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The slow knowledge progression about cancer disease and the high drug clinical failure are mainly due to the inadequacy of the simplistic pre-clinical in vitro and in vivo animal tumor models. To overpass these limits, in recent years many 3D matrix-based cell cultures have been proposed as challenging alternatives, since they allow to better recapitulate the in vitro cells-cells and cells-matrix reciprocal interactions in a more physiological context. Among many natural polymers, alginate has been adopted as an extracellular matrix surrogate to mimic the 3D spatial organization. After their expansion, cancer cells are suspended in an alginate solution and dropped within a crosslinking solution enabling gelification. The result is the generation of a 3D hydrogel embedding a single cell suspension: Cells are equally distributed throughout the gel, and they are free to proliferate generating clonal spheroids. Moreover, according to the hydrogel matrix stiffness that can be easily tuned, tumor cells can spread within the 3D structure and migrate outside, where they may become circulating tumor cells and infiltrate secondary tumor sites when these 3D tumor tissues are cultured in a fluid dynamic environment (i.e., organ on chip).

Natural killer (NK) cells are cytotoxic lymphoid cells that play a key role in defenses against tumors. However, their function may be severely impaired in patients with pancreatic adenocarcinoma (PA). Indeed, PA cells release soluble factors, thereby generating an immunosuppressive environment that dysregulates NK-cell cytolytic function and favors tumor immune evasion. Here, we analyzed the interactions between NK and PA cells using the PANC-1 and CAPAN-1 cell lines derived from a ductal PA and metastatic lesion, respectively. Metastatic and nonmetastatic cell lines were both able to impair NK cytolytic activity. An analysis of the effect of NK cells and NK-cell-derived exosomes revealed substantial differences between the two cell lines. Thus, NK cells displayed higher cytotoxicity against nonmetastatic PA cells than metastatic PA cells in both 2D cultures and in a 3D extracellular matrix cell system. In addition, NK-derived exosomes could penetrate only PANC-1 spheroids and induce cell killing. Remarkably, when PANC-1 cells were exposed to NK-derived soluble factors, they displayed substantial changes in the expression of genes involved in epithelial-to-mesenchymal transition (EMT) and acquired resistance to NK-mediated cytolysis. These results, together with their correlation with poor clinical outcomes in PA patients, suggest that the induction of resistance to cytolysis upon exposure to NK-derived soluble factors could reflect the occurrence of EMT in tumor cells. Our data indicate that a deeper investigation of the interaction between NK cells and tumor cells may be crucial for immunotherapy, possibly improving the outcome of PA treatment by targeting critical steps of NK-tumor cell crosstalk.

In recent years, immunotherapy has emerged as a promising novel therapeutic strategy for cancer treatment. In a relevant percentage of patients, however, clinical benefits are lower than expected, pushing researchers to deeply analyze the immune responses against tumors and find more reliable and efficient tools to predict the individual response to therapy. Novel tissue engineering strategies can be adopted to realize in vitro fully humanized matrix-based models, as a compromise between standard two-dimensional (2D) cell cultures and animal tests, which are costly and hardly usable in personalized medicine. In this review, we describe the main mechanisms allowing cancer cells to escape the immune surveillance, which may play a significant role in the failure of immunotherapies. In particular, we discuss the role of the tumor microenvironment (TME) in the establishment of a milieu that greatly favors cancer malignant progression and impact on the interactions with immune cells. Then, we present an overview of the recent in vitro engineered preclinical three-dimensional (3D) models that have been adopted to resemble the interplays between cancer and immune cells and for testing current therapies and immunotherapeutic approaches. Specifically, we focus on 3D hydrogel-based tools based on different types of polymers, discussing the suitability of each of them in reproducing the TME key features based on their intrinsic or tunable characteristics. Finally, we introduce the possibility to combine the 3D models with technological fluid dynamics platforms, reproducing the dynamic complex interactions between tumor cells and immune effectors migrated in situ via the systemic circulation, pointing out the challenges that still have to be overcome for setting more predictive preclinical assays.

A novel green method for graphene oxide (GO) reduction via ascorbic acid has been adopted to realize bio-friendly reduced graphene oxide (RGO)/polycaprolactone (PCL) nanofibrous meshes, as substrates for bone tissue engineering applications. PCL fibrous mats enriched with either RGO or GO (0.25 wt%) were fabricated to recapitulate the fibrillar structure of the bone extracellular matrix (ECM) and the effects of RGO incorporation on the structural proprieties, biomechanics and bioactivity of the nano-composites meshes were evaluated. RGO/PCL fibrous meshes displayed superior mechanical properties (i.e. Young's Modulus and ultimate tensile strength) besides supporting noticeably improved cell adhesion, spreading and proliferation of fibroblasts and osteoblast-like cell lines. Furthermore, RGO-based electrospun substrates enhanced in vitro calcium deposition in the ECM produced by osteoblast-like cells, which was paralleled, in human mesenchymal stem cells grown onto the same substrates, by an increased expression of the osteogenic markers mandatory for mineralization. In this respect, the capability of graphene-based materials to adsorb osteogenic factors cooperates synergically with the rougher surface of RGO/PCL-based materials, evidenced by AFM analysis, to ignite mineralization of the neodeposited matrix and to promote the osteogenic commitment of the cultured cell in the surrounding microenvironment.

INTRODUCTION:The integration of 3D human tumor organoids with fluidic cell culture chambers allows to mimic the in vivo organ-organ fluidic connections and drug systemic administration, finally offering a reliable and fast approach for novel molecules prescreening tests. In this work, 3D cell-laden hydrogels have been properly realized as ovarian tumor models and cultured within a fluidic Multi In vitro Organ-MIVO®-device for in vitro resembling the cancer environment and carry out drug efficacy assays, alternative to animal preclinical models, by monitoring the cancer regression over time. METHODS:Human ovarian cancer cell line (SKOV-3) has been used to realize alginate-based 3D cell-laden hydrogel-based tumor models, as reported (1). 3D tumor tissues have been cultured within MIVO® chamber under fluid flow resembling the capillary blood flow feeding the tumor. Drug (cisplatin) efficacy have been evaluated in MIVO resembling the in vivo systemic drug transport mechanisms and compared with (i) the in vitro tissue culture under static condition, and with (ii) the use of xenograft mouse model. In particular, SKOV-3 viability was quantitatively assessed through Alamar Blue Assay at different time points. Immunostaining for apoptosis and cell proliferation was also carried out. For in vivo tests, SKOV-3 derived tumors were established via subcutaneous injection into the right flank of nude mice. Tumor size was monitored over time. After 10 days, mice were randomized into two treatment groups when tumor volumes increased to 50 mm3. Cisplatin was administered intravenously every seven days for 3 weeks. Tumor growth was quantified three times a week by using a digital caliper. RESULTS:After one week of drug treatment, the viability of SKOV-3 cultured within the 3D hydrogels in MIVO® decreased up to 50%; caspase positive cells were observed homogeneously distributed within the 3D gels, while very few proliferating cells were observed after 1 week of drug treatment. On the contrary, under static conditions the cells were still viable (higher than 80%) and proliferating until 7 days of drug treatment highlighting chemoresistance. Exceptionally under MIVO® tissue culture, the in vitro tumor regression kinetics resembles exactly the tumor mass reduction measured in vivo. DISCUSSION & CONCLUSIONS:These results highlight that the in vivo-like dynamic environment provided by the MIVO® device allows to properly resemble the 3D tumor tissue perfusion, its culture under a capillary circulation and the systemic drug transport mechanism, suggesting its potential as reliable and cruelty-free platform for preclinical drug efficacy tests. References:(1) Alessandra Marrella et al. Frontiers in immunology 10 (2019): 1876

Metastasis is a dynamic process involving the dissemination of circulating tumour cells (CTCs) through the bloodstream to distant tissues within the body. An improved understanding of the fluid flow induced mechanical forces experienced by CTCs in the blood flow is crucial for fully explicating the metastatic steps and defining vulnerable CTC conditions for therapeutic intervention. Nevertheless, the development of an in vitro platform mimicking able to mimic the dynamic stimuli occurring during metastasis still remains a challenge. We here developed a 3D hydrogel-based tissue model provided with an inner channel resembling the vascularization of the metastatic tumor tissue. The mechanical properties of two different hydrogels (i.e. fibrin and alginate) were measured; glucose mass transport resembling the blood flow through the hydrogels was analyzed by performing a computational fluiddynamic (CFD) analysis. Moreover, this system has been used to evaluate the shear forces inducing fluid flow effects on high metastatic breast cancer cells (MDA-MB-231) circulating within the hydrogel-based channel.

Metastasis is a dynamic process involving the dissemination of circulating tumor cells (CTCs) through blood flow to distant tissues within the body. Nevertheless, the development of an in vitro platform that dissects the crucial steps of metastatic cascade still remains a challenge. We here developed an in vitro model of extravasation composed of (i) a single channel-based 3D cell laden hydrogel representative of the metastatic site, (ii) a circulation system recapitulating the bloodstream where CTCs can flow. Two polymers (i.e., fibrin and alginate) were tested and compared in terms of mechanical and biochemical proprieties. Computational fluid-dynamic (CFD) simulations were also performed to predict the fluid dynamics within the polymeric matrix and, consequently, the optimal culture conditions. Next, once the platform was validated through perfusion tests by fluidically connecting the hydrogels with the external circuit, highly metastatic breast cancer cells (MDA-MB-231) were injected and exposed to physiological wall shear stress (WSS) conditions (5 Dyn/cm2) to assess their migration toward the hydrogel. Results indicated that CTCs arrested and colonized the polymeric matrix, showing that this platform can be an effective fluidic system to model the first steps occurring during the metastatic cascade as well as a potential tool to in vitro elucidate the contribution of hemodynamics on cancer dissemination to a secondary site.

Recently, 3D in vitro cancer models have become important alternatives to animal tests for establishing the efficacy of anticancer treatments. In this work, 3D SKOV-3 cell-laden alginate hydrogels were established as ovarian tumor models and cultured within a fluid-dynamic bioreactor (MIVO®) device able to mimic the capillary flow dynamics feeding the tumor. Cisplatin efficacy tests were performed within the device over time and compared with (i) the in vitro culture under static conditions and (ii) a xenograft mouse model with SKOV-3 cells, by monitoring and measuring cell proliferation or tumor regression, respectively, over time. After one week of treatment with 10 µM cisplatin, viability of cells within the 3D hydrogels cultured under static conditions remained above 80%. In contrast, the viability of cells within the 3D hydrogels cultured within dynamic MIVO® decreased by up to 50%, and very few proliferating Ki67-positive cells were observed through immunostaining. Analysis of drug diffusion, confirmed by computational analysis, explained that these results are due to different cisplatin diffusion mechanisms in the two culture conditions. Interestingly, the outcome of the drug efficacy test in the xenograft model was about 44% of tumor regression after 5 weeks, as predicted in a shorter time just in the fluid-dynamic in vitro tests carried out within MIVO® device. These results indicate that the in vivo-like dynamic environment provided by the MIVO® device allows to better model the 3D tumor environment and predict in vivo drug efficacy than a static in vitro model.

Porous multiphase scaffolds have been proposed in different tissue engineering applications because of their potential to artificially recreate the heterogeneous structure of hierarchically complex tissues. Recently, graded scaffolds have been also realized, offering a continuum at the interface among different phases for an enhanced structural stability of the scaffold. However, their internal architecture is often obtained empirically and the architectural parameters rarely predetermined. The aim of this work is to offer a theoretical model as tool for the design and fabrication of functional and structural complex graded scaffolds with predicted morphological and chemical features, to overcome the time-consuming trial and error experimental method. This developed mathematical model uses laws of motions, Stokes equations, and viscosity laws to describe the dependence between centrifugation speed and fiber/particles sedimentation velocity over time, which finally affects the fiber packing, and thus the total porosity of the 3D scaffolds. The efficacy of the theoretical model was tested by realizing engineered graded grafts for osteochondral tissue engineering applications. The procedure, based on combined centrifugation and freeze-drying technique, was applied on both polycaprolactone (PCL) and collagen-type-I (COL) to test the versatility of the entire process. A functional gradient was combined to the morphological one by adding hydroxyapatite (HA) powders, to mimic the bone mineral phase. Results show that 3D bioactive morphologically and chemically graded grafts can be properly designed and realized in agreement with the theoretical model. Biotechnol. Bioeng. 2016;113: 2286-2297. © 2016 Wiley Periodicals, Inc.

The fast replacement of native functionality (i.e., biological, chemical, mechanical) of damaged skeletal tissues currently represents an important challenge of clinical surgery. Through an accurate study of natural tissues and biomimesis, advanced biomaterials can be designed in the form of porous scaffolds to create hierarchical porous structures with the desired mechanical performances and mass transport properties (permeability and diffusion) while reproducing the complex 3D anatomical shapes. Recently, bioinspired composite scaffolds have been successfully used for the regeneration of bone and osteochondral tissue due to their unique ability to accurately mimic tissue functions and organization at different size scale levels. As a function of the filler/matrix coupling, they may ensure biochemical affinity with the host tissue through a judicious mix of specific morphological and chemical cues, also mimicking the response under load exhibited by natural tissue through a complex organization of material phases. This chapter provides an overview of recently developed composite porous and nonporous platforms used for repair/regeneration of bone and osteochondral tissue. In particular, we have classified composite scaffolds by distinguishing between those with biodegradable and bioresorbable matrices. Moreover, we describe different strategies to functionalize them with organic or inorganic phases by remarking, case by case, their passive or active reinforcement role as a function of intrinsic properties (i.e., degradation, magnetism) and different shapes (i.e., long or short fibers, particles with different shape ratios). Different manufacturing protocols are described, emphasizing their main advantages and disadvantages, and can be adopted to realize functionally and mechanically performed tissue substitutes.

Intestinal permeability is crucial in regulating the bioavailability and, consequently, the biological effects of drugs and compounds. However, systematic and quantitative studies of the absorption of molecules are quite limited due to a lack of reliable experimental models able to mimic human in vivo responses. In this work, we present an in vitro perfused model of the small intestinal barrier using a 3D reconstructed intestinal epithelium integrated into a fluid-dynamic biore­actor (MIVO®) resembling the physiological stimuli of the intestinal environment. This platform was investigated in both healthy and induced pathological conditions by monitoring the absorption of two non-metabolized sugars, lactulose and mannitol, frequently used as indicators of intestinal barrier dysfunctions. In healthy conditions, an in vivo-like plateau of the percentage of absorbed sugars was reached, where mannitol absorption was much greater than lactulose absorption. Moreover, a model of pathologically altered intestinal permeability was generated by depleting extracellular Ca2+, using a calcium-specific chelator. After calcium depletion, the pattern of sugar passage observed under pathological conditions was reversed only in dynamic conditions in the MIVO® chamber, due to the dynamic fluid flow beneath the membrane, but not in static conditions. Therefore, the combination of the MIVO® with the EpiIntestinal™ platform can rep­resent a reliable in vitro model to study the passage of molecules across the healthy or pathological small intestinal barrier by discriminating the two main mechanisms of intestinal absorption.

Graphene-derived materials, such as graphene oxide (GO), have been widely explored for biomedical and biological applications, including cancer research. Despite some recent works proving that GO inhibits the migration and invasion of different cancer cells, so far most of these in vitro studies have been conducted using GO sheets dispersed in solution or as a planar film. On the contrary, little is known about cellular activities, such as cell viability, adhesion, and spreading, when cancer cells interface with GO functionalized hydrogel-based surfaces, biomechanically and structurally more similar to the tumor environment. Here, we evaluate the interactions of human breast cancer cells (MDA-MB-231) with alginate (Alg)/GO hydrogel-based substrates, and compare them with a cancer cell line from human osteosarcoma (HOS) and healthy murine fibroblasts (3T3). We observed that GO addition selectively inhibits malignant breast cancer cell adhesion efficiency and spreading area, while promotes HOS and 3T3 adhesive processes. Furthermore, we did not observe the same results over Alg substrates with GO nanosheets dispersed in the medium, without embedment into the Alg. This suggests that cancer (MDA-MB-231 and HOS) and healthy (3T3) cell adhesion efficacy does not depend on the cellular tumoral nature and it is driven by the topographical cues provided by the GO-based substrates, whose physical-mechanical characteristics better mimic those of the cell native tissue. We envision that this study can provide a rational for future design and use of graphene-based nanomaterials for cancer research by deepening the knowledge of graphene-cancer cell specific interactions.

Purpose of this study was the development of a 3D material to be used as substrate for breast cancer cell culture. We developed composite gels constituted by different concentrations of Alginate (A) and Matrigel (M) to obtain a structurally stable-in-time and biologically active substrate. Human aggressive breast cancer cells (i.e. MDA-MB-231) were cultured within the gels. Known the link between cell morphology and malignancy, cells were morphologically characterized and their invasiveness correlated through an innovative bioreactor-based invasion assay. A particular type of gel (i.e. 50% Alginate, 50% Matrigel) emerged thanks to a series of significant results: 1. cells exhibited peculiar cytoskeleton shapes and nuclear fragmentation characteristic of their malignancy; 2. cells expressed the formation of the so-called invadopodia, actin-based protrusion of the plasma membrane through which cells anchor to the extracellular matrix; 3. cells were able to migrate through the gels and attach to an engineered membrane mimicking the vascular walls hosted within bioreactor, providing a completely new 3D in vitro model of the very precursor steps of metastasis.

Il documento descrive il processo di stampa 3D attraverso hardware fornito da Formlabs.

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 μm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.

The development of novel 3D systems is crucial for engineering artificial tissues since the behavior of cells growth on 2D cell culture substrates does not accurately reflect that of the physiological microenvironment. In this regard, desirable 3D composites should offer tunable structural and functional properties to support appropriate cellular growth and biomechanical loads. In this work, we realized 3D alginate hydrogels functionalized with graphene oxide (GO) nanosheets for the creation of cell laden hybrid materials with proper mechanical properties for tissue engineering applications. We monitored the mechanical proprieties of 2 wt% GO/Alg hydrogels up to one month demonstrating a significant improvement of the compressive elastic modulus reaching values of 300 KPa (6 times higher stiffness), which are close to those of articular tissues. This finding has been correlated to increased intermolecular hydrogen bonds over time between GO and Alg, observed through FT-IR analysis. Interestingly, we show that 3D GO/Alg hydrogels trigger cellular activity in vitro, as demonstrated by the statistically significant improvement of the viability of fibroblasts encapsulated in GO/Alg hydrogels and by the absence of cytotoxicity of suspended GO flakes. All these findings indicate that GO/Alg hydrogel is a promising material for articular tissue engineering, where biomechanical requirements are crucial.

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin-soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow mesenchymal stem cells (MSCs) and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell-mediated contraction, as compared against non-methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition, and histology. In particular, methacrylated cartilage hydrogels supported minimal chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell-matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non-methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan, and collagen type II) but increased collagen type I expression. Non-methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non-methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogenous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin-solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal- and light-induced polymerization but compromises chondrogenic differentiation of seeded MSCs.

The reconstruction of complex joints represents one of the major challenges in Tissue Engineering, whose aim is to realize bioactive 3D grafts interacting with the articular environment while providing structural and mechanical functionality. Due to the complex hierarchical structure and the co-existence of several architectural organizations of natural articular tissue, a series of chemical-physical-biological features have to be carefully controlled and defined for a best tuning of the mechanical and functional properties of osteochondral tissues. However, the control over scaffold architecture using conventional manufacturing techniques is highly process dependent rather than design dependent. As a result, in the last years Rapid Prototyping (RP) techniques are proposed as promising alternative for 3D porous scaffolds fabrication, opening the possibility to realize engineered grafts with defined and reproducible complex internal structures, for an enhanced cellular response in vivo; moreover, implantable personalized articular tissue materials may be created individually for each patient according to the orthopedic requirements. In this Chapter, we will expose the major RP-based techniques, among them laser-, nozzled- and printed-based RP methods, with particular reference to the most cogent works in the field of joints repair.

Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D “architecture” for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK) and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP) motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1). The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate.

Treatment of full-thickness cartilage defects relies on osteochondral bilayer grafts, which mimic the microenvironment and structure of the two affected tissues: articular cartilage and subchondral bone. However, the integrity and stability of the grafts are hampered by the presence of a weak interphase, generated by the layering processes of scaffold manufacturing. We describe here the design and development of a bilayer monolithic osteochondral graft, avoiding delamination of the two distinct layers but preserving the cues for selective generation of cartilage and bone. A highly porous polycaprolactone-based graft was obtained by combining solvent casting/particulate leaching techniques. Pore structure and interconnections were designed to favour in vivo vascularization only at the bony layer. Hydroxyapatite granules were added as bioactive signals at the site of bone regeneration. Unconfined compressive tests displayed optimal elastic properties and low residual deformation of the graft after unloading (< 3%). The structural integrity of the graft was successfully validated by tension fracture tests, revealing high resistance to delamination, since fractures were never displayed at the interface of the layers (n = 8). Ectopic implantation of grafts in nude mice, after seeding with bovine trabecular bone-derived mesenchymal stem cells and bovine articular chondrocytes, resulted in thick areas of mature bone surrounding ceramic granules within the bony layer, and a cartilaginous alcianophilic matrix in the chondral layer. Vascularization was mostly observed in the bony layer, with a statistically significant higher blood vessel density and mean area. Thus, the easily generated osteochondral scaffolds, since they are mechanically and biologically functional, are suitable for tissue-engineering applications for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.

Bio-inspired materials with controlled topography have gained increasing interest in regenerative medicine, because of their ability to reproduce the physical features of natural extracellular matrix, thus amplifying certain biological responses both in vitro and in vivo, such as contact guidance and differentiation. However, information on the ability to adapt this high cell potential to 3D scaffolds, effective to be implanted in clinical bone defect, is still missing. Here, we examine the pattern of bone tissue generated within the implant in an ectopic model, seeding bone marrow progenitor cells onto PCL-MgCHA scaffolds. This composite material presented a porous structure with micro/nanostructured surfaces obtained by combining phase inversion/salt leaching and electrospinning techniques. Histological analysis of grafts harvested after 1-2-6 months from implantation highlights an extent of lamellar bone tissue within interconnected pores of fibre coated PCL-MgCHA composites, whereas uncoated scaffolds displayed sparse deposition of bone. Pure PCL scaffolds did not reveal any trace of bone for the overall 6 months of observation. In conclusion, we show that a structural modification in scaffold design is able to enhance bone regeneration possibly mimicking some physiological cues of the natural tissue.

Preparation of tissue engineered bone constructs to repair large size defects is limited by the difficult supply of oxygen and nutrients to cells deep inside the constructs. Hollow fibre membrane bioreactors (HFMBs) with medium flowing in membrane lumen have structural and functional analogy with cortical bone. In fact, membranes resemble the Haversian canals and provide for a distributed and delocalized source of nutrients and oxygen to surrounding cells. HFMBs where oxygen and nutrients diffuse to cells have been proposed for bone tissue engineering. HFMBs may also be so operated as to promote spontaneous Starling flows to enhance nutrients transfer to cells. In this study, the effect of low and high Starling flows was investigated on sheep mesenchymal stem cells (shMSC) cultured in the extracapillary space of HFMBs with respect to cell distribution, proliferation and early differentiation. After 12 days of culture, at low Starling flows cells formed thin layers around the membranes uniformly along the bioreactor. At high Starling flows, cells proliferated well and formed thick multilayer aggregates filling the space among membranes at the bioreactor outlet. These results make it possible to postulate the use of HFMBs for obtaining tissue engineered constructs for the repair of large bone defects.

Alternative drug delivery approaches to treat cardiovascular diseases are currently under intense investigation. In this domain, the possibility to target the heart and tailor the amount of drug dose by using a combination of magnetic nanoparticles (NPs) and electromagnetic devices is a fascinating approach. Here, an electromagnetic device based on Helmholtz coils was generated for the application of low-frequency magnetic stimulations to manage drug release from biocompatible superparamagnetic Fe-hydroxyapatite NPs (FeHAs). Integrated with a fluidic circuit mimicking the flow of the cardiovascular environment, the device was efficient to trigger the release of a model drug (ibuprofen) from FeHAs as a function of the applied frequencies. Furthermore, the biological effects on the cardiac system of the identified electromagnetic exposure were assessed in vitro and in vivo by acute stimulation of isolated adult cardiomyocytes and in an animal model. The cardio-compatibility of FeHAs was also assessed in vitro and in an animal model. No alterations of cardiac electrophysiological properties were observed in both cases, providing the evidence that the combination of low-frequency magnetic stimulations and FeHAs might represent a promising strategy for controlled drug delivery to the failing heart.

The combination of synthetic polymers and calcium phosphates represent an improvement in the development of scaffolds for bone-tissue regeneration. Ideally, these composites provide both mechanically and architecturally enhanced performances; however, they often lack properties such as osteoconductivity and cell bioactivation. In this study we attempted to generate a composite bone substitute maximizing the available osteoconductive surface for cell adhesion and activity. Highly porous scaffolds were prepared through a particulate leaching method, combining poly-ε-caprolactone (PCL) and hydroxyapatite (HA) particles, previously coated with a sucrose layer, to minimize their embedding by the polymer solution. Composite performances were evaluated both in vitro and in vivo. In PCL-sucrose-coated HA samples, the HA particles were almost completely exposed and physically distinct from the polymer mesh, while uncoated control samples showed ceramic granules massively covered by the polymer. In vivo results revealed a significant extent of bone deposition around all sucrose-coated HA granules, while only parts of the control uncoated HA granules were surrounded by bone matrix. These findings highlight the possibility of generating enhanced osteoconductive materials, basing the scaffold design on physiological and cellular concepts.