Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

Roberts, Michael S.; Dancik, Yuri; Prow, Tarl W.; Thorling, Camilla A.; Lin, Lynlee L.; Grice, Jeffrey E.; Robertson, Thomas A.; König, Karsten; Becker, Wolfgang

doi:10.1016/j.ejpb.2010.12.023

Cited by 145 publications

(122 citation statements)

References 80 publications

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“…Rapid developing additional nonlinear optical microscopy techniques may further broaden the promising first results for the application of engineered skin grafts. 27,28 Our findings support the use of TPLSM as a platform technology as described by Vielreicher et al 25 Based our statistical analysis on data acquired by TPLSM, we can show that hdF-supported angiogenesis in our experimental setup is generally not altered by keratinocyte coverage in a significant manner. In contrast to Liu et al, 18 we could not report an increase of vessel length or total capillary-like structures.…”

Section: Discussionsupporting

confidence: 69%

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

Helmedag

Weinandy

Marquardt

et al. 2015

Tissue Engineering Part A

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Organotypic full-thickness skin grafts (OTSG) are already an important technology for treating various skin conditions and are well established for skin research and development. These obvious benefits are often impaired by the need of laborious production, their noncomplete autologous composition, and, most importantly, their lack of included vasculature. Therefore, our study focused on combining a prevascularized dermal layer with an epidermis to cultivate full-thickness skin grafts incorporating capillary-like networks. It has been shown that prevascularization accelerates ingrowth of tissue-engineered grafts, and it is a prerequisite to circumvent diffusion limits due to graft thickness. To obtain such a graft, we chose a dermal layer incorporating human umbilical vein endothelial cells (HuVEC) amid human dermal fibroblasts within a fibrin-based scaffold, seeded apically with human foreskin keratinocytes (hfKC). Our research investigated the used concept's feasibility, as well as the effect of hfKC addition on the development of a well-connected capillary-like network after approximately 21 days. In addition, we evaluated the utilization of a custom-made constant flow bioreactor for simplified cultivation of these grafts, therefore possibly easing graft production and presumably increasing their cost effectiveness. Skin grafts were assessed by conventional two-dimensional histology. In addition, software-assisted three-dimensional evaluation of the capillary-like structure networks was performed by twophoton laser scanning microscopy (TPLSM) and subsequent image processing was done with ImagePro Ò Analyzer 7.0 software, thereby evaluating its platform technology power in the field of prevascularized skin grafts. All samples showed a capillary-like structure network, but we could report a significant reduction of its total length after 14 days of tri-culture with 5 · 10 5 /cm 2 seeded hfKC, possibly indicating nutritional deficiencies for this particular high cell density experimental setup. Lower concentrations of hfKC did not affect the formation of the capillary-like structures significantly. The developed bioreactor simplified cultivation of prevascularized OTSG. However, a flow-dependent reduction of capillary-like structures in 1 and 5 mL/min flow conditions occurred. We conclude that our technique for creating prevascularized OTSG is feasible. In addition, TPLSM is well suited for analyzing the prevascularization process. We hypothesize that the handling benefits of our bioreactor can be preserved by using considerably lower flow rates while not impairing the forming of capillary-like structure networks.

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Section: Discussionsupporting

confidence: 69%

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

Helmedag

Weinandy

Marquardt

et al. 2015

Tissue Engineering Part A

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“…The ratio is an indicator of the redox state of the cells, representing the proportion of free and protein-bound NAD(P)H within the cell. 10 While we observed a tendency for the redox ratio to be elevated, this difference was not statistically significant (Fig. 4).…”

Section: Discussionmentioning

confidence: 62%

“…Fluorescence emission was spectrally resolved between three linearly arranged photon counters through the use of three dichroic filters in the beam path, spectrally dividing the emission light into three channels for each photon counter: 350 to 450 nm {reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H]}, 450 to 515 nm [NAD(P)H, flavin adenine dinucleotide (FAD)], and 515 to 620 nm (FAD, fluorescein). 10,31 Each FLIM image was collected at an exposure of 13.4 s and acquisition image size of 214 × 214 μm.…”

Section: Multiphoton Intravital Imagingmentioning

confidence: 99%

“…9 For example, autofluorescent nicotinamide adenine dinucleotide (NADH) is a direct measure of cellular metabolic state and can be used to study cellular damage. [10][11][12] The free and bound forms of NADH are involved in the anaerobic and aerobic production of adenosine triphosphate (ATP), respectively. 13,14 Accordingly, a change in the free/bound ratio gives insight into the metabolic state of a cell.…”

Section: Introductionmentioning

confidence: 99%

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Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

et al. 2013

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Abstract. The liver is important in the biotransformation of various drugs, where hepatic transporters facilitate uptake and excretion. Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery, and the developing oxidative stress can lead to graft failure. We used intravital multiphoton tomography, with fluorescence lifetime imaging, to characterize metabolic damage associated with hepatic I/R injury and to model the distribution of fluorescein as a measure of liver function. In addition to measuring a significant increase in serum alanine transaminase levels, characteristic of hepatic I/R injury, a decrease in the averaged weighted lifetime of reduced nicotinamide adenine dinucleotide phosphate was observed, which can be attributed to a changed metabolic redox state of the hepatocytes. I/R injury was associated with delayed uptake and excretion of fluorescein and elevated area-under-the-curve within the hepatocytes compared to sham (i.e., untreated control) as visualized and modeled using images recorded by intravital multiphoton tomography. High-performance liquid chromatography analysis showed no differences in plasma or bile concentrations of fluorescein. Finally, altered fluorescein distribution was associated with acute changes in the expression of liver transport proteins. In summary, multiphoton intravital imaging is an effective approach to measure liver function and is more sensitive in contrasting the impact of I/R injury than measuring plasma and bile concentrations of fluorescein.

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“…Topical delivery has not been optimized. Intact skin is a natural barrier for nanoparticles [14][15][16]. Among all known compounds that enhance the transdermal diffusion, including low-molecular, the most currently effective are those organosulfur compounds such as dimethylsulfoxide and tiofansulfoxide.…”

Section: Introductionmentioning

confidence: 99%

The Technique of Enhancing the Transdermal Penetration for the Gold Nanoparticles and Perspectives of Application

Khayrullin¹,

Terentyuk²,

Savenkova³

et al. 2013

JCT

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Background: In recent years, worldwide attention of researchers focused on the practical implementation of nanoscale materials in biomedical technology. It's proved that intravenous injected gold nanoparticles are accumulated in tumor tissues. However, when gold nanoparticles injected intravenously negative effects in the internal organs of experimental animals are observed. 160 nm diameter particles affect the wall of blood vessels, resulting in vacuolar degeneration of endothelial cells. Particles with a diameter of 50 nm lead to more expressed changes in the internal organs. Injection of the particles diameter of 15 nm causes moderate degeneration of parenchymal cells of internal organs and circulatory disorders. Materials and Methods: In current research, for the first time using the methods of experimental pathology the permeability of intact and damaged skin for nanoscale gold particles in combination with organosulfur compoundsdimethylsulfoxide and tiofansulfoxide were studied. We used 140 male outbred white rats with an average weight 150 -200 grams. All the animals were divided into one control and three experimental groups. Results: Laser microperforation skin with ultrasound treatment can provide good skin permeability, but in contrast to use of agents with organosulfur compounds inflammatory reaction, the destruction of superficial and deep skin tissue structural elements are observed. The comparative efficacy of dimethylsulfoxide and tiofansulfoxide for transdermal permeability of gold nanoparticles was studied. It's proved that in topical application solution of nanoparticles with organosulfur compounds negative effects of the accumulation of nanoparticles in the internal organs, disorders of organ and tissue microcirculation, development in the degenerative changes are not observed. We found that the depth of penetration of damaged skin (contact dermatitis) for the gold nanoparticles in combination with organosulfur compounds, and ultrasound exposure is substantially higher than the penetration of intact skin.

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Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

Cited by 145 publications

References 80 publications

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

The Technique of Enhancing the Transdermal Penetration for the Gold Nanoparticles and Perspectives of Application

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