(114f) Acellular and Growth Factor Free Hydrogel Promotes Arteriole Growth and Prevents Tissue Damage in a Murine Model of Critical Limb Ischemia

Statement of purpose: Critical limb ischemia (CLI) occurs when plaque buildup in the arteries becomes so severe, blood cannot reach the extremities, resulting in painful ulcers and non- healing wounds. Patients suffering from CLI often do not qualify for revascularization procedures, leaving amputation as the primary treatment option. This has led to a push for non- invasive treatments that can promote growth of collateral arterioles to restore blood flow and relieve ischemia. Within this space, hydrogels have been employed as carriers for exogenous biologics, such as growth factors and stem cells. However, less research has focused on how the hydrogels themselves can be engineered to promote collateral arteriole development. Here, we present a gelatin hydrogel, modified with a bioactive peptide, as an off-the-shelf vascular therapy that prevented tissue damage in a murine model of CLI. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins, which stimulates growth of smooth muscle cells for arteriole formation in both ex vivo and in vivo assays. The peptide-functionalized gelatin, termed “GelCad,” restored perfusion after 14 days in BALB/c mice subjected to a femoral artery ligation. Additionally, preliminary data shows GelCad prevented necrosis in the same femoral artery ligation model applied to 22-month-old C57BL/6 mice. These studies offer a localized, easily translatable vascular therapy that does not require exogenous growth factors or cells.

Methods: The modified gelatin biomaterial is synthesized by dissolving porcine gelatin in PBS and using EDC-NHS chemistry to attach the carboxylate end group of the cadherin peptide (HAVDIGGCE) to primary amines on the gelatin backbone. The peptide attachment is confirmed using H-NMR. For experimental use, the biomaterials are reconstituted as a 10% solution in PBS and crosslinked by mixing with an equal volume of a 10% solution of 20 kDa 4-arm polyethylene glycol succinimidyl glutarate (PEG-SG).

Hydrogels were injected into the fat pad of young C57BL/6 mice. After seven days, the mice were cardiac perfused with Microfil polymer to visualize vasculature in the hydrogel plugs by microCT imaging. MicroCT vascular networks were reconstructed and quantified using Vesselucida software. Separate hydrogels were harvested for cryosectioning and antibody staining to confirm the presence of arterioles.

Femoral artery ligation studies were conducted using 3-month-old BALB/c mice or 22-month-old C57BL/6 mice. In this model, blood flow was restricted by tying double knots in the femoral artery at two locations. Hydrogels were applied on top of the exposed tissue in the subcutaneous space. After surgery, at different time points, laser doppler imaging was performed to assess perfusion in both legs and ischemic indices were used to score physical appearance. At the final endpoint, Microfil was used to cast the vessels for microCT imaging of the vascular network within the hydrogels. Histology was also performed on the fixed tissue by a blinded pathologist to assess ischemic damage.

Results: GelCad hydrogels injected into fat pads showed larger vessel diameters compared to unmodified gelatin hydrogels. Additionally, arterioles were identified by alpha-smooth muscle actin (⍺-SMA)+ cells lining CD31+ vessels in the GelCad hyrogels that were not present in the gelatin control.