MICRO-AND NANOENGINEERING APPROACHES TO DEVELOPING GRADIENT BIOMATERIALS SUITABLE FOR INTERFACE TISSUE ENGINEERING - Micro and Nanotechnologies in Engineering Stem Cells and Tissues - page 64

gradient structure can be generated using liquids with decreasing opacity. 83 Thus,
Wang et al. used a gray mask technology to generate a protein concentration gradient
on an aminated glass coverslip. 84 Using a conventional photomask, Li et al. built a
laminin density gradient on a poly(ethylene terephthalate) (PET) substrate by a two-
step UV irradiation method. 85 These authors first generated peroxides on the PET
surface with a first UV irradiation step and then grafted poly(acrylic acid) (PAA) onto
the PET surface with a second UV irradiation step. This process was completed by
covalently coupling the amino terminal groups of laminin proteins to the carboxyl
groups of PAA. The gradient was generated by moving the substrate below the UVat
a controlled speed during the first 3 min of the first UV irradiation, with the result that
different areas on the substrate received different amounts of UV and therefore had
different amounts of peroxides. When the coverslips were loaded with pheochro-
mocytoma PC12 cells and cultured for 2 days, a cell density gradient matching the
laminin density gradient was observed. Toh et al. built a single-density gradient of
biotinylated lectin concanavalin A (ConA-biotin) and a double-density gradient of
polysaccharide mannan and glycoprotein P-selectin on benzophenone (BP)-coated
glass coverslips. 86 The process involved BP-diradical generation via the UV
exposure of the substrate through a photomask with a simultaneous flow of
biomolecules. BP diradicals then formed covalent bonds with proximal biomole-
cules. A ConA-biotin gradient was generated by a shutter with a controlled closing
speed during UV irradiation, and the double gradients were generated by a controlled
rotating shutter. When the resulting materials were loaded with promyelocyte HL-60
cells and cultured for 2 h, a cell density gradient was observed following the
P-selectin gradient. When cells in suspension were flowed perpendicularly to the
P-selectin gradient, a cell rolling velocity gradient matching the P-selectin gradient
was observed. Photolithography can also be used to photopolymerize hydrogels, 87
which could provide a cross-linking density gradient 88 or a patterned gradient with
different molecules such as RGD or particles. Based on numerous research studies,
photolithography plays an important role in designing scaffold substrates suitable for
basic cell studies.
3.3.7 Microfluidics
Microfluidics allows for the patterning of 3D structures suitable for controlling
cellular functions. This patterning technique is closely related to microcontact
printing. Instead of stamping a polydimethylsiloxane (PDMS) mold with a relief
pattern of a master, a microfluidic network is stamped onto a substrate. In this
method, the microchannels are used to deliver fluids to selected areas of a substrate,
and the substrate is exposed to the flow, resulting in the patterning of the material.
This method is frequently used to pattern multiple components on a single substrate
and allows for the directed delivery of cells and soluble factors onto the substrate,
making it important for applications in cell biology, drug screening, and tissue
engineering. Unlike conventional in vitro cell culture methods, microfluidics can
produce miniature and complex structures mimicking the in vivo cellular environ-
ment, which is one of the merits of this technique.