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

TISSUE REPAIR (3)

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue

engineering – Lecture 19

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

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 therapies

Heart 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 devices

In targets of heart transplantation:

• Bridges the time until a donor is found

• In itself enhances the regeneration of the damaged heart muscle

• Improves life quality

In patients not fitting for transplantation:

• Palliative therapy

• Improves life quality

Complications 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 vessel

Endothelial progenitor

cells (hemangioblasts)

Heart

SP cells

Kit+ cells

Sca-1+ cells

Bone marrow

Mesenchymal stem

cells

Hematopoietic stem

cells

SP cells

Skeletal muscle

Satellite cells

SP cells

Fusion-dependent and

fusion-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 muscles

Intravenous infusion

Selective intracoronary infusion

Direct intramyocardial injection

↓Cardiomyocyte apoptosis

Recruitment of resident stem cells

Cardiomyocyte proliferation

Matrix:

Scar composition

Granulation tissue

Pro-angiogenic cytokines

Angiogenic 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

cardiomyocytes

Perivascular

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 stage

2.Epithelial cup (Encloses the

mesenchyme)

3.Bell stage

4.Crown stage

Dentin

Odontoblast

Root

Periodontal

membrane

Cementum

Enamel

Crown

Blood vessel

Sharpey fiber

Gingival fiber

Pulp

Alveolar

bone

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 concepts

Screening of

tooth-forming cells

3D manipulation of

single cells

Transplantation of a

bioengineered tooth germ

Patient derived

stem cells

Epithelial cells

Mesenchymal cells

Transplantation

Bioengineered tooth,

prepared by in vitro culture

Bioengineered tooth

germ development

Bioengineered

tooth germ

TÁMOP-4.1.2-08/1/A-2009-0011

De novo tooth engineering I

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

TÁMOP-4.1.2-08/1/A-2009-0011

De novo tooth engineering II

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

TISSUE REPAIR (4)

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue

engineering – Lecture 20

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

TÁMOP-4.1.2-08/1/A-2009-0011

Major causes of urogenital injuries

Injuries 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

organs

Autologous non-urogenital

tissues

• Skin

• Gastrointestinal segments

• Mucosa from multiple

body sites

Allogen

• Kidney graft for

transplantation (cadaver

or living)

• Cadaver fascia

Xenogenic materials

• Bovine collagen

Arteficial 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 II

Naturally 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

TÁMOP-4.1.2-08/1/A-2009-0011

Bladder reconstruction I

Most 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 II

Augmentation 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 III

Non-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 reconstruction

Animal 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 therapy

Currently 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 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

TÁMOP-4.1.2-08/1/A-2009-0011

Bioartificial kidney

Pump 3

70-80 ml/min

Ultrafiltrate

reservoir

Heat

exchanger

Heat

exchanger

Ultrafiltrate

(into RAD luminal space)

Hem

ofilter

Pressure monitor

Post hemofilter blood

(into RAD ECS)

Replacement

fluid

RAD cartridge

Processed

ultrafiltrate

(urine)

Pump 2

5-7 ml/min

Pump 1

80 ml/min

5-10

mm Hg

10-25

mm Hg

Venous blood

Post RAD

blood

Luminal space

Proximal tubule cells

Extracapillary space

Fiber wall

TÁMOP-4.1.2-08/1/A-2009-0011

Tissue engineered kidney

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

TÁMOP-4.1.2-08/1/A-2009-0011

In vitro engineered murine kidney

Wolff duct

Metanephric

mesenchyme

4-6 days

Bud

Cells

Cells

Bud