Shriners Hospitals for Children - Cincinnati

Because of their extensive wounds, burn patients are chronically exposed to inflammatory mediators. Thus, burn patients, by definition, already have "systemic inflammatory response syndrome." Current definitions for sepsis and infection have many criteria (fever, tachycardia, tachypnea, leukocytosis) that are routinely found in patients with extensive burns, making these current definitions less applicable to the burn population. Experts in burn care and research, all members of the American Burn Association, were asked to review the literature and prepare a potential definition on one topic related to sepsis or infection in burn patients. On January 20, 2007, the participants met in Tucson, Arizona to develop consensus for these definitions. After review of the definitions, a summary of the proceedings was prepared. The goal of the consensus conference was to develop and publish standardized definitions for sepsis and infection-related diagnoses in the burn population. Standardized definitions will improve the capability of performing more meaningful multicenter trials among burn centers.

Growth factors have many activities that make them attractive agents for stimulating tissue repair. Growth factors attract cells into the wound, stimulate their proliferation, and have profound influence on extracellular matrix deposition. Since developing the ability to mass-produce these cytokines by recombinant techniques, hundreds of studies have demonstrated that growth factors can augment all aspects of tissue repair in normal and impaired healing models. After demonstrating that growth factors augment healing, investigators have started to detect and measure growth factors in wounds and have found that wounding initiates the expression of various growth factors. Impaired healing has also been linked to altered growth factor production. These findings have prompted great interest in the use of growth factors to augment clinical healing. Preliminary clinical trials have not produced the results expected. Growth factor treatment has occasionally led to statistically significant improvements in tissue repair, but whether the results are clinically significant can be debated. It appears that to be cost effective, clinical trials must focus on targeting growth factors for specific types of impaired healing. Although growth factors have not been the panacea that was originally expected, they have the potential for making significant clinical improvements when targeted for specific problem wounds.

A modular tube feeding recipe (MTF) was designed to meet the unique nutritional needs of burn patients, applying principles previously documented in our burned guinea pig model. MTF, a high-protein, low-fat, linoleic acid-restricted formulation is enriched with omega-3 fatty acids, arginine, cysteine, histidine, vitamin A, zinc, and ascorbic acid. Fifty patients, 3 to 76 years of age with burns ranging from 10 to 89% total body surface area were prospectively randomized into three groups which blindly compared MTF to two enteral regimens widely utilized in the nutritional support of burns. Age, percent total and third-degree burn, resting energy expenditure, and calorie and protein intake were similar in all groups. Data analysis demonstrated significant superiority of MTF in the reduction of wound infection (p less than 0.03) and length of stay/percent burn (p less than 0.02). MTF was also associated with a decreased incidence of diarrhea, improved glucose tolerance, lower serum triglycerides, reduced total number of infectious episodes and trends toward improved preservation of muscle mass, although statistical significance was not achieved. Seventy percent of deaths occurred in the group supported with an inherently large dose of fat and linoleic acid. Combining these observations, it is believed that MTF is effective in modulating an improved response to burn injury.

Toxic epidermal necrolysis (TEN) is a potentially fatal disorder that involves large areas of skin desquamation. Patients with TEN are often referred to burn centers for expert wound management and comprehensive care. The purpose of this study was to define the presenting characteristics and treatment of TEN before and after admission to regional burn centers and to evaluate the efficacy of burn center treatment for this disorder. A retrospective multicenter chart review was completed for patients admitted with TEN to 15 burn centers from 1995 to 2000. Charts were reviewed for patient characteristics, non-burn hospital and burn center treatment, and outcome. A total of 199 patients were admitted. Patients had a mean age of 47 years, mean 67.7% total body surface area skin slough, and mean Acute Physiology and Chronic Health Evaluation (APACHE II) score of 10. Sixty-four patients died, for a mortality rate of 32%. Mortality increased to 51% for patients transferred to a burn center more than one week after onset of disease. Burn centers and non-burn hospitals differed in their use of enteral nutrition (70 vs 12%, respectively, P < 0.05), prophylactic antibiotics (22 vs 37.9%, P < 0.05), corticosteroid use (22 vs 51%, P < 0.05), and wound management. Age, body surface area involvement, APACHE II score, complications, and parenteral nutrition before transfer correlated with increased mortality. The treatment of TEN differs markedly between burn centers and non-burn centers. Early transport to a burn unit is warranted to improve patient outcome.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Engineered human skin is commonly fabricated using collagen scaffolds that often have poor mechanical properties. To improve the strength of collagen-based scaffolds, poly(caprolactone) (PCL) was blended with collagen and formed into submicron fibers using electrospinning. At concentrations < 10% PCL (M(PCL)/[M(Collagen) + M(PCL)] x 100), the PCL component was evenly distributed within the collagen matrix. Increasing the PCL component to 30% caused separation of the collagen and PCL phases forming local domains of PCL within the collagen matrix. Tensile testing indicated that 10-100% PCL concentrations significantly improved the strength and stiffness of the acellular scaffolds. Engineered skin (ES) made with blended collagen-PCL at a concentration of up to 10% PCL did not significantly alter the stratification of the cells, cell proliferation, or epidermal differentiation compared to the 100% collagen group. Ultimate tensile strength of ES fabricated with the collagen-PCL blends was not significantly greater than that of ES made with 100% collagen scaffolds (0% PCL). The 30% PCL group had the least amount of mechanical strength likely caused by poor epidermal formation and reduced cell viability. These results indicate that minimal additions of PCL can be blended with collagen to produce scaffolds suitable for tissue engineering of human skin. However, the increase in scaffold strength with higher PCL concentrations did not result in significantly stronger ES, indicating that high cell viability and proper development of the epidermis are important factors for developing ES with high strength.

OBJECTIVE: The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. BACKGROUND: Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. METHODS: Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. RESULTS: Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. CONCLUSION: This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism.

OBJECTIVE: Comparison of cultured skin substitutes (CSSs) and split-thickness autograft (STAG) was performed to assess whether the requirement for autologous skin grafts may be reduced in the treatment of massive burns. SUMMARY BACKGROUND DATA: Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been demonstrated to close full-thickness skin wounds in athymic mice and to express normal skin antigens after closure of excised wounds in burn patients. METHODS: Data were collected from 17 patients between days 2 and 14 to determine incidence of exudate, incidence of regrafting, coloration, keratinization, and percentage of site covered by graft (n = 17). Outcome was evaluated on an ordinal scale (0 = worst; 10 = best) beginning at day 14, with primary analyses at 28 days (n = 10) and 1 year (n = 4) for erythema, pigmentation, epithelial blistering, surface roughness, skin suppleness, and raised scar. RESULTS: Sites treated with CSSs had increased incidence of exudate (p = 0.06) and decreased percentage of engraftment (p < 0.05) compared with STAG. Outcome parameters during the first year showed no differences in erythema, blistering, or suppleness. Pigmentation was greater, scar was less raised, but regrafting was more frequent in CSS sites than STAG. No differences in qualitative outcomes were found after 1 year, and antibodies to bovine collagen were not detected in patient sera. CONCLUSIONS: These results suggest that outcome of engrafted CSSs is not different from STAG and that increased incidence of regrafting is related to decreased percentage of initial engraftment. Increased rates of engraftment of CSSs may lead to improved outcome for closure of burn wounds, allow greater availability of materials for grafting, and reduce requirements for donor skin autograft.

Engineering of biologic skin substitutes has progressed over time from individual applications of skin cells, or biopolymer scaffolds, to combinations of cells and scaffolds for treatment, healing, and closure of acute and chronic skin wounds. Skin substitutes may be categorized into three groups: acellular scaffolds, temporary substitutes containing allogeneic skin cells, and permanent substitutes containing autologous skin cells. Combined use of acellular dermal substitutes with permanent skin substitutes containing autologous cells has been shown to provide definitive wound closure in burns involving greater than 90% of the total body surface area. These advances have contributed to reduced morbidity and mortality from both acute and chronic wounds but, to date, have failed to replace all of the structures and functions of the skin. Among the remaining deficiencies in cellular or biologic skin substitutes are hypopigmentation, absence of stable vascular and lymphatic networks, absence of hair follicles, sebaceous and sweat glands, and incomplete innervation. Correction of these deficiencies depends on regulation of biologic pathways of embryonic and fetal development to restore the full anatomy and physiology of uninjured skin. Elucidation and integration of developmental biology into future models of biologic skin substitutes promises to restore complete anatomy and physiology, and further reduce morbidity from skin wounds and scar. This article offers a review of recent advances in skin cell thrapies and discusses the future prospects in cutaneous regeneration.

OBJECTIVE: Comparison of cultured skin substitutes (CSS) and split-thickness skin autograft (AG) was performed to assess whether donor-site harvesting can be reduced quantitatively and whether functional and cosmetic outcome is similar qualitatively in the treatment of patients with massive cutaneous burns. SUMMARY BACKGROUND DATA: Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been shown to close full-thickness skin wounds in preclinical and clinical studies with acceptable functional and cosmetic results. METHODS: Qualitative outcome was compared between CSS and AG in 45 patients on an ordinal scale (0, worst; 10, best) with primary analyses at postoperative day 28 and after about 1 year for erythema, pigmentation, pliability, raised scar, epithelial blistering, and surface texture. In the latest 12 of the 45 patients, tracings were performed of donor skin biopsies and wounds treated with CSS at postoperative days 14 and 28 to calculate percentage engraftment, the ratio of closed wound:donor skin areas, and the percentage of total body surface area closed with CSS. RESULTS: Measures of qualitative outcome of CSS or AG were not different statistically at 1 year after grafting. Engraftment at postoperative day 14 exceeded 75% in the 12 patients evaluated. The ratio of closed wound:donor skin areas for CSS at postoperative day 28 was significantly greater than for conventional 4:1 meshed autografts. The percentage of total body surface area closed with CSS at postoperative day 28 was significantly less than with AG. CONCLUSIONS: The requirement for harvesting of donor skin for CSS was less than for conventional skin autografts. These results suggest that acute-phase recovery of patients with extensive burns is facilitated and that complications are reduced by the use of CSS together with conventional skin grafting.

One critical factor for the transmission of microorganisms from person to person or from the environment to a person (patient or health care worker) is the ability of the microbe to survive on an environmental surface. The purpose of this study was to determine the length of survival of various gram-negative bacteria on fabrics and plastics commonly used in hospitals. Seven materials were tested: smooth cotton (clothing), cotton terry (towels), 60% cotton-40% polyester blend (scrub suits and lab coats), polyester (drapes), 75% nylon-25% spandex (pressure garments), polyvinyl (splash aprons), and polyurethane (keyboard covers). The following bacteria were tested: Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Acinetobacter species, and Enterobacter species. Swatches of the materials were inoculated with defined amounts of bacteria and assayed at regular intervals. Survival was dependent on the bacterium, its inoculum size, and the material tested. At 102 microorganisms per swatch, bacteria survived from less than 1 hour to 8 days. At 10(4) to 10(5) bacteria per swatch, survival ranged from 2 hours to more than 60 days. These findings emphasize the need for careful disinfection and conscientious contact control procedures in areas that serve immunosuppressed individuals, such as patients with burn injuries.

Sepsis is associated with a pronounced catabolic response in skeletal muscle, mainly reflecting degradation of the myofibrillar proteins actin and myosin. Recent studies suggest that sepsis-induced muscle proteolysis may reflect ubiquitin-proteasome-dependent protein breakdown. An apparently conflicting observation is that the ubiquitin-proteasome pathway does not degrade intact myofibrils. Thus, it is possible that actin and myosin need to be released from the myofibrils before they can be ubiquitinated and degraded by the proteasome. We tested the hypothesis that sepsis results in disruption of Z-bands, increased expression of calpains, and calcium-dependent release of myofilaments in skeletal muscle. Sepsis induced in rats by cecal ligation and puncture resulted in increased gene expression of micro-calpain, m-calpain, and p94 and in Z-band disintegration in the extensor digitorum longus muscle. The release of myofilaments from myofibrillar proteins was increased in septic muscle. This response to sepsis was blocked by treating the rats with dantrolene, a substance that inhibits the release of calcium from intracellular stores to the cytoplasm. The present results provide evidence that sepsis is associated with Z-band disintegration and a calcium-dependent release of myofilaments in skeletal muscle. Release of myofilaments may be an initial and perhaps rate-limiting component of sepsis-induced muscle breakdown.

Glutamine is essential for the function of lymphocytes and macrophages, where it serves, among other things, as a source of energy. Little information is available concerning the fuel that polymorphonuclear cells use for their metabolic and bactericidal functions. It was the purpose of this study to determine whether glutamine would enhance the in vitro bactericidal function of normal neutrophils and whether the amino acid would restore the observed impaired function in burn patients to or above the normal level. Twelve burn patients with total body surface area burns ranging from 32% to 87% were studied. At various postburn times, neutrophils were isolated and their ability to kill Staphylococcus aureus in the presence and absence of glutamine was determined and compared with that in normal subjects. Glutamine enhanced the bactericidal function of normal neutrophils. In every patient, at all but two postburn times, glutamine caused an improvement in the observed abnormal neutrophil bactericidal function and often restored it to or slightly above the normal level. Glutamine had no effect on the expression of C3b receptors (CR1 or CD35) or on phagocytosis by the cells. This study confirms the beneficial effects of glutamine in at least one arm of the immune system and adds evidence for the possible advantage of including this amino acid in the diets of burn and other trauma patients.

In this study, we tested the contribution of flagellar motility, flagellin structure, and its glycosylation in Pseudomonas aeruginosa using genetically defined flagellar mutants. All mutants and their parent strains were tested in a burned-mouse model of infection. Motility and glycosylation of the flagellum appear to be important determinants of flagellar-mediated virulence in this model. This is the first report where genetically defined flagellar variants of P. aeruginosa were tested in the burned-mouse model of infection.

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Four burned children suffering complications from elevated intra-abdominal pressures prompted initiation of a prospective study to determine the value of intra-abdominal pressure measurements in 30 children with large burns. Intra-abdominal pressures were measured every 4 hours during burn shock or sepsis, or daily during periods of stability. Patients were arbitrarily divided into those having one or more measurements > or = 30 mm Hg or all values < 30 mm Hg. Patients in the > or = 30 mm Hg group had significantly larger burns, higher mortality, and increased instances of sepsis. Five patients had elevated intra-abdominal pressures during burn shock, with two requiring abdominal escharotomies. Seven were at > 30 mm Hg during sepsis, with three requiring paralysis, and one each requiring placement of a peritoneal catheter or laparotomy. Significant intra-abdominal pressure elevations may occur in patients with extensive burns and are associated with a poorer prognosis. Elevation of intra-abdominal pressure should be considered in severely burned patients with oliguria, hypoventilation, or hypotension.

Integrins are important signal transducers for virtually all neutrophil functions. Although a variety of signals ultimately result in integrin activation, the intracellular targets of integrin-initiated signals are poorly delineated to date. Polymorphonuclear (PMN) leukocyte responses to inflammation are dependent on both the stimulants and the extracellular environment encountered. Integrin ligation, by cell-cell or cell-matrix interactions, activates a variety of signaling cascades. These events dictate the nature of PMN responses to the encountered stimulus. The complex system of effector molecule recruitment and permissive signaling by integrins serves to strictly regulate PMN functions such as cell adhesion, motility, oxidant production, and protein synthesis. Moreover, there is evidence that cross-talk between integrins exists to prime integrin populations for subsequent functioning. This review summarizes the current understanding of signaling mechanisms for integrin priming and activation. In this connection, the role of specific signaling molecules in key PMN functions are examined.

Burn rehabilitation is an essential component of successful patient care. In May 2008, a group of burn rehabilitation clinicians met to discuss the status and future needs of burn rehabilitation. Fifteen topic areas pertinent to clinical burn rehabilitation were addressed. Consensus positions and suggested future research directions regarding the physical aspects of burn rehabilitation are shared.

Multiple surgical procedures necessitated by thermal trauma traditionally require withholding nutritional support during the perioperative period. Significant caloric deficits develop with subsequent catabolism of body tissues to provide energy and amino acids for the synthesis of protein. Eighty patients, matched for age and total body surface area burn, were enrolled in a study to evaluate the safety and efficacy of providing enteral support throughout operative procedures. All patients had duodenal feeding tubes placed under fluoroscopy and were provided with isonitrogenous nutritional support calculated to meet measured energy needs (indirect calorimetry). Forty patients received enteral support throughout 161 surgical procedures, and 40 had enteral support withheld during 129 procedures. Age, incidence of inhalation injury, percentage of total body surface area, and postburn day of admission were similar in both groups. Nutritional parameters, calorie counts, and infectious complications were recorded during the first 4 weeks after burn. No patient in either group experienced aspiration. The unfed group demonstrated a significant caloric deficit (p < 0.006) and increased incidence of wound infection (p < 0.02) and required more albumin supplementation to maintain serum levels at a minimum of 2.5 gm/dl (p < 0.04). Enteral nutrition can be provided safely during the perioperative period and provides the additional benefits of reducing caloric deficits, wound infections, and exogenous albumin supplementation.

Cultured skin substitutes (CSS) consisting of autologous fibroblasts and keratinocytes combined with biopolymers are an adjunctive treatment for large excised burns. CSS containing two cell types are limited by anatomical deficiencies, including lack of a vascular plexus, leading to slower vascularization after grafting than split-thickness autograft. To address this limitation, CSS were prepared containing human keratinocytes, fibroblasts, and dermal microvascular endothelial cells (HDMEC) isolated from a single skin sample. After 16 days in culture, control CSS and CSS containing HDMEC (CSS+EC) were grafted to full-thickness wounds in athymic mice. In CSS+EC in vitro, HDMEC persisted in the dermal substitutes and formed multicellular aggregates. One wk after grafting, HDMEC in CSS+EC organized into multicellular structures, some containing lumens. By 4 wk after grafting, HDMEC were found in linear and circular organizations resembling vascular analogs associated with basement membrane deposition. In some cases, colocalization of HDMEC with mouse perivascular cells was observed. The results demonstrate HDMEC transplantation in a clinically relevant cultured skin model, persistence of HDMEC after grafting, and HDMEC organization into vascular analogs in vitro and in vivo. All cells were derived from the same donor tissue, indicating the feasibility of preparing CSS containing autologous HDMEC for grafting to patients.