of recombinant collagen in presence of magnesium and dynamic culture of hMSC as a promising strategy to closely mimic bone ECM., EU Marie Curie Project "Bio-Inspired Bone Regeneration" 607051, Spanish Ministry of Science, Innovation and Universities (MCIU) RYC-2016-21042 RTI-2018-095794A-C22, Interdisciplinary Center for Clinical Research (IZKF) at the University ofWuerzburg D-361, MCIU

Biomaterial science increasingly seeks more biomimetic scaffolds that functionally augment the native bone tissue. In this paper, a new concept of a structural scaffold design is presented where the physiological multi-scale architecture is fully incorporated in a single-scaffold solution. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) bioceramic scaffolds with different bioinspired porosity, mimicking the spongy and cortical bone tissue, were studied. In vitro experiments, looking at the mesenchymal stem cells behaviour, were conducted in a perfusion bioreactor that mimics the physiological conditions in terms of interstitial fluid flow and associated induced shear stress. All the biomaterials enhanced cell adhesion and cell viability. Cortical bone scaffolds, with an aligned architecture, induced an overexpression of several late stage genes involved in the process of osteogenic differentiation compared to the spongy bone scaffolds. This study reveals the exciting prospect of bioinspired porous designed ceramic scaffolds that combines both cortical and cancellous bone in a single ceramic bone graft. It is prospected that dual core shell scaffold could significantly modulate osteogenic processes, once implanted in patients, rapidly forming mature bone tissue at the tissue interface, followed by subsequent bone maturation in the inner spongy structure.

In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs). Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated. In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3) and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA. Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold. Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA). OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control. This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.

Calcium phosphates (CaPs) are widely accepted biomaterials able to promote the regeneration of bone tissue. However, the regeneration of critical-sized bone defects has been considered challenging, and the development of bioceramics exhibiting enhanced bioactivity, bioresorbability and mechanical performance is highly demanded. In this respect, the tuning of their chemical composition, crystal size and morphology have been the matter of intense research in the last decades, including the preparation of composites. The development of effective bioceramic composite scaffolds relies on effective manufacturing techniques able to control the final multi-scale porosity of the devices, relevant to ensure osteointegration and bio-competent mechanical performance. In this context, the present work provides an overview about the reported strategies to develop and optimize bioceramics, while also highlighting future perspectives in the development of bioactive ceramic composites for bone tissue regeneration.

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

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

Many studies show how biomaterial properties like stiffness, mechanical stimulation and surface topography can influence cellular functions and direct stem cell differentiation. In this work, two different natural materials, gelatin (Gel) and cellulose nanofibrils (CNFs), were combined to design suitable 3D porous biocomposites for soft-tissue engineering. Gel was selected for its well-assessed high biomimicry that it shares with collagen, from which it derives, while the CNFs were chosen as structural reinforcement because of their exceptional mechanical properties and biocompatibility. Three different compositions of Gel and CNFs, i.e., with weight ratios of 75:25, 50:50 and 25:75, were studied. The biocomposites were morphologically characterized and their total- and macro- porosity assessed, proving their suitability for cell colonization. In general, the pores were larger and more isotropic in the biocomposites compared to the pure materials. The influence of freeze-casting and dehydrothermal treatment (DHT) on mechanical properties, the absorption ability and the shape retention were evaluated. Higher content of CNFs gave higher swelling, and this was attributed to the pore structure. Cross-linking between CNFs and Gel using DHT was confirmed. The Young&rsquo, s modulus increased significantly by adding the CNFs to Gel with a linear relationship with respect to the CNF amounts. Finally, the biocomposites were characterized in vitro by testing cell colonization and growth through a quantitative cell viability analysis performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the cell viability analysis was performed by the means of a Live/Dead test with Human mesenchymal stem cells (hMSCs). All the biocomposites had higher cytocompatibility compared to the pure materials, Gel and CNFs.

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl−/− mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl−/− mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl−/− mice that carried scaffolds with either WT or LVhsRL-transduced Rankl−/− MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl−/− mice. Stem Cells Translational Medicine 2019;8:22–34

Scaffolds for bone regeneration have been engineered by a plethora of manufacturing technologies and biomaterials. However, the performance of these systems is often limited by lack of robustness in the process design, that hampers their scalability to clinical application. In the present study, Design of Experiment (DoE) was used as statistical tool to design the biofabrication of hybrid hydroxyapatite (HA)/collagen scaffolds for bone regeneration and optimize their integration in a multilayer osteochondral device. The scaffolds were synthesized via a multi-step bioinspired process consisting in HA nano-crystals nucleation on the collagen self-assembling fibers and ribose glycation was used as collagen cross-linking method to modulate the mechanical and physical properties. The process design was performed by selecting hydrogel concentration, HA/collagen ratio and cross-linker content as key variables and the fabrication was carried out basing on a full factorial design. Scaffold performances were tested by evaluating porosity, swelling ratio, degradation rate and mechanical behavior as model output responses while physicochemical properties of the constructs were evaluated by TGA, ICP, FT-IR spectroscopy, and XRD analysis. Physicochemical characterizations confirmed the nucleation of a biomimetic inorganic phase and the interaction of the HA and collagenic components. The DoE model revealed a significant interaction between HA content and collagen cross-linking in determining porosity, swelling and mechanical properties of the scaffolds. The combined effect of hydrogel concentration and mineral phase played a key role on porosity and swelling while degradation resulted to be mainly affected by the HA loading and ribose content. The model was then used to determine the suitable input parameters for the synthesis of multi-layer scaffolds with graded mineralization rate, that can be used to mimic the whole cartilage-bone interface. This work proved that experimental design applied to complex biofabrication processes represents an effective and reliable way to design hybrid constructs with standardized and tunable properties for osteochondral tissue engineering.

Biomineralization consists of a complex cascade of phenomena generating hybrid nano-structured materials based on organic (e.g., polymer) and inorganic (e.g., hydroxyapatite) components. Biomineralization is a biomimetic process useful to produce highly biomimetic and biocompatible materials resembling natural hard tissues such as bones and teeth. In detail, biomimetic materials, composed of hydroxyapatite nanoparticles (HA) nucleated on an organic matrix, show extremely versatile chemical compositions and physical properties, which can be controlled to address specific challenges. Indeed, different parameters, including (i) the partial substitution of mimetic doping ions within the HA lattice, (ii) the use of different organic matrices, and (iii) the choice of cross-linking processes, can be finely tuned. In the present review, we mainly focused on calcium biomineralization. Besides regenerative medicine, these multifunctional materials have been largely exploited for other applications including 3D printable materials and in vitro three-dimensional (3D) models for cancer studies and for drug testing. Additionally, biomineralized multifunctional nano-particles can be involved in applications ranging from nanomedicine as fully bioresorbable drug delivery systems to the development of innovative and eco-sustainable UV physical filters for skin protection from solar radiations.

Bioceramics are widely considered as elective materials for the regeneration of bone tissue, due to their compositional mimicry with bone inorganic components. However, they are intrinsically brittle, which limits their capability to sustain multiple biomechanical loads, especially in the case of load-bearing bone districts. In the last decades, intense research has been dedicated to combining processes to enhance both the strength and toughness of bioceramics, leading to bioceramic composite scaffolds. This review summarizes the recent approaches to this purpose, particularly those addressed to limiting the propagation of cracks to prevent the sudden mechanical failure of bioceramic composites.

The need of synthetic bone grafts that recreate from macro- to nanoscale level the biochemical and biophysical cues of bone extracellular matrix has been a major driving force for the development of new generation of biomaterials. In this study, synthetic bone substitutes have been synthesized via biomimetic mineralization of a recombinant collagen type I-derived peptide (RCP), enriched in tri-amino acid sequence arginine-glycine-aspartate (RGD). Three-dimensional (3D) isotropic porous scaffolds of three different compositions are developed by freeze-drying: non-mineralized (RCP, as a control), mineralized (Ap/RCP), and mineralized scaffolds in the presence of magnesium (MgAp/RCP) that closely imitate bone composition. The effect of mineral phase on scaffold pore size, porosity, and permeability, as well as on their in vitro kinetic degradation, is evaluated. The ultimate goal is to investigate how chemical (i.e., surface chemistry and ion release from scaffold) together with physical signals (i.e., surface nanotopography) conferred via biomimetic mineralization can persuade and guide mesenchymal stem cell (MSC) interaction and fate. The three scaffold compositions showed optimum pore size and porosity for osteoconduction, without significant differences between them. The degradation tests confirmed that MgAp/RCP scaffolds presented higher reactivity under physiological condition compared to Ap/RCP ones. The in vitro study revealed an enhanced cell growth and proliferation on MgAp/RCP scaffolds at day 7, 14, and 21. Furthermore, MgAp/RCP scaffolds potentially promoted cell migration through the inner areas reaching the bottom of the scaffold after 14 days. MSCs cultured on MgAp/RCP scaffolds displayed higher gene and protein expressions of osteogenic markers when comparing them with the results of those MSCs grown on RCP or Ap/RCP scaffolds. This work highlights that mineralization of recombinant collagen mimicking bone mineral composition and morphology is a versatile approach to design smart scaffold interface in a 3D model guiding MSC fate.

The present work describes the synthesis and characterization of new apatite phases co-doped with gallium, magnesium and carbonate, exhibiting osteogenic and antibacterial ability. The apatites are synthesized at low temperature to retain nanocrystallinity and controlled doping with the various bioactive foreign ions, as assessed by physico-chemical and crystallographic analyses, reporting the achievement of single phases with reduced crystal ordering. The analysis of single and multi-doped apatites reports to different mechanisms acting in the incorporation of gallium and magnesium ions in the apatite structure. The release of bioactive ions is correlated to the behaviour of human mesenchymal stem cells and of different bacterial strands, selected among the most frequently affecting surgical procedures. Enhanced osteogenic and antibacterial ability is assessed in multi-doped apatites, thus suggesting potential future applications as new smart biomaterials integrating a significant boosting of bone regeneration with adequate protection against bacteria. This article is protected by copyright. All rights reserved.

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

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl−/− mice, and found that Rankl−/− BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl−/− BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics.

Bones and hard tissues including the cartilage and teeth are of key importance for the human life, in terms of mobility, manipulation, chewing, and thus it is today one of the most demanding issues in medicine. In the last decades, a great deal of research effort has been devoted to the development of synthetic scaffolds, aiming to drive and sustain the bone regeneration process. Particularly relevant is the concept of "biomimetics", that is the intrinsic ability of a synthetic material to closely reproduce the chemical composition, physical properties, and architecture of native tissues, with the purpose to create 3-D environments able to deliver signals stimulating cell chemotaxis and specific differentiation of autologous stem cells. In the last decades, the research on regenerative bioceramics has greatly expanded, with the purpose to achieve the optimal composition, multi-scale structure, and mechanical performance. In this respect, the present article describes the main features requested for ceramic bone scaffolds and the most recent approaches to develop regenerative bioceramics, with particular respect to bioactive ceramic nanoparticles and their functionalization, 3-D porous ceramics and self-hardening injectable ceramic pastes and cements. Furthermore, the article explores recent advances in additive manufacturing techniques that, in spite of technological difficulties, are considered as a major approach to obtain implantable, personalized devices with organized and tailored porosity. Finally, the article gives an overview on some clinical results encouraging further research in the field.

Osteoporosis stems from an unbalance between bone mineral resorption and deposition. Among the numerous cellular players responsible for this unbalance bone marrow (BM) monocytes/macrophages, mast cells, T and B lymphocytes, and dendritic cells play a key role in regulating osteoclasts, osteoblasts, and their progenitor cells through interactions occurring in the context of the different bone compartments (cancellous and cortical). Therefore, the microtopography of immune cells inside trabecular and compact bone is expected to play a relevant role in setting initial sites of osteoporotic lesion. Indeed, in physiological conditions, each immune cell type preferentially occupies either endosteal, subendosteal, central, and/or perisinusoidal regions of the BM. However, in the presence of an activation, immune cells recirculate throughout these different microanatomical areas giving rise to a specific distribution. As a result, the trabeculae of the cancellous bone and endosteal free edge of the diaphyseal case emerge as the primary anatomical targets of their osteoporotic action. Immune cells may also transit from the BM to the depth of the compact bone, thanks to the efferent venous capillaries coursing in the Haversian and Volkmann canals. Consistently, the innermost parts of the osteons and the periosteum are later involved by their immunomodulatory action, becoming another site of mineral reabsorption in the course of an osteoporotic insult. The novelty of our updating is to highlight the microtopography of bone immune cells in the cancellous and cortical compartments in relation to the most consistent data on their action in bone remodeling, to offer a mechanist perspective useful to dissect their role in the osteoporotic process, including bone damage derived from the immunomodulatory effects of endocrine disrupting chemicals.

Bone is the third most frequent site of metastasis, with a particular incidence in breast and prostate cancer patients. For example, almost 70% of breast cancer patients develop several bone metastases in the late stage of the disease. Bone metastases are a challenge for clinicians and a burden for patients because they frequently cause pain and can lead to fractures. Unfortunately, current therapeutic options are in most cases only palliative and, although not curative, surgery remains the gold standard for bone metastasis treatment. Surgical intervention mostly provides the replacement of the affected bone with a bioimplant, which can be made by materials of different origins and designed through several techniques that have evolved throughout the years simultaneously with clinical needs. Several scientists and clinicians have worked to develop biomaterials with potentially successful biological and mechanical features, however, only a few of them have actually reached the scope. In this review, we extensively analyze currently available biomaterials-based strategies focusing on the newest and most innovative ideas while aiming to highlight what should be considered both a reliable choice for orthopedic surgeons and a future definitive and curative option for bone metastasis and cancer patients.

In the present work, hydroxyapatite (HA) nanoparticles doped with Mg2+, Sr2+, and Zn(2+)ions are developed by wet neutralization method and then sintered at 1,250 degrees C to obtain bulk consolidated materials. Physicochemical and microstructural analyses show that the presence of doping ions in the HA structure induced the formation of beta TCP as secondary phase, during the sintering process, and we found that this effect is depending on the stability of the various doping ions in the hydroxyapatite lattice itself. We also found that the formation of beta TCP as secondary phase, in turn, confines the grain growth of HA induced by the high-temperature sintering process, thus leading to a strong increase of the flexural strength of the bulk materials, according to Hall-Petch-like law. Furthermore, we found that the doping ions enter also in the structure of the beta TCP phase; besides the grain growth confinement, also the solubility and ion release ability of the final materials were enhanced. In addition to ameliorate the mechanical performance, the described phenomena also activate multiple biofunctionalities: (i) ability to upregulate various genes involved in the osteogenesis, as obtained by human adipose stem cells culture and evaluated by array technology; (ii) enhanced resistance to the adhesion and proliferation of Gram+ and Gram- bacterial strains. Hence, our results open a perspective for the use of sintered multiple ion-doped HA to develop ceramic biodevices, such as plates, screws, or other osteosynthesis media, with enhanced strength, osteointegrability, and the ability to prevent post-surgical infections.

In an attempt to mimic the outstanding mechanical properties of wood and bone, a 3D heterogeneous chemistry approach has been used in a biomorphic transformation process (in which sintering is avoided) to fabricate ceramics from rattan wood, preserving its hierarchical fibrous microstructure. The resulting material (called biomorphic apatite ​[BA] henceforth) possesses a highly bioactive composition and is characterised by a multiscale hierarchical pore structure, based on nanotwinned hydroxyapatite lamellae, which is shown to display a lacunar fractal nature. The mechanical properties of BA are found to be exceptional (when compared with usual porous hydroxyapatite and other ceramics obtained from wood through sintering) and unique ​as they occupy a zone in the Ashby map previously free from ceramics, but not far from wood and bone. Mechanical tests show the following: (i) the strength in tension may exceed that in compression, (ii) failure in compression involves complex exfoliation patterns, thus resulting in high toughness, (iii) unlike in sintered porous hydroxyapatite, fracture does not occur ‘instantaneously,’ ​but its growth may be observed, and it exhibits tortuous patterns that follow the original fibrillar structure of wood, thus yielding outstanding toughness, (iv) the anisotropy of the elastic stiffness and strength show unprecedented values when situations of stresses parallel and orthogonal to the main channels are compared. Despite being a ceramic material, BA displays a mechanical behavior similar on the one hand to the ligneous material from which it was produced (therefore behaving as a ‘ceramic with the signature of wood’) and on the other hand to the cortical/spongy osseous complex constituting the structure of compact bone. Keywords: Hydroxyapatite, Mechanical properties, Strength, Fracture, Mechanical tests, Fractal porosity

Citrate and fluoride ions are two constituents of dental enamel hydroxyapatite (HA) nanocrystals. Their individual effect on HA crystallization has already been studied, and it was proven that both citrate and fluoride ions regulate HA crystal growth. However, the combined effect of citrate and fluoride ions on HA nanocrystals has never been reported so far. In this work, we have prepared citrate-fluoride-HA (citrate-FHA) nanoparticles in mild conditions, and we have studied the evolution of morphology and composition upon maturation. We have proven that even in the presence of citrate, fluoride ions are incorporated in the apatitic structure (replacing hydroxyl ions) and accelerate the crystallization process. Interestingly, citrate-FHA nanoparticles exhibit a flattened hexagonal rod-like morphology in contrast to the needle-like platelet morphology of citrate-HA. The density of citrate ions bound on the citrate-FHA surface is higher than that on citrate-HA. Moreover, the relative amount of unidentate citrate-Ca2+ adducts versus the ionic-like ones is higher for citrate-FHA than for citrate-HA. Our results provide a deeper understanding of the combined effect of citrate and fluoride ions on HA nanocrystals that can be used for the design of advanced biomaterials with tailored features, for a better comprehension of the enamel biomineralization process, and for the synthesis of enamel-like nanocrystals.

Many medical-related scientific discoveries arise from trial-error patterns where the processes involved must be refined and modified continuously before any product could be able to reach the final costumers. One of the elements affecting negatively these processes is the inaccuracy of two-dimension (2D) standard culture systems, carried over in plastic plates or similar, in replicating complex environments and patterns. Consequently, animal tests are required to validate every in vitro finding, at the expenses of more funds and ethical issues. A possible solution relies in the implementation of three-dimension (3D) culture systems as a fitting gear between the 2D tests and in vivo tests, aiming to reduce the negative in vivo outcomes. These 3D structures are depending from the comprehension of the extracellular matrix (ECM) and the ability to replicate it in vitro. In this article a comparison of efficacies between these two culture systems was taken as subject, human mesenchymal stem cells (hMSCs) was utilized and a hybrid scaffold made by a blend of chitosan, gelatin and biomineralized gelatin was used for the 3D culture system. [Figure not available: see fulltext.].

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.

Purpose:This prospective study is aimed at investigating clinically and histologically the effectiveness of a biomimetic magnesium-enriched-hydroxyapatite (MgHA)/collagen-based bone substitute for alveolar socket preservation. Materials:Patients scheduled for posterior single tooth extraction were included. The alveolar socket was filled either with MgHA or deproteinized bovine bone matrix (DBBM). In DBBM group, a punch of mucosa was taken from the palate and used to cover the graft. Vertical and horizontal dimensional changes of the alveolar process were assessed clinically with a periodontal probe and with 3-dimensional (3D) analysis of a cast model. Postoperative quality of life was assessed through a questionnaire. After 6 months of healing, an alveolar tissue biopsy was taken for histologic and histomorphometric analysis of the newly formed tissue. After checking normality of the distributions, parametric or nonparametric tests were used for statistical comparisons. Results:Twenty patients (12 males, 8 females, mean age 42.8 ± 5.1 years, range 33-50 years) were treated. After 6 months, vertical and horizontal alveolar ridge resorption was similar in the 2 groups. The 3D analysis of the models showed a significantly higher resorption at the buccal side than at the palatal/lingual side. Histomorphometric analysis showed similar new bone formation for MgHA group (23.07 ± 10.3%) and DBBM (22.77 ± 6.95%), and a significantly higher residual material% for DBBM (15.77 ± 1.95%) than MgHA (5.01 ± 1.04%). Significantly less pain was reported in the first 3 days after surgery in patients of the MgHA group. Conclusion:The MgHA was as safe and effective as DBBM and may represent a feasible bone substitute for alveolar socket preservation.

Hydroxyapatite represents the natural inorganic component of the bone and may be considered an essential element required for the development of bone substitutes in the field of regenerative medicine. Hydroxyapatite bone substitutes own biomimetic, osteoconductive, and osteoinductive properties thanks to their chemical-physical properties and nanostructure that play a critical role for the reconstruction of calcified tissues. Controlling the structure of hydroxyapatite nanocrystals is vital for obtaining a sustained product, and it should be an advantage on the final materials suitable for bone replacement, in terms of adsorptive activity, drug delivery system, etc. Compared to other synthesis techniques, hydrothermal processing (refers to a synthesis in aqueous solution at elevated pressure and temperature, in a closed system) has the ability to precipitate the hydroxyapatite from overheated solution, regulating the rate and uniformity of nucleation, growth, and maturation, which affect size, morphology, and aggregation of the crystals. This chapter wants to provide an overview of realization of nanosized hydroxyapatite-based bioceramics (e.g., powder and 3D structures) with desired morphology of crystallites, by hydrothermal processing. In this way, some critical hydrothermal parameters fundamental on the control of the crystal shape and dimension (pH, temperature, starting precursors, etc.) are discussed.

Synthetic osteoinductive materials that mimic the human osteogenic niche have emerged as ideal candidates to address this area of unmet clinical need. In this study, we evaluated the osteoinductive potential in a rabbit orthotopic model of a magnesium-doped hydroxyapatite/type I collagen ​(MHA/Coll) composite. The composite was fabricated to exhibit a highly fibrous structure of carbonated MHA with 70% (±2.1) porosity and a Ca/P ratio of 1.5 (±0.03) as well as a diverse range of elasticity separated to two distinct stiffness peaks of low (2.35 ​± ​1.16 ​MPa) and higher (9.52 ​± ​2.10 ​MPa) Young's Modulus. Data suggested that these specific compositional and nanomechanical material properties induced the deposition of de novo mineral phase, while modulating the expression of early and late osteogenic marker genes, in a 3D in vitro model using human bone marrow-derived mesenchymal stem cells (hBM-MSCs). When tested in the rabbit orthotopic model, MHA/Col1 scaffold induction of new trabecular bone mass was observed by DynaCT scan, only 2 weeks after implantation. Bone histomorphometry at 6 weeks revealed a significant amount of de novo bone matrix formation. qPCR demonstrated MHA/Coll scaffold full cellularization in vivo and the expression of both osteogenesis-associated genes (Spp1, Sparc, Col1a1, Runx2, Dlx5) as well as hematopoietic (Vcam1, Cd38, Sele, Kdr) and bone marrow stromal cell marker genes (Vim, Itgb1, Alcam). Altogether, these data provide ​evidence of the solid osteoinductive potential of MHA/Coll and its suitability for multiple approaches of bone regeneration., Highlights • We tested magnesium-doped hydroxyapatite/type I collagen (MHA/Coll) in a biologics-free orthotopic bone regeneration model. • MHA/Coll allowed bone formation within 6 weeks in a rabbit orthotopic model. • MHA/Coll supported the formation of both an osteogenic and hematopoietic-like niche.

Material science is a relevant discipline in support of regenerative medicine. Indeed, tissue regeneration requires the use of scaffolds able to guide and sustain the natural cell metabolism towards tissue regrowth. This need is particularly important in musculoskeletal regeneration, such as in the case of diseased bone or osteocartilaginous regions for which calcium phosphate-based scaffolds are considered as the golden solution. However, various technological barriers related to conventional ceramic processing have thus far hampered the achievement of biomimetic and bioactive scaffolds as effective solutions for still unmet clinical needs in orthopaedics. Driven by such highly impacting socioeconomic needs, new nature-inspired approaches promise to make a technological leap forward in the development of advanced biomaterials. The present review illustrates ion-doped apatites as biomimetic materials whose bioactivity resides in their unstable chemical composition and nanocrystallinity, both of which are, however, destroyed by the classical sintering treatment. In the following, recent nature-inspired methods preventing the use of high-temperature treatments, based on (i) chemically hardening bioceramics, (ii) biomineralisation process, and (iii) biomorphic transformations, are illustrated. These methods can generate products with advanced biofunctional properties, particularly biomorphic transformations represent an emerging approach that could pave the way to a technological leap forward in medicine and also in various other application fields.

Magnetic scaffolds have recently attracted significant attention in tissue engineering due to the prospect of improving bone tissue formation by conveying soluble factors such as growth factors, hormones, and polypeptides directly to the site of implantation, as well as to the possibility of improving implant fixation and stability. The objective of this study was to compare bone tissue formation in a preclinical rabbit model of critical femoral defect treated either with a hydroxyapatite (HA)/magnetite (90/10 wt %) or pure HA porous scaffolds at 4 and 12 weeks after implantation. The biocompatibility and osteogenic activity of the novel magnetic constructs was assessed with analysis of the amount of newly formed bone tissue and its nanomechanical properties. The osteoconductive properties of the pure HA were confirmed. The HA/magnetite scaffold was able to induce and support bone tissue formation at both experimental time points without adverse tissue reactions. Biomechanically, similar properties were obtained from nanoindentation analysis of bone formed following implantation of magnetic and control scaffolds. The results indicate that the osteoconductive properties of an HA scaffold are maintained following inclusion of a magnetic component. These provide a basis for future studies investigating the potential benefit in tissue engineering of applying magnetic stimuli to enhance bone formation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 546-554, 2018.