Temple University, Lewis Katz School of Medicine, United States
Introduction:: The bone marrow (BM) is a complex tissue responsible for hematopoiesis that is involved in several diseases including leukemia, anemia, and HIV infection1. There is a need to develop new models of the BM to study disease pathology, test novel therapeutics and bridge the translation gap between the bench and bedside. Microfluidic devices offer the potential to generate complex 3D microenviroments of varying mechanical, biochemical and physical properties2. Recently, digital light processing (DLP) 3D printing has emerged as a favorable technique to synthesize these devices due to high resolution, cellular viability and biocompatibility with the generation of complex branching designs3-5. As such, we proposed 3D bioprinting with DLP microfluidic devices to mimic bone marrow tissues using a combination of polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), and hyaluronic acid methacrylate (HAMA). PEGDA, GelMA and HAMA were chosen for their mechanical tunability, printability, biocompatibility, biodegradability, and similarity to components of the BM6-10. We investigated the contribution of PEGDA molecular weight and DLP print settings on microchannel formation, scaffold porosity, device mechanical properties, and cell morphology11. Overall, these studies lay the foundation for the generation of a novel bone marrow-on-a-chip microfluidic device.
Materials and Methods:: 15 wt% PEGDA with different molecular weights (1000, 3400, and 6000 Da) were dissolved in PBS using the thermal energy effect until fully cleared. The solution was then mixed with 1 wt% HAMA, 4 mM LAP, 1.2 mM tartrazine, and 3 wt% GelMA were added to the solution and then incubated at 37 0C overnight. The solution was mixed using a rocker and then incubated again. The three constant printing parameters were power (20 mW/cm2), burn-in layer (4x), and printing layer of 50 µm. A model, Figure 1A, was used to investigate the lumen formation with the 3D printer DLP scaffold. Throughout the printing, the bioink was exposed to different exposure times (10 to 20 sec). The properties were calculated as a ratio of the lumen diameter to the expected dimension (600 µm). 20 sec exposure time was used to test the physical properties of the 3D disk. First, gels were imaged in SEM and the pore diameter was measured using Fiji. Next, the gels were compressed up to 30% strain of the initial height. Young’s modulus was calculated as a slope of the line between 10% and 20% strain. Lastly, the weight of the disks was measured over 14 days. Bone Marrow MSC-laden hydrogel (1 million cells/mL) were printed using 20-sec exposures, then cultured for 3 and 7 days. The cells were fixed, stained with Phalloidin and Hoechst (DAPI nuclei), and imaged using Nikon Microscope. The cellular volume and sphericity were calculated using Fiji.
Results, Conclusions, and Discussions:: The model, Figure 1A, was 3D printed using DLP with 4 different molecular weight groups (1000 Da, 3400 Da, 6000 Da, equal combination of the three) with three different exposures time (10, 15, 20 sec). The % printability was calculated as the ratio of the experimental dimension to the theoretical dimension (600 µm). Figure 1B showed that, while there is a significant difference among the exposure times, the combination of the three molecular weights had the highest printability % among the three times, especially for 15 and 20 sec. This may be due to the presence of all three different PEGDA molecular weights strengthening the lumen properties. Scaffolds were printed at an exposure time of 20 sec to explore the extreme physical properties (lowest pore size obtained, highest Young’s Modulus, and degradability properties from hydrolysis) of the gels. It was found, Figure 1C, that the lower the molecular weight of PEGDA had the higher the pore size (46.78 ±5.6 µm for 1000 Da vs 28.13 ± 2.907 µm for 6000 Da). It was found that the porosity of 3400 Da vs 6000 Da PEDGA scaffolds were not statistically significant and scaffolds which had PEGDAs of all three molecular weights had the lowest pore sizes (~10 µm). When mechanical properties were assessed, all the groups were extremely stiff ( > 100 kPa), Figure 1D with the highest modulus for the 3400 Da group. This may be due to presence of more unreacted macromer than photoinitiator which led to a reduction in the stiffness12. The weight of the gels found to be reduced by up to 38% in a period of two weeks due to the hydrolysis of PEGDA. We next analyzed the cellular morphology of bioprinted cells. The cellular volume increased by 2-fold and the sphericity of the cells decreased by 1.15-fold by 7 days in culture indicating the ability of the cells to reorganize/degrade the scaffold13. Future studies will examine the co-culture of stem cells and endothelial cells for the assembly of the bone marrow-on-a-chip microenvironment.
Acknowledgements (Optional): : The authors gratefully acknowledge use of facilities and instrumentation supported by the Materials Science and Engineering Departmental Laboratory at the University of Pennsylvania and NSF through the University of Pennsylvania Materials Research Science and Engineering Center (MRSEC) (DMR-1720530). This work was carried out in part at the Singh Center for Nanotechnology, which is supported by the NSF National Nanotechnology Coordinated InfrastructureProgram under grant NNCI-2025608. Funding support by NIH DA056172
References (Optional): : 1
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