Rapid and efficient in vivo angiogenesis directed by electro-assisted bioprinting of alginate/collagen microspheres with human umbilical vein endothelial cell coating layer

Abstract

Rapid reconstruction of functional microvasculature is the urgent challenge of regenerative medicine and ischemia therapy development. The purpose of this study was to provide an alternative solution for obtaining functional blood vessel networks in vivo, through assessing whether hydrogel-based microspheres coated by human umbilical vein endothelial cells (HUVECs) can direct rapid and efficient in vivo angiogenesis without the addition of exogenous growth factors or other supporting cells. Uniform alginate microspheres with adjustable diameter were biofabricated by electro-assisted bioprinting technology. Collagen fibrils were evenly coated on the surface of alginate microspheres through simple self-assembly procedure, and collagen concentration is optimized to achieve the highest HUVECs adhesion and proliferation. Immunofluorescence staining and gene analysis confirmed the formation of the prevascularized tubular structure and significantly enhanced endothelial gene expression. HUVECs-coated hydrogel microspheres with different diameters were subcutaneously injected in immune-deficient mice, which demonstrated rapid blood vessel regeneration and functional anastomosis with host blood vessels within 1 week. Besides, microsphere diameter demonstrated influence on blood vessel density with statistical differences but showed no obvious influence on the area occupied by blood vessels. This study provided a powerful tool for rapid and minimal-invasion angiogenesis of bioprinting constructs and a potential method for vascularized tissue regeneration and ischemia treatment with clinically relevant dimensions.

Keywords: Angiogenesis; Human umbilical vein endothelial cells; Microspheres; Minimal-invasive; Vascular tissue engineering.

Figure 1

Schematic presentation of this study. Alginate microspheres with tunable properties were fabricated through the electro-assisted inkjet printing technology and coated with different concentrations of collagen fibrils. Human umbilical vein endothelial cells (HUVECs) adhered and proliferated on the surface of alginate/collagen microspheres to form an endothelial cell layer. Alginate/collagen-HUVECs constructs were subcutaneously injected into immune-deficient mice. After 7 days, implants were harvested and in vivo angiogenesis was evaluated.

Figure 2

Characteristics of three groups of alginate microspheres. (A) Morphology of Group A alginate microspheres. (B) Diameter distribution of Group A alginate microspheres. (C) Morphology of Group B alginate microspheres. The magnified image showed the microstructure of microspheres examined by SEM (scale bar: 100 µm). (D) Diameter distribution of Group B alginate microspheres. (E) Morphology of Group C alginate microspheres. The magnified image showed microsphere morphology after extrusion through a 27-gauge needle (scale bar: 500 µm). (F) Diameter distribution of Group C alginate microspheres.

Figure 3

Collagen coating, human umbilical vein endothelial cells (HUVECs) seeding, and in vitro prevascularized tissue formation of Group B microspheres. (A) scanning electron microscopy (SEM) examination showing overall morphology of alginate/collagen microspheres. (B) Magnified SEM image showing collagen fibril coating on the alginate microspheres. (C) HUVECs adhesion and proliferation of alginate/collagen microspheres determined by cell viability testing. Data of test groups were compared with the control group where no collagen was added. (D) Optic microscope image of alginate/collagen-HUVECs constructs 72 h after HUVECs seeding, where arrows indicate HUVECs on the microsphere surface. (E) Anti-human PECAM-1/CD31 immunofluorescence staining demonstrating prevascularized tissue formation 72 h after HUVECs seeding, where arrows indicated lumen-like HUVECs structure. (F) Endothelial gene (CD31, VE-Cadherin, vWF, hypoxia-inducible factor1a, vascular endothelial growth factor [VEGF], VEGF-releasing) expression of HUVECs in the three-dimensional construct compared with 2D culture. *indicates P<0.5, **indicates P<0.01, ***indicates P<0.001.

Figure 4

Subcutaneous injection and in vivo angiogenesis. (A) The appearance of immune-deficient mice immediately after injection. The black arrow indicates the injection site. (B) The appearance of injection site 7 days after injection. The black arrow indicates blood vessel ingrowth into the implants. (C) Hematoxylin-eosin (HE) staining of Group A implants 7 days after injection. Black arrows indicate functional blood vessels. “M” indicates non-degraded microspheres. (D) HE staining of Group B implants 7 days after injection. (E) HE staining of Group C implants 7 days after injection. (F) HE staining of the control Group 1. (G) HE staining of the control Group 2. (H) High-magnification image of HE staining, where arrows indicated functional blood vessels indicated by red blood cells. The magnified image showed anti-human PECAM-1 immunofluorescence staining of harvested tissue. White arrows indicated HUVECs lumen structure. Scale Bar: 25 µm. (I) Blood vessel density of the test groups and control groups. (J) Percentage of the area occupied by blood vessels of the test groups and control groups. **indicates P<0.01, ***indicates P<0.001.