A Comparative Study of Spray Keratinocytes and Autologous Meshed Split-Thickness Skin Graft in the Treatment of Acute Burn Injuries

Background. ReCell (Avita Medical, Northridge, CA) is an autologous cell harvesting (ACH) device that enables a thin split-thickness skin biopsy to be processed to produce a cell population that includes a mixed population of keratinocytes, melanocytes, Langerhans cells, and papillary dermal fibroblasts for immediate delivery via a spray applicator onto a prepared skin surface. Materials and Methods. In this Institutional Review Board-approved US Food and Drug Administration phase 2 study, the authors prospectively evaluated the treatment of partial-thickness burns in patients with two 320 cm² areas, 1 area treated with the ACH device and the other with a meshed split-thickness skin graft (MSTSG) as a control. The authors compared the treatment areas for graft take, pigmentation, and color match to surrounding healthy tissue, scarring, and pain. Results. In this preliminary study, 10 patients were treated with this protocol. Eight patients had 100% take to both treatment areas and 2 patients had significant non-take and graft loss attributable to underexcised wound beds and difficulty with the spray applicator. Pigmentation and color match ratings were identical at week 52 and the Modified Vancouver Scar Scale scores were comparable. One subject rated the autologous cell harvesting site as having a better appearance, while the remaining subjects rated their ACH and MSTSG sites’ appearances as being comparable. In early follow-up visits, pain ratings were slightly elevated in the ACH group due to graft healing; however, in visits following week 2, pain ratings at the ACH and MSTSG sites were rated similarly by all patients. Conclusion. This preliminary report describes an early experience with the ACH device and the treatment of partial-thickness burn injuries. In this 10-patient series, patients benefitted from having a decreased donor site size and comparable outcomes with MSTSG treatment. While this preliminary underpowered study has provided positive results, there is a learning curve with choosing the proper wound for treatment with the ACH device, as well as with using the device.

  Early excision of burn wounds and prompt resurfacing with skin grafts is the mainstay of surgical treatment of extensive burn injuries.¹ Studies of these treatments have clearly shown early excision and grafting will improve survival, reduce hypertrophic scarring, decrease pain duration, shorten hospital stay, and reduce infectious complications.^2-7 With this principle in mind, a wide variety of techniques and methods have been applied for coverage of an open burn wound, including meshed and nonmeshed autografts and homografts or a combination of the 2, human amniotic membrane, xenograft, synthetic skin substitutes, and cultured epithelial autografts (CEA).⁸ However, early excision with autograft coverage may be difficult to achieve in patients with extensive burns due to the limited size of donor sites. Definitive coverage of all burn wounds may take several weeks while waiting for donor sites to epithelialize for repeated harvesting or for CEA to grow. Delays may also be due to systemic illness or local wound infection that can develop while waiting for CEA. These delays in autograft coverage have lead to the development of a noncultured epithelial autograft which enables the surgeon to greatly expand the amount of coverage they can obtain from small donor sites with immediate application.⁹ Additionally, early difficulty with transferring CEA to the wound beds lead to the development of a spray-on technique which allows for easy transfer of proliferating keratinocytes to the wound.¹⁰

  An autologous cell harvesting (ACH) system (ReCell, Avita Medical, Northridge, CA) enables the surgeon to immediately process a small split-thickness biopsy and deliver keratinocytes, melanocytes, fibroblasts, and Langerhans cells harvested from the epidermal-dermal junction to the wound bed using a spray-on technique (Figure 1). This technique can cover up to 80 times the area of the split-thickness skin biopsy. This study was part of an Institutional Review Board-approved US Food and Drug Administration phase II clinical trial (NCT01476826) aimed to provide a comparison of percentage graft take, appearance of graft, and pain between meshed split-thickness skin grafts (MSTSG) and ACH.

  All patients admitted to the Richard M. Fairbanks Burn Center at Eskenazi Health, Indianapolis, IN, were pre-screened for study participation. Patients with a partial-thickness to deep partial-thickness burn of 4%-25% total body surface area (TBSA) requiring surgical debridement and skin grafting were asked to consider participating in the study. Inclusion criteria were that patients be 18-85 years of age and have a 4%-25% TBSA acute partial-thickness to deep partial-thickness injury requiring skin grafting that could be divided into 2 separate 320 cm² treatment areas. Patients were excluded from participation if a microbiologically proven pre-existing local or systemic bacterial infection was present; antibiotics were administered for more than 48 hours prior to grafting; the patient was taking medications known to have an effect on wound healing or skin pigmentation; presence of a pre-existing condition that may interfere with wound healing (eg, malignancy, diabetes, or autoimmune disease); or a known hypersensitivity to trypsin or compound sodium lactate (CSL) solution existed. Patients who met all inclusion and exclusion criteria were enrolled into the study. Patients were informed about the potential risks and benefits of the procedure and signed consent was obtained from each of them.

  All patients underwent general anesthesia for a synchronous wound debridement and skin grafting procedure. Two treatment sites were designated as site A and site B and were then randomized to either treatment with spray keratinocytes procured with the ACH device or MSTSG. All wounds were tangentially excised to healthy dermis with brisk punctate bleeding. Hemostasis was obtained using thrombin spray, epinephrine-soaked laparotomy pads, and electrocautery.

  The ACH donor site was a noninjured area, and a dermatome depth setting of 0.006 mm-0.008 mm was used. A 2 cm x 2 cm sample was trimmed from the harvested split-thickness graft. Following the required initial set-up of the ACH unit, the biopsy was placed into the trypsin solution to allow for separation of the dermis and epidermis (Figure 2). The sample was removed after 15 minutes for a test separation of the dermis and epidermis. If the sample did not readily separate it was placed back into the trypsin for an additional 5-10 minutes. Once the dermis and epidermis began to easily separate, the sample was rinsed in the CSL solution and separation was completed providing access to the dermal-epidermal junction. A total of 5 ml of CSL solution was used to intermittently irrigate the device’s work surface while the dermal-epidermal junction of the sample was teased with a scalpel to form a plume of cells that was then aspirated into a 5 ml syringe to form a single suspension. Following a filtering process, the suspension was aspirated into a new 5 ml syringe, the spray nozzle was attached to the syringe, and the suspension was delivered to the field for application (Figure 3).

  The remainder of the skin was harvested from a separate noninjured area with a dermatome setting of 0.010-0.012 and meshed 2:1 to cover the control site. The MSTSG was secured with staples. The graft was primarily dressed with a nonadherent dressing (Telfa Clear Wound Dressing, Covidien, Mansfield, MA), and then covered with a secondary dressing of mesh gauze (Xeroform Petrolatum Gauze, Covidien, Mansfield, MA), and an outer gauze dressing that was secured with a gauze roll dressing (Kerlix or Curity, Covidien, Mansfield, MA).

  Prior to applying the keratinocyte suspension, the primary dressing was secured along the entire length of the dependent edge of the wound with a skin adhesive (Dermabond, Ethicon, Somerville, NJ) and staples. The cell suspension was then evenly sprayed starting at the dependent edge of the wound progressing steadily to the superior wound edge. As the suspension was sprayed, the primary dressing was lifted and the suspension was applied directly to the wound bed. Upon completion of coverage of the wound with the spray suspension, the primary dressing was secured on all edges with the skin adhesive and staples. The secondary dressing was applied over the primary dressing followed by a large gauze dressing secured by the previously noted gauze roll dressing. (Figures 4A-4E).

  The outer dressings for the ACH and MSTSG sites were changed only for excessive drainage accumulation during the first postoperative week. On postoperative day 7, dressings were removed from both the ACH and MSTSG sites. The primary dressings were slowly lifted away from the wound. If the primary dressing had adhered to the wound bed, mineral oil was used to saturate the dressing to free it from the underlying bed. At this time, the percentage of reepithelialization and pigment and color match of the area were evaluated. All unhealed areas were redressed daily with a nonstick dressing and a topical antimicrobial ointment until fully healed. Following complete reepithelialization to the sites, topical antimicrobial therapy was discontinued and lotion was placed on the sites for skin hydration and compressive dressings for scar management. Patient follow-up evaluations were scheduled at postoperative weeks 1, 2, 3, 6, 12, and 52.

  Follow-up visits on postoperative weeks 1, 2, 3, and 6 included assessment of the ACH and MSTSG sites for reepithelialization, pigmentation and color match, pain, and aesthetic appearance. Percentage of reepithelialization was evaluated by using a portable wound measurement device (Visitrak, Smith and Nephew, St. Petersburg, FL). All nonhealed areas were traced on the wound measurement grid and then the nonhealed area was calculated into square centimeters using the measurement device. All sites were compared for color and pigment match to immediately adjacent uninjured skin. Patients were asked to provide their objective assessments of pain as related to the ACH and MSTSG site.

  At visits on postoperative weeks 12 and 52, all of the above evaluations were performed as well as objective assessments by the patient of the appearance of both treatment sites. A scar assessment of the ACH and MSTSG sites was performed using the Modified Vancouver Scar Scale (MVSS). Photographs were obtained of each treatment area at each visit.

  Between October 2007 and September 2008, 10 patients consented and were treated with spray keratinocytes harvested with the ACH device. Six patients completed their 52-week follow-up and 4 were lost to follow-up. The first patient was lost following the postoperative week 3 visit, the second patient was lost following the postoperative week 6 visit, and the third and fourth patients were lost following the postoperative week 12 visit.

  A summary of patient demographic information, injury characteristics, and treatment data is available in Table 1. Ninety percent of the participating patients had contiguous wounds that had 2 designated treatment areas separated by an area of MSTSG not included in the MSTSG study site. The ACH and MSTSG treatment sites of 2 patients were 28 cm² smaller than the required 320 cm² for the study. A protocol deviation report was filed with the sponsor and the patients continued in the study.

  The percentage of graft take was evaluated at each scheduled follow-up visit. Comparable graft take was seen in both treatment sites by postoperative week 3 (Table 2). Two patients experienced graft loss to their ACH treatment sites and required a regrafting procedure to portions of their ACH treated areas. The first patient with graft loss was patient 2 in this series; by postoperative week 2, the ACH site had a 25% graft loss and the MSTSG site had a 19% graft loss. All graft loss was determined by planimetry and required repeat debridement and autografting for wound closure. The graft loss to both sites was attributed to inadequate wound bed debridement at the initial operation. The second patient with graft loss was patient 5 in this series; by postoperative week 2, planimetry determined a 38% graft loss to the center of this patient’s posterior thigh ACH site, and redebridement and autografting was required. This graft loss was attributed to difficulties with the spray applicator, the location of the treatment site, and the deep dermal nature of the injury. Removal of the 2 patients with graft loss from the graft take data set improves the ACH graft take from 91.3% to 97.1% in postoperative week 2.

  Pigmentation and color match of each treated area was rated by the investigators as being matched, mildly mismatched, or grossly mismatched when compared to immediately adjacent uninjured skin (Tables 3 and 4). Eight of the 10 patients had identical pigmentation and color match ratings in the ACH and MSTSG treatment areas on their final follow-up visits, while the remaining 2 patients had comparable pigmentation and color match ratings. During early follow-up visits in the first 6 weeks following the procedure, treatment areas were found to be more mismatched in pigmentation and color when compared to adjacent uninjured skin but were noted to be milder in later visits (Figure 5).

  Pain was evaluated by the patient using a visual analog scale. At each study follow-up visit, the patient was asked to place a mark on the scale that represented their level of pain at that time related to the ACH site and the MSTSG site. The scales were measured to determine the rating on a scale of 0-100. In follow-up visits during postoperative week 1 and 2, elevated pain ratings were directly related to recipient site graft healing. In subsequent study follow-up visits, pain related to the ACH and MSTSG sites was rated similarly by all patients (Table 5). When comparing patients’ pain ratings related to the ACH site and the MSTSG site, the authors found no statistically significant difference between the rating of pain related to either site.

  At follow-up visits during postoperative weeks 12 and 52, patients were asked to rate the appearance of the ACH and MSTSG sites. As with the rating of their pain, a visual analog scale was utilized to document the patients’ opinions on the aesthetic appearance of their treatment sites. The scales were measured to determine the patients’ rating on a scale of 0-100. All patients with the exception of 1 rated the appearance of the ACH and MSTSG sites as being comparable to one another. Patient 8 in the series rated his ACH-treated site as having a better appearance than his MSTSG site (Table 6). The patients’ ratings of the appearance of the ACH and MSTSG sites were compared statistically and no statistical significance was found between the ratings.

  A scar assessment was also performed on both ACH and MSTSG sites at follow-up visits during postoperative weeks 12 and 52 using a scale with a total rating of 14. All scar assessments were performed by the same physical therapist at the burn center. The difference in MVSS score assessed at week 12 was found to be statistically significant, with the MSTSG site measuring at a lower MVSS score than the ACH site. At the week 52 visit, comparable MVSS scores were noted when comparing the ACH site and the MSTSG site with no statistical difference between measurements (Table 7). As expected, most MVSS scores improved from the week 12 visit to the week 52 visit.

  Wound closure after extensive burn wound injury can be an extremely challenging task. Early excision of burn wounds and prompt coverage is the mainstay of surgical treatment of extensive burn injuries.¹ Various options for coverage described in literature are autograft,¹¹ allograft,¹² combinations of autograft and allograft (eg, Sandwich technique),¹³ xenograft,¹⁴ human amniotic membrane,¹⁵ and synthetic skin substitutes.¹⁶ Cultured keratinocytes have been in clinical use for the more than 20 years, with the first application of CEA performed in 1981.⁹ The use of CEA has become one of the common methods in the treatment of severe burn wounds; however, a few disadvantages, such as difficulty in handling the fragile sheets, variable take rates, and 3-4 week growth time have led to recent investigation of different techniques to deliver keratinocytes to the wound bed.^10,11 The authors’ clinical practice group recently reported a large series of patients treated with cultured keratinocytes with standardization of protocols and good to excellent results.¹⁷ Despite the good results with CEA, it is necessary to speed up the time to definitive coverage and healing of the wound beds. Wounds that take longer to heal can increase the morbidity and mortality in patients. This leads to increased costs and utilization of resources, with significant impact on the patient, hospital, and the community at large.¹⁸ Dietch and colleagues¹⁸ have demonstrated the need to speed healing to avoid the formation of hypertrophic scars. They found that a burn wound that takes 21 days or more to heal has a 78% or greater risk of developing a significant scar. A burn wound that heals in less than 10 days has only a 4% risk of developing scar hypertrophy.¹⁸ The authors of the current study set out to evaluate the advantages or disadvantages of the ACH system vs MSTSG.

  The ACH system, first introduced in 2005, enables the surgeon to immediately process a small split-thickness biopsy and deliver keratinocytes, melanocytes, fibroblasts, and Langerhans cells harvested from the epidermal-dermal junction to the wound bed using a spray-on technique.¹⁹ Tissue collection, cell segregation, and preparation of the cell suspension takes approximately 20-30 minutes total, during which time the treatment area is prepared. Once processed, the cell suspension is available for immediate use and can cover a treatment area up to 80 times the area of the donor biopsy site.²⁰ Wood and colleagues²¹ characterized the cell suspension as having a 75% cell viability in which the cells retained their proliferative potential and included melanocytes which could help to restore pigmentation. The ACH system has been utilized in a wide variety of wounds; plastic, reconstructive, burn, and cosmetic procedures including burns and scalds; donor sites; glabrous injuries; mild to moderate scars; hypopigmentation (eg, hypopigmented scars, iatrogenic hypopigmentation, and vitiligo); large congenital melanotic nevus; and in aesthetic rejuvenation procedures.^22-27 Navarro et al²³ demonstrated increased epidermal thickness, confluence, keratin cysts, and blood vessels on histological analysis of full-thickness wounds treated with the ACH device as compared to those treated with culture medium in the control models. Wounds sprayed with the processed ACH suspension have demonstrated faster and better quality of epithelialization than control wounds.²⁸ Additionally, these cell suspensions have demonstrated viable and proliferating melanocytes which have the potential to treat hypopigmented lesions.²² Replacing like tissue with like tissue ensures optimized outcomes.

  The overall results between the ACH system and the MSTSG are comparable for graft take, color match, pigmentation, pain, patient-rated appearance, and scar quality. One advantage to this prospective study is the authors were able to use both MSTSG and the ACH system on the same patient and then make a comparison. This removes the variables that go along with comparing patient to patient, or even site to site, healing. The aesthetic results were noted to be similar between the MSTSG site and the ACH site. Gravante and colleagues,²⁹ who compared ACH vs classic skin grafts for the treatment of deep partial-thickness burns, also found no significant differences in the aesthetic quality of the healing wound. The study did find that postoperative pain was significantly less in the ACH group. The one advantage with the ACH system in the Gravante et al study was that the scar was devoid of a meshed pattern, which may play an important role in the patients’ self-awareness of his or her burn injury.

  Patient satisfaction was noted to be better with the ACH system due to the fact that the donor site is smaller and therefore less painful. The ACH system site has an expansion ratio of 1:80; therefore, even when grafting sites are available, it may be advantageous to minimize the area of the donor site to decrease pain, increase the ease of wound care, and decrease the risk of donor site infection.

  The ACH device has been used in patients worldwide to treat a variety of tissue injuries and can be used alone to treat partial-thickness injuries or in conjunction with dermal reconstruction technology to treat deep dermal injuries or full-thickness injuries.³⁰

  The ACH system does not change the surgical indications and/or principles of epidermal replacement in patients with burns. The authors convey that the series presented is their early experience with ACH and is insufficiently powered to show efficacy or noninferiority over MSTSG; however, this small 10-patient series presents an option to obtain epithelial coverage in a less invasive manner. It should be stressed that the use of keratinocytes for coverage of any wound requires a healthy dermal component (Figure 5). Currently, a large multicenter phase 2 clinical trial similar to this preliminary study is being conducted with the use of the ACH system device compared to MSTSG.

The authors are from Indiana University, Indianapolis, IN.

Address correspondence to:
David Edward Roggy
820 Eskenazi Ave
Indianapolis, IN 46202
davidroggy@gmail.com

Disclosure: This study was a phase II clinical trial sponsored by Avita Medical, Northridge, CA.

These findings were previously presented at the British Burns Association Annual Meeting; March 22-25, 2011; Salisbury, Wiltshire, UK.