Probing endogenous collagen by laser‐induced autofluorescence in burn wound biopsies: A pilot study

The focus of the current study was to interrogate the predictive potential of laser‐induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He‐Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.      

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT)

The present study focuses on the evaluation of the effect of He‐Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post‐wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non‐illuminated controls. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Inhibition by tranilast of collagen accumulation in hypersensitive granulomatous inflammation in vivo and of morphological changes anf functions of fibroblasts in vitro

We examined the effects of tranilast, an anti-allergic agent, on hypersensitive inflammation and on morphology and functions of fibroblasts. In vivo, tranilast suppressed the content of collagen in granulation tissue of hypersensitive granulomatous inflammation induced by methylated bovine serum albumin (m-BSA) in rats. In culture, tranilast inhibited the TGF-β-independent inflammatory exudate-induced stimulation of morphological changes of fibroblasts to myofibroblast-like cells and their proliferation. Collagen gel contraction by myofibroblast-like cells and fibroblasts was also inhibited by tranilast. Flow cytometric analysis revealed that tranilast suspended the cell cycle of fibroblasts at the G0/G1 phase. These results suggest that tranilast modulates the fibrosis and contraction of granulation tissue by inhibiting the growth of myofibroblast-like cells and fibroblasts.     

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80–90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young’s were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.     

Intradermal indocyanine green for in vivo fluorescence laser scanning microscopy of human skin: a pilot study

Background

In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach.

Methodology/Principal Findings

Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin.

Conclusions/Significance

Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders.     

CDH1 is Identified as A Therapeutic Target for Skin Regeneration after Mechanical Loading

Rationale: Mechanical stimuli in the microenvironment are considered key regulators of cell function. Clinically, mechanical force (tissue expander) is widely used to regenerate skin for post-burn or trauma repair, implying that mechanical stretching can promote skin cell regeneration and proliferation. However, the underlying mechanism remains unknown.Methods: Microarray analysis was utilized to detect the hub gene. The expression of Cdh1 as examined in cells and tissues by western blot, q-PCR and immunohistochemistry staining respectively. Biological roles of Cdh1 was revealed by a series of functional in vitro and in vivo studies.Results: Microarray analysis identified Cdh1 as a hub gene related to skin regeneration during rat cutaneous mechanical loading. In vitro studies suggested that both mechanical loading and Cdh1 interference induced keratinocyte dedifferentiation and enhanced stemness, promoting cell proliferation and prevent apoptosis. Furthermore, the forkhead box O1/Krüppel-like factor 4 (FOXO1/KLF4) pathway was activated and contributed to the keratinocyte dedifferentiation. In vivo studies showed that mechanical loading and Cdh1 interference facilitated epidermal dedifferentiation and promoted dermal collagen deposition, and that Cdh1 overexpression could block such influence.Conclusions: In this study, we show that E-cadherin (CDH1), a well-known cell-cell adhesion molecule, plays a crucial role in mechanical stretch-induced skin cell regeneration and proliferation. We have shown for the first time the process by which mechanical stress is transmitted to the epidermis and induces a downstream signaling pathway to induce epidermal cells to differentiate. These findings demonstrate that Cdh1-induced keratinocyte dedifferentiation is a crucial event in mechanical stretch-mediated skin regeneration and that Cdh1 may serve as a potential therapeutic target for promoting skin regeneration.     

Comparative Experimental Study of Wound Healing in Mice: Pelnac versus Integra

Strategies for skin regeneration have been developed to provide effective treatment for cutaneous wounds and disease. Dermal substitutes have been used to cover the lesion to facilitate cell colonization, thereby promoting dermal regeneration. However, very little is known about Pelnac matrix especially at histological level. Therefore, the present work carried out an experimental in vivo comparative analysis between Pelnac and Integra, the most used dermal templates, in a mouse model of full-thickness skin wounds. Histological sections performed at the 3^rd, 6^th and 9^th days after surgery were analyzed with regard to inflammatory response and vascularization. Both templates were completely incorporated in all animals at the end of the analyzed period. Pelnac-treated animals displayed reduced granulation tissue during the first 6 days of treatment compared to the animals treated with Integra at the same time period. The number of inflammatory cells (neutrophils) was similar in both groups during the period, significantly reducing at the end of inflammatory phase (9^th day of treatment) consistent with the progression of healing process. In addition, the density of blood vessels was also statistically similar in both matrices. Therefore, the two dermal templates displayed comparable biological behavior in tissue repair. It is noteworthy that this is the first experimental study comparing Pelnac and Integra dermal templates with focus on full-thickness skin wounds.     

Validation model for Raman based skin carotenoid detection

Raman spectroscopy holds promise as a rapid objective non-invasive optical method for the detection of carotenoid compounds in human tissue in vivo. Carotenoids are of interest due to their functions as antioxidants and/or optical absorbers of phototoxic light at deep blue and near UV wavelengths. In the macular region of the human retina, carotenoids may prevent or delay the onset of age-related tissue degeneration. In human skin, they may help prevent premature skin aging, and are possibly involved in the prevention of certain skin cancers. Furthermore, since carotenoids exist in high concentrations in a wide variety of fruits and vegetables, and are routinely taken up by the human body through the diet, skin carotenoid levels may serve as an objective biomarker for fruit and vegetable intake. Before the Raman method can be accepted as a widespread optical alternative for carotenoid measurements, direct validation studies are needed to compare it with the gold standard of high performance liquid chromatography. This is because the tissue Raman response is in general accompanied by a host of other optical processes which have to be taken into account. In skin, the most prominent is strongly diffusive, non-Raman scattering, leading to relatively shallow light penetration of the blue/green excitation light required for resonant Raman detection of carotenoids. Also, sizable light attenuation exists due to the combined absorption from collagen, porphyrin, hemoglobin, and melanin chromophores, and additional fluorescence is generated by collagen and porphyrins. In this study, we investigate for the first time the direct correlation of in vivo skin tissue carotenoid Raman measurements with subsequent chromatography derived carotenoid concentrations. As tissue site we use heel skin, in which the stratum corneum layer thickness exceeds the light penetration depth, which is free of optically confounding chromophores, which can be easily optically accessed for in vivo RRS measurement, and which can be easily removed for subsequent biochemical measurements. Excellent correlation (coefficient R = 0.95) is obtained for this tissue site which could serve as a model site for scaled up future validation studies of large populations. The obtained results provide proof that resonance Raman spectroscopy is a valid non-invasive objective methodology for the quantitative assessment of carotenoid antioxidants in human skin in vivo.     

Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25 mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.     

Minimally invasive non‐thermal laser technology using laser‐induced optical breakdown for skin rejuvenation

We describe a novel, minimally invasive laser technology for skin rejuvenation by creating isolated microscopic lesions within tissue below the epidermis using laser induced optical breakdown. Using an in‐house built prototype device, tightly focused near‐infrared laser pulses are used to create optical breakdown in the dermis while leaving the epidermis intact, resulting in lesions due to cavitation and plasma explosion. This stimulates a healing response and consequently skin remodelling, resulting in skin rejuvenation effects. Analysis of ex‐vivo and in‐vivo treated human skin samples successfully demonstrated the safety and effectiveness of the microscopic lesion creation inside the dermis. Treatments led to mild side effects that can be controlled by small optimizations of the optical skin contact and treatment depth within the skin. The histological results from a limited panel test performed on five test volunteers show evidence of microscopic lesion creation and new collagen formation at the sites of the optical breakdown. This potentially introduces a safe, breakthrough treatment procedure for skin rejuvenation without damaging the epidermis with no or little social down‐time and with efficacy comparable to conventional fractional ablative techniques. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The effect of silica-treated macrophages on the synthesis of collagen and other proteins in vitro

Treatment with SiO₂ releases from peritoneal macrophages a soluble factor which stimulates the synthesis of collagen and other proteins in incubated slices of experimental granulation tissue. This factor can also be obtained by SiO₂-treatment from certain subcellular particles of intact macrophages. A similar agent is released from the macrophages by incubation with rheumatoid synovialtissue extract. Macrophages induced by paraffin or thioglycollate medium cannot be stimulated further by SiO₂. The SiO₂-treated macrophages have no effect on detached matrix-free cells from embryonic-chick tendon or granulation tissue. Another factor from macrophages, present in the 100000 g-supernatant of the homogenate, inhibits the synthesis of collagen in granuloma slices. The synthesis of DNA and RNA in slices is suppressed by the extract from intact macrophages but not affected by preparations obtained with SiO₂. The possible relevance of these findings to lysosomal actions, to the regulation of granuloma formation and to inflammation are discussed.     

Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms

The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.     

Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of ²H₂O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.     

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons^1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser⁵. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response^1,3,7.We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.     

BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

Microporous acellular extracellular matrix combined with adipose-derived stem cell sheets as a promising tissue patch promoting articular cartilage regeneration and interface integration

BackgroundAcute or chronic injury of articular cartilage leads to localized destruction. Difficulties with interface integration between the implant and native cartilage tissue can lead to an undesirable outcome. To improve cartilage repair and interface integration, we explored the therapeutic efficacy of microporous acellular extracellular matrix (ECM) combined with adipose-derived stem cell (ASC) sheets.MethodsMethods for fabricating ASC sheets and microporous acellular ECM were explored before transplanting the constructed ASC sheet/matrix in vivo and in vitro, respectively. After the operation, distal femur samples were collected at 6 and 12 weeks for further analysis.ResultsThe decellularization process removed 90% of the DNA but retained 82.4% of glycosaminoglycans (GAGs) and 82.8% of collagen, which are the primary components of cartilage matrix. The acellular matrix/ASC sheet construct treatment in vivo showed better interface integration, cartilage regeneration, and collagenous fiber arrangement, which resembles the native structure. There was a significant increase in GAG and collagen accumulation at the zone of regeneration and integration compared to other groups. Gene expression analysis showed that the mRNA level associated with cartilage formation significantly increased in the acellular matrix/ASC sheet group (p<0.05), which is consistent with the histological analysis.DiscussionASC sheets promote interface integration between the implant and native tissue. This effect, together with the acellular matrix as a graft, is beneficial for cartilage defect repair, which suggests that acellular matrix/ASC sheet bioengineered cartilage implants may be a better approach for cartilage repair due to their enhanced integration.     

Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro.     

Cold Atmospheric Plasma (CAP) Changes Gene Expression of Key Molecules of the Wound Healing Machinery and Improves Wound Healing In Vitro and In Vivo

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.