L12 3D Bioprinting

Question 1

1. What are the problems associated with traditional methods of scaffold fabrication?

Answer 1

• ability to mimic the complex microstructures of biological tissues
• takes too long to produce the target tissue
• labour-intensive process
• difficult to fabricate tissues with multiple cell types
• difficult to vascularise large, and complex scaffolds
• difficult to control orientation of the blood vessel growth
• cells cannot achieve distribution with high density and precision - often concentrated in periphery of scaffold

Question 2

2. How can the problems associated with traditional methods of scaffold fabrication be addressed using 3D bioprinting techniques and what are the main advantages of these techniques?

Answer 2

Solutions and advantages:

• shortens time and reduced labour costs
  
  • offers automation
• complete control over assembly of tissue
  
  • precise tissue architecture
• produce precise nano/microscopic features that closely mimic native biological tissue ECM
  
  • better tissue function

Question 3

3. Consider the common components of 3D bioprinting techniques.

Regarding the “blueprint”, what are the key considerations for success?

Answer 3

Blueprint = personalised for the patient:

• MRI/CT scans -> create a CAD model appropriate for patient’s needs -> etc. -> to the printer

Question 4

4. Consider the common components of 3D bioprinting techniques.

Regarding “biomaterials”, what are the key considerations for success?

Answer 4

Viscosity - usually use shear-thinning liquids

• biomaterial needs to have low viscosity so that bioink can be extruded through a nozzle, but viscous enough to form a solid gel to ensure integrity of structure
• too high shear stress and high viscosity can damage cells

Gelation methods / setting times

• concentration of polymer
• polymerisation method (if thermal, use low temps or max 37 degrees, physical or chemical crosslinking eg using UV light to cure the polymer
• quick setting times

Biocompatible

• cells need to be suspended in cell culture media or encapsulated inside the gel

Mechanical Properties

• appropriate for cells, maintain structure of gel

Degradation kinetics and byproducts

• specific to target tissue, ensure degradation products are non-toxic

Question 5

5. Consider the common components of 3D bioprinting techniques.

Regarding “cells”, what are the key considerations for success?

Answer 5

• Widely available, easy to expand in culture
  
  • Need to ensure cell viability in the bioprinted construct
• cell type - stem cells, somatic cells, multiple cell types
• proliferation rates and cell density
  
  • too much proliferation leads to apoptosis
  • too little proliferation leads to loss of viability of construct
• robust - withstand shear stresses, crosslinking methods
  
  • somatic cells (?)

Question 6

4. Consider the common components of 3D bioprinting techniques.

Regarding “post-processing”, what are the key considerations for success?

Answer 6

Allow unwanted material to leach out

In situ printing

Bioreactors

(???)

Question 7

7. Consider the common components of 3D bioprinting techniques.

Regarding “vascularisation”, what are the key considerations for success?

Answer 7

Needs to be incorporated into the scaffold

• Allow pathways for smaller blood vessels for form eg sacrificial bioprinting…

Question 8

8. Give some current applications of 3D bioprinting (the techniques) and bioprinted tissues (the products) in Tissue Engineering and Regenerative Medicine.

Answer 8

• current application (technique)*
• bioprinted tissue example (products)

Inket - sprays droplets of ink via piezoelectric or thermal actuator at end of nozzle, causing a build-up of pressure

• skin, cartilage, bone

Laser Assisted - bioink substrate attached to gold (“ribbon”) -> shine laser through the substrate -> hot air bubble is formed -> deposits a droplet on the substrate

• skin - keratinocytes and fibroblasts in collagen bone

Extrusion - force bioink through a nozzle using pressure to produce a continuous filament

• skin, blood vessels, liver, heart valves, bone, cartilage, tumour

Most of these bioprinted tissues are robust and can deal with large amounts of various mechanical forces (including shear stress)

Question 9

9. What challenges remain to be overcome before 3D bioprinting can be employed as a reliable and efficient method for the large-scale production of tissue engineered constructs for clinical use?

Answer 9

• vascularisation
• innervation
• bioprinter technology - we want increased resolution, high precision and accuracy but also ensure cell viability (eg too small nozzle diameter can lead to higher shear stresses)
• biocompatible
• geometry of organ type - easier to print 2D structures (eg skin), but complexity increases with increasing sizes and structures, eg hollow tubes (eg blood vessel) vs hollow organs (eg bladder) vs solid organs (eg kidney)
• maturation - place construct in bioreactor (?)
• gradients (?)
• transplanting printed constructs into the patients - in situ (?)