An industrially scalable small molecule gelator with applications in tissue engineering and regenerative medicine

Abstract

INTRODUCTION Recently, a lot of research has been invested in the use of hydrogels in the endeavour of tissue engineering and regenerative medicine (TERM) as scaffolds mimicking the extracellular matrix (ECM).1 Polymer based hydrogels are being extensively investigated in the field;2 however, in the class of low molecular weight gelators (LMWGs), mainly peptide based materials are used.3 These are very interesting because of the functionality of the peptides as well as the smart material capabilities, well-defined structure, and ability to form dynamic systems of the LMWGs. Because of the many advantages of LMWGs but the costly and elaborate synthesis of peptides, this work focuses on the development of non-peptide based low molecular weight hydrogelators with applications in TERM. EXPERIMENTAL METHODS The synthesis of the gelators was performed in batch and in a planetary ball milling setup. The compounds were analysed using 1H NMR, 13C NMR, FTIR, ESI, and DSC. The gelation ability of the compounds was assessed by dissolving the gelators in water or cell culture medium at 100 °C followed by cooling the supersaturated solution to room temperature under irradiation with ultrasound. The mechanical properties of the gels were determined by performing strain and frequency sweep measurements as well as a recoverability study on a rheometer. The morphology of the gelator network was studied using AFM and SEM. The cytocompatibility of the gels was assessed by encapsulating L929 cells at a concentration of 1x106 cells/mL in gels made with complete cell culture medium and culturing them for 72 hours at 37 °C in a humidified incubator. Cell proliferation was assessed by counting the cells at 1, 24, 48, and 72 hours, and cell morphology was evaluated by fixing the cells within the gels and staining them with phalloidin and DAPI. RESULTS AND DISCUSSION The gelators were synthesized in quantitative yields, and the scalability of the synthesis was shown using the green industrial technique of ball milling. The minimal gelation concentration was determined to be 0.3 wt. % in both water and cell culture medium. The maximum strain of 0.8 wt. % gels was determined to be between 3.16 – 3.98 %, and the G’ and G” values were 1.7x104 Pa and 3.7x103 Pa, respectively, at 0.08 % strain and an angular frequency of 6.28 rad/s. A recoverability study (Figure 1) showed that the material was thixotropic and thus suitable for delivery via injection. Figure 1 AFM and SEM imaging confirmed that the gelator network consisted of ribbon-like nanofibers, with average fiber heights of 2 nm and fiber lengths varying from 500 to 20000 nm. The cytocompatibility assays showed that cells remained viable in the hydrogels for several days and that the cells proliferated during the first 48 hours of the experiment. CONCLUSION In this work, a non-peptide based LMWG was developed for use in TERM. The compound was shown to have a robust and scalable synthesis. The thixotropic property of the material showed that the hydrogels would be injectable. The fiber morphology was studied, and ribbon-like nanofibers formed the gel network. Last but not least, cells could survive in the material for several days, and they further proliferated. Thus, we believe this material could provide a low cost and cytocompatible hydrogel scaffold for cell expansion and minimally invasive delivery. REFERENCES 1. Berthiaume, F., et al., Annu. Rev. Chem. Biomol. Eng. 2:403-430, 2011 2. Van Vlierberghe, S., et al., Biomacromolecules 12:1387-1408, 2011 3. Hirst, A. R., et al., Angew. Chem. Int. Ed. 47:8002-8018, 2008status: publishe