Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Human-Induced Pluripotent Stem Cells in Plastic and Reconstructive Surgery.

A hallmark of plastic and reconstructive surgery is restoring form and function. Historically, tissue procured from healthy portions of a patient's body has been used to fill defects, but this is limited by tissue availability. Human-induced pluripotent stem cells (hiPSCs) are stem cells derived from the de-differentiation of mature somatic cells. hiPSCs are of particular interest in plastic surgery as they have the capacity to be re-differentiated into more mature cells, and cultured to grow tissues. This review aims to evaluate the applications of hiPSCs in the plastic surgery context, with a focus on recent advances and limitations. The use of hiPSCs and non-human iPSCs has been researched in the context of skin, nerve, vasculature, skeletal muscle, cartilage, and bone regeneration. hiPSCs offer a future for regenerated autologous skin grafts, flaps comprised of various tissue types, and whole functional units such as the face and limbs. Also, they can be used to model diseases affecting tissues of interest in plastic surgery, such as skin cancers, epidermolysis bullosa, and scleroderma. Tumorigenicity, immunogenicity and pragmatism still pose significant limitations. Further research is required to identify appropriate somatic origin and induction techniques to harness the epigenetic memory of hiPSCs or identify methods to manipulate epigenetic memory.

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols in vivo. Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization.

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 µM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 µM.

Ionic liquid treatment for efficient sample preparation of hydrated bone for scanning electron microscopy.

This study presents a new protocol for preparing bone samples for scanning electron microscopy (SEM) using a room temperature ionic liquid (RTIL) treatment method. RTIL-based solutions can be adopted as an alternative to lengthy and laborious traditional means of preparation for SEM due to their unique low-vapour pressure and conductive properties. Applied to biological samples, RTILs can be used quickly and efficiently to observe hydrated, unfixed structures in typical SEM systems. This first-time feasibility study of the optimization of this protocol for bone was explored through various SEM modalities using two distinct ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF4]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF4]), at varying concentrations of 5, 10, and 25 % v/v in aqueous solution through an addition-based method. Based on qualitative observations in the SEM, a 60-second solution addition treatment of 10 % v/v [BMI][BF4] performed the best in imaging hydrated, unfixed bone samples, resulting in minimal charge buildup and no solution pooling on the surface. The treatment was applied effectively to a variety of bone samples, notably flat and polished, as well as highly topographical bone fracture surfaces of both healthy and osteoporotic human bone samples. In comparison to conventionally dehydrated bone, the RTIL treatment better preserved the natural bone structure, resulting in minimal microcracking in observed structures.

Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review.

For biological imaging using electron microscopy (EM), the use of room-temperature ionic liquids (RTILs) has been proposed as an alternative to traditional lengthy preparation methods. With their low vapor pressures and conductivity, RTILs can be applied onto hard-to-image soft and/or wet samples without dehydration - allowing for a more representative, hydrated state of material and opening the possibility for visualization of in situ physiological processes using conventional EM systems. However, RTILs have yet to be utilized to their full potential by microscopists and microbiologists alike. To this end, this review aims to provide a comprehensive summary of biological applications of RTILs for EM to bridge the RTIL, in situ microscopy, and biological communities. We outline future research avenues for the use of RTILs for the EM observation of biological samples, notably i) RTIL selection and optimization, ii) applications for live cell processes and iii) electron beam and ionic liquid interaction studies.