Significant skin-tightening by closure of fractional ablative laser holes.

BACKGROUND AND OBJECTIVE: Ablative fractional laser treatment uses thousands of very small laser beam wounds to damage a fraction of the skin, which stimulates tissue remodeling. Each open micro-wound heals without scarring, but the amount of skin tightening achieved is limited. This animal study was performed to test the hypothesis that immediate temporary closure of fractional laser wounds could increase skin tightening after fractional ablative laser treatment. MATERIALS AND METHODS: Four adult swine were used for the study; 98 square test sites (3 × 3 cm) were tattooed on the abdomen and flanks of each pig. An ablative fractional Erbium:YAG laser (Sciton Profile, Sciton Inc, Palo Alto, CA) was used to treat the test areas. A laser micro-spot fluence of 375 J/cm2 was delivered in 150-250 microseconds pulses, resulting in an array of ablation channels extending 1.5 mm deep into the skin, with a spot size of 250 µm, with 10% treatment density. Immediately following laser exposure the resulting holes were closed using a stretched elastic adhesive dressing, which, when applied, recoiled and compressed the diameter of the ablation holes. The compressive dressings were removed after 7 days. This procedure was compared to removing the same amount of skin (10%) mechanically by specially designed 19 gauge coring needles, as well as to the same laser and coring methods without compression closure. Area and shape of test sites were measured by digital photography before and 28 days after treatment. Data analysis included compensation for animal growth, as measured by increase in the area of the untreated control sites. RESULTS: All treated and control sites healed within a week, without scarring evident at 28 days. Laser treatment combined with compressive wound closure caused significant shrinkage at 28 days compared with untreated control sites. The treated skin area was reduced by 11.5% (P = 0.0001). Needle coring with wound closure produced similar, significant shrinkage (8%, P < 0.0021), whereas laser and needle coring treatment without closure did not result in significant area reduction (P = 0.1289) compared with untreated control sites. CONCLUSION: Significant skin tightening can be achieved by immediate temporary non-invasive wound closure after short pulse Er:YAG fractional ablative laser treatment, as well as after mechanically removing skin with a coring needle. This approach may improve skin tightening after ablative laser treatments. Further clinical studies are necessary to confirm successful application in humans. Lasers Surg. Med. 50:64-69, 2018. © 2017 Wiley Periodicals, Inc.

Micro-fractional, directional skin tightening: A porcine model.

BACKGROUND AND OBJECTIVE: Skin changes are among the most visible signs of aging. Fractional ablative lasers improve skin quality by making small skin wounds that heal rapidly without scarring. While they improve skin texture and discoloration, there is minimal effect on skin laxity. This study was performed to assess skin shrinkage performed by removing multiple small full-thickness skin columns with coring needles combined with wound closure. MATERIALS AND METHODS: In 5 swine 116 squares (3 cm(2) ) were demarcated for treatment and control sites. In treatment sites 10% of the skin was removed by full-thickness skin coring needles (19 gauge) and afterwards closed and compressed with an elastic adhesive dressing. This procedure was compared to puncturing the skin with standard hypodermic needles (without tissue removal) and subsequent closure with compressive dressing. Area and shape of sites were measured before and 28 days after treatment. RESULTS: Test and control sites healed within a week without scarring. Coring with wound closure caused significant shrinkage after 28 days. The treated skin area was reduced by 9% (P < 0.0001) and the direction of shrinkage was influenced by the direction of wound closure. Coring without wound closure and puncturing the skin without tissue removal produced an insignificant 3% decrease in area. CONCLUSION: Significant minimally invasive skin tightening in a preferred direction can be achieved by removing skin with coring needles followed by wound closure. The direction of shrinkage is influenced by the direction of micro-hole closure, irrespective of the skin tension lines. This approach may allow reshaping the skin in a desired direction without scarring.

Fractional laser-assisted drug delivery: Active filling of laser channels with pressure and vacuum alteration.

BACKGROUND AND OBJECTIVE: Ablative fractional laser (AFXL) is rapidly evolving as one of the foremost techniques for cutaneous drug delivery. While AFXL has effectively improved topical drug-induced clearance rates of actinic keratosis, treatment of basal cell carcinomas (BCCs) has been challenging, potentially due to insufficient drug uptake in deeper skin layers. This study sought to investigate a standardized method to actively fill laser-generated channels by altering pressure, vacuum, and pressure (PVP), enquiring its effect on (i) relative filling of individual laser channels; (ii) cutaneous deposition and delivery kinetics; (iii) biodistribution and diffusion pattern, estimated by mathematical simulation. METHODS: Franz diffusion chambers (FCs) were used to evaluate the PVP-technique, comparing passive (AFXL) and active (AFXL + PVP) channel filling. A fractional CO2-laser generated superficial (225 µm;17.5 mJ/channel) and deep (1200 µm; 130.5 mJ/channel) channels, and PVP was delivered as a 3-minutes cycle of 1 minute pressure (+1.0 atm), 1 minute vacuum (-1.0 atm), and 1 minute pressure (+1.0 atm). Filling of laser channels was visualized with a colored biomarker liquid (n = 12 FCs, n = 588 channels). Nuclear magnetic resonance quantified intracutaneous deposition of topically applied polyethylene glycol (PEG400) over time (10 minutes, 1 hour, and 4 hours), investigated with (n = 36 FCs) and without (n = 30 FCs) PVP-filling. Two-dimensional mathematical simulation was used to simulate intradermal biodistribution and diffusion at a depth of 1,000 µm. RESULTS: Active filling with application of PVP increased the number of filled laser channels. At a depth of 1,000 µm, filling increased from 44% (AFXL) to 94% with one PVP cycle (AFXL + PVP; P < 0.01). Active filling greatly enhanced intracutaneous deposition of PEG400, resulting in a rapid delivery six-folding uptake at 10 minutes (AFXL 54 µg/ml vs. AFXL + PVP 303 µg/ml, P < 0.01). AFXL alone generated an inhomogeneous uptake of PEG400, which greatly improved with active filling, resulting in a uniform uptake within the entire tissue. CONCLUSION: Active filling with PVP secures filling of laser channels and induces a deeper, greater, more rapid delivery than conventional AFXL. This delivery technique has promise to improve treatment efficacy for medical treatments of dermally invasive lesions, such as BCCs.

Pretreatment with ablative fractional laser changes kinetics and biodistribution of topical 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL).

BACKGROUND AND OBJECTIVES: 5-Aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) are porphyrin precursors used topically for photodynamic therapy (PDT). Previous studies have established that ablative fractional laser (AFXL) increases topical drug uptake. We evaluated kinetics and biodistribution of ALA- and MAL-induced porphyrins on intact and disrupted skin due to AFXL. MATERIALS AND METHODS: Two Yorkshire swine were exposed to CO2 AFXL (10.6 µm, 1,850 µm ablation depth) and subsequent topical application of ALA and MAL cream formulations (20%, weight/weight). Porphyrin fluorescence was quantified by digital fluorescence photography (30, 90, and 180 minutes) and fluorescence microscopy at specific skin depths (180 minutes). RESULTS: Porphyrins gradually formed over time, differently on intact and AFXL-disrupted skin. On intact skin (no AFXL), fluorescence photography showed that MAL initially induced higher fluorescence than ALA (t = 30 minutes MAL 21.1 vs. ALA 7.7 au, t = 90 minutes MAL 39.0 vs. ALA 26.6 (P < 0.009)) but reached similar intensities for long-term applications (t = 180 minutes MAL 56.6 vs. ALA 52 au, P = ns). AFXL considerably enhanced porphyrin fluorescence from both photosensitizers (P < 0.05). On AFXL-exposed skin, MAL expressed higher fluorescence than ALA for short-term application (t = 30 minutes, AFXL-MAL 26.4 vs. AFXL-ALA 14.1 au, P < 0.001), whereas ALA over time overcame MAL and induced the highest fluorescence intensities obtained (t = 180 minutes, AFXL-MAL 98.6 vs. AFXL-ALA 112.0 au, P < 0.001). In deep skin layers, fluorescence microscopy showed higher fluorescence in hair follicle epithelium for ALA than MAL (t = 180 minutes, 1.8 mm, AFXL-MAL 35.3 vs. AFXL-ALA 46.7 au, P < 0.05). CONCLUSIONS: AFXL changes kinetics and biodistribution of ALA and MAL. It appears that AFXL-ALA favors targeting deep structures.

Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity.

BACKGROUND: Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity. METHODS: The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG). RESULTS: After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the "fish-net" patterning caused by STSG. CONCLUSION: Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing.

Fractional laser-assisted delivery of methyl aminolevulinate: Impact of laser channel depth and incubation time.

BACKGROUND AND OBJECTIVES: Pretreatment of skin with ablative fractional lasers (AFXL) enhances the uptake of topical photosensitizers used in photodynamic therapy (PDT). Distribution of photosensitizer into skin layers may depend on depth of laser channels and incubation time. This study evaluates whether depth of intradermal laser channels and incubation time may affect AFXL-assisted delivery of methyl aminolevulinate (MAL). MATERIALS AND METHODS: Yorkshire swine were treated with CO2 AFXL at energy levels of 37, 190, and 380 mJ/laser channel and subsequent application of MAL cream (Metvix) for 30, 60, 120, and 180 minutes incubation time. Fluorescence photography and fluorescence microscopy quantified MAL-induced porphyrin fluorescence (PpIX) at the skin surface and at five specific skin depths (120, 500, 1,000, 1,500, and 1,800 µm). RESULTS: Laser channels penetrated into superficial (∼300 µm), mid (∼1,400 µm), and deep dermis/upper subcutaneous fat layer (∼2,100 µm). Similar fluorescence intensities were induced at the skin surface and throughout skin layers independent of laser channel depth (180 minutes; P < 0.19). AFXL accelerated PpIX fluorescence from skin surface to deep dermis. After laser exposure and 60 minutes MAL incubation, surface fluorescence was significantly higher compared to intact, not laser-exposed skin at 180 minutes (AFXL-MAL 60 minutes vs. MAL 180 minutes, 69.16 a.u. vs. 23.49 a.u.; P < 0.01). Through all skin layers (120-1,800 µm), laser exposure and 120 minutes MAL incubation induced significantly higher fluorescence intensities in HF and dermis than non-laser exposed sites at 180 minutes (1,800 µm, AFXL-MAL 120 minutes vs. MAL 180 minutes, HF 14.76 a.u. vs. 6.69 a.u. and dermis 6.98 a.u. vs. 5.87 a.u.; P < 0.01). CONCLUSIONS: AFXL pretreatment accelerates PpIX accumulation, but intradermal depth of laser channels does not affect porphyrin accumulation. Further studies are required to examine these findings in clinical trials.

Selective photothermolysis to target sebaceous glands: theoretical estimation of parameters and preliminary results using a free electron laser.

BACKGROUND AND OBJECTIVES: The success of permanent laser hair removal suggests that selective photothermolysis (SP) of sebaceous glands, another part of hair follicles, may also have merit. About 30% of sebum consists of fats with copious CH(2) bond content. SP was studied in vitro, using free electron laser (FEL) pulses at an infrared CH(2) vibrational absorption wavelength band. METHODS: Absorption spectra of natural and artificially prepared sebum were measured from 200 to 3,000 nm, to determine wavelengths potentially able to target sebaceous glands. The Jefferson National Accelerator superconducting FEL was used to measure photothermal excitation of aqueous gels, artificial sebum, pig skin, human scalp, and forehead skin (sebaceous sites). In vitro skin samples were exposed to FEL pulses from 1,620 to 1,720 nm, spot diameter 7-9.5 mm with exposure through a cold 4°C sapphire window in contact with the skin. Exposed and control tissue samples were stained using H&E, and nitroblue tetrazolium chloride staining (NBTC) was used to detect thermal denaturation. RESULTS: Natural and artificial sebum both had absorption peaks near 1,210, 1,728, 1,760, 2,306 and 2,346 nm. Laser-induced heating of artificial sebum was approximately twice that of water at 1,710 and 1,720 nm, and about 1.5× higher in human sebaceous glands than in water. Thermal camera imaging showed transient focal heating near sebaceous hair follicles. Histologically, skin samples exposed to ~1,700 nm, ~100-125 milliseconds pulses showed evidence of selective thermal damage to sebaceous glands. Sebaceous glands were positive for NBTC staining, without evidence of selective loss in samples exposed to the laser. Epidermis was undamaged in all samples. CONCLUSIONS: SP of sebaceous glands appears to be feasible. Potentially, optical pulses at ~1,720 or ~1,210 nm delivered with large beam diameter and appropriate skin cooling in approximately 0.1 seconds may provide an alternative treatment for acne.

Intracutaneous ALA photodynamic therapy: dose-dependent targeting of skin structures.

BACKGROUND: Photodynamic therapy (PDT) using topical aminolevulinic acid (ALA) depends on local drug uptake, metabolism to porphyrins, and depth of light penetration using different wavelengths. Topical ALA-PDT has limited depth of drug penetration. We studied induced porphyrin distribution and PDT after intradermal ALA administration using different drug concentrations followed by high-fluence red light irradiation. MATERIALS AND METHODS: Intradermal injections (∼2 mm deep) of ALA concentrations from 0.0005% to 1% were studied in swine to evaluated porphyrin fluorescence before PDT and clinical and histological damage 24 hours after PDT. Porphyrin accumulation was measured by fluorescence microscopy of frozen section. PDT was performed 3 hours after intradermal injections using a 635 nm LED array at a fluence of 200 J/cm2 . Skin responses to PDT were observed grossly and by histology (blind evaluation). RESULTS: Intradermal ALA caused porphyrin accumulation in epidermis, hair follicles (HF), sebaceous glands (SG), sweat glands (eccrine glands, EG and apocrine glands, AG), and subcutaneous fat. Significant differences of fluorescence intensity were observed between different skin structures (P < 0.05), but there was no significant difference comparing HF to SG; epidermis with either HF or SG; and dermis with fat (P > 0.05). Intradermal ALA is potent. ALA concentrations ≥0.25% followed by red light exposures caused a very intense vascular PDT reaction. Moderate doses of injected ALA concentration (∼0.06%), selectively targeted EG. Low doses (≤0.016%) targeted fat; producing fat necrosis with minimal inflammation, manifested both clinically and histologically. In contrast to topical ALA-PDT, intradermal ALA-PDT can effectively photosensitize deep skin structures. CONCLUSION: Potentially, intradermal ALA-PDT using various ALA concentrations may be useful for treating vascular lesions (malformations, hemangiomas, tumors), EG/AG disorders, fat or deep targets in skin.

Fractional CO(2) laser-assisted drug delivery.

BACKGROUND AND OBJECTIVES: Ablative fractional resurfacing (AFR) creates vertical channels that might assist the delivery of topically applied drugs into skin. The purpose of this study was to evaluate drug delivery by CO(2) laser AFR using methyl 5-aminolevulinate (MAL), a porphyrin precursor, as a test drug. MATERIALS AND METHODS: Two Yorkshire swine were treated with single-hole CO(2) laser AFR and subsequent topical application of MAL (Metvix(R), Photocure ASA, Oslo, Norway), placebo cream and no drug. MAL-induced porphyrin fluorescence was measured by fluorescence microscopy at skin depths down to 1,800 microm. AFR was performed with a 10.6 microm wavelength prototype CO(2) laser, using stacked single pulses of 3 millisecond and 91.6 mJ per pulse. RESULTS: AFR created cone-shaped channels of approximately 300 microm diameter and 1,850 microm depth that were surrounded by a 70 microm thin layer of thermally coagulated dermis. There was no porphyrin fluorescence in placebo cream or untreated skin sites. AFR followed by MAL application enhanced drug delivery with significantly higher porphyrin fluorescence of hair follicles (P<0.0011) and dermis (P<0.0433) versus MAL alone at skin depths of 120, 500, 1,000, 1,500, and 1,800 microm. AFR before MAL application also enhanced skin surface (epidermal) porphyrin fluorescence. Radial diffusion of MAL from the laser-created channels into surrounding dermis was evidenced by uniform porphyrin fluorescence up to 1,500 microm from the holes (1,000, 1,800 microm depths). Skin massage after MAL application did not affect MAL-induced porphyrin fluorescence after AFR. CONCLUSIONS: Ablative fractional laser treatment facilitates delivery of topical MAL deeply into the skin. For the conditions of this study, laser channels approximately 3 mm apart followed by MAL application could produce porphyrins throughout essentially the entire skin. AFR appears to be a clinically practical means for enhancing uptake of MAL, a photodynamic therapy drug, and presumably many other topical skin medications.

Porphyrin distribution after topical aminolevulinic acid in a novel porcine model of sebaceous skin.

BACKGROUND AND OBJECTIVE: Aminolevulinic acid photodynamic therapy (ALA-PDT) depends on drug metabolism into porphyrins. Clinically, ALA-PDT has been used with a wide range of protocols for treating both epidermal and dermal targets, despite limited understanding of porphyrin biodistribution over time. We studied porphyrin accumulation after topical application of ALA in vivo, and also describe the porcine ear as a new animal model to study adnexal glands. STUDY DESIGN/MATERIALS AND METHODS: The microanatomy of anterior ear skin of swine was measured. Topical 20% ALA in water/ethanol was applied under occlusion. Biopsies taken after 5, 10, 15, and then every 15 minutes for a total of 3 hours were examined by fluorescence microscopy of frozen sections to assess accumulation and distribution of porphyrins. RESULTS: Porphyrin fluorescence of digital photomicrograph images was not visually apparent until 30-45 minutes after application, although quantitative pixel analysis showed a statistically significant increase in epidermal fluorescence only 15 minutes after ALA application. From 30 to 120 minutes, epidermis, hair follicles (HF), and sebaceous glands (SG) became progressively more fluorescent. Eccrine gland fluorescence began to be detected after 30 minutes; SG showed fluorescence starting at 45-75 minutes. Fluorescence in all sites reached maximum intensity from 75 to 180 minutes of incubation. There was a trend for HF and SG to express stronger fluorescence compared with epidermis and eccrine glands. CONCLUSION: Anterior pig ear skin is microanatomically similar to human sebaceous skin. The time-dependent accumulation of porphyrins in pilosebaceous units and eccrine glands in this model suggests other routes of uptake of topical ALA in addition to the trans-epidermal route. Apparently, time interval between ALA application and light exposure could be optimized for different uses of ALA-PDT.

Photodynamic treatment of endodontic polymicrobial infection in vitro.

We investigated the photodynamic effects of methylene blue on multispecies root canal biofilms comprising Actinomyces israelii, Fusobacterium nucleatum subspecies nucleatum, Porphyromonas gingivalis, and Prevotella intermedia in experimentally infected root canals of extracted human teeth in vitro. The 4 test microorganisms were detected in root canals by using DNA probes. Scanning electron microscopy showed the presence of biofilms in root canals before therapy. Root canal systems were incubated with methylene blue (25 microg/mL) for 10 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). Light was delivered from a diode laser via a 250-microm diameter polymethyl methacrylate optical fiber that uniformly distributed light over 360 degrees. Photodynamic therapy (PDT) achieved up to 80% reduction of colony-forming unit counts. We concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized.

Photodynamic inactivation of Enterococcus faecalis in dental root canals in vitro.

BACKGROUND AND OBJECTIVES: We previously reported the use of a flexible fiber optic that uniformly distributed light in the root canal space for targeting bacteria after their sensitization with methylene blue (MB). In the present study, we investigated the photodynamic effects of MB on Enterococcus faecalis species in experimentally infected root canals of extracted teeth after their sensitization with a concentration of MB that exhibits reduced dark toxicity. STUDY DESIGN/MATERIALS AND METHODS: In a model of root canal infection, 64 root canal specimens were prepared from extracted, single-rooted teeth and inoculated with E. faecalis (ATCC 29212). Three days later root canal infection was confirmed by scanning electron microscopy. The root canal systems were then incubated with 6.25 microg/ml MB for 5 minutes followed by exposure to light at 665 nm (60 J/cm(2)) that was delivered from a diode laser via a fiber optic with a diameter of 500 microm. Following photodynamic therapy (PDT) the canal content was sampled by flushing the root canals, serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units. High-performance liquid chromatography (HPLC) was employed to determine the porphyrins content of E. faecalis. RESULTS: Scanning electron microscopy confirmed the presence of bacteria in the root canal system. PDT achieved 77.5% reduction of E. faecalis viability. MB alone and light alone reduced bacterial viability by 19.5% and 40.5%, respectively. HPLC did not reveal any porphyrin patterns expressed by E. faecalis. CONCLUSION: The results of this study support the need to determine the optimum MB concentration and light parameters to maximize bacterial killing in root canals.

Photodynamic therapy for endodontic disinfection.

The aims of this study were to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens in planktonic phase as well as on Enterococcus faecalis biofilms in experimentally infected root canals of extracted teeth. Strains of microorganisms were sensitized with methylene blue (25 microg/ml) for 5 minutes followed by exposure to red light of 665 nm with an energy fluence of 30 J/cm2. Methylene blue fully eliminated all bacterial species with the exception of E. faecalis (53% killing). The same concentration of methylene blue in combination with red light (222 J/cm2) was able to eliminate 97% of E. faecalis biofilm bacteria in root canals using an optical fiber with multiple cylindrical diffusers that uniformly distributed light at 360 degrees. We conclude that PDT may be developed as an adjunctive procedure to kill residual bacteria in the root canal system after standard endodontic treatment.

Irritant contact dermatitis induced by a common household irritant: a noninvasive evaluation of ethnic variability in skin response.

OBJECTIVE: This study was undertaken to investigate the ethnic susceptibility to irritant contact dermatitis induced by a common dishwashing liquid using noninvasive technologies. METHODS: A total of 30 participants (15 Caucasian, 15 African American) were patch tested to graded concentrations of a common household irritant and evaluated using clinical scoring, reflectance confocal microscopy, transepidermal water loss, and fluorescence excitation spectroscopy. RESULTS: At 24 hours, the concentration thresholds for clinically perceptible irritancy were significantly higher for African American compared with Caucasian participants. Reflectance confocal microscopy showed stratum corneum disruption, parakeratosis, and spongiosis; these features were more severe in Caucasian participants (P < or = .002). Mean values for transepidermal water loss were significantly higher in the Caucasian group at comparable clinical scores (P < or = .005). Fluorescence excitation spectroscopy showed a broad excitation band at 300 nm (emission 340 nm) and values in both groups returned to baseline by day 7. LIMITATIONS: This pilot study was limited in scope and larger studies are needed to further evaluate ethnic differences in irritant contact dermatitis and to demonstrate the applicability of our findings for other irritants. CONCLUSION: Clinical evaluation, reflectance confocal microscopy, and transepidermal water loss showed significant differences in the cutaneous irritant response between both groups suggesting a superior barrier function of African American skin. Fluorescence excitation spectroscopy on the other hand demonstrated no differences in the hyperproliferative response after irritant exposure and indicated similar kinetics for the two groups.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Non-invasive evaluation of the kinetics of allergic and irritant contact dermatitis.

Reflectance confocal microscopy (RCM) allows non-invasive visualization of human skin in vivo. It has been used to describe the histopathological features of acute contact dermatitis (CD). This work was designed to investigate the kinetics of both allergic and irritant CD (ACD and ICD) in vivo. Eighteen subjects with a prior diagnosis of ACD were patch tested with the specific allergen sodium lauryl sulfate as an irritant, and appropriate controls. RCM, transepidermal water loss (TEWL), and fluorescence excitation spectroscopy (FES) were performed at several time points within 2 wk after patch removal. After removal of the Finn chambers at 48 h, superficial epidermal changes, primarily involving the stratum corneum, and increased epidermal thickness were mainly present in ICD. ACD, on the other hand, showed microvesicle formation peaking at 96 h following patch removal. Both ACD and ICD showed exocytosis and similar degrees of spongiosis on RCM. TEWL and FES demonstrated a significant difference between ACD and ICD. RCM, TEWL, and FES are valuable non-invasive tools to quantitatively study the kinetics of the pathophysiology of acute CD reactions in vivo and monitor the changes at a cellular level.

Transdermal drug delivery with a pressure wave.

Pressure waves, which are generated by intense laser radiation, can permeabilize the stratum corneum (SC) as well as the cell membrane. These pressure waves are compression waves and thus exclude biological effects induced by cavitation. Their amplitude is in the hundreds of atmospheres (bar) while the duration is in the range of nanoseconds to a few microseconds. The pressure waves interact with cells and tissue in ways that are probably different from those of ultrasound. Furthermore, the interactions of the pressure waves with tissue are specific and depend on their characteristics, such as peak pressure, rise time and duration. A single pressure wave is sufficient to permeabilize the SC and allow the transport of macromolecules into the epidermis and dermis. In addition, drugs delivered into the epidermis can enter the vasculature and produce a systemic effect. For example, insulin delivered by pressure waves resulted in reducing the blood glucose level over many hours. The application of pressure waves does not cause any pain or discomfort and the barrier function of the SC always recovers.

Photodestruction of human dental plaque bacteria: enhancement of the photodynamic effect by photomechanical waves in an oral biofilm model.

BACKGROUND AND OBJECTIVES: Periodontal disease results from the accumulation of subgingival bacterial biofilms on tooth surfaces. There is reduced susceptibility of these biofilms to antimicrobials for reasons that are not known. The goals of this study were to investigate the photodynamic effects of a conjugate between the photosensitizer (PS) chlorin(e6) (c(e6)) and a poly-L-lysine (pL) with five lysine residues on human dental plaque bacteria as well as on biofilms of the oral species Actinomyces naeslundii after their exposure to photomechanical waves (PW) generated by a laser in the presence of the conjugate. STUDY DESIGN/MATERIALS AND METHODS: Subgingival plaque samples from 12 patients with chronic destructive periodontitis were divided in 3 groups that were incubated for 5 minutes with 5 microM c(e6) equivalent from the pL-c(e6) conjugate in the presence of fresh medium (group I), PBS (group II), and 80% PBS/20% ethylenediaminetetra-acetic acid (EDTA) (group III) and were exposed to red light. Also, biofilms of A. naeslundii (formed on bovine enamel surfaces) were exposed to PW in the presence of 5 microM c(e6) equivalent from the pL-c(e6) conjugate and were then irradiated with red light. The penetration depth of the conjugate was measured by confocal scanning laser microscopy (CSLM). In both cases, after illumination serial dilutions were prepared and aliquots were spread over the surfaces of blood agar plates. Survival fractions were calculated by counting bacterial colonies. RESULTS: The PS/light combination achieved almost 90% killing of human dental plaque species. In biofilms of A. naeslundii, CSLM revealed that PW were sufficient to induce a 50% increase in the penetration depth of the pL-c(e6) conjugate into the biofilm. This enabled its destruction (99% killing) after photodynamic therapy (PDT). CONCLUSIONS: PW-assisted photodestruction of dental plaque may be a potentially powerful tool for treatment of chronic destructive periodontal disease.

Ultrastructural evidence of stratum corneum permeabilization induced by photomechanical waves.

Photomechanical waves (high amplitude pressure transients generated by lasers) have been shown to permeabilize the stratum corneum in vivo and facilitate the transport of macromolecules into the viable epidermis. The permeabilization of the stratum corneum is transient and its barrier function recovers. Sites on the volar forearm of humans were exposed to photomechanical waves and biopsies were obtained immediately after the exposure and processed for electron microscopy. Electron microscopy showed an expansion of the lacunar spaces within the stratum corneum lipid bilayers but no changes in the organization of the secreted lamellar bodies at the stratum corneum-stratum granulosum boundary. The combination of photomechanical waves and sodium lauryl sulfate enhances the efficiency of transdermal delivery and delays the recovery of the barrier function of the stratum corneum. Electron microscopy from sites exposed to photomechanical waves and sodium lauryl sulfate showed that the lacunar spaces expanded significantly more and the secreted lamellar bodies also appeared to be altered. In either case, there were no changes in the papillary dermis. These observations support the hypothesis that the photomechanical waves induce the expansion of the lacunar spaces within the stratum corneum leading to the formation of transient channels.

Nuclear transport by laser-induced pressure transients.

PURPOSE: Control of the transport of molecules into the nucleus represents a key regulatory mechanism for differentiation, transformation, and signal transduction. Permeabilization of the nuclear envelope by physical methods can have applications in gene therapy. Laser-induced pressure transients can produce temporary aqueous pores analogous to those produced by electroporation and that the cells can survive this procedure. In this study, we examine the role of the pressure transients in creating similar pores in the nuclear envelope. METHODS: The target human peripheral blood mononuclear cells in a 62 microM 72 kDa fluoresceinated dextran solution were exposed to the pressure transients generated by laser ablation. An in vitro fluorescence confocal microscope was used to visualize and quantify the fluoresceinated dextran in the cytoplasmic and nuclear compartments. RESULTS: In contrast to electroporation, the pressure transients could deliver 72 kDa fluoresceinated dextrans, which are normally excluded by the nucleus, across the nuclear envelope into the nucleus. In addition to creating pores in the plasma membrane, temporary pores were also created in the nuclear envelope following exposure to pressure transients. CONCLUSION: The production of temporary nuclear pores could provide a unique resource for drug-delivery and gene therapy.