Design, Stereolithographic 3D Printing, and Characterization of TPMS Scaffolds.

Anatomical and functional tissue loss is one of the most debilitating problems and involves a great cost to the international health-care sector. In the field of bone tissue, the use of scaffolds to promote tissue regeneration is a topic of great interest. In this study, a combination of additive manufacturing and computational methods led to creating porous scaffolds with complex microstructure and mechanical behavior comparable to those of cancellous bone. Specifically, some representative models of triply periodic minimal surfaces (TPMSs) were 3D-printed through a stereolithographic technique using a dental resin. Schwarz primitive and gyroid surfaces were created computationally: they are characterized by a complex geometry and a high pore interconnectivity, which play a key role in the mechanism of cell proliferation. Several design parameters can be varied in these structures that can affect the performance of the scaffold: for example, the larger the wall thickness, the lower the elastic modulus and compressive strength. Morphological and mechanical analyses were performed to experimentally assess the properties of the scaffolds. The relationship between relative density and elastic modulus has been analyzed by applying different models, and a power-law equation was found suitable to describe the trend in both structures.

The unexplored role of alkali and alkaline earth elements (ALAEs) on the structure, processing, and biological effects of bioactive glasses.

Bioactive glass has been employed in several medical applications since its inception in 1969. The compositions of these materials have been investigated extensively with emphasis on glass network formers, therapeutic transition metals, and glass network modifiers. Through these experiments, several commercial and experimental compositions have been developed with varying chemical durability, induced physiological responses, and hydroxyapatite forming abilities. In many of these studies, the concentrations of each alkali and alkaline earth element have been altered to monitor changes in structure and biological response. This review aims to discuss the impact of each alkali and alkaline earth element on the structure, processing, and biological effects of bioactive glass. We explore critical questions regarding these elements from both a glass science and biological perspective. Should elements with little biological impact be included? Are alkali free bioactive glasses more promising for greater biological responses? Does this mixed alkali effect show increased degradation rates and should it be employed for optimized dissolution? Each of these questions along with others are evaluated comprehensively and discussed in the final section where guidance for compositional design is provided.

Micro-CT imaging and finite element models reveal how sintering temperature affects the microstructure and strength of bioactive glass-derived scaffolds.

This study focuses on the finite element simulation and micromechanical characterization of bioactive glass-ceramic scaffolds using Computed micro Tomography ([Formula: see text]CT) imaging. The main purpose of this work is to quantify the effect of sintering temperature on the morphometry and mechanical performance of the scaffolds. In particular, the scaffolds were produced using a novel bioactive glass material (47.5B) through foam replication, applying six different sintering temperatures. Through [Formula: see text]CT imaging, detailed three-dimensional images of the scaffold's internal structure are obtained, enabling the extraction of important geometric features and how these features change with sintering temperature. A finite element model is then developed based on the [Formula: see text]CT images to simulate the fracture process under uniaxial compression loading. The model incorporates scaffold heterogeneity and material properties-also depending on sintering temperature-to capture the mechanical response, including crack initiation, propagation, and failure. Scaffolds sintered at temperatures equal to or higher than 700 [Formula: see text]C exhibit two-scale porosity, with micro and macro pores. Finite element analyses revealed that the dual porosity significantly affects fracture mechanisms, as micro-pores attract cracks and weaken strength. Interestingly, scaffolds sintered at high temperatures, the overall strength of which is higher due to greater intrinsic strength, showed lower normalized strength compared to low-temperature scaffolds. By using a combined strategy of finite element simulation and [Formula: see text]CT-based characterization, bioactive glass-ceramic scaffolds can be optimized for bone tissue engineering applications by learning more about their micromechanical characteristics and fracture response.

Computational models for the simulation of the elastic and fracture properties of highly porous 3D-printed hydroxyapatite scaffolds.

Bone scaffolding is a promising approach for the treatment of critical-size bone defects. Hydroxyapatite can be used to produce highly porous scaffolds as it mimics the mineralized part of bone tissue, but its intrinsic brittleness limits its usage. Among 3D printing techniques, vat photopolymerization allows for the best printing resolution for ceramic materials. In this study, we implemented a Computed micro-Tomography based Finite Element Model of a hydroxyapatite porous scaffold fabricated by vat photopolymerization. We used the model in order to predict the elastic and fracture properties of the scaffold. From the stress-strain diagram of a simulated compression test, we computed the stiffness and the strength of the scaffolds. We found that three morphometric features substantially affect the crack pattern. In particular, the crack propagation is not only dependent on the trabecular thickness but also depends on the slenderness and orientation of the trabeculae with respect to the load. The results found in this study can be used for the design of ceramic scaffolds with heterogeneous pore distribution in order to tailor and predict the compressive strength.

Special Issue: Porous Ceramics, Glasses and Composites, Volume II.

This Special Issue, titled "Porous Ceramics, Glasses and Composites, Volume II", aims to present an up-to-date overview of the synthesis/fabrication, characterization, and applications of porous materials, with a special focus on ceramics, glasses, and composites [...].

Cobalt-Doped Bioactive Glasses for Biomedical Applications: A Review.

Improving angiogenesis is the key to the success of most regenerative medicine approaches. However, how and to which extent this may be performed is still a challenge. In this regard, cobalt (Co)-doped bioactive glasses show promise being able to combine the traditional bioactivity of these materials (especially bone-bonding and osteo-stimulatory properties) with the pro-angiogenic effect associated with the release of cobalt. Although the use and local delivery of Co2+ ions into the body have raised some concerns about the possible toxic effects on living cells and tissues, important biological improvements have been highlighted both in vitro and in vivo. This review aims at providing a comprehensive overview of Co-releasing glasses, which find biomedical applications as various products, including micro- and nanoparticles, composites in combination with biocompatible polymers, fibers and porous scaffolds. Therapeutic applications in the field of bone repair, wound healing and cancer treatment are discussed in the light of existing experimental evidence along with the open issues ahead.

Effects of Bioactive Glasses (BGs) on Exosome Production and Secretion: A Critical Review.

There is an increasing trend toward the application of bioactive glasses in different areas of biomedicine, including tissue engineering and oncology. The reason for this increase is mostly attributed to the inherent properties of BGs, such as excellent biocompatibility, and the ease of tailoring their properties by changing, for example, the chemical composition. Previous experiments have demonstrated that the interactions between BGs and their ionic dissolution products, and mammalian cells, can affect and change cellular behaviors, and thereby govern the performance of living tissues. However, limited research exists on their critical role in the production and secretion of extracellular vesicles (EVs) such as exosomes. Exosomes are nanosized membrane vesicles that carry various therapeutic cargoes such as DNA, RNA, proteins, and lipids, and thereby can govern cell-cell communication and subsequent tissue responses. The use of exosomes is currently considered a cell-free approach in tissue engineering strategies, due to their positive roles in accelerating wound healing. On the other hand, exosomes are known as key players in cancer biology (e.g., progression and metastasis), due to their capability to carry bioactive molecules between tumor cells and normal cells. Recent studies have demonstrated that the biological performance of BGs, including their proangiogenic activity, is accomplished with the help of exosomes. Indeed, therapeutic cargos (e.g., proteins) produced in BG-treated cells are transferred by a specific subset of exosomes toward target cells and tissues, and lead to a biological phenomenon. On the other hand, BGs are suitable delivery vehicles that can be utilized for the targeted delivery of exosomes to cells and tissues of interest. Therefore, it seems necessary to have a deeper understanding of the potential effects of BGs in the production of exosomes in cells that are involved in tissue repair and regeneration (mostly mesenchymal stem cells), as well as in those that play roles in cancer progression (e.g., cancer stem cells). This review aims to present an updated report on this critical issue, to provide a roadmap for future research in the fields of tissue engineering and regenerative medicine.

Nanostructured bioactive glasses: A bird's eye view on cancer therapy.

Bioactive glasses (BGs) arewell known for their successful applications in tissue engineering and regenerative medicine. Recent experimental studies have shown their potential usability in oncology, either alone or in combination with other biocompatible materials, such as biopolymers. Direct contact with BG particles has been found to cause toxicity and death in specific cancer cells (bone-derived neoplastic stromal cells) in vitro. Nanostructured BGs (NBGs) can be doped with anticancer elements, such as gallium, to enhance their toxic effects against tumor cells. However, the molecular mechanisms and intracellular targets for anticancer compositions of NBGs require further clarification. NBGs have been successfully evaluated for use in various well-established cancer treatment strategies, including cancer hyperthermia, phototherapy, and anticancer drug delivery. Existing results indicate that NBGs not only enhance cancer cell death, but can also participate in the regeneration of lost healthy tissues. However, the application of NBGs in oncology is still in its early stages, and numerous unanswered questions must be addressed. For example, the impact of the composition, biodegradation, size, and morphology of NBGs on their anticancer efficacy should be defined for each type of cancer and treatment strategy. Moreover, it should be more clearly assessed whether NBGs can shrink tumors, slow/stop cancer progression, or cure cancer completely. In this regard, the use of computational studies (in silico methods) is highly recommended to design the most effective glass formulations for cancer therapy approaches and to predict, to some extent, the relevant properties, efficacy, and outcomes. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Mechanical characterization of miniaturized 3D-printed hydroxyapatite parts obtained through vat photopolymerization: an experimental study.

Hydroxyapatite is one of the materials of choice for tissue engineering bone scaffolds manufacturing. Vat photopolymerization (VPP) is a promising Additive Manufacturing (AM) technology capable of producing scaffolds with high resolution micro-architecture and complex shapes. However, mechanical reliability of ceramic scaffolds can be achieved if a high fidelity printing process is obtained and if knowledge of the intrinsic mechanical properties of the constituent material is available. As the hydroxyapatite (HAP) obtained from VPP is subjected to a sintering process, the mechanical properties of the material should be assessed with specific reference to the process parameters (e.g. sintering temperature) and to the specific characteristic size of the microscopic features in the scaffolds. In order to tackle this challenge the HAP solid matrix of the scaffold was mimicked in the form of miniaturized samples suitable for ad hoc mechanical characterization, which is an unprecedented approach. To this purpose small scale HAP samples, having a simple geometry and size similar to that of the scaffolds, were produced through VPP. The samples were subjected to geometric characterization and to mechanical laboratory tests. Confocal laser scanning and Computed micro-Tomography (micro-CT) were used for geometric characterization; while, micro-bending and nanoindentation were used for mechanical testing. Micro-CT analyses have shown a highly dense material with negligible intrinsic micro-porosity. The imaging process allowed quantifying the variation of geometry with respect to the nominal size showing high accuracy of the printing process and identifying printing defects on one specific sample type, depending on the printing direction. The mechanical tests have shown that the VPP produces HAP with an elastic modulus as high as approximately 100GPa and flexural strength of approximately 100MPa. The results of this study have shown that vat photopolymerization is a promising technology capable of producing high quality HAP with reliable geometric fidelity.

Zinc- and Copper-Doped Mesoporous Borate Bioactive Glasses: Promising Additives for Potential Use in Skin Wound Healing Applications.

In this study, zinc (Zn)- and copper (Cu)-doped 13-93B3 borate mesoporous bioactive glasses (MBGs) were successfully synthesized using nitrate precursors in the presence of Pluronic P123. We benefited from computational approaches for predicting and confirming the experimental findings. The changes in the dynamic surface tension (SFT) of simulated body fluid (SBF) were investigated using the Du Noüy ring method to shed light on the mineralization process of hydroxyapatite (HAp) on the glass surface. The obtained MBGs were in a glassy state before incubation in SBF. The formation of an apatite-like layer on the SBF-incubated borate glasses was investigated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The incorporation of Zn and Cu into the basic composition of 13-93B3 glass led to changes in the glass transition temperature (Tg) (773 to 556 °C), particle size (373 to 64 nm), zeta potential (−12 to −26 mV), and specific surface area (SBET) (54 to 123 m2/g). Based on the K-means algorithm and chi-square automatic interaction detection (CHAID) tree, we found that the SFT of SBF is an important factor for the prediction and confirmation of the HAp mineralization process on the glasses. Furthermore, we proposed a simple calculation, based on SFT variation, to quantify the bioactivity of MBGs. The doped and dopant-free borate MBGs could enhance the proliferation of mouse fibroblast L929 cells at a concentration of 0.5 mg/mL. These glasses also induced very low hemolysis (<5%), confirming good compatibility with red blood cells. The results of the antibacterial test revealed that all the samples could significantly decrease the viability of Pseudomonas aeruginosa. In summary, we showed that Cu-/Zn-doped borate MBGs can be fabricated using a cost-effective method and also show promise for wound healing/skin tissue engineering applications, as especially supported by the cell test with fibroblasts, good compatibility with blood, and antibacterial properties.

Bioactive Glass-Ceramic Scaffolds Coated with Hyaluronic Acid-Fatty Acid Conjugates: A Feasibility Study.

Promoting bone healing is a key challenge in our society that can be tackled by developing new implantable biomaterials provided with regenerative properties. In this work, the coating of three-dimensional porous glass-derived scaffolds with hyaluronic acid (HA)-fatty acids was investigated for the first time. The starting scaffolds, based on bioactive silicate glass, were produced by foam replication followed by sintering; then, HA-palmitate and HA-oleate conjugate coatings were deposited on the scaffold struts through a dipping procedure. FT-IR analysis confirmed the successful deposition of the coatings on the surface and struts of the scaffolds, the foam-like architecture of which was maintained as assessed by SEM investigations. The in vitro bioactivity of the HA-fatty-acid-coated scaffolds was studied by immersion tests in simulated body fluid and the subsequent evaluation of hydroxyapatite formation. The deposition of the polymeric coating did not inhibit the apatite-forming ability of scaffolds, as revealed by the formation of nanostructured hydroxyapatite agglomerates 48 h from immersion. These promising results motivate further investigation of these novel bioactive systems, which are expected to combine the bone-bonding properties of the glass with the wound-healing promotion carried out by the polymeric conjugates.

PVA-Based Nanofibers Containing Chitosan Modified with Graphene Oxide and Carbon Quantum Dot-Doped TiO2 Enhance Wound Healing in a Rat Model.

Electrospun nanofibrous constructs based on nanoparticles and biopolymers have recently been used in tissue engineering because of their similarity to the extracellular matrix in nature. In this study, electrospun chitosan-carbon quantum dot-titanium dioxide-graphene oxide (CS-CQD-TiO2-GO) nanofibrous mats were synthesized for use as wound dressings by the electrospinning method. To increase the biodegradation rate and water resistance, the fabricated nanofibrous mats were cross-linked. SEM images showed a uniform and coherent structure of CS-CQD-TiO2-GO nanocomposites and CS-CQD-TiO2-GO electrospun nanofibers mats. FTIR analysis, XRD pattern, SEM mapping, and EDS spectrum demonstrate the accuracy of the synthesis as well as the elemental and chemical structure of the nanofibrous mat. The water contact angle indicated that the nanofibrous mat had a hydrophilic property, which is essential for controlling wound exudates. The tensile strength and elongation tests showed that the nanofibrous mat has suitable mechanical properties for wound dressing, including significant flexibility and strength. Interestingly, antimicrobial testing illustrated that the fabricated nanofibrous mat had antibacterial activity against Gram-negative and Gram-positive bacteria. Appropriate cell viability and cytocompatibility of treated mouse fibroblast NIH3T3 cells with the nanofibrous mat were determined using an MTT assay. The animal study results confirmed the proper potential of the nanofibrous mat in wound dressing applications.

Additive manufacturing of bioactive glass biomaterials.

Tissue engineering (TE) and regenerative medicine have held great promises for the repair and regeneration of damaged tissues and organs. Additive manufacturing has recently appeared as a versatile technology in TE strategies that enables the production of objects through layered printing. By applying 3D printing and bioprinting, it is now possible to make tissue-engineered constructs according to desired thickness, shape, and size that resemble the native structure of lost tissues. Up to now, several organic and inorganic materials were used as raw materials for 3D printing; bioactive glasses (BGs) are among the most hopeful substances regarding their excellent properties (e.g., bioactivity and biocompatibility). In addition, the reported studies have confirmed that BG-reinforced constructs can improve osteogenic, angiogenic, and antibacterial activities. This review aims to provide an up-to-date report on the development of BG-containing raw biomaterials that are currently being employed for the fabrication of 3D printed scaffolds used in tissue regeneration applications with a focus on their advantages and remaining challenges.

Radiopaque Crystalline, Non-Crystalline and Nanostructured Bioceramics.

Radiopacity is sometimes an essential characteristic of biomaterials that can help clinicians perform follow-ups during pre- and post-interventional radiological imaging. Due to their chemical composition and structure, most bioceramics are inherently radiopaque but can still be doped/mixed with radiopacifiers to increase their visualization during or after medical procedures. The radiopacifiers are frequently heavy elements of the periodic table, such as Bi, Zr, Sr, Ba, Ta, Zn, Y, etc., or their relevant compounds that can confer enhanced radiopacity. Radiopaque bioceramics are also intriguing additives for biopolymers and hybrids, which are extensively researched and developed nowadays for various biomedical setups. The present work aims to provide an overview of radiopaque bioceramics, specifically crystalline, non-crystalline (glassy), and nanostructured bioceramics designed for applications in orthopedics, dentistry, and cancer therapy. Furthermore, the modification of the chemical, physical, and biological properties of parent ceramics/biopolymers due to the addition of radiopacifiers is critically discussed. We also point out future research lacunas in this exciting field that bioceramists can explore further.

pH-Responsive PVA-Based Nanofibers Containing GO Modified with Ag Nanoparticles: Physico-Chemical Characterization, Wound Dressing, and Drug Delivery.

Site-specific drug delivery and carrying repairing agents for wound healing purposes can be achieved using the intertwined three-dimensional structure of nanofibers. This study aimed to optimize and fabricate poly (vinyl alcohol) (PVA)-graphene oxide (GO)-silver (Ag) nanofibers containing curcumin (CUR) using the electrospinning method for potential wound healing applications. Fourier Transform Infrared (FTIR) spectrophotometry, X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential were used to characterize the nanostructures. The mechanical properties of the nanostructures were subsequently examined by tensile strength and elongation test. As shown by MIC analysis of E. coli and S. aureus bacteria, the fabricated nanofibers had superior inhibitory effects on the bacteria growth. Ag nanoparticles incorporation into the nanofibers resulted in increased loading and encapsulation efficiencies from 21% to 56% and from 61% to 86%, respectively. CUR release from PVA/GO-Ag-CUR nanofiber at pH 7.4 was prevented, while the acidic microenvironment (pH 5.4) increased the release of CUR from PVA/GO-Ag-CUR nanofiber, corroborating the pH-sensitivity of the nanofibers. Using the in vitro wound healing test on NIH 3T3 fibroblast cells, we observed accelerated growth and proliferation of cells cultured on PVA/GO-Ag-CUR nanofibers.

Antioxidant Effects of Bioactive Glasses (BGs) and Their Significance in Tissue Engineering Strategies.

Elevated levels of oxidative stress are usually observed following injuries, leading to impaired tissue repair due to oxidation-related chronic inflammation. Several attempts have been made to manage this unfavorable situation, and the use of biomaterials with antioxidant activity is showing great promise in tissue engineering and regenerative medicine approaches. Bioactive glasses (BGs) are a versatile group of inorganic substances that exhibit an outstanding regenerative capacity for both hard and soft damaged tissues. The chemical composition of BGs provides a great opportunity for imparting specific biological activities to them. On this point, BGs may easily become antioxidant substances through simple physicochemical modifications. For example, particular antioxidant elements (mostly cerium (Ce)) can be added to the basic composition of the glasses. On the other hand, grafting natural antioxidant substances (e.g., polyphenols) on the BG surface is feasible for making antioxidant substitutes with promising results in vitro. Mesoporous BGs (MBGs) were demonstrated to have unique merits compared with melt-derived BGs since they make it possible to load antioxidants and deliver them to the desired locations. However, there are actually limited in vivo experimental studies on the capability of modified BGs for scavenging free radicals (e.g., reactive oxygen species (ROS)). Therefore, more research is required to determine the actual potential of BGs in decreasing oxidative stress and subsequently improving tissue repair and regeneration. The present work aims to highlight the potential of different types of BGs in modulating oxidative stress and subsequently improving tissue healing.

Robocasting of advanced ceramics: ink optimization and protocol to predict the printing parameters - A review.

Direct-Ink-Writing (or robocasting) is a subset of extrusion-based additive manufacturing techniques that has grown significantly in recent years to design simple to complex ceramic structures. Robocasting, relies on the use of high-concentration powder pastes, also known as inks. A successful optimization of ink rheology and formulation constitutes the major key factor to ensure printability for the fabrication of self-supporting ceramic structures with a very precise dimensional resolution. However, to date achieving a real balance between a comprehensive optimization of ink rheology and the determination of a relevant protocol to predict the printing parameters for a given ink is still relatively scarce and has been not yet standardized in the literature. The current review reports, in its first part, a detailed survey of recent studies on how ink constituents and composition affect the direct-ink-writing of ceramic parts, taking into account innovative ceramic-based-inks formulations and processing techniques. Precisely, the review elaborates the major factors influencing on ink rheology and printability, specifically binder type, particle physical features (size, morphology and density) and ceramic feedstock content. In the second part, this review suggests a standardized guideline to effectively adapt a suitable setting of the printing parameters, such as printing speed and pressure, printing substrate, strut spacing, layer height, nozzle diameter in function of ink intrinsic rheology.

Microencapsulation of Bacillus velezensis Using Alginate-Gum Polymers Enriched with TiO2 and SiO2 Nanoparticles.

Bacillus bacteria are a group of plant growth stimulants that increase plant growth and resistance to plant pathogens by producing various metabolites. With their large surface area and small size, nanoparticles can be used in controlled-release formulations and increase the efficiency of the desired product. Encapsulation of biological agents in combination with nanoparticles can be an essential step in increasing the performance of these agents in adverse environmental conditions. In this study, which is the result of a collaboration between scientists from Italy and Iran, Bacillus velezensis was encapsulated in alginate combined with whey protein and zedo, mastic, and tragacanth gums in the presence of silica and titania nanoparticles to obtain two-layer and multilayer assemblies acting as novel, smart micro-encapsulation systems. The results of laboratory studies showed that the B. velezensis could produce protease, lipase, siderophore, auxin, and a dissolution of mineral phosphate. Scanning electron microscopy images (SEM) showed that the studied microcapsules were almost spherical. Moisture affinity, swelling, and efficiency of each microcapsule were examined. The results showed that the highest encapsulation efficiency (94.3%) was related to the multilayer formulation of alginate-whey protein-zedo. XRD and FTIR spectroscopy showed that the alginate, whey protein, and zedo were mixed properly and no incompatible composition occurred in the reaction. This study aimed to provide a suitable formulation of biofertilizers based on biodegradable compounds as an alternative to chemical fertilizers, which is low cost and very effective without harming humans and the environment.

Mechanical Properties of Robocast Glass Scaffolds Assessed through Micro-CT-Based Finite Element Models.

In this study, the mechanical properties of two classes of robocast glass scaffolds are obtained through Computed micro-Tomography (micro-CT) based Finite Element Modeling (FEM) with the specific purpose to explicitly account for the geometrical defects introduced during manufacturing. Both classes demonstrate a fiber distribution along two perpendicular directions on parallel layers with a 90∘ tilting between two adjacent layers. The crack pattern identified upon compression loading is consistent with that found in experimental studies available in literature. The finite element models have demonstrated that the effect of imperfections on elastic and strength properties may be substantial, depending on the specific type of defect identified in the scaffolds. In particular, micro-porosity, fiber length interruption and fiber detaching were found as key factors. The micro-pores act as stress concentrators promoting fracture initiation and propagation, while fiber detachment reduces the scaffold properties substantially along the direction perpendicular to the fiber plane.

An automated 3D-printed perfusion bioreactor combinable with pulsed electromagnetic field stimulators for bone tissue investigations.

In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools. Moreover, when equipped with automation, their use allows reducing user intervention and dependence, increasing reproducibility and the overall quality of the culture process. In this study, an automated uni-/bi-directional perfusion bioreactor combinable with pulsed electromagnetic field (PEMF) stimulation for culturing 3D bone tissue models is proposed. A user-friendly control unit automates the perfusion, minimizing the user dependency. Computational fluid dynamics simulations supported the culture chamber design and allowed the estimation of the shear stress values within the construct. Electromagnetic field simulations demonstrated that, in case of combination with a PEMF stimulator, the construct can be exposed to uniform magnetic fields. Preliminary biological tests on 3D bone tissue models showed that perfusion promotes the release of the early differentiation marker alkaline phosphatase. The histological analysis confirmed that perfusion favors cells to deposit more extracellular matrix (ECM) with respect to the static culture and revealed that bi-directional perfusion better promotes ECM deposition across the construct with respect to uni-directional perfusion. Lastly, the Real-time PCR results of 3D bone tissue models cultured under bi-directional perfusion without and with PEMF stimulation revealed that the only perfusion induced a ~ 40-fold up-regulation of the expression of the osteogenic gene collagen type I with respect to the static control, while a ~ 80-fold up-regulation was measured when perfusion was combined with PEMF stimulation, indicating a positive synergic pro-osteogenic effect of combined physical stimulations.