A Novel Non-Thermal Non-Ablative LED Photomodulation® Device for Reversal of Photoaging: Digital Microscopic & Clinical Results in Various Skin Types

Abstract.

Photomodulation is a process which manipulates

or regulates cell activity using light sources without ther-

mal effect. Previous studies of LED Photomodulation®

have shown skin textural improvement accompanied by

increased collagen deposition with reduced MMP-1

(collagenase) activity in the papillary dermis. The pur-

pose of this study was to investigate a cohort of patients

(N=93) with a wide range of Fitzpatrick skin types

treated by LED Photomodulation® using the Gentle-

waves® full panel 590nm high energy LED array with a

specific sequence of pulsing. Results showed improve-

ment of signs of photoaging in 90%. No side effects were

noted. LED Photomodulation® is a safe and effective

non-painful non-ablative modality for improvement of

photoaging.

Clinical Use of GentlewavesClinical Use of GentlewavesClinical Use of Gentlewaves®®® LED Photomodulation LED Photomodulation LED Photomodulation® ® ®

Robert A. Weiss, M.D. Director, Maryland Laser Skin & Vein Institute, Hunt Valley, Maryland Assistant Professor of Derma-tology, Johns Hopkins U School of Medicine, Baltimore, MD Margaret A. Weiss, M.D. Co-Director, Maryland Laser Skin & Vein Institute, Hunt Val-ley, Maryland Assistant Professor of Derma-tology, Johns Hopkins U School of Medicine, Baltimore, MD Roy G. Geronemus, M.D. Director, Laser & Skin Surgery Center of New York Clinical Professor of Dermatol-ogy, New York University Medi-cal Center, New York, New York David H. McDaniel, M.D. Director, Laser Skin & Vein Center of Virginia Assistant Professor of Clinical Dermatology and Plastic Sur-gery, Eastern Virginia Medical School, Virginia Beach, Vir-ginia

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IntroductionIntroductionIntroduction

Photorejuvenation is the process whereby light energy sources are utilized to reverse the process

of sun induced aging or environmental damage to the skin. Non-ablative photorejuvenation accom-

plishes this without disturbance of the overlying epidermis. Categories of non-ablative devices include

those that affect the wound healing cascade by a thermal or photothermolysis type of injury, but we de-

scribe here a new category of non-wounding or non-thermal light based treatments which has been

termed photomodulation (1).

The primary goal of non-ablative rejuvenation is induction of new collagen and dermal extra-

cellular matrix substance which visibly improves the appearance of rhytids without disturbance or

damage to the overlying epidermis. An additional goal includes the reversal of pigmented and vascular

signs of photoaging which include reduction of superficial dyspigmentation (both dermal and epider-

mal), reduction of dermal telangiectasias and the appearance of an overall smoother texture.

Until now our thinking about how to accomplish this has involved primarily thermal mecha-

nisms, whether it is heating of the dermis to stimulate fibroblast proliferation or heating blood vessels

for photocoagulation. (2;3) Although some interest in non-thermal low-intensity laser therapy (LILT)

or cold laser (very low doses of laser) has occurred, key elements required for understanding mecha-

nisms and successful application for cell growth stimulation have been lacking. In fact red laser wave-

lengths have been reported as failures or with mixed results for stimulation of wound healing. (4;5)

The first experiments performed with low laser doses were revealing. McDaniel et.al. demon-

strated while using various lasers at lower fluence that fibroblast activity could be regulated. (1) Using a

variety of LED light sources or monochromatic laser sources, his group demonstrated that by varying

fluence, procollagen synthesis could be upregulated in fibroblast culture. A clinical correlation was also

shown. (6) Procollagen was measured by microarray assays. Collagen synthesis was accompanied by

an equal and opposite reaction involving reduction or down regulation of matrix metalloprotease-1

(MMP-1 or collagenase). Figure 1 demonstrates results of several experiments in which procollagen is

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measured by stimulation of fibroblasts in culture with a wide range of operating fluences of the pulsed

dye laser. However, there is also a broad peak of collagen stimulation at fluences previously thought to

be too low to produce an effect. This concept of using low energy, narrow band or coherent light with

specific pulse se-

quences and durations

has been termed photo-

modulation. (6) The

concept that cell activ-

ity can be up or down

regulated by light has

been confirmed by

other groups perform-

ing NASA funded re-

search. (7;8)

Cultured fibro-

blasts can be stimulated by using a narrowband light emtting diode (LED) light source of various wave-

lengths. Based on extensive experiments by McDaniel et al beginning several years ago , LEDs emitting

in the 590nm range were discovered to have the greatest effects. (1) Even more critical were results

demonstrating that unless a specific sequence of pulsation was utilized, there was minimal effect on fi-

broblasts in culture (9) The curve in Figure 1 could be reproduced for LED by using a specific “code”

of on-time and off-time (dark time). Application of continuous LED light had no effect. This mecha-

nism of non-thermal stimulation of fibroblast growth with the specific “codes” of pulsing is now termed

and patented as LED Photomodulation®.

Using many of the parameters developed in the laboratory, a multi-center clinical trial was per-

formed on 90 patients with a series of 8 treatments over 4 weeks.(9;10) Patients were followed using

stereotactic digital images, high resolution ultrasound and biopsy for Masson-trichrome, anti-collagen

Figure 1 Figure 1 Figure 1

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and anti-MMP antibodies as well as digital profilometry. Follow-up of clinical results continued for 1

year. (11,12) This study showed very favorable results with over 90% of patients improving by at least

one Fitzpatrick photoaging category and 65% of the patients demonstrating global improvement in fa-

cial texture, fine lines, background erythema and pigmentation. Results peaked at 4 – 6 months follow-

ing a series of 8 treatments.

The underlying mechanism for LED Photomodulation® was determined to be stimulation of mi-

tochrondrial cell organelles with the proper “packets” of photons. This is similar to photosynthetic elec-

tron transport with a proton gradient in chloroplasts of plant cells. (13) Chloroplasts and mitochrondia

share very similar membrane archi-

tectures. The principle photoreceptor

is chlorophyll which is cyclic tetrapyr-

role, like the heme group of cyto-

chromes, while the molecules responsi-

ble for the absorption of light in mito-

chrondria are the cytochrome species

within the mitochondrial membrane

(13)(shown in Figure 2). Both chloro-

phyll and cytochromes are synthesized

from protoporphyrin IX. The cytochrome molecules best absorb light from 562nm to 600nm.

It is believed that light absorption causes conformation changes in antenna molecules within the

mitochrondrial membrane. Translocation of protons begins a pump which ultimately leads to energy

for conversion of ADP to ATP, essentially recharging the cell battery and providing more energy for

growth. Elegant experiments by McDaniel et al. have demonstrated the rapid production of ATP

within mitochrondia within cultured fibroblasts exposed to LED light only with the proper pulsing

Figure 2 Figure 2 Figure 2

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“code”. (1;10) New ATP production occurs within seconds of LED Photomodulation® and this triggers

subsequent metabolic activity of fibroblasts. (10)

This paper reports the results of an additional clinical study involving another cohort of 93 pa-

tients. They were treated by Gentlewaves® LED Photomodulation® and followed for 6 months by

stereotactic digital imaging and digital microscopy. This is an additional cohort not previously reported

in the 90 patient multi-center clinical trial.

Material and MethodsMaterial and MethodsMaterial and Methods Random patients (N=93) with mild to moderate photoaging were offered

the opportunity to receive 8 LED Photomodulation® treatments over a four week period. Treatments

were given with a minimum of 48 hours separation. Prior to the first treatment, the skin was cleansed

with a topical masque (Gentlewaves® masque, LightBioScience, Virginia Beach, VA) Skin types

ranged from Fitzpatrick type I to V. Use of OTC topicals and topical retinoids was permitted as long as

no changes were made to the topical regimen within 3 months prior to the study and for the duration of

the six month follow-up. All patients were coached on the daily use of sunscreen with SPF of 30 or

higher. Stereotactic images were taken using the Canfield system and a Fuji S1 digital camera at day 0,

30 days, 1, 2, 3 and 6 months. The Fuji S1 CCD has 3.5 million actual photosensitive pixels on its sur-

face, producing an effective 6 megapixel file size. Subtle changes in skin texture and other signs of

photoaging are easily analyzed using high resolution baseline images for comparison.

A region just lateral to the left lateral canthus was imaged using digital microscopy using 30X

and polariscopic light magnification ( DG-2 Digital Microscope, Scalar America, Video Microscopes &

Imaging Systems, Sacramento, CA) An acetate tracing was used with the outline of the lateral eye to

accurate position the digital microscope in the follow-up visits. The position of the examined region was

2.5 cm lateral to the left lateral canthus. The resolution of the digital microscope is 330 lines at 2.3

Megapixels which allowed a detailed analysis of skin texture.

Patients performed a self-assessment of their improvement on a quartile scale and independent

investigators graded images using a LCD monitor at 1280x 1064 resolution in 32 bit color mode.

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Changes were graded on a

quartile scale for peri-ocular

wrinkles, reduction in Fitz-

patrick photoaging classifi-

cation, skin texture, back-

ground erythema and pig-

mentation.

Treatments were

given with the Gentlewaves®

LED Photomodulation®

unit (LightBioScience, Vir-

ginia Beach, VA) with a full face panel device as shown in Figure 3. Patients were positioned 2 centime-

ters away from the light source. Pulsed 590nm diode light was delivered in the standard treatment regi-

men as previous reported. (9) Fluence range is typically from 0.1 to 0.8 Joules/cm2. The pulse se-

quence or “code” is pre-set to the sequence yielding maximal effect in previous clinical trials and use on

fibroblasts in cell culture. No user changes from pre-sets were required or permitted. All adverse

events following treatment were recorded.

ResultsResultsResults

The Fitzpatrick scale evaluations showed a reduction of one

class in over 90 percent of subjects. These results are

shown in Table I.

Figure 3Figure 3Figure 3

Photoaging Class

% of Subjects Reduced One Photoaging Scale Category

Non-responders 9

2 to 1 26

3 to 2 31

4 to 3 17

5 to 4 11

6 to 5 8

Total 100 %

Table ITable ITable I

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The best results clinically were seen in the

category of skin texture with improvement

noted by 87% of patients. (Figure 4).

Notably African-American and Asian skin types responded as well as Caucasian skin types in the tex-

tural improvement category

(Figure 5).

At 6 months, independent

review of images also showed re-

duction of peri-ocular rhytids was

in 56%, reduction of background

erythema in 65% and reduction of

background pigmentation in 62%.

A self-assessment by patients for

global improvement of skin appearance at 3 months revealed that they judged the improvement as none,

mild, moderate and excellent in 8, 14, 41, 38% respectively. At 6 months the percentages fell slightly

(Figure 6) .

Results at 6 months

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Peri-ocularwrinkles

Smoother texture Pigmentation Redness

% P

atie

nts

Show

ing

Impr

ovem

ent

Figure 4Figure 4Figure 4

Figure 5Figure 5Figure 5

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Digital microscopy in 10 patients

revealed a 50 to 90% (mean tex-

tural improvement) as judged by

the investigators evaluation depth

of fine lines. An example of an

excellent response as seen by digi-

tal microscopy is shown in Figure

7.

There were no adverse

events. One patient believed

that she had a flare of acne

while 9 others reported an im-

provement in acne. Two pa-

tients noticed improvement of small patches of atopic eczema. One patient believed her eyebrows grew

more thickly but this was not substantiated by the digital images. No pain or heat sensation was re-

ported during treatment. No adverse events such as skin burning or pigmentation changes were noted

in any of the subjects. Clinical results typical at 3 – 6 months following a series of 8 treatments is shown

in Figure 8.

0

5

10

15

20

25

30

35

40

45

50

None Mild Moderate Excellent

% o

f pat

ient

s (N

=93)

3 Months6 Months

Figure 7Figure 7Figure 7

Figure 6Figure 6Figure 6

Figure 8Figure 8Figure 8

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ConclusionsConclusionsConclusions

LED Photomodulation® performed with this specific device including this array of high energy

LEDs and a very specific sequencing time code is highly effective. The initial starting regimen of 8

treatments over 4 weeks is effective for the treatment of typical signs of photoaging. The need for main-

tenance treatments may begin at 4 -6 months. It is unclear how often maintenance treatments will be

required but our clinical experience to date suggests once a month.

It is interesting that a light source can induce similar findings for collagen synthesis and reduced

MMP activity as reported with retinoic acid. (15-17) The mechanisms of a topical drug versus photo-

modulation are very different yet yield similar

effects. It has been proposed that these effects

may be synergistic, specifically with topical

retinol(14). Further investigation is ongoing

about these additive effects.

An anti-inflammatory effect of LED

Photomodulation® was reported by one patient

in the present investigation. Some preliminary

data now indicates an anti-inflammatory effect

for LED Photomodulation® as well. Using a

solar simulator to create UV erythema, pre-

liminary findings indicate a very noticeable re-

duction in UV erythema when LED Photomodulation® is supplied within hours after UV exposure. In

addition low intensity light therapy with LED Photomodulation® after acute UV exposure produced sig-

nificant downregulation of dermal matrix degrading enzymes which were stimulated by the UV expo-

sure. (11)

Figure 9Figure 9Figure 9

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LED Photomodulation® is painless, safe and easy to administer. It appears that this device suc-

cessfully modulates the activity of fibroblasts resulting in smoother skin without the inherent risks of

other thermal devices which may burn the skin or require complicated application techniques. Our re-

sults with parameters developed by fibroblast stimulation in culture appear to translate into a positive

and noticeable clinical response in all skin types. Multiple trials at multiple centers are now ongoing to

confirm these results. Combination treatments with standard thermal non-ablative techniques such as

intense pulsed light or pulsed dye are being performed to determine whether this device may enhance

results after photothermal treatments by stimulating more collagen synthesis and producing less colla-

gen degradation.

References

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(2) Weiss RA, Goldman MP, Weiss MA. Treatment of poikiloderma of Civatte with an intense pulsed light source. Dermatol Surg 2000 Sep;26(9):823-7.

(3) Fatemi A, Weiss MA, Weiss RA. Short-Term Histologic Effects of Nonablative Resurfacing: Results with a Dynamically Cooled Millisecond-Domain 1320 nm Nd:YAG Laser. Dermatol Surg 2002 Feb;28(2):172-6.

(4) Walker MD, Rumpf S, Baxter GD, Hirst DG, Lowe AS. Effect of low-intensity laser irradiation (660 nm) on a radiation-impaired wound-healing model in murine skin. Lasers Surg Med 2000;26(1):41-7.

(5) Sommer AP, Pinheiro AL, Mester AR, Franke RP, Whelan HT. Biostimulatory windows in low-intensity laser activation: lasers, scanners, and NASA's light-emitting diode array system. J Clin Laser Med Surg 2001 Feb;19(1):29-33.

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(7) Whelan HT, Buchmann EV, Dhokalia A, Kane MP, Whelan NT, Wong-Riley MT, et al. Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice. J Clin Laser Med Surg 2003 Apr;21(2):67-74.

(8) Whelan HT, Connelly JF, Hodgson BD, Barbeau L, Post AC, Bullard G, et al. NASA light-emitting diodes for the prevention of oral mucositis in pediatric bone marrow transplant patients. J Clin Laser Med Surg 2002 Dec;20(6):319-24.

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(9) McDaniel DH, Newman J, Geronemus R, Weiss RA, Weiss MA. Non-ablative non-thermal LED photo-modulation - A multicenter clinical photoaging trial. Lasers Surg.Med. 15, 22. 2003.

(10) Geronemus R, Weiss RA, Weiss MA, McDaniel DH, Newman J. Non-ablative LED photomodulation- Light activated fibroblast stimulation clinical trial. Lasers Surg.Med. 25, 22. 2003.

(11) McDaniel DH, Geronemus R, Weiss RA, Weiss MA, Newman J. LED photomodulation reverses acute UV induced skin damage. Lasers Surg.Med. 16, 30. 2004.

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(13) Voet D, Voet JG. Biochemistry. 2nd ed. New York: John Wiley & Sons, Inc.; 1995.

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(15) Fisher GJ, Voorhees JJ. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultra-violet irradiation induces MAP kinase signal transduction cascades that induce Ap-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Investig Dermatol Symp Proc 1998 Aug;3(1):61-8.

(16) Fisher GJ, Talwar HS, Lin J, Lin P, McPhillips F, Wang Z, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo. J Clin Invest 1998 Mar 15;101(6):1432-40.

(17) Varani J, Perone P, Griffiths CE, Inman DR, Fligiel SE, Voorhees JJ. All-trans retinoic acid (RA) stimu-lates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently. J Clin Invest 1994 Nov;94(5):1747-56.