Characterisation of Cell Adhesion to Substrate Materials and the Resistance to Enzymatic and Mechanical Cell-Removal
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Characterisation of Cell Adhesion to Substrate Materials and the Resistance to Enzymatic and Mechanical Cell-Removal Helen J Griffiths1, John G Harvey1, James Dean1, James A Curran1, Athina E Markaki1,2, and T William Clyne1 1 Department of Materials Science and Metallurgy, University of Cambridge, Pembroke St, Cambridge, CB2 3QZ, United Kingdom 2 Department of Engineering, University of Cambridge, Trumpington St, Cambridge, CB2 1PZ, United Kingdom ABSTRACT Cell-implant adhesive strength is important for prostheses. In this paper, an investigation is described into the adhesion of bovine chondrocytes to Ti6Al4V-based substrates with different surface roughnesses and compositions. Cells were cultured for 2 or 5 days, to promote adhesion. The ease of cell removal was characterised, using both biochemical (trypsin) and mechanical (accelerated buoyancy and liquid flow) methods. Computational fluid dynamics (CFD) modelling has been used to estimate the shear forces applied to the cells by the liquid flow. A comparison is presented between the ease of cell detachment indicated using these methods, for the three surfaces investigated. INTRODUCTION Various materials are used for implants [1]. They must be both mechanically suitable and biocompatible. For some implants, strong adhesion to surrounding tissue is desirable. Others require minimal adhesion. Tailoring of cell-implant adhesion is therefore required. Various techniques have been used to characterise adhesion [2-16]. Assessment methods fall broadly into two categories: global population studies and single cell experiments – see Table I. Table I: A Summary of techniques used to assess cell-substrate adhesion. Technique
Resistance to Enzymatic Removal Normal forces (1) Centrifuge
Global / Individual Cell
Cell type
Species
Substrate
Time of testing
Detachment Forces/ Stresses Measured
G
Osteoblast
Human
Ti6Al4V
24 hr – 21 days
-
Neural Erythrocyte Fibroblast Glioma Neural
Chicken Sheep Hamster Human Chicken
Cell-cell
0 – 30 min
ECM Proteins
0 – 60 min
Osteoblast
Mouse
Glass
I
Erythrocyte
Human
Cell-cell
1-100pN/cell
Evans et al [6, 7]
G
Fibroblast Endothelial Erythrocyte Fibroblast Endothelial
Mouse Human Human Mouse Human
Copolymers HEMA/EMA PS/PET ± ECM proteins Polymeric/glass ± ECMPs Glass ± ECMPs Glass/PET/PTFE ± ECMPs
2 hr 5 – 30 min 10 – 60 min 0.5 – 2 hr 20 min / 1 hr
0 - 0.18 Pa 0 - 9 Pa 0.02 - 1.5 Pa 5 - 10 Pa 0.3 - 20 Pa
Horbett et al [8] Pratt et al [9] Mohandas et al [10]
24 hr
530 – 750 Pa
G (2) Ultrasound Normal Forces (1) Micropipette Shear Forces (1) Spinning Disc (2) Parallel Plate Flow Chamber Shear Forces (1) AFM Focal Adhesion Area / Cell Spreading
I
Fibroblast
Mouse
Glass
I
Osteoblast Endothelial
Human Human
Polycarbonate Glass ± ECMPs
Anselme et al [2, 3]
~ 0.1 nN/cell Initial 0.1 nN/cell Post strengthening 3.6 nN/cell 1.47-1.55 μN/cell
hrs - days
-
McClay et al [4] Debavelaere-Callens et al [5]
Truskey et a [11-13] Yamamoto et al [14] Biggs et al [15] Tzoneva et al [16]
There hav
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