Poster N5 - In situ clickable 3-D cell sheets with biomimetic fibrils for enhancing mechanistic robustness

Introduction: Owing to a comparative structure to the natural extracellular matrix, the fibrous matrices have been extensively studied as bioscaffolds for cell cultivation in a biomimic condition. Nanofibrils (NFs), which are fragments originated from electrospun nanofibers, enable a free migration of cells throughout the fibrous matrix, resulting in better cell infiltration, cell-matrix interaction, and cellular behaviors compared to conventional electrospun nanofibers. Thus, the complexes of cell-NFs have been employed in the treatment of osteochondral defects and peripheral nerve repair. However, due to the poor mechanical property, the implantation process of those complexes while maintaining their original morphology is challenging. The destruction of bioscaffolds may interfere the biological expression of cells and resulting in unexpected therapeutic effects. To overcome this difficulty, click reaction of tetrazine (Tz) and trans-cyclooctene (TCO), a powerful strategy for functional bioconjugation and modulation of crosslinked hydrogel within a short time, has been utilized. NFs are decorated with multi-arm PEG-Tz (PEG-3Tz) before cultivating with cells, then multi-arm PEG-TCO (PEG-8TCO) are added as a crosslinker to enhance to overall mechanical property of the complex.

Materials and

Methods: PEG-4COOH and PEG-8NH2 were conjugated with Tz-NH2 (PEG-3Tz) and TCO-NHS (PEG-8TCO), respectively, by amide coupling reaction. The degree of substitution (DS) of Tz and TCO were confirmed by 1H NMR, MALDI-TOF and fluorescamine assay. Poly(ε-caprolactone) nanofibrils (PCL NFs) were prepared by physical fragmentation of electrospun PCL nanofibrous meshes, and subsequently aminolyzed by 1 M ethylenediamine (EDA)/ MeOH mixture to functionalize the amine groups on the surface of PCL NFs (aNFs), which were further surface-decorated with PEG-3Tz by amide coupling reaction with the remained COOH moieties on PEG-3Tz (Tz@NFs). Tz@NFs were characterized using TGA, DLS for zeta-potential, and fluorescamine assay. The crosslinking between Tz@NFs and PEG-8TCO were investigated by FT-IR and swelling test. For in vitro studies, Tz@NFs were cultivated with human corneal epithelial cells (hCEC) and PEG-8TCO was added to strengthen the mechanical property of the complexes. To determine the enhanced mechanical property of the complexes prepared by different crosslinking conditions, these crosslinked complexes were orbitally shaken at 300 rpm for 45 min. The morphology of the complexes before and after shaken were recorded. After the mechanical test, the remained and broken parts of the complexes were collected separately to quantify the DNA content and total weight. Cell viability and DNA content of hCECs in non-crosslinked and crosslinked complexes were analyzed for 6 days and the expression of corneum-specific genes (keratin12, PAX6, and ALDH3A1) were evaluated by qRT-PCR.

Results, Conclusions, and Discussions: Tz and TCO groups were successfully modified on multi-arm PEG with the DS of 79.3% and 74.6%, respectively, which were confirmed by 1H NMR peaks at 7.6 and 8.4 ppm for Tz, and 5.5 ppm for TCO. MALDI-TOF results also showed that the molecular weight of PEG-3Tz and PEG-8TCO increased after the conjugation of Tz and TCO. After grafting PEG-3Tz on aPCL NFs, the intensities of C-O-C, amide I, and N-H peaks on FT-IR spectra of Tz@NFs at 1173, 1541, and 3439 cm-1, respectively, increased compared to those of aNFs. Meanwhile, the zeta potential and remained amine groups of Tz@NFs significantly decreased, suggesting that the amine groups of aNFs were reacted with COOH groups of PEG-3Tz. The early decomposition peak on TGA graph of Tz@NFs and the change of NFs color from white to light pink also indicated the presence of PEG-3Tz on NFs. After the click reaction between Tz@NFs and PEG-8TCO, the C-O stretching and N-H bending peaks at 1640 and 1530 cm-1 of the crosslinked NFs were clearly appeared on the FT-IR spectra. In addition, swelling ratio of the crosslinked NFs significantly decreased compared to that of non-crosslinked NFs. The double addition of PEG-8TCO to the hCEC-Tz@NFs complex after 12 and 24 h of cultivation showed better mechanical properties among all crosslinking conditions, which was confirmed by the negligible change of morphology after shaking, higher remained weight and DNA content. The viability, DNA content, and the expression level of specific genes of hCEC cultivated in crosslinked complex were higher than those of non-crosslinked one, which indicated that better integrity of the complex resulted in the enhanced cells interaction and cellular behaviors.

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