Engineering biomimetic microvascular meshes for subcutaneous islet transplantation

Engineering biomimetic microvascular meshes for subcutaneous islet transplantation
Fabrication and function of transferrable microvascular meshes via anchored self-assembly. (a) Schematic illustration of organization of vascular endothelial cells into a microvascular mesh, which is transferred and attached to a cellular device. In a poorly vascularized subcutaneous space, microvascular mesh can enhance vascularization and anastomoses with host vasculature to provide oxygen/nutrients to donor cells. (b) Design of micropillar arrangement (blue) and ASA-enabled cell organization (purple): the key to the ASA is that inner micropillars (yellow) provide a geometric template for cell self-assembly to form square mesh and boundary micropillars (green) serve as anchoring points for cell attachment to prevent assembled mesh structure from shrinking. (c) A fluorescent image of HUVECs expressing GFP in fibrin matrix organized into a square mesh (1 × 1 cm) on a micropillar substrate via ASA after 2 days of culture. (d) After 2 days of culture, a HUVEC square mesh (5 × 5 cm) is lifted from the micropillar substrate for transfer. The insert is a magnified image of HUVEC mesh and scale bar is 1 mm. (e) HUVEC microvascular mesh generates angiogenic sprouts (white arrows) when embedded in fibrin matrix during 2 weeks of culture in vitro. (f) Microscopic images of retrieved HUVEC microvascular mesh device show a high degree of vascularization after 2 weeks of subcutaneous implantation in SCID-Beige mice. Credit: Nature Communications, doi: 10.1038/s41467-019-12373-5

To successfully engineer cell or tissue implants, bioengineers must facilitate their metabolic requirements through vascular regeneration. However, it is challenging to develop a broad strategy for stable and functional vascularization. In a recent report on Nature Communications, Wei Song and colleagues in the interdisciplinary departments of Biological and Environmental Engineering, Medicine, Mechanical and Aerospace Engineering, Clinical Sciences and Bioengineering in the U.S. described highly organized, biomimetic and resilient microvascular meshes. The team engineered them using controllable, anchored self-assembly methods to form microvascular meshes that are almost defect-free and transferrable to diverse substrates, for transplantation.

The scientists promoted the formation of functional blood vessels with a density as high as ~200 vessels per mm-2 within the subcutaneous space of SCID-Beige mice. They demonstrated the possibility of engineering microvascular meshes using human induced pluripotent stem-cell (iPSCs) derived (ECs). The technique opens a way to engineer patient-specific type 1 diabetes treatment by combining microvascular meshes for subcutaneous transplantation of rat islets in SCID-beige mice to achieve correction of chemically induced diabetes for 3 months.

Vasculature is an essential component of any organ or tissue, and vascular regeneration is critical to successfully bioengineer implants. For instance, during cell replacement therapy for type 1 diabetes (T1D), transplanted insulin producing cells rely on the vasculature to function and survive. Bioengineers often use vascular endothelial cells such as human umbilical vein endothelial cells (HUVECs) to spontaneously assemble into tubular structures within the extracellular matrix (ECM). But the resulting structures can be random, uncontrollable and less efficient for microvascular regeneration. Scientists have recently developed three-dimensional (3-D) printing techniques to engineer controlled cellular constructs with embedded vessels. However, it remains challenging to 3-D print resilient and transferrable, high-resolution, microvasculature.

Bioengineering the microvascular meshes

In the present work, Song et al. engineered a microcapillar-based, anchored self-assembly (ASA) strategy to create controllable, transferrable and scalable microvascular meshes for T1D cell treatment therapies. During ASA, the researchers implemented the micropillars to guide self-assembly of endothelial cells within a fibrin matrix. The pillars served as anchoring points to prevent structural shrinkage during cellular maturation for controllable and resilient microvascular meshes.

Engineering biomimetic microvascular meshes for subcutaneous islet transplantation
Simulation and characterization of the ASA-enabled microvascular meshes. (a, b) The contraction simulation shows an in-plane displacement contour plot of organized cellular mesh structure (a) and the normal stress distribution in the X (Cauchy stress component 11) direction (b) on a 4 × 4 micropillar substrate. The initial shape of cells and fibrin matrix is displayed in light gray. The micropillar diameter is 400 μm and micropillar-to-micropillar interval is 200 μm. The contracted region is marked as dotted purple ellipse and the junction region is purple circle. The displacement unit is μm and the unit of stress is mN μm−2. (c) Cross-sectional images showing a HUVEC mesh suspended between micropillars. The micropillars are pseudo-colored as blue and HUVECs are pseudo-colored as purple. (d) SEM images of a HUVEC mesh (purple) at the inner and boundary regions on the micropillar substrate (blue). (e) Confocal images of a HUVEC mesh at the contracted and junction regions on the micropillar substrate showing the tubular structures. Human CD31 antibody is green, F-actin is red, and nucleus is blue. (f) Screenshots of a glass pipette poking a HUVEC mesh showing high resilience of the mesh. Credit: Nature Communications, doi: 10.1038/s41467-019-12373-5

The research team tuned the dimension and arrangement of micropillars to control the geometry of the microvascular mesh. On transplantation, the microvascular meshes promoted the formation of new blood vessels (neovascularization) and vascular anastomoses (connection between two vessels). They developed the meshes using both HUVECs and human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) suited for patient-specific microvasculature development. Using both types of microvascular meshes, the research team significantly improved vascularization, and corrected diabetes in chemically induced, i.e., streptozotocin (STZ)-based SCID-beige mice for three months. The work provides proof of concept for the use of microvascular meshes in cell replacement therapies for T1D and other diseases.

To achieve rapid and functional vascularization around a cellular device, the research team attached a preformed vascular structure with sufficient density and resolution to the device. After transplantation, the structure induced angiogenesis and promoted anastomoses (connection between two vessels) with the host vasculature. During the process, the researchers observed HUVECs together with a fibrin matrix to self-assemble into an almost defect-free square mesh after 2 days of cell culture on a micropillar substrate. They adjusted the size and arrangement of micropillars to precisely control the mesh geometry and dimension. The cells remained viable and stable on micropillar substrates for at least 4 weeks and rapidly generated sprouts on transfer to embed within a fibrin matrix. Due to constant remodeling, the mesh network did not preserve their original shape.

Simulating and experimentally testing the microvascular meshes

Engineering biomimetic microvascular meshes for subcutaneous islet transplantation
A large piece of HUVEC mesh (5×5 cm) is being lifted up from micropillar substrate and microvascular mesh can be easily transferred to other devices or substrates. Credit: Nature Communications, doi: 10.1038/s41467-019-12373-5

To understand the ASA process, the researchers conducted finite element analysis simulation of the cell assembly process and further tested the microvascular mesh. The simulation considered the contractility of cells on the fibrin matrix to generate an in-plane displacement contour plot of organized mesh structure and stress/strain distribution on the micropillar substrate. The experimental results were consistent with the simulation to validate that microvascular meshes were tightened and suspended between micropillars instead of settling at the bottom of the culture setup. The work supported the hypotheses that stable cell constructs formed by relying on the micropillars. The meshes were also elastic, although mechanically robust and resilient to even withstand poking with a glass pipette. The scientists used these remarkable properties to manipulate and transfer the mesh to diverse substrates without affecting its integrity.

Improved vascularization of micro meshes in SCID-Beige mice.

The research team investigated how microvascular meshes enhanced vascularization by comparing normal HUVEC meshes with normal human dermal fibroblast (NHDFs) added constructs. The combination supported and enhanced vessel formation. The scientists transplanted the constructs within the subcutaneous space of SCID-Beige mice. The subcutaneous space is a poorly vascularized area offering many advantages for cell replacement therapy, including easy access, minimal invasiveness and high transplant capacity. After two weeks, the scientists retrieved the devices and compared vascularization using histology with hematoxylin/eosin (H&E) staining to quantify the blood vessels surrounding the chambers. They revealed a highly vascularized mesh device compared to the control mesh without cells. Immunohistochemical staining further revealed the newly formed vessels to be chimeric in nature, indicating anastomoses and vascular remodeling during vascularization.

To further confirm anastomoses, Song et al. perfused mice with mesh devices via tail injections using two lectin dyes (labeled with green fluorescence protein dye—GFP and a red dye). The overlap of the two different lectin strains confirmed the formation of blood-perfused human vasculatures that anastomosed with the mouse vascular meshes after implanting the HUVEC-GFP/NHDF within the mouse vascular system at different time frames. The results substantiated the ASA-enabled process to transfer microvascular meshes to vascularize cell delivery devices.

Engineering biomimetic microvascular meshes for subcutaneous islet transplantation
Enhancement of vascularization and anastomoses in subcutaneous space of SCID-Beige mice. (a) Schematics and a digital photo of a Mesh device, which is a diffusion chamber with HUVEC meshes (~25 μm thick, purple) in the fibrin gel (gray) on the top and bottom. The diffusion chamber is a cylindrical cell container with a PDMS ring (blue) as the wall and two nylon grids (green) as the top and bottom. The dimensions of each component are labeled in the schematic. (b) Fluorescent images of randomly mixed cells (top) and microvascular mesh (bottom) placed on diffusion chambers after 2 days of culture in EGM-2 medium. HUVEC:NHDF = 9:1, Human CD31 antibody is green to show HUVEC, α-smooth muscle actin (α-SMA) antibody is red to show NHDF, and nylon grid is blue. (c) Cross-sectional hematoxylin/eosin staining images of retrieved devices after 14 days of implantation. Yellow arrowheads point to blood vessels with erythrocytes inside. (d) Density and area percentage of blood vessels at the interface between the device and panniculus carnosus muscle. n = 6 in the No cell and Random, and n = 8 in Mesh groups. Data are mean ± SEM; **P < 0.01, ***P < 0.001, NS (P > 0.05) no significant difference. One-way analysis of variance. (e) Cross-sectional immunostaining images of human (red) and mouse (green) CD31 antibodies showing the human and mouse blood vessels at the interface between the device and panniculus carnosus muscle. (f) Confocal images of perfused lectins bound to human (UEA-I, green) and mouse (GSL-I, red) endothelial cells, confirming the anastomoses between human and mouse vessels. (g) Blood-perfused human vasculatures anastomosed with mouse vascular system in Mesh device after 10 days of subcutaneous implantation. HUVEC-GFP is green and perfused dye DiI in vessels is red. (h) Representative immunostaining images of mature human vasculatures (human CD31 antibody is green) covered with perivascular cells (α-SMA antibody is red) in retrieved Random and Mesh device after 10 days of subcutaneous implantation. (i) The percentage of perivascular cell (PVC) coverage is 19 ± 9% (n = 3; mean ± SEM) and 65 ± 6% (n = 6) for the Random and Mesh devices, respectively. **P < 0.01. Unpaired two-tailed t test. Credit: Nature Communications, doi: 10.1038/s41467-019-12373-5

Correcting type 1 diabetes (T1D) in SCID-Beige mice using rat islets

Song et al. subsequently investigated if microvascular meshes could improve cell replacement therapy for T1D by loading the rat islets in diffusion chambers and attaching microvascular meshes to them using a fibrin gel mesh device. They then transplanted the constructs subcutaneously in the mouse model with STZ-induced diabetes. After 42 days, the mice showed significantly better blood glucose (BG) control compared to those with control meshes without cells and other random groups. When the research team retrieved the grafts, the BG levels increased in all mice to substantiate the anti-diabetic effects of the devices. The research team performed histological staining of the retrieved devices at day 42 to show significantly increased blood vessels surrounding the islets in the implants from the mesh group; supporting diabetes correction. In total, the results confirmed the effectiveness of transferrable micro-meshes to promote revascularization of donor islets and maintain normoglycemia for up to 3 months in diabetic mice.

Engineering microvascular meshes using human iPSC-ECs.

Song et al. then explored the ASA strategy to other types of ECs (endothelial cells) in a clinical setting using human iPSC-ECs; which offered an unlimited cell source for vascularization with significant clinical potential. The iPSC-ECs behaved similarly to HUVECs in vitro, the research team then implanted the microvascular meshes of diverse geometries within the subcutaneous space of SCID-Beige mice for 2 weeks. They confirmed anastomoses and the formation of blood-perfused iPSC-EC vessels using histology stains.

Engineering biomimetic microvascular meshes for subcutaneous islet transplantation
Improved diabetes correction by the iPSC-EC microvascular meshes in SCID-Beige mice. (a) A microscopic image of rat islets in an iPSC-EC mesh device. The gray color aggregates are rat islets. The white arrow points to iPSC-EC mesh. In all in vivo experiments, NHDFs were mixed with iPSC-ECs (iPSC-EC:NHDF = 9:1). (b) Non-fasting BG concentration of the mice during 91 days of subcutaneous transplantation (n = 5 in No iPSC-EC, n = 6 in Random iPSC-EC and Mesh iPSC-EC). *P < 0.05. ANCOVA, time was treated as continuous covariate. (c) BG concentration during intraperitoneal glucose tolerance test (IPGTT) after 30 and 90 days of transplantation. IPGTT at Day 30 (n = 6 in Normal mice, Random iPSC-EC, and Mesh iPSC-EC, and n = 4 in No iPSC-EC). Data are mean ± SEM. *P < 0.05. NS (P > 0.05) no significant difference. IPGTT at Day 90 (n = 6 in Normal mice, n = 3 in No iPSC-EC, n = 4 in Random iPSC-EC, and n = 6 in Mesh iPSC-EC): *P < 0.05. ANCOVA, time was treated as continuous covariate. (d) Immunostaining images (parallel section) of human (red) and mouse CD31 (green) antibodies indicating the anastomoses between human and mouse blood vessels. (e) Confocal image of perfused blood vessels in a re-vascularized rat islet retrieved from the Mesh group at Day 91. (f) Hematoxylin/eosin staining image of a retrieved iPSC-EC mesh device at Day 91. Black arrow points to a rat islet and yellow arrowheads point to blood vessels containing erythrocytes. (g) Immunostaining image of insulin and blood vessels surrounding rat islets in a retrieved iPSC-EC mesh device at Day 91. Rat insulin antibody is red and mouse CD31 antibody is green. Credit: Nature Communications, doi: 10.1038/s41467-019-12373-5

When the scientists similarly attached the iPSC-EC mesh to a diffusion chamber containing rat islets to support the islets. The mesh group led to significantly better diabetes control compared to the control groups in the rat-to-mouse transplantation model. Based on the results, the researchers intend to enhance the function and vascularization of iPSC-EC meshes to improve BG control and glucose responsiveness in transplanted islets. The immunostaining results confirmed the iPSC meshes to promote anastomoses between human and mouse vessels. The results verified the feasibility of engineering patient-specific microvascular meshes during cell replacement therapies for T1D.

In this way, Wei Song and colleagues used spatially arranged micropillars to engineer high-resolution, resilient and transferable microvascular meshes. The micropillar guided the ECs to form patterns with controllable geometry to prevent and matrix from shrinking. The biomimetic microvascular mesh was within the same size range of an in vivo capillary bed for enhanced vascularization. The resilient and transferable microvascular meshes allowed researchers to vascularize islets subcutaneously for transplantation within delivery devices during cell replacement therapy in a T1D mouse model. Song et al. expect to expand the ASA as a general approach across other fields of bioengineering to construct geometrically defined, high-resolution live materials at the microscale for cell therapeutics and regenerative medicine.

More information: Wei Song et al. Engineering transferrable microvascular meshes for subcutaneous islet transplantation, Nature Communications (2019). DOI: 10.1038/s41467-019-12373-5

Peter Carmeliet. Angiogenesis in life, disease and medicine, Nature (2005). DOI: 10.1038/nature04478

Boyang Zhang et al. Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis, Nature Materials (2016). DOI: 10.1038/nmat4570

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Citation: Engineering biomimetic microvascular meshes for subcutaneous islet transplantation (2019, October 16) retrieved 29 February 2024 from
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