Gene therapy using wound healing-associated growth factor gene has received much attention as a new strategy for improving the outcome of tissue transplantation. We delivered plasmid DNA coding for human hepatocyte growth factor (hHGF) to rat free skin grafts by the use of laser-induced stress waves (LISWs); autografting was performed with the grafts. Systematic analysis was conducted to evaluate the adhesion properties of the grafted tissue; angiogenesis, cell proliferation, and reepithelialization were assessed by immunohistochemistry, and reperfusion was measured by laser Doppler imaging as a function of time after grafting. Both the level of angiogenesis on day 3 after grafting and the increased ratio of blood flow on day 4 to that on day 3 were significantly higher than those in five control groups: grafting with hHGF gene injection alone, grafting with control plasmid vector injection alone, grafting with LISW application alone, grafting with LISW application after control plasmid vector injection, and normal grafting. Reepithelialization was almost completed on day 7 even at the center of the graft with LISW application after hHGF gene injection, while it was not for the grafts of the five control groups. These findings demonstrate the validity of our LISW-based HGF gene transfection to accelerate the adhesion of grafted skins.
In our previous study, we delivered plasmid DNA coding for human hepatocyto growth factor (hHGF) to rat skin grafts
based on laser-induced stress wave (LISW), by which production of CD31-positive cells in the grafted skins was found
to be enhanced, suggesting improved angiogenesis. In this study, we validated the efficacy of this method to accelerate
adhesion of grafted skins; reperfusion and reepithelialization in the grafted skins were examined. As a graft, dorsal skin
of a rat was exsected and its subcutaneous fat was removed. Plasmid DNA expression vector for hHGF was injected
into the graft; on its back surface a laser target with a transparent sheet for plasma confinement was placed, and
irradiated with three nanosecond laser pulses at a laser fluence of 1.2 J/cm2 (532 nm; spot diameter, 3 mm) to generate
LISWs. After the application of LISWs, the graft was transplanted onto its donor site. We evaluated blood flow by
laser Doppler imaging and analyzed reepithelialization based on immunohistochemistry as a function of postgrafting
time. It was found that both reperfusion and reepithelialization were significantly enhanced for the grafts with gene
transfection than for normal grafts; reepithelialization was completed within 7 days after transplantation with the
transfected grafts. These findings demonstrate that adhesion of grafted skins can be accelerated by delivering HGF
gene to the grafts based on LISWs.
We performed multiwavelength photoacoustic (PA) measurement for extensive deep dermal burns in rats to monitor the healing process of the wounds. The PA signal peak at 532 nm, an isosbestic point for oxyhemoglobin (HbO2) and deoxyhemoglobin (HHb), was found to shift to a shallower region of the injured skin tissue with the elapse of time. The results of histological analysis showed that the shift of the PA signal reflected angiogenesis in the wounds. Until 24 h postburn, PA signal amplitude generally increased at all wavelengths. We speculate that this increase in amplitude is associated with dilation of blood vessels within healthy tissue under the injured tissue layer and increased hematocrit value due to development of edema. From 24 to 48 h postburn, the PA signal showed wavelength-dependent behaviors; signal amplitudes at 532, 556, and 576 nm continued to increase, while amplitude at 600 nm, an HHb absorption-dominant wavelength, decreased. This seems to reflect change from shock phase to hyperdynamic state in the rat; in the hyperdynamic state, cardiac output and oxygen consumption increased considerably. These findings show that multiwavelength PA measurement would be useful for monitoring recovery of perfusion and change in local hemodynamics in the healing process of burns.
We attempted to monitor the healing process of burn injuries by multiwavelength photoacoustic (PA) measurement.
Deep dermal burn with 20% total body surface area was made in the dorsal skins of rats. The wavelengths of 532 nm,
556 nm, 576 nm and 600 nm were used: 532 nm is isosbestic point for oxyhemoglobin (HbO2) and deoxyhemoglobin
(HHb); 576 nm is HbO2 absorption dominant; and 556 nm and 600 nm are HHb absorption dominant. At 532 nm, 556
nm and 576 nm, the depths of PA signal peak were shifted to the shallower region of the wound with the elapse of time,
which was found to reflect angiogenesis due to wound healing by histological analysis. The amplitudes of PA signals
increased at all the wavelengths until 24 h postburn time. At 48 h postburn time, the signal amplitude continued to
increase at 532 nm and 576 nm, while it decreased at 556 nm and 600 nm. This is attributable to the change from a
shock phase to the phase of hyperdynamic state, which is accompanied by increases in cardiac output and oxygen
consumption. These results suggest that multiwavelength photoacoustic measurement is useful for monitoring healing
process of burn injuries.
KEYWORDS: In vitro testing, Laser therapeutics, Plasma, Pulsed laser operation, Luminescence, Tissues, Glasses, Temperature metrology, In vivo imaging, Laser energy
Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.
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