Ultrashort Tetrameric Peptide Nanogels Support Tissue Graft Formation, Wound Healing and 3D Bioprinting

Abstract

Tissue engineering is a promising alternative to organ transplantation, where the number of waiting patients is not supported by the number of available donors. Tissue engineering aims to fabricate functional tissue using biocompatible scaffolds. Nanogels made from self-assembling ultrashort peptides are promising scaffold biomaterials. We focus on two compounds of a novel class of rationally designed tetrameric peptides for biomedical applications that have the advantage of being natural but synthetic hydrogels. These compounds have an innate tendency to self-assemble into nanofibrous hydrogels, which can be used for the fabrication of three-dimensional (3D) skin grafts, treating full-thickness wounds in minipigs and skeletal muscle tissue proliferation and differentiation. We were able to produce in situ silver nanoparticles within the peptide nanogels, solely through ultraviolet irradiation, with no reducing agent present. Applying the peptide nanogels on full-thickness minipig wounds demonstrated that the scaffolds were biocompatible, with no notable wound inflammation, and comparable to standard care solutions. Interestingly, the peptide scaffolds revealed a high potential to act as antibacterial agents. Microscopic observation demonstrated the ability of human umbilical vein endothelial cells to form tube-like structures within peptide nanogels. Moreover, we successfully produced artificial 3D vascularized skin substitutes using these peptide scaffolds. Additionally, we could demonstrate that both tetrameric peptides support 3D bioprinting, indicating their possible use as future bioinks. We believe that the results described represent an advancement in the context of engineering skin and skeletal muscle tissue, thereby providing the opportunity to rebuild missing, failing, or damaged parts.

Peptide nanogels as a scaffold for fabricating dermal grafts and 3D vascularized skin models

“… 26 This result corroborated our previous findings on cellular distribution and collagen deposition by H/E and Masson trichrome. 11 …”

“…In a previous study, we successfully fabricated a 3D vascularized dermal layer without any supportive vascular growth factors by co-culturing HDFn with HUVEC using different nanogel scaffolds in a ratio of 1:1 and immunostained with different antibodies: CD31against HUVEC cells and vimentin against fibroblast as previously described. 11 In this study, a novel 3D vascularized skin substitute was fabricated by seeding the epidermal keratinocytes on top of the vascularized dermal construct. Confocal microscopy results revealed epidermal layer-like structure formation with both peptide hydrogels on top of the vascularized dermal construct ( Figure 7 ).…”

“… 2 Furthermore, the diameter of these nanofibers supported their use as scaffolds for skin regeneration, 30 – 33 where the natural ECM diametric range is between 5 and 300 nm, 34 in addition to the mechanical stability of both peptide nanogels. 10 , 11 …”

“…Furthermore, fluorescent staining of the actin cytoskeleton, serving as direct evidence for cellular morphology and cytoskeleton structure, 35 demonstrated that dermal cells proliferated and extended within the scaffolds in their first days of encapsulation, and growth rates rapidly increased. 10 , 11 Moreover, a network was created through cell-to-cell junctions, ultimately forming an extensive network saturating the nanogel matrix. The deposition of collagen in the histological section of the dermal fibroblast layer indicated that fibroblasts were mitotically and functionally active by producing their collagen matrix.…”

“…To fulfill the criteria of a tissue-engineered scaffold in maintaining the in vitro proliferation of human dermal fibroblasts, enhancing collagen deposition, and supporting the pre-revascularization of the dermal construct. 11 Based on these findings, in this study, we fabricated a non-vascularized skin substitute by co-culturing human dermal fibroblasts within the abovementioned novel scaffolds and seeding epidermal keratinocytes on top of the surface of both scaffolds. We found that the direct contact of keratinocytes and fibroblasts enhanced keratinocyte proliferation.…”