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In many cases, several different approaches to a single process are examined, with sample results and analyses given to help users in selecting the approach most suited to the problem at hand. Comprehensive and enlightening, Wound Healing: Methods and Protocols offers both basic and clinical scientists a thought-provoking and highly practical collection of widely used model systems and methods for studying the injury repair process. Subcutaneous Sponge Models.

A Porcine Burn Model. Human Skin Organ Culture. In Vitro Matrigel Angiogenesis Model. Quantification of Wound Angiogenesis. Wound Healing in Airways In Vivo.

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Show all. Paul et al. Show next xx. Read this book on SpringerLink. Recommended for you. Gourdie Tereance A. PAGE 1. Alcohol prep pads were then used to wipe the area clean. Hemostasis was achieved by even compression with sterile gauze. The hind limbs were shaved and disinfected, and marks of mm length were made to guide the incisional wound. Incisional wounds 10 mm long and 3 mm deep were created on both limbs on the gastrocnemius muscle with a sharp blade World Precision Instruments, Inc. Wounds were treated subcutaneously with saline or rhMG53 0. Wounds were left open with no dressing.

Four New Zealand rabbits were used for this study. A digital camera Panasonic DMC-ZS3 was used to capture the wound with a metric ruler inside the field of view to set the scale for measurements. Blinded measurement of wound size was performed using ImageJ software.

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The perimeter of the wound was then traced, and the wound area was recorded. The wound size on day 0 was set to and, on each subsequent day, was reported as a percentage of the initial wound size. Skin samples were pinned to Styrofoam rafts to maintain morphology during the fixation. Washed samples were processed and embedded in paraffin.

Deparaffinization and antigen retrieval was performed using cell conditioning solution CC1, Ventana Medical Systems. Slides were counterstained with hematoxylin, dehydrated, and xylene-cleared. The resulting slides showed MG53 protein stained brown and surrounding tissue stained blue. Hydroxyproline is a major component of collagen that helps stabilize the helical structure of collagen. Therefore, its levels can be used as an indicator of collagen content The tissue hydroxyproline content expressed as micrograms per milligram of skin tissue was analyzed.

Human and mouse keratinocytes were purchased from Zen-Bio Inc. Research Triangle Park, NC and cultured according to the manuals of the manufacturer. To evaluate the effect of rhMG53 on the proliferation of 3T3 cells, an 3- 4,5-dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT assay was used. Measurement of the release of lactate dehydrogenase following microglass bead damage was used as an index of membrane injury 18 , The released lactate dehydrogenase was calculated by subtracting the basal lactate dehydrogenase release from wells without glass beads no damage from the experimental conditions.

Time-dependent cell migration was recorded using a Zeiss Axiovert phase-contrast microscope 0, 6, and 22 h post-injury.

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Hydrogel was synthesized through free radical polymerization of N -isopropylacrylamide, acrylic acid, and hydroxyethyl methacrylate-oligohydroxybutyrate Sigma as described previously 25 , The solution was bubbled in nitrogen gas for 15 min before the degassed benzyl peroxide solution was injected. Cells were then incubated with fluorophore-conjugated secondary antibodies Life Technologies for 2 h at room temperature. F-actin was visualized using Alexa Fluor phalloidin Life Technologies. Images were taken and analyzed using a Zeiss confocal microscope.

Groups were compared by Student's t test and analysis of variance for repeated measures.

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To investigate the biological function of MG53 in repairing skin injury, we used Western blot analysis and immunohistochemical staining to determine the expression of MG53 in skin tissues Fig. Although MG53 was detected in mouse skin, keratinocytes derived either from human or mouse did not express MG53 Fig. Immunohistochemical staining confirmed that MG53 is localized to the panniculus carnosus, a subdermal striated muscle layer present in many rodents Fig.

This result is consistent with our previous observation that MG53 is predominantly expressed in striated muscle tissues 14 , MG53 knockout mice show abnormal skin structure. Desmin was used as a marker for skeletal muscle, and tubulin was used as a protein loading control. Although MG53 is absent from keratinocytes and fibroblasts, circulating MG53 may contribute to the maintenance of normal skin architecture under physiological conditions.

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Our previous studies have shown that MG53 present in the blood stream plays a protective role against tissue injuries 18 — The present data show that genetic ablation of MG53 leads to abnormal skin architecture, hair follicle formation, and collagen overproduction in the dermal layer. Wound size was then monitored over the subsequent 15 days. The extent of interstitial collagen deposition was significantly lower in WT littermates Fig.

D , day. Arrows indicate fibrosis and deposition of collagen. Primary cultured human keratinocytes were transfected with GFP-MG53, a fusion construct with GFP added to the amino-terminal end of MG53, to investigate the extent of MG53 participation in the repair of membrane injury.

As shown in Fig.

Wound regeneration and repair : methods and protocols

MG53 protects human keratinocyte from membrane injury. A live-cell imaging movie of the cell response to injury can be found in supplemental Movie 1. LDH , lactate dehydrogenase. We have shown previously that rhMG53 protein could protect various cell types against cell membrane disruption when applied to the extracellular environment To quantify the effect of rhMG53 protection against injury to keratinocytes, we first employed an in vitro keratinocyte cell membrane damage assay. In this assay, we measured the amount of lactate dehydrogenase leaked from the cell interior into the extracellular solution following cell membrane injury induced by microglass beads 18 , This suggests that rhMG53 protects against mechanical injury to keratinocytes.

The wound healing process involves migration of fibroblasts to wound sites and differentiation of fibroblasts into myofibroblasts 2 , 4 , 5 , 8 , To test whether MG53 plays a role in the modulation of fibroblast migration during wound healing, we performed an in vitro scratch wound assay with cultured 3T3 fibroblast cells Fig. The cells were treated with phosphate-buffered saline Fig.

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Images were captured 0, 6, and 22 h post-injury. Cell migration was quantified by the distance traveled by cells from the scratch edge toward the center of the scratch Fig. Both rhMG53 treatment and myc-MG53 overexpression enhanced the migration of fibroblast cells following scratch wounding. Representative images were captured 0, 6, and 22 h post-injury. Cells were fixed and stained with Alexa Fluor phalloidin.

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The MGmediated enhancement of fibroblast migration was not due to an increase in cell proliferation, as determined by MTT assay because the cell growth rate was not affected by the treatment with rhMG53 Fig. Under confocal microscopy, we found that fibroblasts cultured in serum-free medium displayed a formation of intracellular stress fibers that were larger and more contiguous compared with those treated with rhMG53 these were thinner and dissociated Fig. Other investigators have shown that reduced stress fiber formation is correlated with the enhanced cell migration associated with wound healing 31 , Together, these results show that MGmediated reduction of stress fiber formation may be a contributing factor for the enhancement of cell migration during wound healing.

We have shown previously that systemic administration of rhMG53 provided dose-dependent protection against injury to muscles and the heart, kidneys, and lungs under various injurious conditions 18 — We studied the effect of rhMG53 in protection against dermal injury using established rat models of wound healing 29 , 30 , Excisional wounds were created on the back of rats with a biopsy punch diameter, 6 mm Fig. Vehicle or rhMG53 0. We found that wounds treated with rhMG53 displayed better healing compared with those treated with vehicle Fig. On day 1, rhMGtreated wounds showed improved settlement in the wound bed compared with vehicle-treated wounds despite there being no significant difference in wound closure between the two groups.

Improved wound closure was clearly observed starting on day 5 in the rhMGtreated group. The role of rhMG53 in facilitating wound healing was tested further in an incisional wound model. Consistent with our observations in the excisional wound model, rhMG53 treatment led to significant improvement in healing of the incisional wound starting on day 3 and continuing to wound closure Fig. These studies suggest that subcutaneous delivery of rhMG53 has beneficial effects on wound healing.

Wounds were created on the back skin of rats and treated with saline top row or rhMG53 0.