Tumescent Injections in Subcutaneous Pig Tissue Disperse Fluids Volumetrically and Maintain Elevated Local Concentrations of Additives for Several Hours, Suggesting a Treatment for Drug Resistant Wounds.

PurposeBolus injection of fluid into subcutaneous tissue results in accumulation of fluid at the injection site. The fluid does not form a pool. Rather, the injection pressure forces the interstitial matrix to expand to accommodate the excess fluid in its volume, and the fluid becomes bound similar to that in a hydrogel. We seek to understand the properties and dynamics of externally tumesced (swollen) subcutaneous tissue as a first step in assessing whether tumescent antibiotic injections into wounds may provide a novel method of treatment.MethodsSubcutaneous injections of saline are performed in live and dead pigs and the physical properties (volume, expansion ratio, residence time, apparent diffusion constant) of the resulting fluid deposits are observed with diffusion-weighted magnetic resonance imaging, computed tomography, and 3D scanning.ResultsSubcutaneous tissue can expand to a few times its initial volume to accommodate the injected fluid, which is dispersed thoroughly throughout the tumescent volume. The fluid spreads to peripheral unexpanded regions over the course of a few minutes, after which it remains in place for several hours. Eventually the circulation absorbs the excess fluid and the tissue returns to its original state.ConclusionsGiven the evidence for dense fluid dispersal and several-hour residence time, a procedure is proposed whereby tumescent antibiotic injections are used to treat drug-resistant skin infections and chronic wounds that extend into the subcutaneous tissue. The procedure has the potential to effectively treat otherwise untreatable wounds by keeping drug concentrations above minimum inhibitory levels for extended lengths of time

In vivo investigation of the tissue response to commercial Teflon insulin infusion sets in large swine for 14 days: the effect of angle of insertion on tissue histology and insulin spread within the subcutaneous tissue.

Objective: This study investigated the effects of the inflammatory tissue response (ITR) to an insulin infusion set (IIS) on insulin bolus spread over wear time, as well as the effect of cannula insertion angle on the ITR, bolus shape, and pump tubing pressure. Research design and methods: Angled or straight IISs were inserted every other day for 14 days into the subcutaneous tissue of 11 swine and insulin was delivered continuously. Prior to euthanasia, a 70 µL bolus of insulin/X-ray contrast agent was infused while recording a pressure profile (peak tubing pressure, pmax; area under the pressure curve, AUC), followed by the excision of the tissue-catheter specimen. Bolus surface area (SA) and volume (V) were assessed via micro-CT. Tissue was stained to analyze total area of inflammation (TAI) and inflammatory layer thickness (ILT) surrounding the cannula. Results: A bolus delivered through an angled IIS had a larger mean SA than a bolus delivered through a straight cannula (314.0±84.2 mm2 vs 229.0±99.7 mm2, p\u3c0.001) and a larger volume (198.7±66.9 mm3 vs 145.0±65.9 mm3, p=0.001). Both decreased significantly over wear time, independent of angle. There was a significant difference in TAI (angled, 9.1±4.0 mm2 vs straight, 14.3±8.6 mm2, p\u3c0.001) and ILT (angled, 0.7±0.4 vs straight, 1.2±0.7 mm, p\u3c0.001). pmax (p=0.005) and AUC (p=0.014) were lower using angled IIS. As ILT increased, pmax increased, while SA and V decreased. Conclusions: The progression of the ITR directly affected bolus shape and tubing pressure. Although straight insertion is clinically preferred, our data suggest that an angled IIS elicits lower grades of ITR and delivers a bolus with lower tubing pressure and greater SA and V. The subcutaneous environment plays a crucial role in IIS longevity, and the insertion angle needs to be considered in future IIS designs and clinical trials

Novel application of synchrotron x-ray computed tomography for ex-vivo imaging of subcutaneously-injected polymeric microsphere suspension formulations

Purpose: Subcutaneously or intramuscularly administered biodegradable microsphere formulations have been successfully exploited in the management of chronic conditions for over two decades, yet mechanistic understanding of the impact of formulation attributes on in vivo absorption rate from such systems is still in its infancy. Methods: Suspension formulation physicochemical attributes may impact particulate deposition in subcutaneous (s.c.) tissue. Hence, the utility of synchrotron X-ray microcomputed tomography (μCT) for assessment of spatial distribution of suspension formulation components (PLG microspheres and vehicle) was evaluated in a porcine s.c. tissue model. Optical imaging of dyed vehicle and subsequent microscopic assessment of microsphere deposition was performed in parallel to compare the two approaches. Results: Our findings demonstrate that synchrotron μCT can be applied to the assessment of microsphere and vehicle distribution in s.c. tissue, and that microspheres can also be visualised in the absence of contrast agent using this approach. The technique was deemed superior to optical imaging of macrotomy for the characterisation of microsphere deposition owing to its non-invasive nature and relatively rapid data acquisition time. Conclusions: The μCT method outlined in this study provides a novel insight into the relative distribution of vehicle and suspended PLG microspheres following s.c. injection. A potential application for our findings is understanding the impact of injection, device and formulation variables on initial and temporal depot geometry in pre-clinical or ex-vivo models that can inform product design

Effective method for drug injection into subcutaneous tissue

Subcutaneous injection of drug solution is widely used for continuous and low dose drug treatment. Although the drug injections have been administered for a long time, challenges in the design of injection devices are still needed to minimize the variability, pain, or skin disorder by repeated drug injections. To avoid these adverse effects, systematic study on the effects of injection conditions should be conducted to improve the predictability of drug effect. Here, the effects of injection conditions on the drug permeation in tissues were investigated using X-ray imaging technique which provides realtime images of drug permeation with high spatial resolution. The shape and concentration distribution of the injected drug solution in the porcine subcutaneous and muscle tissues are visualized. Dynamic movements of the wetting front (WF) and temporal variations of water contents in the two tissues are quantitatively analyzed. Based on the quantitative analysis of the experimental data, the permeability of drug solution through the tissues are estimated according to permeation direction, injection speed, and tissue. The present results would be helpful for improving the performance of drug injection devices and for predicting the drug efficacy in tissues using biomedical simulation.112Ysciescopu

Inducción del cierre apical en el diente con necrosis pulpar y el ápice no formado

Se efectúa una revisión del proceso de apicoformación en dientes con necrosis pulpar y ápice no formado, analizando el mecanismo de cierre apical inducido por el hidróxido de calcio y otras substancias, así como la histopatología del mismo

Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis

Vibrio vulnificus can cause severe necrotic lesions within a short time. Recently, it has been reported that the numbers of wound infection cases in healthy hosts are increasing, for which surgical procedures are essential in many instances to eliminate the pathogen owing to its rapid proliferation. However, the mechanisms by which V. vulnificus can achieve wound infection in healthy hosts have not been elucidated. Here, we advance a systematic understanding of V. vulnificus wound infection through genome-wide identification of the relevant genes. Signature-tagged mutagenesis (STM) has been developed to identify functions required for the establishment of infection including colonization, rapid proliferation, and pathogenicity. Previously, STM had been regarded to be unsuitable for negative selection to detect the virulence genes of V. vulnificus owing to the low colonization and proliferation ability of this pathogen in the intestinal tract and systemic circulation. Alternatively, we successfully identified the virulence genes by applying STM to a murine model of wound infection. We examined a total of 5418 independent transposon insertion mutants by signature-tagged transposon mutagenesis and detected 71 clones as attenuated mutants consequent to disruption of genes by the insertion of a transposon. This is the first report demonstrating that the pathogenicity of V. vulnificus during wound infection is highly dependent on its characteristics: flagellar-based motility, siderophore-mediated iron acquisition system, capsular polysaccharide, lipopolysaccharide, and rapid chromosome partitioning. In particular, these functions during the wound infection process and are indispensable for proliferation in healthy hosts. Our results may thus allow the potential development of new strategies and reagents to control the proliferation of V. vulnificus and prevent human infections

The Synthesis, Characterization, and Cell Seeding of a Collagen/Hydroxyapatite Porous Scaffold for Treatment of Osseous Defects

The need for finding a bone graft substitute stems from the fact that approximately 200,000 bone grafting procedures are performed annually in the U.S. alone. Although biological grafting options (i.e. autografts & allografts) exist, they do suffer from inherent problems. These include limited resources, costly processing, and potential for pathogen transfer. Thus, investigators have sought synthetic alternatives. The objective of this research was to utilize hydroxyapatite (HA) and collagen, both analogues to the major constituents of bone, to fabricate an optimal synthetic osteoconductive/osteoinductive bone scaffold. Current attempts by other investigators to combine the two materials have been met with some difficulties, resulting in free or loosely bound particulate HA within the collagen matrix. This may ultimately result in a foreign body response to the disseminated crystals. Yet, this study revealed that through manipulation of the collagen\u27s collodial chemistry, it could be made into a more effective carrier medium for particulate HA. It was found that treating the collagen with either IOOmM NaOH or hydrochloric acid followed by titration with 1 N NaOH to a basic pH ( approximately 11 . 8) would yield an adhesive, paste-like matrix capable of incorporating the HA, following lyophilization of the matrix. Composites made under acidic, neutral, and conditions correlating to pH values below 11 .8 were found to possess free HA particulates and loose, friable collagen fibers. The composites were also frozen at two different temperatures to study their effects on microporosity formation. Porosity is an essential characteristic given that it serves as 111 pathways by which vasculature is established, cellular elements may infiltrate; and by which nutrients are supplied to the graft. It was found that porosity of the composite could be controlled by regulating the ice crystal formation prior to lyophilization of the material. Micron size pores were achieved by freezing the composite to -l 5°C and controlling the amount of particulate HA added to the system. TGF-β 1 was also studied for its efficacy in serving as an osteoinductive catalyst with the collagen/HA composite. It was chosen due to its ability to modulate the growth and differentiation of osteoblast precursor cells. Utilizing 1125 as a tracer, release kinetics of the adsorbed polypeptide from the composite were evaluated. Elution was rapid, with approximately 56 ± 3.5% of the theoretical load release following 24 hours incubation. Improved methods by which to prolong the release of the growth factor may be needed in order to provide for optimal inductive properties. Last, the cytocompatibility of the composite was evaluated. Primary cultures of adult, rabbit and fetal bovine osteoblasts were seeded into two formulations of the collagen/HA composite. Following three days of culture, an acid-to-base titrated composite formulation grafted with fetal bovine cells was the only combination which exhibited the presence of adherent cells. The lack of fetal cells upon a base-only treated collagen/HA composite was speculated to be a result of modifications to the collagen due to processing. Seeding technique and cell donor age were also suggested as possible reasons for the absence of cells utilizing adult rabbit cells on either composite formulation. Together, the findings suggest that the composite could serve as a suitable, porous bone scaffold. Due to its compressive strength values, it would best be reserved for non-load-bearing applications, such as an osseous filler material used, for example, in IV conjunction with plated fractures. Improved TGF-β1 incorporation, in-depth in vitro studies, as well as an in vivo model will need to be assessed to determine the true effectiveness of the composite to replace existing biological sources

In vivo and in vitro studies of adipogenesis with particular reference to adipocyte development in rodent skin.

Skin comprises an epidermis, dermis, skin appendages including hair follicles, and a fat layer. There is a growing interest in the biology of specific fat depots, and skin fat is relatively poorly studied. Importantly, most knowledge about the molecular control of adipocyte differentiation comes from in vitro studies on cell lines. This thesis aimed to provide new insights into adipogenesis in vivo by directly studying development of the skin fat layer and its relationship to the surrounding skin and hair follicles. Work, presented in Chapter 2, investigated the timing and localisation of developing fat cells in back skin of rodent embryos. Analysis of the adipogenic transcription factor C/EBPalpha and lipid accumulation revealed preadipocytes in the lower dermis of embryonic mouse skin at e17 and the start of lipid accumulation by e19. The dermal fat cells then created an adipose layer between and beneath hair follicles apparently independently of subcutaneous fat tissue. In Chapter 3, a combined laser capture microdissection and microarray approach generated gene expression profiles of cells from upper and lower dermis over three time points. Verification of the microarray data by qPCR and immunohistochemistry, and bioinformatics analysis confirmed a subdivision of the lower dermis with enriched fat-related pathways. Comparison of this microarray data with published information on adipogenesis of 3T3-cells in vitro showed important early differences with regard to transcription factor, cell cycle, cytoskeletal and extracellular matrix gene expression. Later time points revealed greater similarities between in vivo and in vitro data involving genes characteristic of mature adipocytes. In Chapter 4, the involvement of the Egfr gene (selected from generated microarray lists) in dermal fat development was investigated functionally using a skin organ culture model. In Chapter 5, a marker gene selected from the arrays (Cd36) was successfully used to develop a method of isolating dermal preadipocytes by fluorescence activated cell sorting. Specialised organ culture techniques, presented in Chapters 4 and 5, allowed the adipogenic capabilities of cells from different mouse embryonic skin compartments to be investigated. This revealed a high plasticity of dermal cells at earlier embryonic time points (e15 - 15.5) and their specialisation into either non-fatty cells (upper dermis) or adipocytes (lower dermis) later (e18.5 - 19). As summarised in Chapter 6, this thesis confirmed that the fat layer that develops from cells of the lower dermis should have a distinct nomenclature (dermal adipose tissue) from the subcutaneous fat depot and could be under different regulatory mechanisms. The work has established a new in situ model of in vivo adipogenesis and the microarray data obtained has provided novel information on molecular control of adipogenesis in general, as well as pointers as to why the lower, but not the upper compartment of the late embryonic dermis turns into fat