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

Epithelialization plays a major role in the reconstitution of a wound surface in partial-thickness burns. Wounds that can reepithelialize within 2 weeks are associated with minimal scarring and decreased morbidity. Oxygen is an essential ingredient to all stages of wound healing, particularly the proliferative phase. This study demonstrated 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.

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 autologous epithelial regeneration, but makes a major advance toward the treatment of burns. This report looks at 2 cases in which an oxygen-delivering hydrogel was used.

A 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-delivering hydrogel (ODH) (under clinical investigation by Ozeion LLC, Wilmington, 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.

A 38-year-old white female placed an electric heating 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 therapy. 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 erythematous halo. The wound was dry and there was no associated 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.

The 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 parameters 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 healing 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 residual, 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) (Figure 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 conclusions reached by Davis et al.⁴ Employing a supersaturated emulsion of oxygen in a perflourocarbon solvent, Davis et al⁴ was able to demonstrate that topical oxygen increased the rate of epithelialization in both second degree burns and partial thickness wounds to nearly twice the normal rate of healing.

Spontaneous 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% total 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 demonstrate a commensurate increase in survival as expected, 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 secondary healing increases scarring and the risk of sepsis. It appears that neither option is optimal or without inherent risks. It follows that the development of an agent which, when applied, accelerates epithelialization would be ideally suited to resolve this dilemma.   The burn wound is characterized by the loss of tissue 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 secondary 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 microcirculation in the papillary dermis. Using LFV, Waxman found the critical flow rate to be 6ml/100gm/min, below 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. Surrounding this perimeter was a zone of marginal viability 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 to or greater than 8ml/100gm/min. It was demonstrated that clinical regimens that promoted angiogenesis not only improved circulation and healing, but minimized 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.

Initially the wounding event and the ensuing hypoxic milieu are beneficial to the healing process. These acute conditions act as a lightning rod stimulating 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 mechanism.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, supplemented 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 requires 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 proliferative 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 remodeling 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 pigmentation changes.¹⁵   Ideally, for a wound to have the best chance of closure, 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 levels are easily achievable; however, sustaining them for long periods of time outside the HBOT chamber presents a challenge. Nevertheless, the evidence from multiple studies demonstrates unequivocally that HBOT promotes neoangiogenesis, epithelialization, and ultimately wound healing.^12,13,16,17 The clinical achievements 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 physiology. 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 occlusion,^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 supplying the dermis were separated far from the overlying 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 layers 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 passive as once thought, but takes an active, albeit, minor role in oxygen uptake. Their investigation showed that epidermally stripped porcine skin was permeable to dissolved oxygen when topically administered.²² Interestingly, this latter model resembles a partial thickness burn where the stratum corneum and epidermal layers are vaporized by the thermal injury exposing the relatively porous dermis. These, and other clinical and laboratory 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 investigative trials to promote angiogenesis, epithelialization, and, ultimately, wound healing.^12,13,21-25

Bioengineered processed water was born out of the collaborative efforts of a nuclear physicist, a microbiologist, and an oceanographer, all of whom are involved in the field of bioremediation. Bioremediation is the branch of earth science that employs microbes to digest environmental pollutants, such as oil and gas, into harmless biodegrable waste byproducts. At the time, these scientists were looking for a means to use oxygen to biostimulate aerobic bacteria buried beneath the earth in a hypoxic locale. After years of investigative research and experimentation, they developed a method to alter the hydrogen adhesive bond in water between hydrogen and oxygen. In essence, they discovered 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 organism 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 homogeneity in nature among heterotroph’s basic cellular function (the respiratory cycle) and using that paradigm, 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 demonstrated in another independent study performed at the University of British Columbia sports medicine department 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 reaching peak levels of 95%. These elevated levels were maintained for an additional 20 minutes post a second imbibition before returning to baseline (90% SaO2). Although small, these incremental changes were thought to be significant when viewed within the context of the S-shaped nature of the oxyhemogloblin dissociation 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, enabling 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 oxygen would be released in a dissolved nascent molecular 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 result, it was felt there was little chance of altering the water’s intrinsic physical properties.   Hydrogels are jelly-like substances suspending water 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. Mostly hydrophyllic, 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, holding 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 dissolution 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’ polymer 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 removal, 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, they can be customized by adding a number of ingredients including alginates, hydrocolloids, salts, or antimicrobials. 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 erythema 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 unadulterated 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.

The 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 collagen synthesis. All of these elements are crucial and essential to the optimal healing of a wound. This is particularly 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 oxygen without the associated risks or side effects of other more costly therapeutic options. Oxygen is considered a mild germicide, especially against anaerobes, in addition to facilitating the janitorial role of polymorphs and macrophages and their abilities to sanitize a wound surface.^12,13,18 Finally, of particular relevance to dermatologists 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 between 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 reepithelializing clinical wounds that are difficult to heal.

The author is from Ozeion LLC, Wilmington, DE.

Address correspondence to: Ralph Almeleh, MD 78-12 Metropolitan Ave Middle Village, NY 11379 DrAlmeleh@Ozeion.com

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