Protein Adsorption Affects Osteoblast- Glass Adhesion Strength as Measured by Colloidal Probe Atomic Force Microscopy Jackson Cahn Whitman College

Introduction Previous Findings Time-resolved Adhesion Cleaning Goals  Measure the adhesion forces between individual bone cells (MC3T3-E1) and different types of glasses (bioglasses)  Perfect the method so that these evaluations can be consistent and easily compared  Identify environmental factors possibly affecting measurements 0.5 mm 10x objective20x objective

Atomic Force Microscopy (AFM)  Usually, this technique is used to scan a surface and generate an image.  However, the ability to correlate displacement of the cantilever to deflection of the laser allows for high precision force measurement. Image: Wikimedia Commons Introduction Previous Findings Time-resolved Adhesion Cleaning

Colloidal Probe AFM  AFM cantilevers are made from silicon nitride.  To investigate other materials, we need to modify the tip.  This also makes for a larger contact surface for the cell. Glass Bead Epoxy ~50 µm Introduction Previous Findings Time-resolved Adhesion Cleaning

A Typical Force Curve  Start with the sphere indenting the cell, leave it in contact for a specified contact time  At first, there will be a strong repulsive force due to the compressive elasticity of the cell  As the sphere leaves the cell surface, adhesion will provide a strong negative force  At a sufficient distance, these contacts will separate and there will no longer be any force  Eventually the piezo reaches its highest point and begins to descend back towards the cell  There is no force on the probe as it descends until it makes contact with the cell surface  As the probe indents the cell again, the constant compliance (linear) region resumes F admax

: NIST borosilicate glass, Potter glass modified with (CH 3 ) 2 Cl 2 Si Previous Findings  When I arrived: 127 Force Curves As they had been collected, the procedure had been continually tweaked 3 variables: ▪ Contact Time (15s, 60s, 300s, 900s) ▪ Sphere type ▪ Culture Medium Composition (α-MEM, Media) (NIST, Hydrophobic) α-MEM has: Salts Glucose Ribonucleosides Vitamins Other Nutrients Media adds: 10% Fetal Bovine Serum (v/v) Kanamycin Antibiotic Introduction Previous Findings Time-resolved Adhesion Cleaning

Results n=

Shapes: Evidence of Contamination Only ever on the first contact with a cell! Introduction Previous Findings Time-resolved Adhesion Cleaning

Previous Data--Contamination Introduction Previous Findings Time-resolved Adhesion Cleaning

Time-resolved Adhesion  Repeat 15s adhesion measurements on different cells using the same tip and plot them sequentially  Observe cleanliness effects  Look for trends due to pH, temperature changes  Account for differences in tip geometry, cantilever k, etc. Introduction Previous Findings Time-resolved Adhesion Cleaning

Sample Data Final Plateau ∆F admax Time to complete coverage Introduction Previous Findings Time-resolved Adhesion Cleaning

Time-resolved Adhesion--Results  Final Plateau: Higher adhesion in α-MEM Lower adhesion on hydrophobic glass Data agree with initial results  Protein Coverage: Adhesion decreases more in α-MEM Adhesion decreases more on NIST glass Hydrophobic glass in media actually shows increased adhesion with coverage Data agree with initial results  Protein Coverage Time: Coverage time increase in α-MEM Coverage time increases with hydrophobic glass Introduction Previous Findings Time-resolved Adhesion Cleaning

Results--Final Plateau Introduction Previous Findings Time-resolved Adhesion Cleaning

Time-resolved Adhesion--Results  Final Plateau: Higher adhesion in α-MEM Lower adhesion on hydrophobic glass Data agree with initial results  Protein Coverage: Adhesion decreases more in α-MEM Adhesion decreases more on NIST glass Hydrophobic glass in α-MEM actually shows increased adhesion with coverage Data agree with initial results  Protein Coverage Time: Coverage time increase in α-MEM Coverage time increases with hydrophobic glass Introduction Previous Findings Time-resolved Adhesion Cleaning

Results--Protein Coverage Introduction Previous Findings Time-resolved Adhesion Cleaning

Results--Coverage--Sample Results  Smaller decrease in force  Faster decrease in force  Increase in force with coverage NIST glass in α-MEMNIST glass in mediumHydrophobic in medium Introduction Previous Findings Time-resolved Adhesion Cleaning

Time-Resolved Adhesion--Conclusions  Decrease is due to protein adsorption  Proteins responsible for adhesion may be hydrophilic in nature  After coverage, results are consistent and significant  Cells under serum starvation have increased adhesion  Cells bond more strongly to hydrophilic glasses Introduction Previous Findings Time-resolved Adhesion Cleaning

Recommendations  For an in vivo application, protein coverage will be immediate and irreversible  Therefore, when evaluating materials the post-coverage results are more significant, and also more consistent  Making recordings in media, make at least 5 contacts before data is considered valid Introduction Previous Findings Time-resolved Adhesion Cleaning