Estrogen is an important regulator and promoter of epithelial wound healing. This is facilitated by increased keratinocyte and fibroblast migration and proliferation, as well as promotion of angiogenesis, matrix deposition and inflammatory response dampening. The potential to target this pathway for therapeutics is highlighted by observations that post-menopausal women on hormone replacement therapy have a significantly lower incidence of venous ulcers. Previous work from this laboratory identified four SNPs (single nucleotide polymorphisms) in the 5’UTR of estrogen receptor beta (ERβ) gene that are associated with venous ulcer predisposition. Disease association is further supported by the identification of ERβ as the main conduit of the beneficial effects of estrogen signalling on wound healing. SNP’s of the 5’UTR can affect transcriptional expression through the modification of transcription factor binding sites, epigenetic modifications and translational efficiency via mRNA localisation and secondary structure alterations. To investigate the possible biological function of these SNPs, we have developed disease relevant cell based assays where primary keratinocyte and fibroblast cells were selected harbouring disease-associated SNPs. We demonstrate that the presence of venous ulcer-associated ERβ SNPs reduced the expression of ERβ in skin cells and reduced their migration and proliferative capabilities. Evidence gathered here suggests that ERβ expression is curtailed by a change in transcription factor binding, likely facilitated by the change in nucleotide sequence brought about by the rs2987983 SNP. Further, we demonstrate that SNP-induced changes in fibroblast expression of growth factors and inflammatory mediators can hinder keratinocyte migration and induce a pro-inflammatory phenotype in human monocytes. Lastly, RNAseq analysis of keratinocytes reveals a SNP-dependant gene expression profile that is detrimental to wound healing. This work provides the first evidence of a direct functional link between venous ulcer-associated ERβ SNPs and dysfunctional wound healing. Investigating ERβ SNPs has provided insight into novel mechanisms of estrogen signalling that can be applied for therapeutic development to treat venous ulcers.

Because of the high cost of some wound management regimens, payors may require that moist wound therapies be used before other treatment approaches, such as negative pressure wound therapy (NPWT), are implemented but few studies have investigated the effect of delayed initiation of NPWT on patient outcomes. To examine the impact of early versus late initiation of NPWT on patient length of stay in home health care, a nonrandomized, retrospective analysis was performed on the Outcome and Assessment Information Set (OASIS) information for home care patients with NPWT-treated Stage III or Stage IV pressure ulcers (N = 98) or surgical wounds (N = 464) gathered between July 2002 and September 2004. Early initiation of NPWT following the start of home care was defined as <30 days for pressure ulcers and <7 days for surgical wound patients. Median duration of NPWT was 31 days (range 3 to 169) for pressure ulcers and 27 days (range 5 to 119) for the surgical wound group. Median lengths of stay in the early treatment groups were 85 days (range 11 to 239) for pressure ulcers and 57 days (range 7 to 119) for the surgical group versus 166 days (range 60 to 657) and 87 days (range 31 to 328), respectively, for the late treatment pressure ulcer and surgical groups (P <0.0001). After controlling demographic patient variables, regression analysis indicated that for each day NPWT initiation was delayed, almost 1 day was added to the total length of stay (β = 0.96, P <0.0001 [pressure ulcers]; β = 0.97, P <0.0001 [surgical wounds]). Early initiation of NPWT may be associated with shorter length of stay for patients receiving home care for Stage III or Stage IV pressure ulcers or surgical wounds. Additional studies to ascertain the cost-effectiveness of treatments and treatment approaches in home care patients are needed.

Objective: This study was undertaken to determine the most appropriate management of the subcutaneous tissue of midline vertical incisions with 3 cm or more of subcutaneous fat. Study design: Patients undergoing surgery within the Division of Gynecologic Oncology at University of South Florida and East Tennessee State University with 3 cm or more of subcutaneous fat were randomly assigned to 1 of 3 groups: suture approximation of Camper's fascia, closed suction drainage of the subcutaneous space, or no intervention as a control group. Participants were evaluated daily during postoperative hospitalization and at 2 and 6 weeks postoperatively as an outpatient. Demographic information, perioperative data, and wound complications were recorded and then analyzed with χ2, t test, analysis of variance, and logistic regression where appropriate. Results: Two hundred twenty-five patients were enrolled with 222 eligible for evaluation. Wound complications were observed in 34 (15.3%) patients, and 25 of these women also had wound disruption. Overall wound complication and wound disruption rates were not significantly different between groups: suture (12.8%, 7.7%), drain (17.9%, 14.9%), control (15.6%, 11.7%); P = .70 and P = .39, respectively. Conclusion: Suture approximation or drainage of the subcutaneous tissues of women with 3 cm or more subcutaneous fat measured in midline vertical incisions resulted in no significant change in the incidence of overall wound complications or superficial wound disruption.

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Our objective was to compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. The study design was a prospective, controlled, experimental study. The animal population included 12 female spayed beagles. We hypothesized there would be a difference between the cefazolin concentrations of wounds treated with negative pressure wound therapy when compared to the cefazolin concentrations of wounds treated with nonadherent dressings. Surgical methods were as follows: Full thickness cutaneous wounds were created on each antebrachium (n=24). Following surgery, cefazolin (22 mg/kg) was administered intravenously to each of the dogs and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II while the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24-hour intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected prior to biopsy sampling. At the time of surgery and at each bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. Our results revealed that after initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. We concluded that using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared to conventional nonadherent bandage therapy / Master of Science

No / It is now recognised that psychosocial factors – anxiety and depression, social isolation, low economic status and pain, for example – are associated with delayed healing of wounds. However, little research has been undertaken to examine how these factors may not only be a consequence of delayed healing, but may also play an important role in delaying healing. It is suggested that an evaluation of a patient’s psychosocial status should therefore be included as part of a general wound assessment.

Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes. / Doctor of Philosophy / Skin wounds frequently result in scars that can range from barely visible to enormous eyesores. Almost everyone will experience at least one skin wound in their lifetime leading to a scar that they wish were less visible, feeding the multi-billion dollar market for anti-scarring agents. However, many of the products on store shelves that claim to reduce scar appearance have not proven those claims. Most of the therapies that do have some degree of scientific evidence to support their claims are difficult to use properly, such as silicone sheeting, and often result in only minor improvements to scar appearance. Alpha Connexin Carboxy-Terminus 1 (αCT1), marketed in clinical trials as Granexin® gel, is a protein-based therapy that works on the cellular level to fundamentally alter the skin's initial reaction to wounding and improving long-term scar appearance. This dissertation explores the link between cellular processes altered by αCT1 and long-term clinical improvements in scar appearance by studying both the extracellular matrix present in the scar in human and animal models and the creation of that extracellular matrix by dermal fibroblasts. In both human and animal models, topical application of αCT1 had no effect on skin surface appearance at early timepoints of 2-6 weeks, correlating with previous research that found scar appearance only improved at 3+ months post-injury. However, deep within the newly constructed tissue of the scar, these studies show the collagen organizational structure of αCT1-treated scars is more similar to unwounded skin and slightly more dense at early timepoints, suggesting αCT1 marginally improved the speed of healing. These findings in humans and animals were also verified in part in cell culture experiments that found dermal fibroblasts increased collagen output in response to αCT1 treatment. A novel wound healing model in the hairless guinea pig, superior at replicating human skin than established models like the rat, is also presented and shown to have effects strongly similar to the human with αCT1 treatment. These results provide a fundamental insight into the mode-of-action by which αCT1 may improve long term scar appearance and identifies early collagen structure as a target for future therapeutics to modify, as well as a new animal model in which to test them.

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