Current status of skin replacements for coverage of extensive burn wounds.
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Biological Dressings↗
Biomedical subjects
J F Hansbrough
Publications and source records attributed to J F Hansbrough.
Head injury: an immunologic deficit in T-cell activation.
Despite progress in the management of increased pressure following head injury, infection remains the most common complication in survivors. This study attempted to better define the immunologic deficit that occurs immediately and in the early post-recovery period following severe head injury. Twenty-seven patients admitted with the primary diagnosis of severe head injury (mean Glasgow Coma Score, 6.75) were studied within 24 hours of injury and at weekly intervals. T-cell proliferative response to mitogen stimulation was assessed. T-cell antigen expression following PHA stimulation was assessed for helper (CD4) and suppressor (CD8) subpopulations: the early T-cell activation antigens interleukin-2 receptor (IL2R) and transferrin receptor (TFR), and the late antigen (HLA-DR) using flow cytometry. Polymorphonuclear leukocyte function was assessed using oxidative burst. There was a reduction in the proliferative response of T cells to mitogen stimulation. The B-cell response seemed unaffected. The diminished proliferative response was accompanied by diminished expression of early activation antigens (IL2R and TFR) and late (following DNA synthesis) (HLA-DR). This was seen primarily on helper/inducer cells (CD4+). Polymorphonuclear leukocyte function was unaffected.
Antigens, Differentiation, T-Lymphocyte↗
Postburn immune suppression: an inflammatory response to the burn wound?
In an effort to elucidate the causes of immune suppression which follows severe burn injury, we studied the immunologic effects of subcutaneous implantation of burned skin, as well as implantation of other materials, in mice. Ten days following the implantation, splenic lymphocyte proliferation and lymphocyte surface expression of activation antigens (IL-2R and Ia) were analyzed following a 3-day culture period. In addition, peritoneal neutrophils were analyzed for oxidative burst activity using flow cytometry and a dye which reacts with intracellular hydrogen peroxide. Implantation of a 2 x 2 cm section of burned/unburned skin as well as implantation of a similar-sized piece of cotton gauze or collagen sheet resulted in subsequent suppression of both lymphocyte activation proliferation and neutrophil oxidative burst activity. An intense local inflammatory response to the burn wound may play a role leading to the profound systemic immune suppression which follows severe burn injury.
Animals↗
Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate.
Improved survival of patients with extensive burn injuries requires the development of better techniques for timely and permanent closure of the full-thickness wound. We developed procedures for establishing confluent, stratified layers of cultured, autologous keratinocytes on the surface of a modified collagen-glycosaminoglycan membrane that contains autologous fibroblasts. We transferred the composite grafts onto limited areas of excised full-thickness burn wounds of four patients with extensive burn injuries. Our results indicate that this composite graft material has acceptable "take," with formation of a basement membrane within 9 days of graft placement. Use of this technique for major wound coverage may offer a significant advance in the care of extensively burned patients and also may be useful for providing skin for reconstructive surgery.
Adult↗
Attachment of peptide growth factors to implantable collagen.
Ingrowth of fibrovascular tissue from the woundbed into collagen-based dermal substitutes and survival of cultured epithelium after transplantation may be enhanced by attachment of heparin binding growth factor 2 (HBGF2) and epidermal growth factor (EGF) to collagen. Biotinylation of collagen and the growth factors allows immobilization of HBGF2 and EGF by high affinity binding of tetravalent avidin. Biotinylated HBGF2 and EGF (B-GF) were exposed to complexes of biotinylated collagen (B-COL)-avidin (A) and detected with peroxidase-labeled avidin (AP) followed by chromagen formation on nitrocellulose paper. Binding of biotinylated HBGF2 and EGF was specific (*, P less than 0.05), proportional to the concentration of biotinylated collagen, and resistant to ionic (NaCl) displacement. Data are expressed as mean percentages of maximum binding +/- SEMs: (table; see text) Growth response of cultured human epidermal keratinocytes to HBGF2 (population doubling time, PDT = 0.70 population doublings (PD)/day) confirmed the retention of mitogenic activity after biotinylation (PDT = 0.80 PD/day). Specific binding of biotinylated HBGF2, EGF, or other biologically active molecules (antibiotics, NSAIDs) to implantable collagen may provide a mechanism for positive therapeutic modulation of wound healing, including repair of full-thickness skin wounds with cultured cell-collagen composite grafts.
Cell Division↗
Defective T-cell surface antigen expression after mitogen stimulation. An index of lymphocyte dysfunction after controlled murine injury.
Murine spleen T-cell activation in lectin-stimulated cultures after 25% body surface area burn injury or hind-limb amputation was studied by measuring the temporal expression of cell surface markers using monoclonal antibodies and two-color flow cytometry. Lymphocyte activation has been shown to be accompanied by the appearance of new surface antigens, including Interleukin-2 (IL-2) deceptor (IL-2R) and Ia, and emergence of cells that coexpress helper (Th) and suppressor (Ts) surface markers. IL-2R has been shown to appear early on stimulated cells, before DNA synthesis, whereas Ia appears later. Surface markers (L3T4, Lyt2, Ia, and IL-2R) were analyzed at time 0 and after 24, 48, and 72 hours of mitogen-stimulated culture. The appearance of IL-2R and Ia on Th (L3T4+) and Ts (Lyt-2+) populations was markedly depressed after burn injury, but minimal changes were seen after musculoskeletal injury. In addition, coexpression of L3T4/Lyt2 antigens was markedly reduced in burn-derived cells. Serum from burn-injured animals caused depression of surface antigen expression by stimulated normal cells. Recombinant IL-2, when added to burn-derived cell cultures, did not increase expression of these surface markers during culture, nor did it improve proliferation.
Animals↗
Massive doses of midazolam infusion for delirium tremens.
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Alcohol Withdrawal Delirium↗
Prospective, randomized trial of hypertonic sodium lactate versus lactated Ringer's solution for burn shock resuscitation.
We prospectively randomized 51 adult burned patients on admission to study fluid, electrolyte, and physiologic parameters during burn resuscitation with the use of hypertonic saline (HSL, Na 250 mEq/L, 514 mOsm) or lactated Ringer's solution (LR, Na 130 mEq/L, 268 mOsm). Patients suffered at least 20% total body surface area burns (BSA); the mean BSA injury was 36.7% BSA, with a range of 20 to 74% BSA. All patients were admitted to our Burn Center within at least 12 hours of injury. Laboratory studies included frequent determinations of serum chemistries including osmolalities, and continuous 24-hour urine collections for electrolytes and osmolality determinations. Fluid requirements (cc/kg/% BSA), urine output (cc/kg/hr), sodium intake and excretion (mEq/kg/% BSA), serum and urine osmolality (mOsm/kg), serum creatinine (mg/dl), body weight (kg), and enteral intake (cc/24 hrs and calories/24 hrs) were analyzed for comparison at 24-hour intervals following burn injury. Using Student's t-test, significance was attributed to a p less than 0.05. Nonparametric methods were used to compare non-normalized data. Regression analysis was used to compare sodium intake (mEq/kg) and fluid intake (cc/kg) between the HSL and the LR groups in relation to % BSA. Our data show no advantage of HSL over conventional therapy with LR for burn resuscitation. We were not able to demonstrate decreased fluid requirements, improved tolerance of feedings, or decrease in per cent weight gain.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Serum osmolar and electrolyte changes associated with hypertonic resuscitation.
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Aorta↗
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.
Amputation, Surgical↗
Structure of a collagen-GAG dermal skin substitute optimized for cultured human epidermal keratinocytes.
Collagen and glycosaminoglycan (GAG) dermal skin substitutes (membranes) were studied as substrates for cultured human epidermal keratinocytes. Structure of dermal substitutes was optimized for pore size to promote ingrowth of fibrovascular tissue from the wound bed and for culture of human keratinocytes of the membrane's surface. Pore size of the freeze-dried material was regulated by control of the temperature of freezing between -50 degrees C and -20 degrees C and by concentration of starting materials between 0.17% and 1.62% wt/vol. A nonporous surface of collagen-GAG was laminated to the membranes to provide a planar substrate for cultured epidermal keratinocytes. Thickness of dermal substitutes was regulated by control of the volume and concentration of starting materials. Biotin was conjugated to solubilized collagen for binding with avidin of specific quantities of biologically active molecules. The optimized membranes are suitable substrates for the culture of human epidermal keratinocytes, and together with the cells yield a composite material that is histologically similar to skin.
Artificial Organs↗
Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: technical considerations and specificities of monoclonal antibodies directed against surface markers.
We performed detailed phenotypic analysis of murine lymphocytes from thymus, spleen, lymph node, and peripheral blood using commercially available monoclonal antibodies, each with specificities for membrane surface markers and dual-color flow cytometry. Erythrocyte lysis techniques were utilized for lymphocyte preparation so that inherent difficulties with gradient techniques would be avoided, such as the potential for loss of abnormally sized cells. These studies demonstrated that the specificities of each monoclonal must be carefully determined; for example, the Lyt-1 monoclonal, frequently utilized to identify helper/inducer T cells, also reacts with suppressor/cytotoxic (Lyt-2+) cells; helper/inducer cells are better studied with a more recently available monoclonal, L3T4. Cells from different tissues may differ greatly not only in the presence of surface markers, but also in the surface density of each marker; this density can be studied and quantitated using appropriate analytic software. We also show that larger and more granular lymphocytes appear to be enriched for surface Ia antigen, indicating that these cells may be activated or regulatory subsets; these large, Ia+ T-cells will be lost from analysis if standard, narrow gate settings are used for analyzing forward and side-scatter characteristics or for cell sorting.
Animals↗
Antibody formation after murine injury.
To determine the effect of burn injury on humoral immunity, we followed the murine primary and secondary antibody responses to sheep erythrocytes (SRBC), a T-cell dependent antigen, and lipopolysaccharide (LPS), a T-cell-independent antigen, after 25% TBSA burn. Splenic B-cell-specific antibody synthesis was measured by a hemolytic plaque assay. Simultaneous measurements of specific and nonspecific immunoglobulins were performed by a hemagglutination assay and radial immunodiffusion, respectively. Numbers of splenic primary anti-SRBC-plaque forming cells (PFC) were generally equal in burn and control groups. Numbers of splenic secondary IgG anti-SRBC and anti-LPS PFCs were significantly increased in the burn group with a peak at 5 and 16 days after secondary immunization. There were no differences in serum anti-SRBC or anti-LPS antibody titers between burn-injured and control mice. Nonspecific serum IgG levels were depressed on Days 5 and 10 following injury but had returned to normal by Day 10. Due to the discrepancy between increased secondary PFCs and normal or decreased serum immunoglobulin levels, we measured clearance of exogenously administered IgG. The half-life of IgG in burn mice was 2.5 days compared to 7.1 days in control (P less than 0.05). In conclusion, the antibody response is enhanced after burn injury in the mouse, but this is not reflected in serum levels of specific antibody or immunoglobulins probably due to increased clearance from the circulation.
Animals↗
Trauma peptide induction of lymphocyte changes predictive of sepsis.
Post-trauma immunosuppression is characterized by T-cell subpopulation changes and the presence of a low molecular weight suppressive active peptide (SAP), which suppresses T-cell blastogenesis and neutrophil chemotaxis. This study evaluated post-trauma T-cell antigens and suppressive active peptide/T-cell interactions to determine if the suppressive active peptide concentrations predictive of sepsis can cause changes in antigen expression predictive of sepsis. Human lymphocyte markers and differentiation antigens were analyzed post-trauma using flow cytometry for markers predictive of sepsis. Changes induced by purified suppressive active peptide incubated with normal human lymphocytes were similarly analyzed by flow cytometry. SAP concentrations for incubation were chosen which correlated with concentrations in patients developing clinical sepsis. Significant T-cell changes in patients who developed sepsis include: decreased total T-cells, decreased helper cells, decreased natural killer cells, increased Ia expressing mononuclear cells, increased activated T-cells, (L22) and increased IL-2 expressing cells (TAC). Suppressive active peptide can activate T-cells and cause significant increased expression of IL-2 receptors and natural killer cells. Other T-cell changes following trauma predictive of sepsis seem to occur independent of in vitro incubation with suppressive active peptides. IL-2 expressing cells are known to be more readily suppressed by the suppressive peptide. Suppressive peptide activation and subsequent inhibition of T-cells suggests a potential way to explain suppressive peptide-induced immunosuppression following trauma.
Adult↗
Comparison of wick and fibreoptic catheters in measurement of interstitial pressures in burned extremities.
We have performed simultaneous compartmental pressure monitoring in 14 burned extremities using both the established wick catheter and the recently developed fibreoptic catheter system. Initial pressures recorded by both systems correlated well. Discrepancies in subsequent values were considered to be either due to improper limb positioning or because of catheter damage or obstruction. Both systems are position sensitive, but the problem can be minimized by careful anchoring of the catheters. The fibreoptic catheter is more fragile, but the wick catheter, despite a constant slow infusion of heparinized fluid, is liable to obstruct. While it has proven difficult to compare cost-effectiveness for the two systems, it is our impression that the fibreoptic system offers advantages in terms of ease of placement and maintenance and long-term reliability of measurements. Experience from other burn groups with the two systems will be important. Finally, the parallel use of the two catheters emphasizes the potential fallibility of either system, and the necessity of applying clinical judgement in evaluating circumferentially burned extremities even though direct pressure monitoring is employed.
Adult↗
Warfarin-induced skin necrosis: an entity occasionally requiring burn wound expertise.
We describe a severe case of warfarin-induced skin necrosis with extensive full-thickness tissue loss involving approximately 12 per cent of the body surface area. The early management was conservative, with wound observation only, and no aggressive débridement was attempted. Wound and associated systemic sepsis followed, and the patient was transferred to our burn unit. The wounds were then managed with aggressive therapy including surgical excision, temporary coverage with allograft, and final closure with split-thickness autograft. This case illustrates important principles of appropriate surgical management of extensive skin lesions of this type.
Burn Units↗
Video recording trauma resuscitations: an effective teaching technique.
Since the initial hour after injury is the most crucial time for trauma patients, resuscitation technique is of vital importance. Standardized courses for first-hour management (ATLAS) have been widely accepted. A teaching format based upon video recording of every resuscitation has been developed. Tapes are reviewed by the staff and by the individuals involved in a particular resuscitation. In a weekly resuscitation review conference, actual footage is presented to the trauma team members, specific aspects of a resuscitation are critiqued, and supplemental didactic information is presented. Legal problems have been avoided by making the review and conference a part of the quality assurance process. Patient anonymity is preserved by positioning the video camera at the foot of the resuscitation bed. Tapes are erased after each conference. Video recording allows analysis of: 1) priorities during the resuscitation; 2) cognitive integration of the workup by the team leader; 3) physical integration of the workup by the team leader; 4) team member adherence to assigned responsibilities, resuscitation time, errors or breaks in technique; and 5) behavior change over time. In 3 1/2 years, more than 2,500 resuscitations have been recorded. Over a 3-month period, average resuscitation time to definitive care decreased for age- and injury severity-matched patient groups cared for by one team. Resuscitations have become more efficient and adherence to assigned responsibilities better. Weekly review of resuscitation contributes to improved technique and trauma care.
Critical Care↗
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.
Burns↗