Search

Despite decades of efforts, state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal. Current dressings adhere to the wound and necessitate debridement. This work describes the first “supramolecular hybrid hydrogel (SHH)” burn dressing that is biocompatible, self-healable, and on-demand dissoluble for easy and trauma-free removal, prepared by a simple, fast, and scalable method. These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution. The SHHs show high mechanical strength (>50 kPa), self-heal rapidly in ∼1 min, and dissolve quickly (4–6 min) using an amantadine hydrochloride (AH) solution that breaks the supramolecular interactions in the SHHs. Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro. The SHHs also do not elicit any significant cytokine response in vitro. Furthermore, in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls. Thus, these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

Objective Chronic skin wounds are one of the most devastating complications in diabetic patients due to the formation of advanced glycation end products (AGEs) resulting from non-enzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger pro-inflammatory signals that impair wound healing in diabetes and contribute to the pathology of chronic skin wounds. Approach We previously developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that acts as a competitive inhibitor of AGEs, and another ELP fusion protein containing stromal cell-derived factor 1 (SDF1) that promotes revascularization. Herein, we report the effects of protein coacervates incorporating both vRAGE-ELP and SDF1-ELP on wound healing in an in vitro diabetes-mimicking cell culture system, and in in vivo in full-thickness wounds on diabetic mice. Results The combination of vRAGE-ELP and SDF1-ELP increased cell metabolic activity in AGE-stimulated endothelial cells, promoted in vitro tube formation and accelerated healing in an in vitro cell migration assay. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure in the combination groups reached almost 100% on post-wounding day 35, compared to 62% and 85% on the same days in animals treated with fibrin gel control and vehicle control consisting of ELP alone. Innovation To our knowledge, this is the first study that attempts to reverse the AGE-RAGE-mediated signaling as well as to promote cell proliferation and vascularization in one single treatment. Conclusion The co-delivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.

We describe the design, generation, and in vitro evaluation of targeted amphipathic fusion peptides and immunoconjugates for the ablation of prostate cancer cells. The overexpression of the prostate-specific membrane antigen (PSMA) was exploited as means to specifically deliver cytotoxic peptides to prostate cancer cells. Cationic amphipathic lytic peptides were chosen as cytotoxic agents due to their ability to depolarize mitochondrial membranes and induce apoptosis. Specific delivery of the lytic peptide was facilitated by PSMA-targeting peptides and antibodies. Our results indicate that although the use of PSMA-targeted peptides only modestly enhanced the cytotoxic activity of the lytic peptide, peptide-antibody conjugates were two orders of magnitude more potent than untargeted peptide. In addition to quantifying the cytotoxic activities of the individual constructs, we also investigated the mechanisms of cell death induced by the fusion peptides and immunoconjugates. Although fusion peptides induced oncotic/necrotic death in cells, treatment with immunoconjugates resulted in apoptotic death. In summary, immunoconjugates based on lytic peptides are a promising class of therapeutics for prostate cancer therapy and warrant further investigation. [Cancer Res 2007;67(13):6368–75]

Supplementary Figures 1-2 from Amphipathic Peptide-Based Fusion Peptides and Immunoconjugates for the Targeted Ablation of Prostate Cancer Cells

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA dictates human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA targeting of FOXA1 and FOXA2 in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p < 0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < 0.05), all consistent with a partial/transient cell reprogramming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During hPSC-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate complex effects of FOXA1/2 on hepatic GRN effecting de-differentiation and metabolism with applications in studies of cancer, differentiation, and organogenesis.

Elastin-like peptides (ELPs) are a versatile platform for tissue engineering and drug delivery. In this study, micelle forming ELP chains are genetically fused to three therapeutic molecules, keratinocyte growth factor (KGF), stromal cell-derived growth factor 1 (SDF1), and cathelicidin (LL37), to be used in wound healing. Chronic wounds represent a growing problem worldwide. A combinatorial therapy approach targeting different aspects of wound healing would be beneficial, providing a controlled and sustained release of active molecules, while simultaneously protecting these therapeutics from the surrounding harsh wound environment. The results of this study demonstrate that the conjugation of the growth factors KGF and SDF1, and the antimicrobial peptide LL37 to ELPs does not affect the micelle structure and that all three therapeutic moieties retain their bioactivity in vitro. Importantly, when the combination of these micelle ELP nanoparticles are applied to wounds in diabetic mice, over 90% wound closure is observed, which is significantly higher than when the therapeutics are applied in their naked forms. The application of the nanoparticles designed in this study is the first report of targeting different aspect of wound healing synergistically.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Background: High-powered pulsed electric fields (PEF) may be used for tissue debridement and disinfection, while lower PEF intensities may stimulate beneficial cellular responses for wound healing. We investigated the dual effects of nonuniform PEF on cellular death and stimulation. Methods: Dermal fibroblast or keratinocyte monolayers were exposed to PEF induced by two needle electrodes (2 mm apart). Voltages (100–600 V; 1 Hz; 70 micros pulse width; 90 pulses/cycle) were applied between the two electrodes. Controls consisted of similar monolayers subjected to a scratch mechanical injury. Results: Cell growth and closure of the cell-free gap was faster in PEF-treated cell monolayers versus scratched ones. Media conditioned from cells pre-exposed to PEF, when applied to responder cells, stimulated greater proliferation than media from scratched monolayers. Conclusions: PEF treatment causes the release of soluble factors that promote cell growth, and thus may play a role in the accelerated healing of wounds post PEF.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

Medical robots have demonstrated the ability to manipulate percutaneous instruments into soft tissue anatomy while working beyond the limits of human perception and dexterity. Robotic technologies further offer the promise of autonomy in carrying out critical tasks with minimal supervision when resources are limited. Here, we present a portable robotic device capable of introducing needles and catheters into deformable tissues such as blood vessels to draw blood or deliver fluids autonomously. Robotic cannulation is driven by predictions from a series of deep convolutional neural networks that encode spatiotemporal information from multimodal image sequences to guide real-time servoing. We demonstrate, through imaging and robotic tracking studies in volunteers, the ability of the device to segment, classify, localize and track peripheral vessels in the presence of anatomical variability and motion. We then evaluate robotic performance in phantom and animal models of difficult vascular access and show that the device can improve success rates and procedure times compared to manual cannulations by trained operators, particularly in challenging physiological conditions. These results suggest the potential for autonomous systems to outperform humans on complex visuomotor tasks, and demonstrate a step in the translation of such capabilities into clinical use. Getting safe and fast access to blood vessels is vital to many methods of treatment and diagnosis in medicine. Robot-assisted or even fully autonomous methods can potentially do the task more reliably than humans, especially when veins are hard to detect. In this work, a method is tested that uses deep learning to find blood vessels and track the movement of a patient’s arm.

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.

Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases. Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

Objective: The incidence of severe infectious complications after burn injury increases mortality by 40%. However, traditional approaches for managing burn infections are not always effective. High-voltage, pulsed electric field (PEF) treatment shortly after a burn injury has demonstrated an antimicrobial effect in vivo; however, the working parameters and long-term effects of PEF treatment have not yet been investigated. Approach: Nine sets of PEF parameters were investigated to optimize the applied voltage, pulse duration, and frequency or pulse repetition for disinfection of Pseudomonas aeruginosa infection in a stable mouse burn wound model. The bacterial load after PEF administration was monitored for 3 days through bioluminescence imaging. Histological assessments and inflammation response analyses were performed at 1 and 24 h after the therapy. Results: Among all tested PEF parameters, the best disinfection efficacy of P. aeruginosa infection was achieved with a combination of 500 V, 100 μs, and 200 pulses delivered at 3 Hz through two plate electrodes positioned 1 mm apart for up to 3 days after the injury. Histological examinations revealed fewer inflammatory signs in PEF-treated wounds compared with untreated infected burns. Moreover, the expression levels of multiple inflammatory-related cytokines (interleukin [IL]-1α/β, IL-6, IL-10, leukemia inhibitory factor [LIF], and tumor necrosis factor-alpha [TNF-α]), chemokines (macrophage inflammatory protein [MIP]-1α/β and monocyte chemoattractant protein-1 [MCP-1]), and inflammation-related factors (vascular endothelial growth factor [VEGF], macrophage colony-stimulating factor [M-CSF], and granulocyte–macrophage colony-stimulating factor [G-CSF]) were significantly decreased in the infected burn wound after PEF treatment. Innovation: We showed that PEF treatment on infected wounds reduces the P. aeruginosa load and modulates inflammatory responses. Conclusion: The data presented in this study suggest that PEF treatment is a potent candidate for antimicrobial therapy for P. aeruginosa burn infections.

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, i.e. hypoxia, can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous micro-tissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3-6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability, in this platform, decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in reactive oxygen species (ROS) accumulation and hypoxiainducible factor-1α (HIF1α) expression was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions.

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at –6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at –4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood. Preservation of human livers at subzero temperatures with ice-free supercooling extends ex vivo organ life.

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin (Hb)-based oxygen carriers have shown potential in increasing oxygen delivery to aid wound healing. Macrophages also take up Hb, thus altering their phenotype and the regulation of inflammation. Herein, we compared the effect of Hb and polymerized Hbs (PolyHbs) on the phenotype of human macrophages. Macrophages were incubated with Hb or different forms of PolyHbs, and the inflammatory secretion profile was analyzed. PolyHbs were produced by polymerizing Hb in the relaxed (R) or tense (T) quaternary state and by varying the molar ratio of the glutaraldehyde crosslinking agent to Hb. Hb decreased the secretion of most measured factors. PolyHb treatment led to generally similar secretion profiles; however, Hb had more similar trends to R-state PolyHb. Ingenuity pathway analysis predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb prepared with a 30:1 (glutaraldehyde:Hb) polymerization ratio. When tested in diabetic mouse wounds, T-state PolyHb resulted in the greatest epidermal thickness and vascular endothelial CD31 staining. Thus, the effects of PolyHb on macrophages are affected by the polymerization ratio and the quaternary state, and T-state PolyHb yields secretion profiles that are most beneficial in wound healing.

Here we present a model to estimate the interaction free energy contribution of each amino acid residue of a given protein. Protein interaction energy is described in terms of per-residue interaction factors, [Formula: see text]. Multibody interactions are implicitly captured in [Formula: see text] through the combination of amino acid terms ([Formula: see text]) guided by local conformation indices ([Formula: see text]). The model enables construction of an interaction factor heat map for a protein in a given fold, allows prima facie assessment of the degree of residue–residue interaction, and facilitates a qualitative and quantitative evaluation of protein association properties. The model was used to compute thermal stability of T4 bacteriophage lysozyme mutants across seven sites. Qualitative assessment of mutational effects provides a straightforward rationale regarding whether a particular site primarily perturbs native or non-native states, or both. The presented model was found to be in good agreement with experimental mutational data ([Formula: see text]) and suggests an approach by which to convert structure space into energy space.

Irreversible electroporation of cell membrane with pulsed electric fields is an emerging physical method for disinfection that aims to reduce the doses and volumes of used antibiotics for wound healing. Here we report on the design of the IGBT-based pulsed electric field generator that enabled eradication of multidrug resistant Pseudomonas aeruginosa PAO1 on the gel. Using a concentric electric configuration we determined that the lower threshold of the electric field required to kill P. aeruginosa PAO1 was 89.28 ± 12.89 V mm−1, when 200 square pulses of 300 µs duration are delivered at 3 Hz. These parameters disinfected 38.14 ± 0.79 mm2 area around the single needle electrode. This study provides a step towards the design of equipment required for multidrug-resistant bacteria disinfection in patients with pulsed electric fields.

Donation after circulatory death (DCD) liver grafts are known to be predisposed to primary nonfunction and ischemic cholangiopathy. Many DCD grafts are discarded because of older donor age or long warm ischemia times. Thus, it is critical to improve the quality of DCD liver grafts. Here, we have tested whether an enriched oxygen carrier added to the preservation solution can prolong graft survival and reduce biliary damage.We assessed the adenosine triphosphate (ATP) content decay of mouse liver grafts after cold ischemia, warm ischemia, and combined warm+cold ischemia. In addition, we used a rat model of liver transplantation to compare survival of DCD grafts preserved in high-oxygen solution (preoxygenated perfluorocarbon [PFC] + University of Wisconsin [UW] solution) versus lower oxygen solution (preoxygenated UW solution).Adenosine triphosphate levels under UW preservation fall to less than 10% after 30 minutes of warm ischemia. Preoxygenated UW solution with PFC reached a significantly higher PaO2. After 45 minutes of warm ischemia in oxygenated UW + PFC solution, grafts showed 63% higher levels of ATP (P = 0.011). In addition, this was associated with better preservation of morphology when compared to grafts stored in standard UW solution. Animals that received DCD grafts preserved in higher oxygenation solution showed improved survival: 4 out of 6 animals survived long-term whereas all control group animals died within 24 hours.The additional oxygen provided by PFC during static cold preservation of DCD livers can better sustain ATP levels, and thereby reduce the severity of ischemic tissue damage. PFC-based preservation solution extends the tolerance to warm ischemia, and may reduce the rate of ischemic cholangiopathy.