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

TISSUE REPAIR (3)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 19

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

TÁMOP-4.1.2-08/1/A-2009-0011

Heart failure• One of the most frequent conditions• Major cause of morbidity and mortality in

developed countries• Causes:

– Congenital malformations– Hypertension– Myocardial infarction– Toxic– Infectious

TÁMOP-4.1.2-08/1/A-2009-0011

Heart regenerative therapiesHeart regenerative therapies are in focus of investigation:• The occurence of heart failure (HF) is

increasing with age• Population of developed countries are

increasingly aged• Number of patients surviving myocardial

infarction (MI) is increasing• Most of them have chronic HF (CHF)

TÁMOP-4.1.2-08/1/A-2009-0011

Left ventricle assist device (LVAD)• Aids the pumping function

of the (left) ventricle• Pulsatile pumping or• Continous pumping• Longest bearing of an

implanted LVAD was 7 years

TÁMOP-4.1.2-08/1/A-2009-0011

Ventricular assist devicesIn targets of heart transplantation:• Bridges the time until a donor is found• In itself enhances the regeneration of the damaged heart

muscle• Improves life qualityIn patients not fitting for transplantation:• Palliative therapy• Improves life qualityComplications may involve:• Risk of infection• Risk of clotting disorders• Risk of embolization

TÁMOP-4.1.2-08/1/A-2009-0011

Bone marrow cells in cardiac repair

Blood vesselEndothelial

progenitor cells (hemangioblasts)

HeartSP cellsKit+ cells

Sca-1+ cells

Bone marrowMesenchymal stem

cellsHematopoietic stem

cellsSP cells

Skeletal muscleSatellite cells

SP cells

Fusion-dependent andfusion-independent

differentation

TÁMOP-4.1.2-08/1/A-2009-0011

Cellular therapies in cardiac repair I• Bone marrow cells (BMC)• Hemopoetic stem cells may contribute to

heart repair• Extensively studied in animal models with

variously labelled BMC• Sex-mismatched human heart transplant

patients• After injury, homing to the injured region can

be detected• GCSF mobilisation of BMC does not reproduce

the results with injection

TÁMOP-4.1.2-08/1/A-2009-0011

Cellular therapy of cardiac musclesIntravenous infusionSelective intracoronary infusionDirect intramyocardial injection

↓Cardiomyocyte apoptosisRecruitment of resident stem

cellsCardiomyocyte proliferation

Matrix:Scar

compositionGranulation

tissue

Pro-angiogenic cytokinesAngiogenic ligands

↑Cardiac performance

↑Number of functional

cardiomyocytes↑Perfusion

Secretion of paracrine factors

Differentiation to components of vascular wall

Differentiation to a cardiac phenotype

Fusion with resident

cardiomyocytesPerivascular incorporation

TÁMOP-4.1.2-08/1/A-2009-0011

Cellular therapies in cardiac repair II• No direct evidence of BMC

transdifferentiation to cardiomyocytes• If it occurs, it is a rare event• Maybe the obviously present benefit is the

increased vascularization of the injured heart muscle which enhances intrinsic regeneration capacity

TÁMOP-4.1.2-08/1/A-2009-0011

Cellular therapies in cardiac repair III• Evidence for dividing cardiomyocytes in the

human heart• Multyple types of proliferating cells in the

myocardium was observed bearing both SC markers (Sca-1, CD31) and cardiomyocyte markers upon triggered injury (5-azacytidine)

• Present in rodents and humans• Marked proliferative capacity

TÁMOP-4.1.2-08/1/A-2009-0011

Cellular therapy of cardiac muscle

Cardiomyocite• Single nuclei (central)• Gap junction (+)• Cx43 expression (+)

Myotube• Multinucleated• Gap junction (-)• Cx43 expression (-)

Skeletal muscle• Multinucleated (peripheral)• Gap junction (-)• Cx43 expression (-)

Myoblast (satellite cell)• Single nucleus• Gap junction (+)• Cx43 expression (+)• Proliferation (+)

Fusion and differentiation

???

TÁMOP-4.1.2-08/1/A-2009-0011

Skeletal myoblasts• Early studies used cultured SMBs from muscle

biopsies• Improvement of cardiac performance and life quality:

– Reduced NO consumption– Improvement in NYHA class– Better excercise tolerance

• Patients showed ventricular arhyithmias• Sometimes ICD use was necessary• However, the number of patients treated was low• No untreated control group was used in these studies

TÁMOP-4.1.2-08/1/A-2009-0011

Embryonic stem cells• Cardiogenic potential is assured• Injury repair: hESC needed to be differentiated

before application• Injury itself is not enough to trigger growth and

functional replacement, moreover, inflammatory citokines damage the grafted cells

• Anti-inflammatory treatment and protective agents needed for graft support (IGF-1, pan-caspase inhibitors and NO blockers)

• Differentiated cardiomyocytes trigger an immunoresponse in immunocompetent mice

• Problem: teratoma risk! Translation to the clinic is recently questionable

TÁMOP-4.1.2-08/1/A-2009-0011

Tissue engineering in tooth regeneration/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

TÁMOP-4.1.2-08/1/A-2009-0011

Tooth development• Reciprocal signaling events

between the epithelium and underlying mesenchyme

• Initiation, morphogenesis and terminal differentiation

1.Bud stage2.Epithelial cup (Encloses

the mesenchyme)3.Bell stage4.Crown stage

Dentin

Odontoblast

Root

Periodontalmembrane

Cementum

Enamel

Crown

Blood vessel

Sharpey fiber

Gingival fiberPulp

Alveolarbone

Neural fiber

TÁMOP-4.1.2-08/1/A-2009-0011

Dental pulp stem cells (DPSC)• DPSC are multipotent cells in the dental pulp• Regeneration of dentin after tooth injury• Odontoblasts emerge close to the site of

injury• Undifferentiated mesenchymal cells are

constantly migrating from deeper tooth layers to the dentin differentiating into odontoblasts

• Evidence suggest that these are DPSC

TÁMOP-4.1.2-08/1/A-2009-0011

Differentiation capacity of DPSC• Human DPSC cultured under mineralization-

enhancing conditions • Cells form odontoblast-like cells producing

dentin and expressing nestin• DPSCs phenotypically resembles to MSC but

its capacity to produce dentin is unique

TÁMOP-4.1.2-08/1/A-2009-0011

Bioengineered tooth conceptsScreening of

tooth-forming cells3D manipulation of

single cellsTransplantation of a

bioengineered tooth germ

Patient derivedstem cells

Epithelial cells

Mesenchymal cells

Transplantation

Bioengineered tooth,prepared by in vitro culture

Bioengineered toothgerm development

Bioengineeredtooth germ

TÁMOP-4.1.2-08/1/A-2009-0011

De novo tooth engineering IScaffold-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

TÁMOP-4.1.2-08/1/A-2009-0011

De novo tooth engineering IIReproduction 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

TISSUE REPAIR (4)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 20

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

TÁMOP-4.1.2-08/1/A-2009-0011

Major causes of urogenital injuriesInjuries or loss of function of the urogenital organs:• Congenital malformations• Trauma• Infection, inflammation• Iatrogenic injury

TÁMOP-4.1.2-08/1/A-2009-0011

Repair possibilities of the urogenital organsAutologous non-urogenital tissues• Skin• Gastrointestinal

segments• Mucosa from multiple

body sitesAllogen• Kidney graft for

transplantation (cadaver or living)

• Cadaver fascia

Xenogenic materials• Bovine collagenArteficial materials• Silicone• Polyurethane• Teflon

TÁMOP-4.1.2-08/1/A-2009-0011

Obtaining cells for tissue regeneration• Autologous or allogenic• End stage organ damage restricts cell

availability for tissue repair• In vitro culturing results are different

– In vitro cultured bladder SMC: lower contractility

• Low cell number may hinder possibilities• Stem cells can be the solution• Therapeutic cloning is also might be feasible

TÁMOP-4.1.2-08/1/A-2009-0011

Biomaterials for genitourinary reconstruction I• Arteficial materials• Replacement of ECM functions:

– Providing 3D structure of tissue formation– Regulation and stimulation of cell

differentiation via the storage and release of bioactive factors

– Injecting cells without scaffold support is not effective

TÁMOP-4.1.2-08/1/A-2009-0011

Biomaterials for genitourinary reconstruction IINaturally derived biomaterials:• Collagen• Alginate• Acellular tissue matrices:

– Bladder submucosa– Small intestinal submucosa (SIS)

Synthetic polymers:• PLA, PGA, PLGA

TÁMOP-4.1.2-08/1/A-2009-0011

Uroepithel – unique features• Excretion not absorption• Recent methods favor intestinal autografts

for urethra, ureter or bladder repair• The different structure and function of

uroepithel and intestinal epithel often lead to complications which may be severe

TÁMOP-4.1.2-08/1/A-2009-0011

Urethra reconstruction I 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

TÁMOP-4.1.2-08/1/A-2009-0011

Urethra reconstruction II 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

TÁMOP-4.1.2-08/1/A-2009-0011

Urethra reconstruction III Decellularized and tubularized matrices seeded with autologous urothelium:

• 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

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Bladder reconstruction IMost commonly intestinal-derived mucosal sheets are used for reconstruction:

• Intestinal epithelium is different from urothelium

• Designed to absorb and secrete mucus• Complications: infection, urolithiasis, metabolic

disorders, perforation, increased mucus production, malignancies

Because of disappointing results, attempts for alternative treatments are performed

TÁMOP-4.1.2-08/1/A-2009-0011

Bladder reconstruction IIAugmentation of bladder:• Progressive dilatation of native bladder tissue

in animal experiments• Augmentation cystoplasty in animals and

humans with dilated urethral segments• Better than the usage of GIT-derived

segments

TÁMOP-4.1.2-08/1/A-2009-0011

Bladder reconstruction IIINon-seeded acellular matrices:• Xenogenic SIS → decellularized collagen-based

tissue matrix → no musclular layer• Epithelization of the graft construct did occur• Non-compliance because of the lack of

muscularis layer

Matrices seeded with epithel and SMC:• Successful muscular layer formed, compliance is

fair• Scaffolds: combination of PGA and collagen

TÁMOP-4.1.2-08/1/A-2009-0011

Ureter reconstructionAnimal studies for urether reconstruction:• Non-seeded matrices facilitated the re-

growth of the urethral wall 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 urethral segment in dogs

• Cell seeded biodegradable scaffolds gave more satisfying results in dogs

TÁMOP-4.1.2-08/1/A-2009-0011

Kidney replacement therapyCurrently two options are available for the treatment of end-stage renal failure (ESRF):• Dialysis • Kidney transplantation

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

Kidney transplantation• 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

TÁMOP-4.1.2-08/1/A-2009-0011

Tissue engineered kidneyBioartificial 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

TÁMOP-4.1.2-08/1/A-2009-0011

Bioartificial kidney

Pump 370-80 ml/min

Ultrafiltratereservoir

Heatexchanger

Heatexchanger

Ultrafiltrate(into RAD luminal space)

Hem

ofilte

r

Pressure monitor

Post hemofilter blood(into RAD ECS)

Replacementfluid

RAD cartridge

Processedultrafiltrate

(urine)

Pump 25-7 ml/min

Pump 180 ml/min

5-10mm Hg

10-25 mm Hg

Venous blood

Post RADblood

Luminal spaceProximal tubule cells

Extracapillary spaceFiber wall

TÁMOP-4.1.2-08/1/A-2009-0011

Tissue engineered kidneyIn 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

TÁMOP-4.1.2-08/1/A-2009-0011

In vitro engineered murine kidney

Wolff duct

Metanephricmesenchyme

4-6 days

Bud

Cells

Cells

Bud