The effect of the perfluorodecalin patch on particle emission and skin temperature during laser-induced tattoo removal.

Laser-based procedures for tattoo removals are popular due to high efficacy and a relatively moderate insult. However, it often requires multiple sessions to achieve a satisfactory effect. The perfluorodecalin (PFD) patch utilizes an optical clearing agent to speed up the removal process and may decrease skin insult and harmful particles emission during treatment. This study assessed in pigs the effect of the PFD patch in connection with laser treatment of skin with and without tattoos to determine whether the PFD patch provides benefit in lowering thermal skin insult and particle emission during treatment. Skin temperature measured by infrared thermometer during laser treatment or shortly thereafter showed a significant (approximately 40%) decrease with the PFD patch in sites with tattoos. For laser-treated sites without tattoos, there was a significant decrease of particles observed with the PFD patch. In laser-treated sites with tattoos, a strong trend was seen (approximately a twofold decrease) but did not reach statistical significance due to high variability. The present data show that the PFD patch limits the increase in skin temperature produced with laser during treatment. Moreover, it decreases the emission of particles in sites without tattoos and is suggestive of a similar effect in sites with tattoos.

Devising tissue ingrowth metrics: a contribution to the computational characterization of engineered soft tissue healing.

The paradigm shift brought about by the expansion of tissue engineering and regenerative medicine away from the use of biomaterials, currently questions the value of histopathologic methods in the evaluation of biological changes. To date, the available tools of evaluation are not fully consistent and satisfactory for these advanced therapies. We have developed a new, simple and inexpensive quantitative digital approach that provides key metrics for structural and compositional characterization of the regenerated tissues. For example, metrics provide the tissue ingrowth rate (TIR) which integrates two separate indicators; the cell ingrowth rate (CIR) and the total collagen content (TCC) as featured in the equation, TIR% = CIR% + TCC%. Moreover a subset of quantitative indicators describing the directional organization of the collagen (relating structure and mechanical function of tissues), the ratio of collagen I to collagen III (remodeling quality) and the optical anisotropy property of the collagen (maturity indicator) was automatically assessed as well. Using an image analyzer, all metrics were extracted from only two serial sections stained with either Feulgen & Rossenbeck (cell specific) or Picrosirius Red F3BA (collagen specific). To validate this new procedure, three-dimensional (3D) scaffolds were intraperitoneally implanted in healthy and in diabetic rats. It was hypothesized that quantitatively, the healing tissue would be significantly delayed and of poor quality in diabetic rats in comparison to healthy rats. In addition, a chemically modified 3D scaffold was similarly implanted in a third group of healthy rats with the assumption that modulation of the ingrown tissue would be quantitatively present in comparison to the 3D scaffold-healthy group. After 21 days of implantation, both hypotheses were verified by use of this novel computerized approach. When the two methods were run in parallel, the quantitative results revealed fine details and differences not detected by the semi-quantitative assessment, demonstrating the importance of quantitative analysis in the performance evaluation of soft tissue healing. This automated and supervised method reduced operator dependency and proved to be simple, sensitive, cost-effective and time-effective. It supports objective therapeutic comparisons and helps to elucidate regeneration and the dynamics of a functional tissue.

Computerized histomorphometric study of the splenic collagen polymorphism: A control-tissue for polarization microscopy.

Previous articles have pointed out the presence of type III collagen within the extracellular structure of the parenchymatous organs. This study aimed to quantitatively characterize the collagen polymorphism at the capsule and parenchymal trabeculae of the largest lymphoid organ of the body i.e., the spleen, in mouse, rat, and rabbit models. Following a Picrosirius Red-Polarization procedure and computer assisted image analysis of paraffin sections, the results showed (1) a predominant and significantly higher amount of type III collagen in the trabeculae area compared to the capsule area in the three species, (2) no statistical difference among the three species concerning the parenchymal collagen polymorphism or the type I/type III collagen ratio, (3) a heterogeneous type I/type III collagen ratio varying from 0.86 (mouse) to 6.62 (rabbit) in the fibromuscular capsule region. A qualitative analysis corroborated these histomorphometric results. In conclusion, the spleen may be used as (1) a control tissue to qualitatively visualize type I and III collagen under polarization microscopy and to validate the quality of PSR staining (2) an aid to accurately calibrate the angle of polarization before quantitative measurements of type I and type III collagen. Among the studied species, the rabbit spleen appeared to be the most appropriate control tissue as it showed the highest amount of type I collagen in the capsule and a similarly high amount of type III collagen in the parenchymal trabeculae.

Translating cell-based regenerative medicines from research to successful products: challenges and solutions.

The Tissue Engineering & Regenerative Medicine International Society-Europe (TERMIS-EU) Industry Committee as well as its TERMIS-Americas (AM) counterpart intend to address the specific challenges and needs facing the industry in translating academic research into commercial products. Over the last 3 years, the TERMIS-EU Industry Committee has worked with commercial bodies to deliver programs that encourage academics to liaise with industry in proactive collaborations. The TERMIS-EU 2013 Industry Symposium aimed to build on this commercial agenda by focusing on two topics: Operations Management (How to move a process into the good manufacturing practice [GMP] environment) and Clinical Translation (Moving a GMP process into robust trials). These topics were introduced by providing the synergistic business perspective of partnering between the multiple regenerative medicine stakeholders, throughout the life cycle of product development. Seven industry leaders were invited to share their experience, expertise, and strategies. Due to the complex nature of regenerative medicine products, partnering for their successful commercial development seems inevitable to overcome all obstacles by sharing experiences and expertise of all stakeholders. When ideally implemented, the "innovation quotient" of a virtual team resulting from the combination of internal and external project teams can be maximized through maximizing the three main dimensions: core competences, technology portfolio, and alliance management.

Evaluation of a novel nanocrystalline hydroxyapatite paste Ostim in comparison to Alpha-BSM - more bone ingrowth inside the implanted material with Ostim compared to Alpha BSM.

BACKGROUND: The purpose of this study was to evaluate the performance a newly developed nanocrystalline hydroxyapatite, OSTIM following functional implantation in femoral sites in thirty-eight sheep for 1, 2 or 3 months. Ostim 35 was compared to an established calcium phosphate, Alpha BSM. METHODS: Biomechanical testing, micro-CT analysis, histological and histomorphological analyses were conducted to compare the treatments including evaluation of bone regeneration level, material degradation, implant biomechanical characteristics. RESULTS: The micro-computed tomography (microCT) analysis and macroscopic observations showed that Ostim seemed to diffuse easily particularly when the defects were created in a cancellous bone area. Alpha BSM remained in the defect.The performance of Ostim was good in terms of mechanical properties that were similar to Alpha BSM and the histological analysis showed that the bone regeneration was better with Ostim than with Alpha BSM. The histomorphometric analysis confirmed the qualitative analysis and showed more bone ingrowth inside the implanted material with Ostim when compared to Alpha BSM at all time points. CONCLUSIONS: The successful bone healing with osseous consolidation verifies the importance of the nanocrystalline hydroxyapatite in the treatment of metaphyseal osseous volume defects in the metaphyseal spongiosa.

ESEM evaluations of muscle/nanoparticles interface in a rat model.

In order to examine the influence that shape and chemistry of different materials have on the incitement of a tissue reaction, we implanted five materials (the two metals Ni and Co, the two ceramics TiO2 and SiO2, and the polymer poly vinyl-chloride) as nanoparticles or bulk, in the dorsal muscles of 50 rats. After 6 or 12 months, rats were euthanized and the implanted materials were excised together with the surrounding tissue. After a first histological evaluation, the specimens were prepared for environmental scanning electron microscopy (ESEM) and for energy dispersive spectroscopy (EDS), in order to analyse the chemical composition of the implanted material after the biological interaction had occurred, and to evaluate the possible corrosion and diffusion of the materials at tissue interface. The results indicate that the metals at nanoscale size have a carcinogenic effect, while the bulk materials only induce a foreign-body reaction. The ESEM observations show a chemical transformation of the materials. Corrosion of the metals and subsequent recombination of the released ions in a sort of organic-inorganic crystals is showed and verified by the EDS analyses. Finally, our hypotheses of the involved pathological mechanism are suggested.

In high-risk patients, combination of antiinflammatory procedures during cardiopulmonary bypass can reduce incidences of inflammation and oxidative stress.

Previous studies showed that cardiopulmonary bypass (CPB) was directly associated with a global activation of the inflammatory response, production of oxygen free radicals, and signs of myocardial injury. We therefore evaluated, in the weakest patients, the biological and clinical benefits of a therapeutic optimization of CPB through the combination of several antiinflammatory procedures. High-risk patients undergoing cardiac surgery under CPB were included in this prospective randomized study. Control patients (n = 14) underwent conventional CPB, and treated patients (n = 13) underwent a CPB with Baxter Duraflo II heparin-coated circuits, high doses of aprotinin, and pre-CPB hemofiltration. Usual clinical hemodynamic and biological criteria, inflammation, and oxidative stress markers were measured before, during, and to the second postoperative day. Free radicals were quantified using electronic spin resonance spectroscopy with a spin trap. Significantly lower concentrations of C-reactive protein, interleukin-6, creatine kinase-MB, I-troponin, lactic acid, and systemic free radicals were observed in the plasma of treated patients. These patients had a reduction of postoperative complications and of the length of stay in the intensive care unit. Therefore, pre-CPB therapeutic optimization can reduce the inflammatory response, lower the level of oxidative stress, and help to ameliorate clinical outcome in high-risk patients.

Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: sarcoma development by nanoparticles.

In order to study the pathobiological impact of the nanometre-scale of materials, we evaluated the effects of five different materials as nanoparticulate biomaterials in comparison with bulk samples in contact with living tissues. Five groups out of 10 rats were implanted bilaterally for up to 12 months with materials of the same type, namely TiO2, SiO2, Ni, Co and polyvinyl chloride (PVC), subcutaneously with bulk material on one side of the vertebral column and intramuscularly with nanoparticulate material on the contralateral side. At the end of each implantation time, the site was macroscopically examined, followed by histological processing according to standard techniques. Malignant mesenchymal tumours (pleomorphic sarcomas) were obtained in five out of six cases of implanted Co nanoparticle sites, while a preneoplastic lesion was observed in an animal implanted with Co in bulk form. In the Ni group, all animals rapidly developed visible nodules at the implanted sites between 4 and 6 months, which were diagnosed as rhabdomyosarcomas. Since the ratio of surface area to volume did not show significant differences between the Ni/Co group and the TiO2/SiO2/PVC group, we suggested that the induction of neoplasia was not mediated by physical effects, but was mediated by the well-known carcinogenic impact of Ni and Co. The data from the Co group show that the physical properties (particulate versus bulk form) could have a significant influence on the acceleration of the neoplastic process.

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy.

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.

Evidence for the extrapulmonary localization of inhaled nitric oxide.

Inhaled nitric oxide (NO) has emerged as a promising pulmonary vasodilator to treat pulmonary hypertension associated with heart disease and ventilation/perfusion mismatching. However, the pharmacokinetics of inhaled NO still remains obscure and its cardiopulmonary selectivity appears to be increasingly under debate. In the present study measured NO content and levels of cyclic guanosine 3',5'monophosphate (cGMP), a mediator of NO-induced vasodilation, in a variety of organs from rats subjected to NO inhalation. Electron spin resonance spectroscopy associated to a spin trapping technique using N-methyl D-glucamine dithiocarbamate (FeMGD) was used to directly quantify NO levels in the lung, kidney, liver, aorta, and heart from anesthetized Wistar rats subjected to various doses (0, 20, 50, 100, or 200 ppm) and various times (0, 30, 45, or 75 minutes) of inhaled NO. Inhaled NO at a dose of 100 and 200 ppm significantly increased the NO-FeMGD complex in all organs studied. An increase of cGMP was detected in the lung and the aorta after inhaled NO for 45 minutes at the dose of 50 ppm. No changes in NO levels and its metabolites were shown between 30 and 75 minutes of inhaled NO. The results show that inhaled NO at a dose of 100 ppm or more increases NO levels in other organs beside the lung, strongly suggesting that inhaled NO would be more than a pulmonary vasodilator and its selectivity remains to be reconsidered when used for therapeutic purposes.

Direct demonstration of nitric oxide formation in organs of rabbits treated by transdermal glyceryl trinitrate using an in vivo spin trapping technique.

Glyceryl trinitrate (GTN) is commonly delivered by a patch for the treatment of angina pectoris. The idea is now generally accepted that GTN requires a biotransformation process that activates the drug, in particular through nitric oxide (NO) generation. However, the pharmacokinetics of NO delivery from GTN still remains obscure. The objective of this study was to assess GTN-derived NO formation in vascular tissues and organs in rabbit given GTN patches. NO levels were evaluated in rabbits after 3 h of treatment with a 10 mg GTN patch (GTN group; n = 7) or a placebo patch (CTL; n = 7). Nitrosylhaemoglobin (HbNO) was evaluated by electron spin resonance (ESR) spectroscopy in red cell suspension. In vivo spin trapping technique using FeMGD as a spin trap, associated with ESR was used to quantify NO in tissues. The NO-spin trap complex, which is a relatively stable product, has been measured in several tissues. The ESR spectrum corresponding to HbNO was not found in red cell of GTN or CTL rabbits. The spectrum corresponding to the NO-spin trap complex was observed in all analysed tissues of CTL rabbits. The signal was significantly increased in liver, renal medulla, heart left ventricle and spleen of GTN-treated rabbits, and to a lesser extent in right ventricle and lung. No difference was shown between NO-spin trap levels measured in aorta or inferior vena cava from GTN or CTL rabbits. These data suggest that GTN patch treatment induced NO release, and that tissue-specific differences in transdermal GTN-derived NO exist. The GTN-NO pathway appears to be largely involved in organs such as the liver, kidney and heart.

Postischemic recovery and oxidative stress are independent of nitric-oxide synthases modulation in isolated rat heart.

During myocardial ischemia and reperfusion, nitric oxide ((.)NO) was shown to exert either beneficial or detrimental effects. Uncoupled (.)NO synthases (NOS) can generate superoxide anion under suboptimal concentrations of substrate and cofactors. The aim of our study was to investigate the role of NOS modulation on 1) the evolution of functional parameters and 2) the amount of free radicals released during an ischemia-reperfusion sequence. Isolated perfused rat hearts underwent 30 min of total ischemia, followed by 30 min of reperfusion in the presence of N(G)-nitro-D- or L-arginine methyl ester (NAME, 100 microM) or of D- or L-arginine (3 mM). Functional parameters were recorded and coronary effluents were analyzed with electron spin resonance to identify and quantify the amount of alpha-phenyl-N-tert-butylnitrone spin adducts produced during reperfusion. The antioxidant capacities of the compounds were determined with the oxygen radical absorbance capacity test. L-NAME-treated hearts showed a reduction of coronary flow and contractile performance, although neither L-NAME nor L-arginine improved the recovery of coronary flow, left end diastolic ventricular pressure, rate pressure product, and duration of reperfusion arrhythmia, compared with their D-specific enantiomers. A large and long-lasting release of alkyl/alkoxyl radicals was detected upon reperfusion, but no differences of free radical release were observed between D- and L-NAME or D- and L-arginine treatment. These results may indicate that, in our experimental conditions, cardiac NOS might not be a major factor implicated in the oxidative burst that follows a global myocardial ischemia.

Systemic free radical activation is a major event involved in myocardial oxidative stress related to cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) can induce deleterious effects that could be triggered in part by radical oxygen species; however, their involvement in the course of surgery has been elusive. The aim of this study was to evaluate the time course and origin of radical oxygen species release, myocardial or not, in patients undergoing coronary artery surgery involving CPB. METHODS: Blood samples were taken from periphery and coronary sinus of patients during CPB, and oxidative stress was evaluated by direct and indirect approaches. Direct detection of alkyl and alkoxyl radicals was assessed by electron spin resonance spectroscopy associated with the spin-trapping technique using alpha-phenyl-N-tert-butylnitrone. RESULTS: The authors showed that the spin adduct concentration was not influenced by anesthesia and pre-CPB surgery. A rapid systemic increase of plasma spin adduct concentration occurred after starting CPB, and it stayed at a high concentration until the end of CPB. At the beginning of reperfusion period, radical oxygen species release was accelerated in the coronary sinus; however, it was not significant. A positive correlation was found between alpha-phenyl-N-tert-butylnitrone adduct concentrations and (1) the duration of CPB and (2) concentration of postoperative creatine phosphokinase of muscle band (CPK MB). Plasma vitamin E and C, ascorbyl radical, uric acid, thiol, plasma antioxidant status, and thiobarbituric acid reacting substances were also measured but did not give relevant indications, except for uric acid, which seemed to be consumed by the heart during reperfusion. CONCLUSION: The results indicate that a systemic production of free radicals occurs during CPB that may overwhelm the production related to reperfusion of the ischemic heart. This systemic oxidative stress is likely to participate in secondary myocardial damage.