Angela Gibson

Phase I/II Clinical Evaluation of StrataGraft: A Consistent, Pathogen-Free Human Skin Substitute

BACKGROUND Large wounds often require temporary allograft placement to optimize the wound bed and prevent infection until permanent closure is feasible. We developed and clinically tested a second-generation living human skin substitute (StrataGraft). StrataGraft provides both a dermis and a fully-stratified, biologically-functional epidermis generated from a pathogen-free, long-lived human keratinocyte progenitor cell line, Neonatal Immortalized KeratinocyteS (NIKS). METHODS Histology, electron microscopy, quantitative polymerase chain reaction, and bacterial growth in vitro were used to analyze human skin substitutes generated from primary human keratinocytes or NIKS cells. A phase I/II, National Institute of Health-funded, randomized, safety, and dose escalation trial was performed to assess autograft take in 15 patients 2 weeks after coverage with StrataGraft skin substitute or cryopreserved cadaver allograft. RESULTS StrataGraft skin substitute exhibited a fully stratified epidermis with multilamellar lipid sheets and barrier function as well as robust human beta defensin-3 mRNA levels. Analysis of the primary endpoint in the clinical study revealed no differences in autograft take between wound sites pretreated with StrataGraft skin substitute or cadaver allograft. No StrataGraft-related adverse events or serious adverse events were observed. CONCLUSIONS The major finding of this phase I/II clinical study is that performance of StrataGraft skin substitute was comparable to cadaver allograft for the temporary management of complex skin defects. StrataGraft skin substitute may also eliminate the risk for disease transmission associated with allograft tissue and offer additional protection to the wound bed through inherent antimicrobial properties. StrataGraft is a pathogen-free human skin substitute that is ideal for the management of severe skin wounds before autografting.

Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.

When skin is compromised, a cascade of signals initiates the rapid repair of the epidermis to prevent fluid loss and provide defense against invading microbes. During this response, keratinocytes produce host defense peptides (HDPs) that have antimicrobial activity against a diverse set of pathogens. Using nonviral vectors we have genetically modified the novel, nontumorigenic, pathogen-free human keratinocyte progenitor cell line (NIKS) to express the human cathelicidin HDP in a tissue-specific manner. NIKS skin tissue that expresses elevated levels of cathelicidin possesses key histological features of normal epidermis and displays enhanced antimicrobial activity against bacteria in vitro. Moreover, in an in vivo infected burn wound model, this tissue results in a two log reduction in a clinical isolate of multidrug-resistant Acinetobacter baumannii. Taken together, these results suggest that this genetically engineered human tissue could be applied to burns and ulcers to counteract bacterial contamination and prevent infection.

Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.

The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human beta defensin‐3 (hBD‐3), in keratinocytes to generate a three‐dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis‐specific promoter driving hBD‐3 (NIKShBD‐3) using nonviral methods. Levels of hBD‐3 mRNA and protein in three‐dimensional skin tissue produced from NIKShBD‐3 were determined using quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three‐dimensional full thickness skin tissues containing epidermal NIKShBD‐3 or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKShBD‐3 tissue contained approximately fivefold more hBD‐3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKShBD‐3 tissue produced a significant reduction in the growth of Staphylococcus aureus multiple peptide resistance factor (mprF) compared with control tissue. In an in vivo infected murine burn model, NIKShBD‐3 tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD‐3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third‐degree burn wounds.

Readmission after delayed diagnosis of surgical site infection: a focus on prevention using the American College of Surgeons National Surgical Quality Improvement Program

BACKGROUND Surgical site infection (SSI) is a costly complication leading to increased resource use and patient morbidity. We hypothesized that postdischarge SSI results in a high rate of preventable readmissions. METHODS We used our institutional American College of Surgeons National Surgical Quality Improvement Program database to identify patients undergoing general surgery procedures from 2006 to 2011. RESULTS SSIs developed in 10% of the 3,663 patients who underwent an inpatient general surgical procedure. SSI was diagnosed after discharge in 48% of patients. Patients with a diagnosis of SSI after discharge were less likely to have a history of smoking (15% vs 28%, P = .001), chronic obstructive pulmonary disease (3% vs 9%, P = .015), congestive heart failure (0% vs 3%, P = .03), or sepsis within 48 hours preoperatively (17% vs 32%, P = .001) compared with patients diagnosed before discharge. Over 50% of the patients diagnosed with SSI after discharge required readmission. CONCLUSIONS A diagnosis of SSI after discharge is associated with a high readmission rate despite occurring in healthier patients. We propose discharge teaching improvements and a wound surveillance clinic within the first week may result in a decreased readmission rate.

Chimeric Composite Skin Substitutes for Delivery of Autologous Keratinocytes to Promote Tissue Regeneration

Objective:We hypothesize that the pathogen-free NIKS human keratinocyte progenitor cell line cultured in a chimeric fashion with patients primary keratinocytes would produce a fully stratified engineered skin substitute tissue and serve to deliver autologous keratinocytes to a cutaneous wound. Summary of Background Data:Chimeric autologous/allogeneic bioengineered skin substitutes offer an innovative regenerative medicine approach for providing wound coverage and restoring cutaneous barrier function while delivering autologous keratinocytes to the wound site. NIKS keratinocytes are an attractive allogeneic cell source for this application. Methods:Mixed populations of green fluorescent protein (GFP)-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic and autologous components in chimeric monolayer and organotypic cultures. Results:In monolayer coculture, GFP-labeled NIKS had no effect on the growth rate of primary keratinocytes and cell-cell junction formation between labeled and unlabeled keratinocytes was observed. In organotypic culture employing dermal and epidermal compartments, chimeric composite skin substitutes generated using up to 90% GFP-labeled NIKS exhibited normal tissue architecture and possessed substantial regions attributable to the primary keratinocytes. Tissues expressed proteins essential for the structure and function of a contiguous, fully-stratified squamous epithelia and exhibited barrier function similar to that of native skin. Furthermore, chimeric human skin substitutes stably engrafted in an in vivo mouse model, with long-term retention of primary keratinocytes but loss of the GFP-labeled NIKS population by 28 days after surgical application. Conclusions:This study provides proof of concept for the use of NIKS keratinocytes as an allogeneic cell source for the formation of bioengineered chimeric skin substitute tissues, providing immediate formal wound coverage while simultaneously supplying autologous cells for tissue regeneration.

Oxygen deprivation inhibits basal keratinocyte proliferation in a model of human skin and induces regio-specific changes in the distribution of epidermal adherens junction proteins, aquaporin-3, and glycogen

It is generally accepted that hypoxia and recovery from oxygen deprivation contribute to the breakdown and ulceration of human skin. The effects of these stresses on proliferation, differentiation and expression of cell–cell adhesion molecules were investigated for the first time in an organotypic model of human skin. Fully stratified tissues were exposed to a time course of oxygen deprivation and subsequent reoxygenation. Regional changes in keratinocyte morphology, glycogen stores and cellular junctions were observed, with more differentiated layers of the epidermis exhibiting the first evidence of oxygen deprivation. Cellular swelling within the granular layer was concurrent with aquaporin‐3 depletion. The keratinocyte adherens junction proteins E‐cadherin and β‐catenin were dramatically decreased in a regio‐specific manner throughout the epidermis following oxygen deprivation. In contrast, P‐cadherin and the desmosomal proteins desmoplakin and desmoglein‐1 were refractory to oxygen deprivation. Relative to normoxic controls, hypoxic tissues exhibited increased mRNA levels of the transcriptional repressor Slug; however, mRNA levels of the related transcriptional factor Snail were unaffected. All cellular and molecular changes were reversible upon reoxygenation. These results show that oxygen deprivation and reoxygenation exert differential effects on epidermal adhesion proteins and suggest a novel role for cadherins, β‐catenin, and Slug in hypoxia‐induced junctional changes occurring in stratified squamous epithelium.

Comparison of therapeutic antibiotic treatments on tissue-engineered human skin substitutes.

For regenerative medicine to gain clinical acceptance, the effects of commonly used treatment regimens on bioengineered organs must be considered. The antibiotics mafenide acetate (mafenide) and neomycin plus polymyxin (neo/poly) are routinely used to irrigate postoperative skin grafts on contaminated wounds. The effects of these clinically used antibiotics were investigated using tissue-engineered human skin substitutes generated with primary human keratinocytes or the near-diploid human keratinocyte cell line, Near-diploid Immortal Keratinocytes. Following topical or dermal treatment, the skin substitutes were assayed for viability, tissue morphology, glycogen content, and the expression of active caspase 3. Mafenide, but not neo/poly, induced morphological and biochemical changes in tissue-engineered skin substitutes. Keratinocytes in all histological layers of mafenide-treated skin substitutes exhibited ballooning degeneration and glycogen depletion. Mafenide-treatment also triggered separation of basal keratinocytes from the underlying dermis. None of the antibiotic treatments induced apoptosis, as measured by active caspase 3 immunostaining. The results demonstrate that mafenide, but not neo/poly, is detrimental to the viability and structural integrity of tissue-engineered human skin substitutes. These findings highlight the need to identify treatment regimens that are compatible with and hence enable the therapeutic efficacy of first-generation bioengineered organs such as skin.

Variations in Burn Excision and Grafting: A Survey of the American Burn Association

It is unknown whether variations in burn care affect outcomes or affect the success of emerging therapeutics. The purpose of this study was to assess burn surgeons’ preferences in excision and grafting to determine if surgical technique affects outcomes. A 71-item survey evaluating skin grafting techniques and preferences was emailed to members of the American Burn Association in July and August 2015. The survey was anonymous and voluntary. Relationships between variables were evaluated using Fisher’s exact test. A P-value of ⩽.05 was deemed statistically significant. The survey was sent to 607 burn surgeons, and the response rate was 24%. Clinical judgment is the most widely used method to determine depth of injury. Surgeons who practice in the United States and surgeons who are board certified in general surgery are more likely to determine depth of the burn based on clinical judgment alone (P < .001). Fifty-six percent of surgeons will perform excision as early as postburn day 1 and 73% will excise greater than 20% TBSA in one setting. Surgeons at centers with bed number of ⩽10 (P = .024) or surgeons with board certification in plastic surgery (P = .008) are more likely to excise deep partial-thickness burns with an attempt to retain viable dermis. Geographic location, board certification, and burn unit size all contribute to variations in practice. Strong individual preferences make standardization of therapies challenging and may affect the success of new technologies. Burn surgery continues to be an art as much as a science, and accurate documentation of techniques and outcomes is essential for optimizing successes and documenting failures of new treatment methods.

Damage-induced reactive oxygen species regulate vimentin and dynamic collagen-based projections to mediate wound repair

Tissue injury leads to early wound-associated reactive oxygen species (ROS) production that mediate tissue regeneration. To identify mechanisms that function downstream of redox signals that modulate regeneration, a vimentin reporter of mesenchymal cells was generated by driving GFP from the vimentin promoter in zebrafish. Early redox signaling mediated vimentin reporter activity at the wound margin. Moreover, both ROS and vimentin were necessary for collagen production and reorganization into projections at the leading edge of the wound. Second harmonic generation time-lapse imaging revealed that the collagen projections were associated with dynamic epithelial extensions at the wound edge during wound repair. Perturbing collagen organization by burn wound disrupted epithelial projections and subsequent wound healing. Taken together our findings suggest that ROS and vimentin integrate early wound signals to orchestrate the formation of collagen-based projections that guide regenerative growth during efficient wound repair.

Improving the Histologic Characterization of Burn Depth

Visual assessment of burn wound appearance is the standard of care to determine the depth of thermal injury but often incorrectly predicts wound healing potential. Histologic evaluation of hematoxylin and eosin (H&E) stained burn tissue is prone to subjectivity and is challenging for the novice. Lactate dehydrogenase (LDH) staining may offer a simplified and consistent technique to identify burn tissue viability.