Science, Practice and education regenerative medicine in burn wound healing: Aiming for the perfect skin

The healing of full thickness wounds such as burn wounds remains complicated by hypertrophic scar formation and contraction. The standard treatment is transplantation with autologous split thickness skin grafts. For extensive burns, these grafts are widely meshed due to limited donor sites, which often results in a poor functional and cosmetic outcome. The application of cultured autologous keratinocytes may enhance wound closure and improve scars. The first epidermal substitute, a confluent epithelial sheet, was developed in 1979. These cultured epidermal autografts (CEA) have been used in burn patients with variable success. Due to the variation in the efficacy of CEAs, however, new strategies have been employed. Currently, the application of preconfluent proliferating keratinocytes is considered a better strategy for burn wound treatment. In addition to improvements in epidermal grafts, the healing outcome may improve with the application of dermal substitutes. Over the past several decades, several scaffolds have been developed to mimic the dermis. These substitutes can be supplemented with growth factors and cells. In particular, the application of mesenchymal stem cells (MSCs) is thought to be a promising perspective for cell-based tissue engineering

Increased expression of a homologue of drosophila tissue polarity gene "frizzled" in left ventricular hypertrophy in the rat, as identified by subtractive hybridization

The molecular mechanisms that govern the development of left ventricular hypertrophy are not fully elucidated. We performed a subtractive hybridization procedure to identify genes controlling this adaptive process. Using this approach, we isolated a rat homologue of Drosophila tissue polarity gene "frizzled" 2 (fz-2). The expression of this gene was quantified by competitive reverse transcriptase polymerase chain reactions. The expression was higher in hypertrophic left ventricles at all time points tested, reaching statistical significance at days 1 and 10. We conclude that the fz-2 gene, a highly conserved gene for which a role in intra-and intercellular communication has been described, may be involved in the spatial control of ventricular remodeling

Safety and bactericidal efficacy of cold atmospheric plasma generated by a flexible surface Dielectric Barrier Discharge device against Pseudomonas aeruginosa in vitro and in vivo

BACKGROUND: Cold atmospheric plasma (CAP), which is ionized gas produced at atmospheric pressure, could be a novel and potent antimicrobial therapy for the treatment of infected wounds. Previously we have shown that CAP generated with a flexible surface Dielectric Barrier Discharge (sDBD) is highly effective against bacteria in vitro and in ex vivo burn wound models. In the current paper, we determined the in vitro and in vivo safety and efficacy of CAP generated by this sDBD device. METHODS: The effect of CAP on DNA mutations of V79 fibroblasts was measured using a hypoxanthine-guanine-phosphoribosyltransferase (HPRT) assay. Furthermore, effects on cell proliferation, apoptosis and DNA damage in ex vivo burn wound models (BWMs) were assessed using immunohistochemistry. Next, 105 colony forming units (CFU) P. aeruginosa strain PAO1 were exposed to CAP in a 3D collagen-elastin matrix environment to determine the number of surviving bacteria in vitro. Finally, rat excision wounds were inoculated with 107 CFU PAO1 for 24 h. The wounds received a single CAP treatment, repeated treatments on 4 consecutive days with CAP, 100 µL of 1% (wt/wt) silver sulfadiazine or no treatment. Wound swabs and punch biopsies were taken to determine the number of surviving bacteria. RESULTS: Exposure of V79 fibroblasts to CAP did not increase the numbers of mutated colonies. Additionally, the number of proliferative, apoptotic and DNA damaged cells in the BWMs was comparable to that of the unexposed control. Exposure of PAO1 to CAP for 2 min resulted in the complete elimination of bacteria in vitro. Contrarily, CAP treatment for 6 min of rat wounds colonized with PAO1 did not effectively reduce the in vivo bacterial count. CONCLUSIONS: CAP treatment was safe but showed limited efficacy against PAO1 in our rat wound infection model

Isoenzymes of vitamin-K-dependent carboxylase

Vitamin-K-dependent carboxylase was prepared from bovine liver, kidney, lung and testis and it was checked that these systems obeyed the laws of normal enzyme kinetics. Four carboxylatable substrates were obtained from different sources and the apparent Michaelis constants of the various carboxylase for these four substrates were measured. From the results thus obrained we conclude that carboxylase is a group name for a number of isoenzymes which are present in heaptic as well as in various non-hepatic tissues

Expression profile of proteins involved in scar formation in the healing process of full-thickness excisional wounds in the porcine model

Scar formation in deep dermal wounds is associated with excessive collagen deposition and contraction. Increased collagen synthesis and decreased collagen degradation are the mechanisms through which this form of fibrosis can occur. Another factor might be a different kind of collagen cross-linking seen in fibrotic skin diseases. This type of cross-linking is dependent on the enzyme lysyl hydroxylase-2b. In this study, we examined the expression profile of the potential key players in scar formation in time in healing of acute wounds. Collagen types I and III, lysyl hydroxylase-2b, α-smooth muscle actin, transforming growth factor βs, and the matrix metalloproteinases and their inhibitor mRNA levels were determined. All genes examined show distinct expression patterns over time. The expression of lysyl hydroxylase-2b peaks at day 7, and precedes collagen types I and III expression. Eight weeks after wounding, the scars showed an increased level of lysyl hydroxylase-2b-mediated collagen cross-linking. This study shows that the fibrosis-specific type of cross-linking of collagen seen in human hypertrophic scarring also plays a role in this animal model of wound healing. Moreover, the expression of the putative gene responsible for this type of cross-linking, the lysyl hydroxylase-2b, is elevated during wound healing

The quantification of gammacarboxyglutamic acid residues in plasma-osteocalcin

In this paper we describe an assay procedure for determining the amount of gammacarboxyglutamic acid (Gla) residues in serum- or plasma-osteocalcin. The test includes removing by ethanol precipitation the majority of the proteins from the plasma (e.g., the Gla-containing coagulation factors) and the specific extraction of osteocalcin with the aid of immobilized immunopurified antibodies. It is demonstrated that the Gla-content of circulating osteocalcin from normal cows is similar to that of osteocalcin obtained from bone. Hence, the fact that part of the newly synthesized osteocalcin does not bind to the hydroxyapatite matrix in bone cannot be explained by an undercarboxylation of the molecule

The presence of tissue renin-angiotensin system components in human burn wounds and scars

Objective: Healing of severe and large surface burn wounds is faced with hurdles such as aberrant wound healing and excessive scar formation. The tissue renin-angiotensin system (tRAS) is involved in dermal wound healing, and fibrosis of other organs. However, little is known about the presence of tRAS during burn wound healing in human skin. This study investigated the presence of tRAS components in human burn wounds and scars. Methods: Dermal tissue biopsies were collected from 39 patients and divided into six categories: burn wounds post burn day (PBD)0–9, PBD11–21 and PBD22–37; young scars (1.5–3.5 months), mature scars (>12 months) and control skin from 9 patients. The tRAS components angiotensin converting enzyme (ACE), chymase, angiotensin receptor 1 (AT1) and Mas receptor were detected via immunohistochemistry. Digital images were acquired and analyzed using image analysis software. Results: Burn wounds from PBD22–37 showed a decreased expression of ACE and chymase compared to earlier time points or control, respectively. In contrast, ACE expression was increased in young scars compared to control skin but was normalized in mature scars. In comparison to control, mature scars showed increased AT1 expression. Conclusions: These results show the presence of components of tRAS in human burn wounds and scars. In addition, they suggest that tRAS has a time-dependent response during burn wound healing. Reduced tRAS might play a role in delayed healing, while an increase during remodeling phase might contribute to scar formation. This research provides a basis for future studies exploring tRAS involvement in burn wounds and scars. Keywords: Tissue renin-angiotensin system, Burn wounds, Scars, Ski

Health effects and time course of particulate matter on the cardiopulmonary system in rats with lung inflammation

Recent epidemiological studies associate health effects and particulate matter in ambient air. Exacerbation of the particle-induced inflammation can be a mechanism responsible for increased hospitalization and death due to cardiopulmonary events in high-risk groups of the population. Systems regulating blood pressure that depend on lung integrity can be involved in progression of cardiovascular diseases. This study focused on the expression levels of various genes involved in cardiovascular and pulmonary diseases to assess their role in the onset of cardiovascular problems due to ambient particulate matter and compared these with the corresponding products. Rats with ozone-induced (1600 μg/m3; 8 h) pulmonary inflammation were exposed to 0.5 mg, 1.5 mg, or 5 mg of particulate matter (PM) from Ottawa Canada (EHC-93) by intratracheal instillation. mRNA levels of various genes and their products were measured 2, 4, and 7 d after instillation. At 2 d after exposures to PM, tumor necrosis factor (TNF)-α levels in bronchoalveolar lavage fluid (BALF) were elevated approximately 4 times for the highest EHC-93 dose. MIP-2 protein levels in BALF were elevated approximately three times during the entire time period studied, whereas IL-6 levels were not affected compared to control groups. The MIP-2 mRNA levels revealed a similar pattern of induction. A twofold increase in endothelin (ET)-1 levels at d 2 and a 20% decrease in angiotensin-converting enzyme (ACE) activity at d 7 were measured in plasma. A 60% decrease of ACE and ET-1 mRNA levels suggested a possible endothelial damage in the lung blood vessels. Inducible nitric oxide synthase (iNOS) mRNA was found to be increased 3.5 times 2 d after instillation of the particles. Therefore, the endothelial damage could have been caused by large amounts of the free radical NO. Also, plasma levels of fibrinogen were elevated (20%), which could presumably increase blood viscosity, leading to decreased tissue blood flow. These changes in hematological and hemodynamic parameters observed in our study are in line with heart failure in high-risk groups of the population after high air pollution episodes