Tissue repair (3)

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 19 Tissuerepair(3)

3. Heartfailure • One of the most frequentconditions • Major cause of morbidityand mortalityindevelopedcountries • Causes: • Congenitalmalformations • Hypertension • Myocardialinfarction • Toxic • Infectious

4. Heartregenerativetherapies • Heartregenerativetherapiesareinfocus of investigation: • The occurence of heartfailure (HF) is increasingwithage • Population of developedcountriesareincreasinglyaged • Number of patientssurvivingmyocardialinfarction (MI) is increasing • Most of themhavechronic HF (CHF)

5. Leftventricleassistdevice(LVAD) Aidsthepumpingfunction of the (left) ventricle Pulsatilepumpingor Continouspumping Longestbearing of an implanted LVAD was 7 years

6. Ventricularassistdevices • Intargets of hearttransplantation: • Bridgesthetimeuntil a donor is found • Initselfenhancestheregeneration of thedamagedheartmuscle • Improves life quality • Inpatientsnotfittingfortransplantation: • Palliativetherapy • Improves life quality • Complicationsmayinvolve: • Risk of infection • Risk of clottingdisorders • Risk of embolization

7. Bonemarrow Mesenchymalstemcells Hematopoieticstemcells SP cells Bonemarrowcellsincardiacrepair Skeletalmuscle Satellitecells SP cells Bloodvessel Endothelialprogenitorcells (hemangioblasts) Heart SP cells Kit+cells Sca-1+cells Fusion-dependent and fusion-independent differentation

8. CellulartherapiesincardiacrepairI Bonemarrowcells (BMC) Hemopoeticstemcellsmaycontributetoheartrepair Extensivelystudiedinanimalmodelswithvariouslylabelled BMC Sex-mismatched human hearttransplantpatients Afterinjury, homingtotheinjuredregioncan be detected GCSF mobilisationof BMC doesnotreproducetheresultswithinjection

9. Cellulartherapy of cardiacmuscles Intravenousinfusion Selectiveintracoronaryinfusion Directintramyocardialinjection Fusionwithresidentcardiomyocytes Differentiationto a cardiacphenotype Secretion of paracrinefactors Differentiationtocomponents of vascularwall Perivascularincorporation ↓Cardiomyocyteapoptosis Recruitment of residentstemcells Cardiomyocyteproliferation Matrix: Scarcomposition Granulationtissue Pro-angiogeniccytokines Angiogenicligands ↑Number of functionalcardiomyocytes ↑Perfusion ↑Cardiac performance

10. CellulartherapiesincardiacrepairII No directevidence of BMC transdifferentiationtocardiomyocytes Ifitoccurs, it is a rareevent Maybe theobviouslypresentbenefit is theincreasedvascularization of theinjuredheartmusclewhichenhancesintrinsicregenerationcapacity

11. CellulartherapiesincardiacrepairIII Evidencefordividingcardiomyocytesinthe human heart Multypletypes of proliferatingcellsinthemyocardiumwasobservedbearingboth SC markers (Sca-1, CD31) and cardiomyocytemarkersupontriggeredinjury (5-azacytidine) Presentinrodents and humans Markedproliferativecapacity

12. Cellulartherapy of cardiacmuscle • Cardiomyocite • Singlenuclei (central) • Gapjunction (+) • Cx43 expression (+) • Skeletalmuscle • Multinucleated (peripheral) • Gapjunction (-) • Cx43 expression (-) ??? • Myotube • Multinucleated • Gapjunction (-) • Cx43 expression (-) • Myoblast (satellitecell) • Singlenucleus • Gapjunction (+) • Cx43 expression (+) • Proliferation (+) Fusion and differentiation

13. Skeletalmyoblasts EarlystudiesusedculturedSMBsfrommusclebiopsies Improvement of cardiac performance and life quality: Reduced NO consumption Improvementin NYHA class Betterexcercisetolerance Patientsshowedventriculararhyithmias Sometimes ICD usewasnecessary However, thenumber of patientstreatedwaslow No untreatedcontrolgroupwasusedinthesestudies

14. Embryonicstemcells • Cardiogenicpotential is assured • Injuryrepair: hESCneededto be differentiatedbeforeapplication • Injuryitself is notenoughtotriggergrowth and functionalreplacement, moreover, inflammatorycitokinesdamagethegraftedcells • Anti-inflammatorytreatment and protectiveagentsneededforgraftsupport (IGF-1, pan-caspaseinhibitors and NO blockers) • Differentiatedcardiomyocytestrigger an immunoresponseinimmunocompetentmice • Problem: teratomarisk! Translationtotheclinic is recentlyquestionable

15. Tissue engineering in toothregeneration/replacement Dentition is important for feeding in vertebrates Aberrations in dentition or poor dental care is not life-threatening in developed countries But damage and loss of teeth may substantially affect quality of life

16. Tooth development Reciprocal signaling events between the epithelium and underlying mesenchyme Initiation, morphogenesis and terminal differentiation Bud stage Epithelial cup (Encloses the mesenchyme) Bell stage Crownstage Crown Dentin Odontoblast Enamel Pulp Cementum Gingivalfiber Periodontal membrane Sharpeyfiber Alveolar bone Root Bloodvessel Neuralfiber

17. Dentalpulpstemcells(DPSC) DPSC aremultipotentcellsinthedentalpulp Regeneration of dentin aftertoothinjury Odontoblastsemergeclosetothe site of injury Undifferentiatedmesenchymalcellsareconstantlymigratingfromdeepertoothlayerstothe dentin differentiatingintoodontoblasts Evidencesuggestthattheseare DPSC

18. Differentiationcapacity of DPSC Human DPSC culturedundermineralization-enhancingconditions Cellsformodontoblast-likecellsproducing dentin and expressingnestin DPSCsphenotypicallyresemblesto MSC butitscapacitytoproduce dentin is unique

19. Bioengineered tooth concepts Screening of tooth-forming cells 3D manipulation of single cells Transplantation of a bioengineered tooth germ Epithelial cells Bioengineered tooth germ Patient derived stem cells Mesenchymal cells Bioengineered tooth germ development Bioengineered tooth, prepared by in vitro culture Transplantation

20. De novo tooth engineeringI Scaffold-based roots: Bio-artificial root implant that supports an artificial (porcelain) crown Cells grow inside the scaffold thus serving as a proper anchor Animal (porcine) model proved the applicability of this solution

21. De novo tooth engineeringII Reproduction of embryonic tooth germs: Fully functional tooth by reproducing the embryonic tooth development Both roots and crown are formed Rodent experiments were successful Not only embryonic or newborn cells but also adult cells were able to recreate tooth Both scaffold and scaffoldless experiments

22. 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 20 Tissuerepair(4)

23. Major causes of urogenitalinjuries Injuries or loss of function of the urogenital organs: Congenital malformations Trauma Infection, inflammation Iatrogenic injury

24. Repairpossibilities of theurogenitalorgans • Autologousnon-urogenitaltissues • Skin • Gastrointestinalsegments • Mucosafrommultiple body sites • Allogen • Kidneygraftfortransplantation (cadaverorliving) • Cadaverfascia • Xenogenicmaterials • Bovinecollagen • Arteficialmaterials • Silicone • Polyurethane • Teflon

25. Obtainingcellsfortissueregeneration Autologousorallogenic End stageorgandamagerestrictscellavailabilityfortissuerepair In vitro culturingresultsaredifferent In vitro culturedbladder SMC: lowercontractility Lowcellnumbermayhinderpossibilities Stemcellscan be thesolution Therapeuticcloning is alsomight be feasible

26. BiomaterialsforgenitourinaryreconstructionI Arteficialmaterials Replacement of ECM functions: Providing 3D structure of tissueformation Regulation and stimulation of celldifferentiationviathestorage and release of bioactivefactors Injectingcellswithoutscaffoldsupport is noteffective

27. BiomaterialsforgenitourinaryreconstructionII Naturallyderivedbiomaterials: Collagen Alginate Acellulartissuematrices: Bladdersubmucosa Smallintestinalsubmucosa (SIS) Syntheticpolymers: PLA, PGA, PLGA

28. Uroepithel – uniquefeatures Excretionnotabsorption Recentmethodsfavorintestinalautograftsforurethra, ureterorbladderrepair The differentstructure and function of uroepithel and intestinalepitheloften lead tocomplicationswhichmay be severe

29. Urethra reconstructionI Strictures, injuries, trauma, congenital abnormalities (hypospadiasis) Most often, buccal mucosa grafts are used for reconstruction: Graft tissue is taken from the inner surface of the cheek or lips The epithelium is thick and the submucosa is highly vascular This graft is resistant for infections

30. UrethrareconstructionII Bladder-derived urothelium: Suitable for reconstruction in rabbits No human tests have been conducted Decellularized collagen matrices: The material is available on-demand Good results in „only” reconstructive surgery Results in strictures when tubularized reconstruction is needed

31. UrethrareconstructionIII Decellularized and tubularized matrices seeded with autologousurothelium: Good results in animal models Constructs seeded with cells developed similar histological structure to that of uroepithelium Collagen matrices without cell seeding resulted in strictures

32. BladderreconstructionI Most commonlyintestinal-derivedmucosalsheetsareusedforreconstruction: Intestinalepithelium is differentfromurothelium Designedtoabsorb and secrete mucus Complications: infection, urolithiasis, metabolicdisorders, perforation, increased mucus production, malignancies Because of disappointingresults, attemptsforalternativetreatmentsareperformed

33. BladderreconstructionII Augmentation of bladder: Progressive dilatation of native bladder tissue in animal experiments Augmentation cystoplasty inanimals and humans with dilated urethral segments Better than the usage of GIT-derived segments

34. BladderreconstructionIII Non-seededacellularmatrices: Xenogenic SIS → decellularizedcollagen-basedtissuematrix→ no musclularlayer Epithelization of thegraftconstructdidoccur Non-compliancebecause of thelackofmuscularislayer Matricesseededwithepithel and SMC: Successfulmuscularlayerformed, compliance is fair Scaffolds: combination of PGA and collagen

35. Ureterreconstruction Animal studies for uretherreconstruction: • Non-seeded matrices facilitated the re-growth of the urethralwall components in rats • Stiff tubes like teflon were un-successful in dogs • Non-seeded acellular matrices proved to be un-successful to replace a 3cm long urethralsegment in dogs • Cell seeded biodegradable scaffolds gave more satisfying results in dogs

36. Kidneyreplacementtherapy Currently two options are available for the treatment of end-stage renal failure (ESRF): Dialysis Kidney transplantation

37. Dialysis • Hemodialysis, hemofiltration • Extracorporeal dialyzer unit: hollow fiber dialyzers are most commonly used • Anticoagulated venous blood is let through the dialyzer, countercurrent of dialysis solution is applied • Peritoneal dialysis • Dialysis solution is applied in the peritoneal cavity • Toxic metabolites and excessive water are removed from the patient via osmotic differences between the blood and dialysis solution • Cardiovascular, metabolic and musculoskeletal complications are frequent

38. Kidneytransplantation Most often transplanted parenchymal organ Cadaver or live donor Offers an improvement in the life quality of dialyzed patients Implantation of allogenic grafts needs immunosuppressive treatment Side effects of immunosuppressive agents involve increased risk of infections and malignancies, kidney and hepatotoxicity, cardiovascular and metabolic side effects

39. Tissue engineered kidney • Bioartificial approach: • Replace dialysis machines with bioartificial kidney • Extracorporeal devices/intracorporeal devices • Preclinical trials on dogs with porcine TE renal tubules: successful BUN and K control • However, the patient is still tied to an extracorporeal machine

40. Bioartificialkidney Heat exchanger Pump 2 5-7 ml/min Luminalspace Proximaltubulecells 5-10 mm Hg Fiberwall RAD cartridge Extracapillaryspace Ultrafiltrate reservoir Pressure monitor Heat exchanger 10-25 mm Hg Processed ultrafiltrate (urine) Post hemofilterblood (into RAD ECS) Post RAD blood Ultrafiltrate (into RAD luminalspace) Replacement fluid Hemofilter Pump 3 70-80 ml/min Pump 1 80 ml/min Venousblood

41. Tissueengineeredkidney • In vivo approach: • Human kidney cells were seeded onto a polycarbonate tubular construct • Upon implantation in nude mice the construct was extensively vascularized • Urine-like fluid production: urea and creatinine content • Epithelial cells showed signs of tubular differentiation

42. In vitro engineered murine kidney Cells Bud Bud Wolff duct Cells Metanephric mesenchyme 4-6 days