Bioprinting of human dermal microtissues precursors as building blocks for endogenousin vitroconnective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricatein vitrotissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (µTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly ofµTPs into predefined shapes. Compared to cell-aggregated bioink, ourµTPs represent a functional unit where cells are embedded in their specific ECM.µTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days,µTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter.µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel atT> 30 °C), ensuringµTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, untilµTPs fused into a continuous connective tissue.µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

A functional 3D full-thickness model for comprehending the interaction between airway epithelium and connective tissue in cystic fibrosis.

Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodeling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodeling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to fully replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodeling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed gland-like structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy.

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Role of the mechanical microenvironment on CD-44 expression of breast adenocarcinoma in response to radiotherapy.

The biological effects of ionizing radiation are exploited in the clinical practice of radiotherapy to destroy tumour cells while sparing the surrounding normal tissue. While most of the radiotherapy research focused on DNA damage and repair, recently a great attention is going to cells' interactions with the mechanical microenvironment of both malignant and healthy tissues after exposure. In fact, the stiffness of the extracellular matrix can modify cells' motility and spreading through the modulation of transmembrane proteins and surface receptors' expression, such as CD-44. CD-44 receptor has held much interest also in targeted-therapy due to its affinity with hyaluronic acid, which can be used to functionalize biodegradable nanoparticles loaded with chemotherapy drugs for targeted therapy. We evaluated changes in CD-44 expression in two mammary carcinoma cell lines (MCF10A and MDA-MB-231) after exposure to X-ray (2 or 10 Gy). To explore the role of the mechanical microenvironment, we mimicked tissues' stiffness with polyacrylamide's substrates producing two different elastic modulus values (0.5 and 15 kPa). We measured a dose dependent increase in CD-44 relative expression in tumour cells cultured in a stiffer microenvironment. These findings highlight a crucial connection between the mechanical properties of the cell's surroundings and the post-radiotherapy expression of surface receptors.

A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-ß Aggregation: Synergism of Mechanisms of Action.

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3]·H2O (1) (D-p-FPhF- = N,N'-bis(4-fluorophenyl)formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aß1-42 peptide and its peculiar fragments, Aß1-16 and Aß21-40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV-vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aß1-42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π-π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compounds.

Unknown cell class distinction via neural network based scattering snapshot recognition.

Neural network-based image classification is widely used in life science applications. However, it is essential to extrapolate a correct classification method for unknown images, where no prior knowledge can be utilised. Under a closed set assumption, unknown images will be inevitably misclassified, but this can be genuinely overcome choosing an open-set classification approach, which first generates an in-distribution of identified images to successively discriminate out-of-distribution images. The testing of such image classification for single cell applications in life science scenarios has yet to be done but could broaden our expertise in quantifying the influence of prediction uncertainty in deep learning. In this framework, we implemented the open-set concept on scattering snapshots of living cells to distinguish between unknown and known cell classes, targeting four different known monoblast cell classes and a single tumoral unknown monoblast cell line. We also investigated the influence on experimental sample errors and optimised neural network hyperparameters to obtain a high unknown cell class detection accuracy. We discovered that our open-set approach exhibits robustness against sample noise, a crucial aspect for its application in life science. Moreover, the presented open-set based neural network reveals measurement uncertainty out of the cell prediction, which can be applied to a wide range of single cell classifications.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Capturing the spatial and temporal dynamics of tumor stroma for on-chip optimization of microenvironmental targeting nanomedicine.

Malignant cells grow in a complex microenvironment that plays a key role in cancer progression. The "dynamic reciprocity" existing between cancer cells and their microenvironment is involved in cancer differentiation, proliferation, invasion, metastasis, and drug response. Therefore, understanding the molecular mechanisms underlying the crosstalk between cancer cells and their surrounding tissue (i.e., tumor stroma) and how this interplay affects the disease progression is fundamental to design and validate novel nanotherapeutic approaches. As an important regulator of tumor progression, metastasis and therapy resistance, the extracellular matrix of tumors, the acellular component of the tumor microenvironment, has been identified as very promising target of anticancer treatment, revolutionizing the traditional therapeutic paradigm that sees the neoplastic cells as the preferential objective to fight cancer. To design and to validate such a target therapy, advanced 3D preclinical models are necessary to correctly mimic the complex, dynamic and heterogeneous tumor microenvironment. In addition, the recent advancement in microfluidic technology allows fine-tuning and controlling microenvironmental parameters in tissue-on-chip devices in order to emulate the in vivo conditions. In this review, after a brief description of the origin of tumor microenvironment heterogeneity, some examples of nanomedicine approaches targeting the tumor microenvironment have been reported. Further, how advanced 3D bioengineered tumor models coupled with a microfluidic device can improve the design and testing of anti-cancer nanomedicine targeting the tumor microenvironment has been discussed. We highlight that the presence of a dynamic extracellular matrix, able to capture the spatiotemporal heterogeneity of tumor stroma, is an indispensable requisite for tumor-on-chip model and nanomedicine testing.

Radiation therapy affects YAP expression and intracellular localization by modulating lamin A/C levels in breast cancer.

The microenvironment of breast cancer actively participates in tumorigenesis and cancer progression. The changes observed in the architecture of the extracellular matrix initiate an oncogene-mediated cell reprogramming, that leads to a massive triggering of YAP nuclear entry, and, therefore, to cancer cell proliferation, invasion and probably to increased radiation-resistance. However, it is not yet fully understood how radiotherapy regulates the expression and subcellular localization of YAP in breast cancer cells experiencing different microenvironmental stiffnesses. To elucidate the role of extracellular matrix stiffness and ionizing radiations on YAP regulation, we explored the behaviour of two different mammary cell lines, a normal epithelial cell line (MCF10A) and a highly aggressive and invasive adenocarcinoma cell line (MDA-MB-231) interacting with polyacrylamide substrates mimicking the mechanics of both normal and tumour tissues (∼1 and ∼13 kPa). We report that X-ray radiation affected in a significant way the levels of YAP expression, density, and localization in both cell lines. After 24 h, MCF10A and MDA-MB-231 increased the expression level of YAP in both nucleus and cytoplasm in a dose dependent manner and particularly on the stiffer substrates. After 72 h, MCF10A reduced mostly the YAP expression in the cytoplasm, whereas it remained high in the nucleus of cells on stiffer substrates. Tumour cells continued to exhibit higher levels of YAP expression, especially in the cytoplasmic compartment, as indicated by the reduction of nuclear/cytoplasmic ratio of total YAP. Then, we investigated the existence of a correlation between YAP localization and the expression of the nuclear envelope protein lamin A/C, considering its key role in modulating nuclear deformability and changes in YAP shuttling phenomena. As supposed, we found that the effects of radiation on YAP nucleus/cytoplasmic expression ratio, increasing in healthy cells and decreasing in tumour ones, were accompanied by lower and higher lamin A/C levels in MCF10A and MDA-MB-231 cells, respectively. These findings point to obtain a deeper knowledge of the role of the extracellular matrix and the effects of X-rays on YAP and lamin A/C expression that can be used in the design of doses and timing of radiation therapy.

Spatial patterning of PCLµ-scaffolds directs 3D vascularized bio-constructs morphogenesisin vitro.

Modular tissue engineering (mTE) strategies aim to build three-dimensional tissue analoguesin vitroby the sapient combination of cells, micro-scaffolds (µ-scaffs) and bioreactors. The translation of these newly engineered tissues into current clinical approaches is, among other things, dependent on implant-to-host microvasculature integration, a critical issue for cells and tissue survivalin vivo. In this work we reported, for the first time, a computer-aided modular approach suitable to build fully vascularized hybrid (biological/synthetic) constructs (bio-constructs) with micro-metric size scale control of blood vessels growth and orientation. The approach consists of four main steps, starting with the fabrication of polycaprolactoneµ-scaffs by fluidic emulsion technique, which exhibit biomimetic porosity features. In the second step, layers ofµ-scaffs following two different patterns, namely ordered and disordered, were obtained by a soft lithography-based process. Then, the as obtainedµ-scaff patterns were used as template for human dermal fibroblasts and human umbilical vein endothelial cells co-culture, aiming to promote and guide the biosynthesis of collagenous extracellular matrix and the growth of new blood vessels within the mono-layered bio-constructs. Finally, bi-layered bio-constructs were built by the alignment, stacking and fusion of two vascularized mono-layered samples featuring ordered patterns. Our results demonstrated that, if compared to the disordered pattern, the ordered one provided better control over bio-constructs shape and vasculature architecture, while minor effect was observed with respect to cell colonization and new tissue growth. Furthermore, by assembling two mono-layered bio-constructs it was possible to build 1 mm thick fully vascularized viable bio-constructs and to study tissue morphogenesis during 1 week ofin vitroculture. In conclusion, our results highlighted the synergic role ofµ-scaff architectural features and spatial patterning on cells colonization and biosynthesis, and pave the way for the possibility to create in silico designed vasculatures within modularly engineered bio-constructs.

A switchable light-responsive azopolymer conjugating protein micropatterns with topography for mechanobiological studies.

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However, conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Current methods to fabricate dynamic platforms usually rely on complex chemical strategies or require specialized apparatuses. Also, with these methods, the integration of dynamic signals acting on different length scales is not straightforward, whereas, in some applications, it might be beneficial to act on both a microscale level, that is, cell shape, and a nanoscale level, that is, cell adhesions. Here, we exploited a confocal laser-based technique on a light-responsive azopolymer displaying micropatterns of adhesive islands. The laser light promotes a directed mass migration and the formation of submicrometric topographic relieves. Also, by changing the surface chemistry, the surfacing topography affects cell spreading and shape. This method enabled us to monitor in a non-invasive manner the dynamic changes in focal adhesions, cytoskeleton structures, and nucleus conformation that followed the changes in the adhesive characteristic of the substrate. Focal adhesions reconfigured after the surfacing of the topography, and the actin filaments reoriented to coalign with the newly formed adhesive island. Changes in cell morphology also affected nucleus shape, chromatin conformation, and cell mechanics with different timescales. The reported strategy can be used to investigate mechanotransduction-related events dynamically by controlling cell adhesion at cell shape and focal adhesion levels. The integrated technique enables achieving a submicrometric resolution in a facile and cost-effective manner.

ECM Mechanoregulation in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma is a relatively rare, but devastating tumor, because of the difficulties in providing early diagnosis and effective treatments with conventional chemo- and radiotherapies. Patients usually present pleural effusions that can be used for diagnostic purposes by cytological analysis. This effusion cytology may take weeks or months to establish and has a limited sensitivity (30%-60%). Then, it is becoming increasingly urgent to develop alternative investigative methods to support the diagnosis of mesothelioma at an early stage when this cancer can be treated successfully. To this purpose, mechanobiology provides novel perspectives into the study of tumor onset and progression and new diagnostic tools for the mechanical characterization of tumor tissues. Here, we report a mechanical and biophysical characterization of malignant pleural mesothelioma cells as additional support to the diagnosis of pleural effusions. In particular, we examined a normal mesothelial cell line (Met5A) and two epithelioid mesothelioma cell lines (REN and MPP89), investigating how malignant transformation can influence cellular function like proliferation, cell migration, and cell spreading area with respect to the normal ones. These alterations also correlated with variations in cytoskeletal mechanical properties that, in turn, were measured on substrates mimicking the stiffness of patho-physiological ECM.

PEG-based cleavable hydrogel microparticles with controlled porosity for permiselective trafficking of biomolecular complexes in biosensing applications.

In the last decade, PEG-based hydrogels have been extensively used for the production of microparticles for biosensing applications. The biomolecule accessibility and mass transport rate represent key parameters for the realization of sensitive microparticles, therefore porous materials have been developed, mainly resorting to the use of inert porogens and copolymers with different chain lengths. However, very limited information is reported regarding the addition of cleavable crosslinkers to modulate the network porosity. In this scenario, the aim of this work is to design, synthesize and characterize hydrogel microparticles, based on the copolymerization between PEG-diacrylate and N,N'-(1,2-dihydroxyethylene)-bisacrylamide, a cleavable crosslinker that simultaneously produces pores and reactive groups for bioprobe 3D bioconjugation. The results show great accessibility of these microparticles to antibodies and their complexes, without affecting their diffusion rate. Furthermore, the presence of a well-defined number of reactive aldehydes, produced by the cleavage reaction, allows modulating biosensor sensitivity through a fine control of the conjugation degree. The antibody-conjugated microparticles can efficiently capture the analyte down to a few picograms. These novel microparticles could be used as a highly sensitive platform for biomacromolecule detection in complex fluids, exploiting the combined effects of PEG's anti-fouling properties, large network porosity and interconnections, and three-dimensional bioconjugation.

Biocompatible, photo-responsive layer-by-layer polymer nanocapsules with an oil core: in vitro and in vivo study.

In cancer therapy, stimulus-responsive drug delivery systems are of particular interest for reducing side effects in healthy tissues and improving drug selectivity in the tumoral ones. Here, a strategy for the preparation of a photo-responsive cross-linked trilayer deposited onto an oil-in-water nanoemulsion via a layer-by-layer technique is reported. The system is made of completely biocompatible materials such as soybean oil, egg lecithin and glycol chitosan, with heparin as the polymeric shell. The oil core is pre-loaded with curcumin as a model lipophilic active molecule with anti-tumoral properties. The trilayer cross-linkage is performed via a photoinitiator-free thiol-ene 'click' reaction. In particular, the system is implemented with an o-nitrobenzyl group functionalized with a thiol moiety which can perform both the thiol-ene 'click' reaction and the cleavage meant for controlled drug release at two different wavelengths, respectively. So the preparation and characterization of a photo-responsive natural nanocarrier (PNC) that is stable under physiological conditions owing to the thiol-ene cross-linkage are reported. PNC performance has been assessed in vitro on melanoma cells as well as in vivo on xenograft tumour-induced mice.

Characterization of Hyaluronic Acid-Coated PLGA Nanoparticles by Surface-Enhanced Raman Spectroscopy.

Nanoparticles (NPs) coated with hyaluronic acid (HA) seem to be increasingly promising for targeted therapy due to HA chemical versatility, which allows them to bind drugs of different natures, and their affinity with the transmembrane receptor CD-44, overexpressed in tumor cells. However, an essential aspect for clinical use of NPs is formulation stability over time. For these reasons, analytical techniques capable of characterizing their physico-chemical properties are needed. In this work, poly(lactide-co-glycolide) (PLGA) NPs with an average diameter of 100-150 nm, coated with a few 10 s of nm of HA, were synthesized. For stability characterization, two complementary investigative techniques were used: Dynamic Light Scattering (DLS) and Surface-Enhanced Raman Scattering (SERS) spectroscopy. The first technique provided information on size, polidispersity index, and zeta-potential, and the second provided a deeper insight on the NP surface chemicals, allowing distinguishing of HA-coated NPs from uncoated ones. Furthermore, in order to estimate formulation stability over time, NPs were measured and monitored for two weeks. SERS results showed a progressive decrease in the signal associated with HA, which, however, is not detectable by the DLS measurements.

Cytoskeleton Response to Ionizing Radiation: A Brief Review on Adhesion and Migration Effects.

The cytoskeleton is involved in several biological processes, including adhesion, motility, and intracellular transport. Alterations in the cytoskeletal components (actin filaments, intermediate filaments, and microtubules) are strictly correlated to several diseases, such as cancer. Furthermore, alterations in the cytoskeletal structure can lead to anomalies in cells' properties and increase their invasiveness. This review aims to analyse several studies which have examined the alteration of the cell cytoskeleton induced by ionizing radiations. In particular, the radiation effects on the actin cytoskeleton, cell adhesion, and migration have been considered to gain a deeper knowledge of the biophysical properties of the cell. In fact, the results found in the analysed works can not only aid in developing new diagnostic tools but also improve the current cancer treatments.

Type F mutation of nucleophosmin 1 Acute Myeloid Leukemia: A tale of disorder and aggregation.

Protein aggregation is suggested as a reversible, wide-spread physiological process used by cells to regulate their growth and adapt to different stress conditions. Nucleophosmin 1(NPM1) protein is an abundant multifunctional nucleolar chaperone and its gene is the most frequently mutated in Acute Myeloid Leukemia (AML) patients. So far, the role of NPM1 mutations in leukemogenesis has remained largely elusive considering that they have the double effect of unfolding the C-terminal domain (CTD) and delocalizing the protein in the cytosol (NPM1c+). This mislocalization heavily impacts on cell cycle regulation. Our recent investigations unequivocally demonstrated an amyloid aggregation propensity introduced by AML mutations. Herein, employing complementary biophysical assays, we have characterized a N-terminal extended version of type F AML mutation of CTD and proved that it is able to form assemblies with amyloid character and fibrillar morphology. The present study represents an additional phase of knowledge to deepen the roles exerted by different types of cytoplasmatic NPM1c+ forms to develop in the future potential therapeutics for their selective targeting.

Review on Computer-Aided Design and Manufacturing of Drug Delivery Scaffolds for Cell Guidance and Tissue Regeneration.

In the last decade, additive manufacturing (AM) processes have updated the fields of biomaterials science and drug delivery as they promise to realize bioengineered multifunctional devices and implantable tissue engineering (TE) scaffolds virtually designed by using computer-aided design (CAD) models. However, the current technological gap between virtual scaffold design and practical AM processes makes it still challenging to realize scaffolds capable of encoding all structural and cell regulatory functions of the native extracellular matrix (ECM) of health and diseased tissues. Indeed, engineering porous scaffolds capable of sequestering and presenting even a complex array of biochemical and biophysical signals in a time- and space-regulated manner, require advanced automated platforms suitable of processing simultaneously biomaterials, cells, and biomolecules at nanometric-size scale. The aim of this work was to review the recent scientific literature about AM fabrication of drug delivery scaffolds for TE. This review focused on bioactive molecule loading into three-dimensional (3D) porous scaffolds, and their release effects on cell fate and tissue growth. We reviewed CAD-based strategies, such as bioprinting, to achieve passive and stimuli-responsive drug delivery scaffolds for TE and cancer precision medicine. Finally, we describe the authors' perspective regarding the next generation of CAD techniques and the advantages of AM, microfluidic, and soft lithography integration for enhancing 3D porous scaffold bioactivation toward functional bioengineered tissues and organs.

Geometrical confinement controls cell, ECM and vascular network alignment during the morphogenesis of 3D bioengineered human connective tissues.

Engineered tissues featuring aligned ECM possess superior regenerative capabilities for the healing of damaged aligned tissues. The morphofunctional integration in the host's injury site improves if the aligned ECM elicits the unidirectional growth of vascular network. In this work we used a bottom-up tissue engineering strategy to produce endogenous and highly aligned human connective tissues with the final aim to trigger the unidirectional growth of capillary-like structures. Engineered microtissues, previously developed by our group, were casted in molds featured by different aspect ratio (AR) to obtain final centimeter-sized macrotissues differently shaped. By varying the AR from 1 to 50 we were able to vary the final shape of the macrotissues, from square to wire. We demonstrated that by increasing the AR of the maturation space hosting the microtissues, it was possible to control the alignment of the neo-synthesized ECM. The geometrical confinement conditions at AR = 50, indeed, promoted the unidirectional growth and assembly of the collagen network. The wire-shaped tissues were characterized by parallel arrangement of the collagen fiber bundles, higher persistence length and speed of migrating cells and superior mechanical properties than the square-shaped macrotissues. Interestingly, the aligned collagen fibers elicited the unidirectional growth of capillary-like structures. STATEMENT OF SIGNIFICANCE: Alignment of preexisting extracellular matrices by using mechanical cues modulating cell traction, has been widely described. Here, we show a new method to align de novo synthesized extracellular matrix components in bioengineered connective tissues obtained by means of a bottom-up tissue engineering approach. Building blocks are cast in maturation chambers, having different aspect ratios, in which the in vitro morphogenesis process takes place. High aspect ratio chambers (corresponding to wire-shaped tissues) triggered spontaneous alignment of collagenous network affecting cell polarization, migration and tensile properties of the tissue as well. Aligned ECM provided a contact guidance for the formation of highly polarized capillary-like network suggesting an in vivo possible application to trigger fast angiogenesis and perfusion in damaged aligned tissues.