Assistant Professor University of Massachusetts Amherst, United States
Introduction:: Diabetic foot ulcers (DFU) constitute a major health concern due to their poor healing, which can lead to severe complications including amputation of the affected limb. While DFUs are caused by many factors, two key common issues are poor vasculature and dysregulated wound healing in the patient’s foot. While some current treatments have shown promise, most approaches do not recapitulate the body’s natural wound healing sequence and this results in incomplete vascularization and other complications. Tissue engineering via viral genetic manipulation offers the means to regulate the timing of genetic expression. This can ensure the proper revascularization of a DFU by correctly sequencing protein expression. We propose a hydrogel system loaded with viral vectors whose release can be triggered and sequenced via ultrasonication at chronic DFU sites. To provide a suitable scaffold for tissue growth the hydrogel will be place within pockets in a collagen-gag scaffold that can be individually stimulated by ultrasound. Once established, this platform can be adapted to allow temporal control over several aspects of the wound healing cascade, ensuring improved treatment of DFUs and better patient outcomes.
Materials and Methods:: Hydrogels were prepared by ionically crosslinking alginate solution with a mixture containing calcium sulfate and loading it with adeno-associated virus. After mixing, gels were dispensed into 50 ml volumes. Gels were loaded with 1x10^8 to 1x10^10 genomic copies/gel and the gels modified (1-2% medium molecular weight alginate, between 0-5% oxidized) so that viral release by diffusion, hydrolytic degradation, and ultrasonication of the gel could be controlled. Release studies were performed in a 24 well plate (for diffusion/degradation experiments) or a 15 ml conical tube (for ultrasound experiments) over a period of 14 days. Conditioned media was collected and added to HEK293t cells, and the percentage of cells expressing enhanced Green Fluorescent Protein was confirmed via flow cytometry. Collagen-glycosaminoglycan scaffolds were fabricated with pockets by using an inverted mold design. Following dehydrothermal and chemical crosslinking the AAV-loaded alginate gels were injected into the pockets. Scanning electron microscopy was used to study the microstructure of the scaffolds and alginate gels, and release studies performed.
Results, Conclusions, and Discussions:: AAV was successfully released from the alginate gels using all three methods tested – diffusion, degradation, and ultrasonication. Whereas a large burst release can be triggered via chemical digestion of the hydrogel, a longer sustained release was possible via diffusion. Diffusion can be sustained for up to 9 days, and the viral load affects how much release/transfection occurs from 6% - 100% (See Fig 1). Past studies have looked at virus release from alginate for local delivery but there are fewer explorations on dosage effects. By starting with a low level of baseline diffusion, we successfully triggered viral release with ultrasonication generating a burst of protein expression at a user-defined time (See Fig 1). With the combination of long-term diffusion with triggered short term release we now have a tool that can be modified for vascular gene expression, and more closely mimicking the vascularization pathway, leading to increased vasculature formation at the wound site. Very few papers look at a triggerable release of viruses, and of those groups we did not find anyone working on being able to sequentially release different viruses. This research gives increased control over viral dosing than had previously been achievable. When combined with our collagen-gag scaffold we can deliver viruses to targeted areas at set times. We have already been able to seed and observe gene expression inside the scaffolds. This controlled release shows promise towards completing our goal of developing a DFU treatment for patients with challenging ulcers where traditional therapies have failed.
