Tissue engineering II

• 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
• Describe stirred flask bioreactors

  • Glass or stainless-steel flask containing culture medium and cells (on a scaffold)
  • Impeller or magnetic stir bar provides agitation and mixing of the culture medium
  • Rotation of stir bar causes flow of medium over and through scaffold
  • Suitable for small-scale cultures but may not provide optimal mixing or shear stress control
  • Advantage = encourages mass transfer of nutrients and oxygen to centre of scaffold, and waste away from centre
  • Disadvantage = high shear forces may damage cells, particularly on scaffold's edge

  
• Describe rotary cell culture bioreactors

  • Cells and scaffold materials are placed within a rotating vessel or chamber where gases can be exchanged
  • This vessel suspends the cells in a simulated microgravity condition - cells experience gravity and centrifugal forces
  • Medium flows but developing tissue is stationary
  • Promotes cell aggregation, tissue formation, and extracellular matrix deposition
  • Suitable for tissues with complex three-dimensional architectures
  • Advantage = good mass transfer of fluids with low shear forces acting on it, limited damage to cells and developing tissue

  
• Describe perfusion bioreactors

  • Continuous or intermittent flow of culture medium through the scaffold to deliver nutrients, oxygen, and signalling molecules to the cells and remove waste products
  • Can be designed in many ways - e.g. direct perfusion, parallel plate, or hollow fibre systems
  • Maintain a uniform distribution of nutrients and oxygen throughout the scaffold
  • Suitable for engineering large, dense tissues with high metabolic demands
  • Advantage = good mass transfer and stimulation of growth via. fluid flow

  
• Describe compression bioreactors

  • Apply controlled mechanical compression or strain to the cultured tissue or scaffold - e.g. cyclical compression, setting both magnitude and frequency of compression
  • Stimulates cell differentiation, alignment, and matrix remodeling
  • Mimics mechanical forces experienced by cells in vivo
  • Suitable for load-bearing tissues like cartilage or bone - e.g. stimulates osteogenesis

  
• Describe tension bioreactors

  • Apply mechanical tension or strain to the cultured tissue or scaffold
  • Stimulates cell alignment, differentiation, and tissue remodeling - e.g. generate elastic growth surface
  • Suitable for tissues like tendon, ligament, or muscle, where mechanical forces play a critical role in tissue function - e.g. muscle engineering, line up muscle fibres and fuse them to form multinuclear myotubes extending in the correct direction

  
• Described fluidised bed bioreactors

  • A type of perfusion bioreactor
  • Upward flow of culture medium suspends cells and scaffold particles within the bioreactor chamber
  • Fluid flow creates a dynamic culture that enhances mass transfer and nutrient exchange while minimising shear stress on the cells
  • Suitable for culturing cells in suspension or for supporting 3D cell aggregates
• Describe hollow fiber bioreactors

  • A type of perfusion bioreactor
  • Semi-permeable hollow fibers that serve as a scaffold for cell growth
  • Cells are seeded within the lumens of the hollow fibers, while culture medium is perfused through the extracapillary space surrounding the fibers
  • Allowing for the exchange of nutrients, gases, and waste products across the fiber walls
  • Suitable for high-density cell cultures and can be scaled up for large-scale production
• What are some considerations for bioreactors?

  • Mimic physiological signalling - ensures effective maturation of tissue in vitro, apply different physical and chemical cues
  • Ensure appropriate mass transfer of nutrients/oxygen to cells, and removal of waste away from cells
  • Ensure appropriate mechanical forces (shear forces)

  
• Describe the body as a bioreactor

  • The body can provide the correct environment and physiological conditions for tissue development - i.e. chemical signalling, mechanical forces etc.
  • Implant the scaffold into the host and harness the natural regenerative capacity and physiological processes of the body
• Bone as a bioreactor

  • Calcium alginate hydrogel injected between periosteum and tibia in rabbits
  • Provides right conditions for periosteum SCs to proliferate and generate bone tissue
  • Over time, alginate gel replaced with cellular component (proliferation and differentiation of periosteal cells and then deposition of bone matrix)
  • Bone engineered in vivo in bioreactor is histologically similar to the cortical bone it sits next to

  
• What is the advantages and disadvantages of acellular strategies over engineered tissues and cell-based strategies?

  Advantages

  • Acellular scaffolds can be engineered to have specific structural, mechanical, and biochemical properties
  • Less expensive - compared to culturing large numbers of SCs, acellular scaffolds potentially promote growth and repair processes in the body

  Disadvantages

  • Use in simple reapirs - large tissues and organs need something more complicated
• What are the major challenges in tissue engineering?

  • Vascularisation and anastomosis (forming connections between these tissues and patients own vessels)
  • Innervationa and synaptogenesis (forming nervous connections)
  • Arrangement of multiple cells types in complex 3D patterns and at high resolution
  • Regeneration
• What makes the heart a complex tissue to engineer?

  • Contains many different cell types arranged in specific 3D arrangement
  • Different mechanical properties
  • Complex network of blood vessels which feed the cardiac myocytes - and other smaller/larger vessels
  • Nervous connections feeding into and within the heart
• What parts of the heart can be engineered?

  • Valves - currently using carbon fibre or decellularised pig valve as material and 3D printed scaffolds
  • Blood vessels (smaller) - use endothelial cells on the inside and smooth muscle cells on the outside, use tubular scaffold - but a pulsatile/dynamic culture may increase strength and ECM deposition in produced vessels
  • Cardiac muscle - patches of contractile cardiomyocytes have been made, potential for repairing areas of heart damaged by a heart attack
• What is the current solution to overcome organ complexity?

  Use decellularised tissues/organs as a scaffold to replicate structural complexity, then recellularise with patient's own cells

  • Potentially recellularise vascular network with endothelial cells first to get a nice vasculature, and then rest of the cells
• What is the criteria for vascularisation in engineered tissue?

  • Cells within ~200 um of a blood vessel
  • Integrated capillaries with the patient's pre-existing blood supply - separate networks will not work
  • Important cells recieve nutrients and oxygen while removing waste
• What are the current solutions to overcome vascularisation?

  • Seed scaffold with endothelial cells - can be done randomly (grow/fuse/form microvascular network) or using pre-formed channels within scaffold (via. 3D printing)
  • Incorporate VEGF into scaffold - after implantation, release VEGF to stimulate blood vessel growth around the tissue
  • Build scaffold around vascular bed ex vivo - explant capillary network

  
• Using human platelet lysate and ECFCs to generate vascular networks

  • Cell source: Endothelial Colony Forming Cells (ECFCs), endothelial progenitor gells in blood which form blood vessels when stimulated
  • Scaffold: Human platelet lysate, contains many proteins/GFs that help promote formation of blood vessels, e.g. PDGF, VEGF, FGF-2, EDF
  • Take ECFCs within blood and combine with substrate derived from platelets
  • Platelet-rich plasma is sonicated to destroy platelets and release contents
  • Without membranes, can undergo gelation (+ calcium ions and thrombin) - forms fibrin network with all GFs
• How do human platelet lysate and ECFCs network integrate with patient vessels?

  • ECFCs generate a vascular network
  • GFs in the matrix are secreted (out from sprouting vessels)
  • Potentially attract vessels from a patient and integrate with it

  
• What is the potential of platelet lysate and ECFCs

  • Ready-made, vessel-containing scaffold
  • GFs in gel will stimulate patient's existing vessels to integrate
  • All materials from patient, so no rejection or disease transmission
• How does a bottom-up approach differ from a top-down approach in tissue engineering?

  A bottom-up approach involves building tissues or organs from the level of individual cells or biomaterial components and gradually assembling them into larger structures with increasing complexity. In contrast, top-down approach involves starting with a pre-existing tissue or organ template and modifying or manipulating it to achieve the desired outcome
• What is the easiest bottom-up approach?

  • Seeding cells onto biodegradable (e.g. collagen gel) beads or microcarriers - may want to include endothelial cells for vasculature
  • Encapsulate beads/microcarriers within a mold containing a hydrogel or other biomaterial to fuse them together - shrinkage may occur due to fusing
  • Hydrogel serves as a matrix to support cell growth and tissue formation, while the mold provides a defined shape and structure for the tissue construct
  • Culture under appropriate conditions that promote cell proliferation, differentiation, and tissue formation
• What is a more complex bottom-up approach?

  3D bioprinting - addresses the 3D organisation of cells
• Describe 3D bioprinting

  • 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: droplet/inkjet printing, laser-assisted printing, and extrusion-based printing
  • Suitable for things like skin, bone, cartilage, vascular tissues etc.

  
• Describe laser bioprinting

  • Laser pulse focused onto a donor substrate coated with a thin layer of bioink, causing localised heating and vaporisation - this selectively transfers cells
  • Vaporisation generates a pressure wave, propelling bioink towards the receiving substrate where it forms the desired tissue structure with precise spatial control
  • Suitable for delicate cell types and complex tissue architectures
  • Advantage = high-resolution printing and precise cell positioning without direct contact with the printhead

  
• Describe droplet bioprinting


  • Similar to traditional inkjet printers - uses bioinks containing living cells instead of ink
  • Printer (via. thermal, piezoelectric, or electrostatic printhead) precisely dispenses tiny droplets of bioink onto a substrate/scaffold material - droplets form patterns according the desired tissue structure design, layer-by-layer
  • Advantages = high printing speed, high resolution, and compatibility with a wide range of bioinks
  • Disadvantages = limits in ability to control cell density and viability due to the shear forces exerted during droplet formation/deposition

  
• Describe extrusion bioprinting

  • Bioink (usually in a polymer solution) is continuously pushed or extruded through the nozzle/syringe tip under controlled pressure to form filaments or beads - deposited layer by layer to build desired tissue structure
  • Advantages = versatile (prints various biomaterials) including hydrogels, polymers, and cell-laden bioinks, allows rapid printing of large-scale constructs and enables incorporation of multiple cell types and GFs within the same construct
  • Disadvantage = exert shear forces on cells during extrusion, affecting cell viability and functionality

  
• What type of crosslinking is typical for 3D bioprinted structures?

  1. Crossing linking alginate with calcium ions
  2. Cross linking gelatin methacrylate via. UV
• What methods are used to solidify/structure bioinks during the bioprinting process?

  Chemical crosslinking

  Physical interactions

  Coaxial and microfluidic approaches

  Support bath
• Describe chemical crosslinking

  • Chemical agents to form covalent bonds or UV between biomolecules within the bioink
  • Generates strong and stable bonds
  • Must ensure compatibility with embedded cells and minimise cytotoxicity
• Describe physical interactions

  • Non-covalent forces - e.g. hydrogen bonding, ionic interactions, and hydrophobic interactions
  • Biocompatible and gentle - no harsh chemicals
  • Less robust than chemical crosslinking
  • Requires control of environmental conditions - e.g. temperature, pH, or ion concentration
• Describe coaxial and microfluidic approaches

  • Specialised printing nozzles or microchannels for simultaneous deposition of multiple bioinks with different properties
  • Can generate heterogenous tissues
  • Manipulate the flow of bioinks and create intricate patterns or gradients within the printed structures
• Describe support bath approaches

  • Printing bioink into a temporary support material or bath that can be subsequently removed to reveal the desired tissue construct - e.g. hydrogels, thermoplastics, gelatin-based solutions (can be dissolved or liquefied after)
  • Can generate complex and overhanging structures that would be difficult to achieve using traditional layer-by-layer printing
  • Can generate soft or delicate structures without deformation or collapse during printing
• What are specific types of laser-based bioprinting?

  Stereolithography (SLA)

  • Laser beam selectively solidifies photosensitive resin layer by layer
  • Resin contained in reservoir; build platform gradually moves downward as each layer solidifies
  • Advantage = high resolution and precision
  • Disadvantage = may require post-processing steps

  Digital light processing (DLP)

  • Digital micromirror device (DMD) projects 2D patterns onto a fabrication platform - as platform moves up, different image is projected
  • Built layer-by-layer
  • Advantage = fast printing speeds and high throughput

  
• Combine 3D printing of scaffolds and cells

  • Simultaneously print scaffold (using different biomaterials) and cells directly onto scaffold
  • May not be suitable for all materials but lots of materials can be printed, e.g. thermal polymers