Bone tissue Flashcards
Regenerative medicine and tissue engineering examples of when needed
Arthritis (progressive wear and tear of bone and cartilage)
Bone regeneration (Trauma/accident in bone)
Spinal cord injury/Neurological diseases
Cardiovascular diseases (heart infractions need to replace cardiac tissue)
Why engineer bone tissue (condition, issues with natural healing)
Osteosarcoma: bone tumour
Osteomylitis: bone infection (large chunk of bone may need to be removed to remove infection)
Can’t rely on natural healing as bone will not replace/repair a gap larger than a critical size defect (depends on the bone type and species)
Bone normally repairs by growing on already
present bone; seen from cells on surface of titanium implants (osteoconduction; depends on the action of differentiated bone cells)
Bone only rarely spontaneously appears on/in another (non-bony) tissue (osteoinduction; ability of a material to cause SC differentiation down an osteoblastic lineage). Ex. when a scaffold is implanted in muscle, osteoprogenitor cells are attracted to the scaffold and grow there
How cells interact with biomaterial and what modulates cell response
The interaction of cells with biomaterials is mediated by proteins adsorbed on the material surface.
-Modify surface with proteins/biological recognition molecules on the surface so cells can interact with it (via integrins) and recognise.
Natural scaffolds like collagen/fibronectin/etc naturally has these molecules so no/less modification required.
The surface density, conformation and interaction strength of the ECM proteins dictate cell response. Ex. more unfolded/expanded ECM proteins expose more bioactive sites to the cell’s integrins as well as GF etc
-Modify Topography
-Modify Mechanics (for bone needs to be very stiff)
Signalling cascade occurs (molecular clutch)
3 main components in engineering a tissue, role and important considerations
Biomaterial (scaffold which functions as an artificial ECM to initiate/support cell behaviour through mimicking the characteristics of the physiological ECM).
-Biodegradable (allow cells to respond and differentiate into desired tissue before degrading)
-Porous (influx/efflux of nutrients and allow cell signalling. cells can secrete own material to remodel scaffold)
-Mechanical
properties (high strength for bone due to high mechanical load)
Cells (adult or embryonic SC that can differentiate)
-Autologous (typically from biopsies then cultured to obtain more)
Functional biological molecules
-Biochemically stimulate cell down desired lineage
-Enable cells to grow with nutrients
Properties required of a biomaterial for a bone tissue replacement
Porousity: allows cells to sit within, move around and remodel environment (dynamic system), also allowing passage of fluid and nutrients.
Allows new tissue formation and blood vessel (for cortical bone) growth.
Too much compromise mechanical integrity of scaffold (need enough bulk material to withstand mechanical load)
Bone cells are large so need larger pore sizes than for most other tissues
Pore size:
-100-400 µm for bone
regeneration
-200-350 µm for
osteoconduction
Able to take mechanical loads
Encourages cells to grow
Degradation: too slow doesn’t allow new tissue formation to effectively grow since in the way.
Too fast and gone by the time the cell can respond to material, leading to fracture of material before the new tissue has formed.
Want tissue to degrade as new tissue forms (elderly smokers and diabetics form new tissue slower so needs to be accounted for).
Ex. Bioglass has CaPO4 and other chemicals in bone tissue. Cells recognise bone like environment and are encouraged to differentiated into bone. In isolation however, material is brittle so hard to work with (nondegradable) so not ideal in isolation.
Material for bone scaffolds
Bioglass
-has CaPO4 and other chemicals in bone tissue.
-Cells recognise bone like chemical environment and are encouraged to differentiated into bone.
-In isolation however, material is brittle so hard to work with (nondegradable) so not ideal in isolation.
Polymers (biodegradable) PLA, PGA, PHB
-Degrade to water and CO2
-Ductile
Composites (ideal): Combination of two materials
-Matrix phase: PLA, PGA, PL/GLA, collagen etc (structural support for scaffold, easier to engineer and optimal for cell growth)
-Filler phase (promote bone growth): Hydroxyapatite, tricalcium phosphate, BCP, bioglasses
Bone (composition, organisation, traits)
206 bones in human body and most consists of 2 types of bone:
Cortical bone: solid structure comprising the outside of most bone
-Load bearing component
-Normally less than 3% porous (compact bone, provides structural integrity)
-Made of aligned osteons (Haversian systems)
-Osteons align with the long axis of bone
-Osteons consist of layers of lamallae surrounding blood vessels
-80% mineralised
-Compressive stress is 130 MPa
Red bone marrow in cancellous bone is comprised of:
-Haematopoietic SC (becomes osteoclasts; degrades bone)
-MSC (becomes osteoblasts; produces bone)
Cancellous/trabecular bone: porous material comprising the inner part of bone ends
-Orientation depends on loading direction
-Generally no internal blood vessels as nutrition (provided via bone marrow)
- 80% porosity (absorbs shock)
- Compressive stress: 4-12 MPa
Bone cells and function
Osteoblast (MSC): Bone formation
Secretes:
-Collagen I and small amounts of V
-Osteoclacin
-Osteonectin
-Osteopontin
-RNAKL
-Osteoprotegrin
-Proteoglycans
-Latant proteases
-Growth factors (bone morphogenic proteins; BMPs, TCF-beta, FGF, IGF, PDGF, IL)
Osteoclast: Remodelling of bone tissue
Methods of producing scaffolds
-Include components/processes which produce
gas and thus bubbles
-Solvent casting/Particulate leaching
Polymer is dissolved in non-water solvent, then add porogen. Allow solvent to evaporate and wash with water to dissolve the porogen then dry solvent. Porogens are soluble inclusions manufactured in
materials and then dissolved out (ex. salt and sugar particles)
-Depending on porogen and conc. can obtain different pore sizes and morphology
-Supercritical fluid processing
Polymer solution (ex. PLGA in chloroform solvent, optimised conc. for proper foaming and mechanical strength) has supercritical fluid (ex. CO2) introduced in reactor that will dissolve in the solution and cause phase separation and foam formation with CO2 release
-Electrospinning
Polymer solution is passed through a needle at a controlled rate
An electric field using a high voltage source across the needle and a
grounded collector charges the surface of the polymer droplet and a
thin polymer jet is formed
-3D printing
Synthesize a 3D scaffold from a digital model
Melted polymer (temp. kills cells in 3D so used for 2D scaffolds cells are later seeded on) or hydrogel (gelation induced with UV light or chemical) and is
passed through a nozzle
Layer-by-layer construction of a particular organ structure to form a cell scaffold
Suspension of living cells and a smart gel that provides structure
Currently artificial heart, kidney and liver structures
-Can form microgels by printing droplets and making easily implanted small scaffolds
-Microfluidics
Easily controlled parameters of drops made
Form channels in mould and inject fluids.
E. hydrogel with protein in one channel and oil running through, causing separation of droplets in channel (protein encapsulation)
-Examine pores with scanning electron microscopy
Cell sources for bone engineering and biological properties assessed in vitro, and how
-Embryonic stem cells
-Fresh bone marrow (from surgery like knee replacement)
-Culture expanded mesenchymal stem cells (MSCs)
-Pre-differentiated osteoblasts
Properties:
-Cell seeding, cells evenly distributed through the scaffold by ex. a rotating culture system
-Cell viability and cell infiltration as a function of time with fluorescence microscopy for dead/live staining or cell tracker dye respectively
-Expression of bone markers via PCR or western blot (collagen I,
osteocalcin, osteopontin, osteonectin)
-Levels of alkaline phosphatase, calcium phosphate, phosphate via histology staining
-Biomechanical properties (compressive and tensile testing)
-Assessed by radiography, histology and
scanning electron microscopy
-Looking for bone formation, infiltration with new bone cells and the interaction of the cells with
the scaffold materials
-Cell number growth and cell activity assessed using gene up/down regulation
Advantage/Disadvantages of animal models to test engineered bone
Advantages
* Whole system
* Dynamic system
Disadvantages
* Ethics and cost (Reduction: minimal number used, perform sufficient previous in vitro tests to minimise risk of poor outcome, Replacement: assess if no other animals can be used, and Refinement: minimise harm to animal with most minimally invasive procedure)
* Relevance of species used
* Animal to animal variation (sheep has similar bodyweight to humans)
* Not a person
* National-EU Laws
Types of Animal models to engineer bone. Tests to assess models
Osseointegration model: Simple coating with fibronectin fragment enhances bone growth around stainless steel screw. Performed on healthy and osteoporotic rate and found latter had higher pull out force
Subcutaneous model: screen materials and identify ectopic bone formation and vascularisation.
Useful for identifying material for osteoinduction in a injury with a larger gap than critical size
Examined with histology staining
Segmental bone defect: scaffold of PCL loaded with BMP-7 (range of conc. to confirm this had an effect) implanted in a critical size defect in sheep tibiae and fixed with a 3D printed metal plate and left for 3 months.
-Biomechanical tests performed (better load bearing capacity)
-Bone volume
-Mineral density
Bone segmental defect mouse model: Critical size bone segmental defect is made in a mouse radius (stabilised by ulna). A polyimide sleeve is used that is coated or filled with the material, here it’s fibronectin, PEA (for effective presentation of fibronectin) and BMP2 successfully closed gap.
Without PEA, no bone growth occurred
Epididymal fat pad mouse model
Put material under fat pad (minimally invasive) and evaluate if material can stimulate vascularisation
Implanted VEGF and material
Found more new vessel formation with PEA (compared to PMA control) since fibronectin had better presentation that allowed improved VEGF signalling
Dog’s broken leg:
With an external skeletal fixator they used BMP2, PEA (coated on material via plasma polymerisation) and the defect naturally regenerated the critical size defect that conventional surgeries couldn’t resolve
Implementation and presentation of GF in bone engineering. Animal model examples
Soluble administration:
High doses
Short half life
Requires optimised effective release kinetics over a period of time (unreliable)
Downside: High conc. of BMP has been found to have adverse effects and may be dangerous: difficulty breathing, speaking and swallowing
ECM-bound presentation:
Localised response
Lower concentration required (less toxicity, see bottom)
Potential integrin-GF receptor crosstalk may have additive effect
Presentation examples:
High MW ECM protein with GF and integrin binding sites that can have synergistic effects (can see unfolded structure of domains exposed with TEM and AFM)
PEA induced fibronectin unfolding and fibril like structure with domains exposed
Downside: PEA is synthetic and not recognised by body so slow to degrade and scaffold to clear
Solution is plasma polymerisation: convert PEA into monomer and deposit on scaffold. With plasma generate free radicals that causes polymerisation into PEA causing a 10-100 nm coating of PEA
Animal models:
Bone segmental defect mouse model: Critical size bone segmental defect is made in a mouse radius (stabilised by ulna). A polyimide sleeve is used that is coated or filled with the material, here it’s fibronectin, PEA (for effective presentation of fibronectin) and BMP2 successfully closed gap.
Without PEA, no bone growth occurred
Epididymal fat pad mouse model
Put material under fat pad (minimally invasive) and evaluate if material can stimulate vascularisation
Implanted VEGF and material
Found more new vessel formation with PEA (compared to PMA control) since fibronectin had better presentation that allowed improved VEGF signalling
