Biomaterials Analysis Part I - Mechanical & Physical Characterisation PDF

‭Week 7 - Biomaterials Analysis Part I - Mechanical and Physical Characterisation‬ ‭ echanical‬‭properties‬ M ‭ hysical‬‭properties - what is its physical being; how it‬...

‭Week 7 - Biomaterials Analysis Part I - Mechanical and Physical Characterisation‬ ‭ echanical‬‭properties‬ M ‭ hysical‬‭properties - what is its physical being; how it‬ P ‭- what is the material’s response to load‬ ‭behave internally (thermal, electrical, optical)‬ ‭Characterisation methods - physical + mechanical‬ ‭XRD: x-ray diffraction‬ ‭‬ O ‭ ften done with powders‬ ‭‬ ‭Firing x-ray at different angles - Reflected x-rays will destructively/ constructively‬ ‭interfere‬ ‭‬ ‭Depending on the spacing between atoms in the crystal structure - generates an‬ ‭intensity value at each angle‬ ‭‬ ‭Intensity peaks appear when angles are align with the crystallographic plane‬ ‭○‬ ‭Multiple phases = multiple patterns in a profile‬ ‭‬ ‭Y-axis: intensity; x-axis: angles (degree)‬ ‭‬ ‭Amorphous (no crystallographic planes) materials have a diffuse profile = broad peak‬ ‭‬ ‭Compare sample XRD profiles to database peak profiles - compare what should be‬ ‭expected at certain angle‬ ‭‬ ‭No info on elements (need to know the composition of material prior)‬ F‭ TIR: Fourier transform‬ ‭‬ F‭ iring infrared (various wavelengths) - infrared will be absorbed/ transmitted‬ ‭infrared spectroscopy‬ ‭‬ ‭Depending molecular bond‬ ‭‬ ‭Y-axis: absorbance or transmission; x-axis: wavelength‬ ‭‬ ‭Reassure certain molecular bonds exist in the sample‬ ‭ SC: differential scanning‬ >‭ > Determine the degree of crystallinity of polymer‬ D ‭calorimetry‬ ‭ ‬ ‭By how polymer react in certain temperature and measure heat flow‬ ‭‬ ‭Heat energy applied DOES NOT always result in linearly proportional rise in‬ ‭temperature, i.e.‬ ‭○‬ ‭Heat of transformation - Change in state‬ ‭○‬ ‭Crystalline → amorphous absorbs heat but does not change temperature‬ ‭‬ ‭Through graph:‬ ‭○‬ ‭Melting temperature‬ ‭○‬ ‭Heat required to decrystallise the polymer‬ ‭○‬ ‭% crystallinity - enthalpy of fully crystalline material‬ ‭ niversal testing machine‬ U ‭ ‬ L‭ oad cell converts force into numbers‬ ‭(mechanical testing)‬ ‭‬ ‭Pull up/ push down/ bends, snaps, cracks‬ ‭‬ ‭3 outputs: force, displacement, time‬  ‭ ‬ T‭ o calculate stress & strain, requires geometric data of the sample‬ ‭‬ ‭Then generate stress-strain curve‬ ‭○‬ ‭Stress = force/ cross-sec A‬ ‭○‬ ‭Strain = displacement/ orig length‬ ‭○‬ ‭Some material (i.e. hydrogel) may not have linear profile of stiffness → so use‬ ‭arbitrary point‬ ‭○‬ ‭For viscoelastic material (time-dependent)‬ ‭‬ ‭Creep: apply fix load, observe deformation over time‬ ‭‬ ‭Recovery: remove load, observe how fast it recovers‬ ‭○‬ ‭For elastic materials, strain does not change once load is applied or removed‬ ‭(due to reversible deformation)‬ ‭Bending/ fracture toughness testing‬ ‭‬ ‭Geometry is often standardised (width and thickness)‬ ‭Shear testing‬ ‭‬ ‭A is area parallel to the direction of force‬ S‭ EM: scanning electron‬ ‭‬ ‭ rojecting an electron beam onto the sample‬ P ‭microscopy‬ ‭‬ ‭Detects pattern of electron scattering - dictated by surface characteristics‬ ‭‬ ‭Done in vacuum‬ ‭‬ ‭2 types of electron the machine detect‬ ‭1.‬ ‭Backscattered electrons‬ ‭‬ ‭Expel slightly deeper in‬ ‭‬ ‭>> For elemental analyses‬ ‭‬ ‭Easier to see differences in electron density‬ ‭2.‬ ‭Secondary electrons‬ ‭‬ ‭Does not penetrate deep into surface; expelled from the surface‬ ‭‬ ‭>> Good detail around surface topography‬ ‭‬ ‭Sample preparation‬ ‭○‬ ‭Surface has to be‬‭conductive‬ ‭‬ ‭For non-conductive material - streaking on charge buildup areas →‬ ‭sputter-coated with Au or Pt NPs‬ ‭○‬ ‭Sample has to be‬‭dry‬‭- done under vacuum‬ ‭>> can determine:‬ ‭‬ ‭microporosity/ density‬ ‭‬ ‭Surface features‬ ‭‬ ‭Roughness‬ ‭‬ ‭Diameter of fibers‬ ‭‬ ‭Damage from wear, corrosion, degradation‬ ‭‬ ‭Also: elemental composition, phase, integration of composites & particles, empty‬ ‭& solid space %‬ E‭ DS: electron dispersive‬ ‭>> Determine what elements and atomic constituents in material‬ ‭spectroscopy‬ ‭‬ ‭Often used in combination with SEM‬ ‭‬ ‭Fire x-ray/ electron beam, each element has a unique emission spectrum‬ ‭‬ ‭Can be used in point element analysis or elemental mapping‬ ‭ ther basic‬ O ‭Requires a scale/ caliper/ camera‬ ‭characterisation‬ ‭‬ ‭Density - measure the volume & weight‬ ‭‬ ‭Porosity - Archimedes method (buoyancy)‬ ‭‬ ‭Water content - measure dry weight & at hydrated state‬ ‭‬ ‭Hydrophobicity - image processing for contact angle‬ ‭Week 8 - Biomaterials Analysis Part II - Biological and Chemical Characterisation‬ ‭ iological‬‭properties - how cell behave around the‬ B ‭Chemical‬‭properties - reactions with surrounding‬ ‭material‬ ‭Adherent‬‭Cells‬ ‭‬ U ‭ sed for assessing biocompatibility‬ ‭‬ ‭L-929 murine adipose-derived fibroblasts‬ ‭‬ ‭3T3 murine embryonic fibroblasts (abino mice)‬ ‭○‬ ‭murine/ rodents - easier to access and commercially available‬ ‭‬ ‭(desirable) human cell lines: more clinically relevant, but more expensive and requires strict ethics clearance‬ ‭‬ ‭Tissue-based cells‬‭attach‬‭to a surface in order to function‬ ‭○‬ ‭Prefer the material →‬‭stick flat and fast‬ ‭○‬ ‭Dislike → remains rounded, eventually stick‬ ‭○‬ ‭Stick onto their ECM‬ ‭○‬ ‭Morphology: fibroblast-like, endothelial-like‬ ‭‬ ‭Non-adherent cells (i.e. monocytes, leucocytes, bacteria, etc.) are not used in cytotoxicity/ cytocompatibility‬ ‭testing‬ ‭○‬ ‭As they dont need to attach to surface in order to function‬ ‭○‬ ‭Some can differentiate to become adherent cells tho‬ ‭‬ ‭Purpose: in vitro characterisation of cells under the presence of biomaterial‬ ‭○‬ ‭Qualitative and quantitative check: dead/alive, proliferation rate, how they attach, morphology‬ ‭‬ ‭Replace culture medium (max 7 days) - not to overcrowd and supply enough nutrients‬ ‭Cytotoxicity/ cyticompatibility tests‬ ‭Direct‬ ‭Indirect‬ ‭Cells are exposed in the material’s environment/ its extract‬ ‭‬ C ‭ ells are seeded directly on the material and‬ ‭‬ L‭ eaves material in the solution allow material to‬ ‭placed into the cell culture medium‬ ‭release anything, take the material out and put cells‬ ‭‬ ‭Material topography (mechanobiology) & surface‬ ‭into the extract (then analyse culture medium)‬ ‭chemistry may influence result - need to do control‬ ‭‬ ‭More about the release of degradation byproducts (+‬ ‭(surface polish)‬ ‭chemicals)‬ ‭Qualitative‬ ‭‬ ‭Morphology: stretched/ rounded/ burst (apoptosis)/ absence‬ ‭‬ ‭Cell density: proliferation‬ ‭‬ ‭Results are time-point dependent‬ ‭‬ ‭Usually done ~ 24 hours or in 3-7 days‬ ‭‬ ‭To show cell structures (nucleus, cytoskeleton spread)‬ ‭○‬ ‭Phalloidin‬‭to dye‬‭actin‬‭(cytoskeleton)‬  ‭ ‬ ‭DAPI‬‭to dye‬‭nucleus‬‭(4,6-diamidino-2-phenylindole) - nucleus [adenine-thymine-rich]‬ ○ ‭○‬ ‭Hoescht‬‭binds‬‭DNA‬‭(nucleus)‬ ‭‬ ‭To check dead or alive thru cell membrane damage‬ ‭○‬ ‭Trypan blue‬‭stains‬‭damaged‬‭cells;‬‭leaves healthy cells clear‬ ‭○‬ ‭Usually used in cell counting during set-up, before placing cell on material (k cell conc. and # of cell)‬ ‭○‬ ‭Calcein acetoxymethyl (AM)/ fluorescein diacetatee (FDA)‬‭stains‬‭live cells‬ ‭‬ ‭Permeates cell membrane, fluoresces when reacting with enzyme‬ ‭‬ ‭Healthy cells retain the product within cell (damage cell leaks products out)‬ ‭○‬ ‭Propidium iodide (PI) / ethidium homodimers‬‭stains‬‭dead cells‬ ‭‬ ‭Infiltrate nucleus membrane and binds to DNA‬ ‭‬ ‭Perform‬‭semi-quantitative analysis‬‭by counting live+dead cells (cell viability)‬ ‭○‬ ‭Show difference of %live/dead cells between different material viscosity‬ ‭○‬ ‭Image analysis of cells (size, angle of alignment, spread)‬ ‭All techniques require a translucent substrate‬‭- light transmission of material (as they are light based techniq)‬ ‭‬ ‭>> Opaque materials (metal, ceramics) → use SEM‬ ‭‬ ‭Cell fixation for keeping shape stable prior to SEM using‬‭osmium tetroxide‬‭(toxic), then dehydrated for‬ ‭vacuum environ‬ ‭○‬ ‭Cells could be destroyed by electron beams & vacuum‬ ‭○‬ ‭Could also be done by freeze drying (but may risk damaging cell integrity)‬ ‭‬ ‭No colour - never fully sure cell is dead/alive‬ ‭○‬ ‭But can identify shape and filopodia‬ ‭○‬ ‭Colour is post-processing‬ ‭Quantitative‬ ‭‬ ‭ISO-10993‬ ‭‬ ‭Good for seeing cell condition (not number of cells), limited by field of view‬ ‭‬ ‭MTT assay‬‭- indicates cytocompatibility of material‬ ‭○‬ ‭Add‬‭MTT or MTS‬‭dye to cell,‬‭mitochondrial activity‬‭converts to a purple formazan solution‬ ‭○‬ ‭Level of purple absorbance detected by spectroscopy - proportional to # of cells‬ ‭○‬ ‭Used in direct/ indirect test, done at multiple time points‬ ‭‬ ‭Modified uracil - measure cell proliferation‬ ‭○‬ ‭BrdU Binds to proliferating DNA‬ ‭○‬ ‭Uses fluorescent antibodies tagged on BrdU‬ ‭RT-PCR - real-time polymerase chain reaction‬ ‭‬ ‭Quantitative relative values of # of gene‬ ‭‬ ‭Cells express genes that are relevant to the tissue in which it resides‬ ‭○‬ ‭Cell expresses genes, producing mRNA in transcription‬ ‭‬ ‭Gene can be quantified wrt a control/ housekeeping gene‬ ‭○‬ ‭i.e. a cell expresses more bone genes, genes related to bone expression goes up (e.g. osteopontin,‬ ‭Runx2, osteocalcin, BMP)‬ ‭○‬ ‭Tendon-related gene (tenascin, tenomodulin, scleraxis)‬