In the treatment of severe burn injuries, the shortage of donor sites for split-thickness skin grafts is a significant problem and various methods of wound coverage have been investigated to overcome it.1 Cultured epidermal autografts (CEAs) have been considered as a significant milestone in the treatment of extensive burn wounds. However, the clinical use of CEA has limitations because of lower take rate, especially on an infected bed,2 and mechanical fragility and frequent spontaneous blistering, particularly at the early stages.3 To circumvent these problems, alternative methods of CEA application have been explored. A combination technique using widely expanded split-thickness autografts overlaid by the CEA is now recognized as a useful approach which contributes to prompt epithelialization and provides reliable stability of the resultant epithelium.4,5 As a consequence, definitive wound coverage can be achieved; however, a delay of 3 to 4 weeks required for the generation of a CEA from the patient's skin creates a fundamental clinical problem because it occurs during the life-threatening phase in patients with severe burns.
Meanwhile, a similar technique combining widely expanded split-thickness autografts and allogeneic cultured epidermis (allo-CE) has been reported.6 Although allo-CE is not expected to survive on the wound for a long time after application, it can release a number of growth factors that stimulate the activity of patient’s cells at the application site, promoting wound healing.7,8 In large third-degree burn wounds, all epithelial stem cells are damaged and epithelialization cannot be achieved with allo-CE alone; however, combined with widely expanded split-thickness autografts, allo-CE can enhance reepithelialization in the interstices of the meshed graft.6 Allo-CE can be prepared in advance and cryopreserved so that it is ready to use for the treatment of severely burned patients in the acute phase while CEA is being prepared. Therefore, allo-CE can improve burn wound treatment in the acute phase, making up for the shortcomings of CEA.
Nevertheless, the effectiveness of the method based on the combined application of allo-CE and meshed autogenous skin grafts has not been yet established either in an appropriate controlled study or in an animal model.
In this study, we evaluated the effect of human (h)CE used in combination with a meshed skin graft on wound healing in a xenograft model. We used immunocompetent rats and no immunosuppressive agent to evaluate the wound healing effect of hCE in the condition which would be immunologically rejected similar to the clinical situation. Therefore, it is reasonable to extend the results obtained with a xenograft model in this study to an allogeneic transplantation in clinical use. First, hCE was assessed for the secretion of growth factors in vitro, and then applied with widely meshed (6:1) skin grafts on wounds inflicted on F344 rats. Our results indicate that hCE accelerated wound closure, and induced granulated tissue formation and neovascularization, suggesting the enhancement of the wound healing effect of widely expanded autografts.
MATERIALS AND METHODS
Preparation of hCE
Human cultured epidermis was prepared by Japan Tissue Engineering Co., Ltd. using Green’s method described previously9,10 with some modifications. Briefly, cryopreserved keratinocytes cultured from human neonatal foreskin (C-001-5C; Life Technologies Corporation, Tokyo, Japan) were thawed and disseminated on irradiated 3T3-J2 cells used as a feeder layer. Keratinocytes were cultured in Dulbecco Modified Eagle Medium and Ham’s F12 medium mixed 3:1 and supplemented with 5% fetal calf serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, epidermal growth factor, and antibiotics in an atmosphere of 10% CO2 at 37°C.
To distinguish human and rat keratinocytes on histological sections, the former were labeled with nontoxic lipophilic fluorescent dye PKH26 (Sigma-Aldrich Japan, Tokyo, Japan) according to the manufacturer's protocol. PKH26 is a nontoxic lipophilic fluorescent dye (emission at 567 nm) that stains the membranes of viable cells and is distributed between cells at mitosis; it has a half-life of over 100 days.11–13 For PKH26 staining, keratinocytes cultured for 2 passages were washed with serum-free medium and treated with 4 mL of 2 μM PKH26 solution for 4 minutes at room temperature. Then, the cells were rinsed to remove the dye and further cultured with fresh medium until the next day. Human cultured epidermis was obtained as keratinocyte sheets which were detached after treatment with dispase and aseptically packaged for transportation.
Growth Factor Release By hCE
The secretion of growth factors by hCE sheets into culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, after 2 passages, human keratinocytes were cultured with 30 mL of conditioned medium in a T150-flask for 24 hours and the medium was then harvested and stored at −80°C.
After thawing, the samples were centrifuged at 18,800g for 10 minutes to remove cell debris and analyzed for the release of growth factors. Basic fibroblast growth factor (bFGF), platelet-derived growth factor-AA (PDGF-AA), TGF-α, TGF-β1, and keratinocyte growth factor (KGF) were measured using the Quantikine ELISA kit (R&D Systems, Minneapolis, Minn), and interleukin-1α (IL-1α), IL-1β, and vascular endothelial growth factor (VEGF) were measured using the Invitrogen ELISA kit (Invitrogen Corp., Camarillo, Calif) according to the manufacturers' instructions. The results were expressed as the amount of a growth factor released by 1 hCE sheet after 24-hour incubation compared with the fresh medium of the same composition as control.
Because irradiated 3T3 feeder cells used for keratinocyte culture may also release growth factors, confluent lethally irradiated 3T3 cells were incubated as above without human keratinocytes for 24 hours and their conditioned medium was analyzed by ELISA as described.
F344 8-week-old male (CLEA, Japan) rats were maintained at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. The number of animals used in this study was kept to a minimum, and all possible efforts were made to reduce suffering in compliance with the protocols established by the Animal Research Committee of Kyoto University. Our experimental protocol was approved by the Animal Research Committee (Permit Number: Med Kyo 14570).
Combination Therapy Using hCE With Meshed Skin Grafting
A total of 16 inbred rats were acclimatized in individual cages for 1 week before treatment. They were randomly assigned to the control group and the hCE group according to their body weight. After intraperitoneal injection of sodium pentobarbital (30 mg/kg, Somnopentyl; Kyoritsu Seiyaku Corporation, Tokyo, Japan), the entire dorsum of the animals was clipped and depilated with a depilation cream. General anesthesia (inhalation of 1.5% isoflurane; Wako Pure Chemical Industries Ltd., Osaka, Japan) was also applied when needed.
A 3 × 3-cm full-thickness skin defect was created on the dorsum of each rat. We resected the dorsum skin with a scalpel and scissors leaving pannicles carnosus to prepare a full-thickness skin defect. A piece of split-thickness skin (0.4-mm thick) was harvested from the resected skin using a Padgett drum dermatome (KD-110; Keisei Medical Industrial Co., Ltd., Tokyo, Japan) and expanded at a ratio of 6:1 to prepare meshed skin grafts using a skin graft mesher (MD-11; Keisei Medical Industrial Co., Ltd.). The meshed skin graft was returned to the skin defect area and carefully attached at 8 points at the edges of the area with 5-0 nylon suture to produce the graft of a uniform shape and size on each rat.
After grafting, the wounds in the control group (n = 8) were covered with polyethylene films containing absorbent cotton (Derma-Aid; ALCARE Co., Ltd., Tokyo, Japan), whereas in the hCE group (n = 8), hCE sheets were applied onto the meshed skin grafts and covered with the same dressing (Fig. 1). Then, the site was secured with a surgical bandage (Silkytex; ALCARE Co., Ltd.).
Seven days later, the animals were sacrificed by CO2 inhalation and photographed with a digital camera. Skin specimens were harvested, fixed in 10% neutral-buffered formalin solution, and cut at the center of each wound to obtain cranial and caudal halves. The cranial part was embedded in O.C.T. compound (Tissue-Tek; Sakura Finetek USA, Inc., Calif) and frozen in ethanol-dry ice, whereas the caudal part was embedded in paraffin to prepare 10-μm frozen sections and 6-μm paraffin sections, respectively. The paraffin-embedded sections were then stained with hematoxylin-eosin (HE) and azocarmine and aniline blue (AZAN).
Assessment of the Wound Area
The area where epithelialization was not observed macroscopically in the interstices of the meshed skin graft at day 7 was measured in the photographs using the ImageJ software program, ver. 1.45 (NIH, Bethesda, MD, USA). The results were used to calculate the total nonepithelialized area in each specimen.
Assessment of the Remaining hCE
To confirm the fluorescence of PKH26-labeled hCE before the application to the wound, a piece of hCE was embedded in O.C.T. compound and frozen in ethanol-dry ice at the time of operation (day 0). Frozen sections of hCE alone (day 0) and of wound specimens from the hCE group (day 7) were observed under a fluorescence microscope (KEYENCE BZ-9000 and BZ-II Analyzer ver. 1.42; KEYENCE Japan, Osaka, Japan) using a tetramethylrhodamine isothiocyanate filter. For the comparison, the sections were stained with HE and observed under an optical microscope.
Histological Assessment of Neoepithelium Growth
The length of the neoepithelium was measured starting from the edge of the meshed graft on HE-stained sections under an optical microscope (KEYENCE BZ-9000 and BZ-II Analyzer ver. 1.42). The measurements were performed on both sides of the two strips of grafted skin in the inner part of the wound (4 points in each section), and the total sum was used for the analysis.
Granulation Tissue Formation
To assess the formation of regenerated tissue, the thickness of granulation tissue newly formed in the interstices of the meshed skin graft was evaluated using AZAN-stained sections. In each section, there were 3 areas of granulation tissue; therefore, we measured the distance between the wound surface (excluding the epithelium) and the deep fascia at the center of each granulation area and calculated the mean value.
Immunohistochemical Staining and Evaluation of Newly Formed Capillaries
Immunohistochemical staining with von Willebrand factor (vWF) was used to detect newly formed capillaries. For this, 6-μm sections were dewaxed, rehydrated, and incubated with proteinase K (S3020; Dako Japan, Tokyo, Japan) for 5 minutes at room temperature for antigen retrieval. Anti-vWF rabbit polyclonal antibodies (1:5000, Code No. A0082; Dako) were used as the primary antibody followed by the secondary antibody (K4003, EnVision; Dako). The staining was visualized using 3-3’-diaminobenzidine-4HCl (DAB, Code 725191; Nichirei Biosciences Inc., Tokyo, Japan); the sections were then counterstained with hematoxylin and micrographs were taken under an optical microscope.
In each section, a rectangle of 500 × 300 μm was selected at the center of the granulation area beyond the muscle layer, and the number of newly formed capillaries in the rectangle and the cross-sectioned area of neovascularization were measured using the BZ-II Analyzer imaging software program (version 1.42; KEYENCE Co.).
Measurements and Statistical Analysis
All measurements in this study were performed by 3 plastic surgeons blinded to the group allocation. All data were expressed as the mean ± SD, and statistical significance of the difference was evaluated by Student t test. A P value less than 0.05 was considered statistically significant.
Growth Factor Release by hCE
The secretion of growth factors (bFGF, IL-1α, IL-1β, IL-6, PDGF-AA, TGF-α, TGF- β1, VEGF, and KGF) by hCE is shown in Figure 2.
The conditioned medium of human keratinocytes contained bFGF, IL-1α, PDGF-AA, TGF-α, TGF-β1, and VEGF, whereas IL-1β, IL-6, and KGF were not detected by ELISA (the detection sensitivity was 1.0, 0.7, and 15 pg/mL, respectively, which corresponded to 0.03, 0.02, and 0.45 ng/24 hours per sheet, respectively). The highest secretion was observed for VEGF.
The release of growth factors could not be attributed to 3T3 feeder cells which secreted only TGF-β1 (3.3 ng/24 hours per flask), whereas none of the other growth factors were detected.
Figure 3A shows representative macroscopic images of the wounds 7 days after grafting. The results indicate that the wound area in the hCE group was significantly smaller than that in the control group (P = 0.003; Fig. 3B), suggesting the enhancement of the healing effect by hCE.
Identification of the Epithelium
Fluorescent microphotographs of hCE before application (day 0) and of the wound in the hCE group on day 7 are shown in Figure 4. Fluorescence could be clearly observed in the hCE sheet labeled with PKH26 dye (day 0; Figs. 4A, B). However, no fluorescence was detected in the wound tissue on day 7 postgrafting (Figs. 4C and D), indicating the absence of labeled human keratinocytes in the epithelium covering the wound surface.
Histopathological evaluation of the HE-stained sections of the wound areas at day 7 postgrafting revealed that a separate sheet-shaped structure which would be seen in case of low hCE integration was not observed on the wound surface in the hCE group (Fig. 5A). In the control group, a considerable portion of the graft did not take and some epithelium of the skin graft was necrotized and detached. The neoepithelium in the hCE group was significantly longer than that observed in the control group (P = 0.019; Fig. 5B), indicating that hCE promoted epithelialization in the grafted wound.
Granulation Tissue Thickness
Granulation tissue development in the control and hCE-treated wounds was analyzed in the AZAN-stained sections which showed that the formation of new connective tissue was intensified in the hCE group (Fig. 6A). Granulation tissue was generated on the exposed panniculus carnosus in the interstices of the meshed skin grafts, and its thickness in the hCE-covered wounds was significantly greater than that observed in the control group (P = 0.043; Fig. 6B).
The formation of new blood vessels in the healing wound areas was evaluated by immunostaining with vWF on day 7 posttreatment (Fig. 7A). The number of new capillaries and gross area of neovascularization were significantly greater in the hCE group compared with the control group (P = 0.0003 and 0.008, respectively; Figs. 7B, C), which is consistent with the enhanced formation of the connective tissue in the wound (Fig. 6).
The results of this study indicate that hCE released a number of growth factors and accelerated wound healing in combination with extended meshed grafts.
It has been established that hCE promotes wound healing when used as allografts in deep dermal burns,7,14–17 donor sites,18–21 and chronic ulcers.22–24 The beneficial effect can be attributed to the production of basement membrane proteins (collagen types IV–VII, laminin, and fibronectin) which enhance cell migration, creating a favorable environment for the ingrowth of keratinocytes from the wound bed and edges.14,16 Moreover, keratinocytes in the epidermis can release a number of growth factors,8,25,26 such as bFGF, IL-1α, IL-1β, IL-6, PDGF-AA, TGF-α, TGF-β1, and VEGF27,28; however, there has been no quantitative evaluation of growth factor secretion by keratinocyte sheets cultured on 3T3 feeder cells.
As it is difficult to assess growth factor release by hCE after its application to the wound surface, we performed in vitro analysis of keratinocyte conditioned medium before hCE grafting and detected bFGF, IL-1α, PDGF-AA, TGF-α, TGF-β1, and VEGF. As growth factors have a pivotal role in cell proliferation, differentiation, and metabolism during wound healing stages,13 these results suggest that the released growth factors could account for one of the reasons of the accelerated wound healing with hCE. Besides, keratinocytes interact with fibroblasts in the dermal tissue via a paracrine loop,29 suggesting that hCE-released growth factors may promote intercellular cooperation during wound closure.
The 3T3 feeder cells alone also produced TGF-β1. However, the feeder cells were detached and almost completely removed after human keratinocyte proliferation and the formation of confluent cell sheets; therefore, the contribution of 3T3 cells to the amount of growth factors in hCE conditioned medium was negligible.
Although animal models of meshed skin grafted wounds have been reported,30–33 the variability in size and shape of used skin grafts made them unsuitable for the accurate evaluation of the wound healing process. In this study, we established a standardized model by using uniform skin grafts of identical thickness and shape, wounds of fixed size and location, and raw surfaces in the interstices of controlled size. In this model, the wound area was gradually reduced by epithelialization and wound contraction, and completely healed in 2 weeks, so we could not find any differences between the two groups at that time in a preliminary examination. Therefore, we evaluated wound healing on day 7 postgrafting and found that hCE accelerated epithelialization and granulation tissue formation.
Because of its flexibility, hCE tightly adheres to the uneven surface of the wound and coats the exposed cells and extracellular matrix immediately after application. The interspaces in the expanded graft are covered by the hCE sheet and are filled with plasma from the wound surface. Furthermore, as hCE keratinocytes survive on the wound for some time, they secrete growth factors and basement membrane proteins, creating an ideal environment for cell migration and proliferation. Importantly, hCE produced VEGF which, as a factor critical for angiogenesis in the wound bed, should stimulate the formation of granulation tissue.
However, the absence of PKH26-labeled hCE keratinocytes on day 7 indicates that hCE did not take to the rat wound and may have been detached and lost during sampling, possibly because our xenograft model was based on immunocompetent rats.
Previous studies have also used immunocompetent animals to study hCE effects on wound healing. Thus, it has been shown that the frozen hCE accelerates re-epithelialization and promotes early formation of granulation tissue in a full-thickness skin defect model based on immunocompetent mice.34 Moreover, cultured human keratinocytes enhanced epidermal wound healing in a skin defect model based on pigs.35 Our results are consistent with these data, suggesting that cultured human keratinocytes can exert beneficial effects on the wound healing process in different species by secreting growth factors and other biologically active proteins.
In clinical settings, the approach based on the combination of the allo-CE and meshed autogenous grafts can be very effective in treating patients with severe burns. The beneficial wound healing effect of the combination technique of the allo-CE and autografts may achieve a comparable result to that of the combination method of the CEA and autografts. This combination method of the allo-CE and autografts that can be performed while the CEA is being prepared may promote wound healing and reduce the mortality of severe burn patients.
Our results indicate that hCE produced growth factors (bFGF, IL-1α, PDGF-AA, TGF-α, TGF-β1, and VEGF) and significantly reduced wound area by accelerating granulation tissue formation and angiogenesis if combined with the meshed skin graft in rats. The beneficial effect of hCE in an experimental model suggests that hCE can be potentially used to promote the grafting of widely expanded meshed autografts during the treatment of severe burns. Future studies are required to investigate the destiny of the transplanted keratinocytes when used as allograft.
1. Lumenta DB, Kamolz L, Keck M, et al. Comparison of meshed versus MEEK micrografted skin expansion rate: claimed, achieved, and polled. Plast Reconstr Surg
. 2011;128:40e–41e. doi:10.1097/PRS.0b013e318217463a.
2. Lootens L, Brusselaers N, Beele H, et al. Keratinocytes in the treatment of severe burn injury: an update. Int Wound J
. 2013;10:6–12. doi:10.1111/j.1742-481X.2012.01083.x.
3. Clugston PA, Snelling CF, Macdonald IB, et al. Cultured epithelial autografts: three years of clinical experience with eighteen patients. J Burn Care Rehabil
4. Braye F, Oddou L, Bertin-Maghit M, et al. Widely meshed autograft associated with cultured autologous epithelium for the treatment of major burns in children: report of 12 cases. Eur J Pediatr Surg
. 2000;10:35–40. doi:10.1055/s-2008-1072320.
5. Matsumura H, Matsushima A, Ueyama M, et al. Application of the cultured epidermal autograft “JACE®” for treatment of severe burns: Results of a 6-year multicenter surveillance in Japan. Burns
. 2016;42:769–76. doi:10.1016/j.burns.2016.01.019.
6. Monstrey S, Beele H, Kettler M, et al. Allogeneic cultured keratinocytes vs. cadaveric skin to cover wide-mesh autogenous split-thickness skin grafts. Ann Plast Surg
7. Hefton JM, Madden MR, Finkelstein JL, et al. Grafting of burn patients with allografts of cultured epidermal cells. Lancet
. 1983;2:428–430. doi:10.1016/S0140-6736(83)90392-6.
8. Brain A, Purkis P, Coates P, et al. Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome. BMJ
9. Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci U S A
10. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell
11. Horan PK, Slezak SE. Stable cell membrane labelling. Nature
. 1989;340:167–168. doi:10.1038/340167a0.
12. Horan PK, Melnicoff MJ, Jensen BD, et al. Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol
13. Morimoto N, Saso Y, Tomihata K, et al. Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation. J Surg Res
. 2005;125:56–67. doi:10.1016/j.jss.2004.11.012.
14. Madden MR, Finkelstein JL, Staiano-Coico L, et al. Grafting of cultured allogeneic epidermis on second- and third-degree burn wounds on 26 patients. J Trauma
. 1986;26:955–962. doi:10.1097/00005373-198611000-00001.
15. De Luca M, Albanese E, Bondanza S, et al. Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state. Burns
16. Van der Merwe AE, Mattheyse FJ, Bedford M, et al. Allografted keratinocytes used to accelerate the treatment of burn wounds are replaced by recipient cells. Burns
17. De Luca M, Bondanza S, Cancedda R, et al. Permanent coverage of full skin thickness burns with autologous cultured epidermis
and reepithelialization of partial skin thickness lesions induced by allogeneic cultured epidermis
: a multicentre study in the treatment of children. Burns
. 1992;18(Suppl 1):S16–S19. doi:10.1016/0305-4179(92)90105-4.
18. Teepe RG, Koch R, Haeseker B. Randomized trial comparing cryopreserved cultured epidermal allografts with tulle-gras in the treatment of split-thickness skin graft donor sites. J Trauma
. 1993;35:850–854. doi:10.1097/00005373-199312000-00008.
19. Phillips TJ, Provan A, Colbert D, et al. A randomized single-blind controlled study of cultured epidermal allografts in the treatment of split-thickness skin graft donor sites. Arch Dermatol
20. Rivas-Torres MT, Amato D, Arámbula-Alvarez H, et al. Controlled clinical study of skin donor sites and deep partial-thickness burns treated with cultured epidermal allografts. Plast Reconstr Surg
21. Duinslaeger LA, Verbeken G, Vanhalle S, et al. Cultured allogeneic keratinocyte sheets accelerate healing compared to Op-site treatment of donor sites in burns. J Burn Care Rehabil
. 1997;18:545–551. doi:10.1097/00004630-199711000-00013.
22. Beele H, Naeyaert JM, Goeteyn M, et al. Repeated cultured epidermal allografts in the treatment of chronic leg ulcers of various origins. Dermatologica
23. Lindgren C, Marcusson JA, Toftgård R. Treatment of venous leg ulcers with cryopreserved cultured allogeneic keratinocytes: a prospective open controlled study. Br J Dermatol
24. Leigh IM, Purkis PE, Navsaria HA, et al. Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes. Br J Dermatol
25. Katz AB, Taichman LB. Epidermis as a secretory tissue: an in vitro tissue model to study keratinocyte secretion. J Invest Dermatol
26. Coffey RJ, Derynck R, Wilcox JN, et al. Production and auto-induction of transforming growth factor
-alpha in human keratinocytes. Nature
. 1987;328:817–820. doi:10.1038/328817a0.
27. Myers S, Navsaria H, Sanders R, et al. Transplantation of keratinocytes in the treatment of wounds. Am J Surg
. 1995;170:75–83. doi:10.1016/S0002-9610(99)80258-X.
28. Maas-Szabowski N, Shimotoyodome A, Fusenig NE. Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism. J Cell Sci
. 1999;112(Pt 12):1843–1853.
29. Menon SN, Flegg JA, McCue SW, et al. Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing
processes. Proc Biol Sci
. 2012;279:3329–3338. doi:10.1098/rspb.2012.0319.
30. Chu CS, Matylevitch NP, McManus AT, et al. Accelerated healing with a mesh autograft/allodermal composite skin graft treated with silver nylon dressings with and without direct current in rats. J Trauma
31. Chu CS, McManus AT, Matylevich NP, et al. Integra as a dermal replacement in a meshed composite skin graft in a rat model
: a one-step operative procedure. J Trauma
32. Thilagar S, Jothi NA, Omar AR, et al. Effect of keratin-gelatin and bFGF-gelatin composite film as a sandwich layer for full-thickness skin mesh graft in experimental dogs. J Biomed Mater Res B Appl Biomater
. 2009;88:12–16. doi:10.1002/jbm.b.31024.
33. Branski LK, Mittermayr R, Herndon DN, et al. Fibrin sealant improves graft adherence in a porcine full-thickness burn wound model. Burns
. 2011;37:1360–1366. doi:10.1016/j.burns.2009.08.011.
34. Tamariz E, Marsch-Moreno M, Castro-Muñozledo F, et al. Frozen cultured sheets of human epidermal keratinocytes enhance healing of full-thickness wounds in mice. Cell Tissue Res
35. Svensjö T, Yao F, Pomahac B, et al. Autologous keratinocyte suspensions accelerate epidermal wound healing
in pigs. J Surg Res
. 2001;99:211–221. doi:10.1006/jsre.2001.6197.