Photopolymerization of cell-laden gelatin methacryloyl hydrogels using a dental curing light for regenerative dentistry

Abstract

Photopolymerized hydrogels, such as gelatin methacryloyl (GelMA), have desirable biological and mechanical characteristics for a range of tissue engineering applications.

Objective: This study aimed to optimize a new method to photopolymerize GelMA using a dental curing light (DL).

Methods: Lithium acylphosphinate photo-initiator (LAP, 0.05, 0.067, 0.1% w/v) was evaluated for its ability to polymerize GelMA hydrogel precursors (10% w/v) encapsulated with odontoblast-like cells (OD21). Different irradiances (1650, 2300 and 3700mW/cm²) and photo-curing times (5-20s) were tested, and compared against the parameters typically used in UV light photopolymerization (45mW/cm², 0.1% w/v Irgacure 2959 as photoinitiator). Physical and mechanical properties of the photopolymerized GelMA hydrogels were determined. Cell viability was assessed using a live and dead assay kit.

Results: Comparing DL and UV polymerization methods, the DL method photopolymerized GelMA precursor faster and presented larger pore size than the UV polymerization method. The live and dead assay showed more than 80% of cells were viable when hydrogels were photopolymerized with the different DL irradiances. However, the cell viability decreased when the exposure time was increased to 20s using the 1650mW/cm² intensity, and when the LAP concentration was increased from 0.05 to 0.1%. Both DL and UV photocrosslinked hydrogels supported a high percentage of cell viability and enabled fabrication of micropatterns using a photolithography microfabrication technique.

Significance: The proposed method to photopolymerize GelMA cell-laden hydrogels using a dental curing light is effective and represents an important step towards the establishment of chair-side procedures in regenerative dentistry.

Keywords: Bioengineering; Biomedical and dental materials; Endodontics; Hydrogel; Odontoblast; Regenerative medicine; Visible light.

Figure 1

Example of application of GelMA hydrogel in regenerative dentistry. Synthesis of GelMA macromer (A), cell encapsulation (B), example intracanal hydrogel loading and photopolymerization, (C), and the resulting cell-laden hydrogel material (D).Note that although the schematic depicts an example for regenerative endodontics, the material can be used for any application of intra-oral regeneration, such guided periodontal regeneration, alveolar bone growth and others.

Figure 2

Chemical structures and cleavage of the photoinitiator 1-[4-(2-hydroxyethoxy)-phenyl]-2hydroxy-2-methyl-1-propanone (I2959) (A) and lithium phenyl-2,4,6-trimethyl- benzoylphosphinate (LAP) (B). Spectrum of UV and dental curing lights (C), and measurement of light intensities (D) used in this study. Note that light irradiance is sustained and stable over the time. SP - standard power, HP–high power, XP–extra power, and UV at 1.5 and 8.5 cm distance.

Figure 3

Effect of dental light intensities and exposure time (LAP at 0.067%,). Cell viability of OD21 encapsulated GelMA after 24 hours. The viability assay showed that cells were viable (>80%) when photopolymerized with SP5s (A), HP3s (C) and XP3s (D). The cell viability decreased when photo-curing time was increased to SP20s. Scale bar 400 μm.

Figure 4

Effect of photo initiator concentration. Cell viability of OD21 encapsulated GelMA after 24 hours. The viability assay showed that cells were viable (>80%) when photo-cured with SP5s and 0,05 (A) and 0,067% (B) (w/v) LAP. The cell viability decreased when LAP concentration was increased to 0.1% (C, D). Scale bar 400 μm. p<0.0001 (****).

Figure 5

Comparing DL and UV photopolymerization methods. DL (A, B, C, D) photopolymerized GelMA hydrogels at close distance faster than UV at 8.5 cm (E, F, G, H). GelMA was not photopolymerized after UV exposure to 5s.

Figure 6

Morphology (DL images A to D, UV images F to H) and pore size (E), degradation (I) and elastic modulus (J) of GelMA hydrogels. SEM images showed microporous structure induced during polymerization of GelMA hydrogels. The porous structure of DL photopolymerized hydrogels at different time points are larger than UV photopolymerized hydrogels. UV photopolymerized GelMA hydrogels degraded faster than DL photopolymerized GelMA hydrogels after 10s exposure. The elastic modulus increased with the exposure time for both DL and UV photopolymerized GelMA hydrogels. p<0.05 (*).

Figure 7

Cell viability of 3D GelMA hydrogels encapsulating OD21 cells after exposure to DL and UV and cultured in vitro for 1, 3 and 5 days. Cell viability increased after 5 days in vitro culture (A). Representative uorescent images of OD21 cells stained for live (blue) and dead (green) cells on day 5 (DL images B to E, UV images F to H). UV 5s did not polymerize. p<0.05 (*), p<0.01(**), p<0.001(***), p<0.0001(****).

Figure 8

Effect of UV light intensity. Cell viability of OD21 encapsulated GelMA exposed to UV light (A) at 1560 mW/cm², and DL (B) at 1560 mW/cm² for 5 and 10 seconds after 24 hours. The viability assay (C) showed that cells were viable when photopolymerized with DL for 5 s, and decreased when exposed to UV for 5 s. p<0.0001(****). Scale bar 400 μm.