A nanoenzyme-modified hydrogel targets macrophage reprogramming-angiogenesis crosstalk to boost diabetic wound repair.

Diabetic wounds has a gradually increasing incidence and morbidity. Excessive inflammation due to immune imbalance leads to delayed wound healing. Here, we reveal the interconnection between activation of the NLRP3 inflammatory pathway in endotheliocyte and polarization of macrophages via the cGAS-STING pathway in the oxidative microenvironment. To enhance the immune-regulation based on repairing mitochondrial oxidative damage, a zeolitic imidazolate framework-8 coated with cerium dioxide that carries Rhoassociated protein kinase inhibition Y-27632 (CeO2-Y@ZIF-8) is developed. It is encapsulated in a photocross-linkable hydrogel (GelMA) with cationic quaternary ammonium salt groups modified to endow the antibacterial properties (CeO2-Y@ZIF-8@Gel). CeO2 with superoxide dismutase and catalase activities can remove excess reactive oxygen species to limit mitochondrial damage and Y-27632 can repair damaged mitochondrial DNA, thus improving the proliferation of endotheliocyte. After endotheliocyte uptakes CeO2-Y@ZIF-8 NPs to degrade peroxides into water and oxygen in cells and mitochondria, NLRP3 inflammatory pathway is inhibited and the leakage of oxidatively damaged mitochondrial DNA (Ox-mtDNA, a damage-associated molecular pattern) through mPTP decreases. Futhermore, as the cGAS-STING pathway activated by Ox-mtDNA down-regulated, the M2 phenotype polarization and anti-inflammatory factors increase. Collectively, CeO2-Y@ZIF-8@Gel, through remodulating the crosstalk between macrophage reprogramming and angiogenesis to alleviate inflammation in the microenvironment and accelerates wound healing.

A Sustained-Release Nanosystem with MRSA Biofilm-Dispersing and -Eradicating Abilities Accelerates Diabetic Ulcer Healing.

Introduction: Drug-resistant bacterial infections and biofilm formation play important roles in the pathogenesis of diabetic refractory wounds. Tea tree oil (TTO) exhibits antimicrobial, antimycotic, and antiviral activities, especially against common clinically resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), making it a potential natural antimicrobial for the treatment of acute and chronic wounds. However, TTO is insoluble in water, volatile, light-sensitive, and cytotoxic. While previous macroscopic studies have focused on sterilization with TTO, none have sought to alter its structure or combine it with other materials to achieve sustained release. Methods: Electrospun TTO nanoliposomes (TTO-NLs), arranged linearly via high-pressure homogenization, could stabilize the structure and performance of TTO to achieve slow drug release. Herein, we established a composite nano-sustained release system, TTO-NL/polyvinyl alcohol/chitosan (TTO-NL@PCS), using high-voltage electrospinning. Results: Compared with the control, TTO-NL@PCS exhibits higher concentrations of the active TTO drug components, terpinen-4-ol and 1,8-cineole. Owing to its increased stability and slow release, early exposure to TTO-NL@PCS increases the abundance of reactive oxygen species in vitro, ultimately causing the biofilm to disperse and completely killing MRSA without inducing cytotoxic effects to the host. Moreover, in BKS-Leprem2Cd479/Gpt mice with a whole-layer skin infection, untargeted metabolomics analysis of wound exudates reveals upregulated PGF2α/FP receptor signaling and interleukin (IL)-1ß and IL-6 expression following application of the composite system. The composite also ameliorates the chemotaxis disorder in early treatment and attenuates the wound inflammatory response during the repair stage of diabetic inflammatory wounds, and upregulates VEGF expression in the wound bed. Conclusion: TTO-NL@PCS demonstrates the remarkable potential for accelerating diabetic and MRSA-infected wound healing.

3-D tissue-engineered epidermis against human primary keratinocytes apoptosis via relieving mitochondrial oxidative stress in wound healing.

The tissue-engineered epidermal (TEE), composed of biocompatible vectors and autogenous functional cells, is a novel strategy to solve the problem of shortage of donor skin sources. The human primary keratinocyte (HPK), the major skin components, are self-evident vital in wound healing and was considered as one of the preferred seed cells for TEEs. Since the process of separating HPKs from the skin triggers a stress state of the cells, achieving its rapid adhesion and proliferation on biomaterials remains challenging. The key to the clinical application is to ensure the normal function of cells while improving the proliferation ability in vitro, and to complete the complex mesenchymal epithelialization to achieve tissue remodeling after vivo implantation. Herein, in order to aid HPKs adhesion and proliferation in vitro and promoting wound healing, we developed a three dimensional collagen scaffold with Y-27632 sustainedly released from the nanoplatform, hollow mesoporous organosilica nanoparticles (HMON). The results showed that the porous structure within the TEE supports the implanted HPKs expanding in a three-dimensional mode to jointly construct the tissue-engineered epidermis in vitro and inhibited the mitochondria-mediated cell apoptosis. It was confirmed that the TEEs with suitable degradation rate could maintain drug release after implantation and could accelerate vascularization of wound base and further revealed the involvement of mesenchymal transformation of transplanted HPKs during skin regeneration in a nude mouse model with full-thickness skin resection. In conclusion, our study highlights the great potential of constructing TEE using a nanoparticle platform for the treatment of large-area skin defects.

Construction of an immunorelated protein-protein interaction network for clarifying the mechanism of burn.

BACKGROUND AND AIM: Severe burn is known to induce a series of pathological responses resulting in increased susceptibility to systemic inflammatory response and multiple organ failure, but the underlying molecular mechanism remains unclear at present. The main aim of this study was to expand our understanding of the events leading to circulating leukocyte response after burn by subjecting the gene expression profiles to a bioinformatic analysis. MATERIALS AND METHODS: Comprehensive gene expression analysis was performed to identify differentially expressed genes (DEGs) using the expression profile GSE7404 (Mus musculus, circulating leukocyte, 25% of total body surface area (TBSA), full thickness) downloaded from the Gene Expression Omnibus, followed by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In addition, a postburn protein-protein interaction (PPI) network was constructed to identify potential biomarkers. RESULTS: Maximum changes in the gene expression profile were detected 1 day post burn. Separate Gene Ontology (GO) functional enrichment analysis for upregulated and downregulated DEGs revealed significant alterations of genes related to biological process such as "response to stimuli," "metabolic," "cellular and immune system processes," "biological regulation," and "death" in the leukocyte transcriptome after the burn. The KEGG pathway enrichment analysis showed that the upregulated DEGs were significantly enriched in the nodes of immunorelated and signal transduction-related pathways, and the downregulated genes were significantly enriched for the immunorelated pathways. The PPI network and module analysis revealed that, 1 day after the burn, lymphocyte-specific protein tyrosine kinase (Lck) (downregulated), Jun (upregulated), Cd19 (downregulated), Stat1 (downregulated), and Cdk1 (upregulated) were located centrally in both the PPI network and modules. CONCLUSIONS: Based on an integrated bioinformatic analysis, we concluded that Lck, Jun, Cd19, Stat1, and Cdk1 may be critical 1 day after the burn. These findings expand our understanding of the molecular mechanisms of this important pathological process. Further studies are needed to support our work, focused on identifying candidate biomarkers with sufficient predictive power to act as prognostic and therapeutic biomarkers for burn injury.

[Effects of vacuum sealing drainage combined with irrigation of oxygen loaded fluid on wounds of pa- tients with chronic venous leg ulcers].

OBJECTIVE: To evaluate the therapeutic effects of VSD combined with irrigation of oxygen loaded fluid on the growth of granulation tissue and macrophage polarization in chronic venous leg ulcers. METHODS: Thiry-four patients with chronic venous leg ulcers hospitalized in our department from December 2010 to July 2014 were divided into VSD group ( A, n = 11) , VSD + irrigation group ( B, n = 11) , and VSD + oxygen loaded fluid irrigation group ( C, n = 12) according to the random number table. After admissian, debridement was performed, and granulation tissue in the center of the wound was harvested during the operation. After dehridement, the patients in group A were treated with VSD only (negative pressure from -30 to -25 kPa, the same below) ; the patients in group B were treated with VSD combining irrigation of normal saline; the patients in group C were treated with VSD combining normal saline loaded with oxygen irrigation (flow of 1 L/min) . On post treatment day (PTD) 7, the VSD devices were removed. Cross observation was conducted before debridement and on PTD 7. On PTD 7, the granulation tissue in the center of the wound was harvested for histopathological observation with HE staining and Masson staining, following calculation of granulation tissue coverage rate. After debridement but before the negative pressure therapy (hereinafter referred to as before treatment) and on PTD 7, partial pressure of oxygen of the skin around the wound was measured by transcutaneous tissue oxygen tension survey meter. On PTD 7, expression of vascular endothelial growth factor (VECF) was determined with immunohistochemistry. Before treatment and on PTD 7, cells with double positive expressions of induced nitric oxide synthase plus CD68 ( type I macro- phage) and arginase 1 plus CD68 ( type II macrophage) were observed with immunofluorescence staining and quantified. Data were processed with Fisher's exact test, one-way analysis of variance, covariance analysis, paired test, and LSD test. RESULTS: (1) The gross observation showed that before debridement there was a certain amount of necrotic tissue and little granulation tissue in the wounds of patients in all the 3 groups. On PTD 7, new granulation tissue was found in the wounds of patients in all the 3 groups, and in group C its amount was the largest. (2) On PTD 7, the granulation tissue coverage rate of wounds in pa- tients of group C was higher than that of group A or B ( P <0.05 or P <0.01). (3) On PTD 7, HE staining showed that there appeared more abundant new born microvessels and fibroblasts in the wounds of patients in group C than those in groups A and B; Masson staining showed that there was more abundant fresh collagen distributed orderly in the wounds of patients in group C compared with group A or B. (4) On PTD 7, it was found that partial pressure of oxygen of the skin around the wounds in patients of group C [(40.7 +/- 4.1) mmHg, 1 mmHg = 0.133 kPa] was higher than that of group A [ (35.0 +/- 3.1) mmHg] or B [(35.4 +/- 2.7) mmHg, with P values below 0.01]; the partial pressure of oxygen of the skin around the wounds of patients in all the 3 groups was increased significantly compared with that before treatment (with values from 10.38 to 22.52, P values below 0.01). (5) On PTD 7, the expression of VECF in the wounds of patients in group C was higher than that in group A or B ( P <0.05 or P < 0.01). (6) On PTD 7, the number of type I macrophages in granulation tissue of patients was respectively 14.3 +/- 2.3, 11.5 +/- 3.0, and 10.7 +/- 2.3 per 400 times vision field in groups A , B, and C ( F = 25.14, P < 0.01), while the number in group C was less than that in group A or B ( P < 0.05 or P < 0.01). Compared with that before treatment, the number of type I macrophages was significantly decreased on PTD 7 in all the 3 groups (with values from 14.76 to 23. 73, P values below 0. 01). On PTD 7, the number of type II macrophages in granulation tissue of patients was respectively 32.7 +/- 3.2, 35.1 +/- 3.3 , and 41.3 +/- 3.2 per 400 times vision field in groups A, B, and C ( F = 81.10, P < 0.01), and the number in group C was lager than that in group A or B ( with P values below 0. 01). Compared with that before treatment, the number of type II macrophages in all the 3 groups was significantly increased (with t values from -69.34 to -47.95, P values below 0.01). CONCLUSIONS: VSD combined with irrigation of oxygen loaded fluid can raise the partial pressure of oxygen of the skin around the wounds effectively, promoting the transition of macrophages from type I to type II, thus it may promote the growth of granulation tissue, resulting in a better recipient for skin grafting or epithelization.

[Effects of vacuum sealing drainage combined with irrigation of oxygen loaded fluid on chronic wounds in diabetic patients].

OBJECTIVE: To evaluate the therapeutic effects of VSD combined with irrigation of oxygen loaded fluid on chronic wounds in diabetic patients. METHODS: Twenty-six diabetic patients hospitalized in Nanfang Hospital of Southern Medical University from September 2010 to June 2013, with chronic ulcers on lower extremities conforming to the inclusive criteria, were divided into group VSD (n = 8), VSD + irrigation control group (VSD + IC, n = 9), VSD + oxygen loaded fluid irrigation group (VSD OLI, n = 9) according to the random number table. After gross observation was conducted and wound secretion was sent for bacterial culturing right after admission, debridement was performed. During the debridement, granulation tissue of wound center was harvested for determination of the activity of lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) with ELISA. After debridement, the patients in group VSD were treated with VSD (negative pressure from -30 to -25 kPa, the same below); the patients in group VSD + IC were treated with VSD combining irrigation of normal saline; the patients in group VSD + OLI were treated with VSD combining normal saline loaded with oxygen (flow of 1 L/min ) irrigation. Drainage tube blockage was recorded and its incidence rate was recorded during the treatment. On post treatment day (PTD) 7, tissue exudates were collected and analyzed with blood gas analyzer for determining the partial pressure of oxygen of the exudate. After the VSD was terminated, bacterial culture was conducted as before, and the bacterial clearance rate was calculated. After the calculation of granulation tissue coverage rate, the granulation tissue in the center of the wound was harvested for histopathological observation with HE staining; morphological characteristics and density of mitochondria were observed with transmission electron microscopy; the activity of LDH and SDH was estimated as before; microvascular density (MVD) was counted after CD31 antibody immunohistochemical staining. Then the second stage operation was performed. The method of second stage operation was recorded and survival rate of grafted skin or flap was calculated. Data were processed with one-way analysis of variance, LSD- t test, rank sum test, or Fisher's exact test. RESULTS: (1) The gross observation showed that before debridement there was only necrotic tissue without granulation tissue in the wounds of patients in all the 3 groups. On PTD 7, granulation tissue was found in the wounds of patients in all the 3 groups. HE staining showed that there were more abundant newborn microvessels and regularly arranged fibroblasts in the wounds of group VSD + OLI; less newborn microvessels and relatively sparsely fibroblasts were observed in the wounds of group VSD + IC. There were only sparse newborn microvessels and fibroblasts in the wounds of group VSD. (2) Rates of drainage tube blockage, granulation tissue coverage, and bacterial clearance showed significant differences among the 3 groups (with F values from 10.98 to 770.24, P values below 0.01). The drainage tube blockage rate was significantly lower in groups VSD + IC and VSD + OLI [(2.0 ± 0.4)% and (1.9 ± 0.6)%] than in group VSD [(16.0 ± 1.3)%, with t values respectively 28.77 and 29.20, P values below 0.01]. (3) On PTD 7, the partial pressure values of oxygen of the exudate in groups VSD + IC, VSD + OLI, and VSD were respectively (111 ± 4), (43 ± 4), and (40 ± 4) mmHg (1 mmHg = 0.133 kPa, F = 882.76, P < 0.01). (4) The density of mitochondria in group VSD + OLI was obviously higher than that of the other 2 groups, full in shape, with complete outer membrane and no vacuolization. (5) During debridement, the activity of LDH and SDH in 3 groups showed no significant differences (with F values respectively 0.08 and 1.03, P values above 0.05). On PTD 7, the activity of LDH was lower in group VSD + OLI [(103 ± 15) U/L] than in group VSD + IC [(136 ± 16) U/L, t = 4.49, P < 0.01], while it was higher in group VSD [(155 ± 16) U/L] than in group VSD + IC (t = 2.47, P < 0.05). The activity of SDH was higher in group VSD + OLI [(2.93 ± 0.27) U/L] than that in group VSD + IC [(1.77 ± 0.22) U/L] or group VSD [(1.61 ± 0.19) U/L, with t values respectively 10.21 and 11.65, P values below 0.01]. (6) On PTD 7, there was more positive expression of CD31 in group VSD + OLI than in the other 2 groups. The MVD of groups VSD, VSD + IC, and VSD + OLI were respectively (109 ± 5), (124 ± 5), (141 ± 6) per 400 times visual field (F = 68.78, P < 0.01). (7) The patients in 3 groups mainly received skin or flap grafting as the second stage operation. The survival rates of skin and flap in group VSD + OLI were higher than those of groups VSD + IC and VSD (with t values from 3.32 to 8.26, P < 0.05 or P < 0.01), and the rates were higher in group VSD + IC than in group VSD (with t values respectively 2.67 and 3.18, P values below 0.05). CONCLUSIONS: VSD + OLI is effective in reducing drainage tube blockage, removing necrotic tissue and bacteria, ameliorating ischemia and hypoxia of wound tissue, providing fresh wound bed for wound healing, and improving skin or flap graft survival rates.