Biomaterials Analysis Part I - Mechanical & Physical Characterisation PDF
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This document provides information about mechanical and physical characterization methods for biomaterials. It details techniques such as XRD (X-ray diffraction), FTIR (Fourier-transform infrared spectroscopy), and DSC (differential scanning calorimetry), along with their applications in determining properties like crystallinity and molecular bonds.
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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)
