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. 2022 Aug 4;8(4):599.
doi: 10.18063/ijb.v8i4.599. eCollection 2022.

Thermo-sensitive Sacrificial Microsphere-based Bioink for Centimeter-scale Tissue with Angiogenesis

Mingjun Xie  1   2   3 , Yuan Sun  1   2   3 , Ji Wang  1 , Zhenliang Fu  2   3 , Lei Pan  1 , Zichen Chen  2   3 , Jianzhong Fu  2   3 , Yong He  2   3   4   5
Affiliations

Thermo-sensitive Sacrificial Microsphere-based Bioink for Centimeter-scale Tissue with Angiogenesis

Mingjun Xie et al. Int J Bioprint. .

Abstract

Centimeter-scale tissue with angiogenesis has become more and more significant in organ regeneration and drug screening. However, traditional bioink has obvious limitations such as balance of nutrient supporting, printability, and vascularization. Here, with "secondary bioprinting" of printed microspheres, an innovative bioink system was proposed, in which the thermo-crosslinked sacrificial gelatin microspheres encapsulating human umbilical vein endothelial cells (HUVECs) printed by electrospraying serve as auxiliary component while gelatin methacryloyl precursor solution mixed with subject cells serve as subject component. Benefiting from the reversible thermo-crosslinking feature, gelatin microspheres would experience solid-liquid conversion during 37°C culturing and form controllable porous nutrient network for promoting the nutrient/oxygen delivery in large-scale tissue and accelerate the functionalization of the encapsulated cells. Meanwhile, the encapsulated HUVECs would be released and attach to the pore boundary, which would further form three-dimensional vessel network inside the tissue with suitable inducing conditions. As an example, vascularized breast tumor tissue over 1 cm was successfully built and the HUVECs showed obvious sprout inside, which indicate the great potential of this bioink system in various biomedical applications.

Keywords: Angiogenesis; Bioprinting; Gelatin methacryloyl; Large-scale tissue; Microsphere; Thermo-sensitive material.

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Conflict of interest statement

All authors declare no financial/commercial conflicts of interest.

Figures

Figure 1
Figure 1
Sketch of the preparation of TSM-B and formation of porous centimeter-scale tissue with angiogenesis.
Figure 2
Figure 2
Electrospraying process analysis of TSMs. (A) Post-treatment of the received gelatin microdroplets. (B) Images captured by high-speed camera. (C) Frequency of the droplet generation. (D) Force equilibrium of the gelatin microdroplets in the high voltage electric field. (E) Optical images of TSMs. (F) Diameters of TSMs. (G) Viscosity stabilization duration of electrospraying ink. (H) Shear-thinning profile of the electrospraying ink. (I) Thermo-crosslinking duration of TSMs at low temperature. (J) The stability of the crosslinked TSMs in further bioprinting temperature.
Figure 3
Figure 3
Rheological properties of TSM-B with different recipes. (A) Viscosity stabilization duration of GelMA precursor solution during rapid cooling and recovery process. (B) Shear-thinning profile of the GelMA precursor solution. (C) Results of low amplification oscillation frequency sweep. (D) Shear-thinning profile of TSM-B composed of TSMs with different diameters. (E) Results of low amplification oscillation frequency sweep of TSM-B composed of TSMs with different diameters. (F) Shear-thinning profile of TSM-B composed of TSMs with different volume proportions. (G) Results of low amplification oscillation frequency sweep of TSM-B composed of TSMs with different volume proportions.
Figure 4
Figure 4
Morphology of on-demand nutrient channels in centimeter-scale structure. (A) Solation transferring process of the TSMs in the culturing environment. (B) Confocal fluorescence microscope morphology of the on-demand nutrient channels distribution. (C) SEM morphology of the on-demand nutrient channels distribution.
Figure 5
Figure 5
Printability of TSM-B in extruding bioprinting. (A) Sketch of printability of TSM-B. (B) Printability of TSM-B and GelMA precursor solution. (C) Filament diameter generated by TSM-B. (D) 2D patterns printed by TSM-B. (E) 3D structures printed by TSM-B.
Figure 6
Figure 6
Growing of porous centimeter-scale breast tumor tissue printed by TSM-B. (A) Sketch of the sample observation. (B) Live/Dead testing of MDA-MB-231s. (C) F-actin and nucleus staining of MDA-MB-231s. (D) Viability of the loaded MDA-MB-231s. (E) Proliferation of the loaded MDA-MB-231s tested by CCK-8 kits.
Figure 7
Figure 7
Angiogenesis in the centimeter-scale structures printed with TSM-B. (A) Distribution of GFP-HUVECs at the start of culture. (B) Fluorescence microscope images of 3D sprout of GFP-HUVECs on the 5th day. (C) Optical microscope images of attachment and 3D sprout of GFP-HUVECs on the 2nd and 3rd day.
Figure 8
Figure 8
Confocal fluorescence microscope images of corresponding proteins. (A) CD31, (B) Vinculin, (C) β-tubulin, (D) VE-cadherin.
Figure 9
Figure 9
Bioprinting of centimeter-scale breast tumor tissue with angiogenesis.
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