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
at the University of Pécs and at the University of Debrecen
Identification number: 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
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)
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
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
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
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
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
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
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
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
???
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
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
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
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
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
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
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
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
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
at the University of Pécs and at the University of Debrecen
Identification number: 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
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
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
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
Biomaterials for genitourinary reconstruction II
Naturally derived biomaterials:
• Collagen
• Alginate
• Acellular tissue matrices:
– Bladder submucosa
– Small intestinal submucosa (SIS) Synthetic polymers:
• PLA, PGA, PLGA
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
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
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
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
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
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
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
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
Kidney replacement therapy
Currently two options are available for the treatment of end-stage renal failure (ESRF):
• Dialysis
• Kidney transplantation
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
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
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
Bioartificial kidney
Pump 3 70-80 ml/min Ultrafiltrate
reservoir
Heat exchanger
Heat exchanger
Ultrafiltrate (into RAD luminal space)
Hemofilter
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
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
In vitro engineered murine kidney
Wolff duct
Metanephric mesenchyme 4-6 days
Bud Cells
Cells Bud
