BACKGROUND:StrataGraft® (allogeneic cultured keratinocytes and dermal fibroblasts in murine collagen-dsat) is an FDA-approved viable bioengineered allogeneic cellularized construct for adult patients with deep partial-thickness burns requiring surgery. We characterized the structural and functional properties of StrataGraft to improve product understanding by evaluating extracellular matrix (ECM) molecule distribution and secreted protein factor expression in vitro. METHODS:ECM protein expression was determined using indirect immunofluorescence on construct cross sections using commercial antibodies against collagen III, IV, VI, laminin-332, and decorin. Human collagen I expression was verified by enzyme-linked immunosorbent assay (ELISA) for collagen I C-terminal propeptide. Soluble protein factor secretion was quantified by multiplex biomarker assays and singleplex ELISA in conditioned media from meshed constructs. RESULTS:StrataGraft cellular components produced collagen I, collagen III, collagen VI, and decorin in patterns indicating an organized ECM. Distributions of collagen IV and laminin-332 indicated formation of basement membranes and dermal-epidermal junctions. Soluble protein factors were observed in the pg/cm2/h range from 1 h to the experiment end at 168 h. CONCLUSIONS:The organization of the ECM proteins was like human skin and the viable cellular components provided sustained secretion of soluble wound healing factors, making StrataGraft an attractive option for treating severe burns.

Objective: This phase 3 study evaluated StrataGraft construct as a donor-site sparing alternative to autograft in patients with deep partial-thickness (DPT) burns. Methods: Patients aged >= 18 years with 3-49% total body surface area (TBSA) thermal burns were enrolled. In each patient, 2 DPT areas (<= 2000cm(2) total) of comparable depth after excision were randomized to either cryopreserved StrataGraft or autograft. Coprimary endpoints were: the difference in percent area of StrataGraft treatment site and autograft treatment site autografted at Month 3 (M3), and the proportion of patients achieving durable wound closure of the StrataGraft site without autograft at M3. Safety assessments were performed in all patients. Efficacy and safety follow-up continued to 1 year. Results: Seventy-one patients were enrolled. By M3, there was a 96% reduction in mean percent area of StrataGraft treatment sites that required autografting, compared with autograft treatment sites (4.3% vs 102.1%, respectively; P <.0001). StrataGraft treatment resulted in durable wound closure at M3 without autografting in 92% (95% CI: 85.6, 98.8; n/n 59/64) of patients for whom data were available. The most common StrataGraft-related adverse event was pruritus (15%). Conclusions: Both coprimary endpoints were achieved. StrataGraft may offer a new treatment for DPT burns to reduce the need for autografting. (C) 2021 The Authors. Published by Elsevier Ltd.

2021 WHS Abstract Session List Key Young Investigator Awards Young Investigator Award Competition ..................................................................................................................................................................................................YIA Concurrent Oral Presentations Clinical .................................................................................................................................................................................................................................................................. A Novel Therapies................................................................................................................................................................................................................................................. B Bioengineering/Biomaterials.......................................................................................................................................................................................................................... C Acute Wounds/Inflammation ........................................................................................................................................................................................................................D Fibrosis & Scarring .............................................................................................................................................................................................................................................E Regeneration .......................................................................................................................................................................................................................................................F Chronic Wounds ...............................................................................................................................................................................................................................................G Chronic Wounds/ Epithelialization ..............................................................................................................................................................................................................H Technology ........................................................................................................................................................................................................................................................... I ECM/Bioengineering/Biomaterials ............................................................................................................................................................................................................... J Aging/Inflammation.......................................................................................................................................................................................................................................... K Adipose Tissue/Chronic Wounds .................................................................................................................................................................................................................. L

Abstract Introduction Autograft (AG) is the standard of care for treatment of severe burns. While AG provides effective wound closure (WC), the procedure creates a donor site wound prone to pain and scarring. In a phase 1b trial, no deep partial-thickness (DPT) wound treated with a bioengineered allogeneic cellularized construct (BACC) required AG by Day 28 and WC at the BACC site was achieved in 93% of patients by Month (M) 3. This phase 3 study (NCT03005106) evaluated the efficacy and safety of this BACC in patients with DPT burns. Methods Enrolled patients were aged ≥18 years with 3–49% TBSA thermal burns on the torso or extremities. In each patient, two DPT areas (≤2,000 cm2 total) deemed comparable following excision were randomized to treatment with either cryopreserved BACC or AG. Coprimary endpoints were 1) the difference in percent area of BACC treatment site and AG treatment site autografted at M3 and 2) the proportion of patients achieving durable WC of the BACC treatment site without AG at M3. Ranked secondary endpoints were: 1) the difference between BACC and AG donor sites in average donor site pain intensity through Day 14; 2) the difference between BACC and AG donor site cosmesis at M3; and 3) the difference between BACC and AG treatment site cosmesis at M12. Safety assessments were performed in all patients through M12. Results Seventy-one patients were enrolled. By M3, there was a 96% reduction in mean percent area of BACC treatment sites that required AG, compared with AG treatment sites (4.3% vs 102.1%, respectively; P Conclusions This phase 3 study achieved both coprimary endpoints, including significant autograft sparing and durable WC in DPT burns. Both donor site pain and donor site cosmesis were favorable outcomes of significantly reduced use of AG in BACC-treated patients. M12 POSAS for BACC did not differ significantly from AG. This BACC may offer a new treatment for severe burns to reduce or eliminate the need for AG. Applicability of Research to Practice This BACC has shown clinical benefit in patients with DPT thermal burns, potentially mitigating donor site morbidity. External Funding Stratatech, a Mallinckrodt Company; Funding and technical support for the Phase 3 clinical study were provided by the Biomedical Advanced Research and Development Authority (BARDA), under the Assistant Secretary for Preparedness and Response, within the U.S. Department of Health and Human Services, under Project BioShield Contract No. HHSO100201500027C.

Objective: This open-label, controlled, randomized study assessed the safety, tolerability, and efficacy of StrataGraft tissue compared to autograft in the treatment of deep partialthickness (DPT) burns. Methods: Thirty subjects with DPT thermal burns (3%-43% total body surface area) were treated with StrataGraft tissue as follows: cohort 1, <= 220 cm(2) refrigerated tissue; cohort 2, <= 440 cm(2) refrigerated tissue; and cohort 3, <= 440 cm(2) cryopreserved tissue. On each subject, two comparable areas of DPT burn were randomized to receive StrataGraft tissue or autograft. Coprimary end points were the percent area of the StrataGraft tissue treatment site undergoing salvage autografting by Day 28 and wound closure of treatment sites by 3 months. Results: By Day 28, no StrataGraft tissue treatment sites underwent autografting. By 3 months, 93% and 100% of the StrataGraft tissue and autograft treatment sites achieved complete wound closure, respectively. No significant differences in observer total and overall opinion POSAS scores between StrataGraft tissue and autograft treatment sites were observed at any timepoint. The most common adverse event was pruritus (17%). Conclusions: StrataGraft tissue treatment of DPT thermal burns reduced the need for autograft, resulted in wound closure and treatment-site cosmesis comparable to that of autograft, and was well tolerated. (C) 2019 The Authors. Published by Elsevier Ltd.

Skin degeneration and loss associated with chronic wounds can result in significant morbidity and mortality, due to a disruption in homeostatic function and susceptibility to infection. Numerous biological therapeutics developed to promote the closure of these wounds include acellular dermal analogs, a viable dermal equivalent, a living placental membrane-based wound cover, cadaveric allografts, and a bilayered skin substitute. No product provides a bilayered skin covering addressing the infection risk of the chronic wound environment. The recent development of a bioengineered skin substitute containing human dermal fibroblasts with an epidermal layer of human keratinocytes derived from a stable cell line affords the opportunity to develop genetically modified, indication-specific tissues. Skin tissues featuring the overexpression of human antimicrobial host defense peptides, including the multifunctional cathelicidin protein, were developed and subjected to preclinical testing, and show promise for clinical use to promote the closure of chronic wounds.

The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects.

Pluripotent human embryonic stem (ES) cells provide a consistent developmental model for studying contaminant-induced changes in human cell fate and early developmental processes.Thus far, no studies have investigated the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the growth and differentiation of human ES cells.Here we show that the aryl hydrocarbon receptor (AhR) pathway is functional in human ES cells based on the ability of TCDD to induce the AhR/Arnt-dependent transcription of CYP1A1.No changes in colony morphology were observed in undifferentiated human ES cells following 8 days of treatment with TCDD.However, when human ES cells were allowed to replicate and spontaneously differentiate for 15 days in the presence of TCDD, colonies exhibited less morphological differentiation compared to control human ES cell cultures.This observation was confirmed on the molecular level by lower expression of markers of meso-and endodermal differentiation and higher expression of the pluripotency marker, Oct4, in TCDDtreated cultures.Additionally, TCDD inhibited the fibroblastic morphology resulting from the epithelial to mesenchymal transition (EMT) of spontaneously differentiating ES cells, preserving an undifferentiated cellular phenotype.Furthermore, lower expression of EMT markers as well as a lower incidence of the mesenchymal marker, N-cadherin, was observed around the edges of TCDD-treated cell colonies.These findings suggest that TCDD treatment inhibits human ES cell differentiation, potentially through the inhibition of an EMT-like process.

It has been more than 30 years since the serial cultivation of human keratinocytes in monolayer culture was first described by Rheinwald and Green. Initially, isolation of primary keratinocytes from disaggregated human skin tissue and subsequent propagation was promoted through use of replication-inactivated murine fibroblast feeder layers. Since then numerous advances have been made to the cultivation of human keratinocytes in both two-dimensional monolayer and three-dimensional organotypic culture. Monolayer culture facilitates keratinocyte proliferation, whereas organotypic culturing techniques promote keratinocyte differentiation using conditions permissive for stratification. The protocols presented here describe traditional culturing methods, providing guidance for isolation and serial cultivation of primary human keratinocytes and dermal fibroblasts, as well as the use of these cells types for generation of stratified skin tissue.

BACKGROUNDComplex skin defects, such as burns and acute cutaneous trauma, are life-threatening injuries, often requiring temporary allograft placement to maintain fluid homeostasis and prevent infection until permanent wound closure is possible.THE PROBLEMThe current standard of care for the management of full-thickness wounds that are unable to be closed in a single surgical stage is temporary coverage with cadaver allograft until an acceptable wound bed has been established. This approach has limitations including limited availability of human cadaver skin, the risk of disease transmission from cadaveric grafts, and inconsistent cadaver allograft quality.BASIC/CLINICAL SCIENCENear-diploid neonatal human keratinocyte cell line (NIKS)-based human skin tissue is a full-thickness, living human skin substitute composed of a dermal analog containing normal human dermal fibroblasts and a fully-stratified, biologically and metabolically active epidermis generated from NIKS keratinocytes, a consistent and unlimited source of pathogen-free human epidermal progenitor cells.CLINICAL CARE RELEVANCENIKS-based human skin tissue is a living bioengineered skin substitute (BSS) intended to provide immediate wound coverage and promote wound healing through sustained expression by living cells of wound healing factors.CONCLUSIONA phase I/IIa clinical trial found that NIKS-based BSS was well tolerated and comparable to cadaver allograft in the ability to prepare full-thickness complex skin defects prior to autografting. There were no deaths and no adverse events (AE) associated with this BSS. Exposure of the study subjects to the skin substitute tissue did not elicit detectable immune responses. Notably, this tissue remained viable and adherent in the wound bed for at least 7 days.

Introduction: Notch signaling, specifically Notch1, is known to play a key role in epidermal development; however, the role in tissue repair remains relatively unknown. to study the role of Notch signaling in wound healing, we used NIKS cells, a well-characterized, nontumorigenic human keratinocyte cell line. Western blot and QPCR analysis confirmed similar expression patterns for the Notch family of signaling molecules when comparing NIKS cells to independent primary keratinocytes. These data indicate that NIKS accurately represent the behavior of human keratinocytes and are therefore an ideal model to study Notch signaling during wound healing. Previous research from our lab suggests that DAPT, a widely used Notch inhibitor, causes a reduction in cell proliferation. However, since DAPT inhibits all four isoforms of mammalian Notch, we investigated the role of the specific Notch receptor that regulates keratinocyte proliferation. NIKS grown in 3D cultures form fully functional skin tissue and both Notch1 and 2 preferentially localize in the proliferating basal layer; with Notch1 expression being predominant. Therefore, we hypothesized that Notch1 signaling plays a key role in keratinocyte proliferation during wound healing. Methods: NIKS cells were transiently transfected with Notch1 siRNA to knockdown Notch1 expression or a non-specific siRNA as control; knockdown was confirmed by Western blot. Cell growth was studied using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell lysates of non-specific and Notch1 siRNA treated cells were analyzed for cell cycle arrest markers cyclin B1 and cyclin D1, and the apoptotic markers, activated caspase 3 and activated PARP, by Western blot to determine if reductions in cell growth were due to cell cycle arrest or due to apoptosis. Results: A 20% inhibition of growth was detected in the siRNA transfected cells for up to 72 hours following transient knockdown of Notch1 compared to non-specfic siRNA control (p<0.01). There was also a reduction in cyclin D1 (7-fold) and cylcin B1 (50%) protein levels at 24 hours in NIKS that were treated with Notch1 siRNA compared to control. Levels of total Caspase 3 and total PARP remained unchanged while activated caspase 3 and activated PARP were undetected in both control and Notch1 siRNA transfected NIKS. Conclusions: NIKS cell growth is inhibited when Notch1 signaling is attenuated. the reduction of cyclin D1 and cyclin B1 in Notch1 siRNA transfected cells suggests that Notch1 regulates G1-S and G2-M transition in keratinocyte cell cycle, respectively. in addition, the lack of apoptotic markers activated caspase 3 and activated PARP indicate that mechanism of keratinocyte growth inhibition resulting from diminished Notch1 signaling is due to cell cycle arrest. Understanding the molecular mechanisms of wound healing will help develop techniques that could modify levels of Notch receptor to heal wounds more efficiently.

Roy, Madhuchhanda MD, PhD; Jaraczewski, Taylor J. BSc(Hons); Stofflet, John T. BSc(Hons); Pathak, Priya BSc(Hons); Schlosser, Sandy J. BSc(Hons); Allen-Hoffmann, Lynn B. PhD; King, Timothy W. MD, PhD, FACS Author Information

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 (NIKS(hBD) (-3) ) using nonviral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS(hBD) (-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 NIKS(hBD) (-3) or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKS(hBD) (-3) tissue contained approximately fivefold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKS(hBD) (-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, NIKS(hBD) (-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.

BACKGROUNDComplex skin defects resulting from acute skin trauma and chronic, nonhealing wounds are life-threatening injuries. Infection is one of the most common obstacles to the healing of these types of wounds. Host defense peptides (HDPs) possessing a broad spectrum of activity against microorganisms and serving as innate immune modulators have emerged as potential treatment strategies for infected wounds.THE PROBLEMThe increase in multidrug-resistant clinical bacterial isolates highlights the need for new and innovative anti-infective therapies for the treatment of both acute and chronic skin wounds.BASIC/CLINICAL SCIENCETo address the critical need for new therapeutic options to reduce infection and improve wound healing, a bioengineered skin substitute (BSS) tissue has been created to act as an anti-infective living human skin tissue that provides enhanced expression of the endogenous HDP, cathelicidin. To generate a BSS exhibiting these antimicrobial properties, the clinically tested NIKS progenitor cells were employed to provide a source of genetically uniform, nontumorigenic, pathogen-free human keratinocytes that are amenable to genetic engineering using nonviral means.CLINICAL CARE RELEVANCEPathogenic bacterial strains are increasingly developing antibiotic resistance, thereby forcing the clinician to use potent antibiotics with deleterious effects on keratinocyte viability and migration. Therefore, an urgent need exists for new wound therapies that can circumvent many of the problems associated with current antibiotic treatments.CONCLUSIONEnhanced expression of cathelicidin in a genetically engineered human BSS has been shown to inhibit the bacterial growth of a multidrug-resistant clinical strain of Acinetobacter baumannii in vivo, creating a new and innovative therapeutic option for combating these debilitating wound infections while also promoting healing.

BACKGROUND:For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss.THE PROBLEM:Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites.BASIC/CLINICAL SCIENCE ADVANCES:Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures.CLINICAL CARE RELEVANCE:Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application.CONCLUSION:This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

OBJECTIVE:The goal of this study was to assess the immunogenicity and antigenicity of StrataGraft skin tissue in a randomized phase I/II clinical trial for the temporary management of full-thickness skin loss.BACKGROUND:StrataGraft skin tissue consists of a dermal equivalent containing human dermal fibroblasts and a fully stratified, biologically active epidermis derived from Near-diploid Immortalized Keratinocyte S (NIKS) cells, a pathogen-free, long-lived, consistent, human keratinocyte progenitor.METHODS:Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible. StrataGraft and cadaveric allograft were placed side by side on 15 patients with full-thickness skin defects for 1 week before autografting. Allografts were removed from the wound bed and examined for allogeneic immune responses. Immunohistochemistry and indirect immunofluorescence were used to assess tissue structure and cellular composition of allografts. In vitro lymphocyte proliferation assays, chromium-release assays, and development of antibodies were used to examine allogeneic responses.RESULTS:One week after patient exposure to allografts, there were no differences in the numbers of T or B lymphocytes or Langerhans cells present in StrataGraft skin substitute compared to cadaver allograft, the standard of care. Importantly, exposure to StrataGraft skin substitute did not induce the proliferation of patient peripheral blood mononuclear cells to NIKS keratinocytes or enhance cell-mediated lysis of NIKS keratinocytes in vitro. Similarly, no evidence of antibody generation targeted to the NIKS keratinocytes was seen.CONCLUSIONS:These findings indicate that StrataGraft tissue is well-tolerated and not acutely immunogenic in patients with traumatic skin wounds. Notably, exposure to StrataGraft did not increase patient sensitivity toward or elicit immune responses against the NIKS keratinocytes. We envision that this novel skin tissue technology will be widely used to facilitate the healing of traumatic cutaneous wounds.This study was registered at www.clinicaltrials.gov (NCT00618839).