Deficiency of B vitamins leads to cholesterol-independent atherogenic transformation of the aorta.

Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors, including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in vivo models of atherosclerosis that resemble the human situation. Here, we have developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development, even at low balloon pressure. Our automated system helped to glean vital details of cholesterol-independent changes in the aortic wall of balloon-injured rabbits. We show that deficiency of B vitamins, which are required for homocysteine degradation, leads to atherogenic transformation of the aorta resulting in accumulation of macrophages and lipids, impairment of its biomechanical properties and disorganization of aortic collagen/elastin in the absence of hypercholesterolemia. A combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity compared to any single condition. Our findings suggest that deficiency of B vitamins leads to atherogenic transformation of the aorta even in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.

The Role of Local Inflammation and Hypoxia in the Formation of Hypertrophic Scars-A New Model in the Duroc Pig.

Hypertrophic scars continue to be a major burden, especially after burns. Persistent inflammation during wound healing appears to be the precipitating aspect in pathologic scarring. The lack of a standardized model hinders research from fully elucidating pathophysiology and therapy, as most therapeutic approaches have sparse evidence. The goal of this project was to investigate the mechanisms of scar formation after prolonged wound inflammation and to introduce a method for generating standardized hypertrophic scars by inducing prolonged inflammation. Four wound types were created in Duroc pigs: full-thickness wounds, burn wounds, and both of them with induced hyperinflammation by resiquimod. Clinical assessment (Vancouver Scar Scale), tissue oxygenation by hyperspectral imaging, histologic assessment, and gene expression analysis were performed at various time points during the following five months. Native burn wounds as well as resiquimod-induced full-thickness and burn wounds resulted in more hypertrophic scars than full-thickness wounds. The scar scale showed significantly higher scores in burn- and resiquimod-induced wounds compared with full-thickness wounds as of day 77. These three wound types also showed relative hypoxia compared with uninduced full-thickness wounds in hyperspectral imaging and increased expression of HIF1a levels. The highest number of inflammatory cells was detected in resiquimod-induced full-thickness wounds with histologic features of hypertrophic scars in burn and resiquimod-induced wounds. Gene expression analysis revealed increased inflammation with only moderately altered fibrosis markers. We successfully created hypertrophic scars in the Duroc pig by using different wound etiologies. Inflammation caused by burns or resiquimod induction led to scars similar to human hypertrophic scars. This model may allow for the further investigation of the exact mechanisms of pathological scars, the role of hypoxia and inflammation, and the testing of therapeutic approaches.

Accuracy and stability of an arterial sensor for glucose monitoring in a porcine model using glucose clamp technique.

Intravascular glucose sensors have the potential to improve and facilitate glycemic control in critically ill patients and might overcome measurement delay and accuracy issues. This study investigated the accuracy and stability of a biosensor for arterial glucose monitoring tested in a hypo- and hyperglycemic clamp experiment in pigs. 12 sensors were tested over 5 consecutive days in 6 different pigs. Samples of sensor and reference measurement pairs were obtained every 15 minutes. 1337 pairs of glucose values (range 37-458 mg/dl) were available for analysis. The systems met ISO 15197:2013 criteria in 99.2% in total, 100% for glucose <100 mg/dl (n = 414) and 98.8% for glucose ≥100 mg/dl (n = 923). The mean absolute relative difference (MARD) during the entire glycemic range of all sensors was 4.3%. The MARDs within the hypoglycemic (<70 mg/dl), euglycemic (≥70-180 mg/dl) and hyperglycemic glucose ranges (≥180 mg/dl) were 6.1%, 3.6% and 4.7%, respectively. Sensors indicated comparable performance on all days investigated (day 1, 3 and 5). None of the systems showed premature failures. In a porcine model, the performance of the biosensor revealed a promising performance. The transfer of these results into a human setting is the logical next step.

Author Correction: Systematic in vivo evaluation of the time-dependent inflammatory response to steel and Teflon insulin infusion catheters.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Functional Electrical Stimulation Leads to Increased Volume of the Aged Thyroarytenoid Muscle.

OBJECTIVES/HYPOTHESIS: To reverse sarcopenia and increase the volumes of atrophied laryngeal muscles by functional electrical stimulation (FES) using a minimal invasive surgical procedure in an aged ovine model. STUDY DESIGN: Prospective animal study. METHODS: A stimulation electrode was placed unilaterally near the terminal adduction branch of the recurrent laryngeal nerve (RLN) adjacent to the right cricothyroid joint. The electrode was connected to an implant located subcutaneously at the neck region. Predesigned training patterns were automatically delivered by a bidirectional radio frequency link using a programming device and were repeated automatically by the implant every other day over 11 weeks in the awake animal. Outcome parameters comprised volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM), as well as gene expression analyses. RESULTS: We found significant increases of the volumes of the stimulated TAM of 11% and the TAM diameter at the midmembranous parts of the vocal folds of nearly 40%. Based on gene expression, we did not detect a shift of muscle fiber composition. CONCLUSIONS: FES of the terminal branches of the RLN is a secure and effective way to reverse the effects of age-related TAM atrophy and to increase volumes of atrophied muscles. LEVEL OF EVIDENCE: NA Laryngoscope, 128:2852-2857, 2018.

Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds.

OBJECTIVE: Mesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. METHODS: We topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. RESULTS: AMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm®-alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm®/PMSCs/PLECs co-application compared to Matriderm®/PMSCs treatment. CONCLUSION: Results indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. PRACTICE: Using Matriderm® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. IMPLICATIONS: These promising effects warrant further investigation in clinical trials.

Systematic in vivo evaluation of the time-dependent inflammatory response to steel and Teflon insulin infusion catheters.

Continuous subcutaneous insulin infusion (CSII) catheters are considered the weak link of insulin pump therapy. Wear-time considerably varies between patients and the choice of catheter material is based on personal preferences rather than scientific facts. Therefore, we systematically assessed and quantified the inflammatory tissue response to steel versus Teflon CSII catheters over a maximum wear-time of 7 days in swine. Tissue surrounding catheters was analysed using histopathology and quantitative real-time PCR. The area of inflammation increased significantly over time independent of material which was confirmed by an increase in CD68 expression and an increase in mononuclear and neutrophil cell infiltrate around the catheters. We observed substantially higher fibrin deposition (p < 0.05) around steel on day 4 of wear-time. IL-6 gene expression increased within 24 hours after insertion, returned to normal levels around Teflon (p < 0.05) but remained high around steel (p < 0.05). IL-10 and TGF-ß levels did not resolve over time, indicating impaired wound healing. In conclusion, there was a major temporal effect in the acute inflammatory response to CSII catheters but we found little difference between materials. This study setup presents a robust tool for the systematic analysis of the tissue response to CSII catheters.

Phonation Analysis Combined with 3D Reconstruction of the Thyroarytenoid Muscle in Aged Ovine Ex Vivo Larynx Models.

OBJECTIVE: The aim of the study was to establish a basic data set of combined functional and anatomical measures of aged sheep larynges using ex vivo models. Combining these two approaches in one and the same larynx is an unmet goal so far yet is important as newer treatment strategies aim to preserve the organ structure and new assessment tools are required. Ovine larynges were used as their dimensions, and muscle fiber type distribution highly resemble the human larynx. STUDY DESIGN: Ex vivo animal study. METHODS: Larynges of six sheep (~9 years of age) were subjected to ex vivo functional phonatory experiments. Phonatory characteristics were analyzed as a function of longitudinal vocal fold (VF) prestress. Anatomical measurements of the same larynges comprised micro-computed tomography scans followed by three-dimensional (3D) reconstructions. Using specially adapted radiological scan protocols with subsequent 3D reconstruction, muscle volumes, surface areas, and anatomical measurements were computed. RESULTS: Increasing longitudinal prestress yielded higher subglottal pressure (PS) for the same airflow. Quantitative differences to previous studies-such as the increased PS and increased phonation threshold pressure-were detected. We achieved excellent visualization of the laryngeal muscles and framework, resulting in accurate 3D reconstructions for quantitative analysis. We found no significant intraindividual volume differences of the thyroarytenoid muscles. CONCLUSION: The established protocol allows precise functional and anatomical measures. The data created provide a reference data set for upcoming therapeutic strategies (eg, growth factor therapy, functional electrical stimulation) that target essential structures of the VFs such as the laryngeal muscles and/or the VF mucosa.

Sinus Floor Augmentation Comparing an In Situ Hardening Biphasic Calcium Phosphate (Hydroxyapatite/ß-Tricalcium Phosphate) Bone Graft Substitute with a Particulate Biphasic Calcium Phosphate (Hydroxyapatite/ß-Tricalcium Phosphate) Bone Graft Substitute: An Experimental Study in Sheep.

OBJECTIVE: The aim of the present split-mouth study in sheep was to assess the influence of in situ hardening properties of a biphasic calcium phosphate (BCP) bone graft substitute (BGS) (ratio hydroxyapatite/ß-tricalcium phosphate = 60/40) compared with a particulate BGS with the same biphasic core-granule composition without in situ hardening properties on sinus floor augmentation. MATERIALS AND METHODS: Therefore, bilateral sinus floor augmentation was performed in eight sheep. Poly(lactide-co-glycolide) (PLGA)-coated, in situ hardening biphasic BGS (PLGA-NMP [N-Methyl-2-pyrrolidone]-BCP) was placed at the test site, and a particulate biphasic BGS without PLGA coating (BCP) was used for the contralateral site as a control. Animals were sacrificed after 21 weeks. Sinus augmentation sites were analyzed histologically. The volume was analyzed by computed tomography. Histomorphometric parameters were assessed for the 12 and 21 weeks' time points. Slopes of new bone formation over time were compared with a linear growth regression model. RESULTS: Bone formation after 12 and 21 weeks of healing was 8.94% (±3.74) and 19.82% (±6.29) for PLGA-NMP-BCP and 7.00% (±2.58) and 14.38% (±4.51) for BCP, respectively. The bone growth rate for PLGA-NMP-BCP was higher than the growth rate for BCP (probability 97.5%). The total fraction of calcified hard tissue (% bone fraction + % biomaterial) was around 46% for both tested biomaterials, 21 weeks after sinus floor augmentation. CONCLUSION: The in situ hardening BGS (PLGA-NMP-BCP) performed better than the particulate material (BCP) in terms of bone formation rate. The in situ hardening properties of the PLGA-NMP-BCP material mediated by the PLGA coating and NMP solution as plasticizer had no negative influence on the bone formation.

Comparison of Matrigel and Matriderm as a carrier for human amnion-derived mesenchymal stem cells in wound healing.

Amnion-derived mesenchymal stem cells (AMSC) are a promising tool in regenerative medicine. Here we evaluated the utility of Matrigel and Matriderm as carrier for the topical application of AMSC to mice skin wounds. In both application forms, AMSC promoted neovascularization of the wound area. Matrigel proved as excellent matrix for AMSC and immigrating mouse cells, but the solid Matriderm enabled a more adequate positioning of AMSC into the wound. Although AMSC did not attach to Matriderm, they reliably induced wound reduction. Thus, a combined administration of AMSC/Matriderm could be beneficial to potentiate the encouraging effects on wound healing.

Simultaneous dermal matrix and autologous split-thickness skin graft transplantation in a porcine wound model: a three-dimensional histological analysis of revascularization.

Despite the popularity of a simultaneous application of dermal matrices and split-thickness skin grafts, scarce evidence exists about the process of revascularization involved. In this study, we aimed at analyzing the progression of revascularization by high-resolution episcopic microscopy (HREM) in a porcine excisional wound model. Following the surgical procedure creating 5 × 5 cm(2) full-thickness defects on the back, one area was covered with an autologous split-thickness skin graft alone (control group), the other with a collagen-elastin dermal matrix plus split-thickness skin graft (dermal matrix group). Two skin biopsies per each group and location were performed on day 5, 10, 15, and 28 postoperatively and separately processed for H&E as well as HREM. The dermal layer was thicker in the dermal matrix group vs. control on day 5 and 28. No differences were found for revascularization by conventional histology. In HREM, the dermal matrix did not appear to decelerate the revascularization process. The presence of the dermal matrix could be distinguished until day 15. By day 28, the structure of the dermal matrix could no longer be delineated and was replaced by autologous tissue. As assessed by conventional histology and confirmed by HREM, the revascularization process was comparable in both groups, notably with regard to the vertical ingrowth of sprouting vessels. The presented technique of HREM is a valuable addition for analyzing small vessel sprouting in dermal matrices in the future.

Transdermal uptake and organ distribution of silver from two different wound dressings in rats after a burn trauma.

Silver-containing wound dressings are an integral part of wound therapy in adult and pediatric burn patients. The antimicrobial effect of silver is well known and has been described in numerous studies. Side effects are rarely reported from silver-containing wound care products, even though systemic absorption of silver has been shown by elevated levels of silver in the blood of patients after silver exposure during wound therapy. This animal study investigated the silver levels of blood and in which organs and tissues silver is detectable after long-term application of silver-containing wound dressings after a burn trauma. In male rats, a major full-thickness scald was created on their backs according to a standardized burn model. Two different silver-containing wound dressings (nanocrystalline silver [NCS] and silver sulphate foam [SSF]) were applied initially and changed every 7 days. Weekly blood drawings revealed an increase of blood silver in week three with significant higher values in the SSF compared with NCS group (Ag µg/kg 135.8 vs. 61.7; means; p ≤ 0.05). Thereafter, the NCS group showed significantly higher blood silver levels than the SSF group at week five (Ag µg/kg 192.3 vs. 81.3; means; p ≤ 0.01) and six (Ag µg/kg 168.2 vs. 32.9; means; p ≤ 0.01). After 6 weeks, the animals were sacrificed, and the organs and tissues were analyzed for their silver content by inductively coupled plasma mass-spectrometry. Silver was detectable in all analyzed organs and tissue samples, with higher silver values in parenchymatous organs in the NCS than SSF group (Ag µg/kg; spleen: 3,469 vs. 260; kidney: 3,186 vs. 289; liver: 2,022 vs. 313; means; p ≤ 0.05). Silver was also detectable in brain, testis, lung, heart, and muscle tissue.

Anaesthetic sparing effect of local anaesthesia of the ovarian pedicle during ovariohysterectomy in dogs.

OBJECTIVE: To evaluate the effect of local anaesthesia of the mesovarium on end-tidal isoflurane (Fe'(iso)) concentration and vital parameters during canine ovariohysterectomy. STUDY DESIGN: Prospective, randomized, blinded study. ANIMALS: Twenty client-owned dogs undergoing elective ovariohysterectomy. Mean age 1.7 (+/-0.53, SD) years and mean body weight 21 kg (+/-5.9, SD). METHODS: Pre-medication was with intravenous acepromazine (0.02 mg kg(-1)) and methadone (0.1 mg kg(-1)). Anaesthesia was induced with propofol and maintained with isoflurane in oxygen. One group (n = 10) received local infiltration of the mesovarium with 0.5 mL lidocaine 2% and one group (n = 10) with 0.5 mL NaCl 0.9%. Heart (HR) and respiratory rates (fr), invasive mean arterial blood pressure (MAP) and Fe'(iso)were recorded. The Fe'(iso) was adjusted according to changes in HR, RR and MAP. Time points used for comparison were T1 (after induction of anaesthesia before surgery), T2 (after lidocaine infiltration of the mesovarium) and T3 (surgical manipulation of the ovaries). Data were analysed using a mixed model for repeated measurement anova and the Tukey adjustment. Results are presented as mean +/- SD; p < 0.05 was considered significant. RESULTS: In both groups, HR and fr remained stable at the three time points. Mean values ranged from 84 to 94 beats minute(-1) and from 10 to 14 breaths minute(-1). The Fe'(iso) was significantly lower at T3 compared to T1 and mean values ranged from 0.95% to 1.24%. The mean arterial blood pressure was significantly higher at T3 compared to T1 and mean values ranged from 58 to 96 mm Hg. At none of the time points were there significant differences between the two groups for HR, fr, MAP or Fe'(iso). CONCLUSION: Neither an isoflurane sparing effect nor a difference in autonomic response to surgery was demonstrated following local anaesthesia of the mesovarium. CLINICAL RELEVANCE: There appeared to be minimal benefit from local anaesthesia of the mesovarium during this study.