Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke

Stroke is the primary cause of disability due to the brain’s limited ability to regenerate damaged tissue. After stroke, an increased inflammatory and immune response coupled with severely limited angiogenesis and neuronal growth results in a stroke cavity devoid of normal brain tissue. In the adult, therapeutic angiogenic materials have been used to repair ischemic tissues through the formation of vascular networks. However, whether a therapeutic angiogenic material can regenerate brain tissue and promote neural repair is poorly understood. Here we show that the delivery of an engineered immune-modulating angiogenic biomaterial material directly to the stroke cavity promotes tissue formation de novo, resulting in axonal networks along generated blood vessels. This regenerated tissue produces functional recovery through the established axonal networks. Thus, this biomaterials approach generates a vascularized network of regenerated functional neuronal connections within previously dead tissue, laying the groundwork for the use of angiogenic materials to repair other neurologically diseased tissues.


Sup Fig 1: Therapeutic angiogenic hydrogel concept
This study aims at designing and testing a pro-angiogenic therapy for stroke through a tissue engineering approach where the precise 3D presentation of VEGF to its receptor is controlled through its binding onto a nanoparticle's surface and its packing density onto the particle. For this, we designed an injectable hyaluronic acid (HA) hydrogel encapsulating highly clustered VEGF (hcV) onto nanoparticles of heparin and compared it directly to a condition of a low packing density, or low cluster of VEGF (lcV) where the total amount of heparin and VEGF was kept equal. In order to determine the effect of soluble VEGF (Vs), heparin nanoparticles only (nH), or the hydrogel (Empty gel) components of the hcV treatment, each of these conditions was tested, along with a stroke only (no treatment) as a negative control (For, the composition of each treatment, see Supplementary note 1).

Sup Fig 2: Engineered hydrogel and heparin particles. (A)
Schematic illustration of the hydrogel scaffold made of injectable acrylated hyaluronic acid (HA), MMP degradable motifs and adhesion peptides, as well as heparin nanoparticles and the different VEGF clustering densities. Different clustering densities of VEGF were created by mixing the same amount of VEGF with different amounts of heparin particles, leading to a low (lcV), medium (mcV) and high cluster density (hcV) of the growth factor onto the particle's surface. (B) Characterization of the particles by Transmission electron microscopy (TEM) and (C) Dynamic light scattering (DLS) (95.9 nm, and a PDI of 0.268). The size of nanoparticles was measured after each step of the formation (surfactants, hexane then water) showing an increase in diameter after attachment with VEGF in the 3 cluster conditions. Values represent average values obtained with 3 independent measurements, with standard deviation. (D) Two methods were used to determine the VEGF content in the heparin particles, a direct method that measures the amount of VEGF on the nanoparticle surface using dot blots and an indirect method that measures the amount of VEGF not bound to nH during synthesis using ELISA. The two methods showed the same VEGF concentration per weight of heparin for each of the clustering densities: 20 mg, 2 mg and 200 µg VEGF/mg heparin for high, medium and low cluster, respectively. (E) To ensure that the process of VEGF clustering did not impact the activity of VEGF, the ability of VEGF clusters to enhance endothelial cell (EC) proliferation and (F,G,H) to induce VEGF-receptor-2 (VEGFR-2) phosphorylation on the Y1175 and Y1114 sites and the MAP-Kinase protein p38 and p-42/44 were tested. Data represent the average ± SEM obtained from three independent experiments, and P values were determined by One-way Anova, Tukey's post-hoc test, with *** indicating P < 0.001. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with 1µg heparin nanoparticles (nH), gel + lcV = HA hydrogel with 1µg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test with * and *** indicating P < 0.05 and P < 0.001, respectively. (C1) Images of brain sections showing Evans blue extravasation in the stroke site (white arrow) after intravenous injection of the dye, and (C2) quantification by spectrophotometry of the amount of Evans blue measured per g of brain tissue. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with 1µg heparin nanoparticles (nH), gel + lcV = HA hydrogel with 1µg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with * and ** indicating P < 0.05 and P < 0.01, respectively. Scale bar: 100 µm.

Sup Fig 5: VEGF slow release. (A)
Quantification of the NF200 processes infiltration distance and the positive area for NF200 in the stroke site 2 weeks after gel injection. (B) Quantification of the periinfarct scar thickness and positive area for NF200 around the stroke site. In this set of data, a supplemental group of mice were injected a slow delivery of soluble VEGF and compared with the other VEGF-injected conditions. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH, gel + Vs slow release = HA hydrogel loaded with 200ng of soluble VEGF slowly released from the gel. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with * and **** indicating P < 0.05 and P < 0.0001, respectively. 6: Tail vein bleeding assay. Measurement of the tail vein bleeding time (sec) in mice injected intravenously with saline (PBS), 2µg of heparin or heparin nanoparticles (nH). Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by Oneway ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001.

Sup Fig 7: Angiopoietin-2 secretion. (A) Fluorescent images of vessels (Glut-1, red), Angiopoietin-2 (green) and Dapi (Blue) in the peri-infarct area in the different conditions 2 weeks after stroke. (B)
Quantification of the positive area for Angiopoetin-2 in the peri-infarct area and (C) around vessels (10 µm distance from vessels). No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with 1 µg unloaded nH, gel + lcV = HA hydrogel with 1µg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH, Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's posthoc test, with * and ** indicating P < 0.05 and P < 0.01, respectively. Scale bar: 50 µm. . Empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + lcV = HA hydrogel with 1µg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH, gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001, respectively. Scale bar: 100 µm. P values between gel + hcV and gel + hcV + endostatin were determined by t test, with $ indicating P < 0.05. Data is presented using a min to max box plot, gel + hcV + Endo = gel + hcv + i.p administration ef endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test.

Sup Fig 10: Long-term effect of endostatin on vessel post-stroke vessel formation. (A1)
Quantification of the vascular area in the infarct and (A2) peri-infarct area, Quantification of the vessel morphology: vessel tortuosity (B1) and diameter (B2), Quantification of vessel growth with number of ramifications (C1) and maximum infiltration distance of the vessels into the stroke site (C2) 16 weeks after stroke. Gel + hcV = HA hydrogel with 0.01 µg nH loaded with VEGF and 0.99 µg unloaded nH, Gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with ** and **** indicating P < 0.01 and P < 0.0001, respectively. 11: Role of naked heparin particles in the hcV treatment at 16 weeks post-stroke. Since the injected high and low cluster treatments (respectively hcV and lcV) were designed to contain equal amounts of heparin and VEGF, the VEGF clusterization in the hcV treatment was obtained on a low amount of heparin, leaving a high amount of naked particles. In order to understand the contribution of these naked particles in the pro-repair effect of the hcV, two supplemental group were studied: the hcV -nH where the naked particles from the hcV were removed, and the LcV + nH where additional naked particles were added to the low VEGF cluster condition. (A) Fluorescent images of vessels (Glut-1), astrocytic scar (GFAP), microglia (Iba-1) and axonal neurofilaments (NF200) in and around the stroke site (*) of gel + (hcV -nH) and LcV + nH) conditions, 16 weeks post-stroke. Quantitative assessment of the vascular area in the infarct (B1) and the peri-infarct area (B2), microglial area in the infarct (C1) and the peri-infarct area (C2), axonal area in the infarct (D1) and the peri-infarct area (D2), axonal infiltration distance (E1) and astrocytic scar (E2) at 16 weeks post-stroke. hcV -nH = 0.01 µg nH loaded with 200 ng VEGF, lcV = 1µg nH loaded with 200 ng VEGF and 0.99 µg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.001, respectively. Data represent the average. Scale bar: 100 µm. 12: Brain levels of inflammatory cytokines 3 and 10 days after gel transplantation in the no stroke, no gel and gel + nH conditions. Gel + nH = HA hydrogel with 1µg heparin nanoparticles (nH). Data is presented using a min to max box plot. Each dot in the plots represents one brain sample (concentrations in pg/mL) and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **, indicating p < 0.01.