Verena Kiessig

Acellular human dermis promotes cultured keratinocyte engraftment

In full-thickness skin injury, loss of dermis may result in compromised wound repair, including contracture, hypertrophic scarring, and wound breakdown. This report examines the effect of an acellular dermal matrix on in vivo skin repair. Human keratinocytes cultured onto a synthetic hydrophilic dressing were applied with (N = 9) and without (N = 11) an acellular dermal matrix to full-thickness skin defects on athymic mice. Host cells progressively repopulated the acellular dermal component of the grafts. All animals with dermal matrix revealed fully differentiated epidermis by postoperative day 21. Human keratinocytes persisted in all animals grafted with dermal matrix, compared to only 63.6% of those animals without a dermal component. Planimetric analysis revealed significantly reduced wound contraction (p = 0.016) in animals receiving the dermal matrix. Histologic, immunohistochemical, and electron microscopic analyses also were performed. These studies suggest that an acellular dermal matrix can effectively direct regeneration of normal skin morphology.

Wound closure with human keratinocytes cultured on a polyurethane dressing overlaid on a cultured human dermal replacement

BACKGROUND Burn excision followed by immediate wound coverage has become the clinical standard for managing extensive burn injuries in much of the world. When sufficient autograft skin to achieve permanent wound closure is unavailable, cell culture technology has made the use of cultured human keratinocyte (HK) sheets clinically feasible. Whereas previous techniques have focused on development of multilayered, differentiated HK sheets, our attention has been drawn to using HK in a highly proliferative, less differentiated state. Time requirements for preparation of multistratified cultured HK are high, and preparatory steps may destroy important integrin adhesion molecules. METHODS We describe the use of HK cultured to single layer confluence on a polyurethane membrane(HD), with serum-free medium. HK-HD grafts were transplanted to full-thickness wounds on athymic mice (n = 31). A second group of mice (DG-HK-HD), n = 28) received a living human dermal replacement containing cultured fibroblasts before placement of HK-HD. Control mice received HD alone (n = 4). Basement membrane proteins on healed wounds and surface integrins on cultured HK were identified by means of immunostaining and direct microscopic visualization. RESULTS HK cultured just to the confluent state on polyurethane membrane were positive for integrins alpha(5) and alpha(6), major integrins on proliferating HK. Histologic analysis showed epithelialized wounds in all groups after 21 days. Using an anti-human involucrin antibody we demonstrated the presence of HK in 64.5% of the HK-HD group, 61% of the DG-HK-HD group, and 0% in the HD group. Mice that received the living human dermal replacement containing cultured fibroblasts in combination with HK-HD grafts developed a thick, well-vascularized neodermis. Strong laminin and collagen IV staining was observed in wound areas covered with HK. CONCLUSIONS These data show that full-thickness wounds can be closed by application of a single layer of proliferating HK cultured on a biocompatible polyurethane membrane. This technique is an alternative to the use of multilayered, differentiated HK sheets. Preparation times for HK-HD grafts should be significantly shorter than required for multilayered HK sheets, technical efforts should be less, and more extensive wound areas could be covered.

Role of Melanoma growth stimulatory activity (MGSA/gro) on keratinocyte function in wound healing

Melanoma growth stimulatory activity/groα (MGSA), a member of the α-chemokine family, is produced by a variety of dermal and epidermal cells and can act in a paracrine and autocrine fashion. However, little is known about the importance of MGSA in wound healing. In this study, we quantified the levels of MGSA protein in burn blister and donor site wound fluids. We studied the effects of MGSA on proliferation and migration of primary human keratinocytes and modulation of their integrin expression. Blister fluids contained 0.79 ng/ml (range 0.018 to 4.86 ng/ml) MGSA. Substantial increasing amounts of MGSA were found in donor site fluids from day 1 through day 5 with mean levels ranging from 1.77 to 103 ng/ml at postoperative day 5, which correlated with increasing amounts of tumor necrosis factor α (r = 0.86), a known stimulus for MGSA production. In vitro proliferation experiments revealed a maximum stimulation (2.6-fold) with 10 ng/ ml MGSA for 7 days over unstimulated keratinocyte controls; the ED50 was 0.2 ng/ml. DNA content analysis revealed an increase in S phase with 10 ng/ml MGSA stimulation. In cultured keratinocytes, MGSA enhanced the mean fluorescence intensity for α6, while no significant change was seen for β1, α2 and α5. We also studied the effect of topically applied MGSA (50 ng/cm2) on the healing of meshed split-thickness human skin grafts on athymic mice. In these wounds, MGSA stimulated the rate of epithelialization (P < 0.05) at day 7, and an increased proportion of mitotic keratinocytes was observed. Wound contraction was significantly (P < 0.05) reduced on days 7 and 14 in the MGSA-treated group. These results suggest that MGSA participates in wound healing by stimulating keratinocyte proliferation.

Effects of E-selectin and P-selectin blockade on neutrophil sequestration in tissues and neutrophil oxidative burst in burned rats

OBJECTIVE Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and b) measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury. DESIGN Prospective, controlled, laboratory study. SETTING University research laboratory. SUBJECTS Male Wistar rats (200 to 300 g). INTERVENTIONS After tracheostomy and venous cannulation, rats received 17% total body surface area full-thickness contact burns and were resuscitated with saline (20 mL i.p.). Experimental animals received 2 mg/kg body weight i.v. administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn. Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later. Edema was estimated by measuring extravasated 125 I-labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry. MEASUREMENTS AND MAIN RESULTS Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p < .05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p < .05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn. Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn. Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues. Postburn neutrophil deposition in lungs and liver was blocked (p < .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected. CONCLUSION Burn injury in rats results in accumulation of neutrophils in multiple tissues. Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.

Integrin and matrix molecule expression in cultured skin replacements

Current tissue culture techniques enable human keratinocytes (HK) from a small section of skin to be grown into sheets of epithelium for treating extensive wounds. The additional use of dermal replacements coupled with cultured skin substitutes may improve handling properties and effect ultimate healing results. Adhesion of cultured HK grafts to wounds, and final success rates of HK grafting, are variable and frequently unsatisfactory. To evaluate adhesion molecule (integrin) expression by cultured grafts, as well as matrix molecule distribution, we performed immunohistologic analysis of integrin expression on HK cultured on plastic and a polyurethane membrane, as well as on two dermal substitutes. Multilayered HK sheet grafts were prepared by culturing cells in plastic tissue culture dishes, and HK were cultured to single-layer confluence on polyurethane membranes (Hydroderm; Wilshire Medical Inc., Dallas, Texas). Composite grafts were prepared by seeding proliferating HK on Dermagraft, composed of human neonatal fibroblasts cultured in polyglactin mesh (Advanced Tissue Sciences, La Jolla, Calif.) and on AlloDerm, an acellular human dermis (LifeCell Inc., The Woodlands, Texas). Immunohistologic staining was performed for the integrin subunits alpha 5, alpha 6, and alpha v. Staining for matrix molecules included fibronectin, laminin-1, and laminin-5. HK in cultured epithelial sheets showed integrin alpha 6 expression on basal cells, and only faint alpha 5 and alpha v staining. HK cultured to confluence on Hydroderm reacted with monoclonal antibodies specific for alpha 5, alpha 6, and alpha v. Through HK adhered well to Dermagraft, there was reduced adhesion of HK on AlloDerm that was not accelerated by addition of fibronectin. HK in composite grafts showed distinct reactivity according to the time in culture. HK on Dermagraft lost alpha 5 reactivity by day 17 and only weak alpha v reactivity was seen. Basal keratinocytes on AlloDerm, however, remained alpha 5 and alpha v positive. In both composite grafts, integrin alpha 6 expression was limited to basal keratinocytes.

Growth peptide release from biologic dressings : A comparison

Biologic dressings are believed to stimulate wound healing in a variety of wound types. Cryopreserved allograft skin (CAS) is used as a biologic dressing for excised wounds, partial-thickness wounds, and meshed split-thickness skin grafts, and the use of allogenic or autologous cultured epithelial sheets (CES) has been reported to enhance healing of skin ulcers and deep partial-thickness wounds. However, limitations of allograft skin include bacteriologic and viral safety, limited availability, cost, and ease of handling. Previously we have reported the successful use of human keratinocytes cultured to single-layer confluence on Hydroderm polyurethane membranes (HD/HK) for grafting of full-thickness wounds. In this study we evaluated the release of five different growth peptides (transforming growth factors alpha and beta (TGF-alpha, TGF-beta), interleukin-6, interleukin-8, and melanoma growth stimulatory activity from CAS, CES, and HD/HK grafts. Highest levels of TGF-alpha were found for HD/HK (728 +/- 115 pg/10 cm2 of membrane) followed by CES (491 +/- 137 pg/10 cm2; NS). No TGF-alpha was detectable for CAS, and 3.7-fold, and 25-fold higher levels of interleukin-6 were found for CES (257 +/- 12.7 U/10 cm2) compared with HD/HK and CAS, respectively. Interleukin-8 had similar levels for CES (0.65 +/- 0.7 ng/10 cm2) and HD/HK (0.88 +/- 0.12 ng/10 cm2), whereas melanoma growth stimulatory activity was elevated in CES (2314 +/- 97 pg/10 cm2) compared with HD/HK (1071 +/- 55 pg/10 cm2). TGF-beta was barely detectable for CES and HD/HK. Cryopreserved allograft showed high levels of TGF-beta (5.2 +/- 1.6 ng/10 cm2). Overall mitogenic activity of the supernatants on keratinocyte cultures was assessed. Highest proliferation was seen for CES supernatants followed by HD/HK (NS). Supernatants from CAS had an antiproliferative effect on keratinocytes. We conclude that a single layer of keratinocytes cultured on a polyurethane membrane facilitates keratinocyte proliferation similar to CES, whereas cryopreserved allograft has no mitogenic effect on keratinocytes.

Effects of recombinant bactericidal/permeability-increasing protein (rBPI23) on neutrophil activity in burned rats

Bactericidal/permeability-increasing protein (BPI) is a neutrophil granule protein with potent bactericidal and lipopolysaccharide (LPS)-neutralizing activities. The purpose of this study was to determine if a human recombinant BPI product, rBPI23, would influence neutrophil (PMN) sequestration into various tissues in a rat burn injury model. Leukosequestration may produce local tissue injury from proteases and high-energy oxygen species released from PMNs. Rats received tracheostomy and venous cannulation, then received 17 to 20% total body surface area full-thickness contact burns and resuscitation with 20 ml, of intraperitoneal saline. Ten mg/kg body weight rBPI23 in saline was given by intravenous injection immediately after burn injury, followed by intravenous doses of 2 mg/kg at 2 and 4 hours. Control animals received intravenous saline only. PMN retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (before burn injury) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hours after burn injury, and measuring tissue radioactivity 30 minutes later. Edema was estimated by measuring extravasated 125I-labeled albumin in the various tissues, 30 minutes after injection. Peripheral blood PMNS were analyzed for intracellular H2O2 content by flow cytometry using a fluorescent dye that reacts with H2O2. Radioisotope studies demonstrated significant (p < 0.05) leukosequestration into lung, liver, gut, kidney, and skin tissues at 5 hours after burn injury. Tissue edema, manifested by radiolabeled albumin retention, was not observed in any tissues. Postburn PMN deposition in lungs and skin was decreased (p < 0.05) by the immediate administration of rBPI23 after burn injury. Flow cytometry showed increased intracellular H2O2 content in peripheral blood PMNs 5 hours after burn injury (p < 0.05), which was unaffected by administration of rBPI23. Since sequestration of metabolically active PMNs may induce tissue injury, therapies that block leukosequestration after burn injury may improve clinical outcomes by limiting remote tissue injury.

Effects of early and delayed wound excision on pulmonary leukosequestration and neutrophil respiratory burst activity in burned mice

BACKGROUND Tissue myeloperoxidase (MPO) is a marker of neutrophil (PMN) accumulation in tissues (leukosequestration). We measured MPO in the livers, guts, and lungs of mice after burn injury and studied the additive effect of burn excision on lung MPO. Lung histologic characteristics were also examined. PMN respiratory activity was assessed by measuring intracellular H2O2 content. METHODS Mice received 32% total body surface area (TBSA) burns; some underwent burn excision followed by wound closure with allograft skin, either immediately or 48 hours after burn. Tissue MPO was measured by a colormetric assay, and intracellular H2O2 was quantified by flow cytometry. RESULTS MPO was elevated in lungs 8 to 24 hours after burn (p < 0.05) but not in the liver or ileum. Other burned mice received either immediate or 48-hour-delayed wound excision and allografting. In controls a similar-size area was excised and grafted with normal or burned skin. Burned animals had increased lung MPO compared with nonburned animals (p < 0.05). Highest lung MPO levels were seen after burn/immediate excision (p < 0.001). Lung MPO levels were not different comparing unburned mice undergoing skin excision and grafting with either nonburned or burned skin. When burn excision was delayed 48 hours, lung MPO was increased moderately (p < 0.05) but remained far below levels in mice that were excised immediately after burn. PMN influx into lung tissues was confirmed by histologic examination. PMN H2O2 production was increased in burned mice and was additionally increased after immediate wound excision. CONCLUSIONS Although burn injury produces pulmonary leukosequestration, the phenomenon is unrelated to local effects of burned skin. In this experimental model immediate postburn wound excision increased pulmonary leukosequestration to higher levels than after burn injury alone, and intracellular H2O2 content also increased. Pulmonary leukosequestration may predispose to lung injury, possibly limiting the benefits of wound excision performed extremely early postburn.

Effects of recombinant bactericidal, permeability-increasing protein on bacterial translocation and pulmonary neutrophil sequestration in burned mice.

Burn injury induces bacterial translocation (BT) from the gut in multiple animal models. Etiologic factors contributing to BT may be an ischemia-reperfusion injury to the gut, the release of inflammatory cytokines, oxygen metabolites and other mediators, and cytotoxic effects mediated by endotoxin (lipopolysaccharide). Bactericidal, permeability-increasing protein is a neutrophil granule protein with potent bactericidal and lipopolysaccharide-neutralizing activities. The use of this protein has not been previously reported in a burn-injury model. The purpose of this study was to determine whether recombinant bactericidal, permeability-increasing protein (rBPI23) affects the incidence of BT and myeloperoxidase content in lung tissue (a measure of leukocyte sequestration) in a burn-injury model. Mice received a 32% total body surface area, full-thickness, scald burn, and 10 mg/kg body weight rBPI23 in saline solution was given by intraperitoneal injection at 0, 3, and 6 hours after the burn. Control animals received intraperitoneal saline solution only. All animals received a total of 1 ml saline solution intraperitoneally immediately after burn injury for fluid resuscitation. At 24 hours after burn injury, mesenteric lymph nodes (MLN) were harvested, homogenized, and plated. Lung tissue was harvested and assayed for myeloperoxidase. Burned mice treated with rBPI23 had significantly (p = 0.005, Fishers Exact Test, two-tailed) decreased incidence of BT, compared to burned mouse controls. Leukosequestration into lung tissues was not affected by rBPI23. Postburn administration of rBPI23 reduces but does not abolish the incidence of BT after burn injury in mice, perhaps by reducing intestinal injury during burn shock and the ischemia-reperfusion period by inhibiting the effects of lipopolysaccharide. An alternate explanation may be that rBPI23 could increase clearance and killing of bacteria by host defenses.