Manifestation of Novel Social Challenges in Teaching Material of Medical Biotechnology Master’s Programmes in Hungary

1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 13 Controlledrelease

3. Controlleddrugdeliveryfromscaffolds • Drug release upon matrix degradation • Drug release upon diffusion • Long-term maintenanceof effective local concentration • Localized effects ensured • Limited systemic effects

4. Idealscaffold • 3-dimensional and well defined microstructure • Interconnected pore network • Mechanical properties similar tothose of natural tissues • Biocompatible and bio-resorbable • Controllable degradation and resorption • Local sequestration and controlleddelivery ofspecific bioactive factors • Thusenhancing and guideing the regenerationprocess

5. ECM mimicryas a guideforscaffold design • ECM is the natural medium wherecells proliferate, differentiateand migrate • ECM is a highly organized dynamic biomolecular environmentwheremotifsgoverning cell behavioursare continuously generated and sequestered • Motifs are locally released according tocellular stimuli • Relaseoccurson-demandupondegradation of theadhesion sites binding them to the ECM

6. Growthfactors and the ECM • Growthfactors (GFs) are locally storedby ECM • Storage ininsoluble/latent forms • Specific binding with glycosaminoglycans (e.g. heparins) • Elicit biological activity once released • ECM bindingprovidesconcentrationgradientimportantinmorphogenesis

7. Mimicthefunction of ECM • Future generations of TE scaffolds needtohaveextended functionalityand bioactivity • Synthetic bio-inspiredECM should broadcast specific cellular events • The ability of controlledreleaseofmultiplebioactivemoleculeswillallowthecontrolofcellularbehaviour and successfulregeneration

8. Interspersedsignals • Hydrogels (eithernaturalorsynthetic) havebeensuccesfullyusedforcontrolledrelease of bioactive protein compounds • Moleculesweresimply mixed withthepolymer and wereentrappedupongelation • Natural (collagen, fibrin, hyaluronan) and synthetic (PEG-based, peptide-based) hydrogelshavebeenused • Releasecharacteristicmaymodulatedwithcrosslinkingagents • Solid-statescaffolds: fabricationmethod must be mild (toavoid protein denaturation)

9. Immobilizedsignals • Modification of polymer scaffolds to interact with signaling molecules: immobilization • Prolongeddiffusionout of the scaffoldplatform • Reversible orirreversible binding to the polymer. • Released upon degradation of alinking 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 • Degradation rate ofthe scaffold

10. Signaldeliveryfromcells • Inclusion of nucleicacids (NA) encodingthedesired protein • NA are introduced into targetcells, which thenproduce the desired proteins • Antisenseoligoscan be used to return abnormalgene expression to a certain state • Syntheticpolymerscontainingadhesionsites (RGD) provedto be more effectiveindeliveringtheplasmid

11. Protein deliverysystems(DS) in TE • DS must preventthe protein frominactivationordegradation • Fine-tuning of thereleaseratecan be achievedbymodulatingthecomposition, shape, and architecture of the platform • Continous and pulsatiledelivery • Biodegradable and non-degradableplatforms

12. Non-biodegradablesystems • Ethylene-vinylacetatecopolymers (EVAc) andsilicones: • Mass transport through polymer chains or poresis the only rate-limiting step • Possibleapplicationincellencapsulationpreventingthemtointeractwiththeimmunesystem Time

13. Biodegradablesystems • PLGAis a veryversatile and widelyusedsystem • Poly-orthoestersarenewlyinthe centre of interest (no heatingorsolvents, injectablepolymers) • Polyanhydridesusuallyundergosurfaceerosionwhich has a favorablekinetics Time

14. Controlledreleaseprofilesinbiodegradablesystems Surfaceerosion Typicalrelease profile Corresponding rate Protein orsmall moleculedrug Releaserate Amount of drugreleased dc(t)/dt t t Bulkerosion Typicalrelease profile Corresponding rate Protein orsmall moleculedrug Releaserate Toxicdose Amount of drugreleased dc(t)/dt ceff(t) t t t

15. On-offdrugdeliverysystems • Pulsatile mode of protein and peptide release • Rapid and transient release of acertain amount of drug molecules within a short time-periodimmediately after a pre-determined off-release interval • Classified into “programmed” and “triggered” deliverysystems (DS): • Programmed-DS:the release is governed by theinner mechanism of the device • Triggered-DS:release isgoverned by changes in the physiologic environment of thedevice orby external stimuli • External stimuliinvolvetemperature changes,electric or magnetic fields, ultrasounds orirradiation

16. Programmed and triggereddeliverysystems • Syntheticpolymerscan be engineeredtobeapplicableinprogrammeddelivery • Both surface and bulk-erodingsystemsmay be used • Biggest interest intriggereddelivery is theglucose-sensitiveinsulindelivery • The “intelligent” system consists of immobilized glucose oxidasein a pH-responsive polymeric hydrogel • Inthegel, insulin is enclosed • Uponglucose diffusion intothe hydrogel, glucoseoxidaseconvertsitinto gluconic acid • Loweringof the pH resultsingelswelling and insulin release

17. Inclusion of drugmoleculesintoscaffolds • 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

18. Inclusion of bioactiveproteinsintoscaffolds • VEGF roleintissuevascularization: • Cellsinhypoxictissuessecrete VEGF • Endothelialcellsexpress VEGFR • Stimulatesendothelproliferation • Directsendothelialcellmigration • Tissuevascularization is criticalinnutrition and oxigenization of implanted TE constructs • Controlled VEGF delivery is inthefocus of TE research

19. VEGF supports TE tissuevascularization • Controlled VEGFdeliveryfromalginatemicroparticles: • Bivalentcationsmediatealginatecrosslinking • VEGF encapsulationefficiency and delivery ratio dependsonthecation species (Ca2+or Zn2+) • Zn2+-crosslinkedparticlesprovedto be more toxicthan Zn2+ • Mixture of Ca2+ and Zn2+beadsarethe most favorable

20. Support of tissue differentiation with bioactive proteins • BMP-2: • Key role in regulating osteoblast differentiation • Recombinant hBMP-2 is dissolved in aquaeous solution of polyethylene-oxide (PEO) • rhBMP-2 solution is then added to scaffold material • Scaffold materials include silk fibroin, PCLA, PEG, PLGA, collagen, etc.

21. Experimentalresults with controlled drug delivery scaffolds – VEGF • 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 • Upto 90% of total VEGF vas releasedwithin 30 daysfromsc. implants, 80% withinthefirst 5 days.

22. Clinical results with controlled drug delivery scaffolds – BMP-2 • Use of BMP-2 filled collagen sponges in spinal degenerative diseases to enhance post-operative bone fusion. • BMP-2 treated patientsregain 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.

23. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 14 Biosensors

24. Definition • Biosensor is a devicethattransformsordetectsabiologicalsignal and transformsinto a more easilydetectableone.

25. Concept of an implantableglucosesensor TypeI TypeII Detector (potentially a mobile phone) Glucosesensor Insulinrelease Signal Signal Signal Glucosesensor Implantablepotentiostat Insulincontainer

26. Dexamethasone-loaded PLGA Microspheres 10m

27. Model of biosensor-tissueinteractions Biosensor Interphase Tissue Hydrogels + PEO RBC Sensor Angiogenesis WBC Endothel cell Microsphere fordrug (TRM) release Angiogenicfactororothertissueresponsemodifiers Solubleproteins Fibrin Collagen

28. 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 pHcauses swelling and insulin release.

29. Development of reliableglucosebiosensorsrequire • Novel electrodes are required to decrease invasiveness of the implantable glucose biosensor • Bioactive coatings are necessary to enhance the in vivo life of the implantable glucose sensor • Biosensor coating using electrospinningnanofibres need to be developed • Tissue responses are needed to be studied further to optimize tissue responses to biosensor signals • Angiogenesis around the glucose sensor need to be increased to enhance detection potential of glucose levels and • Finally, novel biostable 3D porous collagen scaffolds need to be developed for tissue compatible biosensors