Bioprinting: Chapter 4

the start of the last push

Biomaterials

• materials that would not work/survive on their own (non-viable)

• materials used in therapeutic and diagnostic systems that are in contact with tissue and biological fluids

• categorized into:

  • polymers

  • ceramics

  • metals

  • glasses

  • carbons

(poppy can make glass cars)

Characteristics of Biomaterials

• nontoxic + noncarcinogenic

• chemically stable

• resistant to corrosion (can withstand stress)\

• can be shaped into complex geometries

Requirements of biomaterials in TE

1. Formability

2. Biocompatibility

3. Suitable mechanical properties

4. Biodegradability

5. Biodegradation product

6. Bioactivity

7. Sterilisation considerations

(four bears send big bald boys soap)

Formability

• each technique requires…

  • viscosity, shear-thinning property, response + transition time, sol-gel transition stimulus

Biocompatibility

• not having toxic effects on biological systems

• being able to perform with an appropriate host

• critical property

• REMEMBER

  • no material is definitely biocompatible (it is process dependent!)

  • no FDA-approved materials

• must continuously perform a certain function

• appropriate host response

Host Response to an implant

• complex physiological reaction of a living body to a synthetic biomaterial

• beings immediately upon implantation

Stages of Host Response

1. Injury and Hemostasis

• blood clots to stop bleeding initiates

2. Protein Adsorption

• proteins in blood/fluids coat the surface of the implant - signaling immune cells

3. Acute Inflammation

• immune cells (eg. neutrophils + macrophages) clean up dead tissue and kill pathogens

4. Chronic Inflammation & Foreign Body Response (FBR)

• when implant cannot be broke down → macrophages fuse into giant cells causing inflammation

5. Fibrous Encapsulation

• body deposits a dense, avascular layer of collagen (scar tisue) around device

Suitable mechanical properties

(key properties of metals)

• tensile yield

• modulus of elasticity

• ultimate strength

• fatigue endurance

(other)

• creep and compressive yield strengths

Biodegradability

• ideally: material degradation rate is synchronized with rate of tissue re-growth

• products:

  • are non-cytotoxic

  • HOWEVER fast degradation leads to acidification

Factors influencing degradation-absorption rate

• degree of crystallinity

• hydrophilicity of the polymer backbone

• volume of porosity

• surface area

• presence of catalysts

Bioactivity

• material makes a positive and advantageous biological response for the body

• bioactive materials include those that …

  • bond to soft tissues

  • materials that release biological stimulants

Sterilisation considerations

• process to destroy living organisms or viruses from the device/material

• depends on material properties (radiation sensitivity + heat resistance)

• SAL

  • tool for quantifying sterility

  • the probability that an implant will remain unsterile

  • accepted value: 10^-6

3 Traditional Sterilisation methods

1. gamma radiation sterilisation

2. ethylene oxide gas sterilisation

3. steam sterilisation

Polymers

• a large molecule consisting of numerous repeated subunits

• hydrogels (for bioprinting)

• synthetic polymers (for tissue engineering)

• types: PLLA, PLGA, PGA, PCL

Poly-L-Lactic Acid, PLLA

• synthetic resorbable and biodegradable polymer

• glass transition temp: 60-65°C

• melting temperatures: 175°C

• slow-degrading polymer (2-5 years)

• suits: load bearing applications

• traits:

  • low extension

  • high tensile strength

  • high modulus (4.8 GPa)

Poly(glycolic acid), PGA

• thermoplastic

• high rigidity and crystallinity (46-50%)

• Tg: 36°C

• Tm: 225°C

• suits: dissolvable sutures

  • the degradation product (glycolic acid) is a natural metabolite

• biodegradation rate: 4 months

• mechanical property degradation rate: 6 weeks

Poly(lactide-co-glycolide), PLGA

• popular (because of its versatility)

• co-polymer

• suits: scaffolds + drug delivery systems

• degradation + mechanical rate: can be tailored

  • by changing ration of lactic acid to glycolic acid

Poly(caprolactone), PCL

• semicrystalline polymer

• Tg: -60°C

• Tm: 59-64°C

• degradation rate: 3 years in vitro (outside the body)

• molecular weight: 50,000

Synthetic Polymers Advantages

• can be tailored

  • includes mechanical, physical, chemical, and thermal properties

• widely available

• manufacture when you need to!

Synthetic Polymers Disadvantages

• could be toxic

• costly

Hydrogels for Bioprinting

• high water contents (up to 99.9%)

• a 3- dimensional network of a series of hydrophilic polymer chains that are crosslinked

• made of natural or synthetic polymers

• can swell or shrink, due to changes in temp, pH, and electric field

Natural polymer hydrogels

• Collagen, gelatin, fibrin, alginate, chitosan and chitin, hyaluronic acid (HA)

Synthetic hydrogels

• poly(2-hydroxethyl methacrylate) / PHEMA, Poly(vinyl alcohol) PVA, Poly(ethylene glycol) PEG

Collagen

• most abundant protein

• main component of natural ECM

• can be naturally degraded

  • done by the enzyme collagenase

  • this means degradation can be controlled by cells!

Gelatin

• protein-based polymer

• derived through partial hydrolysis of collagen

• undergoes gelation during a change temp

Fibrin

• fibrous, non-globular protein

• involved in blood clotting (think glue!)

• consists of thrombin + fibrinogen solutions

Alginate

• a long chain carbohydrate/polymer (polysaccharide)

• contains 2 monomers

  • α-L-guluronic acid

  • β- D-mannuronic acid

• distributed in alternating or repeating blocks

Chitin and Chitosan

Chitin

• polymer

  • made of NAG and linked by beta-glycosidic

  • main sources: shrimp and crab shells

Chitosan

• comes from chitin

• can be used in hydrogel or nano/microparticle solutions

Hyaluronic acid

• found in ECM of all mammalian connective tissues

• lube

  • helpful in the synovial fluid of joints

• low molecular weight

• good for wound healing (angiogenic)

• naturally degraded by hyaluronidase (an enzyme)

• consists of repeating β-D-glucuronic acid and N-
  acetyl-β-D-glucosamine

Natural polymers advantages

• non-toxic

• biocompatible

• safe for humans and the environment

Natural Polymer Disadvantages

• batch by batch variation

• complicated extraction process

Synthetic polymers hydrogels advantages

• highly tunable

• consistent properties

• large-scale production

• pHEMA, PVA, PEG

Poly(2-hydroxethyl methacrylate), pHEMA

• biologically inert

• weak

• high resistance to cell adhesion and protein adsorption

• used for: contact lens

Poly(vinyl alcohol), PVA

• can be photo-cured

• neutral

• non-adhesive to proteins and cells

• low friction coefficient

• similar to PHEMA

  • has available pendant alcohol groups that function as attachment sites for biological molecules

Poly(ethylene glycol), PEG

• the most successful synthetic hydrogels for TE

• biocompatible and hydrophilic material

• used for: cell encapsulations, mediators for immobilising the RGB sequence

Key properties of hydrogel in bioprinting

• rheological properties

• crosslinking mechanisms

• solute transportation

• swelling behaviour

(riya cries so swag)

Rheological properties

• focuses on the flow of matter when applying an external force

• typical parameters

  • viscosity

  • shear thinning

  • yield stress

Viscosity

• the resistance of a fluid to flow under the application of stress

• newtonian fluid

  • depends only on temp

• non-newtonian fluid

  • depends on temp AND shear rate

  • shear thinning

    • viscosity decreased with increased shear rate

  • shear thickening

    • viscosity increases with increased shear rate

Finding the right bioink viscosity

• the higher the viscosity the

  • better the printing

  • the more damage in cells

Shear force

• forces above yield stress

• thinning: high shear weight (fast) → low viscosity (less resistance) → easier to flow

• thickening: low shear weight → high viscosity → harder to flow

• more obvious for polymer solutions with high molecular weight

• Once the fluid is extruded, the shear force is removed → viscosity returns to its original state, helping the bioink maintain its shape

For easy memorization of shear stuff

High shear = thin + flows
Low shear = thick + holds shape

Yield stress

• the stress needed to be overcome to initiate flow

• after stress is removed, network reforms, structure returns

Ideal case of bioprinting for extrusion

printable viscosity (aka shear thinning ) + suitable yield stress

• this bio ink is…

  • easy to print

  • has a quick recovery

  • can maintain its shape

Bioink printability depends on

1. yield stress

2. viscosity

3. storage modulus recovery (how well the bioink regains its structure after printing)

How to design and maintain shape and resolution

• Good shape fidelity

• Sufficient stiffness to maintain shape

• Suitable mechanics for high resolution

• Cell-compatible for growth

Gelation

• used for crosslinking hydrogels

• physical

• chemical

• combination of both

Physical crosslinking

• polymer interactions that form a gel without chemical bonds.

• Caused by polymer entanglement, hydrogen bonds, ionic, or hydrophobic interactions

Thermo-gelation

• temperature change triggers gel formation through hydrophobic interactions

• Common thermo-sensitive hydrogels:

  • PNIPAAm

  • PEO–PPO–PEO (Pluronics)

Physical crosslink disadvantages

• Weak mechanical strength

• poor stability of printed structures

• Increasing polymer molecular weight/crosslink density improves strength

• However, this also increases viscosity → harder to print

Chemical crosslinking

• Forms covalent bonds between polymer chains

• Triggered by heat, pressure, pH changes, or irradiation

• Produces stronger, more stable hydrogels with improved printability and handling

• Common methods: radical polymerisation and functional group reactions

• GOOD BUT can involve bio-incompatible chemicals

Crosslinking by radical polymerisation

• Common method for preparing hydrogels

• Forms hydrogels by polymerising vinyl-containing macromers

• Initiated by redox, heat, or UV light (photopolymerisation)

Chemical crosslinking advantages

• fast crosslinking rates

• better stability and printability

• high mechanical strength

• good handling properties

Combining physical and chemical crosslinking

provides

• faster gelation times

• improved biocompatibility with cells and proteins

• better control on hydrogel properties

Swelling behaviour

• welling = how much water a hydrogel absorbs

• Swelling affects hydrogel properties

• Swelling is controlled by two opposing forces:

  • Osmotic pressure (draws water in)

  • Elastic contraction (resists swelling)

• Water in a swollen hydrogel is either:

  • Bound to polymer chains

  • Free within the polymer network

During Swelling process

• Water enters the hydrogel via hydrogen bonding → creates osmotic pressure

• Elastic contraction resists the swelling

• Swelling pressure = osmotic pressure − elastic contraction

Swelling Continued

• Swelling can be tuned

• affects:

  • Diffusion

  • Molecule movement

  • Optical + mechanical properties

Solute transportation

• determines

  • how cellular products (nutrients + waste) are exchanged within a scaffold

• mainly occurs through diffusion

• Diffusion in ionic gels depends on:

  • Culture conditions

  • Hydrogel mesh size