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 19
TISSUE REPAIR (3)
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
Bone marrow
Mesenchymal stem
cells
Hematopoietic
stem cells
SP cells
Blood vessel
Endothelial
progenitor cells
(hemangioblasts)
Skeletal muscle
Satellite cells
SP cells
Heart
SP cells
Kit+ cells
Sca-1+ 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
Fusion with
resident
cardiomyocytes
Differentiation
to a cardiac
phenotype
↓Cardiomyocyte apoptosis
Recruitment of resident
stem cells
Cardiomyocyte
proliferation
Secretion of
paracrine
factors
Matrix:
Scar
composition
Granulation
tissue
↑Number of
functional
cardiomyocyte
s
Differentiation
to components of
vascular wall
Pro-angiogenic cytokines
Angiogenic ligands
↑Perfusion
↑Cardiac
performance
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
Skeletal muscle
• Single nuclei (central)
• Gap junction (+)
• Cx43 expression (+)
• Multinucleated (peripher
• Gap junction (-)
• Cx43 expression (-)
???
Myotube
Myoblast (satellite
• Multinucleated
• Gap junction (-)
• Cx43 expression (-)
• 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
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, pancaspase inhibitors and NO blockers)
• Differentiated cardiomyocytes trigger an
immunoresponse in immunocompetent mice
• Problem: teratoma risk! Translation to the
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
Crown
Enamel
Cementum
Odontoblast
Pulp
Gingival fibe
Periodontal
membrane
Sharpey fiber
Root
1.Bud stage
2.Epithelial cup
(Encloses the
mesenchyme)
3.Bell stage
Dentin
Blood vessel
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
Epithelial cells
Patient derived
stem cells
Bioengineered
tooth germ
Mesenchymal cells
Bioengineered tooth
Bioengineered tooth,
germ development
prepared by in vitro culture
Transplantation
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
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 20
TISSUE REPAIR (4)
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 nonurogenital 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
Biomaterials for
genitourinary
reconstruction I
TÁMOP-4.1.2-08/1/A-2009-0011
• 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
Biomaterials for
genitourinary
reconstruction II
TÁMOP-4.1.2-08/1/A-2009-0011
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
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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
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
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 unsuccessful in dogs
• Non-seeded acellular matrices proved
to be un-successful to replace a 3cm
long urethral segment in dogs
• Cell seeded biodegradable scaffolds
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
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
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
Heat
exchanger
Pump 2
5-7 ml/min
Luminal space
Proximal tubule cells
5-10
mm Hg
Fiber wall
RAD cartridge
Ultrafiltrate
reservoir
Extracapillary space
Pressure monitor
10-25
mm Hg
Heat
exchanger
Ultrafiltrate
into RAD luminal space)
Hemofilter
Post hemofilter blood
(into RAD ECS)
Processed
ultrafiltrate
(urine)
Post RAD
blood
Replacement
fluid
Pump 3
70-80 ml/min
Pump 1
80 ml/min
Venous blood
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
Cells
Bud
Bud
Wolff duct
Cells
Metanephric
mesenchyme
4-6 days