The positive effect of photobiomodulation in wound healing has previously been reported, however there is a considerable lack of knowledge regarding the molecular mechanisms involved, and no consensus on light parameters. Cytochrome c oxidase (CCO) is established as the main photoreceptor in cells, but light also induces nitric oxide (NO), production of reactive oxygen species (ROS) and activation of ion channels. Emerging new molecular targets include the GPCRs opsins (OPNs) and the circadian clock transcription factors, cryptochromes (CRYs). Localisation of OPN1-SW, OPN3, OPN5, CRY1 and CRY2 was seen in female facial and abdominal human skin. Furthermore, expression of these photoreceptors was retained in primary epidermal keratinocytes and dermal fibroblasts in culture; both cell types expressed OPN1-SW, OPN3, CRY1 and CRY2, at the mRNA and protein level. OPN2 was only expressed in cultured dermal fibroblasts, while in line with in situ expression, OPN5 was only expressed in cultured keratinocytes. The photoreceptor-expressing cultured epidermal keratinocytes demonstrated a dose- and wavelength- dependent response in both metabolic activity and cell migration in a scratch-wound assay. Specifically, low dose (2 J/cm2) blue light (447 nm) increased metabolic activity, but it did not impact keratinocyte migration. In contrast, high dose (30 J/cm2) blue light had no effect on metabolism, but inhibited migration of epidermal keratinocytes. Red light (655 nm) at 30 J/cm2 stimulated metabolic activity but did not modulate migration, while a higher dose of 60 J/cm2 had no effect on keratinocyte metabolic activity. In order to study OPN3 and CRY1 function, they were silenced in keratinocytes using siRNA; additionally 8 μM KL001 was used to stabilize CRY1. KL001 inhibited migration, and induced KRT1 and KRT10, an effect which was abrogated by knockdown of OPN3. Interestingly, knockdown of OPN3 upregulated CRY1 expression, while KL001 upregulated OPN3 expression, indicating a regulation by OPN3 of the molecular epidermal clock. Low levels of blue light increased early differentiation of epidermal keratinocytes, which was mediated by OPN3 and circadian clock mechanisms. However, low levels of blue light decreased keratinocyte DNA synthesis, which was mediated by circadian clock independently of OPN3. Translation of parameters ex vivo showed increasing re-epithelialisation and induction of OPN3 and CRY1 expression following exposure to 2 J/cm2 of blue light; however high doses of blue light inhibited re-epithelialisation. Red light, also increased re-epithelialisation, but had no effect on OPN3 or CRY1 expression. In conclusion, photoreceptors are expressed in human skin and they mediate DNA synthesis, migration and differentiation of epidermal keratinocytes. Furthermore, low dose of blue light interacts with OPN3 to induce epidermal differentiation, through the regulation of the circadian clock. A better understanding of the molecular mechanisms behind the photobiomodulation response in vitro will help to develop light based therapies for human wound healing. Interestingly, selected light parameters translated to human ex vivo skin showed a beneficial effect of low doses of blue (2 J/cm2) and red (30 J/cm2) light in re-epithelialisation. / Marie Curie ... the CLaSSiC project

Clinical management of complex wounds is essential to promote wound healing. Prolonged healing time may lead to longer and more costly hospitalizations and poorer patient outcomes. The removal of nonviable, necrotic tissue via debridement is vital to the healing process. One of the most common debridement techniques, in the United States, is the use of wet-to-dry dressings. There are no defined guidelines or protocols for the timing of dressing changes and subsceequent debridement. The purpose of this study was to perform a review of literature to determine the rationale for the use of wet-to-dry dressings, explore alternative time sequences of treatment, and to identify the risks and benefits for this methodology of debridement in an adult population with acute traumas. Inclusion criteria consisted of peer reviewed, English Language, research articles published within the last 5 years (2007-2012), adults with acute wounds treated by wet-to-dry dressing debridement. This review of literature was conducted using CINAHL and MEDLINE databases using the following search terms: Wound debridement, wet-to-dry dressing', timing, sequencing, schedul', standard', debridement, acute wound', and mechanical debridement. The review of literature yielded zero results meeting the search criteria therefore, a second review of literature was performed using the same search criteria but expanded to include articles published within the past 15 years (1997 -2012). The second review of literature also yielded zero results that met the search criteria. A lack of evidence supporting the use of wet-to-dry dressings for the purpose of debridement suggests that healthcare providers are following tradition rather than evidence based practices. Nurses and healthcare providers need education on best practices in wound care to advocate for their patients to ensure the best possible outcome. Further research on wound care modalities that are clinically efficient is needed.

yes / There are various techniques to enhance tissue regeneration via the application of growth factors to the site of regeneration to induce cells to proliferate, differentiate and regenerate. Generally, direct application of growth factors has little effect [1] because the growth factor diffuses out from the site of regeneration very quickly. This is a problem that can be solved by a controlled release of growth factor at the site of action over a long period of time by use of a bioabsorbable scaffold. Growth factors are protein based molecules in the body which are produced by cells and attach to the cell surface. Growth factors bind to membrane receptors, which in turn activate an intracellular signalling pathway. This will activate or inhibit a gene causing either an up regulation or down regulation of a gene product, which then alters the cells behaviour.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

No description available.

Wounds heal better in skin with terminal hair follicles (large and pigmented) as opposed to those with vellus hair follicles (small and unpigmented), while dermal fibroblasts from different anatomical regions also exhibit phenotypical differences. Tissue repair requires a tight control of cell proliferation, migration and apoptosis, and recent studies have shown the importance of inhibitors of apoptosis proteins (IAPs), which are proteins that prevent the process of apoptosis via their interaction with caspase molecules in wound healing. Oestrogens improve the rate and quality of wound healing, but their relationship with IAPs in human skin has not been studied. Therefore, terminal (scalp) and vellus (facial) hair bearing skin from the same donor was compared in situ and matching primary cultures of dermal fibroblasts were established from terminal (DF(T)) and vellus (DF(V)) hair bearing skin. Using immunofluorescent staining, the expression of IAPs and their antagonists was compared at different stages of the hair cycle following depilation using a murine model and then in terminal and vellus hair bearing human skin. The size and granularity of matching DF(T) and DF(V) cultures was compared by FACS analysis and mRNA and protein expression of Apollon, cIAP2, NAIP and XIAP and their antagonists DIABLO and Xaf1 analysed by qRT-PCR and immunocytochemistry in unwounded and mechanically wounded fibroblast cultures. Differences in proliferation, migration, viability and caspase 3 activity in the presence of 17β-oestradiol and changes in mRNA expression of the oestrogen receptors (GPR30, ERα and ERβ) were compared between the two cell types. IAP protein expression was generally found higher during mid anagen of the hair cycle in murine skin and hair follicles. Overall, expression was slightly higher in human terminal hair bearing skin compared to corresponding vellus hair bearing skin. IAP protein expression was similar in unwounded DF(T) and DF(V) cells with the exception of Apollon which was higher in DF(V) cells. With the exception of XIAP and its direct antagonist Xaf1, mRNA expression was higher in DF(V) cells compared to corresponding DF(T) cells. FACS analysis demonstrated that DF(V) cells were more granular than matching DF(T) cells and proliferated faster. 17β-oestradiol accelerated migration of DF(T) cells only. Mechanical wounding decreased XIAP mRNA in DF(T) and increased it in DF(V) cells, while simultaneously decreasing Xaf1 expression. In unwounded cells, 17β-oestradiol stimulated the expression of XIAP mRNA in both DF(T) and DF(V) cells, but in scratched monolayers, while it also increased expression in DF(T) cells it decreased it in DF(V) cells. A XIAP inhibitor reduced cell viability in both DF(T) and DF(V) cells, which was rescued by 17β-oestradiol in unwounded and mechanically wounded DF(T) cells, but only in unwounded DF(V) cells. 17β-oestradiol decreased caspase 3 activity in the presence of a XIAP inhibitor only in DF(T) cells. These results demonstrate significant differences between dermal fibroblasts cultured from terminal and vellus hair bearing skin of the same individual. The correlation between an increase in XIAP in response to 17β-oestradiol and a higher number of viable cells, along with a reduction in caspase 3 activity suggests that the protective effect of 17β-oestradiol may be modulated via the regulation of XIAP. Further elucidation of these different signalling pathways in dermal fibroblasts from hair bearing skin may lead to improved therapies for chronic non-healing wounds, particularly in postmenopausal females.

Dermal fibroblasts positively contribute throughout the wounding response by secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on the functional, secretory, and molecular properties of these cells. This study aims to understand how the effect of the pro-inflammatory cytokine TNF-α modulates both healthy and diabetic dermal fibroblast phenotype. To mimic a chronic inflammatory environment and assess whether fibroblasts respond similarly in different anatomical sites, donor-matched fibroblasts from face and scalp were pre-incubated for 3 days with different concentrations (2.5, 25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation by day 14 in cells from both sites and stimulated (papillary) metabolic activity at day 14. However, this did not correlate with an increase in papillary cell senescence since this did not appear until passage 17, and then only at a supra pathophysiological concentration. Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α significantly inhibited the cells migration, particularly in diabetic fibroblasts, suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium was collected to assess its effect on other dermal fibroblasts, however, this had no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the secretion of soluble paracrine factors that induce MMP activity in non-diabetic fibroblasts, mirroring previous observations that a pro-inflammatory environment can increase proteolytic activity, and indicating that diabetic fibroblasts were again more sensitive than healthy. No difference was observed with MMP-9 activity and nor did the results with dermal fibroblasts reach statistical significance, perhaps because of a relatively low n-number. The ability of TNF-α to modulate the expression of genes associated with the ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was investigated. There was no change in Sirt1 and Sirt6 expression and no evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α significantly induced mRNA expression of MMP-1 in healthy non-scratched and scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There was also considerable donor variability that prevented statistical significance being achieved under the other conditions. The secretion of various cytokines associated with inflammation was compared in healthy and diabetic fibroblasts in the presence and absence of TNF-α. Seven cytokines were secreted, by healthy and diabetic male and female fibroblasts, although diabetic female fibroblasts did not secrete two of them. TNF-α stimulated secretion of cytokines in healthy and diabetic, male and female cells but the profiles of those released were different between the different groups. There was no TNF-α induced paracrine effect on cytokine secretion by healthy dermal fibroblasts. In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype. Understanding these functional and molecular changes in response to inflammatory cytokines will give a better understanding of the differences between fibroblast activity in normal physiological wound healing and chronic or diabetic non-healing wounds.

No description available.

No description available.

No description available.