Biomaterials Translational ›› 2023, Vol. 4 ›› Issue (2): 115-127.doi: 10.12336/biomatertransl.2023.02.006
• RESEARCH ARTICLE • Previous Articles
Hanyu Chu
), Ying Bai)
Received:2023-06-04
Revised:2023-06-15
Accepted:2023-06-20
Online:2023-06-28
Published:2023-06-28
Contact:
Daping Quan, About author:Daping Quan, cesqdp@mail.sysu.edu.cn;
Figure 1. Schematic diagrams illustrate the preparation of the DM-GelMA bioink for extrusion-based bioprinting. The upper diagram shows the preparation of dECM solution. The middle diagram shows the temperature-controlled flow-focusing microfluidic device to prepare cell-laden DMs. The lower diagram shows the preparation of DM-GelMA composite bioink and its application in extrusion-based bioprinting. Created using 3D Max 2021. dECM: decellularised extracellular matrix; DM: decellularised extracellular matrix microgel; GelMA: gelatin methacryloyl; HFE: HFE: hexane, 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-d odecafluoro-2-(trifluoromethyl); PFO: 1H,1H,2H,2H-perfluoro-1-octanol.
Figure 2. Preparation of the cell-laden DMs. (A) The design of the microfluidic device for high-throughput generation of microgels. Created using AutoCAD 2021. (B) Enlarged view of microfluidic chip during water-in-oil emulsification. (C, D) Photographs of DM collection after gelation (C) and PFO-based transfer into aqueous solution (D). (E) Live/dead staining showing the viability of cells encapsulated in the DMs, green: live cells, red: dead cells. (F) The diameters of the DMs were highly dependent on the flow rate ratio (Qoil/Qaqu) during emulsification. Data are expressed as mean ± SD. (G) Distribution of the DM diameters at Qoil/Qaqu = 10:1. Representative fluorescence micrographs showing the DMs containing the PC12 cells pre-labelled with Cell Tracker green fluorescent dye, when the DMs were dispersed in the (H) oil and (I) aqueous phases, respectively. (J) Fluorescence staining showed the cytoskeletal of PC12 cells encapsulated in the DMs, green: F-actin, blue: DAPI. The dashed lines circle out the DMs with pre-encapsulated PC12 cells. Scale bars: 100 μm. DAPI: 4′,6-diamidino-2-phenylindole; DMs: decellularised extracellular matrix microgels; PFO: 1H,1H,2H,2H-perfluoro-1-octanol; Qoil: the flow rate of the oil phase; Qaqu: the flow rate of the aqueous phase.
Figure 3. Rheological properties of the DM-GelMA composite hydrogel. (A) Rheology assessments showed shear-thinning behaviors evident in both DM-GelMA and GelMA hydrogels. (B) The shear moduli of both DM-GelMA and GelMA hydrogels, UV light was turned on 60 seconds after the rheology test had begun. G' (solid lines): storage modulus, G” (dashed lines): loss modulus. (C) Storage moduli of both DM-GelMA and GelMA hydrogels before and after UV photocrosslinking. Data are presented as means ± SD. **P < 0.01 (Student's t-test). (D) Degradation of both DM-GelMA and GelMA hydrogels presented as the ratio of residual mass during 3 weeks of PBS immersion. m0: the original dry mass, m': the mass of the lyophilised hydrogel at each time point. DM: decellularised extracellular matrix microgel; GelMA: gelatin methacryloyl; PBS: phosphate buffered saline; UV: ultraviolet.
Figure 4. Extrusion of the DM-GelMA hydrogel and preservation of DM morphology. (A) The relationship between different sized needles and their corresponding maximum extrudable DM sizes. DN is the inner diameter of the needles, and Dm is the maximum size of the DMs. The dashed line represents a specific condition when the inner diameter of the needle is equal to the DM size. (B) The schematic diagram illustrates the changes in microgel morphology after extrusion. Created using Microsoft PowerPoint 2020. (C) The circularity of DMs post-extrusion using different sized needles, and representative fluorescence micrographs show the extruded microgels pre-labelled with FITC. Scale bars: 200 μm. Data are expressed as the mean ± SD. **P < 0.01 (one-way analysis of variance followed by Tukey's post hoc analysis). (D) The 3D bioprinted filaments extruded from 410- and 210-μm needles, respectively. (E) The density of microgels in the 3D bioprinted filaments using 410- and 210-μm needles. Data are expressed as the mean ± SD. *P < 0.05 (Student's t-test). DM: decellularised extracellular matrix microgel; FITC: fluorescein isothiocyanate; GelMA: gelatin methacryloyl.
Figure 5. Printability of the DM-GelMA bioink. (A) The DM-GelMA hydrogel was extruded continuously and smoothly, showing a proper-gelation condition for 3D printing. The (B) top-view and (C) side-view of a representative 3D printed grid mesh using DM-GelMA hydrogel. (D) The grid framework 3D printed by DM-GelMA hydrogel, observed by optical microscopy. (E) A representative fluorescence micrograph showed the FITC-labelled DMs (green) embedded in the 3D printed construct. Scale bar: 10 mm (B, C) and 1 mm (D, E). (F) The Pr of the DM-GelMA bioink (“Experiment”) compared with a regular square shape (“Model”, Pr = 1), analysed accordingly to a previously reported semi-quantitative method. 3D: three-dimensional; DM: decellularised extracellular matrix microgel; FITC: fluorescein isothiocyanate; GelMA: gelatin methacryloyl; Pr: printability.
Figure 6. Cell viability in the modular bioinks. (A) Representative fluorescence micrographs after live/dead staining on the PC12 cell-encapsulated bioinks and 3D cultured for 4 days. “GelMA+Cells” represents the bioink that PC12 cells were directly mixed with GelMA solution. “DM/(GelMA+Cells)” represents the bioink consisting of prepared DMs and PC12-cell-encapsulated GelMA solution. “(DM+Cells)/GelMA” denotes the bioink prepared by mixing PC12-cell-encapsulated DMs and GelMA solution. Green: live cells; red: dead cells. (B) Cell viability of the PC12 cells within the abovementioned bioinks based on the fluorescence images shown in A. Data are expressed as the mean ± SD. ***P < 0.001 (one-way analysis of variance followed by Tukey's post hoc analysis). (C) Representative fluorescence micrographs after live/dead staining on the bioprinted filaments using cell-laden GelMA and DM-GelMA bioinks, respectively. Green: live cells; red: dead cells. Scale bars: 500 μm. (D) Post-printing cell viability based on the fluorescence images shown in C. Data are expressed as the mean ± SD. ***P < 0.001 (Student's t-test). DM: decellularised extracellular matrix microgel; GelMA: gelatin methacryloyl; ns: no significance.
Figure 7. 3D bioprinting of multiscale DM-GelMA bioink for RSC96 cells and HUVECs co-culture. (A) Schematic illustration of extrusion-based bioprinting using HUVECs and RSC96 cell-laden DM-GelMA bioink. Created with 3D Max 2021. (B, C) 2D views (B) and 3D views (C) of the bioprinted 3D constructs using the cell-laden DM-GelMA composite bioink, characterised by confocal laser fluorescence microscopy. RSC96 cells and HUVECs were pre-stained with green and orange Cell Tracker Fluorescent Probes, respectively, prior to cell encapsulation. Scale bars: 200 μm. 2D: two-dimensional; 3D: three-dimensional; DM decellularised extracellular matrix microgel; GelMA: gelatin methacryloyl; HUVEC: human umbilical vein endothelial cell.
Additional Figure 1. Cell viability of the PC12 cells encapsulated in the microgels. Data are presented as means ± SD. ns: no significance.
Additional Figure 2. The micrographs and the distribution of the diameters of the microgels for Qoil/Qaqu = 2.5, 5, 7.5, 10, 15, and 20, respectively. Series of DM preparation showed that the microgel size was highly dependent on the corresponding flow rate ratio, Qoil/Qaqu. With increasing the ratio Qoil/Qaqu, the diameter of the generated microgels decreased gradually. The average diameters of the microgels were 128 ± 7, 114 ± 8, 107 ± 8, 101 ± 5, 82 ± 6, and 77 ± 4 μm for Qoil/Qaqu = 2.5, 5, 7.5, 10, 15, and 20, respectively.
Additional Figure 3. The number of cells encapsulated in each microgel and its corresponding frequency of appearance when observed using confocal laser microscopy (n ＞ 300).
Additional Figure 4. The viability of encapsulated PC12 cells decreased with increasing centrifugation time. (A) Representative fluorescence micrographs of the DM encapsulated cells after 0.5, 1, and 2 minutes of centrifugation, respectively. Characterised by live/dead staining. Green: live cells, red: dead cells. Scale bar: 100 μm. (B) Cell viability of the PC12 cells encapsulated in the microgels after 0.5, 1, and 2 minutes of centrifugation, respectively. The cell viability was calculated based on the fluorescence images shown in A. Data are expressed as the mean ± SD. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc analysis). DMs: decellularised extracellular matrix microgels.
Additional Figure 5. 1H-NMR characterisation of gelatin and GelMA. The characteristic peak of the methacrylate vinyl group at 5.4 and 5.7 ppm, as well as the minor peak shown at 2.9 ppm (the protons of methylene of lysine) verified the successful synthesis of GelMA. 1H-NMR: 1H-nuclear magnetic resonance; GelMA: gelatin methacryloyl.
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