Novel methods for generating fractional epidermal micrografts.

BACKGROUND: Epidermal suction blister grafts are an effective treatment for chronic wounds or vitiligo, but this treatment is time consuming and limited to small areas. OBJECTIVES: To compare two novel strategies to create fractional epidermal grafts. METHODS: Epidermal blisters were raised from fresh human skin ex vivo at 38-40 °C, with suction of 380-510 mmHg. In Strategy 1, a 1-cm blister was micromeshed into approximately 500 pieces, transferred to elastic adhesive dressing, then pneumatically expanded to approximately nine times the original blister area. In Strategy 2, a 25-cm(2) array of 100 small blisters was raised, simultaneously harvested and captured directly onto an adhesive dressing. Measurements were taken for the pneumatic expansion limit, the release of microblisters upon hydration of the dressing adhesive, light microscopy, epidermal cell viability and positive L-3,4 dihydroxyphenylalanine melanocyte presence in blisters. RESULTS: Both strategies yielded viable fractional epidermal microblister arrays, carried on a dressing for transfer to graft recipient sites. The microblisters were gradually released upon hydration of the dressing adhesive. Strategy 2 has major advantages as only small blisters are made at the donor site, skilful dissection and physical expansion are not required and the strategy can be scaled to create large-area grafts. CONCLUSIONS: Strategy 2 is the more practical method for fractional epidermal micrografting to treat larger lesions with less donor-site trauma and has recently been commercialized.

Enhanced uptake and photoactivation of topical methyl aminolevulinate after fractional CO2 laser pretreatment.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) of thick skin lesions is limited by topical drug uptake. Ablative fractional resurfacing (AFR) creates vertical channels that may facilitate topical PDT drug penetration and improve PDT-response in deep skin layers. The purpose of this study was to evaluate whether pre-treating the skin with AFR before topically applied methyl aminolevulinate (MAL) could enable a deep PDT-response. MATERIALS AND METHODS: Yorkshire swine were treated under general anesthesia with a fractional CO(2) laser using stacked single pulses of 3 milliseconds, 91.6 mJ per pulse and subsequent topical MAL application for 3 hours (Metvix®). Red light (LED arrays) was then delivered at fluences of 37 and 200 J/cm(2). Fluorescent photography and microscopy was used to quantify MAL-induced porphyrin distribution and PDT-induced photobleaching at the skin surface and five specific depths down to 1,800 µm. RESULTS: Laser-ablated channels were approximately 1,850 µm deep, which significantly increased topical MAL-induced porphyrin fluorescence (hair follicles, dermis, P < 0.0001) and PDT response, both superficially and deep, versus topical MAL application alone. The fraction of porphyrin fluorescence lost by photobleaching was slightly less after 37 J/cm(2) than after 200 J/cm(2) (overall median values 67-90%; 37 vs. 200 J/cm(2), P > 0.05 for all but one comparison). Photobleaching was steady throughout skin layers and did not vary significantly with skin depth at either LED fluence (P > 0.05). CONCLUSIONS: AFR greatly facilitates topical MAL-induced porphyrins and the fraction of photobleached porphyrins is similar for superficial and deep skin. These observations are consistent with AFR-enhanced uptake of MAL, increased porphyrin synthesis, and photodynamic activation of deep porphyrins even at the lower fluence of 37 J/cm(2), widely used in clinical practice. AFR appears to be a clinically practical means for improving PDT deep into the skin. Clinical studies are suggested to evaluate selectivity in targeting dysplastic cell types.

Hair removal with an 800-nm pulsed diode laser.

BACKGROUND AND OBJECTIVE: Laser hair removal is a relatively new procedure. Our purpose was to study the efficacy and safety of a high-power, pulsed diode laser array for removing unwanted hair. METHODS: A total of 38 subjects were treated with a prototype of the 800-nm diode laser system. Fluences ranging from 10 to 40 J/cm(2) (mean, 33.4 J/cm(2)) were used and 1 to 4 treatments (mean, 2.7) were performed. Evaluation of hair loss was performed at least 4 months after the last treatment (mean, 8.7 months) by a blinded assessment of clinical photographs. RESULTS: A total of 59% of the subjects had only sparse hair regrowth at the final follow-up. Higher fluences and multiple treatments produced greater long-term efficacy. Transient pigmentary changes occurred in 29% of the subjects and were more common in darker skin types IV to VI (P =. 047). CONCLUSION: The 800-nm diode laser is an efficient and safe technique for hair reduction. Adverse pigmentary effects occur, but are transient.

Reduction of regrowing hair shaft size and pigmentation after ruby and diode laser treatment.

Laser pulses which selectively damage pigmented hair follicles are a useful treatment for hypertrichosis. Clinically, regrowing hairs are often thinner and lighter after treatment. In this study, hair shaft diameter and optical transmission (700 nm) were measured before and after ruby (694 nm) and diode (800 nm) laser irradiation. Hair was collected from 47 and 41 subjects treated with ruby (0.3 ms and 3 ms) and diode (10-20 ms) lasers, respectively. "Responders" were defined as subjects with significant long-term hair loss as determined by hair counts at 9 and/or 12 months after treatment. In ruby laser responders (34/47), regrowing hairs were significantly both thinner (decreased diameter) and lighter (increased transmission). In "nonresponders" (13/47), regrowing hairs were lighter, but not thinner. The regrowing hair shaft absorption coefficient (as calculated assuming Beer's law) was significantly decreased by 0.3 ms ruby laser treatment, but was not changed by 3 ms ruby laser or diode laser treatment. After diode laser treatment, 38 of the 41 subjects were responders and regrowing hairs were both thinner and lighter. These results show that laser treatments can affect structural recovery (size of hair), follicular pigmentation (hair absorption coefficient), or both. Regrowth of thinner hair (decreased shaft diameter) occurs in conjunction with actual loss of hair. After long pulses (3 ms ruby; diode), regrowing hair was thinner and also lighter to an extent related to the decrease in hair diameter. In contrast, short ruby laser pulses (0.3 ms) appeared to be capable of inhibiting follicular pigmentation per se, in addition to affecting the hair diameter. This may account for the complete regrowth of lighter hair in "nonresponders" treated with 0.3 ms pulses. Laser-induced reduction in hair diameter and/or pigmentation are both long-term responses which confer cosmetic benefits in addition to actual hair loss.

Ruby laser hair removal: evaluation of long-term efficacy and side effects.

BACKGROUND AND OBJECTIVE: Although several studies on laser-assisted hair removal have been published, data on long-term follow-up are few. The present study investigated the long-term efficacy and safety of normal-mode ruby laser pulses on hair removal. STUDY DESIGN/MATERIALS AND METHODS: The normal-mode ruby laser (Epilaser; 694 nm, 3 msec) was used to treat a wide range of body sites in 51 volunteers. The mean follow-up after the last treatment was 8.37 months. RESULTS: Sixty-three percent of the patients had sparse regrowth. The mean fluence used was 46.5 J/cm(2) in patients who had sparse hair regrowth and 39.3 J/cm(2) in patients who had moderate hair regrowth (P = 0.0127). Transient pigmentary changes occurred most frequently in patients with skin type 4. CONCLUSION: The normal-mode ruby laser is an efficient and safe method for long-term hair reduction, especially in fair-skinned individuals with dark hair. Higher fluences produce greater long-term efficacy. Adverse effects are minimal and transient.

Permanent hair removal by normal-mode ruby laser.

OBJECTIVE: To assess the permanence of hair removal by normal-mode ruby laser treatment. METHODS: Hair removal was measured for 2 years after a single treatment with normal-mode ruby laser pulses (694 nm, 270 microseconds, 6-mm beam diameter). OBSERVATIONS: Six test areas on the thighs or backs of 13 volunteers were exposed to normal-mode ruby laser pulses at fluences of 30 to 60 J/cm2 delivered to both shaved and wax-epilated skin. In addition, there was a shaved and wax-epilated control site. Terminal hairs were manually counted before and after laser exposure. Transient alopecia occurred in all 13 participants after laser exposure, consistent with induction of telogen. Two years after laser exposure, 4 participants still had obvious, significant hair loss at all laser-treated sites compared with the unexposed shaved and wax-epilated control sites. In all 4 participants, there was no significant change in hair counts 6 months, 1 year, and 2 years after laser exposure. Laser-induced alopecia correlated histologically with miniaturized, velluslike hair follicles. No scarring and no permanent pigmentary changes were observed. CONCLUSIONS: Permanent, nonscarring alopecia can be induced by a single treatment with high-fluence ruby laser pulses. Miniaturization of the terminal hair follicles seems to account for this response.

Hair growth cycle affects hair follicle destruction by ruby laser pulses.

It has been shown that normal mode ruby laser pulses (694 nm) are effective in selectively destroying brown or black pigmented hair follicles in adult Caucasians. This study investigated how the various stages of the hair follicle growth cycle influence follicle destruction by ruby laser treatment, using a model of predictable synchronous hair growth cycles in the infantile and adolescent mice. A range of ruby laser pulse fluences was delivered during different stages of the hair growth cycle, followed by histologic and gross observations of the injury and regrowth of hair. Actively growing and pigmented anagen stage hair follicles were sensitive to hair removal by normal mode ruby laser exposure, whereas catagen and telogen stage hair follicles were resistant to laser irradiation. Selective thermal injury to follicles was observed histologically, and hair regrowth was fluence dependent. In animals exposed during anagen, intermediate fluences induced nonscarring alopecia, whereas high fluences induced scarring alopecia. The findings of this study suggest treatment strategies for optimal laser hair removal.

Damage to hair follicles by normal-mode ruby laser pulses.

BACKGROUND: Although many temporary treatments exist for hirsutism and hypertrichosis, a practical and permanent hair removal treatment is needed. OBJECTIVE: Our purpose was to study the use of normal-mode ruby laser pulses (694 nm, 270 microseconds, 6 mm beam diameter) for hair follicle destruction by selective photothermolysis. METHODS: Histologically assessed damage in ex vivo black-haired dog skin after the use of different laser fluences was used to design a human study; 13 volunteers with brown or black hair were exposed to normal-mode ruby laser pulses at fluences of 30 to 60 J/cm2, delivered to both shaved and wax-epilated skin sites. An optical delivery device designed to maximize light delivery to the reticular dermis was used. Hair regrowth was assessed at 1, 3, and 6 months after exposure by counting terminal hairs. RESULTS: Fluence-dependent selective thermal injury to follicles was observed histologically. There was a significant delay in hair growth in all subjects at all laser-treated sites compared with the unexposed shaven and epilated control sites. At 6 months, there was significant hair loss only in the areas shaved before treatment at the highest fluence. At 6 months, four subjects had less than 50% regrowth, two of whom showed no change between 3 and 6 months. Transient pigmentary changes were observed; there was no scarring. CONCLUSION: Selective photothermolysis of hair follicles with the normal-mode ruby laser produces a growth delay consistent with induction of prolonged telogen with apparently permanent hair removal in some cases.

Treatment of cafe au lait macules with lasers. A clinicopathologic correlation.

BACKGROUND: Cafe au lait macules (CALMs) respond variably to treatment with different lasers. This study was done to determine whether the type of laser and the individual histologic features of the CALMs could predict clinical response to treatment. Nine CALMs were treated with both the frequency-doubled Q-switched neodymium: YAG laser (wavelength, 532 nm; beam diameter, 2.0 mm) and the Q-switched ruby laser (wavelength, 694 nm; beam diameter, 5.0 mm). Both lasers were used at a fluence of 6.0 J/cm2. Biopsy specimens of the CALMs were obtained before and after treatment. Clinical follow-up was done at 1-, 3-, and 6-month intervals. OBSERVATIONS: Both lasers yielded variable responses to treatment. Two histologic subtypes of CALMs were identified, but these different subtypes did not predict clinical outcome after laser treatment. CONCLUSIONS: Cafe au lait macules respond variably to treatment with both the Q-switched ruby laser and the frequency-doubled Q-switched neodymium:YAG laser. Further research might address the effect of using multiple treatments. In view of these results, clinicians using lasers to treat CALMs should inform their patients of the potential for recurrence or darkening of CALMs.

Thermal relaxation of port-wine stain vessels probed in vivo: the need for 1-10-millisecond laser pulse treatment.

Although thermal relaxation times of cutaneous port-wine stain microvessels have been calculated and used to formulate laser selective photothermolysis, they have never been measured. A scheme to do so was devised by measuring the skin response to pairs of 585-nm dye laser pulses (250-360 microseconds each) as a function of the time interval between the two pulses, in five volunteers with port-wine stains. After a pump pulse delivering 80% of the fluence necessary for causing purpura, the fluence of a second probe pulse necessary to cause purpura was determined and was found to increase with the interval between the two pulses, in a manner consistent with thermal diffusion theory. Biopsy specimens were obtained from four of the five subjects to examine the nature and extent of vessel damage and to measure the port-wine stain vessel diameters. Using diffusion theory, the thermal relaxation time was calculated based on the measured vessel diameters. These calculated values are consistent with the increase in radiant exposure (fluence) of the probe pulse necessary to induce purpura for longer time delays. Two simple models for thermal relaxation of port-wine stain vessels are presented and compared with the data. The data and histologic assessment of the vessel injury strongly suggest that pulse durations for ideal laser treatment are in the 1-10-millisecond region and depend on vessel diameter. No dermatologic lasers presently used for port-wine stain treatment operate in this pulse width domain.

On the mechanism of skin wound "contraction": a granulation tissue "knockout" with a normal phenotype.

This report explores the mechanism of spontaneous closure of full-thickness skin wounds. The domestic pig, often used as a human analogue for skin wound repair studies, closes these wounds with kinetics similar to those in the guinea pig (mobile skin), even though the porcine dermis on the back is thick and nearly immobile. In the domestic pig, as in the guinea pig, daily full-thickness excisions of the central granulation tissue up to but not including the wound edges in both back and flank wounds do not alter the rate or completeness of wound closure or the final pattern of the scar. A purse-string mechanism of closure was precluded by showing that surgical interruption of wound edge continuity does not alter closure kinetics or wound shape. We conclude that "tightness" of skin is not a key factor nor is the central granulation tissue required for normal wound closure. These data imply that in vitro models such as contraction of isolated granulation tissue or of the cell-populated collagen lattice may not be relevant for understanding the cell biology of in vivo wound closure. Implications for the mechanism for wound closure are discussed.

Cosmetic tattoo ink darkening. A complication of Q-switched and pulsed-laser treatment.

BACKGROUND: High-energy, short-pulse lasers, eg, Q-switched lasers, emitting visible and near-infrared light have recently been developed for removing tattoos, with little risk of scarring. The mechanisms of action, and possible adverse effects other than scarring and hypopigmentation, are not fully understood. OBSERVATIONS: We describe five cases of pulsed-laser-induced, immediate, irreversible darkening of cosmetic, white, flesh (skin-color), and pink-red colored tattoos. Irreversible ink darkening can be an insidious complication, because immediate whitening of the skin temporarily obscures the subsequently impressive color change. Among these cases, irreversible ink darkening occurred with Q-switched ruby (694 nm), Q-switched neodymium (Nd):YAG (1064 nm/532 nm), and pulsed green dye (510 nm) lasers. Attempts to remove the darkened ink with further laser treatment failed in two cases, and surgical excision was necessary. In the other three cases, subsequent laser treatments successfully removed the darkened ink. The red cosmetic tattoo ink used in one of the cases was placed in agar in vitro and was converted to a black compound immediately on Q-switched ruby laser exposure. Ferric oxide, a brown-red ingredient commonly used in cosmetic tattoos, was similarly tested and blackened in vitro by Q-switched ruby laser exposures. CONCLUSIONS: Although most tattoos are not darkened by laser treatment, short-pulsed lasers over a wide spectrum can cause immediate darkening of some tattoo inks. Patients should be warned of the potential for irreversible cosmetic tattoo darkening, and test-site exposures should be performed prior to treatment. In some cases, subsequent laser treatments may remove the blackened ink. The mechanism probably involves, at least for some tattoos, reduction of ferric oxide (Fe2O3, "rust") to ferrous oxide (FeO, jet black), but the chemical reaction that is involved remains unknown.

Dye-enhanced laser welding for skin closure.

The use of a laser to weld tissue in combination with a topical photosensitizing dye permits selective delivery of energy to the target tissue. A combination of indocyanine green (IG), absorption peak 780 nm, and the near-infrared (IR) alexandrite laser was studied with albino guinea pig skin. IG was shown to bind to the outer 25 microns of guinea pig dermis and appeared to be bound to collagen. The optical transmittance of full-thickness guinea pig skin in the near IR was 40% indicating that the alexandrite laser should provide adequate tissue penetration. Laser "welding" of skin in vivo was achieved at various concentrations of IG from 0.03 to 3 mg/cc using the alexandrite at 780 nm, 250-microseconds pulse duration, 8 Hz, and a 4-mm spot size. A spectrum of welds was obtained from 1- to 20-W/cm2 average irradiance. Weak welds occurred with no thermal damage obtained at lower irradiances: stronger welds with thermal damage confined to the weld site occurred at higher irradiances. At still higher irradiances, local vaporization occurred with failure to "weld." Thus, there was an optimal range of irradiances for "welding," which varied inversely with dye concentration. Histology confirmed the thermal damage results that were evident clinically. IG dye-enhanced laser welding is possible in skin and with further optimization may have practical application.

Pulsed photothermal radiometry in turbid media: internal reflection of backscattered radiation strongly influences optical dosimetry.

The integrated irradiance (energy fluence rate) within tissue can exceed the incident irradiance due to backscattered and multiply reflected light near the sample surface. This was studied quantitatively using pulsed photothermal radiometry, which measures blackbody radiation emitted by a sample during and after absorption of an optical pulse. Aqueous gels containing absorbing dye with or without various scattering materials were studied using a fast sensitive IR detector system and 1-micros tunable pulsed dye laser. For nonscattering samples, the temperature transient (T-jump) due to absorption of a laser pulse was consistent with Beer's law for homogeneous absorbing media. When scattering was present, increases of up to almost an order of magnitude in the T-jump were observed. For a given absorption coefficient, there was a proportional relationship between the increase in the T-jump and the sample's diffuse reflectance. A model describing the reflectance of diffuse radiation at the sample boundary was derived to explain this result. To test the model, the refractive index was varied with air as the external medium and was also matched to that of BaF(2) as the externalmedium. The subsurface fluence is, to areasonable approximation, given by E congruent with E(0)(l + 2bR),where E(0) is the incident fluence of an infinitely wide collimated beam, b is a coefficient strongly dependent on only the refractive index, and R is the measured diffuse reflectance of the sample. This study shows that irradiance within tissues can greatly exceed the irradiance of incident collimated light, an effect that should be accounted for in photomedical dosimetry or research.

Selective vascular coagulation of rabbit colon using a flashlamp-excited dye laser operating at 577 nanometers.

Previous studies have demonstrated that brief pulses of selectively absorbed optical radiation can be used to confine thermal injury to pigmented targets within tissues. We performed studies in rabbits to assess the usefulness of this technique for selectively coagulating the colonic vasculature. By measuring the optical absorbance of rabbit colon with a spectrophotometer, it was determined that hemoglobin exhibits strong absorption relative to the rabbit colon at a wavelength of 577 nm. Because light must be absorbed to affect tissue, it was hypothesized that laser pulses of this wavelength would selectively damage blood vessels. This hypothesis was tested by examining the effect of 300-microseconds-long 577-nm laser pulses on rabbit colon in vivo. For delivered radiant exposures between 4 and 8 J/cm2, selective coagulation of the colonic vasculature could be produced without damage to the surrounding colon. At greater radiant exposures, vessel hemorrhage was occasionally noted but no transmural thermal injury was produced with delivered radiant exposures as high as 22 J/cm2. This technique may form the basis of a safe and simple treatment of vascular lesions of the colon such as angiodysplasia.

Ultraviolet excimer laser ablation: the effect of wavelength and repetition rate on in vivo guinea pig skin.

Multiple dermatologic conditions that are currently treated with traditional cold-knife surgery are amenable to laser therapy. The ideal surgical treatment would be precise and total removal of abnormal tissue with maximal sparing of remaining structures. The ultraviolet (UV) excimer laser is capable of such precise tissue removal due to the penetration depth of 193 nm and 248 nm irradiation of 1 micron per pulse. This type of ablative tissue removal requires a high repetition rate for efficient lesional destruction. Excimer laser radiation at 193 nm is capable of high repetition rates, which are necessary while 248 nm radiation causes increasing nonspecific thermal injury as the laser repetition rate is increased.

A comparison of the melanocyte response to narrow band UVA and UVB exposure in vivo.

The visible cutaneous pigmentary response to ultraviolet-A (UVA) is immediate and, following sufficient exposure, may persist, whereas ultraviolet-B (UVB)-induced pigmentation appears after a delay of several days. We compared the in vivo response of melanocytes to single and multiple exposures of narrow band UVA and UVB irradiation which produced visibly equal increases in pigmentation. Using a xenon-mercury source matched to a monochromator, human volunteers were exposed to 304 (+/- 5) and 365 (+/- 10) nm radiation. Biopsies were performed 1, 7, and 14 days after irradiation. For each biopsy, the number of melanocytes per square millimeter of epidermis was determined using L-3,4-dihydroxyphenylalanine (dopa)- and tyrosine-incubated split epidermal preparations. Vertical sections were also examined. At days 7 and 14, after both 304 and 365 nm radiation, melanocytes were more intensely dopa-positive than in unirradiated controls, and demonstrated enlarged perikarya and a greater number of enlarged dendrites. Following both 304 and 365 nm radiation the number of dopa-positive melanocytes was increased at days 7 and 14 by 44% and 58%, respectively. Tyrosine positivity, an indicator of enhanced tyrosinase activity and increased melanin formation, was absent in controls and at day 1, and became positive in all but one sample at day 7 and day 14. Therefore, one day after UVA exposure, visible pigmentation but not tyrosinase activity was increased. At day 7, the number of tyrosine-positive melanocytes approximately equaled the number of dopa-positive melanocytes. Although UVA and UVB induce different pigmentary responses, their effects on melanocyte number and function were indistinguishable.

Microsecond-long flash photography of laser-induced ablation of biliary and urinary calculi.

High-speed flash photographs of laser-induced fragmentation of biliary and renal calculi under water were obtained using one-microsecond-long dye-laser pulses for both illumination and ablation. The photographs show the presence of a bubble with irregularities on the surface that suggest the early presence of debris or microbubbles. Fragmentation occurs before the bubble collapses, suggesting that fragmentation is due to laser-induced acoustic transients rather than to collapse of a laser-induced cavitation bubble.

An evaluation of the effectiveness of azelaic acid as a depigmenting and chemotherapeutic agent.

In the past five years, it has been reported that certain dicarboxylic acids (C8-C13) and azelaic acid (C9) (AZA), in particular, have a remarkable effect in the management of lentigo maligna, human malignant melanoma, and certain disorders of hyperpigmentation. Preclinical trials, therefore, were undertaken in order to evaluate the effectiveness of AZA as a depigmenting agent and as a chemotherapeutic agent. Twenty-seven uniformly black pigmented guinea pigs were given topical applications of various concentrations (3, 5, 10, 15, and 20%) of AZA preparations for 8 weeks, and their effects on the melanocytes of epilated skin of the backs and the nonepilated ears of guinea pigs were compared to the effects of well-known depigmenting agents. Whereas 4-isopropylcatechol, monobenzylether of hydroquinone, monoethylether of hydroquinone, hydroquinone, and 4-hydroxyanisole were found to be selectively cytotoxic to melanocytes in black-skinned guinea pigs, AZA has little or no visually recognizable effect on melanocytes in these animals. The therapeutic effect of local s.c. injections of various concentrations of AZA preparations on the development of s.c. implanted B-16 melanoma tumor was evaluated in 96 C57BL/6J mice. In addition, 31 BDF1 mice, implanted i.p. with B-16 melanoma tumor, were used to assess the effect of 100-500 mg/kg concentrations of AZA administered i.p. In both studies, AZA revealed no significant tumoristatic or tumoricidal effect on the size, color, and growth of melanoma. The effect of AZA was also evaluated on S-91A (melanotic or pigmented) and S-91B (amelanotic) human melanoma cells in culture. Low concentrations (10(-5) and 10(-3) M) of AZA had no inhibitory effect on the growth of these cells. Only at higher concentrations (greater than 10(-3) M) was a cytotoxic effect on cell viability observed. These observations indicate AZA is not selectively cytotoxic to normal and proliferative melanocytes and has no apparent inhibitory effect on the formative process of melanin pigmentation.