Table 6.

Methods to Create Surface Density/Concentration Gradients of Peptides and Proteins for Tissue Engineering Applications

Fabrication
Technique	Process driving phenomenon(-a)	Additional processing	Primary gradient-shape controlling parameter(s)	Gradient scale	2D/3D	Gradient-shape control?	References
Soluble factor gradients
Boyden/Zigmond/Dunn chambers	Diffusion (in solution)	–	Time	Macroscale	2Da	Limited	159, 161, 162
Single-source/chamber (of bioactive factor) approaches	Diffusion (in macroporous gels)	–	Time	Macroscale	3D	Limited	157, 167
Dual-/multisource/chamber approaches	Diffusion (in macroporous gel)	–	Time	Macroscale	3D	Limited	150, 151, 160, 163
	Monomer flow, diffusion (in solution)	Microfluidicsb	Channel design, monomer flow	Micro- to macroscale	2D	Yes	168, 169, 171
Microprinting	Convection and diffusion (in macroporous gels)	Contact-less controlled microdispensing	Droplet ejection rate, stage translation rate	Macroscale	2D or 3D	Yes	148, 166
Soluble factor gradients (controlled release approach)b
Phosphatidyl choline–based lipid microtubules (LMTs) in gels	LMT loading in gels	Gelation by cooling	LMT loading profile (the step size)	Macroscale	3D	Limited	153
Microsphere-based scaffolds	Matrix preparation using microspheres loaded with the active factors	Melding the microspheres (using heat-sintering, ethanol-melding, or dichloromethane treatment)	Microsphere size, polymer properties (degradation, molecular weight, etc.)	Macroscale	3D	Limited	172, 173
Immobilized factor gradients
Single-source/chamber approaches	Diffusion (in macroporous scaffolds) (covalently bound)	EDC-NHS chemistry	–	Macroscale	3D	Limited	174
	Diffusion (adsorption on surface)	Capillary method	–	Macroscale	2D	Limited	146
Dual-source/chamber approaches	Diffusion (in gels)	Heterobifunctional crosslinker	–	Micro- to macroscale	3D	Limited	145, 153
Pump-/gravity-driven flow of factor solution	Convection (in macroporous scaffolds)	EDC-NHS chemistry	Suction rate	Macroscale	3D	Yes	174
	Convection (monomer flow)	Photopolymerization (factor entrapped)	Monomer flow rate	Micro- to macroscale	2D or 3D	Yes	149, 152
		Photopolymerization (factor covalently attached)	Monomer flow rate	Micro- to macroscale	2D or 3D	Yes	154–156
	Monomer flow, diffusion	Microfluidics, photopolymerization (factor covalently attached)/factor adsorbed	Channel design, monomer flow	Micro- to macroscale	2D	Yes	42, 147, 170
Capillary force–driven flow of factor solution	Monomer flow, diffusion (adsorption on surface)	Microfluidics (factor adsorbed)	Capillary forces, channel geometry, substrate affinity for the factor	Micro- to macroscale	2D	Limited	175
Grafting/micropatterning	Differential photoexposure (photomask)	Protein conjugation to a photoreactive species	Mask pattern	Micro- to macroscale	2D	Yes	158
	Controlled-time exposure	Gradients of protein conjugated to a photoreactive species	Photoexposure time controlled using a motorized stage	Micro- to macroscale	2D	Yes	66, 164
		Polymer graft density/thickness gradient (using motorized stage/photomask or differential dipping in an initiator solution [ATRP])	Photoexposure time, mask pattern and/or initiator exposure time (filling/removal rate)	Micro- to macroscale	2D	Yes	46, 67, 78, 177
	Computerized printing (inkjet printing)	–	Pattern design, substrate and solution properties	Macroscale	2D	Yes	176

^aSolution-based gradients can be generated in three dimensions. However, cells cannot be kept suspended and eventually attach to the substratum.

^bSome approaches of gradient generation have potential to provide spatial as well as temporal control, such as, microfluidic-based and controlled release approaches.

EDC: ethyl(dimethylaminopropyl) carbodiimide; NHS: N-hydroxysuccinimide; ATRP: atom transfer radical polymerization.