Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice.

This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2-cm wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, and immunohistochemical microscopy. "Take" was determined separately by a direct immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, there was little difference between the two graft types, with the CEG having occasional blister formation. By light microscopy, the CEG had a dissociation of dermis from epidermis until day 42, which was never apparent with the CG. By day 42, the CG had increased dermoepidermal interdigitations similar to rete ridges, with a mature epithelium. Neither of these findings were seen with the CEG. Immunofluorescent and immunohistochemical microscopy for type IV collagen and laminin, as well as electron microscopy, showed similar retardation of basement membrane formation with the CEG. Using this model, the composite graft had significant advantages over the epidermal sheet graft in the closure of full-thickness wounds.

Reconstitution of the histologic characteristics of a giant congenital nevomelanocytic nevus employing the athymic mouse and a cultured skin substitute.

This study addresses the development of an animal model for human giant congenital nevomelanocytic nevi (GCNN). Skin grafts were made from 1) non-involved split-thickness skin from a 12-month-old GCNN patient, 2) nevus split-thickness skin from the same GCNN patient, 3) nevus full-thickness skin, and 4) cadaveric human split-thickness skin. For groups 1) and 2), human epidermal and dermal cells were enzymatically isolated and expanded in tissue culture. Composite grafts were made by placing the cultured dermal cells into a collagen-glycosaminoglycan (GAG) matrix, followed by placement of the epidermal cells onto the opposite, laminated side of the matrix. All grafts were placed onto full-thickness wounds of athymic mice and biopsies were obtained from 6 to 38 weeks later for light microscopy including S-100 immunoperoxidase staining, and electron microscopy. The GCNN cultured skin mice (group 2) developed black, raised skin in the healed wounds. None of the group 1 mice developed lesions, grossly or histologically. All of the nevus full-thickness mice retained the nevus grossly. Histopathologic examination at 38 weeks of the black, raised plaques of group 2 demonstrated a reconstituted dermis similar to group 3. Nevus cells were larger and more epithelioid in the upper dermis, as seen with true GCNN. These nevomelanocytes were not seen in the dermis at 24 weeks, suggesting that the nevus cells migrated from the epidermal component of the cultured graft to the dermis during this time frame (24-38 weeks). The melanocyte identity of these cells was confirmed with S-100 immunoperoxidase staining and electron microscopy. These findings are unique to this composite cultured graft system. The ability to culture specific types of melanocytes and place them int skin substitutes on athymic mice provides a basis for the study of GCNN and melanocyte biology in vivo.

Absence of tumorigenicity in athymic mice by normal human epidermal keratinocytes after culture in serum-free medium.

The very rapid growth rate (1 population doubling/day) of normal human epidermal keratinocytes (HK) cultured in serum-free medium can be utilized for wound closure in burn treatment. However, rapid growth in vitro may present the possibility of neoplastic transformation. To investigate this possibility, HK were cultured from primary isolation to large populations in MCDB 153 medium supplemented with epidermal growth factor (EGF, 10 ng/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), and Bovine Pituitary Extract (BPE, 70 micrograms/ml). HK were studied for their ability to form tumors in athymic mice after subcutaneous inoculation. Sixteen separate HK strains were inoculated from primary cultures, or from secondary cultures either before or after storage in liquid nitrogen. Transformed cell lines, SCC 13 and FL, derived from human epithelial carcinomata were used as controls for tumor formation. HK formed no tumors (0/79) after 26 weeks incubation, SCC 13 formed nodular tumors (3/5) after 20 weeks incubation, and FL formed tumors (5/5) after 4 weeks incubation. HK cells were not found by histological examination of inoculation sites of keratinocyte cultures derived from primary culture from skin. In contrast, palpable tumors from both SCC 13 and FL were returned to tissue culture and continued to proliferate. These results support the conclusion that the rapid growth rate of human epidermal keratinocytes in vitro can be attributed to permissive culture conditions, and not to neoplastic transformation.

High dose intravenous immunoglobulin therapy in burn patients: pharmacokinetics and effects on microbial opsonization and phagocytosis.

Depressed serum immunoglobulin levels following severe burns may lead to subsequent infectious complications following such injuries. In a randomized study we administered multiple doses of Sandoglobulin (500 mg/kg) or albumin intravenously to patients with severe burn injuries and closely monitored serum IgG levels. Patients who received IgG therapy had earlier return of normal serum IgG levels compared to control patients; however, control patients attained normal IgG levels during the second postburn week. Serum half-lives of IgG following infusions were remarkably short (means, 47 hours for infusions within 3 days of injury and 154 hours for infusions in the third postburn week); Sandoglobulin has been reported to have approximately a 21-day half-life in normal individuals. We also measured the opsonic capacity of postburn serum, using fluorescein-labeled microbes and flow cytometry; we identified postburn opsonic defects with certain of the organisms as late as 15 days postinjury, even though serum IgG levels had normalized. These defects were corrected by the in vitro addition of Sandoglobulin to the incubation mixture.

In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh.

The design of a skin-substitute must address the need for a dermal component, as this mesenchymally-derived tissue is important in maintaining the integrity and function of skin. An in vivo study was undertaken to assess the use of two biodegradable meshes, polyglycolic acid and polyglactin-910, as carriers for cultured human fibroblasts in a living dermal replacement. The consistent vascularization and epithelialization of these grafts placed on athymic mice showed that this has potential in re-creating the dermis in a skin-substitute.

Fibroblast sheets enable epithelialization of sounds that do not support keratinocyte migration.

Keratinocyte migration over the wound bed is the single most important parameter for wound epithelialization. Therefore, improvement of the wound bed matrix holds considerable promise for the shortening of hospitalization time in patients with ulcers, burns, and chronic wounds. We investigated wound epithelialization in athymic mice in the presence or absence of a sheet of cultured human fibroblasts. The physiology of keratinocyte growth on fibroblast sheets was investigated in tissue culture using histology, immunofluorescence, and electron microscopy. Keratinocytes from human skin explants were unable to attach or migrate on full-thickness dorsal wounds of athymic mice. Placement of a fibroblast-seeded polyglactin mesh on the wounds resulted in dramatically increased keratinocyte outgrowth. Similarly, human keratinocytes showed good outgrowth on fibroblast sheets at the air/liquid interface in tissue culture. Outgrowth was correlated inversely with fibroblast viability, indicating that the observed effect was due to the complex extracellular matrix secreted by the fibroblasts and matrix-bound growth factors rather than ongoing growth factor release. Collagen IV, a promoter of keratinocyte migration, was found to be abundant in the fibroblast-derived matrix. This study demonstrates that wounds which are unable to support keratinocyte migration can undergo epithelialization if a conducive substrate, supplying appropriate extracellular matrix and/or matrix-bound growth factors, is applied.

Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice.

In patients with extensive full-thickness burns, wound coverage may be accelerated if skin can be expanded to produce a skin replacement that reproducibly supplies blood to the wound and has good structural qualities. In addition, development of skin replacements may benefit patients who require reconstruction or replacement of large areas of abnormal skin. We have developed a composite skin replacement composed of cultured human keratinocytes (HK) and fibroblasts. Cultured human fibroblasts are seeded into the interstices, and cultured HKs are applied to the surface of a matrix composed of type I collagen crosslinked with a glycosaminoglycan, which has a defined physical structure. After HKs reach confluence on the matrix surface, the composite grafts are placed on full-thickness wounds on the dorsum of athymic mice. Graft acceptance, confirmed by positive staining with antibodies specific for human HLA-ABC antigens on HKs, is approximately 90%. A defined skin structure is present histologically by day 10 after grafting, with a differentiated epithelium and a subepidermal layer densely populated by fibroblasts and capillaries without evidence of inflammation. Fluorescent light microscopy to identify laminin and type IV collagen and electron microscopy confirm the presence of basement membrane components by 10 days after grafting. Attachment of the graft to the wound is similar with and without the addition of human basic fibroblast growth factor, a potent angiogenic agent, to the skin replacement before graft placement on wounds.

Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate.

Repair of full-thickness burns requires replacement of both the dermal and the epidermal components of the skin. Use of tissue culture methods allows very large expansions of surface area to be covered by cultured normal human epidermal keratinocytes (HK). Porous and resorbable materials, such as collagen and chondroitin-6-sulfate membranes, may be expected to adhere to wounds and promote fibrovascular ingrowth better than grafts of cultured epidermal keratinocytes alone. This article demonstrates the in vitro formation of biologic attachments between HK and a collagen and chondroitin-6-sulfate dermal skin replacement. Dermal membranes are prepared as generic acellular sheets and stored in the dry state for extended periods. Subconfluent HK cultures in logarithmic phase growth can attach quickly to dermal membranes in vitro, form a confluent epithelial sheet on the surface of each membrane, and exhibit mitotic cells for at least 1 week in vitro. Transmission electron microscopy demonstrates the formation of hemidesmosomes, extracellular matrix, and banded collagen at the interface of the epidermal cells and the dermal membrane. By comparison, HK cultures as confluent sheets released enzymatically with Dispase do not attach to the dermal membranes in vitro, under the conditions tested, although complete coverage of the membrane by the cell sheets is obtained. Growth assays show that subconfluent HK cells retain sufficient growth potential to maintain logarithmic phase growth, but that HK cells disaggregated from confluent sheets become growth arrested in comparison. The composite material has discrete dermal and epidermal compartments, has total thickness comparable to split-thickness skin graft, and can be applied to full-thickness skin defects in a single procedure.

Temporal analysis of murine lymphocyte subpopulations by monoclonal antibodies and dual-color flow cytometry after burn and nonburn injury.

The immune suppression that frequently accompanies severe injury undoubtedly contributes to subsequent infectious complications. Various lymphocyte subpopulations may be identified by surface antigen expression, and alterations in antigen expression by lymphocytes may reflect host immune competence. Using monoclonal antibodies (Moabs) and dual-color flow cytometry, we studied lymphocyte phenotypic expression in mice after either controlled burn injury or hind-limb amputation, with use of peripheral blood, lymph node, and spleen for cell preparation. Moabs were utilized specific for T cells (Lyt-1), helper/inducer cells (L3T4), suppressor/cytotoxic cells (Lyt-2), B cells (IgG), and activated T cells (Ia or IL-2 receptor). The assay techniques called for small amounts of tissue and avoided gradient procedures that might result in selective loss of some lymphocyte populations. The most consistent changes observed were depressions in percentages of L3T4+ and Lyt-2+ cells in spleens of burned mice, accompanied by depression in Ia+ (possibly activated or proliferating) subsets of L3T4+ and Lyt-2+ cells, and the appearance of increased percentages of non-B, non-T lymphocytes. Changes in lymph node cells were minimal. The major alteration seen in peripheral blood was substantial depression of Ia+ subsets, although burned mice had increased circulating Lyt-2+ cells on several late postburn days. Burned mice, unlike limb-trauma mice, had marked splenic hypertrophy with more than a 300% increase in spleen weight after the 30-day postburn period. Eschar excision/implantation experiments indicated that splenic hypertrophy and splenocyte phenotypic changes are related to the presence of burned tissue, which suggests that burned tissue may partially mediate immune changes that accompany severe burn injury.

Arteriovenous fistulas following central venous catheterization.

We report three patients in whom arteriovenous fistulas probably occurred following placement of central venous catheters. Two fistulas apparently followed internal jugular vein catheterization (or attempts), and one was demonstrated angiographically following subclavian vein cannulation. One fistula was repaired operatively, and in this case two separate fistulas in the same anatomical region were found. One patient refused surgery but remained asymptomatic after 6 months of follow-up. In the third patient the fistula probably spontaneously disappeared within one week of its appearance. We discuss two methods of central vein cannulation which should decrease the occurrence of complications following the procedure.

Immunosuppression by hyperbaric oxygen.

Reports from several laboratories have indicated that hyperbaric oxygen might be immunosuppressive in animals. We examined the effect of hyperbaric oxygen on a well-studied model of cell-mediated immunity in the mouse, contact sensitivity to dinitrofluorobenzene (DNFB). Using this model, we showed that daily 5-hour exposure of mice to 2.5 ATA hyperbaric oxygen was markedly immunosuppressive. Immunosuppression occurred when mice were exposed to hyperbaric oxygen (HBOX) for 4 days daily before DNFB sensitization or for 5 days daily after sensitization. The immunosuppression was reversed by intravenous administration of 2 x 10(7) peritoneal exudate cells from syngeneic mice, but was not reversed by 5 x 10(7) lymph node cells intravenously. We showed that daily HBOX exposure resulted in a dramatic decrease in circulating total leukocytes and lymphocytes in spleen weight, and in DNA synthesis in draining lymph nodes of sensitized mice. Serum cortisol levels were only marginally elevated in HBOX-treated mice.

Postburn immunosuppression in an animal model. III. Maintenance of normal splenic helper and suppressor lymphocyte subpopulations by immunomodulating drugs.

Delineation of lymphocyte subpopulations by labeling cells with specific monoclonal antibody now appears to be a reliable means of measuring cellular immunity in various disease states. We determined splenic helper/inducer and suppressor/cytotoxic lymphocyte populations in mice given a 20% to 25% body surface area steam burn injury. The lymphocyte helper: suppressor ratio fell from 3.13 +/- 0.06 in control mice to 1.77 +/- 0.04 in burned animals (p less than 0.0005) 14 days after burn. Immediate postburn eschar removal resulted in improvement in the ratio 14 days later (2.66 +/- 0.14) although not in restoration to normal levels. Postburn treatment of burned mice with intraperitoneal cimetidine, ibuprofen, indomethacin, cyclophosphamide, and topically applied cerium nitrate resulted in substantial restoration of the lymphocyte ratio toward normal values; in animals treated with cimetidine and ibuprofen the resultant lymphocyte ratio was not statistically different from that in control (unburned) mice. These drugs probably inhibit suppressor cell populations or suppress the immunosuppressive effect of toxic materials in the burn wound. Specific pharmacologic therapy improves immune function in burned mice and may result in increased resistance to infection.

Postburn immunosuppression in an animal model: monocyte dysfunction induced by burned tissue.

We studied cell-mediated immunity (CMI) in burned mice using an assay that involves the induction of contact sensitivity to dinitrofluorobenzene (DNFB). Subsequent painting of the ears with DNFB and measurement of ear swelling with calipers is a sensitive and quantifiable assay for CMI. Results may be expressed as mean ear swelling (MS) in units of 10(-4) inches +/- 2 standard errors of the mean. CMI was severely depressed in burned mice over a 2-week period following burn (control MS 48.3 +/- 1.0, 14 days after burn 29.0 +/- 1.0, P less than 0.01). Immediate postburn eschar removal resulted in avoidance of immunosuppression (MS 41.5 +/- 1.0, P less than 0.01) while transfer of burned tissue subcutaneously into unburned mice resulted in severe immunosuppression (MS 33.2 +/- 2.6, P less than 0.01). CMI was restored by intravenous infusion of peritoneal macrophages from unburned mice (MS 41.4 +/- 2.2), but not by infusion of lymphocytes or of macrophages taken from burned mice. This model should prove useful for further study of burn injury-induced immunosuppression.

Development of a temporary living skin replacement composed of human neonatal fibroblasts cultured in Biobrane, a synthetic dressing material.

BACKGROUND: Preferred coverings for excised burn wounds when sufficient autograft skin is not available are fresh or cryopreserved cadaveric skin. Problems with supply, preservation, immune rejection, and potential infection transmission accompanying the use of allograft skin underscore the need for effective alternative temporary skin replacements. METHODS: We cultured human neonatal fibroblasts (HF) for 4 to 6 weeks in nylon mesh of Biobrane, a synthetic dressing consisting of a thin layer of silicone bonded to nylon mesh. Secreted matrix proteins were identified by immunostaining and quantitated, and growth factor-specific messenger RNAs were identified by reverse transcription-polymerase chain reaction. Living grafts (Biobrane/HF) were sutured to full-thickness, excised wounds on athymic mice; control animals received Biobrane alone. Wounds were observed and biopsy specimens were obtained at intervals during the subsequent 40 days. RESULTS: After 3 to 6 weeks of culture in Biobrane the HF proliferated and secreted matrix proteins including type I collagen, fibronectin, and decorin, as well as messenger RNA for several growth factors (acidic fibroblast growth factor, basic fibroblast growth factor, and keratinocyte growth factor). Biobrane/HF grafts were transferred to full-thickness wounds, resulting in rapid fibrovascular ingrowth from the wound and effective wound closure for up to 40 days with minimal inflammatory responses. Biobrane control grafts adhered initially to wounds, but within several days many grafts developed subgraft exudates; histologic sections revealed marked inflammatory responses in these wounds. By 20 days, most BB grafts were separating from the underlying wounds that were closing by epithelialization and contraction. CONCLUSIONS: The Biobrane/HF living skin replacement provides long-term biologic coverage of full-thickness wound defects in mice with rapid incorporation of a living tissue matrix into the wound bed. Because HF have been found to be relatively nonantigenic when transferred to allogeneic hosts, Biobrane/HF grafts could replace the use of cadaveric allograft skin for achieving temporary wound closure after burn wound excision. Biobrane/HF grafts may persist on human wounds for weeks or months, with long-term persistence perhaps primarily dependent on durability of the silicone rubber layer.

Epidermal growth factor limits structural alterations in gastrointestinal tissues and decreases bacterial translocation in burned mice.

BACKGROUND: Burn injury produces acute gastrointestinal derangements that may predispose to bacterial translocation (BT). We studied effects of recombinant human epidermal growth factor (r-HuEGF), a gastrointestinal trophic hormone, on gastrointestinal alterations and BT after murine burn injury. METHODS: r-HuEGF was administered 1 and 12 hours after burn injury in a dose of 4 micrograms per animal subcutaneously after 25% and 32% total body surface area (TBSA) scald burn. Small bowel and gastric weight and histologic factors were studied, and BT was measured by culturing mesenteric lymph nodes. RESULTS: Mice treated with r-HuEGF maintained gastric and small intestine weight measured 24 hours after burn injury, and ileal mucosal height was preserved, whereas burned-untreated mice lost organ weight and mucosal height. BT was decreased significantly in mice with 32% TBSA burn injury treated with r-HuEGF after injury (burn, 64.2% of animals had BT; burn-r-HuEGF, 34.6% had BT; p < 0.05). After 25% TBSA burn injury, BT was also decreased in r-HuEGF-treated animals (burn, 31.4% of animals had BT; burn-r-HuEGF, 14.3% had BT), but the difference was not statistically significant (p < 0.1). CONCLUSIONS: r-HuEGF improves intestinal and gastric structure in mice 24 hours after burn injury and decreases BT after 32% TBSA burn injury.

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.

Biliary complications after liver transplantation: with special reference to the biliary cast syndrome and techniques of secondary duct repair.

In 93 consecutive cases of orthotopic liver transplantation, there were 24 example of biliary obstruction and eight of bile fistula formation. Six of the obstructed livers developed biliary cast formation so extensive that the smaller intrhepatic ducts became plugged to an extent that they could no longer have been treated by surgical mena. In each of the six cases, the most important causative factor was neglected obstruction of the large bile ducts with the intrahepatic lesions apparently being late and secondary. Stone and/or cast formation also occurred in other obstructed livers in the presence of bile fustulas, but these deposits were limited to the large ducts where they could have been or were removed. Although homograft bile undoubtedly has increased lithogenicity at certain posoperative times, the data from the present study have shown that biliary sludge formation essentially is always associated with defective bile duct reconstruction, and the observations have underscored the urgency with which reoperation must be considered. Techniques of secondary intervention have been described, with emphasis on conversion of cholecystojejunostomy to choledochojejunostomy. This operation has permitted salvage of homografts in eight of nine trials and the survival of seven patients.

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.

Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors.

Skin wound closure remains a major problem in acute and reconstructive skin grafting after large burns because of limited availability of donor skin. This report evaluates six protocols for preparation in vitro of skin substitutes composed of cultured human cells, biopolymers, and growth factors for wound closure. Full-thickness wounds in athymic mice treated in a single procedure with cultured skin substitutes were compared directly to treatments with murine skin autograft, human skin xenograft, or no graft. Rectilinear planimetry of healed wounds 6 weeks after surgery showed that skin substitutes cultured in serum-free medium, and for 24 hours before surgery in defined medium with basic fibroblast growth factor (100 ng/ml), were not statistically different (p less than 0.05) in size from treatment with human skin xenograft. Acceptance and persistence of skin substitutes cultured in serum-free media were 70% at 6 weeks after surgery, as determined by staining of healed skin with a fluorescein-labeled monoclonal antibody against human HLA-ABC antigens. Ultrastructural examination of wounds with cultured human skin 6 weeks after treatment showed complete basement membrane, including anchoring fibrils, presence of melanocytes and pigment transfer to keratinocytes, and innervation of healed skin adjacent to basement membrane. These findings demonstrate effectiveness of cultured skin substitutes for closure of skin wounds and illustrate important capabilities to modulate the natural processes of wound repair, to increase supply of materials used for wound repair, and to enhance quality of wound healing.

Postburn immunosuppression in an animal model. IV. Improved resistance to septic challenge with immunomodulating drugs.

We have previously demonstrated that certain pharmacologic agents administered to burned mice will restore cell-mediated immunity, as evidenced by measurement of delayed hypersensitivity responses and determination of splenic helper/suppressor lymphocyte ratios. These drugs are systemic cimetidine, ibuprofen, cyclophosphamide, and topical cerium nitrate. In the studies reported here we performed cecal ligation and puncture (CLP) in burned mice as a measure of resistance to infectious challenge. Survival after CLP with a 23-gauge needle used for puncture was markedly decreased when performed on the tenth postburn day (normal 63.7%, 10 days postburn 20.0%; p less than 0.001), but survival was not decreased when CLP was performed on the fifth (60.0%; p not significant) or twenty-first postburn day (65.3%; p not significant). Animals were then treated with the four agents in carefully defined dosage regimens, and survival was again determined on the tenth postburn day. Survival figures with p values compared to burned, untreated animals: burn plus cimetidine 62.2%, p less than 0.0005; burn plus: ibuprofen 64.7% p less than 0.0003; burn plus cyclophosphamide 68.2%, p less than 0.0001; burn plus cerium nitrate 54.1%, p less than 0.004. Specific pharmacologic therapy in burned mice in dosage regimens that have been shown to improve cell-mediated immunity is also able to significantly improve resistance to subsequent infectious challenge.