The effects of TGF-ß1 and IFN-α2b on decorin, decorin isoforms and type I collagen in hypertrophic scar dermal fibroblasts.

Hypertrophic scars (HTS) develop from an excessive synthesis of structural proteins like collagen and a decreased expression of proteoglycans such as decorin. Previous research has demonstrated that decorin expression is significantly down-regulated in HTS, deep dermal tissue, and thermally injured tissue, reducing its ability to regulate pro-fibrotic transforming growth factor-beta 1 (TGF-ß1) and normal fibrillogenesis. However, treatment of HTS fibroblasts with interferon-alpha 2b (IFN-α2b) has been shown to reduce excessive collagen synthesis and improve HTS by reducing serum TGF-ß1 levels. The expression of decorin isoforms in HTS is currently unknown and the effects of TGF-ß1 and IFN-α2b on decorin, decorin isoform expression and type 1 collagen are of great interest to our group. Dermal fibroblasts were treated with TGF-ß1 and/or IFN-α2b, for 48 h. The expression and secretion of decorin, decorin isoforms and type 1 collagen were quantified with reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assays. The mRNA expression of decorin and each isoform was significantly reduced in HTS fibroblasts relative to normal skin. TGF-ß1 decreased the mRNA expression of decorin and decorin isoforms, whereas IFN-α2b showed the opposite effect. IFN-α2b significantly inhibited TGF-ß1's effect on the mRNA expression of type I collagen alpha 1 in papillary dermal fibroblasts and overall showed relative effects of inhibiting TGF-ß1. These data support that a further investigation into the structural and functional roles of decorin isoforms in HTS pathogenesis is warranted and that IFN-α2b is an important agent in reducing fibrotic outcomes.

Burn Resuscitation Practices in North America: Results of the Acute Burn ResUscitation Multicenter Prospective Trial (ABRUPT).

OBJECTIVES: ABRUPT was a prospective, noninterventional, observational study of resuscitation practices at 21 burn centers. The primary goal was to examine burn resuscitation with albumin or crystalloids alone, to design a future prospective randomized trial. SUMMARY BACKGROUND DATA: No modern prospective study has determined whether to use colloids or crystalloids for acute burn resuscitation. METHODS: Patients ≥18 years with burns ≥ 20% total body surface area (TBSA) had hourly documentation of resuscitation parameters for 48 hours. Patients received either crystalloids alone or had albumin supplemented to crystalloid based on center protocols. RESULTS: Of 379 enrollees, two-thirds (253) were resuscitated with albumin and one-third (126) were resuscitated with crystalloid alone. Albumin patients received more total fluid than Crystalloid patients (5.2 ± 2.3 vs 3.7 ± 1.7 mL/kg/% TBSA burn/24 hours), but patients in the Albumin Group were older, had larger burns, higher admission Sequential Organ Failure Assessment (SOFA) scores, and more inhalation injury. Albumin lowered the in-to-out (I/O) ratio and was started ≤12 hours in patients with the highest initial fluid requirements, given >12 hours with intermediate requirements, and avoided in patients who responded to crystalloid alone. CONCLUSIONS: Albumin use is associated with older age, larger and deeper burns, and more severe organ dysfunction at presentation. Albumin supplementation is started when initial crystalloid rates are above expected targets and improves the I/O ratio. The fluid received in the first 24 hours was at or above the Parkland Formula estimate.

The dynamic changes of monocytes and cytokines during wound healing post-burn injury.

BACKGROUND: Burn injury is a sudden and traumatic injury that affects a large part of the population worldwide, who are placed at high risk of developing hypertrophic scars (HTS). HTS are a fibrotic scar resulting in painful contracted and raised scarring, affecting mobility in joints and work life, as well as cosmetically. The aim of this research was to enhance our understanding of the systematic response of monocytes and cytokines in wound healing after burn injury, in order to develop novel approaches to prevention and treatment of HTS. METHODS: Twenty-seven burn patients and thirteen healthy individuals were recruited in this study. Burn patients were stratified by burn total body surface area (TBSA). Peripheral blood samples were taken post-burn injury. Serum and peripheral blood mononuclear cells (PBMCs) were separated from the blood samples. This research investigated cytokines IL-6, IL-8, IL1RA, IL-10, and chemokine pathways SDF-1/CXCR4, MCP-1/CCR2, RANTES/CCR5 during the wound healing process in burn patients with varying severity of injuries by using enzyme-linked immunosorbent assays. PBMCs were stained for monocytes and the chemokine receptors by flow cytometry. Statistical analysis was done by one-way ANOVA with a Tukey correction, and regression analysis was performed using Pearson's Correlation analysis. RESULTS: The CD14+ CD16- monocyte subpopulation is larger in patients who developed HTS at 4-7 days. The CD14+ CD16+ monocyte subpopulation is smaller in the first week of injury, where it is similar after 8 days. Burn injury increased CXCR4, CCR2, and CCR5 expressions in CD14+ CD16+ monocytes. Increases in MCP-1 at 0-3 days after burn injury was positively correlated with burn severity. IL-6, IL-8, RANTES, and MCP-1 significantly increased with increasing burn severity. CONCLUSIONS: Monocytes and their chemokine receptors, as well as systemic levels of cytokines in wound healing of burn patients and scar development will require ongoing assessment to enhance our understanding of the abnormal wound healing after burn injury.

Stimulation of toll-like receptor pathways by burn eschar tissue as a possible mechanism for hypertrophic scarring.

Hypertrophic scars (HTS) are a common complication following burn injuries with prolonged inflammation. They do not respond well to current treatment options including mechanical, biomolecular and surgical therapies. Toll-like receptor (TLR) 2 and 4 respond to microbes and damaged endogenous ligands to trigger pro-inflammatory pathways, and they are expressed more in HTS fibroblasts compared to normal skin fibroblasts. TLR2 responds to microbial lipoteichoic acid (LTA) while TLR4 responds to microbial lipopolysaccharide (LPS) and endogenous ligands. We investigated the role of burn tissue and small leucine-rich proteoglycans (decorin and biglycan) in the stimulation of TLR2 and TLR4 pathways using cells stably transfected with TLR2 or TLR4 linked to a reporter system. Normal skin (n = 5) was collected post-abdominoplasty, and burn eschar samples (n = 18) were collected from 18 patients between 0 and 14 days post-burn. We found that burn tissue stimulates TLR2 activity significantly more than normal tissue and contains significantly higher levels of LTA. Burn tissue was a stronger stimulator of TLR4 than was normal skin. Burn tissue samples' stimulation of TLR4 and TLR2 correlated. The time post-burn (0-14 days) of wound tissue sampling correlated positively but moderately with TLR2 and TLR4 simulation. In comparison to the dose-dependent effects of natural decorin or biglycan on TLR4 activation, their denatured forms exhibited stronger or weaker stimulation, respectively. They were not potent stimulators of TLR2. TLR2 and TLR4 stimulation is not limited to bacteria in wounds and likely involves multiple endogenous damage-associated molecular patterns. Insight into mechanisms of HTS will facilitate the development of future targeted therapies to modify wound progression and provide benefits to patients suffering with HTS and other fibroproliferative disorders.

Survival of human cadaver skin on severe combined immune deficiency pigs: Proof of concept.

Transplantation of human xenografts onto immunocompromised mice is a powerful research tool for studying wound healing. However, differences in healing between humans and mice and their small size limit this model. We determined whether human cadaver skin xenografts transplanted onto pigs with severe combined immune deficiency (SCID) would survive and not be rejected. Meshed (1:1.5), cryopreserved human cadaver skin was transplanted onto 10 partial thickness dermatome wounds in each of two normal domestic pigs and two SCID pigs. Autografts (n = 2/animal) from the four animals were used as controls. In normal pigs, all autografts were engrafted and healed with a minimal, if any, inflammation and scarring. All human xenografts were rejected by the normal pigs within 5-11 days and associated with an intense T-cell inflammatory response. In contrast, both autografts and xenografts were engrafted and survived the 28-day study in the SCID pigs with a minimal inflammation and no gross scarring.

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property.

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-ß exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-ß- MSCs. Notably, PDGFR-ß+ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 106 GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-ß+ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 105 remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-ß- MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 105) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-ß+ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-ß+ MSCs migrated eightfold more than PDGFR-ß- MSCs toward PDGF-BB. Intriguingly, PDGFR-ß+ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-ß- MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-ß identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.

The Effects of Storage Age of Blood in Massively Transfused Burn Patients: A Secondary Analysis of the Randomized Transfusion Requirement in Burn Care Evaluation Study.

OBJECTIVES: Major trials examining storage age of blood transfused to critically ill patients administered relatively few blood transfusions. We sought to determine if the storage age of blood affects outcomes when very large amounts of blood are transfused. DESIGN: A secondary analysis of the multicenter randomized Transfusion Requirement in Burn Care Evaluation study which compared restrictive and liberal transfusion strategies. SETTING: Eighteen tertiary-care burn centers. PATIENTS: Transfusion Requirement in Burn Care Evaluation evaluated 345 adults with burns greater than or equal to 20% of the body surface area. We included only the 303 patients that received blood transfusions. INTERVENTIONS: The storage ages of all transfused red cell units were collected during Transfusion Requirement in Burn Care Evaluation. A priori measures of storage age were the the mean storage age of all transfused blood and the proportion of all transfused blood considered very old (stored ≥ 35 d). MEASUREMENTS AND MAIN RESULTS: The primary outcome was the severity of multiple organ dysfunction. Secondary outcomes included time to wound healing, the duration of mechanical ventilation, and in-hospital mortality. There were 6,786 red cell transfusions with a mean (± SD) storage age of 25.6 ± 10.2 days. Participants received a mean of 23.4 ± 31.2 blood transfusions (range, 1-219) and a mean of 5.3 ± 10.7 units of very old blood. Neither mean storage age nor proportion of very old blood had any influence on multiple organ dysfunction severity, time to wound healing, or mortality. Duration of ventilation was significantly predicted by both mean blood storage age and the proportion of very old blood, but this was of questionable clinical relevance given extreme variability in duration of ventilation (adjusted r ≤ 0.01). CONCLUSIONS: Despite massive blood transfusion, including very old blood, the duration of red cell storage did not influence outcome in burn patients. Provision of the oldest blood first by Blood Banks is rational, even for massive transfusion.

Transfusion Requirement in Burn Care Evaluation (TRIBE): A Multicenter Randomized Prospective Trial of Blood Transfusion in Major Burn Injury.

OBJECTIVE: Our objective was to compare outcomes of a restrictive to a liberal red cell transfusion strategy in 20% or more total body surface area (TBSA) burn patients. We hypothesized that the restrictive group would have less blood stream infection (BSI), organ dysfunction, and mortality. BACKGROUND: Patients with major burns have major (>1 blood volume) transfusion requirements. Studies suggest that a restrictive blood transfusion strategy is equivalent to a liberal strategy. However, major burn injury is precluded from these studies. The optimal transfusion strategy in major burn injury is thus needed but remains unknown. METHODS: This prospective randomized multicenter trial block randomized patients to a restrictive (hemoglobin 7-8 g/dL) or liberal (hemoglobin 10-11 g/dL) transfusion strategy throughout hospitalization. Data collected included demographics, infections, transfusions, and outcomes. RESULTS: Eighteen burn centers enrolled 345 patients with 20% or more TBSA burn similar in age, TBSA burn, and inhalation injury. A total of 7054 units blood were transfused. The restrictive group received fewer blood transfusions: mean 20.3 ±â€Š32.7 units, median = 8 (interquartile range: 3, 24) versus mean 31.8 ±â€Š44.3 units, median = 16 (interquartile range: 7, 40) in the liberal group (P < 0.0001, Wilcoxon rank sum). BSI incidence, organ dysfunction, ventilator days, and time to wound healing (P > 0.05) were similar. In addition, there was no 30-day mortality difference: 9.5% restrictive versus 8.5% liberal (P = 0.892, χ test). CONCLUSIONS: A restrictive transfusion strategy halved blood product utilization. Although the restrictive strategy did not decrease BSI, mortality, or organ dysfunction in major burn injury, these outcomes were no worse than the liberal strategy (Clinicaltrials.gov identifier NCT01079247).

Alternatively activated macrophages derived from THP-1 cells promote the fibrogenic activities of human dermal fibroblasts.

Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase-1 (MMP-1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP-1 cell line were used to co-culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP-1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti-fibrotic and pro-fibrotic factors, MMP-1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co-cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha-smooth muscle actin expressed cells, expression of the pro-fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti-fibrotic genes and MMP-1 activity. These findings reinforce the pro-fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation.

Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2-like macrophages are pro-fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3-4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty-six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro-fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.

Self-assembling peptide hydrogel scaffolds support stem cell-based hair follicle regeneration.

Recent studies show that designer peptide nanofibers can mimic properties of extracellular matrix molecules, promising great potential as scaffold materials for tissue engineering. However, their ability in supporting organogenesis has not been studied. Here we examined the effect of self-assembling peptide hydrogels in supporting skin derived precursors (SKPs) in hair follicle neogenesis. We found that hydrogels formed by RADA16, PRG which contains RGD, and particularly the combination of RADA16 and PRG (RADA-PRG) enhanced SKP proliferation. Notably, the RADA-PRG hydrogel, which exhibited advantages of RADA16 in adequate nanofiber formation and PRG in providing the integrin binding sequence, exhibited superior effects in enhancing SKP survival, expression of hair induction signature genes such as Akp2 and Bmp6, and more importantly de novo hair genesis in mice. Thus our results suggest that RADA-PRG may serve as a novel scaffold material for stem cell transplantation and tissue engineering.

Urodynamic improvements following oral medical therapy for partial bladder outlet obstruction in an animal model.

PURPOSE: Bladder deterioration after partial outlet obstruction (pBOO) occurs commonly and has significant clinical implications. Our previous animal model results described the progression of pBOO to hypertrophy and fibrosis. We wished to determine if the pathologic process of pBOO can be altered with rationally chosen oral medications. MATERIALS AND METHODS: Female Sprague-Dawley rats underwent controlled surgically induced pBOO. Rats were maintained for a period of 16 weeks at which point urodynamics were performed, and organs harvested. Rats were divided into four groups, each receiving different daily treatment: control (saline), oxybutynin (3 mg/kg), rapamycin (2 mg/kg), and tadalafil (2 mg/kg). Outcomes were assessed after 4,8,12, or 16 weeks. Measures included animal health, urodynamics, histology, mass spectrometry for collagen content, and rtPCR for inflammatory mediators. RESULTS: Rapamycin treated animals exhibited significant mortality at later time points. Oxybutinin and tadalafil treated bladders demonstrated significant improvements in bladder capacity and compliance, with less detrusor hypertrophy than controls. Tadalafil also resulted in a significant down-regulation of HIF-1α, while decorin, biglycan, and TGF-ß were upregulated in treated animals. Tadalafil treated bladders measured lower collagen content towards the end of the study, indicating an antifibrotic effect. CONCLUSIONS: Our study has effectively demonstrated that deleterious changes secondary to pBOO can be altered pharmacologically. Oxybutinin and tadalafil seem to have a time-dependent protective effect on the detrusor muscle, although with different mechanisms of action. Tadalafil treatment in this setting appears to have an antifibrotic effect. This work has the potential to seed important clinical studies and improve clinical practice.

The therapeutic potential of a C-X-C chemokine receptor type 4 (CXCR-4) antagonist on hypertrophic scarring in vivo.

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell-derived factor 1/CXCR4 signaling is up-regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood-borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell-derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE-9908 on dermal fibrosis was determined in vivo using a human HTS-like nude mouse model, in which split-thickness human skin is transplanted into full-thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE-9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down-regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.

Correction: Paracrine Factors of Mesenchymal Stem Cells Recruit Macrophages and Endothelial Lineage Cells and Enhance Wound Healing.

[This corrects the article DOI: 10.1371/journal.pone.0001886.].

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow-derived mesenchymal stem cells.

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow-derived mesenchymal stem cells (BM-MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM-MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell-derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co-culturing layered fibroblasts with BM-MSCs in a transwell insert system, BM-MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM-MSCs in the pathogenesis of hypertrophic scarring.

A nude mouse model of hypertrophic scar shows morphologic and histologic characteristics of human hypertrophic scar.

Hypertrophic scar (HSc) is a fibroproliferative disorder that occurs following deep dermal injury. Lack of a relevant animal model is one barrier toward better understanding its pathophysiology. Our objective is to demonstrate that grafting split-thickness human skin onto nude mice results in survival of engrafted human skin and murine scars that are morphologically, histologically, and immunohistochemically consistent with human HSc. Twenty nude mice were xenografted with split-thickness human skin. Animals were euthanized at 30, 60, 120, and 180 days postoperatively. Eighteen controls were autografted with full-thickness nude mouse skin and euthanized at 30 and 60 days postoperatively. Scar biopsies were harvested at each time point. Blinded scar assessment was performed using a modified Manchester Scar Scale. Histologic analysis included hematoxylin and eosin, Masson's trichrome, toluidine blue, and picrosirius red staining. Immunohistochemistry included anti-human human leukocyte antigen-ABC, α-smooth muscle actin, decorin, and biglycan staining. Xenografted mice developed red, shiny, elevated scars similar to human HSc and supported by blinded scar assessment. Autograft controls appeared morphologically and histologically similar to normal skin. Xenografts survived up to 180 days and showed increased thickness, loss of hair follicles, adnexal structures and rete pegs, hypercellularity, whorled collagen fibers parallel to the surface, myofibroblasts, decreased decorin and increased biglycan expression, and increased mast cell density. Grafting split-thickness human skin onto nude mice results in persistent scars that show morphologic, histologic, and immunohistochemical consistency with human HSc. Therefore, this model provides a promising technique to study HSc formation and to test novel treatment options.

Reduced Intraoperative Blood Loss and Hypothermia in Burn Surgery using Cardiopulmonary Bypass Pumps.

Objective: Patients presenting with total body surface area (TBSA) >40% burns require significant surgical treatment. Two substantial challenges during these surgeries are limiting blood loss and maintaining core temperatures. To overcome these challenges, several techniques have been developed, ranging from the Pitkin syringe method to the pneumatic tourniquet strategy for large-volume hyperthermic insufflation. Here, we compare the pneumatic tourniquet method to a roller pump method for maintenance of intraoperative normothermia and control of bleeding. Methods: We conducted a retrospective chart review of 20 patients presenting with TBSA >40% burns, 10 of whom were treated with the rapid infusion roller pump and 10 of whom were treated with the pneumatic tourniquet technique. Patients from each group were controlled for % TBSA, presence of inhalation injury, age, and date of admission. We reviewed transfusion requirement and the intraoperative temperatures, as well as the average intraoperative drop in temperature. Results: We observed improvement in the infusion volume, operative time, intraoperative temperature drop, minimum intraoperative temperature, estimated blood loss, and amount of required transfusion. Conclusions: Our study suggests that the rapid infusion roller pump technique is capable of achieving superior intraoperative bleeding control and temperature maintenance compared to the pneumatic tourniquet technique, resulting in decreased transfusion requirement.

Objectif: Les patients ayant des brûlures sur plus de 40% de la surface corporelle totale (SCT) nécessitent un traitement chirurgical significatif. Ces interventions chirurgicales sont confrontées à deux défis majeurs: la limitation des pertes sanguines et le maintien de la température corporelle. Pour vaincre ces défis, plusieurs techniques ont été développées, allant de la méthode de la seringue de Pitkin à la stratégie du garrot pneumatique pour insufflation hyperthermique de grand volume. Nous comparons ici la méthode du garrot pneumatique à la méthode de la pompe à galets pour le maintien de la normothermie peropératoire et le contrôle des saignements. Méthodes: Nous avons mené une étude rétrospective des dossiers de 20 patients présentant des brûlures sur > 40 % de la SCT; 10 d'entre eux ont été traités avec la pompe à galets pour perfusion rapide et 10 ont été traités avec la technique du garrot pneumatique. Les patients de chaque groupe ont été contrôlés pour le pourcentage de SCT, la présence de lésions d'inhalation, l'âge et la date de leur hospitalisation. Nous avons revu les besoins en transfusion ainsi que les températures peropératoires et la chute moyenne peropératoire de la température. Résultats: Nous avons observé une amélioration du volume de perfusion, de la durée d'intervention, de la chute peropératoire de la température, de la température peropératoire minimum, de la perte sanguine estimée et de la quantité requise de transfusion. Conclusions: Notre étude suggère que la technique de perfusion rapide avec une pompe à galets permet d'obtenir un meilleur contrôle peropératoire des saignements que la technique avec garrot pneumatique, aboutissant à une diminution des besoins de transfusions.

Serial Casting as an Effective Method for Burn Scar Contracture Rehabilitation: A Case Series.

Guidelines and protocols for orthoses in burn scar contracture rehabilitation are limited. The current study aims to determine the optimal frequency of casting, potentially facilitating the development of a serial casting protocol. Previous literature supporting casting has low generalizability due to methodology limitations. Seven patients with burn scar contracted joints, who did not respond to traditional therapy, were recruited in this study. Patients were serially casted once, three times, or five times a week. Joint range of motion was maximized with stretching and exercise techniques before every new cast application. Across all patients, active range of motion increased from 65.8 ± 27.8° to 108.1 ± 23.3° with casting; or from 57.8 ± 16.2% to 96.7 ± 2.9% of normal. Similarly, scars improved from 9.5 ± 1.5 to 4.9 ± 1.4 on the Modified Vancouver Scar Scale score. This therapeutic effect was achieved within an average of 8.5 ± 3.7 d and 4.0 ± 2.2 new cast applications. Given the study findings, the procedures outlined could be used to develop a standardized serial casting protocol for burn scar contracture rehabilitation.

Injury-induced interleukin-1 alpha promotes Lgr5 hair follicle stem cells de novo regeneration and proliferation via regulating regenerative microenvironment in mice.

BACKGROUND: The hair follicles (HFs) are barely regenerated after loss in injuries in mammals as well as in human beings. Recent studies have shown that the regenerative ability of HFs is age-related; however, the relationship between this phenomenon and the stem cell niche remains unclear. This study aimed to find a key secretory protein that promotes the HFs regeneration in the regenerative microenvironment. METHODS: To explore why age affects HFs de novo regeneration, we established an age-dependent HFs regeneration model in leucine-rich repeat G protein-coupled receptor 5 (Lgr5) + /mTmG mice. Proteins in tissue fluids were analyzed by high-throughput sequencing. The role and mechanism of candidate proteins in HFs de novo regeneration and hair follicle stem cells (HFSCs) activation were investigated through in vivo experiments. The effects of candidate proteins on skin cell populations were investigated by cellular experiments. RESULTS: Mice under 3-week-old (3W) could regenerate HFs and Lgr5 HFSCs, which were highly correlated with the immune cells, cytokines, IL-17 signaling pathway, and IL-1α level in the regeneration microenvironment. Additionally, IL-1α injection induced de novo regeneration of HFs and Lgr5 HFSCs in 3W mouse model with a 5 mm wound, as well as promoted activation and proliferation of Lgr5 HFSCs in 7-week-old (7W) mice without wound. Dexamethasone and TEMPOL inhibited the effects of IL-1α. Moreover, IL-1α increased skin thickness and promoted the proliferation of human epidermal keratinocyte line (HaCaT) and skin-derived precursors (SKPs) in vivo and in vitro, respectively. CONCLUSIONS: In conclusion, injury-induced IL-1α promotes HFs regeneration by modulating inflammatory cells and oxidative stress-induced Lgr5 HFSCs regeneration as well as promoting skin cell populations proliferation. This study uncovers the underlying molecular mechanisms enabling HFs de novo regeneration in an age-dependent model.