Advantages and limitations of using cell viability assays for 3D bioprinted constructs.

Bioprinting shows promise for bioengineered scaffolds and three-dimensional (3D) disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumor cells, with or without normal bone cells). Despite demonstrating 80%-90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell-cell interactions through thez-axis. In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques.

Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs.

Age-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need ofin vivoassay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, currentin vitropreclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes.

Incorporation/Enrichment of 3D Bioprinted Constructs by Biomimetic Nanoparticles: Tuning Printability and Cell Behavior in Bone Models.

Reproducing in vitro a model of the bone microenvironment is a current need. Preclinical in vitro screening, drug discovery, as well as pathophysiology studies may benefit from in vitro three-dimensional (3D) bone models, which permit high-throughput screening, low costs, and high reproducibility, overcoming the limitations of the conventional two-dimensional cell cultures. In order to obtain these models, 3D bioprinting offers new perspectives by allowing a combination of advanced techniques and inks. In this context, we propose the use of hydroxyapatite nanoparticles, assimilated to the mineral component of bone, as a route to tune the printability and the characteristics of the scaffold and to guide cell behavior. To this aim, both stoichiometric and Sr-substituted hydroxyapatite nanocrystals are used, so as to obtain different particle shapes and solubility. Our findings show that the nanoparticles have the desired shape and composition and that they can be embedded in the inks without loss of cell viability. Both Sr-containing and stoichiometric hydroxyapatite crystals permit enhancing the printing fidelity of the scaffolds in a particle-dependent fashion and control the swelling behavior and ion release of the scaffolds. Once Saos-2 cells are encapsulated in the scaffolds, high cell viability is detected until late time points, with a good cellular distribution throughout the material. We also show that even minor modifications in the hydroxyapatite particle characteristics result in a significantly different behavior of the scaffolds. This indicates that the use of calcium phosphate nanocrystals and structural ion-substitution is a promising approach to tune the behavior of 3D bioprinted constructs.

Epigenetic Regulation Mediated by Sphingolipids in Cancer.

Epigenetic changes are heritable modifications that do not directly affect the DNA sequence. In cancer cells, the maintenance of a stable epigenetic profile can be crucial to support survival and proliferation, and said profile can differ significantly from that of healthy cells. The epigenetic profile of a cancer cell can be modulated by several factors, including metabolites. Recently, sphingolipids have emerged as novel modulators of epigenetic changes. Ceramide and sphingosine 1-phosphate have become well known in cancer due to activating anti-tumour and pro-tumour signalling pathways, respectively, and they have recently been shown to also induce several epigenetic modifications connected to cancer growth. Additionally, acellular factors in the tumour microenvironment, such as hypoxia and acidosis, are now recognised as crucial in promoting aggressiveness through several mechanisms, including epigenetic modifications. Here, we review the existing literature on sphingolipids, cancer, and epigenetic changes, with a focus on the interaction between these elements and components of the chemical tumour microenvironment.

Contribution of Mitochondrial Activity to Doxorubicin-Resistance in Osteosarcoma Cells.

Osteosarcoma is considered the most common bone tumor affecting children and young adults. The standard of care is chemotherapy; however, the onset of drug resistance still jeopardizes osteosarcoma patients, thus making it necessary to conduct a thorough investigation of the possible mechanisms behind this phenomenon. In the last decades, metabolic rewiring of cancer cells has been proposed as a cause of chemotherapy resistance. Our aim was to compare the mitochondrial phenotype of sensitive osteosarcoma cells (HOS and MG-63) versus their clones when continuously exposed to doxorubicin (resistant cells) and identify alterations exploitable for pharmacological approaches to overcome chemotherapy resistance. Compared with sensitive cells, doxorubicin-resistant clones showed sustained viability with less oxygen-dependent metabolisms, and significantly reduced mitochondrial membrane potential, mitochondrial mass, and ROS production. In addition, we found reduced expression of TFAM gene generally associated with mitochondrial biogenesis. Finally, combined treatment of resistant osteosarcoma cells with doxorubicin and quercetin, a known inducer of mitochondrial biogenesis, re-sensitizes the doxorubicin effect in resistant cells. Despite further investigations being needed, these results pave the way for the use of mitochondrial inducers as a promising strategy to re-sensitize doxorubicin cytotoxicity in patients who do not respond to therapy or reduce doxorubicin side effects.

Behavioral Voluntary and Social Bioassays Enabling Identification of Complex and Sex-Dependent Pain-(-Related) Phenotypes in Rats with Bone Cancer.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

Endogenous Extracellular Matrix Regulates the Response of Osteosarcoma 3D Spheroids to Doxorubicin.

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Electrospun fibers coated with nanostructured biomimetic hydroxyapatite: A new platform for regeneration at the bone interfaces.

Reconstruction of gradient organic/inorganic tissues is a challenging task in orthopaedics. Indeed, to mimic tissue characteristics and stimulate bone regeneration at the interface, it is necessary to reproduce both the mineral and organic components of the tissue ECM, as well as the micro/nano-fibrous morphology. To address this goal, we propose here novel biomimetic patches obtained by the combination of electrospinning and nanostructured bone apatite. In particular, we deposited apatite on the electrospun fibers by Ionized Jet Deposition, a plasma-assisted technique that allows conformal deposition and the preservation in the coating of the target's stoichiometry. The damage to the substrate and fibrous morphology is a polymer-dependent aspect, that can be avoided by properly selecting the substrate composition and deposition parameters. In fact, all the tested polymers (poly(l-lactide), poly(D,l-lactide-co-glycolide, poly(ε-caprolactone), collagen) were effectively coated, and the morphological and thermal characterization revealed that poly(ε-caprolactone) suffered the least damage. The coating of collagen fibers, on the other hand, destroyed the fiber morphology and it could only be performed when collagen is blended with a more resistant synthetic polymer in the nanofibers. Due to the biomimetic composition and multiscale morphology from micro to nano, the poly(ε-caprolactone)-collagen biomimetic patches coated with bone apatite supported MSCs adhesion, patch colonization and early differentiation, while allowing optimal viability. The biomimetic coating allowed better scaffold colonization, promoting cell spreading on the fibers.

Individual Trajectories of Bone Mineral Density Reveal Persistent Bone Loss in Bone Sarcoma Patients: A Retrospective Study.

Multiagent chemotherapy offers an undoubted therapeutic benefit to cancer patients, but is also associated with chronic complications in survivors. Osteoporosis affects the quality of life of oncologic patients, especially at the paediatric age. However, very few studies have described the extent of loss of bone mineral density (BMD) in bone sarcoma patients. We analysed a retrospective series of children and adolescents with primary malignant bone tumours (52 osteosarcoma and 31 Ewing sarcoma) and retrieved their BMD at diagnosis and follow-up as Hounsfield units (HU). We studied their individual BMD trajectories before and after chemotherapy up to 5 years, using routine chest CT scan and attenuation thresholds on T12 vertebrae ROI. At one year, bone sarcoma patients showed significant bone loss compared to diagnosis: 17.6% and 17.1% less for OS and EW, respectively. Furthermore, a bone loss of more than 49.2 HU at one-year follow-up was predictive of the persistence of a reduced bone mass over the following 4 years, especially in patients with EW. At 4 years, only 26% and 12.5% of OS and EW, respectively, had recovered or improved their BMD with respect to the onset, suggesting a risk of developing morbidities related to a low BMD in those subjects.

Radiodynamic Therapy with Acridine Orange Is an Effective Treatment for Bone Metastases.

Current multimodal treatment of bone metastases is partially effective and often associated with side effects, and novel therapeutic options are needed. Acridine orange is a photosensitizing molecule that accumulates in acidic compartments. After photo- or radiodynamic activation (AO-PDT or AO-RDT), acridine orange can induce lysosomal-mediated cell death, and we explored AO-RDT as an acid-targeted anticancer therapy for bone metastases. We used osteotropic carcinoma cells and human osteoclasts to assess the extracellular acidification and invasiveness of cancer cells, acridine orange uptake and lysosomal pH/stability, and the AO-RDT cytotoxicity in vitro. We then used a xenograft model of bone metastasis to compare AO-RDT to another antiacid therapeutic strategy (omeprazole). Carcinoma cells showed extracellular acidification activity and tumor-derived acidosis enhanced cancer invasiveness. Furthermore, cancer cells accumulated acridine orange more than osteoclasts and were more sensitive to lysosomal death. In vivo, omeprazole did not reduce osteolysis, whereas AO-RDT promoted cancer cell necrosis and inhibited tumor-induced bone resorption, without affecting osteoclasts. In conclusion, AO-RDT was selectively toxic only for carcinoma cells and effective to impair both tumor expansion in bone and tumor-associated osteolysis. We therefore suggest the use of AO-RDT, in combination with the standard antiresorptive therapies, to reduce disease burden in bone metastasis.

FT-IR Spectral Signature of Sensitive and Multidrug-Resistant Osteosarcoma Cell-Derived Extracellular Nanovesicles.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400-4000 cm-1 (resolution 4 cm-1, 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601 cm-1), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs.

The Release of Inflammatory Mediators from Acid-Stimulated Mesenchymal Stromal Cells Favours Tumour Invasiveness and Metastasis in Osteosarcoma.

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and 18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

3D Printing and Bioprinting to Model Bone Cancer: The Role of Materials and Nanoscale Cues in Directing Cell Behavior.

Bone cancer, both primary and metastatic, is characterized by a low survival rate. Currently, available models lack in mimicking the complexity of bone, of cancer, and of their microenvironment, leading to poor predictivity. Three-dimensional technologies can help address this need, by developing predictive models that can recapitulate the conditions for cancer development and progression. Among the existing tools to obtain suitable 3D models of bone cancer, 3D printing and bioprinting appear very promising, as they enable combining cells, biomolecules, and biomaterials into organized and complex structures that can reproduce the main characteristic of bone. The challenge is to recapitulate a bone-like microenvironment for analysis of stromal-cancer cell interactions and biological mechanics leading to tumor progression. In this review, existing approaches to obtain in vitro 3D-printed and -bioprinted bone models are discussed, with a focus on the role of biomaterials selection in determining the behavior of the models and its degree of customization. To obtain a reliable 3D bone model, the evaluation of different polymeric matrices and the inclusion of ceramic fillers is of paramount importance, as they help reproduce the behavior of both normal and cancer cells in the bone microenvironment. Open challenges and future perspectives are discussed to solve existing shortcomings and to pave the way for potential development strategies.

Acid Microenvironment in Bone Sarcomas.

In bone sarcomas, extracellular proton accumulation is an intrinsic driver of malignancy. Extracellular acidosis increases stemness, invasion, angiogenesis, metastasis, and resistance to therapy of cancer cells. It reprograms tumour-associated stroma into a protumour phenotype through the release of inflammatory cytokines. It affects bone homeostasis, as extracellular proton accumulation is perceived by acid-sensing ion channels located at the cell membrane of normal bone cells. In bone, acidosis results from the altered glycolytic metabolism of bone cancer cells and the resorption activity of tumour-induced osteoclasts that share the same ecosystem. Proton extrusion activity is mediated by extruders and transporters located at the cell membrane of normal and transformed cells, including vacuolar ATPase and carbonic anhydrase IX, or by the release of highly acidic lysosomes by exocytosis. To date, a number of investigations have focused on the effects of acidosis and its inhibition in bone sarcomas, including studies evaluating the use of photodynamic therapy. In this review, we will discuss the current status of all findings on extracellular acidosis in bone sarcomas, with a specific focus on the characteristics of the bone microenvironment and the acid-targeting therapeutic approaches that are currently being evaluated.

Acid-Induced Inflammatory Cytokines in Osteoblasts: A Guided Path to Osteolysis in Bone Metastasis.

Bone metastasis (BM) is a dismal complication of cancer that frequently occurs in patients with advanced carcinomas and that often manifests as an osteolytic lesion. In bone, tumor cells promote an imbalance in bone remodeling via the release of growth factors that, directly or indirectly, stimulate osteoclast resorption activity. However, carcinoma cells are also characterized by an altered metabolism responsible for a decrease of extracellular pH, which, in turn, directly intensifies osteoclast bone erosion. Here, we speculated that tumor-derived acidosis causes the osteoblast-osteoclast uncoupling in BM by modulating the pro-osteoclastogenic phenotype of osteoblasts. According to our results, a low pH recruits osteoclast precursors and promotes their differentiation through the secretome of acid-stressed osteoblasts that includes pro-osteoclastogenic factors and inflammatory mediators, such as RANKL, M-CSF, TNF, IL-6, and, above the others, IL-8. The treatment with the anti-IL-6R antibody tocilizumab or with an anti-IL-8 antibody reverted this effect. Finally, in a series of BM patients, circulating levels of the osteolytic marker TRACP5b significantly correlated with IL-8. Our findings brought out that tumor-derived acidosis promotes excessive osteolysis at least in part by inducing an inflammatory phenotype in osteoblasts, and these results strengthen the use of anti-IL-6 or anti-IL-8 strategies to treat osteolysis in BM.

Curcumin-Loaded Nanoparticles Impair the Pro-Tumor Activity of Acid-Stressed MSC in an In Vitro Model of Osteosarcoma.

In the tumor microenvironment, mesenchymal stromal cells (MSCs) are key modulators of cancer cell behavior in response to several stimuli. Intratumoral acidosis is a metabolic trait of fast-growing tumors that can induce a pro-tumorigenic phenotype in MSCs through the activation of the NF-κB-mediated inflammatory pathway, driving tumor clonogenicity, invasion, and chemoresistance. Recent studies have indicated that curcumin, a natural ingredient extracted from Curcuma longa, acts as an NF-κB inhibitor with anti-inflammatory properties. In this work, highly proliferating osteosarcoma cells were used to study the ability of curcumin to reduce the supportive effect of MSCs when stimulated by acidosis. Due to the poor solubility of curcumin in biological fluids, we used spherical polymeric nanoparticles as carriers (SPN-curc) to optimize its uptake by MSCs. We showed that SPN-curc inhibited the release of inflammatory cytokines (IL6 and IL8) by acidity-stimulated MSCs at a higher extent than by free curcumin. SPN-curc treatment was also successful in blocking tumor stemness, migration, and invasion that were driven by the secretome of acid-stressed MSCs. Overall, these data encourage the use of lipid-polymeric nanoparticles encapsulating NF-κB inhibitors such as curcumin to treat cancers whose progression is stimulated by an activated mesenchymal stroma.

Exploring Metabolic Adaptations to the Acidic Microenvironment of Osteosarcoma Cells Unveils Sphingosine 1-Phosphate as a Valuable Therapeutic Target.

Acidity is a key player in cancer progression, modelling a microenvironment that prevents immune surveillance and enhances invasiveness, survival, and drug resistance. Here, we demonstrated in spheroids from osteosarcoma cell lines that the exposure to acidosis remarkably caused intracellular lipid droplets accumulation. Lipid accumulation was also detected in sarcoma tissues in close proximity to tumor area that express the acid-related biomarker LAMP2. Acid-induced lipid droplets-accumulation was not functional to a higher energetic request, but rather to cell survival. As a mechanism, we found increased levels of sphingomyelin and secretion of the sphingosine 1-phosphate, and the activation of the associated sphingolipid pathway and the non-canonical NF-ĸB pathway, respectively. Moreover, decreasing sphingosine 1-phosphate levels (S1P) by FTY720 (Fingolimod) impaired acid-induced tumor survival and migration. As a confirmation of the role of S1P in osteosarcoma, we found S1P high circulating levels (30.8 ± 2.5 nmol/mL, n = 17) in the serum of patients. Finally, when we treated osteosarcoma xenografts with FTY720 combined with low-serine/glycine diet, both lipid accumulation (as measured by magnetic resonance imaging) and tumor growth were greatly inhibited. For the first time, this study profiles the lipidomic rearrangement of sarcomas under acidic conditions, suggesting the use of anti-S1P strategies in combination with standard chemotherapy.

Strawberry-Derived Exosome-Like Nanoparticles Prevent Oxidative Stress in Human Mesenchymal Stromal Cells.

Plant-derived exosome-like nanovesicles (EPDENs) have recently been isolated and evaluated as potential bioactive nutraceutical biomolecules. It has been hypothesized that EPDENs may exert their activity on mammalian cells through their specific cargo. In this study, we isolated and purified EPDENs from the strawberry juice of Fragaria x ananassa (cv. Romina), a new cultivar characterized by a high content of anthocyanins, folic acid, flavonols, and vitamin C and an elevated antioxidant capacity. Fragaria-derived EPDENs were purified by a series of centrifugation and filtration steps. EPDENs showed size and morphology similar to mammalian extracellular nanovesicles. The internalization of Fragaria-derived EPDENs by human mesenchymal stromal cells (MSCs) did not negatively affect their viability, and the pretreatment of MSCs with Fragaria-derived EPDENs prevented oxidative stress in a dose-dependent manner. This is possibly due to the presence of vitamin C inside the nanovesicle membrane. The analysis of EPDEN cargo also revealed the presence of small RNAs and miRNAs. These findings suggest that Fragaria-derived EPDENs may be considered nanoshuttles contained in food, with potential health-promoting activity.

Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons.

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.

Unravelling the Effect of Citrate on the Features and Biocompatibility of Magnesium Phosphate-Based Bone Cements.

In the framework of new materials for orthopedic applications, Magnesium Phosphate-based Cements (MPCs) are currently the focus of active research in biomedicine, given their promising features; in this field, the loading of MPCs with active molecules to be released in the proximity of newly forming bone could represent an innovative approach to enhance the in vivo performances of the biomaterial. In this work, we describe the preparation and characterization of MPCs containing citrate, an ion naturally present in bone which presents beneficial effects when released in the proximity of newly forming bone tissue. The cements were characterized in terms of handling properties, setting time, mechanical properties, crystallinity, and microstructure, so as to unravel the effect of citrate concentration on the features of the material. Upon incubation in aqueous media, we demonstrated that citrate could be successfully released from the cements, while contributing to the alkalinization of the surroundings. The cytotoxicity of the materials toward human fibroblasts was also tested, revealing the importance of a fine modulation of released citrate to guarantee the biocompatibility of the material.