Tissue engineering I

• What is tissue engineering?

  Tissue engineering focuses on the development of scaffolds and medical devices to regenerate damaged and diseased parts of the body, constructed for the needs of eaach individual
• What is the general workflow of tissue engineering?

  1. Cell isolation and enrichment/purification of desired cells
  2. Expansion of cell number in culture - need a sufficiency amount to regenerate the tissue
  3. Seeding on a suitable 3D scaffold - provides three-dimensionality to tissue to support growth and differentiation of cells, can be plain or imparted with small molecules/growth factors
  4. Maturation of the tissue (proliferation and differentiation) until functional - use bioreactors to provide physiological cues for normal development
  5. Implantation in patient

  
• Autologous or allogeneic cell source

  Autologous

  • Cells derived from the patient themselves (self-cells)
  • Stem cells like MSCs and iPSCs are the most common sources, e.g. satellite cells to make muscle cells
  • Differentiated cells, e.g. chondrocytes from non-damaged, non-load bearing joint to replace cartilage defect

  Allogeneic

  • Cells derived from a donor - either a matched related or unrelated donor
  • Stem cells like MSCs and iPSCs are the most common sources
  • ESCs can be used, but less common these days
  • Differentiated cells
• How are cells enriched for tissue engineering?

  Biopsies and cell aspirations may contain numerous cell types - need to isolate and purify/enrich for cell type of interest to ensure as many stem cells in the population, and as few of the contaminating cells

  
  

  Techniques:

  • Differential adhesion - ability to adhere to a surface, engineer a surface to have an antigen on it that causes it to bind
  • Density centrifugation - size
  • Fluorescence-activated cell sorting (FACS) - size, granularity, surface markers
  • Magnetic-activated cell sorting (MACS) - surface markers
• What factors must be considered during expansion for tissue engineering?

  
  

  • Different cells grow at different rates - the tissue should grow at a rate where it is ready by the time of implantation
  • Culture conditions affect growth and function - may want to preserve "stemness" (ability to proliferate) while expanding and just change the culture medium to influence growth/differentiation to the desired phenotype when needed
  • Phenotype is important - if the cell source has pluripotent SCs (ESCs/iPSCs), assess for pluripotency markers (transplanting pluripotent SCs risks teratoma formation)
• How are cells expanded for tissue engineering?

  Purified cell populations need to be expanded before seeding on scaffolds

  • In vitro studies usually grow cells in flasks before seeding
  • Increase the number of layers in a flask to expand the surface area ("cell hotels")
  • Use of flasks may not be appropriate for clinical application and wide-spread adption - to be scaled up, require bioprocessing strategies to enable growth of large cell numbers
• What are bioreactors?

  Bioreactors are any manufactured device or system that supports a biologically active environment. In tissue engineering, they provide a tissue-specific physiological in vitro environment during tissue maturation
• What is the stirred tank bioreactor?

  • Simplest type
  • Stirrer/agitator/impeller rotates at the bottom of the tank - mixes culture medium and upheave adherent mammalian cells
  • Adherent cells grown on small microcarriers - spherical shape gives very large SA:V ratio and provide large SA for growth
• What is the fluidised bed bioreactor?

  • Culture medium flows upwards through column which contains microcarriers on the inside
  • Suitable velocity of fuid flowing over microcarrier will fluidise the particles - thry grow in suspension
  • Fluid flow circulates around the tank - good aeration and
• What is the hollow fibre bioreactor?

  • Cells grown on the inside or outside of the staw
  • Flow of culture medium goes through centre of the fibres which can bundle up
  • Cells grow on both surfaces
  • Extremely high SA:V ratio
• What are scaffolds?

  Scaffolds provide 3D structural support for cell attachment, cell growth, and subsequent tissue development
• What are the ideal properties of a scaffold?

  • Biocompatible - avoid eliciting a reaction
  • Biodegradable - leave behind only the natural tissue
  • Cytocompatible - compatible with the cells being grown on them
  • Porous - cells should reach the centre and be distributed throughout the scaffold
  • Mechanically appropriate - withstand certain loads
  • Architecturally appropriate - necessary to generate correct structures, e.g. guide cells in particular direction
  • Growth promoting - can have controlled drug/GF release

  Not neccesary to acheive all of these
• What types of materials can be used for scaffolds?

  Organic

  • Natural biomaterials
  • Synthetic biomaterials (polymers)
  • Decellularised tissues

  Inorganic

  • Bioceramics and bioactive glassess
• What are the advantages and disadvantages of natural biomaterial scaffolds?

  Advantages

  • Essentially renewable
  • Natural materials that cells already interact with

  Disadvantages - may want to consider synthetic biomaterials

  • Batch-batch variation
  • Not particularly scalable
• What are examples of natural biomaterial scaffolds?

  Polypeptides

  • Collagen, gelatin, fibronectin, fibrin, laminin - ECM molecules, widely used because cells are already used to them
  • Silk fibroin, zein, soy protein - non-mammalian proteins

  Polysaccharides

  • Hyaluronic acid - naturally found in joints and many beauty products
  • Alginate (seaweed)
  • Chitosan (cells crustaceans)
  • Starch (waste vegetable products)

  Decellularised tissues
• What are the advantages and disadvantages of synthetic biomaterial scaffolds?

  Advantages

  • Control over properties - e.g. degradation, strength, chemical functionality, and biological signals, design them to have motifs which cells can recognise and bind to
  • Reproducibility - scalable chemical process, batch-batch consistency
  • Bulk processing
  • Interesting properties - e.g. temperature responsive to phase transition

  Disadvantages

  • Incomplete mimicry of natural tissue - e.g. cell-binding motifs, signalling cues, cell interactions present in natural ECM
  • Foreign body response
• What are examples of synthetic biomaterial scaffolds?

  Polyesters

  • Most commonly found - poly(caprolactone), poly(lactic acid), poly(glycolic acid)
  • Most widely studied = poly(lactic-co-glycolic acid) or PLGA

  PLGA

  • Degrades via. hydrolysis of ester backbone to lactic (LA) and glycolic acid (GA), removed from the body in urine and breath
  • Ratio of LA:GA controls degradation - more hydrophobic backbone = slower rate of ingress of water into the polymer
  • LA has an additional methyl group = more hydrophobic = slows degradation
  • Also control degradation by changing molecular weight of PLGA
• How are tissues decellularised for scaffolds?

  • Human/animal tissue treated with detergent to decellularise
  • Decelluarisation removes cellular components and DNA/protein from the tissue to leave behind only the ECM
  • This decelluarised ECM matrix is used to grow cells on as a scaffold - recreates structure of the tissue
  • Can also use decelluarised plant matrices (cellulose) as scaffolds too
• Describe compression scaffolds

  Compresses powdered biomaterial under high pressure to create a solid structure - can use a mold to generate the desired shape
• Describe solvent casting vs. particle leaching scaffolds

  Particle leaching imparts pores within the scaffold while solvent casting does not

  
  

  Solvent casting

  • Dissolve polymer/biomaterial in solvent to create a solution
  • Cast solution into a mold of desired shape
  • Evaporate solvent off which leaves behind the soldified polymer/scaffold

  Particle leaching

  • Mix polymer solution with sacrificial particles, e.g. salt (known size)
  • Cast solution into a mold of desired shape
  • Once solidified, immerse scaffold in solvent that dissolves the salt
  • Leaves behind interconnected pores within scaffold
• Describe freeze drying (lyophilised) scaffolds

  • Freeze a water-soluble biomaterial
  • Subject it to vacuum-induced sublimation to remove frozen water under vacuum conditions
  • Leaves behind a porous scaffold structure with interconnected pores due to the ice crystals
  • However, with water-soluble polymers (particular matrix molecules), need chemical cross-linking to stop redissolving in aqueous environment
• Describe spinning scaffolds

  • Extrude a polymer solution through a spinneret or nozzle to form continuous fibres
  • Collect and assemble extruded fibres into desired scaffold shape
  • Generate fibrous scaffolds with aligned or random fibre orientations
  • Different techniques: wet spinning, dry spinning, melt spinning, electrospinning

  A) Wet spinning

  
• Describe electrospinning scaffolds

  • A high voltage is applied to a polymer solution or melt dispensed from a syringe tip
  • This causes the polymer to form a jet that undergoes stretching and elongation as it travels towards a grounded collector
  • Resulting fibers are collected on the collector surface to form a nonwoven mesh or mat
• Describe 3D bioprinting scaffolds


  • Layer-by-layer deposition of bioinks containing cells and biomaterials to create complex 3D structures
  • Bioinks are dispensed from a printer nozzle or extruder in a controlled manner, guided by computer-aided design (CAD) models
  • Different technqiues: inkjet printing, extrusion-based printing, and laser-assisted printing
• Describe hydrogel scaffolds

  • 3D networks of hydrophilic polymer chains, capable of absorbing and retaining large amounts of water
  • Structurally similar to natural ECM - able to support cell growth and tissue regeneration
  • Different techniques to initiate gelation: thermal (phase transitions), ionic (crosslinking polymers with an ion), UV (crosslinking polymer backbone), enzymatic (reactions between functional groups and polymer chains), covalent (covalent bonds between polymers)
• 2D vs. 3D scaffolds effect on morphology

  Collagen-coated glass (2D)

  • Glass slide surface = very stiff
  • Forced apical-basal polarity - cells only interact with flat 2D surface, restricting focal-adhesions to xy-plane
  • Continous layered matrix
  • Bulk of culture medium sat on the cell - no soluble gradients

  Collagen gel (3D)

  • Hydrogel matrix (ECM-like) = softer
  • No forced polarity - cells are surrounded by ECM fibrils, adhesions in all dimensions
  • Discontinuous matrix - fibrils surrounding cells
  • Culture medium surrounds - have gradients of GFs, oxygen, etc.
• What is a major disadvantage of collagen gel/hydrogel scaffolds in vivo?

  The mechanical properties of collagen gel or hydrogel scaffolds may not be sufficient to withstand the mechanical stresses and loading conditions experienced in vivo. As a result, these scaffolds may undergo deformation, collapse, or fragmentation
• How can PCL/collagen electrospinning be used to engineer skeletal muscle?

  • PCL mixed with collagen (1:1) - blended together and electrospun into nanofibrous scaffolds
  • Skeletal muscle cells, such as myoblasts or satellite cells, are seeded onto the electrospun PCL/collagen scaffolds
  • Cells adhere to the scaffold fibers and begin to proliferate and differentiate
  • Form aligned myotubes and muscle fibers that closely resemble the architecture of native skeletal muscle tissue
  • PCL/collagen scaffolds can be functionalised with GFs

  
• How are nanofibres of electrospun scaffolds aligned?

  Use different speeds to align nanofibres on collector - increase the speeds until most of nanofibers at highest speed are all oriented in direction of rotation
• What else can be used to engineer muscle?

  Grass

  • Fine lines on the surface of grass can be used to guide myoblast growth
  • Grass must be decellularised

  
• What is the role of scaffold stiffness in myotubes

  Optimised stiffness (elastic properties) can optimise maturation and growth of tissue

  
  

  e.g. Generating multinuclear myotubes

  • Striation (stripes) of myotubes = indication of myotube maturity (more = better)
  • Striation is dependent on elasticity of growth substrate
  • Myotubes grown directly on glass (stiff) = little striation
  • Myotubes grown on other myotubes (softer elastic) = many striations
• What is the role of scaffold stiffness in stem cell fate (neuronal vs. cardiac)

  Mechanical properties of the growth surface can influence stem cell fate, i.e. promote self-renewal or influence lineage

  
  

  e.g. Soft gel = neuronal differentiation

  • 1 kPA soft surface mimics CNS

  Optimised elastic gel = cardiac differentiation

  • 20 kPA elastic surface - formation of particular colonies
• What is the difference between scaffold topography vs. patterning?

  Scaffold topography refers to the overall physical characteristics and surface properties of the scaffold, while scaffold patterning involves the intentional manipulation or arrangement of specific features or elements within the scaffold structure to create spatially defined patterns or designs
• What is the role of scaffold topography

  Can influence stem cell fate into a particular phenotype (differentiation)

  
  

  e.g. Disordered square topography stimulates osteogenesis (green marker)

  
• What is the role of scaffold patterning

  Rearrangement of cyoskeleton (e.g. through stamped patterns on scaffold surface) can influence gene expression of the cells growing in the patterns

  
  

  e.g. Promotion of adipogeneis in flower shape and promotion of osteogenesis in star shape

  
• What is the role of scaffold modification

  Impart additional motifs or qualities into scaffold that were not already present to guide cell function and fate

  e.g. Electrospinning

  • Use a blend solution for the nanofibres - one biomaterial and another biologically/therapeutically functional agent
  • Immobilise growth promoting fators on the surface of the electrospun scaffold
  • Can also use plasma to functionalise/activate surface or wet chemical treatment (enhance biocompatibility or stick other biofunctional molecules on)

  
• Polyester example of scaffold modification

  • Polyesters modified using NaOH - partially hydrolses polyester backbone and generates alcohols and carboxylic acid
  • Polyesters modified using primary amines - aminylases polyester backbone and forms amino groups of surface
  • Subsequent reaction with coupling reagents can be used to attach bioactive motifs (or whole proteins) - potential for scaffold patterning

  e.g. impart chemical handles for adhesive capabilities

  
• Polydopamine example of scaffold modification

  • Inspired by shellfish mussel and mussel foot (rock) interaction - mussel foot proteins are rich in catechol and amines, reminiscent of dopamine
  • Found that you can take any substrate (e.g. scaffold) and incubate in solution of dopamine (usually around pH 8.5, overnight) - dopamine polymerases and forms thin coating
  • This increases adhesion directly so cells grow on it - can also stick other biomolecules like collagen which has its own advantageous properties

  
• What are other methods of scaffold modification?

  • Adsorption - incubate in mixture of serum or ECM proteins which adsorb onto scaffold
  • Direct ligation - requires chemical functionality on scaffold, e.g. amines and carboxylic acids (attach adhesive motif)
  • Surface entrapment - incubate in partial solvent to cause swelling of outer layers of scaffold, then use antisolvent to collapse and trap ECM molecules on scaffold surface
  • Ionic interactions - incubate +vely charged surface in solution of -vely charged molecules and coat it (can layer)
  • Mineralisation - incubate in simulated body fluid