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Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 13 CONTROLLED RELEASE TÁMOP-4.1.2-08/1/A-2009-0011 Controlled drug delivery from scaffolds • Drug release upon matrix degradation • Drug release upon diffusion • Long-term maintenance of effective local concentration • Localized effects ensured • Limited systemic effects TÁMOP-4.1.2-08/1/A-2009-0011 Ideal scaffold • 3-dimensional and well defined microstructure • Interconnected pore network • Mechanical properties similar to those of natural tissues • Biocompatible and bio-resorbable • Controllable degradation and resorption • Local sequestration and controlled delivery of specific bioactive factors • Thus enhancing and guideing the TÁMOP-4.1.2-08/1/A-2009-0011 ECM mimicry as a guide for scaffold design • ECM is the natural medium where cells proliferate, differentiate and migrate • ECM is a highly organized dynamic biomolecular environment where motifs governing cell behaviours are continuously generated and sequestered • Motifs are locally released according to cellular stimuli • Relase occurs on-demand upon degradation of the adhesion sites binding them to the ECM TÁMOP-4.1.2-08/1/A-2009-0011 Growth factors and the ECM • Growth factors (GFs) are locally stored by ECM • Storage in insoluble/latent forms • Specific binding with glycosaminoglycans (e.g. heparins) • Elicit biological activity once released • ECM binding provides concentration gradient important in morphogenesis TÁMOP-4.1.2-08/1/A-2009-0011 Mimic the function of ECM • Future generations of TE scaffolds need to have extended functionality and bioactivity • Synthetic bio-inspired ECM should broadcast specific cellular events • The ability of controlled release of multiple bioactive molecules will allow the control of cellular behaviour and successful regeneration TÁMOP-4.1.2-08/1/A-2009-0011 Interspersed signals • Hydrogels (either natural or synthetic) have been succesfully used for controlled release of bioactive protein compounds • Molecules were simply mixed with the polymer and were entrapped upon gelation • Natural (collagen, fibrin, hyaluronan) and synthetic (PEG-based, peptide-based) hydrogels have been used • Release characteristic may modulated with crosslinking agents • Solid-state scaffolds: fabrication TÁMOP-4.1.2-08/1/A-2009-0011 Immobilized signals • Modification of polymer scaffolds to interact with signaling molecules: immobilization • Prolonged diffusion out of the scaffold platform • Reversible or irreversible binding to the polymer. • Released upon degradation of a linking tether or the matrix itself • Determinants of the amount of bound signal and release profile: – The number of binding sites – Affinity of the signal for sites TÁMOP-4.1.2-08/1/A-2009-0011 Signal delivery from cells • Inclusion of nucleic acids (NA) encoding the desired protein • NA are introduced into target cells, which then produce the desired proteins • Antisense oligos can be used to return abnormal gene expression to a certain state • Synthetic polymers containing adhesion sites (RGD) proved to be more effective in delivering the plasmid TÁMOP-4.1.2-08/1/A-2009-0011 Protein delivery systems (DS) in TE • DS must prevent the protein from inactivation or degradation • Fine-tuning of the release rate can be achieved by modulating the composition, shape, and architecture of the platform • Continous and pulsatile delivery • Biodegradable and non-degradable platforms TÁMOP-4.1.2-08/1/A-2009-0011 Non-biodegradable systems Ethylene-vinyl acetate copolymers (EVAc) and silicones: • Mass transport through polymer chains or pores is the only rate-limiting step • Possible application in cell encapsulation preventing them to interact with the immune system Time TÁMOP-4.1.2-08/1/A-2009-0011 Biodegradable systems • PLGA is a very versatile and widely used system • Poly-ortho esters are newly in the centre of interest (no heating or solvents, injectable polymers) • Polyanhydrides usually undergo surface erosion which has a favorable kinetics Time TÁMOP-4.1.2-08/1/A-2009-0011 Controlled release profiles in biodegradable systems Amount of drug released Protein or small molecule drug Corresponding rate dc(t)/dt Release rate t t Typical release profile Corresponding rate Release rate t Toxic dose ceff(t) Bulk erosion Typical release profile dc(t)/dt Protein or small molecule drug Amount of drug released Surface erosion t t TÁMOP-4.1.2-08/1/A-2009-0011 On-off drug delivery systems • Pulsatile mode of protein and peptide release • Rapid and transient release of a certain amount of drug molecules within a short time-period immediately after a predetermined off-release interval • Classified into “programmed” and “triggered” delivery systems (DS): – Programmed-DS: the release is governed by the inner mechanism of the device – Triggered-DS: release is governed by changes in the physiologic environment of the device or by external stimuli TÁMOP-4.1.2-08/1/A-2009-0011 Programmed and triggered delivery systems • Synthetic polymers can be engineered to be applicable in programmed delivery • Both surface and bulk-eroding systems may be used • Biggest interest in triggered delivery is the glucose-sensitive insulin delivery • The “intelligent” system consists of immobilized glucose oxidase in a pHresponsive polymeric hydrogel • In the gel, insulin is enclosed • Upon glucose diffusion into the hydrogel, glucose oxidase converts it into gluconic acid TÁMOP-4.1.2-08/1/A-2009-0011 Inclusion of drug molecules into scaffolds Poly-methyl-methacrylate (PMMA) beads with antibiotics (mostly aminoglycosides): • Orthopedic and trauma surgery • Treatment of chronic osteomyelitis and/or ulcers • Bones and joints are „blind spots” of systemic antibiotic therapy because the limited blood supply • PMMA beads release antibiotics gradually • High local antibiotic concentration can be achieved • Limited systemic side effects TÁMOP-4.1.2-08/1/A-2009-0011 Inclusion of bioactive proteins into scaffolds VEGF role in tissue vascularization: • • • • • Cells in hypoxic tissues secrete VEGF Endothelial cells express VEGFR Stimulates endothel proliferation Directs endothelial cell migration Tissue vascularization is critical in nutrition and oxigenization of implanted TE constructs • Controlled VEGF delivery is in the focus of TE research TÁMOP-4.1.2-08/1/A-2009-0011 VEGF supports TE tissue vascularization Controlled VEGF delivery microparticles: from alginate • Bivalent cations mediate alginate crosslinking • VEGF encapsulation efficiency and delivery ratio depends on the cation species (Ca2+ or Zn2+) • Zn2+-crosslinked particles proved to be more toxic than Zn2+ • Mixture of Ca2+ and Zn2+ beads are the most favorable Support of tissue differentiation with bioactive proteins TÁMOP-4.1.2-08/1/A-2009-0011 BMP-2: • Key role in regulating osteoblast differentiation • Recombinant hBMP-2 is dissolved in aquaeous solution of polyethyleneoxide (PEO) • rhBMP-2 solution is then added to scaffold material • Scaffold materials include silk fibroin, PCLA, PEG, PLGA, collagen, etc. Experimental results with controlled drug delivery scaffolds – VEGF TÁMOP-4.1.2-08/1/A-2009-0011 • Half-life of VEGF is 50 min, therefore controlled release is critical • Controlled release is based on electrostatic attractions between the carrier (acidic gelatine, IEP=5.0) and VEGF (IEP=8.6) • Extent of gelatin cross-linking also influences release • Up to 90% of total VEGF vas released within 30 days from sc. implants, 80% within the first 5 days. Clinical results with controlled drug delivery scaffolds – BMP-2 TÁMOP-4.1.2-08/1/A-2009-0011 • Use of BMP-2 filled collagen sponges in spinal degenerative diseases to enhance post-operative bone fusion. • BMP-2 treated patients regain the ability to self-care and mobility faster, their pain scores are significantly lower. • Their mood and emotional control is also significantly better than that of control patients. Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 14 BIOSENSORS TÁMOP-4.1.2-08/1/A-2009-0011 Definition Biosensor is a device that transforms or detects a biological signal and transforms into a more easily detectable one. TÁMOP-4.1.2-08/1/A-2009-0011 Concept of an implantable glucose sensor Type I Detector (potentially a mobile phone) Signal Glucose sensor Implantable potentiostat Type II Glucose sensor Insulin release Signal Signal Insulin container TÁMOP-4.1.2-08/1/A-2009-0011 Dexamethasone-loaded PLGA Microspheres 10m TÁMOP-4.1.2-08/1/A-2009-0011 Model of biosensor-tissue interactions Biosensor Interphase Tissue Hydrogels + PEO RBC Sensor Angiogenesis WBC Endothel cell Microsphere for drug (TRM) release Angiogenic factor or other tissue response modifiers Soluble proteins Fibrin Collagen TÁMOP-4.1.2-08/1/A-2009-0011 The “intelligent” system • Consists of immobilized glucose oxidase in a pH-responsive polymeric hydrogel, enclosing a saturated insulin solution. • As glucose diffuses into the hydrogel, glucose oxidase catalyzes its conversion to gluconic acid, thereby lowering the pH in the microenvironment of the membrane. • Low pH causes swelling and insulin release. TÁMOP-4.1.2-08/1/A-2009-0011 Development of reliable glucose biosensors require 1. Novel electrodes are required to decrease invasiveness of the implantable glucose biosensor 2. Bioactive coatings are necessary to enhance the in vivo life of the implantable glucose sensor 3. Biosensor coating using electrospinning nanofibres need to be developed 4. Tissue responses are needed to be studied further to optimize tissue responses to biosensor signals 5. Angiogenesis around the glucose sensor need to be increased to enhance detection