Chemical Engineering University of New Hampshire durham, New Hampshire, United States
Introduction: Microgel-based injectable hydrogels, which are produced by crosslinking (or annealing) the microgels, have recently received a lot of attention due to their ability to induce rapid cell adhesion and promote cell-cell interactions within the interstitial space. Although these approaches showed significantly improved cellular responses compared to the conventional nonporous injectable hydrogels, porosity of the hydrogel is limited by the interstitial pore space and the cellular growth is mostly excluded from the microgel phase. Use of microporous microgels and their assembly can increase porosity of the hydrogel and serve as better scaffolds for tissue engineering. However, most microgels are thermally unstable and the pore structures are lost during the cell encapsulation process at physiological temperature. In this research, we report a novel method of producing thermally stable microporous microgels that can be crosslinked to form a highly porous hydrogel. Cellular growth in interstitial space and within the microgels is demonstrated using human dermal fibroblasts (hDFs).
Materials and
Methods: Nonporous microgels of gelatin/alginate composite were produced using the water-in-oil emulsion method. These microgels were swelled in deionized water, followed by freeze-drying to obtain microporous microgels. The microporous microgels were pre-crosslinked in CaCl2 solution which ionically crosslinks alginate within the microgels. These microgels were crosslinked by microbial transglutaminase (mTG) at 37 C to form a bulk hydrogel. The presence of micropores within the microgels after the crosslinking process was confirmed by the use of fluorescently labeled microgels and 3D imaging by confocal microscopy. Cell viability and morphologies encapsulated in the hydrogel were monitored using live/dead assay on day 1 and 7 post-encapsulation. More detailed structures and distribution of cells were obtained by actin staining.
Results, Conclusions, and Discussions: results Confocal microscope images clearly showed the presence of micropores in the crosslinked assembly of microgels (Figure 1a). These microporous structures of microgels remained stable at 37 C during the 7 days period of cell culture. Without the ionic crosslinking by Ca2+, the microgels partially melted during the mTG crosslinking process and entirely lost the microporous structure (Figure 1b). When hDFs were encapsulated in the microgel assembly, the cells fully spread in the pore space and proliferated. Some cells were seen to grow within the pores of microgels (Figure 1c). Live/dead assay showed excellent cell viability with minimal cell death over 7 days. (data not shown) Conclusion We demonstrated the successful production of thermally stable microporous microgels which could be further crosslinked by mTG to form a bulk hydrogel without losing the original microporous structure. When hDFs were encapsulated in this microgel assembly, the cells fully spread and proliferated using both the interstitial pores and the pores within the microgels. These novel microgels are expected to demonstrate improved cellular response compared to the nonporous microgel-hydrogels and serve as excellent scaffolds for tissue engineering.
