Biomedical and bio-nanotechnological approach of degenerative diseases

Osteoarthritis is a degenerative disease of the joints, that progressively leads to destruction and loss of function of the articular cartilage. Within the framework of this doctoral thesis, studies were carried out to find biomarkers in the blood that would allow the diagnosis of the disease with nanotechnology and molecular biology techniques, as well as studies to construct an "intelligent" biomaterial capable of inducing in vivo chondrogenesis and contributing to regeneration of the articular cartilage. The study of the expression of COMP, ADAMTS-5 and TIMP-3 genes in the blood of patients and healthy donors, while as the detection of miR-93 in the blood serum of patients and healthy donors using gold nanoparticles in solution and silicon surfaces, showed that these molecules can be used as diagnostic biomarkers of the disease. In addition, smaller functional areas of the C-terminal domain of transforming growth factor TGF-β1 that may induce in vitro chondrogenesis have been designed and synthesized. The results showed that the shorter protein regions of the C-terminal domain of TGF-β1 increased in vitro chondrogenesis in three-dimensional cultures compared to the one-dimensional ones, with the best results being achieved by addition of peptide 8 of the C-terminal domain of TGF-β1. At the same time, the shorter protein regions of the C-terminal domain of TGF-β1 reduce the expression of the aggrecanase ADAMTS-5 gene, which is positively regulated by the exogenous addition of TGF-β1, thus promoting cell differentiation and extracellular matrix formation. In this case, the lower expression was demonstrated by the peptide 6 addition and immediately after the peptide 8 addition, indicating that the two small peptides could be used to construct a biomaterial for articular cartilage regeneration.Η οστεoαρθρίτιδα (OA) αποτελεί μια εκφυλιστική νόσο των αρθρώσεων που προοδευτικά οδηγεί σε καταστροφή του αρθρικού χόνδρου και απώλεια της λειτουργίας του. Στα πλαίσια της παρούσας διδακτορικής διατριβής πραγματοποιήθηκαν μελέτες με τεχνικές νανοτεχνολoγίας και μοριακής βιολογίας για την εύρεση βιοδεικτών στο αίμα που θα επιτρέψουν τη διάγνωση της νόσου καθώς και μελέτες κατασκευής ενός «έξυπνου» βιοϋλικού ικανού να επάγει την in vivo χονδρογένεση και να συμβάλει στη αποκατάσταση του κατεστραμμένου αρθρικού χόνδρου. Η μελέτη της έκφρασης των γονιδίων COMP, ADAMTS-5 και ΤΙΜΡ-3 στο αίμα ασθενών και υγιών δοτών καθώς η ανίχνευση του miR-93 σε όρο αίματος ασθενών και υγιών δοτών με τη χρήση νανοσωματιδίων χρυσού σε διάλυμα αλλά και σε επιφάνειες πυριτίου, έδειξαν πως τα μόρια αυτά μπορούν να χρησιμοποιηθούν ως βιοδείκτες για τη διάγνωση της νόσου. Επιπρόσθετα, σχεδιάστηκαν και συντέθηκαν μικρότερες λειτουργικές επικράτειες της C-τελικής περιοχής του αυξητικού παράγοντα μετασχηματισμού TGF-β1 που δύναται να επάγουν την in vitro χονδρογένεση. Τα αποτελέσματα έδειξαν πως οι βραχύτερες πρωτεϊνικές επικράτειες της C-τελικής περιοχής του TGF-β1 επάγουν σε μεγαλύτερο βαθμό την in vitro χονδρογένεση στις τριδιάστατες καλλιέργειες σε σχέση με τις μονοδιάστατες, με τα καλύτερα αποτελέσματα να επιτυγχάνονται στην περίπτωση χρήσης του πεπτιδίου 8 της C-τελικής περιοχής του TGF-β1. Ταυτόχρονα, οι βραχύτερες πρωτεϊνικές επικράτειες της C-τελικής περιοχής του TGF-β1 μειώνουν την έκφραση του γονιδίου της αγκρεκανάσης ADAMTS-5, το οποίο ρυθμίζεται θετικά από την εξωγενή προσθήκη του TGF-β1, προάγοντας έτσι τη διαφοροποίηση των κυττάρων και τον σχηματισμό της εξωκυττάριας μήτρας. Στη προκειμένη περίπτωση τη χαμηλότερη έκφραση επέδειξε το πεπτίδιο 6 και αμέσως μετά το πεπτίδιο 8, υποδεικνύοντας ότι τα δύο μικρότερα πεπτίδια θα μπορούσαν να χρησιμοποιηθούν για την κατασκευή ενός βιοϋλικού που αποσκοπεί στην αναγέννηση του αρθρικού χόνδρου

Translational Framework Predicting Tumour Response in Gemcitabine-Treated Patients with Advanced Pancreatic and Ovarian Cancer from Xenograft Studies

The aim of this evaluation was to predict tumour response to gemcitabine in patients with advanced pancreas or ovarian cancer using pre-clinical data obtained from xenograft tumour-bearing mice. The approach consisted of building a translational model combining pre-clinical pharmacokinetic–pharmacodynamic (PKPD) models and parameters, with dosing paradigms used in the clinics along with clinical PK models to derive tumour profiles in humans driving overall survival. Tumour growth inhibition simulations were performed using drug effect parameters obtained from mice, system parameters obtained from mice after appropriate scaling, patient PK models for gemcitabine and carboplatin, and the standard dosing schedules given in the clinical scenario for both types of cancers. Tumour profiles in mice were scaled by body weight to their equivalent values in humans. As models for survival in humans showed that tumour size was the main driver of the hazard rate, it was possible to describe overall survival in pancreatic and ovarian cancer patients. Simulated tumour dynamics in pancreatic and ovarian cancer patients were evaluated using available data from clinical trials. Furthermore, calculated metrics showed values (maximal tumour regression [0–17%] and tumour size ratio at week 12 with respect to baseline [− 9, − 4.5]) in the range of those predicted with the clinical PKPD models. The model-informed Drug Discovery and Development paradigm has been successfully applied retrospectively to gemcitabine data, through a semi-mechanistic translational approach, describing the time course of the tumour response in patients from pre-clinical studies.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEpu

Aza-Reversine Promotes Reprogramming of Lung (MRC-5) and Differentiation of Mesenchymal Cells into Osteoblasts

Reversine or 2-(4-morpholinoanilino)-N6-cyclohexyladenine was originally identified as a small organic molecule that induces dedifferentiation of lineage-committed mouse myoblasts, C2C12, and redirects them into lipocytes or osteoblasts under lineage-specific conditions (LISCs). Further, it was proven that this small molecule can induce cell cycle arrest and apoptosis and thus selectively lead cancer cells to cell death. Further studies demonstrated that reversine, and more specifically the C2 position of the purine ring, can tolerate a wide range of substitutions without activity loss. In this study, a piperazine analog of reversine, also known as aza-reversine, and a biotinylated derivative of aza-reversine were synthesized, and their potential medical applications were investigated by transforming the endoderm originates fetal lung cells (MRC-5) into the mesoderm originated osteoblasts and by differentiating mesenchymal cells into osteoblasts. Moreover, the reprogramming capacity of aza-reversine and biotinylated aza-reversine was investigated against MRC-5 cells and mesenchymal cells after the immobilization on PMMA/HEMA polymeric surfaces. The results showed that both aza-reversine and the biofunctionalized, biotinylated analog induced the reprogramming of MRC-5 cells to a more primitive, pluripotent state and can further transform them into osteoblasts under osteogenic culture conditions. These molecules also induced the differentiation of dental and adipose mesenchymal cells to osteoblasts. Thus, the possibility to load a small molecule with useful “information” for delivering that into specific cell targets opens new therapeutic personalized applications

SpAD Biofunctionalized Cellulose Acetate Scaffolds Inhibit Staphylococcus aureus Adherence in a Coordinating Function with the von Willebrand A1 Domain (vWF A1)

Staphylococcus aureus is one of the major pathogens causing and spreading hospital acquired infections. Since it is highly resistant to new generation antibiotics, novel strategies have to be developed such as the construction of biofunctionalized non-adherent surfaces that will prevent its tethering and subsequent spread in the hospital environment. In this frame, the domain D of protein A (SpAD) of S. aureus has been immobilized onto cellulose acetate scaffolds by using the streptavidin/biotin interaction, in order to study its interaction with the A1 domain of von Willebrand factor (vWF A1), a protein essential for hemostasis, found in human plasma. Subsequently, the biofunctionalized cellulose acetate scaffolds were incubated with S. aureus in the presence and absence of vWF A1 at different time periods and their potential to inhibit S. aureus growth was studied with scanning electron microscopy (SEM). The SpAD biofunctionalized scaffolds perceptibly ameliorated the non-adherent properties of the material, and in particular, the interaction between SpAD and vWF A1 effectively inhibited the growth of S. aureus. Thus, the exhibition of significant non-adherent properties of scaffolds addresses their potential use for covering medical equipment, implants, and stents

Freeze-Drying Process for the Fabrication of Collagen-Based Sponges as Medical Devices in Biomedical Engineering

This paper presents a systematic review of a key sector of the much promising and rapidly evolving field of biomedical engineering, specifically on the fabrication of three-dimensional open, porous collagen-based medical devices, using the prominent freeze-drying process. Collagen and its derivatives are the most popular biopolymers in this field, as they constitute the main components of the extracellular matrix, and therefore exhibit desirable properties, such as biocompatibility and biodegradability, for in vivo applications. For this reason, freeze-dried collagen-based sponges with a wide variety of attributes can be produced and have already led to a wide range of successful commercial medical devices, chiefly for dental, orthopedic, hemostatic, and neuronal applications. However, collagen sponges display some vulnerabilities in other key properties, such as low mechanical strength and poor control of their internal architecture, and therefore many studies focus on the settlement of these defects, either by tampering with the steps of the freeze-drying process or by combining collagen with other additives. Furthermore, freeze drying is still considered a high-cost and time-consuming process that is often used in a non-optimized manner. By applying an interdisciplinary approach and combining advances in other technological fields, such as in statistical analysis, implementing the Design of Experiments, and Artificial Intelligence, the opportunity arises to further evolve this process in a sustainable and strategic manner, and optimize the resulting products as well as create new opportunities in this field