Altering CO2 Laser Pulse Energy Settings Did Not Influence Differential Gene Transcription in Normal Skin in a Red Duroc Pig Model.

OBJECTIVES: Fractional ablative CO2 lasers are used clinically to treat cutaneous burn scars with reported varying degrees of effectiveness. It was hypothesized that different laser pulse energy settings may lead to differential gene transcription in a porcine model. METHODS: Uninjured skin from red Duroc pigs was treated with a fractional ablative CO2 laser set to 70, 100, or 120 mJ across the abdomen (n = 4 areas per treatment). Punch biopsies of both treated and untreated sites were taken before treatment (baseline), at 30 min, and at each hour for 6 h and stored in All-Protect tissue reagent. The biopsies were then used to isolate RNA, which was subsequently used in qRT-PCR for eight genes associated with wound healing and the extracellular matrix: CCL2, IL6, FGF2, TIMP1, TIMP3, COL1A2, MMP2, and DCN. RPL13a was used as a housekeeping gene to normalize the eight genes of interest. One-way ANOVA tests were used to assess for differences among laser pulse energies and two-way ANOVA tests were used to assess the differences between treated and untreated areas. RESULTS: While six of the eight genes were upregulated after treatment (p < 0.05), there were no significant differences in gene expression between the different laser pulse energies for any of the eight genes. CONCLUSION: While laser treatment is correlated with a positive and significant upregulation for six of the eight genes 4 h after intervention, the pulse energy settings of the laser did not lead to a statistically significant difference in gene transcription among the treatment areas. Different laser pulse energies may not be required to induce similar cellular responses in a clinical setting.

Addressing Burn Hypertrophic Scar Symptoms Earlier: Laser Scar Revision May Begin as Early as 3-6 Months After Injury.

OBJECTIVES: Fractional ablative CO2 laser (FLSR) is used to treat hypertrophic scars (HTSs) resulting from burn injuries, which are characterized by factors, such as erythema, contracture, thickness, and symptoms of pain and itch. Traditionally, waiting a year after injury for scar maturation before starting laser treatment has been recommended; however, the potential benefits of earlier intervention have gained popularity. Still, the optimal timing for beginning laser intervention in patients with HTSs remains uncertain. This study aims to evaluate the ideal timing for the initiation of FLSR for HTSs using several qualitative and quantitative assessment measures. It was hypothesized that early intervention would lead to similar improvement trends as later intervention, however, would be more ideal due to the shortened time without symptom relief for patients. METHODS: Patients who received three or more laser treatment sessions and completed both pre- and posttreatment evaluations were included in this analysis (n = 69). FLSR treatment was administered at 4-8-week intervals. Patients starting treatment before 6 months after injury were classified as the early-stage intervention group and those beginning treatment at 6-12 months after injury were classified as the late-stage intervention group. Demographic data, including the age of patients at the time of first treatment, age of scars at the time of first treatment, biological sex, ethnicity, Fitzpatrick skin type, and use of laser-assisted drug delivery, were collected by retrospective chart review. Patients were evaluated on six subjective scales and objectively for scar stiffness with durometry. For all scales, higher scores indicate worse scars. A two-way ANOVA, Student's t-test, and Mann-Whitney U-test were used to compare scores from the pre- to posttreatment evaluations. RESULTS: There were no significant differences between the groups for any of the demographic or scar-specific variables; thus, differences in outcome can be attributed to the timing of intervention. Both groups demonstrated an improvement in scars with treatment over time (p < 0.05). Both early- and middle-stage initiation showed scar symptom improvement in five out of six scales. In the late-stage intervention, the Patient and Observer Scar Assessment Scale-Patient average score did not show improvement. In the early-stage intervention, the Vancouver Scar Scale total did not show improvement. Quantitative evaluation of scar stiffness by durometry did not show symptom improvement in either group. The Scar Comparison Scale demonstrated the greatest improvement across groups. CONCLUSION: Laser treatment led to scar improvement in at least one scale at each stage of initiation. Both intervention timelines resulted in equivalent outcomes, and early intervention should be considered when initiating FLSR treatment in burn scars to alleviate symptoms earlier.

Treatment of hypopigmented burn hypertrophic scars with short-term topical tacrolimus does not lead to repigmentation.

OBJECTIVES: Dyschromia is an understudied aspect of hypertrophic scar (HTS). The use of topical tacrolimus has successfully shown repigmentation in vitiligo patients through promotion of melanogenesis and melanocyte proliferation. It was hypothesized that HTSs treated with topical tacrolimus would have increased repigmentation compared to controls. METHODOLOGY: Full-thickness burns in red Duroc pigs were either treated with excision and meshed split-thickness skin grafting or excision and no grafting, and these wounds formed hypopigmented HTSs (n = 8). Half of the scars had 0.1% tacrolimus ointment applied to the scar twice a day for 21 days, while controls had no treatment. Further, each scar was bisected with half incurring fractional ablative CO2 laser treatment before topical tacrolimus application to induce laser-assisted drug delivery (LADD). Pigmentation was evaluated using a noninvasive probe to measure melanin index (MI) at Days 0 (pretreatment), 7, 14, and 21. At each timepoint, punch biopsies were obtained and fixed in formalin or were incubated in dispase. The formalin-fixed biopsies were used to evaluate melanin levels by H&E staining. The biopsies incubated in dispase were used to obtain epidermal sheets. The ESs were then flash frozen and RNA was isolated from them and used in quantitative reverse transcription polymerase chain reaction for melanogenesis-related genes: Tyrosinase (TYR), TYR-related protein-1 (TYRP1), and dopachrome tautomerase (DCT). Analysis of variance test with Sídák's multiple comparisons test was used to compare groups. RESULTS: Over time, within the grafted HTS and the NS group, there were no significant changes in MI, except for Week 3 in the -Tacro group. (+Tacro HTS= pre = 685.1 ± 42.0, w1 = 741.0 ± 54.16, w2 = 750.8 ± 59.0, w3 = 760.9 ± 49.8) (-Tacro HTS= pre = 700.4 ± 54.3, w1 = 722.3 ± 50.7, w2 = 739.6 ± 53.2, w3 = 722.7 ± 50.5). Over time, within the ungrafted HTS and the NS group, there were no significant changes in MI. (+Tacro HTS= pre = 644.9 ± 6.9, w1 = 661.6 ± 3.3, w2 = 650.3 ± 6.2, w3 = 636.3 ± 7.4) (-Tacro HTS= pre = 696.8 ± 8.0, w1 = 695.8 ± 12.3, w2 = 678.9 ± 14.0, w3 = 731.2 ± 50.3). LADD did not lead to any differential change in pigmentation compared to the non-LADD group. There was no evidence of increased melanogenesis within the tissue punch biopsies at any timepoint. There were no changes in TYR, TYRP1, or DCT gene expression after treatment. CONCLUSION: Hypopigmented HTSs treated with 0.1% tacrolimus ointment with or without LADD did not show significantly increased repigmentation. This study was limited by a shorter treatment interval than what is known to be required in vitiligo patients for repigmentation. The use of noninvasive, topical treatments to promote repigmentation are an appealing strategy to relieve morbidity associated with dyschromic burn scars and requires further investigation.

Examining Obesity and Its Association With Burn Injury: A Secondary Analysis of the Transfusion Requirement in Burn Care Evaluation Study.

INTRODUCTION: Literature examining the connection between obesity and burn injuries is limited. This study is a secondary analysis of a multicenter trial data set to investigate the association between burn outcomes and obesity following severe burn injury. MATERIALS AND METHODS: Body mass index (BMI) was used to stratify patients as normal weight (NW; BMI 18.5-25), all obese (AO; any BMI>30), obese I (OI; BMI 30-34.9), obese II (OII; BMI 35-39.9), or obese III (OIII; BMI>40). The primary outcome examined was mortality. Secondary outcomes included hospital length of stay (LOS), number of transfusions, injury scores, infection occurrences, number of operations, ventilator days, intensive care unit LOS, and days to wound healing. RESULTS: Of 335 patients included for study, 130 were obese. Median total body surface area (TBSA) was 31%, 77 patients (23%) had inhalation injury and 41 patients died. Inhalation injury was higher in OIII than NW (42.1% versus 20%, P = 0.03). Blood stream infections (BSI) were higher in OI versus NW (0.72 versus 0.33, P = 0.03). Total operations, ventilator days, days to wound healing, multiorgan dysfunction score, Acute Physiology and Chronic Health Evaluationscore, hospital LOS, and intensive care unit LOS were not significantly affected by BMI classification. Mortality was not significantly different between obesity groups. Kaplan-Meier survival curves did not significantly differ between the groups (χ2 = 0.025, P = 0.87). Multiple logistic regression identified age, TBSA, and full thickness burn as significant independent predictors (P < 0.05) of mortality; however, BMI classification itself was not predictive of mortality. CONCLUSIONS: No significant association between obesity and mortality was seen after burn injury. Age, TBSA, and percent full- thickness burn were independent predictors of mortality after burn injury, while BMI classification was not.

Treatment of burn hypertrophic scar with fractional ablative laser-assisted drug delivery can decrease levels of hyperpigmentation.

BACKGROUND: Laser treatments have been used to treat a variety of scar symptoms, including the appearance of scars following burn injury. One such symptom is hyperpigmentation. There are several qualitative and quantitative measures of assessing improvement in hyperpigmentation over time. The Patient and Observer Scar Assessment Scale (POSAS) and Vancouver Scar Scale (VSS) are two scales that describe characteristics of scar such as pigmentation level. These scales are limited by their qualitative nature. On the other hand, spectrophotometers provide quantitative measures of pigmentation. Prior studies have reported that laser can change scar pigmentation, but no quantitative values have been reported. The current study examines changes in scar melanin index after CO2 fractional ablative laser scar revision (FLSR) via noninvasive probe measurement in patients of various Fitzpatrick skin types (FST). MATERIALS AND METHODS: Patients with scars of various sizes and etiologies were treated with FLSR. A database was constructed including 189 patients undergoing laser treatment. From this pool, individuals were selected based on the criteria that they completed at least two laser sessions and had Melanin index measurements for both of these sessions and the pre-operative visit. This criteria resulted in 63 patients of various FST in the cohort. Melanin index, POSAS-Observer (O) and -Patient (P) pigmentation and color scores and VSS-pigmentation scores were examined over time. Demographic information (age of patient at time of first treatment, age of scar at time of first treatment, use of laser-assisted drug delivery (LADD), gender, FST, and Ethnicity) were collected from the medical record. Patients were grouped as "responder" if their Melanin index indicated decreased levels of hyperpigmentation after FLSR treatment in more than half of their total number of visits and "nonresponder" if it did not. RESULTS: The majority of patients were responders (41/63). In responder patients, measurements of Melanin index showed significantly improved levels of hyperpigmentation in hypertrophic scars after two FLSR sessions (p < 0.05). Age of patient, gender, FST, age of scar, ethnicity, or type of drug delivered by LADD did not predict responder grouping. POSAS-O and -P pigmentation/color scores showed improved scores after two FLSR sessions within the responder group. POSAS-P color scores showed improved scores after two and three FLSR sessions in the nonresponder group. VSS pigmentation scores showed improved scores after three FLSR sessions in the responder group only. CONCLUSION: Based on Melanin index values, FLSR leads to improvements in hyperpigmentation in certain patients.

Laser-assisted drug delivery of synthetic alpha melanocyte stimulating hormone and L-tyrosine leads to increased pigmentation area and expression of melanogenesis genes in a porcine hypertrophic scar model.

OBJECTIVES: One symptom of hypertrophic scar (HTS) that can develop after burn injury is dyschromia with hyper- and hypopigmentation. There are limited treatments for these conditions. Previously, we showed there is no expression of alpha melanocyte stimulating hormone (α-MSH) in hypopigmented scars, and if these melanocytes are treated with synthetic α-MSH in vitro, they respond by repigmenting. The current study tested the same hypothesis in the in vivo environment using laser-assisted drug delivery (LADD). METHODS: HTSs were created in red Duroc pigs. At Day 77 (pre), they were treated with CO2 fractional ablative laser (FLSR). Synthetic α-MSH was delivered as a topical solution dissolved in  l-tyrosine (n = 6, treated). Control scars received LADD of  l-tyrosine only (n = 2, control). Scars were treated and examined weekly through Week 4. Digital images and punch biopsies of hyper, hypo-, and normally pigmented scar and skin were collected. Digital pictures were analyzed with ImageJ by tracing the area of hyperpigmentation. Epidermal sheets were obtained from punch biopsies through dispase separation and RNA was isolated. qRT-PCR was run for melanogenesis-related genes: tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), and dopachrome tautomerase (DCT). Two-way ANOVA with multiple comparisons and Dunnett's correction compared the groups. RESULTS: The areas of hyperpigmentation were variable before treatment. Therefore, data is represented as fold-change where each scar was normalized to its own pre value. Within the LADD of NDP α-MSH + l-tyrosine group, hyperpigmented areas gradually increased each week, reaching 1.3-fold over pre by Week 4. At each timepoint, area of hyperpigmentation was greater in the treated versus the control (1.04 ± 0.05 vs. 0.89 ± 0.08, 1.21 ± 0.07 vs. 0.98 ± 0.24, 1.21 ± 0.08 vs. 1.04 ± 0.11, 1.28 ± 0.11 vs. 0.94 ± 0.25; fold-change from pre-). Within the treatment group, pretreatment, levels of TYR were decreased -17.76 ± 4.52 below the level of normal skin in hypopigmented scars. After 1 treatment, potentially due to laser fractionation, the levels decreased to -43.49 ± 5.52. After 2, 3, and 4 treatments, there was ever increasing levels of TYR to almost the level of normally pigmented skin (-35.74 ± 15.72, -23.25 ± 6.80, -5.52 ± 2.22 [p < 0.01, Week 4]). This pattern was also observed for TYRP1 (pre = -12.94 ± 1.82, Week 1 = -48.85 ± 13.25 [p < 0.01], Weeks 2, 3, and 4 = -34.45 ± 14.64, -28.19 ± 4.98, -6.93 ± 3.05 [p < 0.01, Week 4]) and DCT (pre = -214.95 ± 89.42, Week 1 = -487.93 ± 126.32 [p < 0.05], Weeks 2, 3, and 4 = -219.06 ± 79.33, -72.91 ± 20.45 [p < 0.001], -76.00 ± 24.26 [p < 0.001]). Similar patterns were observed for scars treated with LADD of  l-tyrosine alone without NDP α-MSH. For each gene, in hyperpigmented scar, levels increased at Week 4 of treatment compared to Week 1 (p < 0.01). CONCLUSIONS: A clinically-relevant FLSR treatment method can be combined with topical delivery of synthetic α-MSH and l-tyrosine to increase the area of pigmentation and expression of melanogenesis genes in hypopigmented HTS. LADD of  l-tyrosine alone leads to increased expression of melanogenesis genes. Future studies will aim to optimize drug delivery, timing, and dosing.

Assessing Factor V Antigen and Degradation Products in Burn and Trauma Patients.

INTRODUCTION: Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. METHODS: Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann-Whitney U test, and logistic regression. RESULTS: Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = -0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). CONCLUSIONS: Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical.

Modeling Burn Progression Using Comb Burns: The Impact of Thermal Contact Duration on Model Outcomes.

BACKGROUND: Burn progression is a phenomenon that remains poorly characterized. The mechanisms of burn conversion are not completely understood, and consequently, both predictive diagnostic tools and interventions are limited. The rat comb burn model is a commonly used approach to study horizontal burn conversion. However, there is significant variability in how the model is performed. Skin contact duration, comb device heating method, comb heating duration, amount of pressure applied, the weight of the comb, and associated depth of burn are all variables that are heterogeneous in studies utilizing the model. MATERIALS AND METHODS: Here, contact duration was examined to determine the impact the duration of burn delivery has on the conversion of interspaces in this model. Data from multiple experiments consisting of 10, 15, 20, 30, 40, and 45 s comb burns were compiled and assessed. Burns were made using combs heated in a 100°C dry bath and then monitored for 2 d. Interspace viability was assessed by digital and laser doppler imaging and biopsy procurement. RESULTS: Laser Doppler Imaging and viable interspace measurements showed that as burn duration increased, the percentage of the viable interspace and interspace perfusion decreased. Additionally, a contact time of 30 s or greater was required to result in 100% interspace conversion. CONCLUSIONS: These results demonstrate a need to better characterize and potentially standardize the rat comb burn model to reduce variation and maintain it as a valuable tool for controlled studies of the pathophysiology of burn wound progression.

A Pilot Study of Negative Pressure Therapy with Autologous Skin Cell Suspensions in a Porcine Model.

BACKGROUND: Negative pressure wound therapy (NPWT) is an option for securing meshed split thickness skin grafts (mSTSGs) after burn excision to optimize skin graft adherence. Recently, the use of autologous skin cell suspension (ASCS) has been approved for use in the treatment of burn injuries in conjunction with mSTSGs.To date, limited data exists regarding the impact of NPWT on healing outcomes when the cellular suspension is utilized. It was hypothesized that NPWT would not negatively impact wound healing of ASCS+mSTSG. MATERIALS AND METHODS: A burn, excision, mSTSG, ASCS ± NPWT model was used. Two Duroc pigs were utilized in this experiment, each with 2 sets of paired burns. Four wounds received mSTSG+ASCS+NPWT through post-operative day 3, and 4 wounds received mSTSG+ACSC+ traditional ASCS dressings. Cellular viability was characterized prior to spraying. Percent re-epithelialization, graft-adherence, pigmentation, elasticity, and blood perfusion and blood vessel density were assessed at multiple time points through 2 weeks. RESULTS: All wounds healed within 14 days with minimal scar pathology and no significant differences in percent re-epithelialization between NPWT, and non-NPWT wounds were observed. Additionally, no differences were detected for pigmentation, perfusion, or blood vessel density. NPWT treated wounds had less graft loss and improved elasticity, with elasticity being statistically different. CONCLUSIONS: These data suggest the positive attributes of the cellular suspension delivered are retained following the application of negative pressure. Re-epithelialization, revascularization, and repigmentation are not adversely impacted. The use of NPWT may be considered as an option when using ASCS with mSTSGs for the treatment of full-thickness burns.

Galectin-1 production is elevated in hypertrophic scar.

Upon healing, burn wounds often leave hypertrophic scars (HTSs) marked by excess collagen deposition, dermal and epidermal thickening, hypervascularity, and an increased density of fibroblasts. The Galectins, a family of lectins with a conserved carbohydrate recognition domain, function intracellularly and extracellularly to mediate a multitude of biological processes including inflammatory responses, angiogenesis, cell migration and differentiation, and cell-ECM adhesion. Galectin-1 (Gal-1) has been associated with several fibrotic diseases and can induce keratinocyte and fibroblast proliferation, migration, and differentiation into fibroproliferative myofibroblasts. In this study, Gal-1 expression was assessed in human and porcine HTS. In a microarray, galectins 1, 4, and 12 were upregulated in pig HTS compared to normal skin (fold change = +3.58, +6.11, and +3.03, FDR <0.01). Confirmatory qRT-PCR demonstrated significant upregulation of Galectin-1 (LGALS1) transcription in HTS in both human and porcine tissues (fold change = +7.78 and +7.90, P <.05). In pig HTS, this upregulation was maintained throughout scar development and remodeling. Immunofluorescent staining of Gal-1 in human and porcine HTS showed significantly increased fluorescence (202.5 ± 58.2 vs 35.2 ± 21.0, P <.05 and 276.1 ± 12.7 vs 69.7 ± 25.9, P <.01) compared to normal skin and co-localization with smooth muscle actin-expressing myofibroblasts. A strong positive correlation (R = .948) was observed between LGALS1 and Collagen type 1 alpha 1 mRNA expression. Gal-1 is overexpressed in HTS at the mRNA and protein levels and may have a role in the development of scar phenotypes due to fibroblast over-proliferation, collagen secretion, and dermal thickening. The role of galectins shows promise for future study and may lead to the development of a pharmacotherapy for treatment of HTS.

Reactive Oxygen Species Scavenging Potential Contributes to Hypertrophic Scar Formation.

BACKGROUND: Reactive oxygen species (ROS) can damage macromolecules if not appropriately neutralized by ROS scavengers. The balance between ROS and ROS scavengers is essential to prevent the accumulation of damage in healthy tissues. This balance is perturbed in hypertrophic scar (HTS). MATERIALS AND METHODS: Full-thickness wounds were created on the flanks of Duroc pigs at day 0 that developed into HTS (n = 4). Wounds and HTSs were biopsied weekly for 135 d. Total transcriptome microarrays were conducted with focused ROS scavenger analysis. Confirmatory quantitative reverse transcription polymerase chain reaction and immunofluorescence of ROS scavengers: superoxide dismutase 1, microsomal glutathione S-transferase 1, and peroxiredoxin 6 were performed throughout wound healing and HTS development. RESULTS: Total transcriptome microarray analysis identified over 25 ROS scavenger genes that were significantly downregulated in HTS at all time points compared with basal level controls (BL) (FDR<0.01; fold change > or <2). Ingenuity pathway analysis identified multiple ROS scavenging pathways involved in HTS (P < 0.01). Quantitative reverse transcription polymerase chain reaction of representative scavengers confirmed and expanded this finding to the initial phases of wound healing (P < 0.05, n = 4). The protein products of the genes were lower in wound and HTS tissues compared with BL. CONCLUSIONS: A balance between ROS production and scavenging must be maintained for normal wound healing, which is perturbed in wounds that heal to form HTSs. We postulate that endogenous scavengers can be administered as a prophylactic or post-treatment to rebalance ROS and attenuate symptoms of scar.

Plasma Ameliorates Endothelial Dysfunction in Burn Injury.

BACKGROUND: A complex inflammatory response mediates the systemic effects of burn shock. Disruption of the endothelial glycocalyx causes shedding of structural glycoproteins, primarily syndecan-1 (SDC-1), leading to endothelial dysfunction. These effects may be mitigated by resuscitative interventions. MATERIALS AND METHODS: Sprague-Dawley rats were used to create small, medium, and large burns and uninjured controls. Three different intravenous resuscitation protocols were applied within each group: Lactated Ringer's (LR) alone, LR plus fresh frozen plasma (FFP), or LR plus albumin. Blood was serially collected, and plasma SDC-1 was quantified with enzyme-linked immunosorbent assay. In one cohort, Evan's Blue Dye (EBD) was administered and quantified in lung by spectrophotometry as a functional assay of vascular permeability. In a second cohort, intact SCD-1 was quantified by immunohistochemistry in lung tissue. Statistical analysis employed two-way analysis of variance with multiple comparisons and Student's t-test. RESULTS: EBD extraction from lung was significantly greater with higher injury severity versus controls. Extraction decreased significantly in large-burn animals with addition of FFP to LR versus LR-only; addition of albumin to LR did not decrease EBD extraction. Plasma SCD-1 increased in injured animals compared with controls, and changes correlated with injury severity in all resuscitation groups (significance, P < 0.05). Lung SCD-1 staining reflected the results in the EBD assay. CONCLUSIONS: Addition of FFP, not of albumin, to post-burn resuscitation diminishes vascular leakage associated with large burns. Addition of colloid does not affect SDC-1 shedding as measured in plasma. Ongoing work will further define pathophysiologic mechanisms and potential therapeutic interventions to mitigate injury and promote repair of the endothelial glycocalyx.

Heat transfer analysis and resolution quantification of active dynamic thermography through human skin.

OBJECTIVES: Active dynamic thermography (ADT) is a non-contact imaging technique that characterizes non-homogeneities in thermal conductance through objects as a response to applied energy stimulus. The aim of this study was to (i) develop a heat transfer model to define the relationship between thermal stimulation and resolution and (ii) empirically quantify the resolution an ADT imaging system can detect through a range of depths of human skin. MATERIALS AND METHODS: A heat transfer model was developed to describe a thermally non-conductive object below a sheet of skin. The size and depth of the object were varied to simulate wound conditions, while the intensity and duration of thermal stimulation were varied to define stimulation parameters. The model was solved by numerical analysis. For ex vivo experimentation, freshly excised human pannus tissue was cut into sheets of thickness 2.54-6.35 × 10-4 m (0.010-0.025vinches) for a total of 48 grafts from 12 patients. Grafts were placed over a 3D printed resolution target with objects ranging from 0.445-0.125 LP/mm. Stimulation from a 300 W halogen lamp array was applied for 0.5-14 seconds for a total of 480 experiments. RESULTS: ADT resolved a peak of 0.428 ± 0.025 LP/mm for 2.54 × 10-4 m (0.010 inches) skin thickness, 0.384 ± 0.030 LP/mm for 3.81 × 10-4 m (0.015 inches), 0.325 ± 0.042 LP/mm for 5.08 × 10-4 m (0.020 inches) and 0.249 ± 0.057 LP/mm for 6.35 × 10-4 m (0.025 inches) skin thickness. Additionally, it was determined that the ideal duration of stimulation energy with a 300 W stimulation system was 4 seconds for 2.54 × 10-4 m, 6 seconds for 3.81 × 10-4 m, 8 seconds for 5.08 × 10-4 m, and 14 seconds for 6.35 × 10-4 m skin thickness. CONCLUSIONS: This study has characterized the correlation between thermal stimulus input and resolvable object size and depth for ADT. Through ex vivo experimentation it has also quantified the functional imaging depth to below the sub-cutis, beyond that of conventional imaging techniques. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Reference ranges for rotational thromboelastometry in male Sprague Dawley rats.

BACKGROUND: A functional coagulation assay was used to investigate the extrinsic pathway of coagulation on citrated whole blood samples from healthy adult male Sprague Dawley rats using the mini cup and pin system. METHODS: Reference values for coagulation parameters from forty-three animals were calculated using data obtained from the ROTEM® delta hemostasis analyzer with the EXTEM test. RESULTS: The following ranges, presented as the 2.5-97.5 percentiles, were established: CT [18-77], CFT [20-80], α [73-86], MCF [53-70], and ML [1-22], along with others. CONCLUSIONS: These reference ranges can be used in future studies in rats to identify clinically significant coagulopathies.

Disruption of Biofilms and Neutralization of Bacteria Using Hypochlorous Acid Solution: An In Vivo and In Vitro Evaluation.

OBJECTIVES: The aims of this study were to assess the effectiveness of a hypochlorous acid-based wound cleanser (Vashe Wound Solution [VWS], SteadMed Medical, Fort Worth, Texas) in disrupting methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms relative to other cleansers using an in vitro collagen biofilm model and to evaluate cleansers' cytotoxicity. The bioburden reduction of venous stasis wounds by VWS and another cleanser was evaluated. METHODS: Plates coated with collagen films incubated with active bacteria cultures to yield biofilm mimics were treated with VWS, 1% and 10% povidone-iodine (PI), 0.05% chlorhexidine wound solution (CWS), or normal saline for 3 or 10 minutes. Biofilms were then analyzed for biomass density using a crystal violet assay, quantitative cultures, and fluorescent microscopy. Cytotoxicity was measured using neutral red uptake by primary human dermal fibroblasts. Pre- and postcleansing exudates and swab samples obtained from venous stasis wounds of patients were processed and plated on a series of selective agar plates for bacteria typing and quantification. RESULTS: All agents tested significantly neutralized methicillin-resistant S aureus and P aeruginosa biofilms compared with saline control as assessed by crystal violet assay and fluorescent microscopy assays. Undiluted VWS was significantly less cytotoxic compared with 1% PI, CWS, and 10% PI (in increasing order of cytotoxicity). There was no significant difference in bacterial reduction in wounds after treatment with VWS or CWS for any type of bacteria examined using selective media. In wounds that were treated with VWS or CWS, there was a similar percentage reduction in bacterial colony-forming units from precleansing levels when plated on tryptic soy agar, MacConkey, streptococcal, and mannitol salt agar plates. Plates treated with CWS trended toward higher bacterial reduction on nonselective and gram-negative agars, whereas VWS trended toward higher bacterial reduction in Streptococcus-selective agars. CONCLUSIONS: These findings support the use of VWS in the treatment of wounds with biofilms and to reduce the bioburden of venous stasis ulcers. While VWS-treated biofilms had higher biomass than CWS- and saline-treated biofilms, most of the cellular component was not viable. Ultimately, VWS had a similar effectiveness to CWS in eliminating bacteria but with lower cytotoxicity.

Compression therapy affects collagen type balance in hypertrophic scar.

BACKGROUND: The effects of pressure on hypertrophic scar are poorly understood. Decreased extracellular matrix deposition is hypothesized to contribute to changes observed after pressure therapy. To examine this further, collagen composition was analyzed in a model of pressure therapy in hypertrophic scar. MATERIALS AND METHODS: Hypertrophic scars created on red Duroc swine (n = 8) received pressure treatment (pressure device mounting and delivery at 30 mm Hg), sham treatment (device mounting and no delivery), or no treatment for 2 wk. Scars were assessed weekly and biopsied for histology, hydroxyproline quantification, and gene expression analysis. Transcription levels of collagen precursors COL1A2 and COL3A1 were quantified using reverse transcription-polymerase chain reaction. Masson trichrome was used for general collagen quantification, whereas immunofluorescence was used for collagen types I and III specific quantification. RESULTS: Total collagen quantification using hydroxyproline assay showed a 51.9% decrease after pressure initiation. Masson trichrome staining showed less collagen after 1 (P < 0.03) and 2 wk (P < 0.002) of pressure application compared with sham and untreated scars. Collagen 1A2 and 3A1 transcript decreased by 41.9- and 42.3-fold, respectively, compared with uninjured skin after pressure treatment, whereas a 2.3- and 1.3-fold increase was seen in untreated scars. This decrease was seen in immunofluorescence staining for collagen types I (P < 0.001) and III (P < 0.04) compared with pretreated levels. Pressure-treated scars also had lower levels of collagen I and III after pressure treatment (P < 0.05) compared with sham and untreated scars. CONCLUSIONS: These results demonstrate the modulation of collagen after pressure therapy and further characterize its role in scar formation and therapy.

A Nude Mouse Model of Xenografted Hypertrophic Scar Cells to Test Therapeutics in the Skin.

BACKGROUND: Existing animal models for testing therapeutics in the skin are limited. Mouse and rat models lack similarity to human skin in structure and wound healing mechanism. Pigs are regarded as the best model with regards to similarity to human skin; however, these studies are expensive, time-consuming, and only small numbers of biologic replicates can be obtained. In addition, local-regional effects of treating wounds that are closely adjacent to one-another with different treatments make assessment of treatment effectiveness difficult in pig models. Therefore, here, a novel nude mouse model of xenografted porcine hypertrophic scar (HTS) cells was developed. This model system was developed to test if supplying hypo-pigmented cells with exogenous alpha melanocyte stimulating hormone (α-MSH) will reverse pigment loss in vivo. METHODS: Dyschromic HTSs were created in red Duroc pigs. Epidermal scar cells (keratinocytes and melanocytes) were derived from regions of hyper-, hypo-, or normally pigmented scar or skin and were cryopreserved. Dermal fibroblasts (DFs) were isolated separately. Excisional wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0 and formed a dermis. On day 3, epidermal cells were seeded onto the dermis. The grafting dome was removed on day 7 and hypo-pigmented xenografts were treated with synthetic α-MSH delivered with microneedling. On day 10, the xenografts were excised and saved. Sections were stained using hematoxylin and eosin hematoxylin and eosin (H&E) to assess xenograft structure. RNA was isolated and quantitative real-time polymerase chain reaction (qRT-PCR) was performed for melanogenesis-related genes TYR, TYRP1, and DCT. RESULTS: The seeding of HTSDFs formed a dermis that is similar in structure and cellularity to HTS dermis from the porcine model. When hyper-, hypo-, and normally-pigmented epidermal cells were seeded, a fully stratified epithelium was formed by day 14. H&E staining and measurement of the epidermis showed the average thickness to be 0.11 ± 0.07 µm vs. 0.06 ± 0.03 µm in normal pig skin. Hypo-pigmented xenografts that were treated with synthetic α-MSH showed increases in pigmentation and had increased gene expression of TYR, TYRP1, and DCT compared to untreated controls (TYR: 2.7 ± 1.1 vs. 0.3 ± 1.1; TYRP1: 2.6 ± 0.6 vs. 0.3 ± 0.7; DCT 0.7 ± 0.9 vs. 0.3 ± 1-fold change from control; n = 3). CONCLUSIONS: The developed nude mouse skin xenograft model can be used to study treatments for the skin. The cells that can be xenografted can be derived from patient samples or from pig samples and form a robust dual-skin layer containing epidermis and dermis that is responsive to treatment. Specifically, we found that hypo-pigmented regions of scar can be stimulated to make melanin by synthetic α-MSH in vivo.

Endothelial dysfunction is dampened by early administration of fresh frozen plasma in a rodent burn shock model.

BACKGROUND: Endothelial dysfunction has been implicated in the pathogenesis of burn shock affecting patients with large thermal injury. In response to injury, glycocalyx components like Syndecan-1 (SDC-1) are shed into circulation and have been used as markers of endothelial damage. Previous work in our lab has shown plasma inclusive resuscitation (PIR) with fresh frozen plasma (FFP) ameliorates endothelial damage. However, there remains a paucity of information regarding optimal timing and dosing of PIR as well as organ specific endothelial responses to shock. We aimed to examine the impact of PIR on endothelial dysfunction using clinically-translatable timing and dosing. METHODS: Spraque-Dawley rats were used to create thermal burns. Rats were subjected to 40% TBSA scald burns and were resuscitated with LR only, LR + albumin and LR + early 1 ml boluses of FFP at 0,2,4 and 8 hours post-injury. A late group also received LR + FFP starting at hour 10 post-injury. SDC-1 levels were quantified by ELISA and qRT-PCR analysis characterized transcription of glycocalyx components and inflammatory cytokines in the lung and spleen. Evan's blue dye (EBD) was used to quantify amount of vascular leakage. RESULTS: LR + early FFP reduced EBD extravasation when compared to LR only groups while late FFP did not. When comparing LR only vs LR + early FFP, SDC-1 levels were reduced in the LR + early FFP group at hour 8, 12 and 24 (5.23 vs. 2.07, p < 0.001, 4.49 vs. 2.05, p < 0.01, 3.82 vs 2.08, p < 0.05, respectively). LR only groups had upregulation of Exostosin-1 (EXT-1) and SDC-1 in the lung compared to LR + early FFP groups (p < 0.01 and p < 0.05) as well as upregulation of cytokines IL-10 and IFN-Ƴ (p < 0.001 and p < 0.001). CONCLUSIONS: Early administration of LR + FFP reduces the magnitude of SDC-1 shedding and dampens the cytokine response to injury. The upregulation of glycocalyx components as a response to endothelial injury is also decreased in the lung and spleen by LR + early FFP administration.

Rete ridges are decreased in dyschromic burn hypertrophic scar: A histological study.

Dyschromic hypertrophic scar (HTS) is a common sequelae of burn injury, however, its mechanism has not been elucidated. This work is a histological study of these scars with a focus on rete ridges. Rete ridges are important for normal skin physiology, and their absence or presence may hold mechanistic significance in post-burn HTS dyschromia. It was posited that hyper-, and hypo-pigmented areas of scars have different numbers of rete ridges. Subjects with dyschromic burn hypertrophic scar were prospectively enrolled (n = 44). Punch biopsies of hyper-, hypo-, and normally pigmented scar and skin were collected. Biopsies were paraffin embedded, sectioned, stained with H&E, and imaged. The number of rete ridges were investigated. Burn hypertrophic scars that healed without autografts were first investigated. The number of rete ridges was higher in normal skin compared to HTS that was either hypo- (p < 0.01) or hyper-pigmented (p < 0.001). This difference was similar despite scar pigmentation phenotype (p = 0.8687). Autografted hyper-pigmented scars had higher rete ridge ratio compared to non-autografted hyper-pigmented HTS (p < 0.0001). Burn hypertrophihc scars have fewer rete ridges than normal skin. This finding may explain the decreased epidermal adherence to underlying dermis associated with hypertrophic scars. Though, contrary to our hypothesis, no direct link between the extent of dyschromia and rete ridge quantity was observed, the differences in normal skin and hypertrophic scar may lead to further understanding of dyschromic scars.

Cold atmospheric plasma is bactericidal to wound-relevant pathogens and is compatible with burn wound healing.

Burn wound healing can be significantly delayed by infection leading to increased morbidity and hypertrophic scarring. An optimal antimicrobial agent would have the ability to kill bacteria without negatively affecting the host skin cells that are required for healing. Currently available products provide antimicrobial coverage, but may also cause reductions in cell proliferation and migration. Cold atmospheric plasma is a partially ionized gas that can be produced under atmospheric pressure at room temperature. In this study a novel handheld Aceso Plasma Generator was used to produce and test Aceso Cold Plasma (ACP) in vitro and in vivo. ACP showed a potent ability to eliminate bacterial load in vitro for a number of different species. Deep partial-thickness and full-thickness wounds that were treated with ACP after burning, after excision, after autografting, and at days 5, 7, and 9 did not show any negative effects on their wound healing trajectories. On par with in vitro analysis, bioburden was decreased in treated wounds vs. control. In addition, metrics of hypertrophic scar such as dyschromia, elasticity, trans-epidermal water loss (TEWL), and epidermal and dermal thickness were the same between the two treatment groups.It is likely that ACP can be used to mitigate the risk of bacterial infection during the phase of acute burn injury while patients await surgery for definitive closure. It may also be useful in treating wounds with delayed re-epithelialization that are at risk for infection and hypertrophic scarring. A handheld cold plasma device will be useful in treating all manner of wounds and surgical sites in order to decrease bacterial burden in an efficient and highly effective manner without compromising wound healing.