Student University of Cincinnati Middletown, Ohio, United States
Introduction:: Each year, approximately half a million patients in the US require treatment for burns, with nearly 40,000 hospitalizations and 3,400 deaths. Over 160,000 skin grafts are transplanted annually and unfortunately, not all patients regain full sensation [1]. Sensory recovery typically takes months or even years to return to normal. Additionally, grafted skin lacks innervated blood vessels and sweat glands, which further indicates diminished sensation and negatively affects the capacity of grafted skin to dissipate heat through cutaneous vasodilation and perspiration [1]. Previous work demonstrates graft reinnervation of patient’s native sensory neurons (SNs) is not only imperative for regaining sensation, but also supports the healing of damaged tissue [2]. Tissue engineered skin (TES) has become a popular alternative to traditional wound healing by producing skin constructs that aim to offer complete regeneration of functional skin. Yet, innervation and functional sensory recovery within surrounding native skin remains a challenge. In this work, we activated biocompatible and piezoelectric polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) scaffolds via ultrasound stimulation to promote Schwann cell (SC) and SN alignment, elongation and extension, as well as scaffold perforation. The purpose of this study is to investigate neurite outreach for application in experimental TESs by inducing electrical activation of piezoelectric PVDF-TrFE with the mechanical deformations caused by the applied ultrasound waves.
Materials and Methods:: Aligned and unaligned PVDF-TrFE scaffolds were electrospun according to our previous work [3] and seeded with RT4-D6P2T SCs and primary mouse SNs separately and in co-culture. Scaffolds were stimulated with ultrasound (0.08 W/cm2), stained, and imaged via confocal microscopy. Images were analyzed in Nikon Elements for cell alignment, elongation, and integration into scaffolds.
Results, Conclusions, and Discussions:: Confocal images of SC-SN cocultures on PVDF-TrFE with and without applied ultrasound (Fig. 1A) show alignment, SC elongation, and neurite extension. Neurite length (Fig. 1B) indicates ultrasound promotes neurite extension on aligned scaffolds and neurite integration on unaligned scaffolds (Fig. 1C). Perforation depth suggests SCs fully integrate into the scaffold whereas SNs elongate across fibers on aligned scaffolds with applied ultrasound. Cocultures display greater integration of neurites into scaffolds, likely because neurites are tracking SCs. The integration of neurites after stimulation gives rise to incorporating this noninvasive therapeutic modality for enhancing wound healing strategies. Piezoelectric material activation advances the regenerative response by inducing phenotypic changes in the cells that relocate to damaged skin tissue. Looking forward, the degree of SN functionality via responsiveness to capsaicin must be examined for verification of functional nociception. In conclusion, ultrasound stimulation of neurites on PVDF-TrFE showed integration into unaligned scaffolds and extension across fibers on aligned scaffolds. This holds great potential for investigating nerve integration and functionality for application in experimental TESs.
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References (Optional): : 1. Nedelec B, Hou Q, Sohbi I, Choinière M, Beauregard G, Dykes RW. Sensory perception and neuroanatomical structures in normal and grafted skin. Burns, 2005. 31(7): p. 817-30. 2. Weng T, Wu P, Zhang W, Zheng Y, Li Q, Jin R, Chen H, You C, Guo S, Han C, Wang X. Regeneration of skin appendages and nerves: current status and further challenges. J Transl Med, 2020. 18(1): p. 53. 3. Orkwis JA., Wolf AK, Shahid SM, Smith C, Esfandiari L, Harris GM. Development of a Piezoelectric PVDF-TrFE Fibrous Scaffold to Guide Cell Adhesion, Proliferation, and Alignment. Macromol Biosci, 2020: p. e2000197.
