TISSUE ENGINEERING & REGENERATION

- bone can undergo effective regeneration as long as the defect is not too extensive.
- Cartilage – little or no propensity for regeneration.
- Diseases which can be potentially treated with stem cells –
o Skin – burns and skin defects
o Cardiac Muscle – Heart failure
o Heart Valves – congenital and acquired
o Cartilage – degenerative and traumatic joint disorders
o Trachea and Bronchus – congenital and acquired stenosis and resection for malignancy
o Bladder – congenital bladder malformation, cystectomy
o Anal/Bladder sphincter – Incontinence
o Pancreatic Islets – DM1
o Large Blood vessels – Atheromatous, Aneurysmal or Traumatic arterial disease.
o Esophagus – Benign stenosis, resection for malignancy
o Small intestine – Intestinal failure after surgical resection, for Crohn’s disease, cancer or
ischemia.
o Spinal cord injury and neurodegenerative conditions
o Degenerative retinal conditions.

Areas underpinning the science –

1. Stem cell biology
2. Cell Signalling biology
3. Scaffold Design
4. Bioreactor Design

Bioreactors – provide physical environment for growth of engineered tissues in vitro.

Areas where regenerative therapies have already been introduced – damaged cartilage.

SOURCE OF CELLS:
Somatic Cells SSC hESC Fetal Cells iPSC
Ease of availability Limited Good Moderate Moderate Good
Expansion in vitro Limited Good Excellent Good Excellent
Pluripotency No Limited Excellent Limited Excellent
Ethical concerns No No Yes Yes Yes
Risk of Malignancy None Low Moderate Moderate Moderate
Autologous Yes Yes No No Yes
Future Limited High Limited Limited High
SSC – somatic stem cells, hESC – Human Embryonic Stem cells, iPSC – Induced Pluripotent Stem Cells.

SOMATIC CELLS
- fully differentiated specialized cells obtained from normal tissues
- examples
o Skin has been engineered using cultured epithelial cells in vitro for burn patients
o Chondrocytes have been isplated, expanded in vitro and implanted into areas of deficient
cartilage – Autologous chondrocyte implantation
o Bladder wall – combination of smooth muscle cells and uroepithelial cells expanded in vitro
and grown on a scaffold before re-implantation.
- Cells obtained from the recipient itself by tissue biopsy – autologous cells
- Cells can also be from deceased unrelated donors – allogeneic cells.
- Not practical for most situations –
o Not readily available in adequate numbers
o Limited proliferation – cannot be expanded sufficiently

STEM CELLS –
- Undifferentiated cells capable of self-renewal and also differentiate into other specialized cells.
 - Classified according to their source –
o hESC – human embryonic stem cells – early embryo
o Fetal stem cells
o Somatic stem cells – adults
o Induced Pluripotent stem cells – by reprogramming adult specialized cells.

SOMATIC STEM CELLS

- aka – adult tissue resident stem cells
- Bone marrow, Gut – regularly replace damaged or senescent cells in blood and GI mucosa.
- Differentiate in limited number of specialized cell types – multipotent
- Examples –
o Hematopoeitic stem cells – Widely used for Rx of hematological malignancies.
o Mesenchymal stem or stromal cells – most widely used SSC in tissue engineering.
o Endothelial progenitor cells
o Neural Stem cells

Mesenchymal stem and stromal cells

- Mutlipotent
- Sourced from a variety of tissues – bone marrow, adipose, umbilical cord.
- Morphologically similar to fibroblast
- Adherent to plastic – this property is used to separate them from other cells in vitro.
- Express markers – CD105, CD73, CD90
- They donot express markers of hematopoietic stem cells – CD34, CD45.
- Mainly differentiate into mesodermal lineages – osteoblasts, chondrocytes, adipocytes, tenocytes,
myocytes
- Recently shown to differentiate into endodermal and ectodermal lineages also.
- Potent trophic and anti-inflammatory properties
- Produce VEGF, IGF, HGF, PGE2
- Isolated from bone marrow – Iliac Crest aspiration
- Isolated from subcutaneous fat – Liposuction – less invasive and higher yield.
- Can be used immediately upon isolation or after their expansion in vitro or after differentiation into
desired lineage.
- Uses –
o Repair defects of cartilages
- mechanism of action –
o Unclear – whether direct contribution or via immunomodulatory/paracrine effects of trophic
mediators released by them.

EMBRYONIC STEM CELLS

- obtained form inner cell mass of early human blastocyst – days 4-5 after fertilization
- using embryos that have been created through in vitro fertilization and are surplus.
- James Thompson – developed the technique for isolation and growing.
- Totipotent
- The surplus embryos need to be destroyed to obtain ESCs
- Allogenic cells – risk of rejection.
- Somatic Cell Nuclear Transfer – transfer the nucleus of a somatic cell of the recipient into the oocyte
that has its nucleus removed – theoretically possible – to produce autogenic ESCs.

FETAL STEM CELLS –

- from blood, bone marrow and other tissues of aborted fetuses.
- Proliferate in vitro as efficiently as Embryonic Stem Cells.
- Pluripotent
- Used in - parkinson’s disease, Diabetes, Spinal Cord Injury.
- Less ethical issues.

Induced Pluripotent Stem cells

- Cells reprogrammed using genetic manipulation to become embryonic like iPSCs
 - Uses retroviral or lentiviral transfection to introduce a combination of transcription factors – OCT 3
/4, SOX2, NANOG, LIN28, OSKM reprogramming factor (Kruppel like factor + c-MYC)
- Specialized somatic cells are transfected.
- Proliferate in vitro as efficiently as ESCs
- Pluripotent
- Autologous -  risk of rejection.
- Allogenic – from voluntary donors on the basis of their HLA types to create national or international
tissue banks of iPSCs – and then provide fully or partially matched cell transplant.
- Genomic integration of viruses  oncogenes, tumorigenesis.
- Non-retroviral vectors – to reduce the risk of genomic integration – Adenovirus, Sandai Virus,
Plasmids, Episomal Vectors, synthetic RNA
- New molecules and growth factors that obviate the need for vectors.
- Process of obtaining adequate iPSCs – may take several weeks.

In vitro Differentiation of stem cells to specialized tissue cells –

- Addition of critical amounts of activin, BMP-4, FGF-2
- iPSC  exposed to BMP-4, Retinoic acid  ectoderm  keratinocyte lineage.
- iPSC  cultured on Matrigel scaffold + Ascorbic acid  exposed to Glycogen Synthase Kinase
Inhibitor –f/b inhibitor of Wnt Signalling  Immature Cardiomyocyte.
- Surface characteristics of the scaffold – also promote stem cell differentiation along a particular
lineage.
- Mechanical stress also influences cell fate decisions.
- Sequential exposure used to differentiate the ESCs or iPSCs into endodermal, mesodermal and
ectodermal lineages f/b further differentiation into specific lineages.
- Cells must be fully phenotyped and their function confirmed before they are used for therapeutic
purposes.

Scaffolds for tissue Engineering –

- stem cells cultured under conventional tissue culture conditions  sheets or small 3-D collections
called organoids.
- To assume complex anatomical relations – need appropriate scaffold – that gives physical support and
shape to engineered tissues, mimicking extracellular matrix.
o Allows cells to attach – provide physical support
o Delivers cells signals as necessary – guide growth, differentiation and migration
- Rigid and semi-rigid scaffolds are used – that are porous and act as templates to seed donor cells.
- Scaffolds – derived from –
o Intact human tissue – Natural Scaffolds
o Engineered implantable biomaterials – Artificial Scaffolds
- Requirements of a scaffold –
o Provide structural support to cells
o Allow them to attach, migrate and proliferate
o Enable oxygen, nutrients and regulatory factors access to all cells
o Deliver signals – promote migrate and proliferation
o Biocompatible
o Non-Immunogenic
o Ideally – biodegradable.

NATURAL SCAFFOLDS –
- obtained by treatment of human tissues or organs to remove the resident cell types, leaving behind
ECM that preserves the intricate architecture of the tissue or organ.
- Allow natural architecture of the tissue to be maintained.
- May also provide key cell signals that guide growth and differentiation
- Obtained by –
o Immersing in detergents
 o Perfusing with detergents via arteries.
- Effectively destroys cellular elements and leaves the collagen rich ECM.
- Optimal approach – depends on the type of tissue or organ being used to create the scaffold.
- Useful – engineering length of trachea.
- Using natural scaffolds to engineer whole organs – kidneys, liver – extremely intricate 3D structure of
the organ is preserved – extremely challenging to achieve using artificial materials.
- Decellularized organ scaffolds – allow the scaffold to be repopulated with autologous cells – prevent
rejection.
- Disadvantage – use a whole human organ for each organ to be engineered.

ARTIFICIAL SCAFFOLDS –
- All scaffolds must be biocompatible – in most settings they must be biodegradable and bioresorbable.
- Natural materials –
o Polysaccharides
o Collagen o Gelatin
o Fibrin o Cellulose
- Synthetic materials – Major advantage – reproducible characteristics can be ensured.
o Polylactide (PLA)
o Polyglycolide (PGA)
o Graphene
- Bioactive ceramics (Hydroxypatite) and Bioactive Glasses – used for skeletal repair.
- Blend of natural and synthetic materials can also be used.
- Porous 3D structure – most scaffolds.
- Hydrogel Scaffolds – cross linked hydrophilic polymers – favorable physical and chemical
characteristics.
o Absorb large amounts of water while maintaining their 3D structure and integrity.
o Fabricated from Collagen and Fibrin or PLA.
o Can be impregnated with GF
- Electrospinning Technology – produce scaffolds composed of fibers with a diameter at the nanoscale
level.
o Blends of different synthetic or synthetic+natural polymers can be used.
- Microsphere based Scaffolds –
- Smart Scaffolds – have biomimetic properties – provide biophysical cues and cellular signals to
instruct and guide cell behavior.
- Recent development allow a vascular network to be created by 3D printing.
o Have the pore size and diffusion coefficients needed to provide necessary cues for cell
differentiation and migration.
- Artificial scaffolds can be engineeredto incorporate molecules – VEGF.

APPROACHES TO CELL SEEDING OF SCAFFOLDS AND BIOREACTORS –

- achieved in vitro
- Most appropriate seeding system – depends on the tissue being engineered.
- Approaches are –
o Static Cell Seeding – most simple, least effective. Concentrated Cell suspension is placed in
direct contact with the scaffold. Efficiency and penetration – Low. Scaffolds can be coated
with special agents to increase the cell attachment.
o Dynamic Cell Seeding – scaffold is either rotated in the medium or both the scaffold and
suspension are rotated together. Some systems – high speed rotation to generate centrifugal
forces.  efficiency,  culture duration,  penetration.
o Magnetic Cell Seeding – Cells labeled with supramagnetic beads coated with a ligand that
specifically binds to molecules on their surface OR by culturing cells with cationic liposomes
that contain supramagnetic ferrous particles. Concerns regarding potential side effects of the
supramagnetic particles.
o Pressure and Vacuum Cell Seeding - reduces the time for culture, but pressure and vacuum
may affect cell viability.
 o Photopolymerized hydrogels– Cells are suspended in an aqueous monomer solution and
UV light is used to promote polymerization of the hydrogel scaffold. Arginine-Glycine-
Aspartic Acid adhesion peptide can be incorporated to increase the adhesion of the cells to
the scaffold.
o Bioreactor perfusion systems – can also be used as whole organ perfusion systems in the
case of decellularized organ scaffold.
- To achieve a rapid seeding of viable cells with high seeding efficiency and uniform and effective
penetration of cells into the scaffold.

CHALLENGES TO ENGINEERING TISSUE IN VITRO –

- Delivery of Oxygen and Nutrients to 3D tissue constructs.
- Growth requirement of different cell types are different.
- Flat tissues – cornea, skin and cartilage – less problematic.
- Hollow Organs – gut, bladder, trachea, bronchus – greater challenge
- Complex Solid Organs – greatest challenge of all.
- Obtaining adequate number of cells and maintaining their viability and function.

IMPLANTATION -
- Only effective if it becomes fully integrated into surrounding tissue and be remodeled appropriately
by the recipient.
- Considerable mechanical stress in musculoskeletal and cardiovascular tissues – and engineered
tissues are not subject to these mechanical stresses during their in vitro fabrication.

SAFETY CONCERNS –
- tumour formation
o most serious concerns
o relates specifically to ESC and iPSC. Little risk with SSC.
o Ability of stem cells to form teratomas is one of the hallmarks of pluripotency
o Risk reduced by
 implanting cells that show full differentiation only.
 Viral vectors that did not integrate into the genome or non-viral approaches.
 Transdifferentiation - use of techniques to reprogram somatic cells to adopt the
function of a different cell type without having to make them first revert back to
pluripotent state.
- Genetic and Epigenetic Abnormalities
- Transmission of infection –
o Good manufacturing practices guidelines – to avoid bacterial infections.
- Poor viability and loss of function
- Differentiation to undesired cell types
- Rejection (Allogenic cells)
- Side effects of immunosuppression