Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

Abstract

The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water‐based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well‐defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass‐transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme‐laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…This hydrogel network can be Reaction Chemistry & Engineering Review specifically designed by a careful selection of monomers and cross-linkers. 190,193 Next to others, this methodology benefits from high flexibility, easy automatization and fast implementation, especially compared to covalent immobilization techniques. Another positive aspect is the fact that hydrogels are stable at high temperatures and can operate in the presence of harsh reaction conditions.…”

Advances in 3D Gel Printing for Enzyme Immobilization

“…As already mentioned, there has been a need for developing 3D printed hydrogels with smaller and finer structures to reduce mass transfer limitations and improve the effectiveness factor of entrapped fast-acting enzymes [ 85 ]. Lahann and co-workers [ 80 ] developed a new 3D printing technique named “3D jet writing” to create micro-sized polymer hydrogel fibers oriented in a self-supporting 3D scaffold. At a glance, the equipment set-up looks like a combination of DIW and electrospinning, with a print head nozzle attached to a high voltage power supply and the platform grounded.…”

“…The success of 3D printed immobilized enzymes for biocatalysis hinges heavily on minimizing diffusion limitations that limit catalytic rates, which could partially be overcome by improving the printing resolution of popular economical techniques, namely hydrogel-based direct ink writing. There are already a number of developments, such as 3D jet writing [ 80 ] and melt electrowriting [ 81 ] for creating micro-sized enzyme-entrapped polymer fibers with well-defined 3D patterns with fine structural features to mitigate diffusion limitations and aid mixing. General progress in 3D printing technology development, i.e., higher resolution and speed, and lowering of the cost of the most advanced 3D printing technologies will also provide advances for enzyme immobilization.…”

“… ( A ) The 3D jet writing of hydrogel fibers yielding oriented hydrogel fibers loaded with enzymes (reproduced with permission from Ref. [ 80 ]). ( B ) Melt electrowriting of PCL with entrapped electrostatically stabilized enzymes/proteins: ( a ) photo of the fabric with area of 3 cm 2 ; ( b ) widefield microscopy view of the fine structures of the printed fabric with fiber thickness <5 μm; ( c ) enzymatic activity after the melt electrowriting process as shown by the fluorescent emission from the enzyme catalyzed hydrolysis reaction; ( d ) fluorescent plastics created by entrapping superfolder green fluorescent protein into PCL; ( e ) printed ordered grid structure with fluorescent PCL; and ( f ) the fluorescent plastics were used to print fabrics with repetitive tangles (reproduced with permission from Ref.…”

3D Bioprinted Scaffolds for Tissue Repair and Regeneration

“…Enzymes are proteins or RNAs produced by living cells that are highly specific and catalytically efficient for their substrates. Printing enzymes mixed with other polymer materials is a common method to improve their stability, activity and biomedical applications (Steier et al, 2020). A bioink prepared by mixing gelatin methacrylamide, tyrosinase and collagen has been studied for in vivo skin tissue bioprinting.…”