Chitosan-polygalacturonic acid complex dressing improves diabetic wound healing and hair growth in diabetic mice.

Chronic skin wounds decrease the quality of life of millions of diabetic patients worldwide. Chitosan has previously been shown to possess hemostatic properties, decrease inflammation, promote fibroblast proliferation, and hair growth. We developed a relatively low-cost polyelectrolyte complex (PEC) film dressing made of chitosan and polygalacturonic acid and tested it for its ability to accelerate diabetic wound healing. Genetically diabetic male mice were shaved on the dorsum, and one day later a 1 cm diameter full-thickness excisional wound was created. The PEC film was applied immediately after wounding and left in place for 14 days. Controls consisted of wounds treated with a fibrin gel. Wounds covered with the PEC film had closed completely by post-wounding day 42, while untreated wounds were only half-way closed. Histological analysis of wounds confirmed that PEC-treated wounds had fully re-epithelialized, while control wounds lacked a continuous epidermis at the wound center. We also observed that the area of skin under the PEC film experienced much more rapid hair growth. Histologically, there were significantly more hair follicles around the scar area (p < 0.05) in the PEC-treated group as compared to the control group. Thus, chitosan-polygalacturonic acid PEC films can accelerate both wound healing and hair growth in diabetic mice, and should be further investigated as a potential future treatment for diabetic chronic wounds.

High-Voltage, Pulsed Electric Fields Eliminate Pseudomonas aeruginosa Stable Infection in a Mouse Burn Model.

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/ß, IL-6, IL-10, leukemia inhibitory factor [LIF], and tumor necrosis factor-alpha [TNF-α]), chemokines (macrophage inflammatory protein [MIP]-1α/ß 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.

Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

BACKGROUND & AIMS: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.

Neural lineage differentiation of embryonic stem cells within alginate microbeads.

Cell replacement therapies, using renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, they are often limited by lack of control and low yields of differentiated cells. Previously, we developed a scalable murine embryonic stem cell differentiation environment which maintained cell viability and supported ES cell differentiation to hepatocyte lineage cells. Differentiated hepatocyte function was contingent upon aggregate formation within the alginate microbeads. The present studies were designed to determine the feasibility of adapting the alginate encapsulation technique to neural lineage differentiation. The results of our studies indicate that by incorporating the soluble inducer, retinoic acid (RA), into the permeable microcapsule system, cell aggregation was decreased and neural lineage differentiation enhanced. In addition, we demonstrated that even in the absence of RA, differentiation could be directed away from the hepatocyte and toward the neural lineage by physical cell-cell aggregation blocking. In conjunction with the mechanical and physical characterization of the alginate crosslinking network, we determined that 2.2% alginate microencapsulation can be optimally adapted to ES neural differentiation. This study offers insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages, and could potentially be adaptable to differentiation of other stem cell types given the correct inducible factors and material properties.