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BackgroundIn an era of precision and stratified medicine, homogeneity in population-based cohorts, stringent causative entry, and pattern analysis of datasets are key elements to investigate medical treatments. Adhering to these principles, we collected in vivo and in vitro data pointing to an insulin-sensitizing/insulin-mimetic effect of myo-inositol (MYO) relevant to cell regeneration in dentistry and oral surgery. Confirmation of this possibility was obtained by in silico analysis of the relation between in vivo and in vitro results (the so-called bed-to-benchside reverse translational approach).ResultsFourteen subjects over the 266 screened were young adult, normal weight, euglycemic, sedentary males having normal appetite, free diet, with a regular three-times-a-day eating schedule, standard dental hygiene, and negligible malocclusion/enamel defects. Occlusal caries were detected by fluorescence videoscanning, whereas body composition and energy balance were estimated with plicometry, predictive equations, and handgrip. Statistically significant correlations (Pearson r coefficient) were found between the number of occlusal caries and anthropometric indexes predicting insulin resistance (IR) in relation to the abdominal/visceral fat mass, fat-free mass, muscular strength, and energy expenditure adjusted to the fat and muscle stores. This indicated a role for IR in affecting dentin reparative processes. Consistently, in vitro administration of MYO to HUVEC and Swiss NIH3T3 cells in concentrations corresponding to those administered in vivo to reduce IR resulted in statistically significant cell replication (ANOVA/Turkey tests), suggesting that MYO has the potential to counteract inhibitory effects of IR on dental vascular and stromal cells turnover. Finally, in in silico experiments, quantitative evaluation (WOE and information value) of a bioinformatic Clinical Outcome Pathway confirmed that in vitro trophic effects of MYO could be transferred in vivo with high predictability, providing robust credence of its efficacy for oral health.ConclusionOur reverse bed-to-benchside data indicate that MYO might antagonize the detrimental effects of IR on tooth decay. This provides feasibility for clinical studies on MYO as a regenerative factor in dentistry and oral surgery, including dysmetabolic/aging conditions, bone reconstruction in oral destructive/necrotic disorders, dental implants, and for empowering the efficacy of a number of tissue engineering methodologies in dentistry and oral surgery.

The present study focuses on tailoring the relative content of Fe3+ and Fe2+ ions incorporation into hydroxyapatite (HA) lattice, employing a hydrothermal approach in a closed vessel to minimize Fe2+ oxidation and secondary phase formation. Citrate molecules are used to regulate nanoparticle formation/stability, creating a mild reducing environment, while the impact of a stronger reducing agent, hydroxylamine, is explored. Fe3+ insertion was found to be less favoured than Fe2+, possibly due to charge imbalance. Iron doping significantly alters stoichiometry and crystallinity of HA, with Fe3+ enhancing OH− depletion. Morphological analysis reveals differences among samples, as induced by the different Fe ions incorporation: particularly Fe2+ ion incorporation is found to maintain rod-like structures, which changes upon Fe3+ presence. Overall, this study provides insights into controlled doping of HA with iron ions, vital for developing stable, redox-responsive nanomaterials applicable in cancer therapy and other applications where surface activity plays a relevant role.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

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.

Abstract 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.

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 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.

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.

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.

Abstract 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

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.

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.

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.

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.

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.

Tooth loss is a common consequence of a huge number of causes and can decrease the quality of humans’ life. Tooth is a complex organ composed of soft connective tissues and mineralized tissues of which dentin is the most voluminous component whose formation is regulated by a very complex process displaying several similarities with osteogenesis. Calcium phosphates, in particular hydroxyapatite (HA), is the phase present in higher amount into the structure of dentin, characterized by microscopic longitudinal dentinal tubules. To address the challenge of dental tissue regeneration, here we propose a novel biomimetic approach, to design hybrid scaffolds resembling the physico-chemical features of the natural mineralized tissues, suitable to recreate an appropriate microenvironment that stimulates cell colonization and proliferation, therefore effective for improving regenerative approach in dental applications. Biomineralization is the adopted synthesis as a nature inspired process consisting in the nucleation of magnesium-doped-hydroxyapatite (MgHA) nanocrystals on the gelatin (Gel) matrix generating hybrid flakes (Gel/MgHA) featured by a Gel:MgHA weight ratio close to 20:80 and size of 50–70 μm. Chemical and topotactic constrains affect the formation of MgHA mineral phase on the organic template, generating quasi-amorphous MgHA as revealed by XRD analysis and Ca/P ratio lower than 1.67, resembling the chemical and biological features of the natural apatite. The Gel/MgHA was then merged into the polymeric blend made of chitosan (Chit) and Gel to obtain a 3D porous scaffold with polymers: MgHA weight ratio of 40:60 and featured by an aligned porous structure as obtained by controlled freeze-drying process. The overall composite shows a swelling ratio of about 15 times after 6 h in PBS. The chemical stability was assured by means of a dehydrothermal cross-linking treatment (DHT) keeping the degradation lower than 20% after 28 days, while cell adhesion and proliferation were evaluated using a mouse fibroblast cell line.

In the present work, hydroxyapatite (HA) nanoparticles doped with Mg2+, Sr2+, and Zn2+ ions are developed by wet neutralization method and then sintered at 1,250°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 βTCP 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 βTCP 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 βTCP 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.

Abstract 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

Abstract The surface structure and hydrophilicity of synthetic nanocrystalline apatite with strongly bound citrates on their surface are here investigated at the molecular level, by combining advanced IR spectroscopy, microgravimetry and adsorption microcalorimetry. Citrate are found to form unidentate-like and ionic-like complexes with surface Ca2+ ions, with a surface coverage closely resembling that present in bone apatite platelets (i.e., 1 molecule/(n nm)2, with n ranging between 1.4 and 1.6). These surface complexes are part of a hydrated non-apatitic surface layer with a sub-nanometre thickness. Noticeably, it is found that the hydrophilicity of the nanoparticles, measured in terms of adsorption of water molecules in the form of multilayers, decreases in a significant extent in relation to the presence of citrates, most likely because of the exposure toward the exterior of –CH2 groups. Our findings provide new insights on the surface properties of bio-inspired nano-apatites, which can be of great relevance for better understanding the role of citrate in determining important interfacial properties, such as hydrophobicity, of bone apatite platelets. The evaluation and comprehension of surface composition and structure is also of paramount interest to strictly control the functions of synthetic biomaterials, since their surface chemistry strongly affects the hosting tissue response.

The regeneration of bone fractures, resulting from trauma, osteoporosis or tumors, is a major problem in our super-aging society. Bone regeneration is one of the main topics of concern in regenerative medicine. In recent years, stem cells have been employed in regenerative medicine with interesting results due to their self-renewal and differentiation capacity. Moreover, stem cells are able to secrete bioactive molecules and regulate the behavior of other cells in different host tissues. Bone regeneration process may improve effectively and rapidly when stem cells are used. To this purpose, stem cells are often employed with biomaterials/scaffolds and growth factors to accelerate bone healing at the fracture site. Briefly, this review will describe bone structure and the osteogenic differentiation of stem cells. In addition, the role of mesenchymal stem cells for bone repair/regrowth in the tissue engineering field and their recent progress in clinical applications will be discussed.

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.

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.

Stem cell homing, namely the recruitment of mesenchymal stem cells (MSCs) to injured tissues, is highly effective for bone regeneration in vivo. In order to explore whether the incorporation of mimetic peptide sequences on magnesium-doped (Mg-doped) hydroxyapatite (HA) may regulate the homing of MSCs, and thus induce cell migration to a specific site, we covalently functionalized MgHA disks with two chemotactic/haptotactic factors: either the fibronectin fragment III1-C human (FF III1-C), or the peptide sequence Gly-Arg-Gly-Asp-Ser-Pro-Lys, a fibronectin analog that is able to bind to integrin transmembrane receptors. Preliminary biological evaluation of MSC viability, analyzed by 3-(4,5-dimethyl­thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, suggested that stem cells migrate to the MgHA disks in response to the grafted haptotaxis stimuli.

Calcium phosphate cements (CPCs) have been extensively studied in last decades as nanostructured biomaterials for the regeneration of bone defects, both for dental and orthopedic applications. However, the precise control of their handling properties (setting time, viscosity, and injectability) still represents a remarkable challenge because a complicated adjustment of multiple correlated processing parameters is requested, including powder particle size and the chemical composition of solid and liquid components. This study proposes, for the first time, a multifactorial investigation about the effects of powder and liquid variation on the final performance of Sr-doped apatitic CPCs, based on the Design of Experiment approach. In addition, the effects of two mixing techniques, hand spatula (low-energy) and planetary shear mixing (high-energy), on viscosity and extrusion force were compared. This work aims to shed light on the various steps involved in the processing of CPCs, thus enabling a more precise and tailored design of the device, based on the clinical need.

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’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.

Background: Many bone substitutes have been applied for sinus regeneration procedures, such as autogenous bone, inorganic bovine bone, porous and resorbable hydroxyapatite, tricalcium phosphate, bioactive glass, and blood clots. The aim of the present study was a tomographic, histological and histomorphometrical evaluation in humans, of specimens retrieved from sinuses augmented with MgHA/collagen-based scaffolds, after a healing period of 6 months.Materials and Methods: Eleven healthy patients and a total of 15 sinuses were included in this study. The maxillary sinuses were filled with commercial MgHA/collagen-based scaffolds (RegenOss) with a porous three-dimensional (3D) structure (Fin-Ceramica Faenza S.p.A., Faenza, Italy). These grafts have a composite design, that replicate the organization of bone structure, obtained by a technique in which a specific hybrid organic–inorganic composite is spontaneously built by a biological mechanism. The CBCT scans were done before the procedure, after the surgical protocol (T1), and 6 months after sinus surgery (T2) for implantology. Bone specimens were stored in 10% formalin solution, embedded in a glycolmethacrylate resin and sectioned by a high-precision diamond disc. Histologic and histomorphometric analysis were carried out to evaluate the graft reabsorption and bone healing.Results: The mean volume after graft elevation, calculated for each of the 15 sinuses, was 2,906 mm3 in the immediate postoperative period (5–7 days), ranging from 2,148.8 to 3,146.4 mm3. In the late postoperative period (6 months) it was 2,806.7 mm3, ranging from 2,010.9 to 3,008.9 mm3. The sinuses were completely healed and no residual MgHA/collagen-based scaffolds were visible. Osteoblasts appeared actively secreting bone matrix and marrow spaces contained moderate numbers of stromal cells and vascular network. Osteoblasts were observed actively secreting osteoid matrix. The tissues present in the samples were composed of 1.9 ± 1.9% of lamellar bone, 36 ± 1% of woven bone and 58 ± 3.8% of marrow spaces.Conclusion: Mg-MgHA/collagen-based scaffolds can successfully be used for sinus augmentation procedures.

OBJECTIVE: To determine the effect of laser irradiation at a low dose on human osteoblastlike cells. Materials and methods: 32 porous hydroxyapatite scaffolds currently used for bone tissue engineering were seeded with MG63 cells and irradiated or not with a GaAlAs diode laser (wavelength 915 nm, dose 2 J/cm²) using different power density and exposure duration. RESULTS: After 72-h incubation, cells showed well spread morphology and good adhesion on both laser-treated and untreated scaffolds. Laser irradiation did not interfere in cell viability and proliferation as compared with the non-irradiated controls. CONCLUSION: This study suggests that there is no effect of 915 nm laser irradiation at a dose of 2 J/cm² on the proliferation rate of MG63 cells. Future investigations are needed to compare different dose and wavelength regimens in order to determine the optimal set of laser parameters for maximum cell yield and safe clinical application.

Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

Traditionally regarded as single cell organisms, bacteria naturally and preferentially build multicellular communities that enable them to react efficiently to external stimuli in a coordinated fashion and with extremely effective outcomes. These communities are bacterial biofilms, where single cells or microcolonies are embedded in self-built Extracellular Polymeric Substance (EPS), composed of different macromolecules, e.g., polysaccharides, proteins, lipids, and extracellular DNA (eDNA). Despite being the most common form in nature and having many biotechnologically useful applications, biofilm is often regarded as a life-threatening form of bacterial infection. Since this form of bacterial life is intrinsically more resistant to antibiotic treatment and antimicrobial resistance is reaching alarming levels, we will focus our attention on how nanotechnology made new tools available to the medical community for the prevention and treatment of these infections. After a brief excursus on biofilm formation and its main characteristics, different types of nanomaterials developed to prevent or counteract these multicellular forms of bacterial infection will be described. A comparison of different classifications adopted for nanodrugs and a final discussion of challenges and future perspectives are also presented.

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