BE 450 – Physical Property Characterization & Testing of Biomaterials

Introduction to BE 450: Biomaterials & Biocompatibility

• Course focus: relationships among material analysis, biocompatibility, and biomaterial design.
• Central theme: understanding how physical and chemical properties dictate in-vivo performance and safety.
• Three broad analytical pillars introduced:
  • Physical properties
  • Chemical properties
  • Biocompatibility metrics (toxicity, protein adsorption, hemocompatibility, cellular & animal response)

Aspects of Biomaterial Testing

• Physical Properties
  • Mechanical behavior, hydrophobicity, crystallinity, porosity, surface area, surface topography.
• Chemical Properties
  • Functional groups (amine, carboxyl), surface charge, elemental makeup, degradability/erosion.
• Biocompatibility
  • Cytotoxicity, protein adsorption profiles, blood compatibility, tissue/cellular response, small- & large-animal studies.

Physical Property Testing – Overview

• Six primary laboratory categories underscored:
  1. Mechanical testing
  2. Hydrophobicity & surface energy
  3. Degree of crystallinity
  4. Surface topography/roughness
  5. Porosity characterization
  6. Surface area measurements

Mechanical Testing – Rationale & Context

• Systematic engineering process to gauge performance under external forces & environments.
• Critical for:
  • Informing design decisions (geometry, thickness, reinforcement).
  • Guaranteeing safety/reliability (avoid fracture, fatigue, creep).
  • Accelerating new-material development (compare to benchmarks, iterate formulations).

Core Mechanical Test Modes

• Tensile / Compressive / Shear
  • Output: E (elastic modulus), ext{UTS} (ultimate tensile strength), strain-to-failure, ductility, toughness.
• Torsional – rotational stress.
• Cyclic / Fatigue – repeated load–unload to reveal endurance limit.
• Biaxial – simultaneous orthogonal loading; mimics tissues like skin, myocardium.
• 3- or 4-Point Bending – combined tension/compression; common for beams, bone plates.
• Rheological (viscometry) – viscous & viscoelastic response, esp. hydrogels.
• Wear/abrasion – resistance to repeated contact or articulating motion (joint implants).

Stress & Strain Fundamentals

• Stress ((\sigma)): load normalized by cross-sectional area.
• Strain ((\epsilon)) – deformation intensity.
  • Normal strain: \epsilon = \Delta L / L (dimensionless).
  • Shear strain: \gamma = d / h (radians).
  • Sign convention: tensile \epsilon > 0, compressive \epsilon < 0.
• Hooke’s Law in linear elastic region: \sigma = E \epsilon.
• Generic stress–strain curve terms:
  • Elastic region, plastic region, yield point, ultimate tensile strength (UTS), fracture point.

Instrumentation

• Instron (universal testing machine)
  • Interchangeable load cells (mN to kN ranges).
  • Accurate linear actuator; multiple clamps/fixtures.
• Biaxial testers
  • Use fiducial markers + optical tracking.
  • Often integrate temperature control & water bath for tissue samples.

Hydrophobicity

• Definition: tendency of a surface/material to repel water, influencing protein adsorption, cell attachment, drug loading/release & degradation kinetics.

Contact-Angle Methodology

• Contact angle ((\theta)): angle between tangent to liquid droplet and solid surface in air/vapor.
  • \theta > 90^{\circ} → hydrophobic.
  • \theta < 90^{\circ} → hydrophilic.
• Instrument: contact-angle goniometer (sessile drop, tilting plate, captive bubble variants).
• Example dataset referenced: corona-treated poly(ethylene terephthalate) (PET) analysis; showed surface-energy changes.

Crystallinity

• Degree & organization of crystalline regions impact:
  • Mechanical stiffness and brittleness.
  • Thermal transitions (glass transition, melting).
  • Chemical resistance & degradation rate.

Differential Scanning Calorimetry (DSC)

• Measures heat flow vs temperature to quantify latent transitions.
• Key temperatures:
  • T_g (glass transition) – onset of rubbery mobility.
  • T_c (cold crystallization) – exothermic crystal formation.
  • T_m (melting point) – endothermic fusion.
• Crystallinity % typically: \%Xc = \frac{\Delta Hm - \Delta Hc}{\Delta Hm^{\circ}} \times 100 where \Delta H_m^{\circ} = heat of fusion for 100 % crystalline reference.
• PLLA thermogram discussed; area under peaks translates to energy/gram.

X-Ray Diffraction (XRD)

• Probes atomic lattice by constructive interference of X-rays ((\lambda = 0.5{-}50\,\text{Å})).
• Supplies:
  • Miller indices (hkl) – crystallographic “address”.
  • Unit cell dimensions, lattice type (cubic, tetragonal, etc.).
• Diffraction pattern intensity vs 2θ distinguishes amorphous vs crystalline regions.
• Example: calcium molybdate (CaMoO(4)) shown; revealed dodecahedral (CaO(8)) & tetrahedral clusters reconstructed via Miller planes.

Surface Topography

• Surface architecture modulates host response, integration, and opportunities for chemical functionalization.
• Key metrologies: Surface Probe Microscopy (SPM), Electron Microscopy (EM).

Scanning Tunneling Microscopy (STM)

• Quantum tunneling current between conductive tip and sample enables atomic-scale mapping ((<0.1\,\text{nm}) Z-resolution).
• Operates in constant-height or constant-current modes.
• Requires conductive/semiconductive surfaces; provides crystallographic real-space images (e.g., graphene lattice).

Atomic Force Microscopy (AFM)

• Measures cantilever deflection due to tip–sample van-der-Waals, electrostatic, or chemical forces.
• Laser beam reflected into photodiode for pico-Newton force sensitivity.
• Variable tips:
  • Sharp/narrow → high lateral resolution.
  • Wide/blunt → broader, faster scans.
• Scan speed/resolution trade-off.
• Bioengineering applications:
  • Tip functionalization with ligands, antibodies, integrin-specific peptides.
  • Detect single-molecule binding events, quantify unbinding forces, observe enzymatic processes.
  • Micromechanical indentation: F = -k d, where k = cantilever spring constant, d = deflection.
  • High-speed AFM enables near-real-time biomolecular imaging.

Scanning Electron Microscopy (SEM)

• Electron beam accelerated toward specimen; electromagnetic lenses focus beam.
• Interaction types:
  • Secondary electrons → topographical contrast.
  • Backscattered electrons → atomic-number contrast (Z-contrast).
  • EDX (energy-dispersive X-ray) → elemental composition.
• Representative images: moth pollen; collagen/chitosan scaffolds.
• Limitations: high vacuum, charging of non-conductive/soft samples (necessitates conductive coatings like Au), difficulty with live cells.

Microscopy Scorecard (condensed)

Porosity & Surface Area (briefly referenced)

• Porosity: influences nutrient transport, tissue in-growth; measured by mercury intrusion, micro-CT, or BET gas adsorption.
• Surface area: critical for catalysis/drug loading; determined by BET or dye adsorption techniques.

Integrated Example – Tissue-Engineered Aortic Valve

• Multi-layer scaffold replicating valve architecture:
  1. Fibrosa analog (outer)
  2. Spongiosa analog (middle)
  3. Ventricularis analog (inner)
• Desired properties & corresponding assays:
  • Mechanical durability → uniaxial/biaxial tensile, cyclic fatigue.
  • Crystallinity tuning → DSC/XRD to modulate stiffness.
  • Cellular adhesion → surface hydrophobicity adjustment, ligand grafting; measured with contact-angle + protein adsorption studies.
  • Porosity mapping → SEM/µCT for interconnectivity, infiltration potential.
  • Biocompatibility → in-vitro cell culture, perfusion bioreactor, followed by in vivo implantation.

Cross-link to Chemical & Biocompatibility Themes

• Surface chemistry (amine/carboxyl functionalization) often optimized after physical profiles are confirmed.
• Hydrophobicity & topography jointly dictate protein adsorption layer, which in turn orchestrates cell behavior – the Vroman effect.
• Ethical/practical point: thorough bench-top validation minimizes animal usage by filtering inadequate formulations early.

Key Equations & Numerical References

• Normal strain: \epsilon = \Delta L / L.
• Shear strain: \gamma = d / h (radians).
• Hooke’s Law: \sigma = E \epsilon.
• AFM force: F = -k d.
• DSC crystallinity: \%Xc = \frac{\Delta Hm - \Delta Hc}{\Delta Hm^{\circ}} \times 100.
• Contact-angle hydrophobic range: \theta > 90^{\circ}.

Practical Take-Home Messages

• No single test suffices; multimodal characterization is compulsory.
• Matching in-vitro mechanical & surface behavior to in-vivo physiological loads/chemistry reduces failure risk.
• Choose microscopy/analytical modality balancing resolution, sample environment, and preparation constraints.
• Iterative feedback between material formulation (chemistry) and property measurements (physics) underpins successful biomaterial design.