C11 - Colloidal Fibrillar Composites to Augment Chronic Wound Healing in Human and Veterinary Settings

Introduction: : Chronic non-healing wounds pose a significant clinical problem in both human and veterinary settings due to clotting deficiency and persistent inflammation. This deficiency in clotting results in a poorly formed fibrin matrix, which can contribute to the formation of non-healing wounds. In such cases, it is important to speed up the wound healing process. The Brown Lab has previously developed colloidal fibrin-based nanoparticles (FBNs), that are pre-polymerized and use physiologically relevant thrombin and fibrin concentrations, unlike traditional fibrin glues that are typically used to treat non-healing wounds. To enhance wound healing and promote cell activity, we developed colloidal composites using pro-healing precursors to treat chronic non-healing wounds in human and equine patients. Our initial work with FBNs used adult human fibrinogen to create particles (aFBN). Prior work from the Brown Lab has shown that neonatal fibrinogen has pro-healing properties. We wanted to leverage this for diabetic non-healing wounds in humans; therefore, we developed neonatal fibrin based nanoparticles (nFBNs) to augment wound healing in diabetic wounds. Simultaneously, we were interested in incorporating native pro-healing factors for treating equine distal limb wounds. Current equine therapeutics for distal limb wounds are expensive and are not applicable across all stages of wound healing. Therefore, we also developed a new class of FBNs made from equine platelet poor plasma (pFBN) to treat non-healing distal limb wounds in horses.

Materials and

Methods: : FBNs are synthesized through a series of simple mechanical perturbation steps. For nFBNs, clots are made with 2 mg/mL fetal fibrinogen and 1 U/mL thrombin. For pFBNs, clots are made by combining filtered equine platelet poor plasma (PPP) with 1 U/mL thrombin. Once the clots are polymerized, they go through a series of filtration and sonication steps to create colloidal nanoparticles that can be lyophilized and stored at room temperature for further use. Our goal was to assess the pro-healing capabilities of both the nFBN and pFBNs in delayed wound healing settings. We assessed the impact of the different types of FBNs into the native fibrin matrix by incorporating varying concentrations of the particles into fibrin clots and determining clot density via confocal microscopy. To validate impact on clot polymerization metrics, nFBNs and pFBNs were incorporated into clots and clotting was measured via an absorbance-based assay and rheology. Clot polymerization kinetics was also determined using activated partial thromboplastin time tests. Cellular response was measured through a series of fibroblast attachment, spreading, and migration assays. For pFBNs, dermal explants were isolated from the distal limb of equine patients and cultured within clots pFBNs in 3D. Outgrowth was measured to assess wound healing potential. nFBNs efficacy was test in vivo in a full thickness dermal wound healing model, and transcriptomic analysis was done to determine stage and quality of wound healing.

Results, Conclusions, and Discussions:: nFBNs can successfully be synthesized to comparable sizes of aFBNs created from adult fibrinogen (Figure 1A). As compared to aFBNs, nFBNs are more effective increasing fiber density within a fibrin clot (Figure 1C). When incorporated into fibrin clots, nFBNs significantly impacted overall clot structure resulting in decreased pore area and increased number of overall pores. Incorporation of nFBNs into fibrin clots did not significantly impact the stiffness of the clots, however differences in clotting time were seen between the aFBNs and nFBNs. Overall, aFBNs clotted faster and showed a dose dependent increase in clotting speed; no such relationship was seen with the nFBNs (Figure 1B). nFBNs also promote cell attachment in a fibrin clot. Additionally, nFBNs enhance cell spreading and migration within a fibrin clot, as compared to aFBNs. To assess the wound healing capabilities of the nFBNs in vivo, full thickness dermal wounds were created on diabetic mice and assessed over nine days (Figure 1D). nFBNs increased the rate of wound healing and led to better healing outcome confirmed by transcriptomic analysis (Figure 1E).
Using equine PPP as the base of the FBNs did not significantly impact particle size, and the resultant pFBNs were comparable in size to previously developed FBNs (Figure 2A). There was a distinct dose dependent increase in fiber density when the pFBNs were incorporated into equine fibrin clots (Figure 2C). Incorporation of the pFBNs into equine plasma resulted in stiffer clots that clotted faster than control clots (Figure 2B). A stark cell response was also observed with the addition of pFBNs to clots. With increasing concentrations of pFBNs into fibrin clots, we observed increased cell attachment and spreading (Figure 2D). Preliminary explant studies support in vitro results. In these explant studies, we once again observed an increase in cells migration out of the explant into fibrin matrix with increasing pFBN concentration. Using pro-healing precursors allows us to fine tune the healing properties of FBNs and leverage specific healing factors to treat chronic non-healing wounds in human and veterinary settings.

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