The viable bioengineered allogeneic cellularized construct StrataGraft® synthesizes, deposits, and organizes human extracellular matrix proteins into tissue type-specific structures and secretes soluble factors associated with wound healing.

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.

A phase 3, open-label, controlled, randomized, multicenter trial evaluating the efficacy and safety of StrataGraft® construct in patients with deep partial-thickness thermal 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 total) of comparable depth after excision were randomized to either cryopreserved StrataGraft or autograft.

Chimeric autologous/allogeneic constructs for skin regeneration.

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.

TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin.

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization.

Classical human epidermal keratinocyte cell culture.

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.

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 (NIKS(hBD) (-3) ) using nonviral methods.

Clinical Evaluation of NIKS-Based Bioengineered Skin Substitute Tissue in Complex Skin Defects: Phase I/IIa Clinical Trial Results.

Background: Complex 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 Problem: The 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.

A Bioengineered Human Skin Tissue for the Treatment of Infected Wounds.

Background: Complex 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.

Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

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.

StrataGraft skin substitute is well-tolerated and is not acutely immunogenic in patients with traumatic wounds: results from a prospective, randomized, controlled dose escalation trial.

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.

The StrataTest® human skin model, a consistent in vitro alternative for toxicological testing.

Three-dimensional in vitro skin models provide an alternative to animal testing for assessing tissue damage caused by chemical or physical agents and for the identification and characterization of agents formulated to mitigate this damage. The StrataTest® human skin model made with pathogen-free NIKS® keratinocyte progenitors is a fully-stratified tissue containing epidermal and dermal components that possesses barrier function as determined by measurements of electrical impedance. Independent batches of skin tissues responded consistently to known chemical irritants even after refrigerated storage for up to 7 days.

Visualization of morphological and molecular features associated with chronic ischemia in bioengineered human skin.

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure.

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 patient's 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.

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.

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).

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.

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.

Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures.

Background: Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues.

Generation and differentiation of human embryonic stem cell-derived keratinocyte precursors.

Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum-free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin causes accelerated terminal differentiation in fetal mouse skin.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental toxin, has been shown to cause a human skin pathology called chloracne. The majority of laboratory mouse strains, with the exception of mice bearing a mutation in thehairless gene, fail to display overt signs of chloracne upon exposure to TCDD. As a result, only minimal data exist on the effects of TCDD in adult haired mice and no data exist on the effects of TCDD in developing mouse skin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture.

Human exposure to the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a severe skin pathology known as chloracne. In these studies we employed a three-dimensional, organotypic model system to study the effects of TCDD on human skin. This model uses the spontaneously immortalized human keratinocyte cell line NIKS and recapitulates both the three-dimensional microenvironment and epithelial-mesenchymal interactions found in intact human skin.

Treatment of normal human keratinocytes with 2,3,7,8-tetrachlorodibenzo-p-dioxin causes a reduction in cell number, but no increase in apoptosis.

We have examined the effect of TCDD on the growth of normal human keratinocytes. TCDD is a ubiquitous environmental toxicant that causes a severe dermatopathology in humans, which is known as chloracne. The cell biological basis of this pathology remains unknown.

The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle.

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed.

Normal growth and differentiation in a spontaneously immortalized near-diploid human keratinocyte cell line, NIKS.

We report the isolation and characterization of a spontaneously immortalized human keratinocyte cell line, NIKS. The cell line is not tumorigenic in athymic nude mice and maintains cell-type-specific requirements for growth in vitro. NIKS cells express steady-state levels of transforming growth factor-alpha, transforming growth factor-beta1, epidermal growth factor receptor, c-myc, and keratin 14 mRNAs comparable with the parental BC-1-Ep keratinocyte strain.

Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult.