Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cells response to extracellular matrix

摘要

Introduction Periodontitis is an inflammatory disease that causes destruction in tooth supporting structure, periodontal defect and eventually tooth loss, negatively affecting up to 15% of adults worldwide, Although existing clinical therapies for periodontal disease (e.g., root surface conditioning, guided tissue regeneration) have shown benefits for controlling local inflammation of periodontium and the progression of periodontitis, they can not produce desirable tissue regeneration, Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). Materials and Methods Bioprinting-based approach was proposed to generate nano-liter sized 3D cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. Results and Discussion We first checked the viability of human PDLSCs in hydrogels with different GelMA/PEG ratio after 3-day culture. From live/dead staining, we observed that human PDLSCs encapsulated in GelMA/PEG hydrogels remained viable for all six compositions. We then quantified the cell viability by counting the number of live and dead cells. We found that cell viability decreased significantly with increasing volume ratio of PEG. Cell viability at day 3 after printing was 82.5±4.1 % in hydrogels with the GelMA/PEG volume ratio of 5/0, which was obviously higher than that of 0/5 (30%) hydrogel samples. Our observation is in agreement with the previous study where embryoid bodies encapsulated in PEG hydrogels reduced cell growth, while in GelMA hydrogels, higher call viability was obtained, This may be attributed to that GelMA and PEG exhibit different bioactivities, where the biodegradable GelMA is capable of mediating cell adhesion, proliferation and differentiation while PEG hydrogels are nondegradable and chemically inert. In addition, after culture for 3 days, human PDLSCs spread and elongated in first three hydrogels (GelMA/PEG volume ratio: 5/0, 4/1 and 3/2), which varied inversely with the increase in volume ratio of PEG. Especially, in 5/0 (GelMa/PEG, v/v) hydrogel, cells spread, elongated and formed interconnected networks with neighboring cells. While in the last three hydrogels (GelMA/PEG volume ratio: 2/3,1/4 and 0/5), human PDLSCs did not spread and elongate any more, just appeared in round shape. We also quantified cell spreading area using ImageJ and found that cell area reduced dramatically with decrease in GelMA and increase in PEG volume ratio concurrently. Conclusion In this study, we proposed a bioprinting-based approach to generate cell-laden hydrogel array with gradient composition for screening cell-ECM interaction in 3D. As a first verification, we printed GelMA and PEG sequentially to obtain hydrogel array with gradient composition by tuning the volume ratios of GelMA-to-PEG. The viability and multipotency of human PDLSCs were maintained during isolation and printing process. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratio of GelMA-to-PEG, where cell viability and spreading area decreased along with increasing ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.