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Abstract: Epithelialization plays a major role in the reconstitution of a wound surface in partial-thickness burns. Wounds that can reepithe-lialize within 2 weeks are associated with minimal scarring and de-creased morbidity. Oxygen is an essential ingredient to all stages of wound healing, particularly the proliferative phase. This study demon-strated accelerated spontaneous neoepithelialization in 2 patients with second degree burns at approximately twice the normal rate. This rapid autologous epithelial regeneration was attributed to a hydrogel which delivers oxygen to a hypoxic wound bed.

Key words: burn, biology of wound healing, accelerated healing

WOUNDS 2013;25(10):E18-E25

From Ozeion LLC, Wilmington, DE

Address correspondence to:Ralph Almeleh, MD78-12 Metropolitan AveMiddle Village, NY 11379DrAlmeleh@Ozeion.com

Disclosure: The author discloses he is chief executive officer of Ozeion LLC, Wilmington, DE.

Burns, specifically second-degree burns, provide the clinical setting in which a wound heals by secondary intention. If epithelialization is delayed beyond 10 days, hypertrophic scarring and pigmentation

may result. The goal therefore, is to accelerate epithelialization within this time frame to avoid this and other complications. Oxygen has been shown to promote wound healing.1 A hydrogel that can deliver oxygen topically would provide a safe, natural “growth factor” that not only stimulates au-tologous epithelial regeneration, but makes a major advance toward the treatment of burns. This report looks at 2 cases in which an oxygen-deliv-ering hydrogel was used.

Case OneA 23-year-old white female in good health sustained a deep dermal

burn as a result of a curling iron inadvertently touching her left cheek. She presented on postinjury day 4 with a 3.5 cm x 1.5 cm wound that overlaid the suborbital malar aspect of the left cheek. The wound displayed typical characteristics of a deep second-degree thermal injury with tiny islands of granulation tissue interspersed among yellowish-white fibrinous exudate and cellular debris. The patient had self-treated the wound with topical antibiotic ointment. There was no evidence of gross infection or cellulitis. Following initial assessment the patient was advised to wash daily with an antibacterial soap and begin applying a newly formulated oxygen-deliv-ering hydrogel (ODH) (under clinical investigation by Ozeion LLC, Wilm-

Spontaneous Accelerated Epithelialization in Deep Dermal Burns Using an Oxygen-Delivering Hydrogel: A Report of Two Cases

Ralph Almeleh, MD

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ington, DE) made from biogenerated ‘processed’ water, twice daily (bid). The patient was followed biweekly for 1 month, at which time she was discharged with no apparent evidence of her initial burn injury.

Case TwoA 38-year-old white female placed an electric heat-

ing pad on her elbow to relieve pain which resulted in a second degree burn measuring 2.5 cm x 1.25 cm, just distal to the right elbow. The patient denied any serious medical condition except for hypothyroidism, controlled successfully with thyroid replacement ther-apy. She had a 23-year history of smoking 1 pack of cigarettes daily. An overlying scab sealed the wound, which was surrounded by a circumferential erythema-tous halo. The wound was dry and there was no associ-ated discharge or cellulitis. She was seen on postinjury day 2 and started on a mild antibacterial cleanser and ODH twice daily. The wound went on to heal within 1 week, and the patient was followed up twice during the subsequent month before being discharged.

ResultsThe first case demonstrates a deep dermal burn

of the face which reepithelialized in just 3 days. The second patient’s response did not initially appear as dramatic until a closer examination of the healing pa-rameters was completed. Several models were utilized to assess the progress of reepithelialization, including the length of the neoepithelial tongue from the wound margin, the change in wound perimeter, the percent change in wound area, or the change in area/change in perimeter.3 Winter2 found epithelialization occurred at a rate of approximately ≥ 7 mm/day depending on local wound conditions. He further demonstrated that the imposition of a dry scab to a wound impeded the pace of reepithelialization, as keratocytes diverted precious energy to scab dissolution and lysis.2 Winter concluded that wounds heal optimally in a moist or gel-like environment. Furthermore, if a moist dressing is initially employed a scab will not form and the heal-ing rate will increase as well as the oxygen tension in the wound.1,2 Using these criteria the cheek burn in the first case reepithelialized very quickly. The faint re-sidual, nearly imperceptible, scar present on the cheek at 27 days post-treatment is further confirmation of the propulsive pace of epithelialization (Figure 1).

The second patient had a reduction in surface area from 3.05 cm2 to 0.375 cm2, resurfacing 87% of the

wound bed in 5 days and almost healed completely by day 6 demonstrate the optimal maximal rate at which this wound progressed to restoration (2 mm/day) (Fig-ure 2). The difference between the 2 cases could be explained in part by the physical resistance barrier imposed by the scab to the diffusion of oxygen and keratinocyte migration as previously elucidated by Winter.2 Both these results reinforced earlier conclu-sions reached by Davis et al.4 Employing a supersatu-rated emulsion of oxygen in a perflourocarbon solvent, Davis et al4 was able to demonstrate that topical ox-ygen increased the rate of epithelialization in both second degree burns and partial thickness wounds to nearly twice the normal rate of healing.

DiscussionSpontaneous epithelialization of burn wounds may

take as long as 21 days before the wound is resurfaced. This was the rationale for early tangential excision and split-thickness skin grafting proposed by Janzekovic5

40 years ago. The idea was to not only prevent the conversion to a deeper thermal injury but to restore skin continuity and protect against infection. The risk to this blanket approach is that many patients would heal quickly and spontaneously without the need for surgery, avoiding associated blood loss and prolonged anesthesia with its potential risks and complications.

Burdge et al6 found that as little as a 10%-14% to-tal body surface area (TBSA) burn was associated with 50% mortality in the elderly population (individuals > 60 years of age), presumably because of their multiple associated comorbidities. Unfortunately, early excision and skin grafting in this subset of patients did not dem-onstrate a commensurate increase in survival as expect-ed, although it did decrease morbidity.7 Consequently the argument for a more conservative approach in this cohort is often proposed.8 In practice, however, this route also has its objections and drawbacks, as second-ary healing increases scarring and the risk of sepsis. It appears that neither option is optimal or without

Keypoints

•Typically, burns cannot be repaired primarily, andthus, are the classic example of a wound healing by secondary intention.

•Inanattempttorepairitself,theburnwoundacti-vates the kaleidoscope of biochemical and cellular events that encompass the 3 phases of wound heal-ing: inflammation, proliferation, and maturation.

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inherent risks. It follows that the development of an agent which, when applied, accelerates epithelializa-tion would be ideally suited to resolve this dilemma.

The burn wound is characterized by the loss of tis-sue from either a thermal, chemical, or electrical source. Typically, burns cannot be repaired primarily, and thus, are the classic example of a wound healing by second-ary intention. In an attempt to repair itself, the burn wound activates the kaleidoscope of biochemical and cellular events that encompass the 3 phases of wound healing: inflammation, proliferation, and maturation. It is the purview of the treating physician to carefully ascertain the depth of injury before recommending the appropriate corrective action. Unfortunately, burn wounds are notoriously difficult to assess clinically, with practitioners being wrong about the degree of the burn as much as 30% of the time.9 Waxman10 turned

to instrumentation to more accurately determine the severity of a burn, popularizing the use of laser flow velocimetry (LFV) or flowmetry to measure the micro-circulation in the papillary dermis. Using LFV, Waxman found the critical flow rate to be 6ml/100gm/min, be-low which the chance of spontaneous healing would be poor and the likelihood of hypertrophic scarring increased. These measurements appeared to precisely quantify numerically what was clinically observed by Jackson in 1947, and described as 3 concentric circles or zones of injury.11 The central core area demonstrated no flow on LFV or thrombosis, equivalent to a zone of maximal tissue destruction, necrosis, and anoxia. Sur-rounding this perimeter was a zone of marginal viabil-ity or hypoxia and stasis, corresponding to flow rates of 6-8ml/100gm/min. Finally in the outermost zone lies an area of hyperemia and normoxia with flow rates equal

Figure 1. The patient presented with a deep dermal burn as a result of a curling iron touching her left cheek. A)Initialinjury;B)3daysafterstartingtreamentwiththeoxygen-deliveringhydrogel,thewoundsurfacewasreepithelial-ized; C) 9 days postinjury; and D) 27 days postinjury, there was no visual evidence of the burn.

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to or greater than 8ml/100gm/min. It was demonstrat-ed that clinical regimens that promoted angiogenesis not only improved circulation and healing, but mini-mized scarring as well.10 It is in the intermediate zone where survivability is in question and salvageability a treatment priority. It is here where the astute physician can exert his influence with decisions ensuring a more favorable outcome for the burn patient.

The Role of Oxygen in Wound HealingInitially the wounding event and the ensuing hy-

poxic milieu are beneficial to the healing process. These acute conditions act as a lightning rod stimu-lating residual macrophages to induce angiogenesis (from endothelial buds via VEGF) and vasodilatation from neighboring blood vessels. However, if prolonged and not transitory, the hypoxic environment becomes detrimental to skin resurfacing and the healing mecha-nism.12 It’s an established tenet that for the wound to heal properly, the early hypoxic condition be reversed and normoxia restored. In fact, numerous studies, sup-plemented by the recent advent of hyperbaric oxygen therapy (HBOT), have demonstrated and validated the critical need of oxygen in all 3 phases of the healing calendar of events.13

Following coagulation, the inflammatory phase re-quires oxygen for phagocytosis and the enzymatic cleansing of wound debris and bacteria by neutrophils and macrophages.12 The adequate supply of oxygen is particularly essential during the subsequent prolif-erative phase where fibroblasts are enticed to secrete collagen and extra cellular matrix, the biologic mortar of the healing wound. In addition, both angiogenesis and epithelialization cannot proceed normally in the absence of oxygen. Finally, the maturation and remod-eling phase requires oxygen as a cofactor for collagen crosslinking by proline and hydroxylysine.12,13

Epithelialization is a particularly crucial event for the resurfacing of the burn wound in the process of healing by secondary intention. When epithelialization is robust and rapid it proceeds as a result of mitosis occurring at 17 times the normal rate from multiple sites including nests of intact basal epithelial cells, dermal appendages, and wound edges.14 This burst of activity is highly energy-dependent, requiring oxygen as a substrate for the production of ATP. Furthermore, the quicker the epithelial cells migrate to resurface the wound, the less chance of residual scarring and pig-mentation changes.15

Ideally, for a wound to have the best chance of clo-sure, the partial pressure of oxygen in the tissue bed should be a minimum of 30 mm Hg and preferably more than 40 mm Hg.12,16 The author’s experience from the use of hyperbaric oxygen demonstrated these lev-els are easily achievable; however, sustaining them for long periods of time outside the HBOT chamber pres-ents a challenge. Nevertheless, the evidence from mul-tiple studies demonstrates unequivocally that HBOT promotes neoangiogenesis, epithelialization, and ul-timately wound healing.12,13,16,17 The clinical achieve-ments garnered with HBOT have brought into question the firmly held belief that the skin receives its oxygen requirements only internally through its blood supply. Two recent articles, one by Schreml et al12 and another by Ladzinsky and Roe13 review skin and wound physiol-ogy. They summarize with clarity, backed by scientific data, the case for external delivery of oxygen to the epidermis and superficial dermis. This may explain 2 findings: 1) that oxygen levels to the superficial layers of the skin were maintained in spite of arterial limb oc-clusion,13,18 and 2) the morphology of the circulatory pattern of the skin. Studying the anatomy of the skin’s blood supply, Ryan19 noted that the capillary loops sup-plying the dermis were separated far from the over-lying epidermis, which led him to surmise that the oxygen and metabolic requirements of this superficial layer were minimal. In retrospect, the author wonders whether these observations, although accurate, were incomplete. It has since been shown that oxygen can penetrate the stratum corneum and the superficial lay-ers of the skin. Therefore, it is quite possible this area’s oxygen requirements are satisfied in part externally, and not just by dermal capillary loops.12,13,20,21 Atrux-Tallau et al22 supports the claim that the skin is not as pas-sive as once thought, but takes an active, albeit, minor role in oxygen uptake. Their investigation showed that epidermally stripped porcine skin was permeable to

Keypoints

•Evidence from multiple studies demonstrates un-equivocallythathyperbaricoxygentherapy(HBOT),promotes neoangiogenesis, epithelialization, and ultimately wound healing.12,13,16,17

•Topically administered oxygen therapy, unlikeHBOT, can be locally administered at normobaricpressures in a portable device without the associ-ated risks and possible systemic complications seen withHBOT.12

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dissolved oxygen when topically administered.22 Inter-estingly, this latter model resembles a partial thickness burn where the stratum corneum and epidermal layers are vaporized by the thermal injury exposing the rela-tively porous dermis. These, and other clinical and labo-ratory findings, have armed the wound care specialist with another therapeutic tool, topically administered oxygen therapy (TOT). Topically administered oxygen therapy, unlike HBOT, can be locally administered at normobaric pressures in a portable device without the associated risks and possible systemic complications seen with HBOT.12 Furthermore, TOT allows oxygen to be delivered directly to the surface of the wound, independent of its microcirculation. To be effective, however, topically delivered gaseous oxygen must be converted into a dissolved form for it to be biologically active and utilized by the target cell.13 Nevertheless,

despite this physiologic barrier, TOT has been shown to be safe and efficacious, and proved in early investi-gative trials to promote angiogenesis, epithelialization, and, ultimately, wound healing.12,13,21-25

It’s All About the WaterBioengineered processed water was born out of the

collaborative efforts of a nuclear physicist, a microbiol-ogist, and an oceanographer, all of whom are involved in the field of bioremediation. Bioremediation is the branch of earth science that employs microbes to di-gest environmental pollutants, such as oil and gas, into harmless biodegrable waste byproducts. At the time, these scientists were looking for a means to use oxy-gen to biostimulate aerobic bacteria buried beneath the earth in a hypoxic locale. After years of investiga-tive research and experimentation, they developed a

Figure 2. The patient presented with a second-degree burn on her left elbow from an electric heating pad. A)Initialinjury;B)1daypost-treatmentwiththeoxygen-deliveringhydrogel,theerythematoushalowasdiminished;C)5days postinjury; D) 21 days postinjury,

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method to alter the hydrogen adhesive bond in water between hydrogen and oxygen. In essence, they dis-covered a process to weaken this resting energy bond, allowing oxygen to dissociate and wrench itself free from a water molecule and avail itself to a living organ-ism in oxygen debt.

As a result of their efforts, these microbes were then able to function optimally, metabolizing hydrocarbons even under anaerobic conditions. Aware of the homo-geneity in nature among heterotroph’s basic cellular function (the respiratory cycle) and using that para-digm, the author extrapolated those results and applied them to living cells within the animal kingdom, man included. Furthermore, the special nature of this water was verified when it was independently studied by an outside laboratory.26 These findings indirectly validated the contention of an altered energy state within the water molecule when they discovered that the surface tension was 61 dynes/cm, nearly 20% below normal water. In addition, the water froze at 34°F.

The water’s oxygen-liberating capability was dem-onstrated in another independent study performed at the University of British Columbia sports medicine de-partment in Vancouver.27 In this experiment, 4 healthy volunteers were placed in a hypoxic chamber where their arterial oxygen saturation (SaO2) was lowered and stabilized at 90%. These values were confirmed by continuous pulse oximetry. All 4 subjects were then given 600 cc of the oxygen-liberating water (OLW). Within ten minutes of ingestion all subjects registered a modest increase in their SaO2, with 2 of the 4 reach-ing peak levels of 95%. These elevated levels were maintained for an additional 20 minutes post a second imbibition before returning to baseline (90% SaO2). Al-though small, these incremental changes were thought to be significant when viewed within the context of the S-shaped nature of the oxyhemogloblin dissocia-tion curve.

In summary, taken in aggregate, these additional characteristics were of immense importance. Not only was the water able to deliver oxygen, but because of its lower surface tension it was more permeable, en-abling it to transgress the dermal barrier more readily than other water-based solvents. Consequently, OLW functions in a dual capacity, as a drug delivery system with that “drug” being oxygen. Furthermore, the oxy-gen would be released in a dissolved nascent molecu-lar state ready to be taken up by a targeted hypoxic cell. This is compatible with a number of laboratory

and clinical trials previously cited, validating the utility of TOT for wound healing. Finally, a hydrogel format was settled upon as the dressing of choice primarily because its composition is 90% or more water. As a re-sult, it was felt there was little chance of altering the water’s intrinsic physical properties.

Hydrogels are jelly-like substances suspending wa-ter in a semiliquid colloidal state. The ensuing gel is formed by the interaction of a variety of insoluble poly-mers, both natural and synthetic, and water. Mostly hy-drophyllic, they can absorb thousands of times their dry weight, and by cross linking with one another, can entrap water in this semiliquid environment.

Viewed another way, polymers are a maze of fences that have corralled horses, the water molecules, hold-ing them penned up. Depending on their interaction, the fence posts and railing can be either tightly glued or weakly bonded, making the gel state permanent or reversible. Similarly, the forces that make hydrogel dis-solution impossible are the presence of covalent bonds among the polymer chains as opposed to weaker ionic interactions. As a result, a weaker or more ‘inert’ poly-mer that readily dissolved on contact with the skin was chosen. As such, it is able to donate the entrapped water molecules to the wound surface, providing not only a source of moisture but topical oxygen delivery as well.

Hydrogels are available in 2 forms, as sheets or as amorphous gels with varying viscosity. Six qualities make them particularly suitable for the treatment of burns. First, they are cool and soothing as well as easily applied and adaptable to any wound surface. Second, their gel-like structure contributes to wound healing and epithelialization. Third, they are gentle upon re-moval, an important consideration in easily damaged skin or granulating tissue. Fourth, they can both protect and hydrate a wound and absorb moderate amounts of exudate. Fifth, they can contribute to autolysis, scab dissolution, and debridement of necrotic tissue. Finally,

Keypoints

•Hydrogelsarejelly-likesubstancessuspendingwa-ter in a semiliquid colloidal state. The ensuing gel is formed by the interaction of a variety of insoluble polymers, both natural and synthetic, and water.

•Theirsoothingfeel,gentleremovalandcontribu-tion to epithelialization, among other features, make hydrogels particularly suitable for the treat-ment of burns.

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they can be customized by adding a number of ingredi-ents including alginates, hydrocolloids, salts, or antimi-crobials. In summary, they can be adjusted and tailored to deal with the local needs of a particular wound.

Once the hydrogel was formulated, it was next tested on first degree burns in 6 volunteers at the Skin Study Center, Broomall, PA.28 Each individual had 6 sites, 1”x 1”, marked on their back. Four of the 6 sites received controlled and precisely measured UVB radiation via a 150 watt solar stimulating xenon light source. The radiation received was equivalent to 2 measured ery-thema dosages (MEDs) subsequently confirmed by a minolta chromameter and expert graders the following day. Afterward the gel vs a similar-appearing placebo was applied 2 times a day to 4 of the sites, leaving 2 un-adulterated for comparison. The results demonstrated a 40% reduction in erythema over 48 hours with the ODH when compared with placebo. At this point, the author felt comfortable and justified with proceeding to the next phase, testing the hydrogel clinically on deeper thermal injuries.

ConclusionThe cases presented are unusual and remarkable for

the manner and speed with which the burn wounds epithelialized and resurfaced. The author believes this can be explained by the unique intrinsic properties present in the water-based hydrogel. Foremost of these, is its oxygen releasing capability, which is known to promote epithelialization, angiogenesis, granulation tissue, and ground substance formation, as well as col-lagen synthesis. All of these elements are crucial and essential to the optimal healing of a wound. This is par-ticularly significant for the salvagability of a hypoxic, marginally viable (Jackson type II) burn. Furthermore, the accelerated closure of a wound by epithelialization minimizes scarring and residual pigment changes to the skin. Another byproduct of oxygen-liberating water is its increased absorptive quality, making it an ideal solvent and drug delivery system, especially for water-soluble agents.

Processed from water, which is ubiquitous and readily available, the oxygen-delivering hydrogel is portable, cheap, and safe because it is still chemically H2O. In summary, the author considers this gel a form of TOT, providing topical oxygen continuously to an oxygen-deprived and dependent wound. Moreover, it can satisfy the increased metabolic demand for oxy-gen without the associated risks or side effects of other

more costly therapeutic options. Oxygen is considered a mild germicide, especially against anaerobes, in addi-tion to facilitating the janitorial role of polymorphs and macrophages and their abilities to sanitize a wound sur-face.12,13,18 Finally, of particular relevance to dermatolo-gists and plastic surgeons, it has potential suitability as a soothing postoperative healing balm in cosmetic cases such as dermabrasion, laser surgery, or chemical peels of the face.

The difference in the rate of epithelialization be-tween the 2 second degree burns can be attributed to the presence of a scab in the second case and its consequent effects on wound healing. These 2 cases are reported not only because ODH appears to hasten healing in burns, but also as a novel approach at reepi-thelializing clinical wounds that are difficult to heal.

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