High Throughput Techniques for the Investigation of Cell–Material Interactions - Biointerfaces: Where Material Meets Biology - page 275

the uniform pAl layer in 5% aqueous phosphoric acid using a vertical motion
stage at a constant velocity, resulting in pore sizes ranging from 5 nm to
3 mm. A detailed description of this pAl gradient preparation method is
described below. This controlled immersion technique is arguably more
versatile than other pAl fabrication techniques described above, given the
ease of extending the length and steepness of the pAl gradient.
A general method for pAl gradient formation is:

d n 3 r 4 n g | 7

1. Al foil should be cleaned by sonication in acetone for 30 min followed
by submersion in a 5% NaOH solution for 10 s and rinsing with co-
pious amounts of MilliQ water and drying in a nitrogen stream.
2. Porous alumina (pAl) with a uniform pore size is first generated by
anodic etching in a custom-built reaction cell. The Al foil and cell are
immersed in an oxalic acid solution (0.3 M, 0.0 0.1 1C) and vigorously
stirred. A voltage is applied between the gold back contact and the lead
counter-electrode (100 V) for 2 min.
3. Following etching, the pAl was rinsed with MilliQ water and then in-
cubated in toluene for 20 min and dried with nitrogen. This step sig-
nificantly decreased the degree of wicking observed by the phosphoric
acid solution.
4. Membranes were then converted into pAl gradients via pore enlarge-
ment using chemical etching. 138 This chemical etching was performed
by fixing the pAl membrane to a vertical motion stage and subsequently
slowly dipping them into a solution of phosphoric acid (5%, 25 1C,
gentle stirring) over a period of 180 min at a constant velocity
(0.0012 mm s 1 ).
5. Following complete immersion, the surfaces were immediately washed
with copious amounts of MilliQ water and dried with a gentle nitrogen
stream.
6. pAl substrates were found to be stable in aqueous conditions for at
least 21 days following incubation in PBS at 37 1C without any further
surface modifications.
7. The steepness and pore size range of the pAl gradient can be controlled
through adjustment of the dipping velocity into the phosphoric acid
solution, with a velocity of 1.2 mms 1 (corresponding to a maximum
incubation time of 180 min) proving to be optimal to cover the largest
pore range. Longer incubation times (4200 min) led to complete
pore collapse. Conversely, whilst a shorter maximum incubation time
( o 150 min) led to the successful preparation of a gradient surface, a
smaller pore range was achieved.
8. One of the key advantages of this method is the ability to extend the
length of the gradient by varying the dipping velocity in accordance
with the increased length of the gradient desired.

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It has been shown that the pore size not only influences the attachment
of cells but also the morphology and differentiation of cells. 116,118,139-143