Ultraviolet-A Light and Negative-Pressure Wound Therapy to Accelerate Wound Healing and Reduce Bacterial Proliferation.

BACKGROUND: Ultraviolet (UV)-A therapy is a simple, inexpensive, and effective modality for wound healing, with tremendous potential to improve healing and reduce clinical infections in a number of clinical settings. To date, application of UV-A relies on bulky and hard-to-dose lamps that provide inconsistent therapy, thus making it difficult to apply therapy that is appropriate for the patient. METHODS: This study was designed to test the effectiveness of a novel wound therapy device that combines UV-A with traditional negative-pressure wound therapy (NPWT) to promote wound healing. Furthermore, we tested the ability of fiberoptic UV-A delivery to inhibit bacterial proliferation. Finally, we assayed the level of DNA damage that results from UV-A as compared to established UV-C therapies. Wound healing studies were performed in a porcine model using an articulated therapy arm that allows for continued therapy administration over an extended time course. Negative-pressure wound therapy was administered alone or with UV-A fiberoptic therapy for 2 weeks. Dressings were changed twice a week, at which time wound area was assessed. RESULTS: Data demonstrate that UV-A with NPWT treatment of wounds results in greater healing than NPWT alone. Using the same therapy device, we demonstrate that exposure of Staphylococcus aureus and Pseudomonas aeruginosa to fiberoptic UV-A results in decreased colony area and number of both bacterial strains. Finally, we show that UV-A induces minimal DNA damage in human fibroblasts and no more DNA damage in wound tissue as compare to intact skin. CONCLUSIONS: These data demonstrate that UV-A can decrease bacterial proliferation and promote wound healing when coupled with NPWT.

Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length.

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome.

Adenosine A2A Receptor Activation Enhances Blood-Tumor Barrier Permeability in a Rodent Glioma Model.

The blood-tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma. Poor drug entry across the BTB allows infiltrative glioma stem cells to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood-brain barrier in non-tumor-bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, downregulation of junctional protein expression by 4 hours, and reestablishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide concentrations, yet no increased survival noted with combined temozolomide therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes among glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance central nervous system treatment delivery to aggressive brain tumors. IMPLICATIONS: This study provides insight on the use of a vasoactive agent to increase exposure of the BTB to chemotherapy with intention to improve glioma treatment efficacy.

Author Correction: Her9/Hes4 is required for retinal photoreceptor development, maintenance, and survival.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Her9/Hes4 is required for retinal photoreceptor development, maintenance, and survival.

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. Her9, the zebrafish ortholog of human HES4, is a basic helix-loop-helix-orange transcriptional repressor that regulates neurogenesis in several developmental contexts. We have previously shown that her9 is upregulated during chronic rod photoreceptor degeneration and regeneration in adult zebrafish, but little is known about the role of her9 during retinal development. To better understand the function of Her9 in the retina, we generated zebrafish her9 CRISPR mutants. Her9 homozygous mutants displayed striking retinal phenotypes, including decreased numbers of rods and red/green cones, whereas blue and UV cones were relatively unaffected. The reduction in rods and red/green cones correlated with defects in photoreceptor subtype lineage specification. The remaining rods and double cones displayed abnormal outer segments, and elevated levels of apoptosis. In addition to the photoreceptor defects, her9 mutants also possessed a reduced proliferative ciliary marginal zone, and decreased and disorganized Müller glia. Mutation of her9 was larval lethal, with no mutants surviving past 13 days post fertilization. Our results reveal a previously undescribed role for Her9/Hes4 in photoreceptor differentiation, maintenance, and survival.

Toll-like Receptor 4 Signaling and Downstream Neutrophilic Inflammation Mediate Endotoxemia-Enhanced Blood-Labyrinth Barrier Trafficking.

HYPOTHESIS: Both toll-like receptor 4 (TLR4) and downstream neutrophil activity are required for endotoxemia-enhanced blood-labyrinth barrier (BLB) trafficking. BACKGROUND: Aminoglycoside and cisplatin are valuable clinical therapies; however, these drugs often cause life-long hearing loss. Endotoxemia enhances the ototoxicity of aminoglycosides and cisplatin in a TLR4 dependent mechanism for which downstream proinflammatory signaling orchestrates effector immune cells including neutrophils. Neutrophil-mediated vascular injury (NMVI) can enhance molecular trafficking across endothelial barriers and may contribute to endotoxemia-enhanced drug-induced ototoxicity. METHODS: Lipopolysaccharide (LPS) hypo-responsive TLR4-KO mice and congenitally neutropenic granulocyte colony-stimulating factor (GCSF) GCSF-KO mice were studied to investigate the relative contributions of TLR4 signaling and downstream neutrophil activity to endotoxemia-enhanced BLB trafficking. C57Bl/6 wild-type mice were used as a positive control. Mice were treated with LPS and 24 hours later cochleae were analyzed for gene transcription of innate inflammatory cytokine/chemokine signaling molecules, neutrophil recruitment, and vascular trafficking of the paracellular tracer biocytin-TMR. RESULTS: Cochlear transcription of innate proinflammatory cytokines/chemokines was increased in endotoxemic C57Bl/6 and GCSF-KO, but not in TLR4-KO mice. More neutrophils were recruited to endotoxemic C57Bl/6 cochleae compared with both TLR4 and GCSF-KO cochleae. Endotoxemia enhanced BLB trafficking of biocytin-TMR in endotoxemic C57Bl/6 cochleae and this was attenuated in both TLR4 and GCSF-KO mice. CONCLUSION: Together these results suggest that TLR4-mediated innate immunity cytokine/chemokine signaling alone is not sufficient for endotoxemia-enhanced trafficking of biocytin-TMR and that downstream neutrophil activity is required to enhance BLB trafficking. Clinically, targeting neutrophilic inflammation could protect hearing during aminoglycoside, cisplatin, or other ototoxic drug therapies.

Pilot study to evaluate a novel three-dimensional wound measurement device.

As the burden of diabetes continues to grow and treatment standards require careful tracking of wound progress, clinicians increasingly need to rely on technological improvements in wound measurement technologies to track the progress of their treatments. This study aims to determine the accuracy of a new three-dimensional wound measurement (3DWM) device against laser-assisted wound measurement (LAWM) devices and traditional methods of wound measurement. Using several wound models, we demonstrate that the 3DWM device measures wound area, depth and volume similarly to the other methods tested. This is especially apparent when changes in wound measurements were compared between the two devices. Differences between the two technologies were apparent when analysing wound measurement time and measurement repeatability. There was a significantly lower incidence of error in measurements between the 3DWM device and the LAWM device. Finally, the measurement time was significantly faster with the 3DWM device compared to the LAWM device. Together, these data demonstrate that the 3DWM device provides an accurate and reproducible method for measuring changes in wound healing similar to other available technologies. Further, the use of the 3DWM device provides a faster and more consistent measurement, which is critical for clinical application and use.

Validation of a laser-assisted wound measurement device in a wound healing model.

In the treatment and monitoring of a diabetic or chronic wound, accurate and repeatable measurement of the wound provides indispensable data for the patient's medical record. This study aims to measure the accuracy of the laser-assisted wound measurement (LAWM) device against traditional methods in the measurement of area, depth and volume. We measured four 'healing' wounds in a Play-Doh(®) -based model over five subsequent states of wound healing progression in which the model was irregularly filled in to replicate the healing process. We evaluated the LAWM device against traditional methods including digital photograph assessment with National Institutes of Health ImageJ software, measurements of depth with a ruler and weight-to-volume assessment with dental paste. Statistical analyses included analysis of variance (ANOVA) and paired t-tests. We demonstrate that there are significantly different and nearly statistically significant differences between traditional ruler depth measurement and LAWM device measurement, but there are no statistically significant differences in area measurement. Volume measurements were found to be significantly different in two of the wounds. Rate of percentage change was analysed for volume and depth in the wound healing model, and the LAWM device was not significantly different than the traditional measurement technique. While occasionally inaccurate in its absolute measurement, the LAWM device is a useful tool in the clinician's arsenal as it reliably measures rate of percentage change in depth and volume and offers a potentially aseptic alternative to traditional measurement techniques.

The role of estrogen in the modulation of autologous fat graft outcomes.

BACKGROUND: Autologous fat grafting is a widely used procedure, yet the mechanisms that regulate graft outcomes are poorly understood. Estrogen signaling is a potent regulator of lipid handling, inflammation, fibrosis, and adipocyte progenitor recruitment in adipose tissues. To date, no studies have investigated the effect of circulating estrogens on fat graft outcomes. METHODS: Immunosuppressed (Nu/Nu) mice underwent ovariectomy or sham surgery. Forty-five days later, half the mice (donors) were killed, and adipose tissue was taken and transplanted into the remaining cohort (recipients). Forty-five days after transplantation, grafts were dissected, weighed, and assessed for expression of vascular endothelial growth factor, estrogen receptor-α, and vascular density. RESULTS: Grafts harvested from and transplanted into sham environments are smaller but more highly vascularized compared with ovariectomy environments. The estrogenic effects on grafts are more critical at the site of the donor tissue than the recipient. Finally, expression of estrogen receptor-α in the grafted tissue correlates with the observed graft characteristics, which is altered by both the donor and recipient environments. CONCLUSIONS: Circulating estrogens have significant effects on fat graft outcomes, primarily at the site of the donor tissue. As there are well-established depot-specific estrogenic responses, the choice of adipose depot used as a donor for fat grafting may affect outcomes. In addition, outcomes may be confounded by the patient's hormonal status. Understanding the mechanisms by which estrogen signaling regulates graft outcomes is important in refining this commonly used clinical procedure.

Genome editing of mouse fibroblasts by homologous recombination for sustained secretion of PDGF-B and augmentation of wound healing.

BACKGROUND: Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. "Genome editing" is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation. METHODS: This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression. RESULTS: The application of engineered PDGF-B-expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy. CONCLUSIONS: These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

Comparison of adipocyte viability and fat graft survival in an animal model using a new tissue liquefaction liposuction device vs standard Coleman method for harvesting.

BACKGROUND: The use of autologous fat for augmentation has become common practice among plastic surgeons for both cosmetic and reconstructive procedures. Previously reported data suggest that the method of fat extraction can have profound effects on adipocyte viability and subsequent fat graft survival. OBJECTIVE: The authors describe a pilot study comparing a new tissue liquefaction liposuction device (TLL; HydraSolve Lipoplasty System; Andrew Technologies, Irvine, California) with a standard syringe aspiration method with respect to adipocyte viability, fat graft survivability, and fat graft quality. METHODS: Lipoaspirate from 5 patients was harvested using either TLL or the standard method. Samples were centrifuged and assayed for cell viability. All lipoaspirate samples were grafted into nude rats and harvested 42 and 84 days later. Graft survival and quality were assessed. RESULTS: There was no difference in adipocyte viability between the lipoaspirate conditions. At 42 days, there was no significant difference in fat graft weight and the TLL grafts were more fibrotic than the standard control grafts, but this was improved with the increased centrifuge rate. At 84 days, fat grafts were equivalent with respect to graft weight and histology. CONCLUSIONS: Lipoaspirate harvested with the TLL device and centrifuged at 3000 rpm resulted in fat grafts that were equivalent in weight and histology to those from lipoaspirate harvested with the standard syringe aspiration technique.

Validation of a laser-assisted wound measurement device for measuring wound volume.

BACKGROUND: Accurate and precise wound measurement is an essential part of the medical record when monitoring a patient with a chronic wound. This study was designed to determine if a new device, a laser-assisted wound measurement (LAWM) device, provides valid measurements for wound area, depth, and volume. METHODS: We compared four methods to evaluate the area and volume of 12 wounds of differing size and depth that were created on the dorsum of a sacrificed pig. We evaluated the LAWM device, digital photograph assessment with National Institutes of Health ImageJ software, measurements of depth with a ruler, and weight-to-volume assessment with dental paste. We then sought to cross validate this data with further analyses obtained from these measurements using a Play-Doh®-based wound as a model for constant area with different depths. RESULTS: We demonstrate that the LAWM device measures wound area accurately. Depth (and therefore volume) measurements, however, are artificially low. This inaccuracy is the same for shallow and deep wounds. CONCLUSIONS: The inaccuracy in the depth and volume measurements with the LAWM device results in an artificially low measurement. However, this may not affect percentage difference measurements. Further studies will need to be performed to determine if this device can accurately determine wound changes in the clinical setting.

Simultaneous irrigation and negative pressure wound therapy enhances wound healing and reduces wound bioburden in a porcine model.

Infected foot wounds are one of the most common reasons for hospitalization and amputation among persons with diabetes. The objective of the study was to investigate a new wound therapy system that employs negative pressure wound therapy (NPWT) with simultaneous irrigation therapy. For this study, we used a porcine model with full-thickness excisional wounds, inoculated with Pseudomonas aeruginosa. Wounds were treated for 21 days of therapy with either NPWT, NPWT with simultaneous irrigation therapy using normal saline or polyhexanide biguanide (PHMB) at low or high flow rates, or control. Data show that NPWT with either irrigation condition improved wound healing rates over control-treated wounds, yet did not differ from NPWT alone. NPWT improved bioburden over control-treated wounds. NPWT with simultaneous irrigation further reduced bioburden over control and NPWT-treated wounds; however, flow rate did not affect these outcomes. Together, these data show that NPWT with simultaneous irrigation therapy with either normal saline or PHMB has a positive effect on bioburden in a porcine model, which may translate clinically to improved wound healing outcomes.

The sexually dimorphic role of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion, inflammation, and fibrosis.

Our data demonstrate that estrogens, estrogen receptor-α (ERα), and estrogen receptor-ß (ERß) regulate adipose tissue distribution, inflammation, fibrosis, and glucose homeostasis, by determining that αERKO mice have increased adipose tissue inflammation and fibrosis prior to obesity onset. Selective deletion of adipose tissue ERα in adult mice using a novel viral vector technology recapitulated the findings in the total body ERα null mice. Generation of a novel mouse model, lacking ERα specifically from adipocytes (AdipoERα), demonstrated increased markers of fibrosis and inflammation, especially in the males. Additionally, we found that the beneficial effects of estrogens on adipose tissue require adipocyte ERα. Lastly, we determined the role of ERß in regulating inflammation and fibrosis, by breeding the AdipoERα into the ßERKO background and found that in the absence of adipocyte ERα, ERß has a protective role. These data suggest that adipose tissue and adipocyte ERα protects against adiposity, inflammation, and fibrosis in both males and females.

The comparison of two negative-pressure wound therapy systems in a porcine model of wound healing.

The purpose of this study was to compare two negative-pressure wound healing systems (NPWT), -75 mmHg with a silicone-coated (SC) dressing and -125 mmHg with polyurethane foam dressing (standard of care). In addition, this study compared the effects of two different dressing interfaces, SC dressing and gauze, with -75 mmHg pressure. For both comparisons, two groups of five pigs were evaluated over a 21-day time course. Two excisional wounds were made on each animal and NPWT dressings were applied. A canvas saddle was constructed to hold the NPWT device so the animal had free range of the pen. Dressings were changed twice a week and wound measurements were taken. Specimens for histology and gene expression analyses were taken on day 7 and 21. These data show that there is increased expression in a few genes associated with remodeling and inflammatory processes in the NPWT-125 with polyurethane foam as compared with the NPWT-75 with SC dressing. These two systems, however, are equivalent with respect to wound healing, histology, and gene expression over 21 days of healing. Further, we demonstrate that there is no difference in measure of healing between the SC dressing and a basic gauze dressing.