Wenyi Li1 Jin Guo1 Shengjie Ling2 Ying Chen1 Chunmei Li1 Fiorenzo Omenetto1 David Kaplan1

1, Tufts University, Medford, Massachusetts, United States
2, ShanghaiTech University, Shanghai, , China

Well-designed micropatterns present in native tissues and organs involve changes in extracellular matrix compositions, cell types and mechanical properties to reflect complex biological functions. However, mimicking these micropatterns in vitro remains a challenge and the patterning strategies often showed limited guidance of cell orientation in relatively short culture periods. Silica-based micropatterns are popular in many biomedical fields including in vitro tissue models, due to the biocompatibility and high versatility of silica. Yet, harsh conditions (e.g. extremely high temperature and/or pressures) are often required to create silica patterns, and bonding between substrates is not strong enough. In this work, a de novo design strategy to code functional micropatterns to engineer cell alignment through the integration of aqueous-peptide inkjet printing and site-specific biomineralization is presented. Inkjet printing provides direct writing of macroscopic biosilica selective peptide-R5 patterns, which allow site-specific growth of silica nanoparticles through in situ biomineralization, with micrometer-scale resolution on the surface of a biopolymer (silk) hydrogel to achieve the alignment of human mesenchymal stem cell (hMSCs) and enhanced immobilization of bovine serum albumin (BSA).
To create the micropatterns, peptide-R5 was inkjet printed on the surface of enzymatically crosslinked silk hydrogels, followed by subsequent silicification to induce biosilica deposition. Linewidth and gap distance between each printed line were manipulated by adjusting drop spacing and drop volume during printing. Biomineralization was confirmed by examining silica nanoparticles covering the printed lines but not elsewhere. A 20 µm pattern gap distance and 1 µm linewidth were achieved. Well-defined peptide patterns on the substrate were also evidenced by printing fluorescein isothiocyanate (FITC)-labeled R5 and observed by fluorescence microscopy. hMSCs were cultured on the micropatterned hydrogels and specific alignment along the printed lines was noted, while the response on the unpatterned controls was randomized. Additionally, FTIC-labeled BSA and R5 were printed together and after 6 days of incubation in phosphate buffered solutions (PBS), BSA immobilized and aligned exclusively along the biosilica micropatterns, while the FITC-BSA alone extended over the surface, which suggests improved protein stability and alignment on micropatterns.
A de novo strategy to design functional micropatterns to engineer cell alignment and protein immobilization through inkjet printing and site-specific biomineralization was demonstrated. This cost-effective micropattern design scheme can meet a wide range of needs in the biomedical field with implications for broader material designs.