2. 3D bioprinting
3D bioprinting is an emerging technology for the
fabrication of tissue constructs to repair damaged or
diseased human tissues or as in vitro model systems for
drug screening applications.
2
3. 3
Basis of 3D bioprinting:
• Biomaterial-inks : Systematic combination of polymeric hydrogel
biomaterials and growth factors as optional (polymeric hydrogel
materials devoid of cells)
• Bio-inks : Systematic combination of living cells, polymeric
hydrogels and growth factors as optional; in some cases polymeric
hydrogels are also optional for bioinks (combination of polymeric
hydrogel materials and cells )
5. 5
Sterilization is a process by which all forms of microbial life, such as yeast,
bacteria and viruses are completely eliminated through:
• The denaturing of proteins
• Disruption of cell membranes
• Destruction of nucleic acids
Sterilization
6. 6
Sterilization of medical devices such as implant prosthesis, dental implants, and
surgical aids can be carried out at the terminal stage or before packaging.
However, bioinks for live cell 3D bioprinting must be sterilized at an earlier stage,
typically before the encapsulation of cells within the biomaterial.
• All the precursor materials used in the synthesis of hydrogels as well as the
processes involved in the synthesis of hydrogels must be conducted in sterile
environment to prevent contamination from microbial lifeforms or unwanted
particles.
• Moreover, biomaterials and thus 3D bioprinted constructs are considered as
medical devices and are mandated to be sterilized before use, as they are intended
to be placed within the body and interacts with sterile body fluids
7. 7
Sterilization of biomaterials is of paramount importance, however, literature on the
various methods indicate that sterilization techniques often influence their material,
mechanical, structural, chemical and biological properties like:
Viscosity of bioinks (most important parameters) as the rheological properties affect
printability and mechanical properties printability of 3D bioprinted constructs
Degradation and decomposition of materials
Discolouration
Embrittlement
induction of cytotoXic effects can result
The effect of sterilization process on rheological, mechanical, physiochemical and
cytocompatibility properties on biomaterials
8. 8
Although various sterilization techniques are reported for biomaterial scaffolds,
there is a lack of a specific technique for sterilizing 3D bioprinting processes.
Methods of sterilization
Sterilization techniques can be categorised into two broad categories
Physical
• Syringe filtration
• Autoclaving
• Steam treatment
• Irradiation
• Plasma
Chemical
• Ethylene oXide (EtO)
• Peracetic acid (PAA)
• Ethanol
• Super-critical carbon dioXide (scCO2)
Each sterilization technique has its merits and limitations
9. 9
It is important that choosing the best sterilization technique, which:
Has the best degree of killing or inactivating microorganism
Have minimal adverse influence on the inherent or tailored properties
Biomaterial’s viscoelastic properties
Mechanical properties of biomaterials
Denaturation of protein-based bioinks
Changes in physiochemical properties
Bioink’s printability
Biological activity
Any changes to these properties may result in drastic effects on bioprinting process,
safety and efficacy during in vivo testing.
11. 11
Filter sterilization
Using membrane filters for sterilization of biomaterials may contain heat-labile
components, enzymes or proteins that can be easily destroyed by heat.
These mambrane are made of various types of materials (pore di- ameters of 0.22
μm and 0.45 μm) such as
• Cellulose esters,
• Polymers such as polyvinylidene fluoride (PVDF),
polytetrafluoroethylene(PTFE), polyether sulfone (PES), and nylon
12. 12
Advantages of filter sterilization
• Easy to use
• Available in a variety of materials and membrane pore sizes
• Does not adversely affect biomaterial properties
Limitations of filter sterilization
• The flow rate of permeate is dependent on the viscosity of the liquid.
Viscous gels are more challenging to filter when compared to gels with lower
viscosity.
• this method is not as effective as heat or chemical sterilization.
• The pore size may be ineffective as viruses and mycoplasma can permeate
through the membranes.
• the loss of material during the filtration process ( specially in scale-up) The use
of vacuum for more viscous materials may cause damage to the membrane
13. 13
Commercially, membrane filters are sold as syringe filters where one end can fit
into a standard syringe needle hub and the other can dispense filtered permeate.
The working principle involves passing media or biomaterial in liquid form
through the filter and microorganisms larger than the membrane filter pore size
adhere to the membrane surface
When a force is applied on a syringe
filled with un- sterilized media, the
porous membrane traps contaminants
allowing sterile permeate to flow.
Syringe filtration must be performed in
biosafety cabinets or sterile
environments.
14. 14
Heat treatment
Heat treatment methods can be classified into two categories:
• Dry-heat
• Dteam sterilization
From the perspective of 3D bioprinting, heat sterilization of bio-inks must be carried
out before addition of cells.
Advantages of heat treatment
Simple
fast,
effective with high penetration rates
leaves no toXic residues as water is the main component
Limitations of heat treatment
the high temperatures have detrimental effects on biodegradable polymers ,which
result in lower molecular weight
decrease in mechanical properties such as yield strength, tensile and compression
strength
This can result in the change in viscosity of the gel and thereby alter printability and
stability of the hydrogel.
15. 15
Biomaterial Ink:
Heat treatment significant lead to changes of the properties of the biomaterial inks
( like sodium alginate, alginate, agarose-gelatin composites)
Decrease in molecular weight
Decrease in viscosity (the longer polymeric chains breaking down due to high
thermal fluxes)
Viscosity reduction showed effects on printability → lost their shape fidelity
No change in cell viability
Decrease in polydispersity index (as a results of the uniform cleaving of bonds
due to the homogeneous heat and pressure during autoclave cycle)
Increase in the spreading ratio (the amount of material spread out in a filament
after extrusion) and resulted in discontinuous filaments, depending on the
printing pressure and speed
Increase in metabolic activity ,which attributed to the lower molecular weight
that caused an increase in matriX permeability and hence facilitated better
nutrient exchange
However, there are conflicting reports of thermal effects on biomaterial ink or bioinks.
herefore, further investigations into the type of sterilization and its effect on mechanical,
chemical, physical, and biological properties of the biomaterial inks are required.
16. 16
Pasteurization:
There is a significant lack of studies that focus on the effect of sterilization
methods on printability, cellular activity, and mechanical properties. Additionally,
it was found that most of the studies reported pasteurization as the choice of
sterilization, however recommended pasteurization conditions were not met.
Recently, pasteurization is being used in the
sterilization of bioinks and biomaterial ink
(alginate-gelatin-graphene oxide polymer,
alginate-gelatin, alginate-gelatin-hydroxyapatite,
alginate-gelatin hydrogel, carboxymethyl
chitosan and gelatin solutions, Silk-fibroin
hydrogel)
17. 17
Radiation methods are effective in inactivation of bacterial cells because it breaks
down bacterial DNA resulting in the inhibition of bacterial division. Radiation
sterilization is a convenient method as terminal sterilization is achieved at ambient
temperatures and is regarded as an environmentally safe technology.
Irradiation techniques typically used for sterilization of bioinks are
Gamma (γ)
Ultraviolet (UV) light
Irradiation
18. 18
Gamma irradiation
Gamma irradiation is categorised as ionizing radiation techniques. Gamma rays
are electromagnetic and are obtained from synthetic radioisotopes of Cobalt such
as Cobalt 60 (60Co) and are used to produce radiation in the range of 10–30
kGy/h.
Gamma irradiation works through the transfer of energy from photons to the
target material resulting in the generation of highly excited electrons and highly
reactive free radicals.
The free radicals cleave the phosphodiester backbones of DNA molecules of
microorganisms thus preventing replication resulting in sterility of the material
19. 19
Advantages of gamma irradiation
Simple
Rapid
Effective
Limitations of gamma irradiation
It can cause cross-linking and chain scission in polymers
It can lead to reduction on viscosity and stability
Increase in compressive modulus
Reduction in pore size attributed to higher compressive modulus due to increased
cross-linking
Reduction in cell viability and printability
Irradiation had a significant impact on the sol-gel transition of the hydrogel and
resulted in an increased gelation time
Alter the chemical structure of ink
It have a drastic effect on biomaterials’ chemical characteristics, mechanical properties,
and molecular weights, thus affecting 3D bioprintability
20. 20
Printability of GelMA (a) subjected to different sterilization methods (E-
GelMA – Sterilized by Ethylene oXide, A-Gelma-Autoclave sterilization, γ-
GelMA- Gamma irradiation sterilization. (b) corresponding Pr value of printed
structures.
In terms of printability, radiation treated hydrogels failed to form
consistent grid patterns
Printability is typically assessed by measuring the Pr value which is demonstrated by
whether interconnect grid lines form a square shape
literature has shown that Pr values between 0.9 and 1.1 are considered to demonstrate
good filament morphology as well as me- chanical stability
These results suggest that γ
radiation is unsuitable as a
sterilization method for 3D
bioprinting due to the extreme
changes in the chemical,
mechanical properties and
printability of hydrogels
21. 21
Ultraviolet light
Ultraviolet (UV) light is another common method used in steriliza- tion of
biomaterials before preparation of sterile bioinks.
Lower energy photons are used in wavelengths between 290 and 200 nm, which
is considered optimal for disinfection purposes with a wavelength of 260 nm
considered to be the most lethal.
This method is dependent on the duration and proXimity of the UV source and
the cleaving of the thymine bond destroys DNA irreparably causing the cells to
become inert.
UV sterilization is not considered as a completely effective method for terminal
stage sterilization of biological life.
UV light is capable of cleaving bonds; hence this method affects mechanical
properties of natural polymer-based biomaterials.
22. 22
Effect of different sterilization methods on sodium alginate in various forms. (i)
the types of sterilization methods used. (ii) the effect on cellular activity across
the sterilization methods. (iii) the effect of various sterilization methods on the
rheological properties.
Cell viability was not
significantly affected
among the sterilization
methods on sodium
alginate as a biomaterial
ink
23. 23
The effect of various sterilization methods on 3D bioprinting fidelity. (A, G) varying
line pattern to be printed, (B,H) pristine unsterilized alginate, (C,I) UV treatment,
(D,J) Syringe filtered, (E,K) autoclaved as solution, (F,L) autoclaved as powder.
Bioprinting outcomes also indicated that print structures and shape
were retained with biomaterials sterilized via UV as seen in below
figure
24. 24
Plasma
The use of plasma to inactivate microorganisms and sterilize biodegradable
scaffolds is an area of academic interest.
When a gas is subjected to thermal energy, one or more electron is ejected from
its ground state in a process known as ionisation.
While ionisation results in a higher number of protons than electrons, a plasma
can be defined as an ionised or energised gas with an equal number of
positively and negatively charged particles and are categorised as being either
high-temperature or low-temperature
