Realization and characterization of bioactive composite materials for locomotor tissue regeneration

Tissue Enginnering emerges as a potential and alternative therapeutic process to treat severely injured patients with minimally invasive techniques. Cartilage and bone injuries occur due to several reasons and they compromise quality of life. This thesis was focused on development of biomaterials that could mimic cartilage and bone tissue, using only natural substances, gelatin and/or collagen, crosslinked with genipin (GP) and hydroxyapatite (HA) . These materials are cheap and easy to handle, and in particular collagen, represents a chemo- attractor factor, that may help cellular colonization. First of all genipin reaction was studied to establish reaction rate constant and crosslinking degree as function of genipin concetrations. The optimal genipin concentration was decided primarly assuring that it was not cytotoxic, meseauring its release in acqueous enviroment. Then elastic moduli of scaffolds prepared with different GP concentrations, different protocols and different HA concentrations were measured taking into account that scaffolds had to present mechanical properties suited to the implant site. Roughness surface of scaffolds, was also investigated with SEM, to ensure an optimal integration with implant site and to verify the presence of a right porosity to allow its cell colonisation. Bone scaffolds were arranged to reproduce a HA gradient, an important bone feature, and their anisotropy was valueted. Biocompatibility tests, in vitro tests, for cartilage and bone biomaterials were performed using primary and immortalised cells. Finally preliminary in vivo tests using small animal models, rats with a femur lesion, were performed for cartilage and bone substitute, selected on the basis of their mechanical properties. The aim was to follow bone and cartilage regeneration, after injection of biomaterials, and to compare it with the physiological regeneration

A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells' morphogenesis, and they range in a three-dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffnes

Genipin diffusion and reaction into a gelatin matrix for tissue engineering applications

Genipin is a natural low-toxic cross-linker for molecules with primary amino groups, and its use with collagen and gelatin has shown a great potential in tissue engineering applications. The fabrication of scaffolds with a well-organized micro and macro topology using additive manufacturing systems requires an accurate control of working parameters, such as reaction rate, gelling time, and diffusion constant. A polymeric system of 5% w/v gelatin in PBS with 2 mg/mL collagen solutions in a 1:1 weight ratio was used as template to perform measurements varying genipin concentration in a range of 0.1-1.5% w/w with respect to gelatin. In the first part of this work, the reaction rate of the polymeric system was estimated using a new colorimetric analysis of the reaction. Then its workability time, closely related to the gelling time, was evaluated thanks to rheological analysis: finally, the quantification of static and dynamic diffusion constants of genipin across nonreacting and reacting membranes, made respectively by agarose and gelatin, was performed. It was shown that the colorimetric analysis is a good indicator of the reaction progress. The gelling time depends on the genipin concentration, but a workability window of 40 min guaranteed up to 0.5% w/w genipin. The dynamic diffusion constant of genipin in the proposed polymeric system is in the order of magnitude of 10(-7) . The obtained results indicated the possibility to use the genipin, gelatin, and collagen, in the proposed concentrations, to build well-defined hydrogel scaffolds with both extrusion-based and 3D ink-jet system. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015

Design and Validation of an Open-Hardware Print-Head for Bioprinting Application

In the last decades drop-on-demand inkjet technology played an increasing role in industrial and medical applications. This is due to the ability to deposit a small amount of material in precisely defined position. In the field of Biofabrication, inkjet printers are used to build 2D and 3D scaffolds and gels with biological molecules, including living cells. Several works, including seminal papers on inkjet bioprinting, were carried out with modified office printers. These printers have fixed structural characteristics and operating size, especially on the print-head, limiting the range of materials that can be dispensed. The aim of the present work is the design and fabrication of an open-source piezoelectric inkjet print-head, optimized for the bioprinting field. This low-cost, reproducible, reliable, versatile and biocompatible device will enable various research laboratories to work with a shared device; the open source allowing for parts to be modified to suit specific needs. The design was carried out by Finite Element (FE) modelling of the piezoelectric, mechanical, fluid dynamics and their coupling. The design was optimized for shear rate, which we minimized in order to be able to print cells. The mechanical frame of the printer was designed and built using a low-cost 3D printer. The nozzle plate was fabricated from a polycarbonate disc coated with biocompatible silicone, to increase the hydrophobicity of the outer surface of the disc, preventing ink adhesion on the edge of the nozzle; the refilling system, and the electronic control were also part of the project and will be freely available to download. The FE models were validated with ad-hoc experiments, printing water, gelatin solution, and cell culture media, by modulating the wave power in amplitude, frequency and duty cycle. The tests showed a large working window both respect to viscosity and to surface tension. Finally Human Skin Fibroblasts (ATCC-CRL- 2522, Teddington UK), suspended in culture media, were printed. Cell viability, assessed by CellTiter-Blue and LIVE / DEAD tests, resulted comparable with the control, demonstrating the validity of the first open source piezoelectric inkjet print-head for biofabrication

Phantoms in medicine: the case of ophthalmology

Physical and in-silico phantoms have revealed extremely useful in the development of new surgical techniques and medical devices and for training purposes. The fabrication of eye phantoms requires knowledge of anatomy and physical principles beyond the eye physiology and medical instruments used in the clinical scenario. After a proper definition of phantoms and the discussion about their classification, the present work reviews the various phantoms developed in ophthalmology, illustrating the rationale of their design

Bone and gut microbiota crosstalk: A novel 3D in vitro approach

The present research aimed at shedding light on the interplay between the composition of the human gut microbiota and bone cells

Clinical Profiles in Multiple Sclerosis: Cognitive Reserve and Motor Impairment along Disease Duration

(i) Background: Cognitive impairment in people with multiple sclerosis (MS) has been studied in relation to certain clinical variables (e.g., motor disability and disease duration) and lifestyle factors such as cognitive reserve (CR). However, only very few studies have considered the interaction of clinical variables and cognitive reserve in preserving the integrity of the neuropsychological profile. In this paper, we hypothesised that a higher level of CR might predict good cognitive efficiency by modulating the clinical outcome of the disease. (ii) Methods: A sample of 100 participants with MS (age range 30–74), was recruited and assessed remotely with a questionnaire to measure CR and a cognitive screening test. Data were analysed through generalized additive models. (iii) Results: We found that the model analysing the interaction between CR and disease duration, and between CR and motor disability, was able to explain a significant percentage of cognitive performance. In particular, higher levels of CR predicted a better cognitive performance despite a long disease duration, unless the motor disability was severe. (iv) Conclusion: This study highlights the crucial role of CR in modulating cognitive efficiency in people with MS

Study of the Adhesion of the Human Gut Microbiota on Electrospun Structures

Although the adhesion of bacteria on surfaces is a widely studied process, to date, most of the works focus on a single species of microorganisms and are aimed at evaluating the antimicrobial properties of biomaterials. Here, we describe how a complex microbial community, i.e., the human gut microbiota, adheres to a surface to form stable biofilms. Two electrospun structures made of natural, i.e., gelatin, and synthetic, i.e., polycaprolactone, polymers were used to study their ability to both promote the adhesion of the human gut microbiota and support microbial growth in vitro. Due to the different wettabilities of the two surfaces, a mucin coating was also added to the structures to decouple the effect of bulk and surface properties on microbial adhesion. The developed biofilm was quantified and monitored using live/dead imaging and scanning electron microscopy. The results indicated that the electrospun gelatin structure without the mucin coating was the optimal choice for developing a 3D in vitro model of the human gut microbiota

Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) 3D bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold which underwent osteogenic differentiation for two weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during two more weeks of culture. We tested three experimental conditions: Condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM):endothelial medium (EM); Condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; Condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In Conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under Condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just four weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development

Antigene MYCN Silencing by BGA002 Inhibits SCLC Progression Blocking mTOR Pathway and Overcomes Multidrug Resistance

: Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene-PNA oligonucleotide (BGA002)-as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC