Adult SC and Biomaterials Flashcards
How do body cells interact with the biomaterial
Biomaterial requires chemically reactive groups (ex. carboxylase) to attract proteins from serum
Serum proteins in the ECM (hydrogel of mostly collagen I and water) are absorbed onto the biomaterial via the chemistry on the surface.
Cells have integrins which detect amino acid motifs (ex. RGD) on serum proteins.
Signalling between integrins and cell nucleus allows cell response. Phenotype is the proteins the cell expresses.
Stem cells (traits and examples)
Stem cells have potency (can differentiate) and can self-renew into 2 identical daughter cells or asymmetrically (one identical and one differentiated), so older people have as many SCs as young, but poorer regenerative capacity. Most of life is quiescent (to prevent exhaustion, division is dangerous).
Potency
Stem cells differentiate into surrounding tissue. ECM and biochemical signals forms the niche (specialised microenvironment) that keeps SCs undifferentiated (ex. MSC and hemopoietic SC in bone marrow)
Totipotent: zygote, can differentiate into all cell types
Pluripotent: Embryonic SC, can make all tissues in body but not placenta (when injected into animals/people forms teratoma)
Multipotent: can differentiate into surrounding tissue. Ex. Haemopoietic SCs (blood cells) and MSCs (sticky; bone, cartilage (on end of bone), ligaments/tendons (connects bone to muscle/bone), fat)
Cells all have all the same genes but regulation and expression of transcription factors stops from being pluripotent and capable of expressing any protein to become any cell
ECM Absorbs water well so most of cells are soft (except bone since sticks to ceramics in hydrogel to obtain it’s stiff phenotype)
Example, cell migration: contractile actin stress fibres need to be stuck to something to pull (attached to linker proteins that interact with integrins, ECM fibril protein then biomaterial). In wound healing fibronectin adheres, contracts, adheres, contracts to travel across wound). When not stuck, cell apoptoses (except blood cells)
Development of field of biomaterials
Biomaterials have evolved.
Began with bioinert materials (no/low immune response). Ex. perspex shards in eyes in injury had no inflammation, so then used in treatment of cataracts.
Second generation of biomaterials include bioactive biomaterials molecules to interact with tissues. Ex. titanium dental implant to promote bone growth.
Third generation (current) is bioactive biomaterials to support and stimulate tissue regrowth before getting degraded by body. Ex. Boom with ear on back of mouse.
Material is:
Chemically treated
Topographically treated
Porous
Biodegradable/permanent
Cartilage (where, cause of damage, treatment, composition, organisation, )
Mainly in form hyaline cartilage (smooth glassy white appearance) in nose, trachea, articular surface of bone, etc
Traits:
Cartilage is flexible and strong to withstand compression (5-10% at ~1Hz) and tensile (stretching) and shearing (sliding) forces. Astronauts (in low gravity, no mechanical load) loses bone mass and stock of osteoblasts/clasts
Damage:
Cartilage is worn down in arthritis. Since it has no nerve supply but bone does, you feel pain via nerves in bone. Cartilage is also damaged via trauma. Cartilage is avascular so can’t heal (ex. unlike bone).
Composition:
Cartilage is comprised of 10% chondrocytes (found in small cavities lacunae) and synthesise/maintain the ECM which is mainly collagen type II (dense fibril network) to provide mechanical
functionality. The proteoglycan aggrecan in cartilage allows it to absorb and retain water (70% of cartilage)
Organisation:
Deep zone (contains small amount of collagen X) attaches to subchondral bone (perpendicular to surface). Transitional zone is random fibres. Superficial zone (fibres parallel to surface).
Treatments:
-Debridement:
Cutting away diseased/damaged tissue and relies on healthy cells producing more matrix to support the remaining tissue, or make more matrix.
-Subchondral drilling:
Drill and penetrate the subchondral bone (just below cartilage surface) to cause bleeding from underlying vessels allow clot formation and it also forms fibrocartilage (mechanically inferior to hyaline cartilage, as it is organised (crimping structure) to resist tensile forces, not compressive.)
//for joint to last longer they wair until worse before replacing.//
-Tissue engineering (typically for athletes)
Biopsy of knee (healthy cartilage) to obtain chondrocytes and culture to expand (use TGF-β to push phenotype back to chondrocyte instead of differentiating down line into fibrocartilage). However TGF-β as well as inducing expression of collagen II, also collagen X (deep zone phenotype).
Seed chondrocytes into collagen hydrogel matrix (mimics natural environment), add to wound area and suture on periocostal flap (contains SC for further regeneration) to cover.
Issue: deep zone develops and others (heterogenous), and doesn’t have the same overall organisation as natural.
Features influencing differentiation pathway and experiments performed as evidence
Provide biomaterial/scaffold environment similar to the natural environment that the desired mature cells reside in.
Environment influences signalling which directs phenotype. Changing signalling will change phenotype, even if the environment isn’t one that would induce that phenotype (ex. increasing ROCK signalling in cell will induce tension and osteoblast phenotype.)
Identify phenotype with differentiation markers (RUNX2 for osteoblast/bone, PPARγ for adipocytes/fat)
Chemistry (ability to adhere):
-Form different size sticky islands (lots of RGD). Via down/up regulation of Rho/ROCK pathway, small islands induce adipocyte (since they are small initially before filling with lipid), and large islands induce osteoblasts
-Cell can form adhesions better on pointy corners (star), inducing osteoblasts than round (flower), inducing adipocytes. Note same size and overall shape.
-t-butyl is high in adipose) and phosphate in bone (calcium phosphate and collagen makes it strong but not brittle).
Stiffness (ability to gather chemistry):
Brain is softest, then fat (3 kPa), muscle (10 kPa), cartilage (20 kPa), precalcified bone (40 kPa, too heavy as an implant), calcified bone (40 GPa).
Culture MSC on different stiffness. On stiffness similar to tissue, differentiates into that tissue (β3 tubulin as bone marker, myoD for muscle and RUNX2 for bone)
Soft material (up to ~20 kPa) deforms when pulled on, reducing tension and downregulating ERK
Topography (presentation of chemistry):
Electron beam lithography to pattern things (etch) very precisely. Etched grid of circles that were ordered, disordered, or random.
In some disorder induced bone formation (similar to the small errors in pattern in natural biology)
ERK (drives growth, inhibits RUNX2, induces PPARγ via phosphorylation). Negative feedback loop drives ERK into nucleus?
same genes in all cells. can’t regulate genes the same as when embryonic stem cell. sox2 oct4 nano induces pluripotency (ex. in fibroblasts turns into embryonic like stem cells)
MSC: runx2, sox9, ppar-gamma,
How to quicken biomaterial discovery
Necessary to develop new tissue engineered constructs. Preferably quicker for new developments.
Chemochip
-Culture embryonic SC expressing Oct4-GFP and select those definitively with embryonic SC phenotype (green) and that the phenotype is later maintained.
-Culture on array of microspots of polymers (homo and hetero) on a slide.
-Use high-throughput methods to characterise each polymer (roughness, stiffness, drop ball of water to assess hydrophobicity via how much it spreads. Surface chemistry analysis with mass spectrometry).
-Quantify cell response (ex. stain and image size
-Map relationship between cell response and polymer/material characteristics
-Allows development of cell specific surfaces (ex. found for embryonic SC, since a little hydrophobic allows proteins to adhere, but not too much else denature.)
For MSC used library of polymers and tested different timepoints
Cultured on microarrays and used surface markers (use antibody and flow cytometry, ex. CD markers)
Select 3 best polymers and culture long-term on petri dish.
Maintained stemness and differentiation potential.
Topochip
-Similar to Chemochip but instead use computer algorithm generate different features from primitive shapes
-Used alkaline phosphatase as marker for osteogenesis
-Identified topography that best induced osteogenesis
-Micrometer scale
Chemotopochip
-Use chemistry library (similar to that in chemochip) on the topochip
Nanotopochip
-Etch patterns onto silicon mould with electron beam lithography
-Make master stamp (negatives are made in metal shims that can fit injection moulders)
-Pattern can be stamped onto plastic (ex. 24 well plate)
-Different cell responses can be tested
-Ex. pillar height was tested for chondrogenic properties (tall things feel soft to cells). Found optimal pillar height to support chondrocyte phenotype in culture (cell sits on top of one pillar)
-Ex. pillar bendiness was screened by changing the aspect ratio (width to height ratio, more bendy with more height). Identified 16 kPa induced osteogenesis best (cell sits on layer of many pillars)
Push stamp into hydrogel to form well reaction chambers by adding proteins with inject printer
Ex. Higher cell density (affects spreading) induces adipocyte
