Publications by authors named "Robert J Christy"

65 Publications

Evaluation of KP-1199: a novel acetaminophen analog for hemostatic function and antinociceptive effects.

Transfusion 2021 07;61 Suppl 1:S234-S242

United States Army Institute of Surgical Research, JBSA Fort Sam Houston, San Antonio, Texas, USA.

Background: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples.

Methods: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 μg/ml) and 10× (2140 μg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation.

Results: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested.

Conclusion: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.
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http://dx.doi.org/10.1111/trf.16497DOI Listing
July 2021

Minimal Effects of Intravenous Administration of Xenogeneic Adipose Derived Stem Cells on Organ Function in a Porcine 40%TBSA Burn Model.

J Burn Care Res 2021 May 31. Epub 2021 May 31.

Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America.

Adipose stem cells (ASCs) have shown therapeutic promise for various conditions, including burn injury. While ASCs have immunomodulatory properties, concerns exist over pro-coagulant activity after intravenous (IV) administration. In the present study, we examined IV human ASC delivery in terms of coagulation, organ function, and inflammation in a 40% total body surface area (TBSA) swine burn model. Anesthetized female Yorkshire swine were burned and randomized to receive 15ml/kg Lactated Ringer's containing: no ASCs; a low dose (5x10 5 ASCs/kg), or a high dose (5x10 6 ASCs/kg). For biochemical analysis, blood was collected at baseline (BL), 3, 6, 12, and 24 hours post-burn, while kidney and liver tissue was collected post-euthanasia. A significant, but transient, effect of ASCs was seen on prothrombin times and INR, wherein low doses revealed slight hypercoagulation. Burns increased partial thromboplastin time, fibrinogen, and d-dimer levels, which was unchanged with ASC administration. ASCs tended to exacerbate increases in bilirubin at 3 hours, but this didn't reach statistical significance. A significant effect of ASCs on creatinine and BUN was seen, wherein low doses elevated levels at 24 hours (creatinine, p=0.0012; BUN, p=0.0195). Hepatic and renal TUNEL staining were similar for all groups. A dose-dependent decrease in IL-8 was observed, while low doses significantly increased IL-1RA at 3 (p=0.050), IL-12 at 12 (p=0.021) and IL-6 at 24 hours post-burn (p=0.035). IV administration of xenogeneic ASCs slightly increased coagulation, but effects on burn-induced renal and hepatic dysfunction effects were minimal. Despite some significant immunomodulation, organ dysfunction effects were modest. Collectively, this study provides evidence to be skeptical about xenogeneic ASC administration in regards to burn.
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http://dx.doi.org/10.1093/jbcr/irab094DOI Listing
May 2021

ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging.

Stem Cell Res Ther 2021 05 6;12(1):270. Epub 2021 May 6.

United States Army Institute of Surgical Research, JBSA Fort Sam Houston, 3698 Chambers Pass, San Antonio, TX, USA.

Background: Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue.

Methods: ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer).

Results: There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P < 0.01) of passaging on mitochondrial ROS production, with increased ROS in BH ASCs at later passages. Oxidative phosphorylation capacities (leak and maximal respiration) increased significantly in BH ASCs (P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs.

Conclusions: In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.
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http://dx.doi.org/10.1186/s13287-021-02327-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100366PMC
May 2021

Accelerated Wound Closure of Deep Partial Thickness Burns with Acellular Fish Skin Graft.

Int J Mol Sci 2021 Feb 4;22(4). Epub 2021 Feb 4.

Burn and Soft Tissue Injury Research Department, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston, TX 78234, USA.

Thermal injuries are caused by exposure to a variety of sources, and split thickness skin grafts are the gold standard treatment for severe burns; however, they may be impossible when there is no donor skin available. Large total body surface area burns leave patients with limited donor site availability and create a need for treatments capable of achieving early and complete coverage that can also retain normal skin function. In this preclinical trial, two cellular and tissue based products (CTPs) are evaluated on twenty-four 5 × 5 deep partial thickness (DPT) burn wounds. Using appropriate pain control methods, DPT burn wounds were created on six anesthetized Yorkshire pigs. Wounds were excised one day post-burn and the bleeding wound beds were subsequently treated with omega-3-rich acellular fish skin graft (FSG) or fetal bovine dermis (FBD). FSG was reapplied after 7 days and wounds healed via secondary intentions. Digital images, non-invasive measurements, and punch biopsies were acquired during rechecks performed on days 7, 14, 21, 28, 45, and 60. Multiple qualitative measurements were also employed, including re-epithelialization, contraction rates, hydration, laser speckle, and trans-epidermal water loss (TEWL). Each treatment produced granulated tissue (GT) that would be receptive to skin grafts, if desired; however, the FSG induced GT 7 days earlier. FSG treatment resulted in faster re-epithelialization and reduced wound size at day 14 compared to FBD (50.2% vs. 23.5% and 93.1% vs. 106.7%, < 0.005, respectively). No differences in TEWL measurements were observed. The FSG integrated into the wound bed quicker as evidenced by lower hydration values at day 21 (309.7 vs. 2500.4 µS, < 0.05) and higher blood flow at day 14 (4.9 vs. 3.1 fold change increase over normal skin, < 0.005). Here we show that FSG integrated faster without increased contraction, resulting in quicker wound closure without skin graft application which suggests FSG improved burn wound healing over FBD.
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http://dx.doi.org/10.3390/ijms22041590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915828PMC
February 2021

Clinical Translational Potential in Skin Wound Regeneration for Adipose-Derived, Blood-Derived, and Cellulose Materials: Cells, Exosomes, and Hydrogels.

Biomolecules 2020 09 27;10(10). Epub 2020 Sep 27.

Obatala Sciences Inc., New Orleans, LA 70148, USA.

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.
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http://dx.doi.org/10.3390/biom10101373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650547PMC
September 2020

Spatial Frequency Domain Imaging (SFDI) of clinical burns: A case report.

Burns Open 2020 Apr 19;4(2):67-71. Epub 2020 Feb 19.

Beckman Laser Institute and Medical Clinic, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92617, United States.

While visual assessment by a clinician is the standard of care for burn severity evaluations, new technologies at various stages of development are attempting to add objectivity to this practice by quantifying burn severity. Assessment accuracy generally improves after the burn injury has progressed, but early assessments that correctly identify superficial partial and deep partial burns have the potential to lead to more prompt treatments and shorter recovery times. To date, Spatial Frequency Domain Imaging (SFDI) has only been used in animal models of burns, but has shown the potential to categorize burns accurately at earlier time points. Here we examine the potential for SFDI to assess burn severity in clinical patients. We also utilize Laser Speckle Imaging (LSI), an FDA cleared non-invasive imaging technology that typically measures blood perfusion in order to evaluate burns in clinical patients. We present a case series of two patients, both with partial thickness burns of varying severity. Partial thickness burns are often difficult for clinicians to categorize based on visual appearance alone. SFDI and LSI were both performed on each patient at approximately 24 and 72 h after their respective burn incidents. Each technique was able to render spatially resolved information that enabled improved assessment accuracy for each burn. This represents the first publication of SFDI applied to clinical burn patients after being successfully utilized in animal models, and highlights the potential for SFDI as a feasible tool for the timely categorization of burn severity.
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http://dx.doi.org/10.1016/j.burnso.2020.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442210PMC
April 2020

Enzymatic Debridement of Porcine Burn Wounds via a Novel Protease, SN514.

J Burn Care Res 2020 09;41(5):1015-1028

Department of Burn and Soft Tissue Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

Necrotic tissue generated by a thermal injury is typically removed via surgical debridement. However, this procedure is commonly associated with blood loss and the removal of viable healthy tissue. For some patients and contexts such as extended care on the battlefield, it would be preferable to remove devitalized tissue with a nonsurgical debridement agent. In this paper, a proprietary debridement gel (SN514) was evaluated for the ability to debride both deep-partial thickness (DPT) and full-thickness burn wounds using an established porcine thermal injury model. Burn wounds were treated daily for 4 days and visualized with both digital imaging and laser speckle imaging. Strip biopsies were taken at the end of the procedure. Histological analyses confirmed a greater debridement of the porcine burn wounds by SN514 than the vehicle-treated controls. Laser speckle imaging detected significant increases in the perfusion status after 4 days of SN514 treatment on DPT wounds. Importantly, histological analyses and clinical observations suggest that SN514 gel treatment did not damage uninjured tissue as no edema, erythema, or inflammation was observed on intact skin surrounding the treated wounds. A blinded evaluation of the digital images by a burn surgeon indicated that SN514 debrided more necrotic tissue than the control groups after 1, 2, and 3 days of treatment. Additionally, SN514 gel was evaluated using an in vitro burn model that used human discarded skin. Treatment of human burned tissue with SN514 gel resulted in greater than 80% weight reduction compared with untreated samples. Together, these data demonstrate that SN514 gel is capable of debriding necrotic tissue and suggest that SN514 gel could be a useful option for austere conditions, such as military multi-domain operations and prolonged field care scenarios.
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http://dx.doi.org/10.1093/jbcr/iraa111DOI Listing
September 2020

Quantifying the effects of wound healing risk and potential on clinical measurements and outcomes of severely burned patients: A data-driven approach.

Burns 2020 03 10;46(2):303-313. Epub 2019 Dec 10.

U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, United States. Electronic address:

Introduction: Given recent advances in computational power, the goal of this study was to quantify the effects of wound healing risk and potential on clinical measurements and outcomes of severely burned patients, with the hope of providing more insight on factors that affect wound healing.

Methods: This retrospective study involved patients who had at least 10% TBSA% "burned" and three burn mappings each. To model risk to wounds, we defined the variable θ, a hypothetical threshold for TBSA% "open wound" used to demarcate "low-risk" from "high-risk" patients. Low-risk patients denoted those patients whose actual TBSA% "open wound" ≤θ, whereas high-risk patients denoted those patients whose actual TBSA% "open wound" >θ. To consider all possibilities of risk, 100 sub analyses were performed by (1) varying θ from 100% to 1% in decrements of 1%, (2) grouping all patients as either "low-risk" or "high-risk" for each θ, and (3) comparing all means and deviations of variables and outcomes between the two groups for each θ. Hence, this study employed a data-driven approach to capture trends in clinical measurements and outcomes. Plots and tables were also obtained.

Results: For 303 patients, median age and weight were 43 [29-59] years and 85 [72-99]kg, respectively. Mean TBSA% "burned" was 25 [17-39] %, with a full-thickness burn of 4 [0-15] %. Average crystalloid volumes were 4.25±2.27mL/kg/TBSA% "burned" in the first 24h. Importantly, for high-risk patients, decreasing θ was matched by significant increases in PaO2-FiO2 ratio, platelet count, Glasgow coma score (GCS), and MAP. On the other hand, increasing their risk θ was also matched by significant increases in creatinine, bilirubin, lactate, blood, estimated blood loss, and 24-h and total fluid volumes. As expected, for low-risk patients, clinical measurements were more stable, despite decreasing or increasing θ. At a θ of 80%, statistical tests indicated much disparity between high-risk and low-risk patients for TBSA% "burned", full thickness burn, bilirubin (1.66±1.16mg/dL versus 0.83±0.65mg/dL, p=0.005), GCS (7±2 versus 12±3, p<0.001), MAP (42±22mm Hg versus 59±22mm Hg, p=0.004), 24-h blood, estimated blood loss, 24-h fluid, total fluid, and ICU length of stay (81±113 days versus 24±27 days, p=0.002). These differences were all statistically significant and remained significant down to θ=10%.

Conclusion: Wound healing risk and potential may be forecasted by many different clinical measurements and outcomes and has many implications on multi-organ function. Future work will be needed to further explain and understand these effects, in order to facilitate development of new predictive models for wound healing.
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http://dx.doi.org/10.1016/j.burns.2019.11.017DOI Listing
March 2020

Evaluation of Three-Dimensional Printed, Keratin-Based Hydrogels in a Porcine Thermal Burn Model.

Tissue Eng Part A 2020 03 9;26(5-6):265-278. Epub 2020 Jan 9.

Fischell Department of Bioengineering, University of Maryland, College Park, Maryland.

Keratin is a natural material that can be derived from the cortex of human hair. Our group had previously presented a method for the printed, sequential production of three-dimensional (3D) keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin-based constructs through ultraviolet crosslinking in a lithography-based 3D printer. In this study, we have used this bioink to produce a keratin-based construct that is capable of delivering small molecules, providing an environment conducive to healing of dermal burn wounds , and maintaining stability in customized packaging. We characterized the effects of manufacturing steps, such as lyophilization and gamma irradiation sterilization on the properties of 3D printed keratin scaffolds prepared for testing. Keratin hydrogels are viable for the uptake and release of contracture-inhibiting Halofuginone, a collagen synthesis inhibitor that has been shown to decrease collagen synthesis in fibrosis cases. This small-molecule delivery provides a mechanism to reduce scarring of severe burn wounds . data show that the Halofuginone-laden printed keratin is noninferior to other similar approaches reported in literature. This is indicative that the use of 3D printed keratin is not inhibiting the healing processes, and the inclusion of Halofuginone induces a more organized dermal healing after a burn; in other words, this treatment is slower but improves healing. These studies are indicative of the potential of Halofuginone-laden keratin dressings in dermal wound healing. We aim to keep increasing the complexity of the 3D printed constructs toward the production of complex scaffolds for the treatment and topographical reconstruction of severe burn wounds to the face.
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http://dx.doi.org/10.1089/ten.TEA.2019.0181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476396PMC
March 2020

PEGylated Platelet-Free Blood Plasma-Based Hydrogels for Full-Thickness Wound Regeneration.

Adv Wound Care (New Rochelle) 2019 Jul 2;8(7):323-340. Epub 2019 Jul 2.

Combat Trauma and Burn Injury Research, U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas.

To develop a cost-effective and clinically usable therapy to treat full-thickness skin injuries. We accomplished this by preparing a viscoelastic hydrogel using polyethylene glycol (PEG)-modified platelet-free plasma (PEGylated PFP) combined with human adipose-derived stem cells (ASCs). PEGylated PFP hydrogels were prepared by polymerizing the liquid mixture of PEG and PFP±ASCs and gelled either by adding calcium chloride (CaCl) or thrombin. Rheological and studies were performed to assess viscoelasticity and the ability of hydrogels to direct ASCs toward a vasculogenic phenotype, respectively. Finally, a pilot study evaluated the efficacy of hydrogels±ASCs using an athymic rat full-thickness skin wound model. Hydrogels prepared within the range of 11 to 27 mM for CaCl or 5 to 12.5 U/mL for thrombin exhibited a storage modulus of ∼62 to 87 Pa and ∼47 to 92 Pa, respectively. The PEGylated PFP hydrogels directed ASCs to form network-like structures resembling vasculature, with a fourfold increase in perivascular specific genes that were confirmed by immunofluorescent staining. Hydrogels combined with ASCs exhibited an increase in blood vessel density when applied to excisional rat wounds compared with those treated with hydrogels (110.3 vs. 95.6 BV/mm;  < 0.05). Furthermore, ASCs were identified in the perivascular region associated with newly forming blood vessels. This study demonstrates that PFP modified with PEG along with ASCs can be used to prepare cost-effective stable hydrogels, at the bed-side, to treat extensive skin wounds. These results indicate that PEGylated plasma-based hydrogels combined with ASCs may be a potential regenerative therapy for full-thickness skin wounds.
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http://dx.doi.org/10.1089/wound.2018.0844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855295PMC
July 2019

Spatial frequency domain imaging: a quantitative, noninvasive tool for in vivo monitoring of burn wound and skin graft healing.

J Biomed Opt 2019 07;24(7):1-9

University of California, Beckman Laser Institute and Medical Clinic, Irvine, California, United States.

There is a need for noninvasive, quantitative methods to characterize wound healing in the context of longitudinal investigations related to regenerative medicine. Such tools have the potential to inform the assessment of wound status and healing progression and aid the development of new treatments. We employed spatial frequency domain imaging (SFDI) to characterize the changes in optical properties of tissue during wound healing progression in a porcine model of split-thickness skin grafts and also in a model of burn wound healing with no graft intervention. Changes in the reduced scattering coefficient measured using SFDI correlated with structural changes reported by histology of biopsies taken concurrently. SFDI was able to measure spatial inhomogeneity in the wounds and predicted heterogeneous healing. In addition, we were able to visualize differences in healing rate, depending on whether a wound was debrided and grafted, versus not debrided and left to heal without intervention apart from topical burn wound care. Changes in the concentration of oxy- and deoxyhemoglobin were also quantified, giving insight into hemodynamic changes during healing.
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http://dx.doi.org/10.1117/1.JBO.24.7.071615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630099PMC
July 2019

Relationship Between Burn Wound Location and Outcomes in Severely Burned Patients: More Than Meets the Size.

J Burn Care Res 2019 08;40(5):558-565

U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

We hypothesized that burn location plays an important role in wound healing, mortality, and other outcomes and conducted the following study to test this multifold hypothesis. We conducted a study to retrospectively look at patients with burns ≥10% TBSA. Demographics, TBSA, partial/full thickness burns (PT/FT) in various wound locations, fluids, inhalation injury, mortality, ICU duration, and hospital duration were considered. Initial wound healing rates (%/d) were also calculated as a slope from the time of the first mapping of open wound size to the time of the third mapping of open wound size. Multivariate logistic regression and operating curves were used to measure mortality prediction performance. All values were expressed as median [interquartile range]. The mortality rate for 318 patients was 17% (54/318). In general, patients were 43 years [29, 58 years] old and had a TBSA of 25% [17, 39%], PT of 16% [10, 25%], and FT of 4% [0, 15%]. Between patients who lived and did not, age, TBSA, FT, 24-hour fluid, and ICU duration were statistically different (P < .001). Furthermore, there were statistically significant differences in FT head (0% [0, 0%] vs 0% [0, 1%], P = .048); FT anterior torso (0% [0, 1%] vs 1% [0, 4%], P < .001); FT posterior torso (0% [0, 0%] vs 0% [0, 4%], P < 0.001); FT upper extremities (0% [0, 3%] vs 2% [0, 11%], P < .001); FT lower extremities (0% [0, 2%] vs 6% [0, 17%], P < .001); and FT genitalia (0% [0, 0%] vs 0% [0, 2%], P < .001). Age, presence of inhalation injury, PT/FT upper extremities, and FT lower extremities were independent mortality predictors and per unit increases of these variables were associated with an increased risk for mortality (P < .05): odds ratio of 1.09 (95% confidence interval [CI] = 1.61-1.13; P < .001) for mean age; 2.69 (95% CI = 1.04-6.93; P = .041) for inhalation injury; 1.14 (95% CI = 1.01-1.27; P = .031) for mean PT upper extremities; 1.26 (95% CI = 1.11-1.42; P < .001) for mean FT upper extremities; and 1.07 (95% CI = 1.01-1.12; P = .012) for mean FT lower extremities. Prediction of mortality was better using specific wound locations (area under the curve [AUC], AUC of 0.896) rather than using TBSA and FT (AUC of 0.873). Graphs revealed that initial healing rates were statistically lower and 24-hour fluids and ICU length of stay were statistically higher in patients with FT upper extremities than in patients without FT extremities (P < .001). Burn wound location affects wound healing and helps predict mortality and ICU length of stay and should be incorporated into burn triage strategies to enhance resource allocation or stratify wound care.
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http://dx.doi.org/10.1093/jbcr/irz098DOI Listing
August 2019

Identifying Plasma Derived Extracellular Vesicle (EV) Contained Biomarkers in the Development of Chronic Neuropathic Pain.

J Pain 2020 Jan - Feb;21(1-2):82-96. Epub 2019 Jun 19.

United States Army Institute of Surgical Research, Fort Sam Houston, San Antonio, Texas.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. PERSPECTIVE: This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers.
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http://dx.doi.org/10.1016/j.jpain.2019.05.015DOI Listing
June 2021

Delivery of silver sulfadiazine and adipose derived stem cells using fibrin hydrogel improves infected burn wound regeneration.

PLoS One 2019 13;14(6):e0217965. Epub 2019 Jun 13.

Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, Ft. Sam Houston, TX, United States of America.

Infection control is necessary for improved burn wound regeneration. In this study contact burn wounds were induced on the dorsum of the rats and were infected with Pseudomonas aeruginosa (107cfu/ml of saline) and left overnight (12-14 hours) to establish the infection. After 12 hours, the wounds were treated with PEGylated fibrin hydrogel containing 50 mgs of silver sulfadiazine (SSD) loaded chitosan microsphere (SSD-CSM-FPEG). On day 9, SSD-CSM-FPEG treated burn wounds further received adipose derived stem cell (5×104 ASCs cells/ml) embedded in PEGylated fibrin hydrogel. Wounds were assessed for the healing outcomes such as neovascularization, granulation tissue formation, wound closure and collagen maturation. Analysis of bacterial load in the burn wound biopsies, demonstrated that SSD-CSM-FPEG significantly reduced bacterial infection, while overt infection was still observed in the untreated groups on day 14. Sequential treatment of infected wounds with SSD-CSM-FPEG followed by ASC-FPEGs (SSD-CSM-ASC-FPEG) significantly reduced bacterial colonization (9 log reduction) and pro-inflammatory cytokine (TNF-α) expression. A significant increase in neovascularization markers; NG2 and vWF was also observed. Histological analysis indicated the wounds treated with SSD-CSM-ASC-FPEG increased amount of dermal collagen matrix deposition, a thicker granulation tissue on day 21 and more mature collagen on day 28. This work demonstrates that the sequential treatment of infected burn wounds with SSD-CSM-FPEG followed by ASC-FPEG reduces bacterial infection as well as promotes neo-vascularization with improved matrix remodeling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217965PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563979PMC
March 2020

Burn wound classification model using spatial frequency-domain imaging and machine learning.

J Biomed Opt 2019 05;24(5):1-9

University of California, Irvine, Beckman Laser Institute and Medical Clinic, Irvine, California, United States.

Accurate assessment of burn severity is critical for wound care and the course of treatment. Delays in classification translate to delays in burn management, increasing the risk of scarring and infection. To this end, numerous imaging techniques have been used to examine tissue properties to infer burn severity. Spatial frequency-domain imaging (SFDI) has also been used to characterize burns based on the relationships between histologic observations and changes in tissue properties. Recently, machine learning has been used to classify burns by combining optical features from multispectral or hyperspectral imaging. Rather than employ models of light propagation to deduce tissue optical properties, we investigated the feasibility of using SFDI reflectance data at multiple spatial frequencies, with a support vector machine (SVM) classifier, to predict severity in a porcine model of graded burns. Calibrated reflectance images were collected using SFDI at eight wavelengths (471 to 851 nm) and five spatial frequencies (0 to 0.2  mm  -  1). Three models were built from subsets of this initial dataset. The first subset included data taken at all wavelengths with the planar (0  mm  -  1) spatial frequency, the second comprised data at all wavelengths and spatial frequencies, and the third used all collected data at values relative to unburned tissue. These data subsets were used to train and test cubic SVM models, and compared against burn status 28 days after injury. Model accuracy was established through leave-one-out cross-validation testing. The model based on images obtained at all wavelengths and spatial frequencies predicted burn severity at 24 h with 92.5% accuracy. The model composed of all values relative to unburned skin was 94.4% accurate. By comparison, the model that employed only planar illumination was 88.8% accurate. This investigation suggests that the combination of SFDI with machine learning has potential for accurately predicting burn severity.
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http://dx.doi.org/10.1117/1.JBO.24.5.056007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536007PMC
May 2019

Involvement of brain-derived neurotrophic factor (BDNF) in chronic intermittent stress-induced enhanced mechanical allodynia in a rat model of burn pain.

BMC Neurosci 2019 04 24;20(1):17. Epub 2019 Apr 24.

Battlefield Pain Management Research Group, United States Army Institute of Surgical Research, 3698 Chambers Pass, JBSA Fort Sam Houston, San Antonio, TX, 78234-4504, USA.

Background: Reports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury.

Results: Unstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia (P < 0.001). Increased expression of BDNF mRNA in the PFC (P < 0.05), and elevated TrkB and p-TrkB (P < 0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats (P < 0.001).

Conclusion: These results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state.
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http://dx.doi.org/10.1186/s12868-019-0500-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480655PMC
April 2019

Impact of hemoglobin breakdown products in the spectral analysis of burn wounds using spatial frequency domain spectroscopy.

J Biomed Opt 2019 02;24(2):1-4

University of California Irvine, Beckman Laser Institute and Medical Clinic, Irvine, California, United States.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000 nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24 h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.
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http://dx.doi.org/10.1117/1.JBO.24.2.020501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398280PMC
February 2019

Peroxisome proliferator-activated receptor-α agonist and all-trans retinoic acid induce epithelial differentiation of subcutaneous adipose-derived stem cells from debrided burn skin.

J Cell Biochem 2019 06 16;120(6):9213-9229. Epub 2018 Dec 16.

Combat Trauma and Burn Injury Research, United States Army Institute of Surgical Research, Fort Sam Houston, Texas.

This study demonstrates that adipose-derived stem cells from debrided skin (dsASCs) of burn patients can be isolated in sufficient quantities and differentiated into cytokeratin-expressing cells by treating them with all-trans retinoic acid (ATRA) and the peroxisome proliferator-activated receptor-α (PPARα) specific activator fenofibrate. Differentiation of dsASCs with ATRA and a combination of growth factors induced expression of simple epithelial markers (KRT7, KRT8, KRT18, and KRT19), along with low levels of stratified epithelial markers (KRT5, KRT10, KRT13, and KRT14). We have optimized a condition to induce dsASCs differentiation to epithelial cells by treatment with ATRA and fenofibrate alone. Real-time polymerase chain reaction analysis showed a significant increase in transcript levels (>75-fold) for basal (KRT5 and KRT14), suprabasal (KRT10), and cornified envelope markers (involucrin [IVL] and Loricrin [LOR]) with this treatment. Expression of the proteins encoded by these transcripts was confirmed by immunocytochemical analysis. Further, we show that dsASCs differentiated to a skin epithelial cell phenotype through activation of nuclear hormone receptors PPARα and RXRγ. Collectively this study shows that dsASCs can be differentiated to skin epithelial cells, without the requirement for exogenous growth factors. This differentiation protocol using dsASCs in combination with an appropriate biocompatible scaffold can be adapted to develop epithelial skin substitute for burn wound treatment.
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http://dx.doi.org/10.1002/jcb.28197DOI Listing
June 2019

Evaluating clinical observation versus Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging for the assessment of burn depth.

Burns 2019 03 14;45(2):450-460. Epub 2018 Oct 14.

Beckman Laser Institute and Medical Clinic, University of California, 1002 Health Sciences Road East, Irvine, CA 92617, United States; Department of Biomedical Engineering, University of California, 3120 Natural Sciences II, Irvine, CA 92697, United States. Electronic address:

While clinical examination is needed for burn severity diagnosis, several emerging technologies aim to quantify this process for added objectivity. Accurate assessments become easier after burn progression, but earlier assessments of partial thickness burn depth could lead to earlier excision and grafting and subsequent improved healing times, reduced rates of scarring/infection, and shorter hospital stays. Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging are three non-invasive imaging modalities that have some diagnostic ability for noninvasive assessment of burn severity, but have not been compared in a controlled experiment. Here we tested the ability of these imaging techniques to assess the severity of histologically confirmed graded burns in a swine model. Controlled, graded burn wounds, 3cm in diameter were created on the dorsum of Yorkshire pigs (n=3, 45-55kg) using a custom-made burn tool that ensures consistent pressure has been employed by various burn research groups. For each pig, a total of 16 burn wounds were created on the dorsal side. Biopsies were taken for histological analysis to verify the severity of the burn. Clinical analysis, SFDI, LSI and thermal imaging were performed at 24 and 72h after burn to assess the accuracy of each imaging technique. In terms of diagnostic accuracy, using histology as a reference, SFDI (85%) and clinical analysis (83%) performed significantly better that LSI (75%) and thermography (73%) 24h after the burn. There was no statistically significant improvement from 24 to 72h across the different imaging modalities. These data indicate that these imaging modalities, and specifically SFDI, can be added to the burn clinicians' toolbox to aid in early assessment of burn severity.
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http://dx.doi.org/10.1016/j.burns.2018.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420831PMC
March 2019

PEG-Plasma Hydrogels Increase Epithelialization Using a Human Ex Vivo Skin Model.

Int J Mol Sci 2018 10 13;19(10). Epub 2018 Oct 13.

Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, San Antonio, TX 78234-6315, USA.

In vitro cell culture methods are used extensively to study cellular migration, proliferation, and differentiation, which play major roles in wound healing but the results often do not translate to the in vivo environment. One alternative would be to establish an ex vivo model utilizing human discarded skin to evaluate therapies in a more natural setting. The purpose of this study was to institute such a model by creating 'wounds' in the center of a piece of discarded skin and treating them with three different biomaterials: collagen, polyethylene glycol (PEG)-fibrin, or PEG-platelet free plasma (PFP). Explants were cultured for 14 days with supernatant and microscopy images collected every 3 days to assess cytotoxicity and epithelialization. After 14 days, the explants were fixed, sectioned, and stained for cytokeratin-10 (CK-10), alpha-smooth muscle actin (α-SMA), and wheat germ (WG). Compared to controls, similar levels of cytotoxicity were detected for 12 days which decreased slightly at day 14. The PEG-PFP hydrogel-treated wounds epithelialized faster than other treatments at days 6 to 14. A 6-8 cell layer thick CK-10+ stratified epidermis had developed over the PEG-PFP hydrogel and cells co-stained by WG and α-SMA were observed within the hydrogel. An ex vivo model was established that can be used practically to screen different therapies exploring wound healing.
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http://dx.doi.org/10.3390/ijms19103156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213988PMC
October 2018

The Cutaneous Microbiome and Wounds: New Molecular Targets to Promote Wound Healing.

Int J Mol Sci 2018 Sep 11;19(9). Epub 2018 Sep 11.

United States Army Institute of Surgical Research, 3650 Chambers Pass, JBSA Fort Sam Houston, TX 78234, USA.

The ecological community of microorganisms in/on humans, termed the microbiome, is vital for sustaining homeostasis. While culture-independent techniques have revealed the role of the gut microbiome in human health and disease, the role of the cutaneous microbiome in wound healing is less defined. Skin commensals are essential in the maintenance of the epithelial barrier function, regulation of the host immune system, and protection from invading pathogenic microorganisms. In this review, we summarize the literature derived from pre-clinical and clinical studies on how changes in the microbiome of various acute and chronic skin wounds impact wound healing tissue regeneration. Furthermore, we review the mechanistic insights garnered from model wound healing systems. Finally, in the face of growing concern about antibiotic-resistance, we will discuss alternative strategies for the treatment of infected wounds to improve wound healing and outcomes. Taken together, it has become apparent that commensals, symbionts, and pathogens on human skin have an intimate role in the inflammatory response that highlights several potential strategies to treat infected, non-healing wounds. Despite these promising results, there are some contradictory and controversial findings from existing studies and more research is needed to define the role of the human skin microbiome in acute and chronic wound healing.
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http://dx.doi.org/10.3390/ijms19092699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164292PMC
September 2018

Advancements in Regenerative Strategies Through the Continuum of Burn Care.

Front Pharmacol 2018 9;9:672. Epub 2018 Jul 9.

Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research San Antonio, TX, United States.

Burns are caused by several mechanisms including flame, scald, chemical, electrical, and ionizing and non-ionizing radiation. Approximately half a million burn cases are registered annually, of which 40 thousand patients are hospitalized and receive definitive treatment. Burn care is very resource intensive as the treatment regimens and length of hospitalization are substantial. Burn wounds are classified based on depth as superficial (first degree), partial-thickness (second degree), or full-thickness (third degree), which determines the treatment necessary for successful healing. The goal of burn wound care is to fully restore the barrier function of the tissue as quickly as possible while minimizing infection, scarring, and contracture. The aim of this review is to highlight how tissue engineering and regenerative medicine strategies are being used to address the unique challenges of burn wound healing and define the current gaps in care for both partial- and full-thickness burn injuries. This review will present the current standard of care (SOC) and provide information on various treatment options that have been tested pre-clinically or are currently in clinical trials. Due to the complexity of burn wound healing compared to other skin injuries, burn specific treatment regimens must be developed. Recently, tissue engineering and regenerative medicine strategies have been developed to improve skin regeneration that can restore normal skin physiology and limit adverse outcomes, such as infection, delayed re-epithelialization, and scarring. Our emphasis will be centered on how current clinical and pre-clinical research of pharmacological agents, biomaterials, and cellular-based therapies can be applied throughout the continuum of burn care by targeting the stages of wound healing: hemostasis, inflammation, cell proliferation, and matrix remodeling.
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http://dx.doi.org/10.3389/fphar.2018.00672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046385PMC
July 2018

Predicting the Ability of Wounds to Heal Given Any Burn Size and Fluid Volume: An Analytical Approach.

J Burn Care Res 2018 08;39(5):661-669

U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

The intrinsic relationship between fluid volume and open wound size (%) has not been previously examined. Therefore, we conducted this study to investigate whether open wound size can be predicted from fluid volume plus other significant factors over time and to evaluate how machine learning may perform in predicting open wound size. This retrospective study involved patients with at least 20% TBSA burned. Various predictive models were developed and compared using goodness-of-fit statistics (R2, error [mean absolute error (MAE), root mean squared error (RMSE)]). Bland-Altman analysis was also performed to determine bias. A total of 121 patients were included in the analysis. Median TBSA burned was 31% (interquartile range: 26-46%). Average crystalloid volumes were 4.0 ± 2.7 ml/kg/TBSA in the first 24 hours. There were 24 (20%) patients who died. Importantly, multivariate analysis identified seven independent predictors of open wound size. Also, machine learning analysis was able to stratify patients based on the 20th day after admission, ~40% TBSA burned, and fluid volumes. Models for predicting open wound size varied in performance (R2 = .79-.90, MAE = 3.97-7.52, RMSE = 7.11-10.69). Notably, a combined machine learning model using only four features (fluid volume, days since admission, TBSA burned, age) performed the best and was sufficient to predict open wound size, with >90% goodness of fit and <4% absolute error. Bland-Altman analysis showed that there were no biases in the models. Open wound size can be predicted reliably using machine learning and fluid volume, days since admission, TBSA burned, and age. Future work will be needed to validate the utility of this study's models in a clinical environment.
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http://dx.doi.org/10.1093/jbcr/iry021DOI Listing
August 2018

Assessment of Ablative Fractional CO2 Laser and Er:YAG Laser to Treat Hypertrophic Scars in a Red Duroc Pig Model.

J Burn Care Res 2018 10;39(6):954-962

Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida.

Hypertrophic scarring is a fibroproliferative process that occurs following a third-degree dermal burn injury, producing significant morbidity due to persistent pain, itching, cosmetic disfigurement, and loss of function due to contractures. Ablative fractional lasers have emerged clinically as a fundamental or standard therapeutic modality for hypertrophic burn scars. Yet the examination of their histopathological and biochemical mechanisms of tissue remodeling and comparison among different laser types has been lacking. In addition, deficiency of a relevant animal model limits our ability to gain a better understanding of hypertrophic scar pathophysiology. To evaluate the effect of ablative fractional lasers on hypertrophic third-degree burn scars, we have developed an in vivo Red Duroc porcine model. Third-degree burn wounds were created on the backs of animals, and burn scars were allowed to develop for 70 days before treatment. Scars received treatment with either CO2 or erbium: yttrium aluminum garnet (YAG) ablative fractional lasers. Here, we describe the effect of both lasers on hypertrophic third-degree burn scars in Red Duroc pigs. In this report, we found that Er:YAG has improved outcomes versus fractional CO2. Molecular changes noted in the areas of dermal remodeling indicated that matrix metalloproteinase 2, matrix metalloproteinase 9, and Decorin may play a role in this dermal remodeling and account for the enhanced effect of the Er:YAG laser. We have demonstrated that ablative fractional laser treatment of burn scars can lead to favorable clinical, histological, and molecular changes. This study provides support that hypertrophic third-degree burn scars can be modified by fractional laser treatment.
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http://dx.doi.org/10.1093/jbcr/iry012DOI Listing
October 2018

Delivery of Allogeneic Adipose Stem Cells in Polyethylene Glycol-Fibrin Hydrogels as an Adjunct to Meshed Autografts After Sharp Debridement of Deep Partial Thickness Burns.

Stem Cells Transl Med 2018 04 18;7(4):360-372. Epub 2018 Feb 18.

Burn Injury Research, United States Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas, USA.

Harvesting of autografts results in donor site morbidities and is limited in scenarios such as large total body surface area burns. In these instances, coverage is increased by meshing grafts at the expense of delayed biologic closure. Moreover, graft meshing increases the likelihood of contraction and hypertrophic scarring, limits range of motion, and worsens cosmesis. Many tissue engineering technologies have touted the promise of adipose-derived stem cells (ASCs) for burn wounds. The primary objective of the current study was to determine feasibility and efficacy of in situ ASC delivery via PEGylated fibrin (FPEG) hydrogels as adjuncts to meshed split thickness skin grafts in a porcine model. Deep partial thickness burns were created on the dorsum of anesthetized Yorkshire pigs, and subsequently debrided on post-burn day 4. After debridement, wounds were treated with: split thickness skin grafts (STSG); meshed STSG (mSTSG); and mSTSG + FPEG with increasing doses of ASCs. We show that FPEG hydrogels can be delivered in situ to prevent the contraction seen after meshing of STSG. Moreover, ASCs delivered in FPEG dose-dependently increase blood vessel size which significantly correlates with CD31 protein levels. The current study reports a dual-action adjunct therapy to autografting administered in situ, wherein FPEG acts as both scaffolding to prevent contraction, and as a delivery vehicle for ASCs to accelerate angiogenesis. This strategy may be used to incorporate other biologics for generating tissue engineered products aimed at improving wound healing and minimizing donor sites or scarring. Stem Cells Translational Medicine 2018;7:360-372.
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http://dx.doi.org/10.1002/sctm.17-0160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866942PMC
April 2018

Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison.

Cell Transplant 2017 12;26(12):1840-1854

4 Transplant Center San Antonio, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized-solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.
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http://dx.doi.org/10.1177/0963689717742157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802637PMC
December 2017

Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells.

Stem Cells Int 2017 23;2017:7108458. Epub 2017 Aug 23.

Combat Trauma and Burn Injury Research, US Army Institute of Surgical Research, San Antonio Military Medical Center, JBSA Ft Sam Houston, San Antonio, TX, USA.

Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95-99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively ( < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.
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http://dx.doi.org/10.1155/2017/7108458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613713PMC
August 2017

A PEGylated platelet free plasma hydrogel based composite scaffold enables stable vascularization and targeted cell delivery for volumetric muscle loss.

Acta Biomater 2018 Jan 8;65:150-162. Epub 2017 Nov 8.

Combat Trauma and Burn Injury Research, Institute of Surgical Research, TX 78234, United States. Electronic address:

Extracellular matrix (ECM) scaffolds are being used for the clinical repair of soft tissue injuries. Although improved functional outcomes have been reported, ECM scaffolds show limited tissue specific remodeling response with concomitant deposition of fibrotic tissue. One plausible explanation is the regression of blood vessels which may be limiting the diffusion of oxygen and nutrients across the scaffold. Herein we develop a composite scaffold as a vasculo-inductive platform by integrating PEGylated platelet free plasma (PFP) hydrogel with a muscle derived ECM scaffold (m-ECM). In vitro, adipose derived stem cells (ASCs) seeded onto the composite scaffold differentiated into two distinct morphologies, a tubular network in the hydrogel, and elongated structures along the m-ECM scaffold. The composite scaffold showed a high expression of ITGA5, ITGB1, and FN and a synergistic up-regulation of ang1 and tie-2 transcripts. The in vitro ability of the composite scaffold to provide extracellular milieu for cell adhesion and molecular cues to support vessel formation was investigated in a rodent volumetric muscle loss (VML) model. The composite scaffold delivered with ASCs supported robust and stable vascularization. Additionally, the composite scaffold supported increased localization of ASCs in the defect demonstrating its ability for localized cell delivery. Interestingly, ASCs were observed homing in the injured muscle and around the perivascular space possibly to stabilize the host vasculature. In conclusion, the composite scaffold delivered with ASCs presents a promising approach for scaffold vascularization. The versatile nature of the composite scaffold also makes it easily adaptable for the repair of soft tissue injuries.

Statement Of Significance: Decellularized extracellular matrix (ECM) scaffolds when used for soft tissue repair is often accompanied by deposition of fibrotic tissue possibly due to limited scaffold vascularization, which limits the diffusion of oxygen and nutrients across the scaffold. Although a variety of scaffold vascularization strategies has been investigated, their limitations preclude rapid clinical translation. In this study we have developed a composite scaffold by integrating bi-functional polyethylene glycol modified platelet free plasma (PEGylated PFP) with adipose derived stem cells (ASCs) along with a muscle derived ECM scaffold (m-ECM). The composite scaffold provides a vasculo-inductive and an effective cell delivery platform for volumetric muscle loss.
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http://dx.doi.org/10.1016/j.actbio.2017.11.019DOI Listing
January 2018

Sound-stress-induced altered nociceptive behaviors are associated with increased spinal CRFR2 gene expression in a rat model of burn injury.

J Pain Res 2017 1;10:2135-2145. Epub 2017 Sep 1.

United States Army Institute of Surgical Research, San Antonio Military Medical Center, Fort Sam Houston, San Antonio, TX, USA.

Sound stress (SS) elicits behavioral changes, including pain behaviors. However, the neuronal mechanisms underlying SS-induced pain behaviors remain to be explored. The current study examined the effects of SS on nociceptive behaviors and changes in expression of the spinal corticotropin-releasing factor (CRF) system in male Sprague Dawley rats with and without thermal pain. We also studied the effects of SS on plasma corticosterone and fecal output. Rats were exposed to 3 days of SS protocol (n = 12/group). Changes in nociceptive behaviors were assessed using thermal and mechanical pain tests. Following the induction of SS, a subgroup of rats (n = 6/group) was inflicted with thermal injury and on day 14 postburn nociceptive behaviors were reassessed. Spinal CRF receptor mRNA expression was analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). In addition, plasma corticosterone and spinal CRF concentrations were quantified using enzyme-linked immunosorbent assay (ELISA). Increased defecation was observed in SS rats. SS produced transient mechanical allodynia in naive rats, whereas it exacerbated thermal pain in thermally injured rats. Spinal mRNA expression was unaffected by stress or thermal injury alone, but their combined effect significantly increased its expression. SS had no effect on plasma corticosterone and spinal CRF protein in postburn rats. To conclude, SS is capable of exacerbating postburn thermal pain, which is linked to increased gene expression in the spinal cord. Future studies have to delineate whether attenuation of CRFR2 signaling at the spinal level prevents stress-induced exacerbation of burn pain.
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http://dx.doi.org/10.2147/JPR.S144055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589110PMC
September 2017

Antinociceptive effects of pluronic lecithin organo (PLO)-opioid gels in rats with thermal injury.

Burns 2017 Dec 1;43(8):1709-1716. Epub 2017 Aug 1.

Burn Injuries Task Area, United States Army Institute of Surgical Research, 3698 Chambers Pass, San Antonio Military Medical Center, Fort Sam Houston, TX 78234, United States. Electronic address:

Opioids are extensively used as analgesics to control burn pain. However, systemic administration of opioids induces multiple adverse effects that are primarily CNS mediated. Alternately, topical application of low dose of opioids directly at the site of injury could attenuate pain while avoiding CNS-mediated side effects. Pluronic lecithin organogels (PLO) have been extensively used as vehicles to deliver topical drugs. In this study, we for the first time assessed the analgesic efficacy of three opioid-PLO formulations (fentanyl, methadone & morphine) in a rat full-thickness thermal injury (FTTI) pain model. Experiments were performed using 44 adult male Sprague-Dawley rats. A single 0.1mL topical application of either morphine (5mg/mL, n=6), fentanyl (10μg/mL, n=8), methadone gel (5mg/mL, n=8), ketamine (50mg/mL, n=6), saline (0.1mL, n=8) or PLO gel alone (0.1mL, n=8) was administered to the plantar surface of the injured hindpaw on days 4 and 7 following thermal injury. The anti-hyperalgesic effects were then measured (5, 15, 30, 60 and 120min post-drug application) using the Hargreaves' thermal test. All three opioids produced statistically significant increases in paw withdrawal latency (PWL), taken as a measure of anti-hyperalgesia, in comparison to saline-treated group (P<0.05), at both 4 and 7days post injury, with fentanyl showing greatest efficacy. Taken together, a low dose of topical application of opioids can reduce thermal hyperalgesia in a rat hindpaw FTTI model, supporting the development of topical formulations of these drugs for burn pain treatment in the clinic.
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http://dx.doi.org/10.1016/j.burns.2017.04.020DOI Listing
December 2017
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