protein adsorption affects osteoblast- glass adhesion strength as measured by colloidal probe atomic...
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Protein Adsorption Affects Osteoblast-Glass Adhesion Strength as Measured by Colloidal Probe Atomic Force Microscopy
Jackson CahnWhitman 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 0.5 mm
10x objective 20x 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
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
Fadmax
:NIST borosilicate glass, Potter glass modified with (CH3)2Cl2Si
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
O
Si
O
Si
O
O OH
O
Si
O
Si OHO
Si
O
Si
O
O O
O
Si
O
Si O Si
Si
CH3
H3CCH3
O
CH3
O
Si
Si
n
CH3
CH3
Cl
Cl
CH3
CH3
Introduction Previous Findings Time-resolved Adhesion Cleaning
Results
n= 8 13 4 6 6 8 3 3 2 7 2 3 1 2 1 1
Shapes: Evidence of Contamination
Only ever on the first contact with a cell!
Introduction Previous Findings Time-resolved Adhesion Cleaning
Previous Data--ContaminationSphere cleanliness-dependence of adhesion at 15s conctact
1
10
100
NIST In Media NIST In MEM HPhob in Media HPhob in MEM
Fad
max
(n
N)
1st Touch Not 1st Touch
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 DataRepeated 15s Adhesion Measurements using a Clean NIST Glass Tip in α-
MEM over 60min
0
10
20
30
40
50
60
0 10 20 30 40 50
Fad
max
(n
N)
Final Plateau
∆Fadmax
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 PlateauFinal Plateau
0
1
2
3
4
5
6
7
8
NIST in Media NIST in MEM Hphob in Media Hphob in MEM
F ad
ma
x (
nN
)
Initial Results
0
1
2
3
4
5
6
7
8
NIST in Media NIST in MEM Hphob in Media Hphob in MEM
F ad
ma
x (
nN
)
Final Plateau
0
1
2
3
4
5
6
7
8
NIST in Media NIST in MEM Hphob in Media Hphob in MEM
Fad
max
(n
N)
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 CoverageEffect of Contamination (∆Fadmax)
-120
-100
-80
-60
-40
-20
0
20
NIST in Media NIST in MEM Hphob in Media Hphob in MEM
∆F ad
max
(n
N)
Effect of Contamination (Time)
0
5
10
15
20
25
NIST in Media NIST in MEM Hphob in Media Hphob in MEM
Tim
e to
Co
mp
let
Co
nta
min
atio
n (
min
)
Introduction Previous Findings Time-resolved Adhesion Cleaning
Repeated 15s Adhesion Measurements using a Clean NIST Glass Tip in α-MEM over 60min
0
10
20
30
40
50
60
0 10 20 30 40 50
Fad
max
(n
N)
Results--Coverage--Sample ResultsRepeated 15s Adhesion Measurements using a Clean NIST Glass Tip in α-
MEM over 60min
0
10
20
30
40
50
60
0 10 20 30 40 50
Fad
max
(n
N)
Repeated 15s Adhesion Measurements using a Clean NIST Glass Tip in Medium over 90min
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80 90
Fad
max
(n
N)
Repeated 15s Adhesion Measurements using a Clean Hydrophobic Glass Tip in Medium over 75min
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70
Fad
max
(n
N)
Smaller decrease in force Faster decrease in force
Increase in force with coverage
NIST glass in α-MEM NIST 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