HYPOTHETICAL ETIOLOGY AND COMPETITIVE ASSESSMENT OF TERAHERTZ LIGHT INDUCED RHYTIDE IMPROVEMENT

BY: JOSEPH TSUN DAW TAN

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

Thesis Advisor: Dr. Christopher Cullis

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

May 2012

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______JOSEPH TSUN DAW TAN______candidate for the Entrepreneurial Biology Masters degree*.

(signed) ______DR. ROBIN SNYDER______(chair of the committee)

______DR. CHRISTOPHER CULLIS______

______DR. JEAN WELTER______

______DR. GERALD MEARINI______

______

(date) ______1/20/2012______

* We also certify that written approval has been obtained for any proprietary material contained therein. 2

Table of Contents

List of Tables 5

Acknowledgements 6

Abstract 7

Introduction 8

1 Characterizing the need 9

1.1 Market overview and geographical perspectives 9

1.2 Market segmentation : clarifying the rhytide improvement market 10

1.3 Identifying the central job to be done : bolstering self-concept 10

2 Current approaches and their limitations 11

2.1 Relevant anatomy and composition of skin 12

2.2 Rhytide formation : aging and photoaging 13

2.3 Primer on infrared interaction with skin 15

2.4 EMR-based rhytide improvement techniques and etiology 18

2.4.1 Ablative skin resurfacing 19

2.4.2 Non-ablative approaches 20

2.4.3 Fractional photothermolysis 23

2.4.4 Radiofrequency (RF) stimulation 23

2.5 Other interventional techniques and their mechanisms 24

2.5.1 Rhytidectomy 25

2.5.2 Topicals 25

2.5.3 Botulinum toxin 25

2.5.4 Soft tissue fillers 26

3

2.6 Market and technical analysis of leaders in aesthetic lasers 26

3 Hypothetical mechanisms of terahertz rhytide improvement 28

3.1 Existing evidence of THz exposure on biological systems 28

3.1.1 Penetration depth on skin and water 29

3.1.2 Primary chromophore – hydrogen bond resonance 30

3.1.3 Membrane effects 31

3.1.4 Biophotomodulatory evidence 31

3.2 Finding THz’s unique value proposition in rhytide improvement 34

4 Testable hypothesis to generate proof of concept and safety data 35

4.1 Available equipment for THz delivery 36

4.2 Discussion of experimental models 37

4.3 Testable hypothesis and planned experimentation 39

4.3.1 Hypothesis 1: Genotoxicity 39

4.3.2 Hypothesis 2: Thermal stress and biophotomodulation 40

4.3.3 Hypothesis 3: Membrane effects/potentiation of topicals 41

5 Conclusion 43

Bibliography 44

4

List of Tables

Table 1. Summary and comparison of various categories of EMR-based skin 19 resurfacing approaches ranked in order of clinical invasiveness.

Table 2. Revenue and product comparison of top six aesthetic laser companies 26

5

Acknowledgements

First and foremost, I would like to thank the members of my thesis committee, Drs. Christopher Cullis, Jean Welter, and Gerald Mearini for taking the time to contribute their respective expertise towards this multidisciplinary project. Without Dr. Cullis’s vision for the department that enabled me to transition into this new career path, Dr. Mearini’s providing me the forum, opportunity and mentorship to pursue this line of research, and Dr. Welter’s patient support in helping me gain confidence in my argument and clarify my scope, I would not be here today. I would like to thank my other mentors who have made an impact on me during my time at Case Western Reserve University: Dr. Hillel Chiel, Dr. Mary Davis, Ed Caner, Bruce Terry and my CGA representative, Dr. Robin Snyder. At every critical juncture of a tribulation that I thought I could not overcome, they provided me with support and perspective outside of the expectations of their position. I hope to be able to do for others as they have done for me, and will always reflect fondly on this institution that I proudly call my alma mater. I would like to thank my family in both generational directions, and without genetic boundary. They are my reason for living, and I hope to be there for them in their time of need as they have been there for me. Finally, to my darling wife, thank you for loving me when I felt like nothing. If that is not true love, I do not know what is. May all who read this stay young at heart.

6

Hypothetical Etiology and Competitive Assessment of Terahertz Light Induced Rhytide Improvement

Abstract

by

JOSEPH TSUN DAW TAN

Aesthetic lasers are a billion dollar industry that continues to grow at a CAGR of

9.0%. The age-resisting rhytide improvement market is driven by consumer consumption of lower-risk, minimal-downtime, and highly effective approaches. The clinical and market success of novel combination techniques has already prompted consolidation in a highly fragmented market, suggesting that continued innovation in this space could yield great dividends. The advent of new technologies that unlock the last unexploited band of electromagnetic radiation offers the prospect of new combinatorial value in a space still ripe for consolidation. Existing evidence suggests that THz exposure on skin can achieve dermal impact, achieve non-genotoxic membrane permeability increases and preferentially initiate production of regeneration recruiting molecules via non-thermal biophotomodulation.

Further work is proposed and must be performed to clarify the best mode to deliver the maximum beneficial bioeffects from THz exposure.

7

Introduction

Our faces are our social passports to the world. Every day, our face is a part of our personal brand, an evolutionarily enforced schema that helps others remember who we are and the physical construct that connects us to the rest of our personhood. But as we age, this external construct so intrinsically tied to self-concept inevitably changes, so it is no surprise that the cosmetic anti-aging and vanity industry is at least a $17 billion industry1, spanning multiple industry segments and occupying a significant portion of discretionary income.

Thus, it is not difficult to understand the driver of these markets, but actually delivering this value to the consumer, reversing the effects of time and biology, is not as simple, and is the holy grail of the vanity industry.

For this study, we will focus on one subset of this market: rhytide improvement, colloquially referred to as wrinkle smoothing or skin rejuvenation, and more specifically, solutions via electromagnetic radiation (EMR). We will characterize the need, review the underlying etiology behind rhytides and current approaches, and introduce the scientific underpinnings of a novel EMR-based approach that utilizes the last unexploited bandwidth of

EMR, terahertz light (THz). After reviewing the current state of understanding of THz phenomenology, we will suggest several product strategies after defining areas of unique value proposition, and provide a technical roadmap of the next steps to reaching market.

8

1 Characterizing the need

1.1 Market overview and geographical perspectives

According to market research firm Frost & Sullivan, in 2005, the aesthetic lasers market had revenues of $589.2 million with a projected compound annual growth rate

(CAGR) of 9.0% based on market data from 2002-2005. Today, this represents an approximately $1 billion market that is highly diversified in approach and market participation.2 Frost & Sullivan correctly predicted the high potential for market consolidation when in 2009, Syneron, making increasing gains on the market with the newest combination technologies, merged with Candela, the incumbent market leader, making

Syneron the largest aesthetic laser company in the world.3

Differing regulatory hurdles necessary to market in North America, Europe, and Asia have made geographic analyses of cosmetic medical products an important consideration.

Past history has shown that North American and Asia/Pacific (APAC) regions delay regulatory approval until European markets generate sufficient safety evidence, demonstrating European markets as a testing ground for worldwide aesthetic laser markets.

However, while Europe is a great indicator of which technologies perform best, the APAC region is the one with the largest growth potential with a project CAGR of 17.6%.4 Much of this growth is attributed to the rise in discretionary income in China, which only contributed

10% of the APAC market. While in 2005, North America held 47% of the world market share, Asia is reportedly the market leader in spite of tightening regulatory control in the region.5

9

1.2 Market segmention : clarifying the rhytide improvement market

While laser hair removal was the largest single application of aesthetic lasers, skin rejuvenation, resurfacing and tightening as a sector comprised 56.5% of the aesthetic laser applications market.6 Because of the ubiquity of aging wrinkles, rhytide improvement is the predominant application within this segment, which also includes treatment of more narrow afflictions of acne scars, vascular and pigmented lesions. Where there were five aesthetic laser companies in 2005 that claimed 60.9% of market share, in the skin rejuvenation market, the top five participants in the skin rejuvenation space claimed only 39.9% of the market share, demonstrating the multitude of participants and techniques in that space. We will explore these techniques and their etiology in section 2.

1.3 Identifying the central job to be done : bolstering self-concept

As with any vanity product, the central job to be done is to bolster one’s self-concept through improvement of one’s appearance. In the case of rhytide improvement, the goal is to improve appearance via smoothing of sun damaged or age-related wrinkles. Whether the ultimate satisfaction comes from merely taking steps to looking younger, or the actual delivery of wrinkle smoothing, both factors are important to take into account as market drivers. Therefore, when accounting for cost, non-monetary costs such as health risks, downtime, and pain are important yet difficult to quantify with monetary costs.

10

The ideal therapy would minimize physical and social pain while still delivering the satisfaction of improved self-concept. This implies that clinical effectiveness is not the end- all be-all, as one might expect. That the total cosmeceuticals market size was projected to be

$16.5 billion in 2010 demonstrates how products that yield minute clinical changes with low intangible costs are what the market seeks. 7 Understanding these outcome expectations of the consumer puts the drivers of market acceptance in perspective for aesthetic EMR-based treatments.

2 Current approaches and their limitations

Over the last two decades, options for wrinkle improvement have greatly expanded beyond surgical and topical approaches. While surgery remains the gold standard with respect to clinical effectiveness, surgery is costly with long, non-cosmetically appealing recovery periods, not to mention the health risks inherent to surgery.8 Conversely, topical treatments often do not yield the effectiveness which many consumers desire. This need to reduce cost, recovery time, and health risk, coupled with a friendly regulatory environment for medical devices, has been a market driver for the development of new non-surgical techniques, as evidenced by the field’s burgeoning growth over the last decade.9 While non- surgical approaches also include dermabrasion, chemical peels/cosmeceuticals, and botulism toxin with fillers, the scope of this review will focus on EMR-centered interventions.

11

2.1 Relevant anatomy and composition of skin

On the macro scale, the skin is comprised of two main layers, the epidermis and the dermis. The epidermis, the outer layer, is made up of several sub-layers and cell types, but predominantly (95%) keratinocytes that arise from the basement membrane and stratify to the stratum corneum, by which point they have lost much of their water content and slough off continuously. As they stratify, they also collect a high concentration of melanin from melanocytes, which darken skin tone. The thickness of the epidermis is between 100-200

µm, with the variance due to columnar fingers called rete pegs that extend into the healthy dermis below.

The dermis, which provides the structural support upon which the epidermis resides, consists mostly of fibroblasts bound to extracellular fibrillar collagen matrix (ECM) anchored to a ground layer underneath the dermis. The fibroblasts are continually producing and regulating pro-collagen, a collagen precursor. To give a sense of the scale of this process, collagen comprises 90% of the protein content in skin as it incorporates into the dermis as the predominant structural protein, thus its importance in determining skin laxity.10

Because the ground layer contains glycosaminoglycans, these polysaccharides common to connective tissue exert strong osmotic pressure that retains water to the bottom of the dermis.

12

Delving deeper, the remaining layers of interest are the subcutaneous hypodermis, comprised largely of fatty tissue, and, since the face is the most common area of cosmetic rhytide concern, the superficial musculoaponeurotic system (SMAS). The SMAS is the outer fascia of the facial muscle whose primary function is to transfer mechanical action of facial musculature to the overlying skin.11 While the SMAS is similar to the dermis in composition and mechanical properties, surgically stretching the SMAS and overlying layers retains the desired facial tension much longer than only stretching the overlying layers.12,13 Thus, the

SMAS is the target of a surgical rhytidectomy, colloquially known as a facelift.

2.2 Rhytide formation: aging and photoaging

Pigmentation changes, deep wrinkles, sallowness, atrophy, leathery appearances are commonly regarded as “age-related” skin changes, but in reality, these symptoms are due to chronic ultraviolet (UV) damage accumulated over time.14 Chronological aging, otherwise known as normal aging, exhibits fine wrinkling and skin laxity that result from atrophy of both layers of skin. Not only does the epidermis atrophy, but the dermal-epidermal junction also flattens, as evidenced by the disappearance of rete pegs.15 Fewer fibroblasts, lower collagen levels, and a thinner dermis are also characteristic of chronologic aging as well.16

13

Chronologic aging is thought to be a result of aggregate action of reactive oxygen species (ROS) on the normal regenerative processes of skin. In keeping the skin fresh and supple, the dermal collagen is broken down and rebuilt constantly. Collagen production is stimulated by the TGF-β class of cytokines, while the transcription factor AP-1 inhibits production and up-regulates matrix metalloproteinase (MMP), a class of enzymes that specialize in breaking down extracellular matrix proteins.17,18 ROS act to subtly inhibit TGF-

β and up-regulate AP-1 over time, resulting in gradual dermal atrophy.19

The prevailing theory on photoaging is that UV insults create acute imbalances in normal collagenic regeneration processes that result in accumulated imperfect reparations of dermal collagen. UV radiation is known to be ionizing, and causes increases of ROS in the skin. In addition, UV also acts independently to up-regulate AP-1 (inhibition and breakdown) and down-regulate TGF-β (regeneration). Within 24 hours of the UV insult, collagen breakdown is observed.20,21 As a single event, these changes in the skin are minute, but accumulated over years, and compounded with the natural course of aging, they result in disorganized and loose collagen structures that manifest the visible and physical imperfections in the skin.

Histologically, the two most prominent characteristics of photoaged skin are solar elastosis, which is the accumulation of an elastin layer at the dermal/epidermal junction, called the Grenz zone, and disorganized collagen in a thinning dermis.22,23 In photo-aged skin, collagen fibers in this region, typically oriented orthogonal to the epidermal plane in normal skin, are instead oriented in parallel.24 The epidermis may also atrophy, but it may also thicken, which is not seen in pure aging processes.25

14

2.1.3 Primer on infrared interaction with skin

From the simplest perspective, EMR is either absorbed or scattered by matter, or else it passes right through it. Absorptive processes are dictated by equivalences in photon frequency and bond frequencies, also referred to as resonant frequencies. Roughly, the higher the bond energy, the higher the resonant frequency, so understanding which bond energies correspond with which frequencies of light is important in translating the ultimate physical effect of a particular frequency of light. The IR-A (near-infrared) and IR-B (mid- infrared) bands correspond to highly energetic intra-molecular bond vibrations. The IR-C band, of which THz also called roughly equivalent to the THz band, interacts primarily with hydrogen-bond vibrations and other high-energy inter-molecular bond vibrations. Longer wavelength/lower energy microwave radiation is absorbed by even lower energy inter- molecular bonds, such as those held together by van der Waal’s forces.26

Any IR light not absorbed is deflected and scattered. It is important to note some analytical techniques rely upon unique scattering processes, such as Raman spectroscopy, to yield relevant information. However, for our purposes, since we are interested in physical impact on skin as opposed to its interrogation, scattering spatially broadens the area and volume of tissue exposed to radiation given an initial spot size. The broadened radiation can be absorbed, or redirected and then absorbed, or simply reflected away from the body.

Due to variances in both absorptive processes and scattering at each wavelength, infrared penetration depth in skin varies broadly. So, understanding the penetration depth at a particular wavelength is vital to inform the nature of biophysical impact. Near-infrared/IR-

A (780 – 1,400 nm) penetrates deeply up to several millimeters, but the deepest mid-

15 infrared/IR-B (1,400 - 3000 nm) can penetrate is under 1 mm. At the long wavelength edge of IR-B (3000 nm), the water absorption peak is the greatest, resulting in a penetration depth of only 1 µm.27 As such, at the short-wavelength end of IR-C (3000 nm - 1 mm), radiation is absorbed completely by the strateum corneum, but approaching the long-wavelength end of

IR-C (~ 1 mm), penetration depth approaches 100-150 µm, reaching the uppermost layers of the dermis.28,29

Absorptive processes are the reason IR-B and IR-C has shallower penetration depths than IR-A. The primary absorptive processes throughout IR-C, which peaks out around a wavelength of 3 µm, are the continuously varying hydrogen bond (H-bond) energies in liquid water. Ignoring a few “window” wavelengths that do not absorb water strongly, nearly every wavelength in the IR-C band corresponds to a H-bond energy/frequency that always exists in the constantly reorganizing environment of liquid water.

Conversely, as one moves through the IR-B band towards the IR-A band, the photon frequency no longer resonates with H-bond vibrational frequencies. Instead, other, higher energy, higher frequency intra-molecular bond energies resonate and absorb the radiation.

These bond energies are unique to each kind of covalent bond, which forms the basis for IR spectroscopy. Thus, at each wavelength, radiation is absorbed by a smaller percentage of matter, which results in deeper penetration and greater scattering.

16

The matter that causes the main absorptive processes at a particular wavelength of radiation is termed a chromophore. In the IR-C band, the predominant chromophore is water. In the IR-A and IR-B bands, other chromophores are hypothesized, but whether they specifically are causal targets is less clear, given the heterogeneous chemical composition of skin. The term selective photothermolysis, which simply refers to the decision of which chromophore(s) to target, is often used to describe the mechanism of how laser energy is absorbed by tissue.

Ultimately, by varying wavelength, pulse duration and/or pulse intensity, several kinds of photo-induced effects are achievable. Most of the time, IR exposure results in a photothermal effects, which simply refers to photon energy being absorbed and converted into heat/temperature rise. Some lasers, such as the Q-switched Nd:YAG laser, can achieve nanosecond pulse widths, which are so short that a photomechanical effect occurs in the form of a plasma shockwave, mechanically disrupting the target. Because of the short pulse duration, bulk thermal effects do not accumulate. Photochemical effects are defined as chromophore absorption that alters chemical composition or initiates a chemical reaction.

Finally, photobiomodulation refers to any combination of photothermal, photomechanical, and photochemical effects that trigger or modulate endogenous biological processes.

17

2.4 EMR-based rhytide improvement techniques and etiology

Two fundamental mechanisms dominate how EMR-based skin resurfacing achieves their outcomes: controlled thermal injury that initiates reconstruction of skin, and non- thermal stimulation of endogenous regenerative cellular processes. EMR-based techniques are roughly classified by their severity of impact on the skin. The ablative CO2 laser, the first to be used on skin, still stands as the most severe. Just as less-invasive laser approaches were sought as a response to the cons of surgery, so too were other laser approaches sought as a response to blunt the cons of the CO2 laser. Yet, ablative CO2 laser treatment remains next to surgery as the most clinically effective approach.30

Severity of impact on the skin is related to the wavelength, intensity, and duration of radiation used. Wavelength choice determines which chromophores are targeted, as well as penetration depth, while higher intensity and greater exposure duration increases the amount of energy delivered to the skin. The following section summarizes the various EMR-based approaches and their theoretical mechanisms of action.

18

Approach Technology Mechanism Advantages Disadvantages Minimum 2-6 wk Surgical stretching of Physical stretching Clinical Gold downtime Surgery SMAS and tightening Standard Risks inherent to all surgery Non-surgical Gold Postoperative Vaporize epidermis CO laser (10,600 nm) Standard complications still 2 + papillary dermis Ablative Er:YAG (2940 nm) Proven results for numerous, possible for neocollagenesis fine/course wrinkles pigmentary changes Faster recovery than Microthermal Same as ablative, with ablative, but more Lower risk but still treatment zones Fractional finer spatial control effective than non- ablating the skin (MTZ) ablative Deeper sub- Monopolar or Bipolar Still painful, epidermal heating arrays of electrodes Dermal/Sub- mitigated by than other Commonly coupled dermal heating contact cooling, RF approaches with with an optical along current paths local anesthetic, similar results as approach and/or vibration fractional approach Various lasers and Non-ablative Minimal to no Benefits can be filtered intense pulse thermal stimulation epidermal injury, subtle and may not Non-ablative light (IPL) spanning Targets bioactive thus minimal last as long as visible, IR-A and IR-B chromophores recovery time ablative approaches Table 1: Summary and comparison of various categories of EMR-based skin resurfacing approaches ranked in order of clinical invasiveness

2.4.1 Ablative skin resurfacing

Three main lasers, all operating in or close to the IR-C band, are used in ablative skin resurfacing: CO2 (10,600 nm), erbium-doped yttrium aluminum garnet (Er:YAG, 2940 nm), and, newly introduced in 2007, the yttrium scandium gallium garnet (YSGG, 2796 nm) laser.

With water as a strong chromophore in this band, the penetration depth is in the tens of microns with the IR light energy being absorbed and converted into heat. Because of the limited penetration depth, the mechanism of action is the complete surface vaporization of the epidermis and top layers of the dermis, which induces a healing response that reorganizes existing collagen and produces new collagen (neocollagenesis).31

19

The procedure is still risky, since the vaporization creates an oozing open wound that takes a few days to reepithelialize. In addition, patients are advised to avoid sunlight post- procedure to minimize a common side effect of discoloration. However, it is the first and oldest form of laser-based skin resurfacing, leaving over a decade of evidence that attests to its comparative efficacy.

2.4.2 Non-ablative approaches

As the name of this group of approaches suggests, non-ablative techniques are an attempt to avoid ablation of the epidermis that is the primary cause of downtime and complications. The market driver for this class of approaches has been the consumer/operator desire for next to no downtime procedures that improve the overall safety, which also speeds market entry by lowering the regulatory barrier of entry. As such, a myriad of approaches that utilize wavelengths from the visible to the IR-B bands have reached market, each with clinical data of effectiveness and minimal invasiveness. However, even the most current reviews acknowledge that current understanding of the underlying mechanisms of action are lacking, and furthermore, correlating histology to clinical effectiveness is a hotly debated topic among investigators.32 This ambiguity is interesting to note from the regulatory standpoint, since clearly, a firm understanding of why clinical effects occur is not necessary to reach market as long as safety and clinical efficacy are demonstrated.

20

Many modalities are classified as non-ablative, but they can be separated into three categories: vascular lasers (532-1,064 nm), IR-B lasers (1,320-1,550 nm), and intense pulsed light (broadband 400-1,200 nm). Nearly all non-ablative modalities rely upon photothermal injury to the dermis, but how photothermal injury manifests in the skin varies with the differing penetration depths and absorptive processes. Hemoglobin or melanin is the target chromophore of the vascular lasers, while IR-B lasers and intense pulsed light (IPL) sources typically either target water, or are absorbed non-specifically by multiple chromophores.

What is constant across modalities is the necessity to reach a minimum epidermal temperature of 45° C to observe any histological changes.33 This epidermal temperature correlates with a dermal temperature of 70° C, which is sufficient to achieve immediate thermal collagen tightening, discussed in greater detail below.34 In addition, the accompanying thermal injury to the microvasculature in the skin is hypothesized to prompt fibroblast proliferation and neocollagenesis.35 Lasers in the visible and IR-A, where hemoglobin is a target chromophore, are thought to potentiate this response, which is why they are referred to as vascular lasers.36 Various frequencies of visible light enable targeting of a range of hemoglobin oxidation, making it possible to target blood vessels of various sizes.37 Vascular absorption is thought to induce cytokine production from the vasculature, initiating an inflammatory response that leads to collagen remodeling.38

The primary chromophore for IR-B lasers is water, just as in IR-C, but scattering is greater with IR-B, which results in greater penetration depth and dermal photothermal impact.39 As such, IR-B lasers impact vascular targets, but also achieve thermal collagen tightening. Thermal collagen tightening is hypothesized to be a result of two processes: immediate thermal shrinkage and dermal fibroblast stimulation that creates new dermal 21 collagen.40 Ligaments, which are comprised predominantly of collagen, are regularly thermally tightened in sports medicine. Literature suggests that collagen be heated to 57-61°

C, but at shorter exposure durations, the shrinkage temperature can be as high as 85° C.41

While reaching the threshold temperature is necessary for immediate tightening, too much heating will irreversibly cause complete denaturing of collagen and cell death.42

Neocollagenesis will still occur, as it does during ablative approaches, but it increases the risk profile, defeating the entire point of non-ablative approaches.

Intense pulsed light (IPL), is a blend of high-intensity visible and IR light with the highly water-absorbing frequencies filtered out to increase the depth of penetration.

Photosensitizers, such as a carbon suspension, can be used in conjunction with IPL exposure to increase absorption and dermal impact.43 Because it is broadband polychromatic light, multiple chromophores are simultaneously targeted, so in addition to the skin rejuvenating action, a smoothing of the pigmentation profile of the skin is also achieved.44

LED treatment bears mentioning as well, since it is one of the few non-thermal light- based techniques, although the clinical effectiveness is questioned. LED treatment relies on photobiomodulatory inhibition of MMP’s, thereby lowering the rate of collagen breakdown, and upregulation of the mitochondrial transport chain, increasing cellular metabolism.45,46

While the approach is based upon evidence of in vitro bioeffects in human cells, properly controlled clinical data is still currently being performed to determine clinical effectiveness.47

22

2.4.3 Fractional photothermolysis

Fractional skin resurfacing, not to be confused with the term selective photothermolysis, utilizes ablative lasers but instead of ablating the entire surface and sub- surface of the skin, only an evenly distributed fraction of the skin is ablated.48 The laser energy is focused onto discrete microscopic points called microthermal treatment zones

(MTZ) that are columns of thermally denatured skin. Photothermal injury similar to the ablative approach is achieved, but the key difference is that healthy, unperturbed skin that surrounds the MTZ’s aids both the removal of dead tissue and the supplying of nutrients to the MTZ, speeding recovery.

Typically, fractional approaches utilize ablative lasers, such as the CO2 (10,600 nm),

Er:YAG (2940 nm), and more recently, YSSG (2790 nm) lasers. However, the technique has now been extended to non-ablative lasers as well (Table 2).

2.4.4 Radiofrequency (RF) stimulation

Put simply, RF stimulation is controlled surface electrocution that concentrates current locally to the sub-surface of the skin with an array of electrode tips of various lengths to optimize sub-epidermal impact. Electric current travels through the electrolyte-laden dermis easily, causing resistive heating of dermal and sub-dermal tissue along current paths.

As such, it is capable of achieving deeper tissue heating without causing excess damage to the outer layers, compared to the other approaches.

23

Tissue heated to a threshold temperature initiates thermal collagen remodeling in two ways. Directly heating the dermal layer causes immediate local collagen shrinkage. Similar to fractional photothermolysis, the desired outcome is distributed thermal lesions in the dermal collagen along current paths that leaves the surrounding tissue intact, resulting in similar benefits as fractional approaches, except non-columnar, resulting in deeper impact and minimal epidermal injury. In fact, the RF approach can be ablative or non-ablative, simply by adjusting the amount of current injected, and both approaches have shown clinical success.49 The procedure is not painless, but surface cooling, topical anesthetics, and in some devices, contact vibration are used to mitigate pain.

2.5 Other interventional techniques and their mechanisms

A myriad of other approaches for rhytide improvement are prevalent, ranging from invasive surgical approaches to non-invasive topicals, and the targeted mechanisms of action of each approach are equally broad. Furthermore, recent reviews of the literature have found that combining synergistic treatments generally outperform singular approaches.50 Thus, as we consider adding the use of high-powered terahertz light to the rhytide arsenal, understanding where those potential synergies may lie will ultimately guide future product development and partnerships.

24

2.5.1 Rhytidectomy

Beginning with what remains the gold standard in terms of efficacy, a rhytidectomy aims to stretch out rhytides and maintain tension to the skin. As previously mentioned, the surgeon dissects down to the SMAS layer, pulls it and the superior skin structures taut, and secures the SMAS to maintain tension. The results are proven, but the procedure carries a great deal of risk inherent to surgery and recovery times of up to 6 months.

2.5.2 Topicals

On the other end of the risk spectrum, various vitamins, peptides, and antioxidants are used as active ingredients in ointments to biochemically stimulate dermal regeneration.

Vitamins A (tretinoin), C, and peptide fragment Pal-KTTS have all been shown to stimulate collagen regeneration and increase collagen levels in the dermis.51,52,53 Antioxidants such as coenzyme Q act to neutralize ROS and allow natural regenerative processes to dominate.54

2.5.3 Botulinum toxin

Tradenamed Botox® (Allergan, Irvine, CA), botulinum toxin injections temporarily paralyze involuntary muscles in the face such that the overlying skin remains more relaxed and wrinkle-free. The mechanism of the toxin is the inhibition of acetylcholine receptors that activate muscles. Reapplication is necessary every 3-6 months. Side effects include pain, bruising, and paralysis of the eyelid.55

25

2.5.4 Soft tissue fillers

Often used in conjunction with Botox®, injectable soft tissue fillers were originally thought to reduce wrinkles by expanding the volume of material underneath the skin, but recently, studies show that fibroblast stretching may actually be responsible for neocollagenesis. The materials injected can be collagen based, e.g. bovine/human collagen, or non-collagen based, e.g., hyaluronic acid (Restylane®, Medicis Pharmaceuticals,

Scottsdale, AZ), calcium hydoxylapatite (Radiesse®, BioForm Medical, Inc., San Mateo,

CA), and poly-L-lactic acid (Sculptra™, Dermik Laboratories, Bridgewater, NJ).

2.6 Market and technical analysis of leaders in aesthetic lasers

TTM λ Company ($ mil) Trade Name ® (nm) Technique/Technology employed Syneron/Candela 337 Matrix IR 915 + RF Fractional diode laser + bipolar RF (NASDAQ:ELOS) CO2RE 10,600 Ablative CO2 laser GentleYAG 1,064 Non-ablative long-pulse Nd:YAG GentleMAX 755+1,064 Non-ablative alexandrite laser + Nd:YAG Solta Medical 113 Fraxel re:fine 1,550 Fractional non-ablative diode laser (NASDAQ:SLTM) Fraxel re:store 10,600 Non-ablative lower power CO2 laser Fraxel re:pair 10,600 Fractional Ablative CO2 laser Thermage RF Monopolar RF Cynosure 99 Affirm 1,320 + Non-ablative Nd:YAG + IPL (NASDAQ:CYNO) 1,440+IPL Palomar 97 Starlux/Artisan/ IPL Nearly identical platforms enabling IPL + (NASDAQ:PMTI) Palomar Icon 2940 Ablative/Fractional Er:YAG 1540 Non-ablative/Fractional Er:Glass 1440 Non-ablative/Fractional Nd:YAG Cutera 57 Pearl Fractional 2790 Ablative/Fractional YSGG laser (NASDAQ: CUTR) Laser Genesis 1064 Non-ablative long pulse Nd:YAG Titan IPL IPL Lumenis Not Ultrapulse Encore 10,600 Ablative/Fractional CO2 laser (Privately held) Known IPL Quantum IPL Intense Pulsed Light

Table 2: Revenue and product comparison of top six aesthetic laser companies. Current revenue data from Morningstar.com. Product data compiled from each company’s respective websites. TTM: trailing twelve months total company revenues, λ: wavelength.

26

Before delving into the technology behind commercial offerings of the top six leading aesthetic laser companies, one piece of data should be apparent: within five years, Syneron has maneuvered itself past all of the market leaders in 2005. While they did merge with one of those leaders, Candela, it is important to note that not only did Syneron acquire Candela, but the two companies also continue to keep separate books and report revenues separately.

That being said, Syneron’s TTM revenue is $221 million compared to Candela’s $116 million, showing that in many respects, Syneron’s elos™ technology platform has drastically outcompeted the market. Of course, TTM data includes revenue from all of a firm’s product offerings, but since much of a firm’s product offerings are dependent on its unique product platform, it is a rough but valid assessment of market acceptance.

Also of note is the market success of RF treatments, offered only by the top two companies on the list. A relatively new platform, RF is beginning to overtake fractional approaches, and, from a manufacturing and cost standpoint, RF is much less expensive than laser systems. Furthermore, Syneron’s combination of RF and a fractional approach is a win for novel technologies and combination techniques, which are reported in independent literature reviews to have greater clinical efficacy without appreciable procedural risk.56

Without knowing the full patent landscape and just examining each firm’s product offering, with exception to older technologies like the CO2 and 1,064 nm Nd:YAG lasers, each firm appears anchored to its own wavelength(s) and lasers. In fact, one of the drivers in the Syneron/Candela merger may have been Candela’s lack of RF techniques in its portfolio.

In an attempt to compete with the market leaders, companies with older legacy platforms such as Palomar and Cynosure have tried to create single systems that can do everything.

While this is convenient for a sole practitioner who does not want to purchase multiple 27 systems, in an already diverse market with many existing technologies to choose from, it may promote an unintended effect of confusing the consumer. Conversely, both Syneron and

Solta Medical place their focus on marketing technology platforms, i.e., elos™ at Syneron,

Fraxel and Thermage at Solta Medical. Just from visiting their respective websites, the clarity of message appears to leverage the innovation advantage previously described.

3 Hypothetical mechanisms of terahertz rhytide improvement

Summarizing the etiology of existing techniques, light-based rhytide improvement is achieved by collagen reorganization or neocollagenesis via photothermal wounding at various depths of skin, modulation of cellular activity, or a combination of both. Considering these mechanisms as we review the existing literature on THz/biological tissue interaction will help define how to strategically utilize THz to provide novel value. We will then define where THz holds unique value proposition to strategically define which hypotheses to test.

3.1 Existing evidence of THz exposure on biological systems

Before reviewing the literature in this section, it is worth noting that only within the last 10 years has there been equipment commercially available to biological researchers to perform experimentation. While there has been an exponential growth of papers in this area, very few are performed under the same conditions, whether it be choice of frequency, length of pulse, type of cells exposed, published specifications on sources and detectors, or proper controls.57 Only within the last two years have researchers begun to collectively move towards a more methodical approach to enable comparison between studies.

28

3.1.1 Penetration depth on skin and water

The penetration depth of THz light in liquid water varies with frequency, with the deepest penetration of roughly 130 µm occurring at the long wavelength end of the band (~ 1 mm), and the shallowest penetration of a few microns occurring by the short wavelength end

(~ 3 µm). To understand the effects of the addition of polar and non-polar substances to water, as are found in biological systems, consider first that THz is strongly absorbed by hydrogen bonds and that non-polar substances are largely transparent to THz. Thus, in the hypothetical situation of a heterogeneous blend of water and non-polar substance, the non- polar substance merely takes up volume where water normally would be, resulting in a lower absorption of THz light compared to an equal volume of water. This is termed THz defect.

Conversely, the addition of polar molecules will increase the H-bonding interactions in solution such that a non-linearly greater amount of THz light is absorbed. This is termed

THz excess.58

The penetration depth of THz on skin, a stratified heterogeneous blend of polar and non-polar molecules held in water, has been experimentally shown to be between 150-200

µm, demonstrating a net THz defect effect. This is due to the dehydrated nature of the outer layers of skin and the predominance of lipids, so strong absorption does not begin until the lower layers of epidermis down to the water-bound collagen in the dermis.59

29

3.1.2 Primary chromophore – hydrogen bond resonance

THz exposure can achieve thermal impact with sufficient focused power because the primary absorptive mechanism of THz on skin is via resonating H-bonds that convert THz energy to intermolecular vibration and ultimately, heat. However, the nature of this resonance is not well understood. Because of the key role of H-bonding interactions in many biochemical processes, several investigators in the last five years have been scrutinizing the topic of THz bioeffects and by extension, safety. At this point, the closest evidence describing the effects of this resonance has been that of membrane effects and photobiomodulation, covered in the following sections. It is important to note that collagen itself does not have a THz signature, but collagen in situ is heavily water bound, which absorbs THz broadly. When water-bound collagen dehydrates only 10% of its original weight, the absorption coefficient drops to 0.2 cm-1. Alternatively put, ~95% of the THz light passes through the dehydrated collagen, showing how transparent collagen is to THz when not bound to water.60

30

3.1.3 Membrane effects

Pulsed THz light has been shown to have effects on artificial cellular membranes called liposomes, intended to mimic the phospholipid bilayer of cell membranes.

Researchers filled liposomes with an enzyme called carbionic anhydrase (CA), which catalyzes the reaction of p-nitrophenyl acetate (p-NPA) to acetic acid and p-nitrophenol (p-

NP). The substrate, p-NPA, was added to the bulk aqueous portion, and researchers examined the reaction rate of the system comparing pulsed THz light and sham exposures.

When controlling for temperature, statistically significant differences in reaction rates were observed, proof that the liposomes experienced permeability changes. 61 The authors hypothesize that the mechanism behind this phenomenon may be due to the changes in vibrational dynamics of the membrane imparted by the THz pulses.

Another notable study to be discussed in greater detail in the next section involves high-powered THz light exposure onto cells in vitro. Confocal microscopy of THz-exposed cells confirmed increased membrane permeability compared to hyperthermic controls.62

3.1.4 Biophotomodulatory evidence

The key distinction to make when considering the ability of THz light to biophotomodulate biological systems is whether observed effects are thermal or non-thermal in nature. As mentioned earlier, it remains difficult to make definitive conclusions even after a comprehensive, less than year old, worldwide review of literature on THz bioeffects performed by the THz Bioeffects Division at the Air Force Research Labs (AFRL). That being said, the authors did conclude that the evidence across biological systems suggests THz imparts non-thermal bioeffects without the genotoxic effects that have been recently modeled 31 and hypothesized.63 In biology, nearly everything is context dependent, so it may not be of use to consider the ultimate bioeffects observed in plants, invertebrates, or even different mammalian cells. As such, we will consider for this work two studies that have direct relevance: an in vivo study performed on burn wounds and a more recent in vitro study performed on human dermal fibroblasts.

Ostrovskiy and coworkers exposed frequencies resonant to gaseous nitric oxide (NO) on patients with superficial and deep burns in combination with traditional burn wound therapies. NO is a known mediator of healing response, and its incorporation into a wound is a common target for other wound healing technologies. The investigators hypothesized that choosing resonant gaseous NO frequencies would enhance uptake of atmospheric NO into the wound. Frequencies of 150, 176-150, and 664 GHz were used with 0.03 mW/cm2 exposure power. After regular exposures during recovery, the results they report were remarkable: THz exposed wounds experienced lowered microbial content, faster epithelization times, lowered time to autodermoplastics application, and patients reporting overall decreased pain without any observed side effects. While the authors concluded that their chosen frequencies caused increased atmospheric NO absorbance that caused the enhanced wound healing, unfortunately, other frequencies of THz light were not used as a comparative control.64 Also, the work was not peer-reviewed and presented as conference proceedings, though this is a fairly common occurrence in the world of biophotonics.

A series of in vitro work on human dermal fibroblasts done by the AFRL provides a well-controlled insight into THz induced bioeffects. Dermal fibroblasts were exposed to continuous wave (CW) 2.52 THz at 84.8 mW/cm2 and 227 mW/cm2 for durations that varied from 5 to 80 minutes.65,66,67 The choice of 2.52 THz was in response to Alexandrov and 32 coworkers modeling result in 2010 that this frequency has resonant effects that may unzip

DNA and disrupt transcription.68 To study the gene transcription that results from

THz/UV/heat exposed cells, mRNA assays using GeneChip® technology (Asuragen Inc.,

Austin, TX), which allows for a broad examination of 47,000 gene transcripts, were performed.69

While genotoxic UV exposure resulted in 40-fold increase in DNA repair transcription, both THz and hyperthermic controls remained unchanged, providing strong empirical evidence against THz genotoxicity. In the high powered exposure studies (227 mW/cm2), THz and hyperthermic controls also exhibited identical transcription of apoptotic, necrotic, and cell stress responses. However, two differences were noted in THz-exposed cells compared to hyperthermic controls. First, as mentioned previously, confocal microscopy confirmed increased membrane permeability. Secondly, THz-exposed cells had statistically significant increases in transcription of several cytokines and interleukins, biomolecules that perform immune recruitment and can trigger regenerative processes.70

This result provides the first potential mechanistic link between THz exposure and wound healing. Another study utilizing mRNA analysis on THz exposed cells was performed on temperature-controlled mouse mesenchymal stem cells, which also demonstrated that THz exposures preferentially increased transcription of adiponectin,

GLUT4, and PPARG, all metabolic regulating proteins.71 Coupled with other examples found in the AFRL’s 2011 review, these results strongly suggest a non-genotoxic, frequency dependent photobiomodulatory effect of both pulsed and CW THz exposures on biological systems.

33

3.2 Finding THz’s unique value proposition in rhytide improvement

To summarize the previous section, the literature supports the following assertions:

1. Long wavelength THz light can achieve thermal dermal impact.

2. THz resonates hydrogen bonds, converting light energy into intermolecular vibrational energy

3. THz exposure can affect membrane permeability.

4. THz exposure may have non-genotoxic biophotomodulatory bioeffects, including the upregulation of immune recruiting and metabolic controlling molecules.

5. THz biophotomodulatory effects may enhance wound healing.

Referring back to the central job to be done, the goal of an aesthetic laser product is to bolster one’s self-concept. Pain or extended downtime can blunt the boosting of self-esteem that came from seeking self-improvement. Conversely, modest improvements coupled with minimal pain and downtime drives repeat business, favorable testimonials, and referrals.

Thus, the market pull is coming from consumers of aesthetic treatments who want higher effectiveness with the lowest possible social and physical costs.

EMR-based techniques mostly rely upon thermal stimulation of the skin to induce varying degrees of immune responses, which triggers collagen reorganization or neocollagenesis. In addition, combining non-ablative treatments hs a synergistic effect while keeping a low risk profile, and has demonstrable success in the marketplace. These conclusions point to the following ways THz could play a role in rhytide improvement:

34

1. Focused, high powered, long-wavelength THz beams deliver thermal stress and immune-recruiting photobiomodulatory effects onto intact skin.

2. Lower powered THz beams that can span across the THz band applied in conjunction with thermal stress caused by existing non-ablative and fractional approach to enhance immune recruitment to the skin surface via THZ photobiomodulation.

3. Long-wavelength THz light used in conjunction with topicals to enhance dermal uptake of active topical ingredients.

4. Lowering recovery time of ablative approaches via burn wound healing

4 Testable hypotheses to generate proof of concept and safety data

Because existing laser systems covered in this review are clearly more capable of delivering effective amounts of thermal stress, it follows that subsequent investigations should focus on THz’s unique value propositions: biophotomodulatory immune recruitment potential and membrane permeability to enhance effects of topicals. Both mechanisms will require THz light imparting a change to the skin, so safety data will be as important as efficacy data. To date, there is no THz predicate medical device, so, despite THz’s known non-ionizing properties, the prospect of THz bioeffects makes it highly likely that the FDA will require an investigational device exemption (IDE) application to generate human safety data through institutional review board (IRB) approved studies. Following the completion of these studies, the device will need to pass through pre-market authorization (PMA) before it is marketable in the United States. However, the device will more likely follow the course of other aesthetic laser devices and achieve first market entry in European markets, where regulatory hurdles are not as stringent.

35

4.1 Available equipment for THz delivery

The backbone of either of these routes is a high-powered THz light delivery system.

To summarize available technology, CO2 laser pumped molecular gas far-IR lasers, quantum cascade lasers, and backward wave oscillators are capable of generating the hundreds of milliwatts necessary to impart thermal effects, but each has its own limitations. Quantum cascade lasers in the far-IR range currently still require cryogenic cooling, conventional backward wave oscillators (BWO) are unreliable with limited lifetimes, and the CO2 laser pumped far-IR laser, while small enough to be the size of a desktop computer, cost hundreds of thousands of dollars. If lower powered THz exposures are sufficient, then solid-state electronic diode sources, photomixers, and laser-pulsed photoconductive switches become possibilities as well.

Teraphysics Corporation is working on a novel family of compact and scalable BWO sources and traveling wave tube (TWT) amplifiers. These sources and amplifiers would be able to achieve the power levels for thermal impact, but run at ambient temperature and have a lowered size and cost profile. Once successful, these devices could play a powerful role in commercializing not only this work, but many other THz-related applications as well.

36

4.2 Discussion of experimental models

Human work will likely not be possible until its safety profile is confirmed, because of the increased scrutiny on the nature of THz bioeffects. Thus, identifying an appropriate model is important to properly assess both the safety and immune-recruitment potential of

THz exposures. The two most commonly used animal models are porcine and mouse skin, but these models are actually problematic in that tissue architecture and immune responses differ enough from humans so that they do not accurately reflect THz impact or the subsequent desired immune response in human skin.72,73 For example, the differences in thickness of the outermost layers of porcine and mouse skin will impact which layer of skin receives the most THz energy. Performing THz exposure studies on mice and pigs will likely be a requested safety study to confirm overall and long-term safety on animal systems, but a better in vitro model that mimics both the architecture of human skin and the subsequent immune response is preferred at this point in research.

Previous works studying the bioeffects of infrared systems have utilized artificial human skin equivalents called “raft” cultures that layer relevant cells to form a stromal and epithelial region. Cell cultures by themselves are not as relevant to inform possible in vivo effects, because they behave differently without the presence of an extracellular matrix.74 To create skin raft cultures, human fibroblasts are grown in a collagenic matrix, mimicking the dermal stroma, and human keratinocytes are grown on top of this stromal layer. When the outer keratinocyte layer is exposed to air, the layer differentiates like normal skin, creating an

“epidermal” region that is similar in architecture to human skin in vivo.

37

With this model, we can test many of the safety/efficacy biomodulatory and membrane questions that exist, which can provide the groundwork towards IDE approval and studies of actual efficacy on human rhytides. The model is very flexible to utilizing specialized cells, such as fibroblasts transfected with bioluminescent reporter genes to monitor, for example, inflammatory cytokine production. Because of the high cost associated with high-throughput transcription assays like GeneChip®, it is not cost effective to perform such analysis on a larger scale investigation. Rather, once gene targets are identified, adenoviruses engineered to transfect firefly luciferase (luc) downstream to the target gene of interest can be propagated and used to transfect cells in the raft model, resulting in a much more cost-effective scaling of experimentation.75

Gene expression can then be monitored non-invasively in situ and across multiple time points after exposure on the same experimental setup. Because THz is known to have a finite penetration depth of up to 200 µm, only a thin layer of the raft model will be needed.

The thin tissue structure of the raft model will be ideal for data acquisition, as there will not be much tissue mass for either the D-luciferin substrate to permeate the construct or the 560 nm light generated by its oxidation by luc to permeate and reach the detector array. In comparison to standard destructive approaches such as histology, reverse transcriptase polymerase chain reactions (RT-PCR) on mRNA transcripts, in situ hybridization, and

Western blots analysis, this model is a flexible, cost-effective, and time saving means to yield desired data.

38

4.3 Testable hypotheses and planned experimentation

Using the human skin raft construct, we can test the following three hypotheses that should characterize more fully the safety profile, minimum thresholds for immune- stimulating bioeffects, and the ability of THz exposure to potentiate neocollagenesis stimulating topicals. While this is not the end of the tests required to reach market, this data will generate the evidence necessary to plan the next round of experimentation. All experiments, unless otherwise noted, should be performed at body temperature to avoid undesired heat or cold shock responses.

4.3.1 Hypothesis 1: THz bioeffects do not cause genotoxic effects

Because of the increased scrutiny on the nature of THz bioeffects, it will be important to confirm the lack of genotoxic effects. The work done at the AFRL provides a roadmap for this experiment. The firefly luciferase gene will be inserted downstream to promoters of

DNA repair genes and transfected via adenovirus into the raft constructs. These constructs will then be exposed to THz light, a heat control, and a positive genotoxic control (UV light) for a series of increasing amounts of time. The heat control should rise in temperature at the same rate as the THz exposed. At multiple time points after initial exposure, D-luciferin will be added prior to bioluminescent imaging. If DNA repair genes are being promoted, luciferase will also be transcribed and translated two hours later. Luciferase that is translated will react with D-luciferin and generate light. Just as in the AFRL study, comparing the THz to the negative heat control and to the positive UV control will determine the degree to which

THz contributes to genotoxicity. The results of this study should determine at a cellular level whether THz exposure has genotoxic effects.

39

4.3.2 Hypothesis 2: THz-induced thermal stress is necessary and sufficient to induce THz- specific biophotomodulation.

In the AFRL study, the cells that were sent for GeneChip® analysis were those that were thermally stressed to the point of cell death to determine whether apoptotic or necrotic processes dominated. Under these conditions, THz-exposed cells were shown to preferentially express cytokine and interleukin mRNA transcripts. Thus, while that experiment demonstrated that thermal stress is sufficient to induce THz-specific biophotomodulation, it does not give any data on whether thermal stress is necessary to induce the same effects. If thermal stress is not necessary, this reduces the need for a high- powered source, and may actually yield synergistic outcomes when coupled with existing modalities that already apply thermal stress.

There are two ways to perform this experiment. In both ways, we will generate rafts in a similar fashion to the experiment in Hypothesis 1, except luc will be transfected downstream to promoters of cytokines found to be upregulated in thermally stressed dermoblasts. The first approach is to apply for a constant amount of time a series of increasing THz power to the raft constructs, and compare cytokine transcription to heat- exposed constructs that rise in temperature at the same rate. The point at which THz-exposed constructs begin to outpace the cytokine transcription of heat-controls will point to when this temperature dependence takes place.

40

The second approach, one that eliminates thermal effects entirely, is to place the raft constructs in a temperature-controlled bath during THz exposure. As a negative control, we would choose a known wavelength of inert light, such as microwave energy. It would also be interesting to run a test on 585 nm light, where clinical results are attributed to light stimulated cytokine production in the microvasculature. However, because this in vitro testing model does relies on diffusion to keep the cells supple, there may not be any hemoglobin vascular targets for the 585 nm to impact. Along the same lines, this experimental setup could test to see if there was non-thermal effects from non-ablative methods currently used.

4.3.3 Hypothesis 3: THz exposure combined with application of vitamins A, C, or peptide fragment Pal-KTTS potentiates neocollagenesis.

Because vitamins A, C, and peptide fragement Pal-KTTS are thought to stimulate production of collagen type-1 production, we can use this same temperature controlled experimental set up in Hypothesis 2 to verify this ability of topicals, whether THz exposure potentiates this response, and whether THz exposure by itself can non-thermally trigger collagen type-1 production. Since we only concerned with fibroblast neocollagenesis activity, only the dermal fibroblasts need be transfected with luc downstream to promoters of the procollagen I gene. The “epidermal” keratinocyte layer will play the role of keeping the experiment relevant to in vivo contexts.

In the THz exposed and sham exposure constructs, half of the raft will have a particular applied topical, and half of the raft will remain uncoated. Since THz membrane effects should be non-thermal in nature, all experimental setups will be temperature controlled to body temperature. The uncoated, sham exposed test should yield our baseline

41 procollagen I transcription rate, which we compare the others against. Theoretically, the sham exposed coated construct should be our positive control, showing increases in procollagen I transcription. If there are no statistically significant differences to our baseline, then the underlying theory of topical action is called into question.

Given that the topical alone does cause increases in procollagen I transcription with time compared to the baseline, then comparing the THz exposed transcription rates to this positive control will determine THz’s ability to stimulate procollagen I transcription by itself, and its ability to potentiate this response.

42

5 Conclusion

Based upon the review of existing rhytide improvement technologies and THz bioeffects literature, the incorporation of the final, unexploited band of light in the electromagnetic spectrum appears to be a promising line of investigation. Coupled with the high demand and consistent growth of the low-risk cosmetic procedure market, one can envision THz being a selling point of a next generation suite of combination therapies that can lead this market space. While the empirical evidence is at a state of infancy, with the advent of new technology platforms such as Teraphysics’ suite of high-powered THz sources and amplifiers, current market leaders ought to be keen on keeping an eye on future innovations. Indeed, for a company such as Solta Medical, which already holds positions in all leading EMR-based methods, acquiring a patent protected novel core capability of a high- powered THz light delivery platform may be the key to staying competitive with

Syneron/Candela. By generating the data proposed in this work and marketing the platform with the right partner, companies like Teraphysics are well positioned to participate in this high-growth multi-billion-dollar market of the future.

43

1 Choi, C.M. and Berson, D.S. (2006) Cosmeceuticals. Semin Cutan Med Surg 25 (3):163- 168. 2 Frost & Sullivan. “Global aesthetic laser markets : F873-54.” Frost & Sullivan Market Report, July 2006. 3 Syneron. “Syneron Medical Ltd. and Candela Corporation to merge creating a leading global aesthetic device company.” http://www.syneron.com/whats-new/syneron- medical-ltd-and-candela-corporation-merge-creating-a-leading-global-aesthetic-devi (accessed Dec. 10, 2011). 4 Frost & Sullivan. “Global aesthetic laser markets : F873-54.” Frost & Sullivan Market Report, July 2006. 5 Medical Grapevine Asia. “Aesthetic laser and light device market expected to grow in Asia Pacific this year.” http://www.medicalgrapevineasia.com/mg/2011/09/08/aesthetic-laser- and-light-device-market-expected-to-grow-in-asia-pacific-this-year/ (accessed Dec. 8, 2011). 6 Frost & Sullivan. “Global aesthetic laser markets : F873-54.” Frost & Sullivan Market Report, July 2006. 7 Choi, C.M. and Berson, D.S. (2006) Cosmeceuticals. Semin Cutan Med Surg 25 (3):163- 168. 8 Finzi E. and Spangler A. (2005) Multipass vector (Mpave) technique with nonablative radiofrequency to treat facial and neck laxity.” Dermatol Surg 31:916-922. 9 Frost & Sullivan. “Global aesthetic laser markets : F873-54.” Frost & Sullivan Market Report, July 2006. 10 Bernstein, E.F., Chen, Y.Q., Kopp, J.B., Fisher, L., Brown, D.B., and Hahn, P.J. (1996) Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun- protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy. J Am Acad Dermatol 34(2 Pt 1):209-218. 11 White M.W., Makin I.R.S., Barthe P.G., Slayton MH, and Iklich R.E. (2007) Selective creation of thermal injury zones in the superficial musculoaponeurotic system using intense ultrasound therapy: a new target for noninvasive facial rejuvenation. Arch Facial Plast Surg 9:22–29. 12 Har-Shai Y., Sele E., Rubinstien I, Lindenbaum E., Mitz V., and Hirshowitz B. (1998) Computerized morphometric quantification of elastin and collagen in SMAS and facial skin and the possible role of fat cells in SMAS viscoelastic properties. Plast Reconstr Surg 102: 2466-2470. 13 Har-Shai, Y., Bodner S.R., Egozy-Golan D., Lindenbaum E.S., Ben-Izhak O., Mitz V., and Hirshowitz B. (1996) Mechanical properties and microstructure of the superficial musculoaponeurotic system. Plast Reconstr Surg 98 (1):59-70 14 Yaar M., Eller M.S., and Gilchrest B.A. (2002) Fifty years of skin aging. J Invest Dermatol 67 (1):209-224. 15 Kurban R.S. and Bhawan J. (1990) Histologic changes in skin associated with aging. J Dermatol Surg Onc 16 (10):908-914. 16 Varani, J., Warner, R.L., Gharee-Kermani, Phan, S.H., Kang, S., and Chung, J.H. (2000). Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinase’s and stimulates collagen accumulation in naturally aged human skin. J Invest Dematol 114(3):480-486. 44

17 Massague, J. (1998) TGF-beta signal transduction. Annu Rev Biochem 67:753-791. 18 Kang, S., Fisher, G.J., and Voorhees, J.J. (1997) Photoaging: Pathogenesis, prevention, and treatment. Clin Geriatr Med 17(4):643-659. 19 Hensley, K., and Floyd, R.A. (2002) Reactive oxygen species and protein oxidation in aging: a look back, a look ahead. Arch Biochem Biophys 397(2):377-383. 20 Fisher, G.J., Datta, S.C., Talwar, H.S., Wang, Z.Q., Varani, J., and Kang, S. (1996) Molecular basis of sun-induced premature skin aging and retinoid antagonism. Nature 379(6563):335-339. 21 Fisher, G.J., Wang, Z.Q., Datta, S.C., Varani, J., Kang, S., and Voorhees, J.J. (1997) Pathophysiology of premature skin aging induced by ultraviolet light. New Engl J Med 337(20):1419-1428. 22 Lavker, R.M. (1995) Cutaneous aging: chronologic versus photoaging. In B Gilchrest (Ed.), Photodamage (Vol. 1, pp. 123-135). Cambridge: Blackwell. 23 Bernstein, E.F., Chen, Y.Q., Kopp, J.B., Fisher, L., Brown, D.B., and Hahn, P.J. (1996) Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun- protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy. J Am Acad Dermatol 34(2 Pt 1):209-218. 24 Alam, M, Hsu, T.S., Dover, J.S., Wrone, D.A., and Arndt, K.A. (2003) Nonablative laser and light treatments: histology and tissue effects – a review. Laser Surg Med 33:30-39. 25 Lavker, R.M. (1995) Cutaneous aging: chronologic versus photoaging. In B Gilchrest (Ed.), Photodamage (Vol. 1, pp. 123-135). Cambridge: Blackwell. 26 Pickwell and Wallace (2006) Biomedical applications of terahertz technology. J Phys D Appl Phys 39(17):R301-R310. 27 International commission on non-ionizing radiation protection. (2006) ICNIRP statement on far infrared radiation exposure. Health Phys 91(6):630-645. 28 Ibid. 29 Wilmink G.J. and Grundt J.E. (2011) Invited review article: current state of research on biological effects of terahertz radiation. J Infrared Milli Terahz Waves 32(10):1074- 1122. 30 Hirsch, R.J., Dayan, S.H. (2004) Nonablative resurfacing: minimally invasive office-based procedures. Facial Plast Surg 20:57-61. 31 Atiyeh, B.S., and Dibo, S.A. (2009) Nonsurgical nonablative treatment of aging skin: radiofrequency technologies between aggressive marketing and evidence-based efficacy. Aesth Plast Surg 33:283-294. 32 Alam, M, Hsu, T.S., Dover, J.S., Wrone, D.A., and Arndt, K.A. (2003) Nonablative laser and light treatments: histology and tissue effects – a review. Laser Surg Med 33:30-39. 33 Fatemi, A., Weiss, M.A., and Weiss, R.A. (2002) Short-term histologic effects of nonablative resurfacing: results with a dynamically cooled millisecond-domain 1,320 nm Nd:YAG laser. Dermatol Surg 28:172-176. 34 Goldberg, D.J. (2000) Full-face nonablative dermal remodeling with a 1,320 nm Nd:YAG laser. Dermatol Surg 26:915-918. 35 Alam, M, Hsu, T.S., Dover, J.S., Wrone, D.A., and Arndt, K.A. (2003) Nonablative laser and light treatments: histology and tissue effects – a review. Laser Surg Med 33:30-39.

45

36 Hantash. B.M. and Banthia, V. (2011) Emerging technologies: nonablative lasers and lights. In Prendergast, P.M. and Shiffman, M.A. (Eds.), Aesthetic Medicine (Vol. 1, pp. 605-615). 37 Ibid. 38 Orringer, J.S., Voorhees, J.J., Hamilton, T., Hammerberg C., Kang, S., Johnson, T.M., Karimipour, D.J., and Fisher, G. (2005) Dermal matrix remodeling after nonablative laser therapy. J Am Acad Dermatol 53:775-782. 39 Hantash. B.M. and Banthia, V. (2011) Emerging technologies: nonablative lasers and lights. In Prendergast, P.M. and Shiffman, M.A. (Eds.), Aesthetic Medicine (Vol. 1, pp. 605-615). 40 Ruiz-Esparza, J. (2006) Painless, nonablative, immediate skin contraction induced by low- fluence irradiation with new infrared device: a report of 25 patients. Dermatol Surg 32:601-610. 41 Ibid. 42 Zelickson, B.D., Kist, D., Bernstein, E., Brown, D.B., Ksenzenko, S., Burn, J., Kilmer, S., Mehregan, D., and Pope, K. (2004) Histological and ultrastructural evaluation of the effects of a radiofrequency-based nonablative dermal remodeling device. Arch Dermatol 140:204-209. 43 Dover, J.S., Bhatia, A.C., Stewart, B., and Arndt, K.A. (2005) Topical 5-amino-levulinic acid combined with intense pulsed light in the treatment of photoaging. Arch Dermatol 141(10):1247-1252. 44 Hantash. B.M. and Banthia, V. (2011) Emerging technologies: nonablative lasers and lights. In Prendergast, P.M. and Shiffman, M.A. (Eds.), Aesthetic Medicine (Vol. 1, pp. 605-615). 45 Narurkar, V.A. (2006) Lasers, light sources, and radiofrequency devices for skin rejuvenation. Semin Cutan Med Surg 25:145-150. 46 Hantash. B.M. and Banthia, V. (2011) Emerging technologies: nonablative lasers and lights. In Prendergast, P.M. and Shiffman, M.A. (Eds.), Aesthetic Medicine (Vol. 1, pp. 605-615). 47 Ibid. 48 Hantash, B.M., and Mahmood, M.B. (2007) Fractional photothermolysis: a novel aesthetic laser surgery modality. Dermatol Surg 33(5):525-534. 49 Ruiz-Esparza, J., and Gomez, J.B. (2003) The medical face-lift: a non-invasive, nonsurgical approach to tissue tightening in facial skin using nonablative radiofrequency. Dermatol Surg 29:325-332. 50 Tierney E.P. and Hanke C.W. (2010) Recent advances in combination treatments for photoaging: review of the literature. Dermatol Surg 36:829-840. 51 Griffiths, C.E., Russman, A.N., Majmuda, G., Singer, T.S., Hamilton, T.A., and Voorhees, J.J. (1993) Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid). New Engl J Med 329(8):530-535. 52 Nusgens, B.V., Humbert, P., Rougier, A., Richard, A., and Lapiere, C.M. (2002) Stimulation of collagen biosynthesis by topically applied vitamin C. Eur J Dermatol 4(12), XXXII-IV.

46

53 Katayama, K., Armendariz-Borunda, J., Raghow, R., Kang, A.H., and Seyer, J.M. (1993) A pentapeptide from type I procollagen promotes extracellular matrix production. J Biol Chem 268(14):9941-9944. 54 McDaniel, D.H., Neudecker, B.A., DiNardo, J.C., Lewis, J.A., and Maibach, H.I. (2005) Clinical efficacy assessment in photodamaged skin of 0.5% and 1.0% idebenone. J Cosm Dermatol 4:167-173. 55 Helfrich, Y.R., Sachs, D.L., and Voorhees, J.J. (2008) Overview of skin aging and photoaging. Dermatol Nurs 20(3):177-83. 56 Shah, G.M., and Kilmer, S.L. (2005) Combined nonablative rejuvenation techniques. Dermatol Surg 31(9):1206-1210. 57 Wilmink G.J. and Grundt J.E. (2011) Invited review article: current state of research on biological effects of terahertz radiation. J Infrared Milli Terahz Waves 32 (10):1074- 1122. 58 Born, B. and Havenith, M. (2009) Terahertz dance of proteins and sugars with water. J Infrared Millim Te 30(12):1245-1254. 59 Tewari, P., Culjat, M.O., Taylor, Z.D., Suen, J.Y., Burn, B.O., Lee, H., Brown, E.R., Grundfest, W.S., and Singh, R.S. (2009) Role of collagen in terahertz absorption in skin. Proc. SPIE – Advanced Biomedical and Clinical Diagnostic Systems VII 7169:71691A1- 71691A-8. 60 Corridon, P.M. and Wilke, I. (2007) Monitoring the dehydration of collagen by time- domain terahertz transmission measurements. Proc. IRMMW-THz Joint 32nd International Conference on Terahertz Electronics 611-612. 61 Ramundo-Orlando, A., Gallerano, G.P., Stano, P., Doria, A., Giovenale, E., Messina, G., Cappelli, M., D’Arienzo, M., and Spassovsky, I. (2007) Permeability changes induced by 130 GHz pulsed radiation on cationic liposomes loaded with carbonic anhydrase. Bioelectromagnetics 28:587-598. 62 Wilmink, G.J., Vincelette, R., Ibey, B.L., Rivest, B.D., Bernhard, J., Roach, W.P., Roth, C.L., Horn, C.B., and Roberson, D. (2010) Determination of death thresholds and identification of terahertz (THz)-specific gene expression signatures. Proc. SPIE Optical Interactions with Tissues and Cells XXI 7462: 75620K. 63 Wilmink G.J. and Grundt J.E. (2011) Invited review article: current state of research on biological effects of terahertz radiation. J Infrared Milli Terahz Waves 32 (10):1074- 1122. 64 Ostrovskiy, N.V., Nikituk, C.M., Kirichuk, V.F., Krenitskiy, A.P., Majborodin, A.V., Tupikin, V.D., and Shub, G.M. (2005) Application of the terahertz waves in therapy of burn wounds. Proc. Infrared and Millimeter Waves Joint 30th Intl. Conf. on Terahertz Electronics TC5-1:301-302. 65 Wilmink, G.J., Rivest, B.D., Ibey, B.L., Berhard, J., Roach, W.P., and Roth, C.L. (2010) Quantitative investigation of the bioeffects associated with terahertz radiation. Proc. SPIE Optical Interactions with Tissues and Cells XXI 7562:75620L. 66 Wilmink, G.J., Rivest, B.D., Roth, C.L., Ibey, B.L., Payne, J.A., Cundin, L.X., Frundt, J.E., Peralta, X., Mixon, D.G., and Roach, W.P. (2010) In vitro investigation of the biological effects associated with human dermal fibroblasts exposed to 2.52 THz radiation. Laser Surg Med p. n/a-n/a

47

67 Wilmink, G.J., Vincelette, R., Ibey, B.L., Rivest, B.D., Bernhard, J., Roach, W.P., Roth, C.L., Horn, C.B., and Roberson, D. (2010) Determination of death thresholds and identification of terahertz (THz)-specific gene expression signatures. Proc. SPIE Optical Interactions with Tissues and Cells XXI 7462: 75620K. 68 Alexandrov, B.S., Gelev, V., Bishop, A.R., Usheva, A., and Rasmussen, K.O. (2010) DNA breathing dynamics in the presence of a terahertz field. Phys Lett A 374(10):1214- 1217. 69 Wilmink, G.J., Vincelette, R., Ibey, B.L., Rivest, B.D., Bernhard, J., Roach, W.P., Roth, C.L., Horn, C.B., and Roberson, D. (2010) Determination of death thresholds and identification of terahertz (THz)-specific gene expression signatures. Proc. SPIE Optical Interactions with Tissues and Cells XXI 7462: 75620K. 70 Ibid. 71 Alexandrov, B.S., Rasmussen, K.O., Bishop, A.R., Usheva, A., Alexandrov, L.B., Chong, S., Dagon, Y., Booshehri, L.G., Mielke, C.H., Phipps, M.L., Martinex, J.S., Chen, H.T., and Rodriguez, G. (2011) Non-thermal effects of terahertz radiation on gene expression in mouse stem cells. Biomed Opt Expr 2(9):2679-2689. 72 Davidson, J.M. (1998) Animal models for wound repair. Arch Dermatol 290:S1-S11. 73 Ross, E.V., Sajben, F.P., Hsia, J., Barnette, D., Miller, C.H., and McKinlay, J.R. (2000) Nonablative skin remodeling: selective dermal heating with a mid-infrared laser and contact cooling combination. Lasers Surg Med 26(2):186-195. 74 Bissell, M.J., Hall, H.G., and Parry, G. (1982) How does the extracellular matrix direct gene expression? J Theor Biol 99(1):31-68. 75 Wilmink, G.J., Opalnik, S.R., Beckham, J.T., Davidson, J.M., and Jansen, E.D. (2006) Assessing laser-tissue damage with bioluminescent imaging. J Biomed Opt 11(4):041114-1-15.

48