Biological Effect of Helium-Neon (He-Ne) Laser Irradiation on Mouse Myeloma (Sp2-Ag14) Cell Line In Vitro

We examined the effect of helium-neon (He-Ne) laser irradiation with a wavelength of 632.8 nm on cell cycle synchronisation of monolayer growing mouse myeloma (Sp2-Ag14) cell line. The monolayer cultures were exposed to repeated doses of different energy densities (4–64 J/cm²). The nuclear DNA content has been studied by flow cytometry to obtain the cell percentage in each cell cycle phase. Results showed that the He-Ne laser irradiation at energy densities of 8–64 J/cm² produced a significative and different effect on the G0–G1 and S phases of cell cycle over control. In contrast, no significant effect in G2–M phase was produced by He-Ne laser irradiation at any energy density compared with non-treated control. These results support previous observations suggesting that He-Ne laser irradiation of low energy density interferes with cell cycling and may inhibit cell proliferation when irradiation is performed at doses of 8 J/cm² or more. Received for publication 22 July 1997; accepted following revision 6 February 1998.     

Helium-Neon (He-Ne) Laser Irradiation Increases the Incidence of Unreduced Bovine Oocytes During the First Meiotic Division In Vitro

Low power He-Ne laser irradiation was applied to immature bovine oocytes to investigate the incidence of unreduced oocytes (diploid oocytes) during the first meiotic division in vitro. Immature bovine oocytes from cows killed at a slaughterhouse were irradiated with He-Ne laser irradiation at 0.05 and 0.25 J/cm² exposures. An oocyte group was left untreated serving as the control group. All oocytes were matured in TCM-199 medium supplemented with 10% fetal calf serum (FCS). Unreduced oocyte percentages obtained in the irradiated oocytes group were significantly higher (p<0.01 and p<0.001, respectively) than those of the control group. Furthermore, the laser-treated oocytes showed a degeneration rate significantly higher (p<0.01 and p<0.001, respectively) than those observed in the control group. It is concluded that the application of He-Ne laser irradiation at 0.05 and 0.25 J/cm² exposures increased the incidence of unreduced oocytes and the percentage of degenerated oocytes during the in vitro meiosis process of immature bovine oocytes. Received 13 October 1997; accepted following revision 13 January 1998     

Inhibitory Effect of Yellow He-Ne Laser Irradiation Mediated by Crystal Violet Solution on Early Plaque Formation in Human Mouth

. The purpose of this study was to evaluate the effect of yellow He-Ne laser irradiation in the presence of crystal violet (CV) solution on dental plaque formation in the human mouth. Four enamel specimens were fixed on a retainer, one of which was placed on both maxillary buccal sites of four subjects. The retainers were assigned randomly to 3- or 7-day experimental periods and the right or left buccal sites for the evaluation of plaque formation on the specimens. Then, the specimens were assigned randomly to four treatments as follows: A, laser irradiation; B, CV application; C, CV application and laser irradiation and D, no treatment (control). At three and seven days after the treatments, the inhibitory effects of plaque formation on the specimens were evaluated by measuring the thickness of the plaque on each section. Analysis of the plaque formation scores revealed that treatment C had a significantly greater inhibitory effect on plaque formation than treatment A, B and D after 3 days, however there were no significant differences among the plaque formation scores of the four treatment groups after 7 days. Analysis of the plaque thickness revealed that at both 3 and 7 days after treatment, the plaque layers after treatment C were significantly thinner than those after treatments A, B and D. These results indicated that yellow He-Ne laser irradiation in the presence of CV had an inhibitory effect of plaque formation in human mouth. Paper received 9 July 1999; accepted after revision 3 February 2000.     

Helium–Neon Laser Irradiation: Effect on the Experimental Pain Threshold

. The aim of this double-blind study was to examine the effects of helium–neon laser irradiation on the mechanical (pressure algometry) and electrical (1 ms monophasic square-wave pulses, 50 Hz) pain threshold. 32 pain-free subjects were randomly assigned to either the experimental group (helium–neon laser stimulation: 5 mW, 10 min) or the placebo group (sham stimulation). Laser or sham stimulation and pain threshold ascertainment were carried out on the dorsal aspect of the forearm area. The contralateral arm served as an untreated control. The groups were compared with each other and with the control arm. No significant differences were found between the laser stimulation and the sham stimulation in changes of either the mechanical or the electrical pain threshold. There were no changes in the mechanical pain threshold through laser stimulation and sham stimulation with respect to the untreated contralateral arm. After laser stimulation electrical pain threshold was significantly higher (p<0.01) in the treated arm than in the untreated contralateral arm, because this threshold decreased in the contralateral arm. This was not the case in sham treatment. The data suggest that helium–neon laser stimulation does not raise the experimental pain threshold in healthy subjects compared to placebo treatment. After helium–neon laser stimulation there was a decrease of the electrical pain threshold in the contralateral arm. To disclose the mechanism of this effect further experimental investigations under strict electrophysiological conditions are required. Paper received 3 December 1999; accepted after revision 6 January 2000.     

Promotion of Bone Repair in the Cortical Bone of the Tibia in Rats by Low Energy Laser (He-Ne) Irradiation

The effect of low energy laser (He-Ne) irradiation on bone repair in the cortical part of the tibia of the rat was investigated using biochemical and radioactive labeling methods. A fixed round hole was created in the lateral aspect of the tibia and the newly formed tissue was collected from the gap in the cortical bone. Alkaline phosphatase (ALP) and calcium progressively accumulated at the site of injury, peaking at 9 and 13 days postinjury, respectively. Direct irradiation of the hole injury with He-Ne laser on days 5 and 6 postinjury altered osteoblastic activity at the injured site as reflected by alkaline phosphatase activity. The laser irradiation also caused a significant increase (∼2-fold) in calcium accumulation at the site of injury for 9–18 days postinjury. The rate of calcium deposition, measured by radioactive calcium, was significantly higher (∼2-fold) in the laser-irradiated rats as compared with controls. It is concluded that the process of bone repair in a hole created in the rat tibia is markedly enhanced by direct He-Ne laser irradiation of the injured site at the optimal energy level and time postinjury. Received: 3 July 1995 / Accepted: 22 December 1995     

Nonthermal Effects of Nd:YAG Laser on Biological Functions of Human Skin Fibroblasts in Culture

Previous studies have indicated that laser can selectively affect the biological functions of cells. In the present study, the role of a thermal component in laser-induced alterations in the biology of human skin fibroblasts was examined. Cells were cultured on 96-well tissue culture plates, subjected to treatment with the Nd.YAG laser (wavelength 1,064 nm), and the temperature of the medium was monitored by a microprobe connected to a telethermometer. For comparison, parallel cultures were heated to the same temperatures by tungsten-halogen lamp. The cell cultures were analyzed for collagen synthesis by incubating the cultures with [³H]proline, and the collagen production was assayed by the synthesis of nondialyzable [³H]hydroxyproline. The rate of DNA replication was also determined by measuring the uptake of [³H]thymidine. A marked decrease of collagen production and thymidine incorporation was noted in the cultures subjected to Nd.YAG laser. No suchdecreases were noted in cultures heated to the corresponding temperatures by tungsten-halogen lamp. The results thus indicate that the biochemical alteration caused by the Nd.YAG laser in human fibroblast functions cannot be explained on the basis of thermal effects.     

Antibacterial Effect of Yellow He-Ne Laser Irradiation with Crystal Violet Solution on Porphyromonas gingivalis: An Evaluation Using Experimental Rat Model Involving Subcutaneous Abscess

. A study was conducted to evaluate the antibacterial effect of yellow He-Ne laser irradiation with crystal violet solution (CV) on Porphyromonas gingivalis (P.g.). Paper points were soaked with a P.g. suspension (10⁹ ml) with 0.8 mg/l CV added, laser-irradiated for 60 s (laser group), and implanted subcutaneously on the back of rats. Three additional groups were studied: CV group: the paper point was soaked with the P.g. suspension plus 0.8 mg/l CV, but laser irradiation was not performed; P.g. group: the paper point was soaked with the P.g. suspension only and laser irradiation was not performed; control group: the paper point was soaked with sterilised isotonic sodium chloride solution and laser irradiation was not performed. Seven days after implantation, block sections of all implanted sites were examined histologically. The abscess area in the laser group was smaller than in the P.g. group or CV group, but larger than in the control group. The number of inflammatory cells was greatest in the P.g. and CV groups, with fewer in the laser group and still fewer in the control group. The results indicate that a yellow He-Ne laser with 0.8 mg/l CV solution exerts an antibacterial effect in vivo. Paper received 12 July 1999; accepted after revision 24 March 2000.     

Helium-Neon laser irradiation inhibits the growth of kidney epithelial cells in culture

We have investigated the in vitro action of helium-neon (He-Ne) laser light on the cell cycle and the growth of rat kidney epithelial cell cultures. Dose-response studies showed that repeated He-Ne irradiation (dose rate 40 mW/cm²) once a day in a dose range between 11.9 and 142 J/cm² significantly inhibited cell growth, while daily irradiation with 4.7 J/cm² had no effect. Microscopic examination of nuclear spreads revealed an increased number of cells in mitosis after a single irradiation with 142 J/cm². These results support previous observations suggesting that laser light of low thermal energy interferes with cell cycling and may inhibit cell growth when irradiation is performed at doses of 11.9 J/cm² or more.     

Photodynamic therapy with photofrin in combination with Buthionine Sulfoximine (BSO) of human glioma in the nude rat

High concentrations of cellular glutathione (GSH) within tumour cells may reduce the ability of photodynamic therapy (PDT) to selectively destroy tumour, consequently, a means of improving the therapeutic ratio of PDT in brain tumour is necessary. Therefore, we hypothesize that PDT in combination with Buthionine Sulfoximine (BSO), an agent which lowers cellular glutathione, can significantly enhance destruction of U87 and U251n tumour cells. PDT was performed using Photofrin as a photosensitiser in combination with BSO administration on male Fisher rats with intracerebral U87 and on non-tumour rats (administered at different optical doses in combination with Photofrin). In vitro experimentation utilising colony forming, cell cytotoxicity, and matrigel artificial basement membrane invasion assays showed significant enhancement of tumour kill and significant reduction of migration in tumour cells treated with BSO in combination with Photofrin PDT in comparison with individual therapies for both U87 and U251n cell lines. In vivo combination PDT-BSO treatment of U87 tumour rats exhibited significantly more tumour necrosis than individual treatments. In conclusion, our data suggests BSO enhances Photofrin PDT treatment of human glioma.     

A Comparative Optical Analysis of Cylindrical Diffuser Fibres for Laser Therapy Using Fluorescence Imaging

. Cylindrical light diffusers are commercially available for clinical applications such as photodynamic therapy (PDT) and interstitial laser photocoagulation (ILP). A fluorescence imaging technique has been used to quantify the light distribution produced by each of six different diffuser fibres. The light distribution produced by each device was found to depend on the distance the light propagated in the fluorescent dye solution. At a distance of 1 mm from the diffuser midline, the measured profiles were found to be consistent with published results obtained in air at a similar distance. The three devices intended for PDT utilised scattering particles and reflectors in their construction. The profiles produced by these applicators revealed peaks that were attributed to the reflectors located at their distal tips. By comparison, the two etched ILP fibres displayed either a strong modulation in the profile due to non-uniform etching or a predominant forward peak associated with the conical shape of the tip. We conclude that it is important to take into account the forward-directed light emitted by the diffusers when considering clinical applications using these devices. Paper received 12 January 1999; Accepted after revision 12 April 1999.     

Effects of combined valproic acid and the epidermal growth factor/vascular endothelial growth factor receptor tyrosine kinase inhibitor AEE788 on renal cell carcinoma cell lines in vitro

OBJECTIVE

To evaluate adhesion and growth inhibiting effects of the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on renal cell carcinoma (RCC) cells.

MATERIALS AND METHODS

Caki‐1 cells were treated with AEE788 and VPA, either alone or in combination, to investigate RCC cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. Tumour cell proliferation was examined by MTT dye reduction assay. Effects of drug treatment on cell signalling pathways were determined by Western blotting. The expression levels of integrin α and β subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression).

RESULTS

RCC cell treatment with AEE788 and VPA in combination resulted in a stronger inhibition of tumour cell proliferation than that caused by either drug alone. There were also additive effects of the combined treatment on tumour cell adhesion to endothelial cells and to immobilized laminin (but not to immobilized fibronectin and collagen). AEE788 alone or combined with VPA reduced Akt expression and histone H3 acetylation. Both compounds altered integrin α and β subtype expression, in particular α1, α3 and β4, and blocked integrin‐dependent integrin‐linked kinase and focal‐adhesion kinase (total and phosphorylated) signalling.

CONCLUSIONS

Both AEE788 and VPA profoundly block the interaction of RCC cells with endothelium and extracellular matrix and reduce tumour growth in vitro. Therefore, this combined regimen warrants further preclinical and possible clinical study for treating advanced RCC.     

强骨宝方对体外培养成骨细胞影响的实验研究

目的:探讨不同浓度强骨宝方提取液直接添加对体外培养成骨细胞增殖、分化与矿化功能的影响。方法:采用胰蛋白酶-Ⅱ型胶原酶消化法从2只1~2日龄SD乳鼠颅盖骨中分离出成骨细胞,鉴定细胞并传代培养后,分为对照组与4个实验组,实验组分别用终浓度为100、50、10、5μg/ml的强骨宝方提取液加入成骨细胞培养体系,对照组用不含强骨宝方提取液的培养基培养,应用MTT比色法、ALP含量测定、矿化结节形成等分别观察其对成骨细胞增殖、分化及矿化能力的影响。结果:100、50及10μg/ml浓度的强骨宝方提取液均具有促进体外成骨细胞增殖、分化与矿化的作用,以100μg/ml与50μg/ml促进作用最强。结论:每毫升含生药2g的强骨宝方提取液,pH=7.0,50μg/ml的添加浓度可能最适合于体外培养成骨细胞的增殖、分化及矿化成骨能力。     

5-氨基乙酰丙酸光动力学疗法对两种人卵巢癌细胞的体外杀伤作用

目的：探讨5-氨基乙酰丙酸（5-ALA）光动力学疗法（photodynamic therapy,PDT）对体外培养人卵巢癌SKOV3和AO细胞的杀伤作用。方法：于体外培养的SKOV3和AO细胞分别加入不同浓度的5-ALA（0．1、0．5、1．0、2．5、12．5mmol／L）孵育4h,以波长为630nm的半导体激光进行照射,照射能量密度分别为0．1、0．5、2．5、12．5J／cm^2。用四唑盐（MTT）比色法测定细胞存活率,并与单纯药物对照组（不同浓度5ALA孵育、无光照）、单纯光照组（无5-ALA孵育、不同能量密度光照）与阴性对照组（无药物孵育、无光照）进行比较。计算5ALA—PDT对SKOV3和AO细胞的半数杀伤浓度（IC50）。结果：5-ALA—PDT处理后两种卵巢癌细胞存活率均明显下降（P〈0．05）,提示5ALA—PDT对体外培养的人卵巢癌SKOV3和AO细胞均有杀伤作用,其杀伤作用随5-ALA的浓度和激光能量密度增加而增加,当5-ALA浓度超过2．5mmol／L或照射能量密度超过2．5J／cm^2时细胞存活率变化不明显。单纯药物对照组、单纯光照组的细胞存活率与阴性对照组相比差异无显著性（P〉0．05）。5-ALA-PDT对两种卵巢癌细胞杀伤效应强弱不同,对SKOV3及AO细胞的半抑制浓度（IC50）分别为0．34mmol／L和2．50mmol／L,两者差异有显著性（P〈0．05）。结论：5-ALA-PDT可有效杀伤体外培养的人卵巢癌细胞SKOV3和AO细胞,且对两种细胞的杀伤作用有差异,对SKOV3细胞株的杀伤效应要强于AO细胞株。     

Picosecond Laser Ablation of Dentine in Endodontics

. The interaction of picosecond laser radiation with human dental tissue was investigated in this study, in order to determine the ablation rates and the surface characteristics of the dentine by using scanning electron microscopy (SEM). Dentine ablation was performed by using tooth sections of different thicknesses (0.5–2.0 mm). Dental tissue samples were irradiated in air with the fundamental wavelength and first harmonic of a regenerative amplifier Nd:YAG laser system, at 1064 nm and 532 nm, respectively, with a pulse duration of 100 ps and a pulse repetition rate of 10 Hz. The results showed very clean craters surrounded by minimum melting of the surface of dentine when the 1064 nm pulses were used. In contrast, when the first harmonic 532 nm pulses were used, the SEM examinations revealed cracks and melting of dentine with irregular surface modification. Consequently, it seems that cleaning and shaping of the root canal walls during endodontic therapy with the picosecond Nd:YAG laser application may be possible in the future. The, as yet unexplored, field of the picosecond laser interaction with hard dental tissue is expected to be a potential alternative for powerful laser processing of biomedical structures. Paper received 24 February 1998; accepted following revision 20 November 1998.     

Enhanced Sensory Reinnervation of Dental Target Tissues in Rats Following Low Level Laser (LLL) Irradiation

. Previous studies have suggested that low level laser (LLL) treatment at specific wavelengths can enhance motor and sensory function in peripheral nerves after injury. The dental pulp is innervated by unmyelinated C fibres and small myelinated Aδ fibres derived from the trigeminal ganglion, both of which are known to contain calcitonin gene related peptide (CGRP). The aim of the present study was to examine the effects of daily LLL treatment on the sensory reinnervation of the first molar pulp 10 days subsequent to inferior alveolar nerve (IAN) axotomy, by counting the number of nerve fibre profiles immunoreactive to CGRP in rats. Axotomy of the IAN was performed via an extraoral buccal incision. The animals (n=11) were divided into two groups receiving either daily LLL treatment with a GaAlAs laser (λ=830 nm), or sham laser treatment over the site of injury for 10 days postoperatively. The animals were transcardially perfused and fixed at death before excising the jaws for further fixation and demineralisation. The jaws were cryosectioned obliquely through the mesial root pulp of the first molar tooth, which was the chosen level for evaluation of reinnervation. The CGRP antigen–antibody binding sites were visualised using a standard avidin biotin peroxidase technique. Both sham and LLL treatment and the evaluation of results were conducted blind. The results were statistically analysed and presented as median with 25%–75% quartiles. A statistically significant (p≤0.0000) greater number of CGRP immunoreactive nerve profiles were seen in the LLL-treated group (median=5, range 4–6) compared to the sham laser treated group (median=2, range 1–3), at the specified area for evaluation. These results suggest that LLL treatment can enhance reinnervation of denervated dental tissues after IAN axotomy in the rat. The enhanced reinnervation could be due to accelerated regeneration of the axotomised nerve, to collateral reinnervation, or a combination of both these nerve responses. The mechanisms whereby these changes are effected are still unknown. Paper received 15 October 1997; accepted following revision 25 November 1998.     

成人甲状旁腺细胞的培养方法及其分泌功能研究

目的探讨成人甲状旁腺细胞的体外培养方法及其分泌功能。方法采用胶原酶法消化成人甲状旁腺腺瘤组织,进行原代和传代细胞培养。每天在显微镜下观察原代细胞和传代细胞的形态变化。测量原代培养1、5、10、15及20 d和传代培养1、5、10及15 d时培养液中甲状旁腺激素（parathyroid hormone,PTH）的水平。结果消化后的细胞形态完整,原代培养2 d时大部分细胞贴壁并展开,3 d时细胞全部展开,4～15 d期间的细胞形态变化不大,16～20 d时部分细胞开始出现老化。传代培养1 d时细胞全部贴壁展开,主要呈梭形,与原代细胞相比体积略有增大;2～15 d期间的细胞形态变化不大。原代细胞上清液中PTH浓度从10 d开始明显降低,15 d时的PTH浓度低于10 d（P〈0.01）。同时点传代细胞上清液中PTH的浓度均低于原代甲状旁腺细胞,且5 d和1 d、10 d和5 d、15 d和10 d比较,上清液中PTH浓度的差异均无统计学意义（P〉0.05）。结论原代培养10 d内的甲状旁腺细胞具有良好的形态结构,分泌功能最强,可作为永久性甲状旁腺功能低下时同种异体细胞移植的细胞来源;传代后的细胞仍具有分泌功能,但远低于原代细胞。     

Dosedependent photodynamic effects of 9-acetoxy-2,7,12,17-tetrakis(\-methoxyethyl)-porphycene in vitro

Porphycenes are chemically pure photosensitizers for topical and systemic photodynamic therapy (PDT). Fast cellular uptake of 9-acetoxy-2,7,12,17-tetrakis-(sB-methoxyethyl)porphycene (ATMPn) has been shown previously. HaCaT human keratinocytes were incubated with ATMPn (1 nmol l^-1 to 1 μmol l^-1 in DMSO or DOPC liposomes). After 1 h, cells were irradiated with different light doses (0, 24, 48J cm^-2) using an incoherent light source (580—740 nm, 40 mW cm^-2). Cytotoxic effects were determined by assessing the mitochondrial activity using the MTT assay 24 h following irradiation. Cytotoxic effects were dependent on ATMPn concentration and light dose. Using 20 nmol 1^-1, a 50% decrease of mitochondrial activity (EC⁵⁰) after irradiation with 24 J cm^-2 was achieved. Lowering the ATMPn concentration (10nmol 1^-1) and increasing the light dose (48 J cm^-2) yielded the same effect (EC⁵⁰). Maximal decrease of mitochondrial activity (90%) was achieved using ATMPn concentrations of 50–100 nmol l^-1 and a light dose of 24 J cm^-2 or 25 nmol l^-1 ATMPn and 48 Jcm^-2. There was no difference regarding the dose-dependent cytotoxic effects using either ATMPn in DMSO or DOPC liposomes. In the control group (incubation with 1 nmol 1^-1 to 1μmol 1^-1 ATMPn, no irradiation), dark toxicity was not observed. Cell photosensitization with ATMPn was very efficient in vitro yielding the maximal cytotoxic effect at very low ATMPn concentrations as compared to other photosensitizers. Since ATMPn in DMSO and DOPC liposomes revealed the same cytotoxic effects without dark toxicity, theDMSO formulation, which is much easier to prepare, will be preferred in future studies.     

Damage to Tumour and Brain by Interstitial Photodynamic Therapy in the 9L Rat Tumour Model Comparing Intravenous and Intratumoral Administration of the Photosensitiser

Summary In the 9L rat brain tumour model the damage to tumour and normal brain by photodynamic therapy after intratumoural photosensitizer administration (intratumoural PDT) was studied. Twenty four rats received an intratumoural injection of 4 or 40 mm³ haematoporphyring deriative (HpD, 5 mg ml^–1), followed by interstitial irradiation with 20 Joule (J) (630 nm) 5 h later. For comparison, seven rats were treated with 20 Joule 24 h after an intravenous injection of 10 mg kg^–1 HpD (intravenous PDT). With the chosen PDT parameters there was no important difference between the damaged areas produced by intratumoural PDT or intravenous PDT. No selective tumour kill was observed. Even though normal brain tissue was heavily damaged, vital tumour parts were still present. Intravenous PDT caused extensive diffuse damage to small blood vessels in tumour and surrounding normal brain. Intratumoural PDT was characterised by an infiltration of polymorphonuclear cells into damaged tissue, dilatation of larger blood vessels and gross haemorrhage. These results suggest an immediate vascular shutdown in the intravenous approach, while in the intratumoural approach the vasculature remained patent initially. Because of the severe side effects observed, the use of HpD seems not advisable for intratumoural PDT of brain tumours.     

Photodynamic therapy of C3H tumours in mice: Effect of drug/light dose fractionation and misonidazole

C3H mammary carcinomas transplanted to the feet of mice were treated with haematoporphyrin derivative (HPD) or Photofrin II(PII) and laser light at 630 nm. While fluence rates lower than 100 mW cm⁻² gave minimal hyperthermic effects, a slight but significant growth delay was observed in unsensitized tumours exposed to a fluence rate of 150 mW cm⁻² which induced tumour temperatures in the range 40–50°C. Different modes of fractionation of the light fluence and of the HPD dose were tested but were found to give poorer rather than better results than the application of a single light exposure 24 h after intraperitoneal injection of HPD. Different PII doses were applied together with different light fluences, keeping the product of the drug dose and light fluence constant. In the dose range 6.25–50 mg/kg body weight the resulting effect on tumours was constant, allowing for a slight effect of hyperthermia at the highest light fluences, and possibly a photodegradation of PII. Misonidazole given before photodynamic treatment (PDT) slightly reduced the effect of PDT on the tumour growth. When given after PDT, however, misonidazole improved the therapeutic results significantly.