In this report, we describe a laser-latex combination system that enables membrane-impermeable molecules to penetrate cell membranes. Laser light (Q-switched Nd:YAG laser, 532.5 nm) was used to irradiate a mixture of commercial latex particles (blue dyed, 1 micro m in diameter) and mouse fibrosarcoma (Meth-A) cells. After irradiation, membrane permeability was evaluated by flow cytometric assaying using propidium iodide (PI) and fluorescein diacetate (FDA). The proportion of permeabilized-resealed cells was affected by changes in the light intensity (approximately 780 mW/cm(2)), the irradiation time (approximately 240 s), and/or the particle concentration (approximately 10(9) particles/ml). The permeability persisted up to 20 min after light irradiation. Near the sites of individual particles, the permeability of the cell membrane is modified, probably due to localized temperature changes. These results suggest that this laser-induced permeabilization strategy constitutes a new means of delivering exogenous materials into living cells.

Low-energy laser irradiation has positive effects on bone fracture healing, osteoblast proliferation, bone nodule formation, and alkaline phosphatase activity. However, the mechanism by which low-energy laser irradiation affects bone is not clearly known. It was recently found that light at a low radiation dosage is absorbed by intracellular chromophores. High-intensity pulsed laser irradiation can produce acoustic waves in the target surface by rapidly heating the tissue. We considered that the acoustic waves induced by high-intensity pulsed laser irradiation, in addition to the photochemical effects that are induced, accelerate bone formation. To clarify whether high-intensity pulsed laser irradiation accelerates bone formation, we investigated bone formation in the irradiated femur of rat, using histomorphometric analysis. Rat femurs were irradiated with a Q-switched Nd: YAG laser, which has a wavelength of 1064 nm, under two conditions: once a day, with the average fluence rate set at 100 mW/cm(2) (LA1), and twice a day, i.e., every 12 h, with the average fluence rate set at 50 mW/cm(2) (LA2). The mean bone volume and mineral apposition rate in the LA1 group were significantly higher than those in the nonirradiated group (control). These values were highest for the LA2 group, and were about 1.52 and 1.25-fold those of the control, respectively. These data demonstrated that the number of pulses, rather than the intensity of the laser irradiation, affects bone formation. Thus, this study indicated that high-intensity pulsed laser irradiation accelerates bone formation in the metaphysis. This bone formation induced by high-intensity pulsed laser irradiation might be due to laser-induced pressure waves.

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SummaryThe effects of various concentrations of the nerve growth factor (NGF) and of dimethylsulfoniopropionate (DMSP) on the outgrowth of neurites from pheochromocytoma (PC12) cells were examined singly or in combination on an RPMI 1640 medium containing 10% horse serum, 5% fetal bovine serum, penicillin and streptomycin in collagen-coated Petri dishes by increasing the incubation times up to 4 d. The results indicated that NGF significantly accelerated the number of neurite-bearing cells, which reached a maximum at concentrations of more than 0.5 ng/mL among the various concentrations of NGF on the 4th day. The combined effects of the various concentrations (10 Ϫ 6 -10 Ϫ 3 M ) of DMSP with NGF (5 ng/mL) on the growth and the number of neurite-bearing cells were then examined, which demonstrated that all the concentrations of DMSP restricted the growth of the cells to various extents but that the concentration of DMSP at 10 Ϫ 4 M with the NGF more highly accelerated the number of neurite-bearing cells than did the NGF alone during the experimental period.

SummaryThe combined effects of dimethylsulfoniopropionate (DMSP) (10 Ϫ 3 , 10 Ϫ 4 and 10 Ϫ 5 M ) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (5 ng/mL) and the nerve growth factor (NGF) (5 ng/mL) on the outgrowth and elongation of neurites from pheochromocytoma (PC12) cells were examined on RPMI medium containing fetal bovine serum and horse serum with penicillin and streptomycin in collagen-coated dishes for 5 d. The growth was higher in increasing order of the DMSP (10 Ϫ 3 M ), MPTP and NGF, the DMSP (10 Ϫ 5 M ), MPTP and NGF, the MPTP and NGF group and the control group up to 3 d, but not in the NGF and the DMSP (10 Ϫ 4 M ), MPTP and NGF groups. The growth in all the experimental groups showed plateaus from days 4 to 5. The appearance of neurites from the cells in all the groups showed maxima on the 3rd day. The administration of NGF significantly stimulated the outgrowth of neurites from the cells, while the supplementation of MPTP noticeably inhibited the appearance of neurites even in the presence of NGF up to 5 d. However, the addition of DMSP (10 Ϫ 3 and 10 Ϫ 4 M ) to the latter group completely prevented the inhibition of the MPTP. These facts were significantly supported by the photographs of neurite-bearing cells on the 3rd day and also by the photometric analyses examining the reaction of MPTP to DMSP, NGF or Collagen IV.