(277c) Development of Pre-Vascularized Lymphatic Grafts with Controlled Sevs Release for Directed Inflammation Modulation and Tissue Repair

Functional wound healing requires tissue hemostasis in which the interstitial flow plays a major role by regulating fibroblast activation, ECM remodeling, and angiogenesis. However, in chronic wounds, excessive interstitial shear stress causes persistent inflammation, fluid accumulation, and fibrosis. Typically, wound dressings are implemented to foster tissue repair. Wound dressings should meet different necessities such as inflammation reduction, protection of the wound from the environment, tissue regeneration stimulation, and chemical stimuli delivery. Nonetheless, conventional wound dressings fail to adequately restore interstitial flow, promote lymphangiogenesis, and provide stage-specific biochemical cues limiting their effectiveness in functional tissue repair. Hence, in this study we integrated lymphangiogenesis-supporting granular gels with tunable small extracellular vesicles (sEVs) delivery to sequentially deliver cargo for different stages of tissue regeneration.

sEVs were produced from mesenchymal stem cells (MSCs) and endothelial colony forming cells (ECFCs) to obtain bioactive cargo with anti-inflammatory and pro-angiogenic properties. Culture media was collected and the sEVs were isolated using asymmetric nanoporous membranes (ANM). sEVs were characterized using nanoparticle tracking analysis (NTA), TEM, and western blot. Monocytes and M1 macrophages were treated with MSC-sEVs to test their differentiation and polarization onto M2 macrophages, respectively. The efficiency of the differentiation/polarization and its perdurability was assessed via FACS. ECFC-sEVs were administered to gestational diabetes mellitus (GDM) ECFCs55 to test their ability to improve cell migration and tube formation in 2D. Angiogenesis was studied using confocal microscopy, and image analysis with the AutoTube software. Moreover, two subpopulations of microgels were fabricated via pipetting using norbornene-modified hyaluronic acid (NorHA) for loading the MSC-sEVs and ECFC-sEVs distinctively. The sEVs release kinetics were adjusted using different ratios of MMP-sensitive/DTT crosslinker to release the MSC-sEVs and ECFC-sEVs during the early stage, and late stage of wound healing, respectively. Released sEVs were quantified using immunojanus particles (IJPs).

The pre-vascularized lymphatic tissue grafts were fabricated using NorHA granular hydrogels. Lymphatic endothelial cells (LECs) were embedded in a degradable NorHA interstitial matrix using MMP-sensitive crosslinker. The interstitial precursor was mixed with the sEVs-loaded microgels in a 1:3 volume ratio. Lymphatic capillaries development was monitored using bright-field microscopy. The grafts were then implanted at day 3 in the dorsum of 10-week-old female db/db mice. Immunohistochemistry assays revealed integration between the tissue grafts capillaries and the hosts vasculature. A synergistic effect was observed for the lymphatic grafts loaded with sEVs with substantial improvements in the wound healing score.

Overall, the developed wound dressing platform showed suitability for inflammation modulation and chronic wound healing. The versatility of the technology allows it to be used for different types of tissue regeneration and repair broadening the landscape of future regenerative medicine therapeutics.