Hyperspectral Imaging for Burn Depth Assessment in an Animal Model.

UNLABELLED: Differentiating between superficial and deep-dermal (DD) burns remains challenging. Superficial-dermal burns heal with conservative treatment; DD burns often require excision and skin grafting. Decision of surgical treatment is often delayed until burn depth is definitively identified. This study's aim is to assess the ability of hyperspectral imaging (HSI) to differentiate burn depth. METHODS: Thermal injury of graded severity was generated on the dorsum of hairless mice with a heated brass rod. Perfusion and oxygenation parameters of injured skin were measured with HSI, a noninvasive method of diffuse reflectance spectroscopy, at 2 minutes, 1, 24, 48 and 72 hours after wounding. Burn depth was measured histologically in 12 mice from each burn group (n = 72) at 72 hours. RESULTS: Three levels of burn depth were verified histologically: intermediate-dermal (ID), DD, and full-thickness. At 24 hours post injury, total hemoglobin (tHb) increased by 67% and 16% in ID and DD burns, respectively. In contrast, tHb decreased to 36% of its original levels in full-thickness burns. Differences in deoxygenated and tHb among all groups were significant (P < 0.001) at 24 hours post injury. CONCLUSIONS: HSI was able to differentiate among 3 discrete levels of burn injury. This is likely because of its correlation with skin perfusion: superficial burn injury causes an inflammatory response and increased perfusion to the burn site, whereas deeper burns destroy the dermal microvasculature and a decrease in perfusion follows. This study supports further investigation of HSI in early burn depth assessment.

Smartphone-based wound assessment system for patients with diabetes.

Diabetic foot ulcers represent a significant health issue. Currently, clinicians and nurses mainly base their wound assessment on visual examination of wound size and healing status, while the patients themselves seldom have an opportunity to play an active role. Hence, a more quantitative and cost-effective examination method that enables the patients and their caregivers to take a more active role in daily wound care potentially can accelerate wound healing, save travel cost and reduce healthcare expenses. Considering the prevalence of smartphones with a high-resolution digital camera, assessing wounds by analyzing images of chronic foot ulcers is an attractive option. In this paper, we propose a novel wound image analysis system implemented solely on the Android smartphone. The wound image is captured by the camera on the smartphone with the assistance of an image capture box. After that, the smartphone performs wound segmentation by applying the accelerated mean-shift algorithm. Specifically, the outline of the foot is determined based on skin color, and the wound boundary is found using a simple connected region detection method. Within the wound boundary, the healing status is next assessed based on red-yellow-black color evaluation model. Moreover, the healing status is quantitatively assessed, based on trend analysis of time records for a given patient. Experimental results on wound images collected in UMASS-Memorial Health Center Wound Clinic (Worcester, MA) following an Institutional Review Board approved protocol show that our system can be efficiently used to analyze the wound healing status with promising accuracy.

Seroma Formation in Rat Latissimus Dorsi Resection in the Presence of Biologics: The Role of Quilting.

BACKGROUND: Seroma formation is a well-recognized complication associated with many operative procedures. Despite its ubiquity, a lack of definitive scientific understanding of the etiology, natural history, and biochemistry of seromas remains. We endeavored to create and examine seromas in a rat model in the setting of commonly used biologic implants and to examine the role of quilting sutures/mechanical fixation in mitigating seroma development. METHODS: Female Sprague-Dawley rats were assigned to either Quilting or Nonquilting groups then subdivided into one of 3 porcine dermal implant groups (Permacol Surgical Implant, Strattice Reconstructive Tissue Matrix, or XCM Biologic Tissue Matrix) or control group. A 5-cm midline back incision was made, the skin reflected and the latissimus dorsi muscle resected bilaterally. Implants were sutured into the surgical bed using a running suture. The skin of nonquilted rats was closed with a running subcuticular suture. Quilted rats underwent placement of absorbable quilting sutures spaced 2 cm apart between the skin and underlying implant or muscle before skin closure. Postoperatively, rats were monitored for seroma formation with fluid aspirated as needed. At 28 or 90 days, rats were euthanized. Seroma and implants were examined grossly and under light microscopy. RESULTS: Of nonquilted rats, 42/54 (78%) developed seromas compared with 19/46 (41%) of quilted rats (P < 0.05), defined by bursa cavity present at necropsy. When a biologic implant was present, 28/35 (80%) of nonquilted rats developed seromas compared with 12/33 (36%) of quilted rats (P < 0.05). In the control group, 14/19 (74%) of nonquilted rats developed seromas compared with 7/13 (54%) of quilted rats. This difference was not statistically significant. Bursa presence was confirmed histologically in all cases, with no difference in bursa character seen between groups. CONCLUSIONS: This study confirms a reliable rat model of seroma formation, with most of the rats exhibiting at least subclinical seromas. There was no difference in seroma formation rate in the presence of biologic implants, and no differences in bursa character between implants. Mechanical fixation with quilting sutures decreased seroma rate significantly in all subgroups. All rats with seromas at necropsy had histological evidence of a bursa with no difference in appearance between groups.

Hyperspectral imaging for early detection of oxygenation and perfusion changes in irradiated skin.

Studies examining acute oxygenation and perfusion changes in irradiated skin are limited. Hyperspectral imaging (HSI), a method of wide-field, diffuse reflectance spectroscopy, provides noninvasive, quantified measurements of cutaneous oxygenation and perfusion. This study examines whether HSI can assess acute changes in oxygenation and perfusion following irradiation. Skin on both flanks of nude mice (n=20) was exposed to 50 Gy of beta radiation from a strontium-90 source. Hyperspectral images were obtained before irradiation and on selected days for three weeks. Skin reaction assessment was performed concurrently with HSI. Desquamative injury formed in all irradiated areas. Skin reactions were first seen on day 7, with peak formation on day 14, and resolution beginning by day 21. HSI demonstrated increased tissue oxygenation on day 1 before cutaneous changes were observed (p<0.001). Further increases over baseline were seen on day 14, but returned to baseline levels by day 21. For perfusion, similar increases were seen on days 1 and 14. Unlike tissue oxygenation, perfusion was decreased below baseline on day 21 (p<0.002). HSI allows for complete visualization and quantification of tissue oxygenation and perfusion changes in irradiated skin, and may also allow prediction of acute skin reactions based on early changes seen after irradiation.

Accelerated porcine wound healing after treatment with α-gal nanoparticles.

BACKGROUND: The α-gal epitope is a carbohydrate antigen that interacts specifically with the natural anti-Gal antibody--the most abundant antibody in humans. Anti-Gal/α-gal epitope interaction activates complement to generate chemotactic factors that induce rapid recruitment of macrophages. The authors hypothesized that α-gal epitopes on nanoparticles can accelerate wound healing by inducing rapid recruitment and activation of macrophages in wounds. METHODS: α-Gal nanoparticles were generated from phospholipids, cholesterol, and α-gal glycolipids. α-Gal nanoparticle treatment of wounds was studied in 12 α1,3galactosyltrasferase knockout pigs. Like humans, these pigs lack α-gal epitopes and produce the natural anti-Gal antibody. Full-thickness wounds (20 × 20 mm) with tattooed borders were created on the back of pigs. α-Gal nanoparticles (10 or 100 mg) were topically applied onto the wounds. Saline-treated wounds served as control. Wound open surface area was measured every 3 to 4 days during dressing changes. Wounds from euthanized pigs were subjected to histological evaluation. RESULTS: Treated wounds displayed many more macrophages and increased angiogenesis than control wounds in the same pig. On day 10, wounds treated with 10 mg and 100 mg displayed 35 and 60 percent decreased open surface area compared with control wounds, respectively, and 80 and 90 percent less than control wounds on day 13 (p < 0.05). No keloid formation or no increase in scar formation was observed on day 60. CONCLUSIONS: α-Gal nanoparticle treatment of wounds accelerates macrophage recruitment, angiogenesis, and wound healing in pigs producing the anti-Gal antibody. As humans produce high titers of anti-Gal antibodies, this treatment may exhibit a similar beneficial effect in the clinical setting.

Engraftment of human HSCs in nonirradiated newborn NOD-scid IL2rγ null mice is enhanced by transgenic expression of membrane-bound human SCF.

Immunodeficient mice engrafted with human HSCs support multidisciplinary translational experimentation, including the study of human hematopoiesis. Heightened levels of human HSC engraftment are observed in immunodeficient mice expressing mutations in the IL2-receptor common γ chain (IL2rg) gene, including NOD-scid IL2rγ(null) (NSG) mice. Engraftment of human HSC requires preconditioning of immunodeficient recipients, usually with irradiation. Such preconditioning increases the expression of stem cell factor (SCF), which is critical for HSC engraftment, proliferation, and survival. We hypothesized that transgenic expression of human membrane-bound stem cell factor Tg(hu-mSCF)] would increase levels of human HSC engraftment in nonirradiated NSG mice and eliminate complications associated with irradiation. Surprisingly, detectable levels of human CD45(+) cell chimerism were observed after transplantation of cord blood-derived human HSCs into nonirradiated adult as well as newborn NSG mice. However, transgenic expression of human mSCF enabled heightened levels of human hematopoietic cell chimerism in the absence of irradiation. Moreover, nonirradiated NSG-Tg(hu-mSCF) mice engrafted as newborns with human HSCs rejected human skin grafts from a histoincompatible donor, indicating the development of a functional human immune system. These data provide a new immunodeficient mouse model that does not require irradiation preconditioning for human HSC engraftment and immune system development.

NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.

BACKGROUND: Transplantation of human skin on immunodeficient mice that support engraftment with functional human immune systems would be an invaluable tool for investigating mechanisms involved in wound healing and transplantation. Nonobese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) readily engraft with human immune systems, but human skin graft integrity is poor. In contrast, human skin graft integrity is excellent on CB17-scid bg (SCID.bg) mice, but they engraft poorly with human immune systems. METHODS: Human skin grafts transplanted onto immunodeficient NSG, SCID.bg, and other immunodeficient strains were evaluated for graft integrity, preservation of graft endothelium, and their ability to be rejected after engraftment of allogeneic peripheral blood mononuclear cells. RESULTS: Human skin transplanted onto NSG mice develops an inflammatory infiltrate, consisting predominately of host Gr1(+) cells, that is detrimental to the survival of human endothelium in the graft. Treatment of graft recipients with anti-Gr1 antibody reduces this cellular infiltrate, preserves graft endothelium, and promotes wound healing, tissue development, and graft remodeling. Excellent graft integrity of the transplanted skin includes multilayered stratified human epidermis, well-developed human vasculature, human fibroblasts, and passenger leukocytes. Injection of unfractionated, CD4 or CD8 allogeneic human peripheral blood mononuclear cell induces a rapid destruction of the transplanted skin graft. CONCLUSIONS: NSG mice treated with anti-Gr1 antibody provide a model optimized for both human skin graft integrity and engraftment of a functional human immune system. This model provides the opportunity to investigate mechanisms orchestrating inflammation, wound healing, revascularization, tissue remodeling, and allograft rejection and can provide guidance for improving outcomes after clinical transplantation.

Use of gauze-based negative pressure wound therapy in a pediatric burn patient.

Negative pressure wound therapy (NPWT) is described as it is used in the treatment of an infant burn victim. This case highlights the ability and techniques used to maintain an airtight dressing seal in the perirectal region. Use of this dressing type post-skin grafting allowed for 100% graft adhesion and no bacterial contamination despite close proximity to the rectum. Favorable experience and outcome with this patient are strong indicators that NPWT should be considered as a viable treatment in pediatric populations and that situations where body contour or fluids may make NPWT difficult to administer should not be a deterrent to therapy.

Remote ischemic preconditioning modulates p38 MAP kinase in rat adipocutaneous flaps.

Ischemic preconditioning has been shown to improve survival of cutaneous flaps. The authors examined the effect of remote ischemic preconditioning (RIPC) on phosphorylation of p38 MAP kinase and related the results to flap survival. Female Wistar rats had 8 x 12-cm abdominal adipocutaneous flaps raised on the medial branch of the superficial epigastric artery. Controls (Group 1) had the flap elevated and the pedicle clamped for 3 hr, then closed with a sheet of plastic between the flap and abdominal wall. Group 2 animals had RIPC by tourniquet on the contralateral hind limb before the flap was dissected. Group 3 animals mimicked Group 2 and also had an infusion of the nitric oxide blocker, N-nitro-L-arginine methyl ester (L-NAME) 5 min prior to the RIPC. Group 4 had the flap elevated prior to the RIPC. All groups except Group 1 had 10 min of RIPC with 30 min of reperfusion, then 3 hr of ischemia. Tissue samples were taken at the distal margins of the flaps before preconditioning and 30 min after preconditioning for detection of p38 MAP kinase and phosphorylated p38 MAP kinase (pp38 MAP kinase). Group 2 flaps (RIPC before flap elevation) exhibited better flap tissue survival and had well-defined phosphorylation of p38 MAP kinase 30 min post RIPC, when compared to the other groups. Pre-infusion with the nitric oxide blocker (Group 3) before RIPC blocked the survival advantage conferred by preconditioning and diminished the phosphorylation of p38 MAP kinase. Tissue from all groups showed very little phosphorylation of p38 MAP kinase following 3 hr of ischemia. Thus, increased tissue survival is correlated with elevated levels of p38 MAP kinase phosphorylation following RIPC. This effect is inhibited by blockade of nitric oxide. Modulation of the p38 MAP kinase pathway may represent a protection pathway for ischemic preconditioning.

Periosteal cell pellet culture system: a new technique for bone engineering.

To treat bone loss that is induced by disease or wounds, bone grafts are commonly used. In dentistry, guided tissue regeneration is effective in the treatment of periodontal diseases. However, bone resorption after implantation is a major problem with the bone graft and guided tissue regeneration technique. This study examines a cell pellet culture system without exogenous scaffolds for bone regeneration. First, we examined the effect of ascorbic acid on cells. Transmission electron microscopic observation revealed that cells formed a three-dimensional structure of multiple cell layers after 5 weeks of culturing in medium containing 50 microg/ ml ascorbic acid with the medium changed every 7 days. A single cell pellet was produced by centrifuging cells that were gathered from 10 tissue culture dishes. Van Gieson staining and collagen type I immunostaining showed that the pellet contained collagen fibers and cells that adhered to the collagen fibers. Several of these cell pellets were implanted subcutaneously on the backs of nude mice for 6 weeks. Histology and immunohistochemistry results indicated new bone formation, vascular invasion, and insular areas of calcification. Bone tissue was surrounded by osteoblasts. The appearance of new bone formation is similar to that seen in intramembranous ossification. The present pellet system is reliable and might solve problems of bone resorption after implantation.

The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium.

Regenerative medicine approaches for the treatment of damaged or missing myocardial tissue include cell-based therapies, scaffold-based therapies, and/or the use of specific growth factors and cytokines. The present study evaluated the ability of extracellular matrix (ECM) derived from porcine urinary bladder to serve as an inductive scaffold for myocardial repair. ECM scaffolds have been shown to support constructive remodeling of other tissue types including the lower urinary tract, the dermis, the esophagus, and dura mater by mechanisms that include the recruitment of bone marrow-derived progenitor cells, angiogenesis, and the generation of bioactive molecules that result from degradation of the ECM. ECM derived from the urinary bladder matrix, identified as UBM, was configured as a single layer sheet and used as a biologic scaffold for a surgically created 2 cm2 full-thickness defect in the right ventricular free wall. Sixteen dogs were divided into two equal groups of eight each. The defect in one group was repaired with a UBM scaffold and the defect in the second group was repaired with a Dacron patch. Each group was divided into two equal subgroups (n = 4), one of which was sacrificed 15 min after surgical repair and the other of which was sacrificed after 8 weeks. Global right ventricular contractility was similar in all four subgroups groups at the time of sacrifice. However, 8 weeks after implantation the UBM-treated defect area showed significantly greater (p < 0.05) regional systolic contraction compared to the myocardial defects repaired with by Dacron (3.3 +/- 1.3% vs. -1.8 +/- 1.1%; respectively). Unlike the Dacron-repaired region, the UBM-repaired region showed an increase in systolic contraction over the 8-week implantation period (-4.2 +/- 1.7% at the time of implantation vs. 3.3 +/- 1.3% at 8 weeks). Histological analysis showed the expected fibrotic reaction surrounding the embedded Dacron material with no evidence for myocardial regeneration. Histologic examination of the UBM scaffold site showed cardiomyocytes accounting for approximately 30% of the remodeled tissue. The cardiomyocytes were arranged in an apparently randomly dispersed pattern throughout the entire tissue specimen and stained positive for alpha- sarcomeric actinin and Connexin 43. The thickness of the UBM graft site increased greatly from the time of implantation to the 8-week sacrifice time point when it was approximately the thickness of the normal right ventricular wall. Histologic examination suggested complete degradation of the originally implanted ECM scaffold and replacement by host tissues. We conclude that UBM facilitates a constructive remodeling of myocardial tissue when used as replacement scaffold for excisional defects.

Tissue-engineered trachea from sheep marrow stromal cells with transforming growth factor beta2 released from biodegradable microspheres in a nude rat recipient.

OBJECTIVE: The purpose of this study was to evaluate the feasibility of using autologous sheep marrow stromal cells cultured onto polyglycolic acid mesh to develop helical engineered cartilage equivalents for a functional tracheal replacement. We also explored the potential benefit of local delivery of transforming growth factor beta 2 with biodegradable gelatin microspheres. METHODS: Bone marrow was obtained by iliac crest aspiration from 6-month-old sheep and cultured in monolayer for 2 weeks. At confluence, the cells were seeded onto nonwoven polyglycolic acid fiber mesh and cultured in vitro with transforming growth factor beta 2 and insulin-like growth factor 1 for 1 week. Cell-polymer constructs were wrapped around a silicone helical template. Constructs were then coated with microspheres incorporating 0.5 microg transforming growth factor beta 2. The cell-polymer-microsphere structures were then implanted into a nude rat. On removal, glycosaminoglycan content and hydroxyproline were analyzed in both native and tissue-engineered trachea. Histologic sections of both native and tissue-engineered trachea were stained with hematoxylin and eosin, safranin-O, and a monoclonal anti-type II collagen antibody. RESULTS: Cell-polymer constructs with transforming growth factor beta 2 microspheres formed stiff cartilage de novo in the shape of a helix after 6 weeks. Control constructs lacking transforming growth factor beta 2 microspheres appeared to be much stiffer than typical cartilage, with an apparently mineralized matrix. Tissue-engineered trachea was similar to normal trachea. Histologic data showed the presence of mature cartilage. Glycosaminoglycan and hydroxyproline contents were also similar to native cartilage levels. CONCLUSIONS: This study demonstrates the feasibility of engineering tracheas with sheep marrow stromal cells as a cell source. Engineering the tracheal equivalents with supplemental transforming growth factor beta 2 seemed to have a positive effect on retaining a cartilaginous phenotype in the newly forming tissue.

Novel technique for peripheral nerve reconstruction in the absence of an artificial conduit.

The purpose of this study is to promote nerve regeneration across a peripheral nerve gap, using a biologic, tissue-engineered nerve (TEN), containing a high density of viable Schwann cells (SCs) in the absence of supportive foreign materials and a tubular system. Isolated SCs from adult rat sciatic nerve were seeded onto biodegradable constructs and implanted into the backs of nude mice to create TENs. Six weeks later, the constructs were harvested, implanted into surgically created sciatic nerve gaps in rats without supportive artificial conduits and compared with both an autograft group and a silicone conduit group using SCs. Two months later, functional assessment was evaluated by walking track analysis and the implanted lesions were imaged by transmission electron microscopy. The axonal number and sciatic function index of the TEN were significantly higher than those of the silicone group and achieved a comparable level to the autograft group. The results indicate that the large number of SCs within their own extracellular matrix appeared sufficient to enable neuronal growth across a nerve gap in the absence of an artificial conduit and that these circumstances may have a positive effect on the supplement of growth factors from the surrounding tissues of implanted TEN.

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea.

BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.