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Promotion of oral surgical wound healing using autologous mucosal cell sheets
Oral Oncology 69 (2017) 84–91
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Promotion of oral surgical wound healing using autologous mucosal cell sheets Jong-Lyel Roh ⇑, Hyejin Jang, Jaewang Lee, Eun Hye Kim, Daiha Shin Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
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Article history: Received 3 January 2017 Received in revised form 11 March 2017 Accepted 19 April 2017
Keywords: Cell sheet Mucosa Fibrin glue Oral wound Graft
a b s t r a c t Objectives: Severe oral mucosal and tissue defects can lead to pain, infection, and later undesirable healing of scarring and adhesion, resulting in a poor quality of life. In vitro-engineered oral mucosal equivalents for covering such defects are an alternative to avoiding the donor site morbidity of conventional skin or tissue grafts. We examined the efficacy of our newly developed three-dimensional mucosal cell sheets in an in vivo tongue wound model mimicking the surgical extirpation of tongue cancer. Materials and methods: Small oral mucosal and autologous fibrin samples were obtained from surgical patients and Sprague-Dawley rats. The fibrin was mixed with fibroblasts and seeded with keratinocytes that had been primarily cultured for in vitro cell expansion. The three-dimensional autologous cell sheets, cultured in air-lift interface inserts, were transplanted into deep wounds of the rat ventral tongue. Gross and microscopic findings of the postsurgical wounds were compared between wound control and cell sheet groups. Results: The cell sheets were flexible, expandable, and easy to transfer, and had histological characteristics similar to that of the normal oral mucosa, with high p63 positivity. They promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in the wound control. The cell sheet-healed tongue had similar histology to that of a normal tongue. Conclusions: Our engineered cell sheets have potential applicability for the rapid healing of oral mucosal and soft tissue defects, without scarring, adhesion, and functional deficits. Condensed abstract: The efficacy of in vitro-engineered mucosal equivalents, using completely autologous mucosa and plasma, was examined. Transplantation of the autologous cell sheets into deep wounds of the rat ventral tongue promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in controls. Healed and normal tongues showed similar histology. Ó 2017 Published by Elsevier Ltd.
Introduction Oral mucosal and soft tissue defects can be caused by surgical extirpation of intraoral pathological lesions, trauma, recurrent ulcers, and irradiation. If not properly treated, the defects lead to pain, infection, and later undesirable healing, such as scarring and adhesion to adjacent tissues. This might cause significant deficits of functions that are essential for daily life, such as deglutition, articulation, and speech, as well as respiration. To prevent undesirable discomfort and sequelae, a skin graft, local or regional flap, or microvascular free flap is currently used to restore the mucosal lining or soft tissue defects in the oral cavity. The procedures com-
⇑ Corresponding author at: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea. E-mail address: [email protected] (J.-L. Roh). http://dx.doi.org/10.1016/j.oraloncology.2017.04.012 1368-8375/Ó 2017 Published by Elsevier Ltd.
monly require harvesting skin or tissues from the same patients, which might result in morbidity and leave aesthetically unacceptable scarring in the donor sites. The current surgical approach for filling a large soft tissue defect is microvascular free flap transfer, which commonly requires a considerable operation time and experienced surgical hands. A superficial intraoral defect without considerable soft tissue deficits can be covered with a split thickness skin graft. In vitro-cultured cell-based regenerative approaches have been introduced as feasible alternative methods to restoring the mucosal lining and tissue defects [1–3]. Epithelial cell sheets prepared from the autografts or allografts of epidermal cells have been applied to the grafting of burns [4–6]. In addition, it has been reported in the literature that cell sheets or equivalents from human mucosa can be in vitro-engineered for potential application to the closure of oral wounds [7–9]. Oral mucosal equivalents consisting of human lamina propria fibroblasts and oral epithelial cells have been shown to mimic the normal
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oral mucosa, with similar histological and immunohistochemical marker expression levels [8]. Furthermore, in vitro-produced oral mucosal equivalents were introduced for intraoral grafting after their production using scaffolds of acellular dermis [7], amniotic membrane [10], and collagen glue [11], etc. These were not autologous, but obtained from animals or cadavers and required pretreatments for eventual immunorejection of the equivalents. Human plasma is an abundant source of autologous fibrin that might be an optimal scaffold for producing skin and mucosal epithelial equivalents [12,13]. Lamina propria fibroblasts can be easily cultured in vitro, mixed in the autologous fibrin glue, and then used to facilitate the growth of keratinocytes seeded on the mixture, without potential immunorejection [13]. Therefore, we developed an in vitro-engineered autologous mucosal cell sheet consisting of keratinocytes and of plasma fibrin containing lamina propria fibroblasts. The keratinocytes and fibroblasts were obtained by in vitro culture using animal-product-free and completely autologous materials. Herein, this study examined the potential efficacy of our newly developed three-dimensional oral mucosal cell sheet in an in vivo tongue wound model mimicking the clinical setting of surgical extirpation of tongue cancer. Methods In vitro culture of mucosal samples A small piece of mucosa was sampled from the normal mucosa adjacent to the resection margins in patients who underwent transoral surgery. This work was approved by the Institutional Review Board of our hospital, and informed consent was obtained from each patient. Plasma was also obtained from the 25 mL blood samples drawn from the same patients using a vacutainer tube (BD Bioscience, Franklin Lakes, NJ, USA) at the time of surgery. The mucosae were sterilized with povidone-iodine solution (SigmaAldrich, St. Louise, MO, USA) and washed 3 times in phosphatebuffered saline solution. All tissues were treated with 1 U/mL dispase (STEMCELL Technologies, Vancouver, BC, Canada) for 2 h at 37 °C, and the epithelial and subepithelial layers were then separated. Both layers were separately treated with trypsin-ethylene diaminetetraacetic acid (ThermoFisher Scientific, Waltham, MA, USA) for 15 min at 37 °C. The cells were separately seeded in culture dishes and grown in the culture medium, which was a 3:1 mixture of Dulbecco’s modified Eagle’s minimal essential medium and Ham’s F12 (ThermoFisher), containing 10% heat-inactivated autologous serum, human recombinant insulin (5 mg/mL), triiodothyronine (1.3 ng/mL), adenine (24 mg/mL), hydrocortisone (0.4 mg/mL), and cholera toxin (8 ng/mL) (all purchased from Sigma-Aldrich), and supplemented with a penicillin-streptomy cin-amphotericin antibiotic-antimycotic solution (ThermoFisher). Human recombinant epidermal growth factor (10 ng/mL; ThermoFisher) was also added to the medium for culturing mucosal keratinocytes. The medium and supplements were replaced at every 3 days. Generation of in vitro-engineered mucosal cell sheet The plasma obtained from each patient’s blood was used to make fibrin glue as the source of the scaffolds. The fibrin glue was composed of a mixture of 0.5 mL of plasma, 1% calcium chloride, 70 mL of tranexamic acid (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and 0.5 mL of medium with 5 105 fibroblasts. The mixture was allowed to solidify in transwell cell culture inserts with a 0.4-mm pore-size polyester membrane (Corning, Inc., Corning, NY, USA) at 37 °C for 60 min. The inserts were placed in the plates with the medium and supplements. Keratinocytes were
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seeded on the mixture of fibrin glue and fibroblasts, and grown under air-liquid interface culture conditions, with the same medium, autologous serum, and supplements as mentioned above. The in vitro culturing of keratinocytes and fibroblasts was also performed using biopsy samples of the oral buccal mucosa from Sprague-Dawley (SD) rats. Blood was also obtained from each rat and used as a source of autologous fibrin glue. The autologous mucosal cell sheet was produced in the same way as described above. About 10–14 days after cell culturing, the fibrin glue and submucosal fibroblasts were mixed, poured, and solidified in the insert well, and keratinocytes were then seeded onto the mixture. Five to 7 days later, the mucosal cell sheets were constructed and prepared for transplantation. Histological examinations of the mucosal cell sheets The in vitro-cultured mucosal cell sheet or tissue samples were harvested, embedded in optimal cutting temperature compound (Sakura Fineteck USA Inc., Torrance, CA, USA), immediately snapfrozen in liquid nitrogen, and stored at –80 °C for subsequent use. The stored sheets or tissue samples were prepared as 5-mmthick frozen sections for histological examination. The sections were stained with hematoxylin and eosin (Sigma-Aldrich). The samples were also examined immunohistochemically for the staining of p63 (1:200; GeneTex, Inc., Irvine, CA, USA), pancytokeratin AE1/AE3 (1:200 dilution, Dako, Glostrup, Denmark), cytokeratin 5/6 (1:200, Dako), and ki-67 (1:200, GeneTex) according to established protocols and examined under a fluorescence microscope (Olympus, Tokyo, Japan). Intraoral grafting of the mucosal cell sheet into a surgically wounded rat tongue All animal study procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of our institution. Male SD rats, weighing 180– 220 g, were purchased from Central Lab Animal Inc. (OO). The animals were anesthetized by intramuscular injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Iris scissors were used to remove the mucosa and submucosal soft tissues of the ventral tongue in each SD rat to create a 50-mm2 deep wound (Fig. 1). The autologous mucosal cell sheet was detached from the culture dish and trimmed to a similar shape and size as the surgical defect. The cell sheet was then overlaid with a silastic sheet (0.01000 thickness; Bentec Medical, Woodland, CA, USA) and grafted onto the defect by use of 5-0 absorbable vicryl sutures. For the wound control group, a silastic sheet without the cultured mucosal sheet was attached to the wound site. All silastic sheets were removed at 3 days after grafting. Each experimental group included 25 rats. Normal tongues were also obtained from 5 unwounded rats. The body weight and food intake of each group were measured at every 3 days. Gross and microscopic examinations of the postsurgical wounds Gross photographs of each rat wound were taken regularly. The wound size at each postoperative day was also measured and compared with that of the original wound in each rat, and comparisons were also made between the wound control and cell sheet groups. Five rats of each group were sacrificed at postsurgical days 3, 7, 14, 21, and 28, and the tissues from the previous oral wounds were harvested and quickly frozen at 80 °C. Thereafter, 5-mm-thick sections were stained with hematoxylin and eosin and Masson’s trichrome stains (Sigma-Aldrich) and observed under a microscope (Nikon Co., Tokyo, Japan). The epithelial and subepithelial thicknesses and collagen density were measured in a blind manner in
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Fig. 1. Transplantation of the autologous mucosal cell sheet after tongue wounding. (A) Deep surgical wounding of the ventral tongue in a Sprague-Dawley rat, as a model mimicking a surgical defect after the extirpation of human tongue cancer. (B and C) Attachment of the in vitro-engineered mucosal cell sheet (asterisks) on the surgical defect. A piece of silastic sheet (arrows) was used to prevent the detachment or injury of the cell sheet after transplantation in the experimental animals.
10 randomly selected fields, using the ImageJ processing program (National Institutes of Health, Bethesda, MD, USA). Statistical analysis The data are presented as the mean ± standard deviation. The statistical significance of the differences among different treatment groups was assessed using the Mann–Whitney U test, and that among different periods in each group were assessed using the Wilcoxon signed-rank test. All tests were two-sided and a value of P < 0.05 was considered as being significant. Statistical analyses were performed using IBM SPSS software version 23.0 (IBM, Armonk, NY). Results Characteristics of the in vitro-engineered mucosal cell sheet Keratinocyte and fibroblast colonies were found in the separately cultured dishes of all oral mucosal samples at 2–4 days after harvesting and culturing. The cells grew to >80% confluency within 7–14 days. Most of the cultured human keratinocytes and fibroblasts were viable, with 80.3 ± 5.2% and 82.2 ± 4.7%, respectively, when examined with vital dye prior to harvesting for generation of the mucosal cell sheet. Fibroblasts from the submucosal layers grew more quickly in culture than did keratinocytes from the mucosal layer. Bacterial and fungal contamination was not found in any of the cultures from all samples. The in vitro culture was also successfully performed from biopsy samples harvested from the buccal mucosa of SD rats. The viability and colony-forming ability of the cells were similar to those of the human samples. Three or more cell sheets of 5-cm2 size from each sample were generated within 2–3 weeks from the time point of mucosal sampling. The three-dimensional cell sheets were flexible enough to be easily stretched up to double their size without tearing or injury. The sheets were also easily detached, using fine-tip forceps and without the need for an enzymatic detachment solution, for transfer to other dishes and to cut for grafting (Fig. 2). Histological examination of the mucosal cell sheets revealed their similarity to the histological characteristics of the oral mucosa (Fig. 3). The mucosal layer of the cell sheet included 2–4 layers of cuboidal-shaped nucleated epithelial cells interspersed with elongated cells. The submucosal layer of the cell sheet included fibroblasts and fibrin embedded in the scaffold, similar to the oral submucosal structures of the extracellular matrix and fibroblasts, except for the interspersed vessels. However, the cell sheet had no evidence of the rete ridges between epithelial and
subepithelial layers observed in normal oral mucosa. The cultured keratinocytes and the epithelial layers of the cell sheet were enriched with p63-positive cells. In addition, immunohistochemical analyses showed that most cells in epithelial layers of cell sheets were positive for cytokeratin 5/6 and AE1/AE2 pancytokeratin, and were negative for ki67 (data not shown). Promotion of oral wound healing using the in vitro-engineered mucosal cell sheet The deep wounds made in the ventral tongue of SD rats were covered with (or without, as the wound control) mucosal cell sheets. Gross photographs taken regularly after the surgery showed earlier closure of the wound defects in the mucosal cell sheet group than that in the wound control group (P < 0.05, Figs. 4 and 5). The wound control had substantial inflammatory reactions and sloughs covering the surgical defect on the ventral tongue surface during the early stage after wounding. In addition, the anterior to posterior length of the tongue was shortened, probably by the devascularization and mutilation of the tongue tip of the injured rats. On the other hand, the viable mucosal cell sheet covering the surgical defect in the cell sheet group promoted wound healing and prevented further injury of the mucosa-deficient tongue. Reepithelialization was completed within 3 weeks in both the wound control and cell sheet groups, but was more rapid in the latter group (P < 0.05, Fig. 5). Microscopic evidence of accelerated wounding healing by mucosal cell sheet grafting Microscopic examinations of the surgical wounds according to postsurgical days supported the observed gross findings and differences between the wound control and cell sheet groups (Fig. 6). Sloughs were found in the denuded mucosal surface of the wound control rats, whereas the surgical defect was covered with the mucosal sheet in the cell sheet group. Inflammatory reactions and muscular disruption of the injured submucosa were also more observed in the wound control than the cell sheet group during the early stage of wound healing (P < 0.05). At the late stage, the mucosal and submucosal compositions of the cell sheet group appeared to be more similar to those of the unwounded normal rat tongue, whereas those of the wound control rats were more thickened and fibrotic (P < 0.05). The characteristics of the healed mucosa and submucosa were evaluated at postsurgical days 14 and 28 and compared between the two experimental groups (Fig. 7). In both groups, the mucosa and submucosa were thickened, and although collagen deposition
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Fig. 2. Photographs showing the in vitro-engineered mucosal cell sheet. (A) Air-lifting culture of the three-dimensional mucosal cell sheet. (B) The mucosal cell sheet in an insert wall. (C) The ease in detaching, transferring, and handling a cell sheet prior to transplantation.
Fig. 3. Microscopic examination of an autologous mucosal cell sheet produced from human and rat oral mucosae. Hematoxylin and eosin stains of the human oral mucosa (A) and in vitro-engineered three-dimensional mucosal cell sheet (B). P63 immunohistochemistry of the mucosal cell sheet (C).
had increased during the early stage of wound healing, this lessened during the later stage. Compared with the cell sheet group, the healed mucosa and submucosa were thicker and collagen was more deposited in the wound control group (P < 0.05). The finally healed mucosa and submucosa compositions of the cell sheet group appeared to be similar to those of the normal ventral rat tongue. Microscopic muscular disruption was found in the ventral tongue of the wound control rats but not in that of the cell sheet group.
Discussion This study showed that an in vitro-engineered oral mucosal equivalent can be produced by use of a completely autologous mucosa and plasma. Epithelial keratinocytes and lamina propria fibroblasts obtained from oral biopsy grew well in the air-liquid interface inserts without a 3T3 feeder layer. The mucosal cell sheet showed similarity to the histological characteristics of the oral
mucosa, with high p63 positivity. The engineered sheets were easy to handle and transfer, and showed flexibility to stretch without tearing. In the in vivo rat model mimicking the surgical extirpation of tongue cancer, the cell sheet promoted oral tongue wound healing with re-epithelialization, and showed microscopic evidence of accelerated wound healing with minimal fibrosis. The cell sheethealed tongues appeared have similar histology to that of a normal tongue. Therefore, our study suggests that our newly developed three-dimensional mucosal cell sheet can be used to restore the mucosal lining or soft tissue defects in the oral cavity. In vitro-tissue engineering has been used to restore tissue or organ defects with or without biodegradable scaffolds [14]. Cell transfer without scaffolds is widely used for a variety of human diseases, but many cells are lost soon after transplantation [15]. Accordingly, tissue engineering methods have been developed to overcome this problem, using biodegradable scaffolds to support tissue formation. In vitro-engineered cell sheets have been made using collagen glue [11], the acellular dermis [7], and the amniotic
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Fig. 4. Comparison of macroscopic wound healing between the wound control and mucosal cell sheet groups. Gross photographs taken at days 0, 3, 7, and 21 after deep wounding of the ventral tongue in the wound control (upper panels) and cell sheet groups (lower panels). *Asterisks indicate the well-adapted and well-survived mucosal cell sheet grafts at the early stage of wound healing.
Fig. 5. Comparison of wound sizes between the wound control and mucosal cell sheet groups. The wound size was measured regularly after deep wounding of the ventral tongue in Sprague-Dawley rats and is represented relative to that of a surgical defect at day 0. *P < 0.05 between the wound control (ctr) and cell sheet groups.
membrane [10], etc. as scaffolds to support the cells. Cell sheet engineering using a temperature-responsive polymer without biodegradable scaffolds has also been introduced [16]. Our in vitro-cultured cell sheets contain autologous fibrin and fibroblasts as well as keratinocyte layers. Autologous fibrin glue can be easily obtained from an abundant source of blood, ensuring safe biocompatibility and avoiding immunorejection.
Fibrin glue, as the first of the natural biomaterials, with autologous transplantation of cultured keratinocytes has been applied to tissue engineering for the closure of chronic wounds and burns [17]. Covering oral surgical wounds with only an autologous fibrin glue without cell components helped to relieve postoperative pain, and prevented viral infections and allergic reactions in patients undergoing partial glossectomy [18]. However, the absence of fibroblasts in the collagen scaffold might result in poor epithelial organization, as previously noted [19]. Another study reported that the death of epithelial cells seeded on an acellular dermal scaffold was due to the absence of fibroblasts [20]. Fibroblasts incorporated in fibrin glue promoted keratinocyte growth in vitro, whereas fibroblasts incorporated within a subepithelial substratum promoted mucosal epithelial maturation and organization in vivo [21]. Therefore, fibroblasts appear to be fundamental for preserving the coherence of the three-dimensional structure, by producing collagen fibers and thereby replacing the fibrin matrix [11,22]. Our in vitro-cultured mucosal cell sheet has the advantages of flexibility to stretch up to double its size, and of ease of handling and transfer to other dishes or to the defect site. The cell sheet can be detached manually using fine-point forceps, without the need for enzymatic detachment solutions. The transfer to other locations was also easy, without the need for carriers for placing the sheet, such as vaseline gauze [23]. Furthermore, our autologous cell sheet adapted well and immediately on the oral mucosal defect. The time from mucosal biopsy to production of the mucosal cell sheet was relatively fast, with completion within 2–3 weeks, which is acceptable in the clinical setting. For patients undergoing surgery for removal of an oral lesion, the biopsy for a small piece of normal tissue can be done simultaneously with the biopsy for the pathological lesion. The in vitro-cultured mucosal cell sheet can be used to cover the tissue or mucosal lining defect after the surgical extirpation of pathological oral lesions. Another advantage of our
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Fig. 6. Comparison of microscopic wound healing between the wound control and mucosal cell sheet groups. Microscopic photographs of tongues stained with hematoxylin and eosin after deep wounding of the ventral tongue in the wound control (ctr, A–C) and cell sheet groups (E and F). Asterisks indicate sloughs covering the unhealed wounds at the early stage (arrows). Open arrows indicate re-epithelialization of the ventral tongue wound (C, E, and F) and the ventral side of the normal tongue (D) in SpragueDawley rats. The bars indicate 500 lm.
autologous mucosal cell sheet is its simple and cheap production, because most of the essential compositions can be obtained from the same patient. Moreover, our cell sheet has histological similarity to the normal oral mucosa and an enrichment of p63-positive cells as an oral stem cell marker [24], giving it high regenerative potential to improve wound healing and restore defects. The present study examined the in vivo efficacy of in vitrocultured autologous mucosal cell sheets in an oral tongue wound model mimicking surgical treatment cases for tongue cancer. After transplantation, the survival and status of grafts are critical to the repair of intraoral defects. The survival of grafts has relied mainly on nutrient diffusion from the wound bed at the early stage of transplantation [25]. The fibroblasts in cultured grafts and wounds might help to adapt the keratinocyte growth and maturation in a defective oral mucosal lining [26]. Cultured fibroblasts provide cytokines and growth factors that stimulate early wound healing [27]. Inflammation at wounds is an important determinant of the wound healing process. An excessively activated inflammatory response might hinder the regeneration process and eventually lead to hypertrophic scar contraction and fibrosis [28]. The present study showed that the autologous mucosal sheet graft adapted well to, and survived in, the surgical tongue wound. The quick recovery of the mucosal layer can provide a barrier to infection from pathogens and maintain water homeostasis within the mucosa. The grafts also prevent excessive inflammatory responses and granulation formation in the wounds, eventually leading to a more natural wound healing process. These findings might be related to proper microvascular formation and less fibrosis in the wounds. At a later stage, this also contributes to the natural healing of the wounded oral mucosa, mimicking the histological characteristics of the normal tongue. However, our present findings will require further validations in other animal models with wounding
in different anatomical sites, and in human cases with surgical extirpation of pathological oral lesions. There is the potential risk of grossly normal but microscopically/genetically abnormal oral mucosa of oral cancer patients that is used for the production of tissue-engineered mucosal cell sheet. This can be solved by the use of other mucosa or low lip mucosa that involves relatively low risk of co-existing mucosal abnormality if the patients had no lip cancers. The site may be the advantages of easy accessibility and minimal morbidity for sampling. The mucosa cell sheets engineered from the nasal cavity may be also used, which will be better for lining surface defects in the upper respiratory tract or other sites requiring ciliated epithelium. The other potential advantage of our mucosal cell sheets is the use of completely autologous sources of cells and matrices, which needs acceptable costs for its production as well as avoids potential post-transplantation immunorejection. However, future studies will be required to ensure no acceleration of residual cancer growth or potential side effects for long-term follow-ups in the surgical animal models with oral cancers and clinical patients.
Conclusions The present study examined the potential efficacy of mucosal equivalents that were in vitro-engineered using completely autologous mucosae and plasma. The mucosal cell sheets proved to be highly efficient at wound healing, fast and simple to produce, highly flexible, and easy to handle and transfer. Autologous cell sheets transplanted into the deep wounds of the rat ventral tongue promoted oral wound healing, with earlier re-epithelialization and less submucosal fibrosis than that seen in the wound control. The cell sheet-healed tongue had similar histology to that of a normal tongue. Our engineered mucosal cell sheets thus have potential
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Fig. 7. Characteristics of the healed mucosa and submucosa. (A–C) Masson’s trichrome staining of the wound control (ctr, A) and mucosal cell sheet groups (B) on postsurgical day 28, and a normal tongue (NT, C). (D and E) Thickness of the epithelial and subepithelial layers on days 14 and 28. (F) Quantification of the collagen intensity in the image. *P < 0.05.
applicability for the rapid healing of oral mucosal lining and soft tissue defects, without scarring, adhesion, and functional deficits. Conflict of interest statement The authors declare no conflicts of interest. Funding This study was supported by the grants (No. HI15C2920 and HI14C23050000) from the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health & Welfare, Seoul, Republic of Korea (J.L. Roh). References [1] Kelm JM, Fussenegger M. Scaffold-free cell delivery for use in regenerative medicine. Adv Drug Deliv Rev 2010;62:753–64. [2] Takagi R, Yamato M, Kanai N, Murakami D, Kondo M, Ishii T, et al. Cell sheet technology for regeneration of esophageal mucosa. World J Gastroenterol 2012;18:5145–50. [3] Bates D, Kampa P. Cell-based regenerative approaches to the treatment of oral soft tissue defects. Int J Oral Maxillofac Implants 2013;28:e424–31. [4] O’Connor NE, Mulliken JB, Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75–8. [5] Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting of burn patients with allografts of cultured epidermal cells. Lancet 1983;2:428–30. [6] Oshima H, Inoue H, Matsuzaki K, Tanabe M, Kumagai N. Permanent restoration of human skin treated with cultured epithelium grafting–wound healing by stem cell based tissue engineering. Hum Cell 2002;15:118–28.
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Promotion of oral surgical wound healing using autologous mucosal cell sheets Jong-Lyel Roh ⇑, Hyejin Jang, Jaewang Lee, Eun Hye Kim, Daiha Shin Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
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Article history: Received 3 January 2017 Received in revised form 11 March 2017 Accepted 19 April 2017
Keywords: Cell sheet Mucosa Fibrin glue Oral wound Graft
a b s t r a c t Objectives: Severe oral mucosal and tissue defects can lead to pain, infection, and later undesirable healing of scarring and adhesion, resulting in a poor quality of life. In vitro-engineered oral mucosal equivalents for covering such defects are an alternative to avoiding the donor site morbidity of conventional skin or tissue grafts. We examined the efficacy of our newly developed three-dimensional mucosal cell sheets in an in vivo tongue wound model mimicking the surgical extirpation of tongue cancer. Materials and methods: Small oral mucosal and autologous fibrin samples were obtained from surgical patients and Sprague-Dawley rats. The fibrin was mixed with fibroblasts and seeded with keratinocytes that had been primarily cultured for in vitro cell expansion. The three-dimensional autologous cell sheets, cultured in air-lift interface inserts, were transplanted into deep wounds of the rat ventral tongue. Gross and microscopic findings of the postsurgical wounds were compared between wound control and cell sheet groups. Results: The cell sheets were flexible, expandable, and easy to transfer, and had histological characteristics similar to that of the normal oral mucosa, with high p63 positivity. They promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in the wound control. The cell sheet-healed tongue had similar histology to that of a normal tongue. Conclusions: Our engineered cell sheets have potential applicability for the rapid healing of oral mucosal and soft tissue defects, without scarring, adhesion, and functional deficits. Condensed abstract: The efficacy of in vitro-engineered mucosal equivalents, using completely autologous mucosa and plasma, was examined. Transplantation of the autologous cell sheets into deep wounds of the rat ventral tongue promoted oral wound healing with earlier re-epithelialization and less fibrosis than that in controls. Healed and normal tongues showed similar histology. Ó 2017 Published by Elsevier Ltd.
Introduction Oral mucosal and soft tissue defects can be caused by surgical extirpation of intraoral pathological lesions, trauma, recurrent ulcers, and irradiation. If not properly treated, the defects lead to pain, infection, and later undesirable healing, such as scarring and adhesion to adjacent tissues. This might cause significant deficits of functions that are essential for daily life, such as deglutition, articulation, and speech, as well as respiration. To prevent undesirable discomfort and sequelae, a skin graft, local or regional flap, or microvascular free flap is currently used to restore the mucosal lining or soft tissue defects in the oral cavity. The procedures com-
⇑ Corresponding author at: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea. E-mail address: [email protected] (J.-L. Roh). http://dx.doi.org/10.1016/j.oraloncology.2017.04.012 1368-8375/Ó 2017 Published by Elsevier Ltd.
monly require harvesting skin or tissues from the same patients, which might result in morbidity and leave aesthetically unacceptable scarring in the donor sites. The current surgical approach for filling a large soft tissue defect is microvascular free flap transfer, which commonly requires a considerable operation time and experienced surgical hands. A superficial intraoral defect without considerable soft tissue deficits can be covered with a split thickness skin graft. In vitro-cultured cell-based regenerative approaches have been introduced as feasible alternative methods to restoring the mucosal lining and tissue defects [1–3]. Epithelial cell sheets prepared from the autografts or allografts of epidermal cells have been applied to the grafting of burns [4–6]. In addition, it has been reported in the literature that cell sheets or equivalents from human mucosa can be in vitro-engineered for potential application to the closure of oral wounds [7–9]. Oral mucosal equivalents consisting of human lamina propria fibroblasts and oral epithelial cells have been shown to mimic the normal
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oral mucosa, with similar histological and immunohistochemical marker expression levels [8]. Furthermore, in vitro-produced oral mucosal equivalents were introduced for intraoral grafting after their production using scaffolds of acellular dermis [7], amniotic membrane [10], and collagen glue [11], etc. These were not autologous, but obtained from animals or cadavers and required pretreatments for eventual immunorejection of the equivalents. Human plasma is an abundant source of autologous fibrin that might be an optimal scaffold for producing skin and mucosal epithelial equivalents [12,13]. Lamina propria fibroblasts can be easily cultured in vitro, mixed in the autologous fibrin glue, and then used to facilitate the growth of keratinocytes seeded on the mixture, without potential immunorejection [13]. Therefore, we developed an in vitro-engineered autologous mucosal cell sheet consisting of keratinocytes and of plasma fibrin containing lamina propria fibroblasts. The keratinocytes and fibroblasts were obtained by in vitro culture using animal-product-free and completely autologous materials. Herein, this study examined the potential efficacy of our newly developed three-dimensional oral mucosal cell sheet in an in vivo tongue wound model mimicking the clinical setting of surgical extirpation of tongue cancer. Methods In vitro culture of mucosal samples A small piece of mucosa was sampled from the normal mucosa adjacent to the resection margins in patients who underwent transoral surgery. This work was approved by the Institutional Review Board of our hospital, and informed consent was obtained from each patient. Plasma was also obtained from the 25 mL blood samples drawn from the same patients using a vacutainer tube (BD Bioscience, Franklin Lakes, NJ, USA) at the time of surgery. The mucosae were sterilized with povidone-iodine solution (SigmaAldrich, St. Louise, MO, USA) and washed 3 times in phosphatebuffered saline solution. All tissues were treated with 1 U/mL dispase (STEMCELL Technologies, Vancouver, BC, Canada) for 2 h at 37 °C, and the epithelial and subepithelial layers were then separated. Both layers were separately treated with trypsin-ethylene diaminetetraacetic acid (ThermoFisher Scientific, Waltham, MA, USA) for 15 min at 37 °C. The cells were separately seeded in culture dishes and grown in the culture medium, which was a 3:1 mixture of Dulbecco’s modified Eagle’s minimal essential medium and Ham’s F12 (ThermoFisher), containing 10% heat-inactivated autologous serum, human recombinant insulin (5 mg/mL), triiodothyronine (1.3 ng/mL), adenine (24 mg/mL), hydrocortisone (0.4 mg/mL), and cholera toxin (8 ng/mL) (all purchased from Sigma-Aldrich), and supplemented with a penicillin-streptomy cin-amphotericin antibiotic-antimycotic solution (ThermoFisher). Human recombinant epidermal growth factor (10 ng/mL; ThermoFisher) was also added to the medium for culturing mucosal keratinocytes. The medium and supplements were replaced at every 3 days. Generation of in vitro-engineered mucosal cell sheet The plasma obtained from each patient’s blood was used to make fibrin glue as the source of the scaffolds. The fibrin glue was composed of a mixture of 0.5 mL of plasma, 1% calcium chloride, 70 mL of tranexamic acid (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and 0.5 mL of medium with 5 105 fibroblasts. The mixture was allowed to solidify in transwell cell culture inserts with a 0.4-mm pore-size polyester membrane (Corning, Inc., Corning, NY, USA) at 37 °C for 60 min. The inserts were placed in the plates with the medium and supplements. Keratinocytes were
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seeded on the mixture of fibrin glue and fibroblasts, and grown under air-liquid interface culture conditions, with the same medium, autologous serum, and supplements as mentioned above. The in vitro culturing of keratinocytes and fibroblasts was also performed using biopsy samples of the oral buccal mucosa from Sprague-Dawley (SD) rats. Blood was also obtained from each rat and used as a source of autologous fibrin glue. The autologous mucosal cell sheet was produced in the same way as described above. About 10–14 days after cell culturing, the fibrin glue and submucosal fibroblasts were mixed, poured, and solidified in the insert well, and keratinocytes were then seeded onto the mixture. Five to 7 days later, the mucosal cell sheets were constructed and prepared for transplantation. Histological examinations of the mucosal cell sheets The in vitro-cultured mucosal cell sheet or tissue samples were harvested, embedded in optimal cutting temperature compound (Sakura Fineteck USA Inc., Torrance, CA, USA), immediately snapfrozen in liquid nitrogen, and stored at –80 °C for subsequent use. The stored sheets or tissue samples were prepared as 5-mmthick frozen sections for histological examination. The sections were stained with hematoxylin and eosin (Sigma-Aldrich). The samples were also examined immunohistochemically for the staining of p63 (1:200; GeneTex, Inc., Irvine, CA, USA), pancytokeratin AE1/AE3 (1:200 dilution, Dako, Glostrup, Denmark), cytokeratin 5/6 (1:200, Dako), and ki-67 (1:200, GeneTex) according to established protocols and examined under a fluorescence microscope (Olympus, Tokyo, Japan). Intraoral grafting of the mucosal cell sheet into a surgically wounded rat tongue All animal study procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of our institution. Male SD rats, weighing 180– 220 g, were purchased from Central Lab Animal Inc. (OO). The animals were anesthetized by intramuscular injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Iris scissors were used to remove the mucosa and submucosal soft tissues of the ventral tongue in each SD rat to create a 50-mm2 deep wound (Fig. 1). The autologous mucosal cell sheet was detached from the culture dish and trimmed to a similar shape and size as the surgical defect. The cell sheet was then overlaid with a silastic sheet (0.01000 thickness; Bentec Medical, Woodland, CA, USA) and grafted onto the defect by use of 5-0 absorbable vicryl sutures. For the wound control group, a silastic sheet without the cultured mucosal sheet was attached to the wound site. All silastic sheets were removed at 3 days after grafting. Each experimental group included 25 rats. Normal tongues were also obtained from 5 unwounded rats. The body weight and food intake of each group were measured at every 3 days. Gross and microscopic examinations of the postsurgical wounds Gross photographs of each rat wound were taken regularly. The wound size at each postoperative day was also measured and compared with that of the original wound in each rat, and comparisons were also made between the wound control and cell sheet groups. Five rats of each group were sacrificed at postsurgical days 3, 7, 14, 21, and 28, and the tissues from the previous oral wounds were harvested and quickly frozen at 80 °C. Thereafter, 5-mm-thick sections were stained with hematoxylin and eosin and Masson’s trichrome stains (Sigma-Aldrich) and observed under a microscope (Nikon Co., Tokyo, Japan). The epithelial and subepithelial thicknesses and collagen density were measured in a blind manner in
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Fig. 1. Transplantation of the autologous mucosal cell sheet after tongue wounding. (A) Deep surgical wounding of the ventral tongue in a Sprague-Dawley rat, as a model mimicking a surgical defect after the extirpation of human tongue cancer. (B and C) Attachment of the in vitro-engineered mucosal cell sheet (asterisks) on the surgical defect. A piece of silastic sheet (arrows) was used to prevent the detachment or injury of the cell sheet after transplantation in the experimental animals.
10 randomly selected fields, using the ImageJ processing program (National Institutes of Health, Bethesda, MD, USA). Statistical analysis The data are presented as the mean ± standard deviation. The statistical significance of the differences among different treatment groups was assessed using the Mann–Whitney U test, and that among different periods in each group were assessed using the Wilcoxon signed-rank test. All tests were two-sided and a value of P < 0.05 was considered as being significant. Statistical analyses were performed using IBM SPSS software version 23.0 (IBM, Armonk, NY). Results Characteristics of the in vitro-engineered mucosal cell sheet Keratinocyte and fibroblast colonies were found in the separately cultured dishes of all oral mucosal samples at 2–4 days after harvesting and culturing. The cells grew to >80% confluency within 7–14 days. Most of the cultured human keratinocytes and fibroblasts were viable, with 80.3 ± 5.2% and 82.2 ± 4.7%, respectively, when examined with vital dye prior to harvesting for generation of the mucosal cell sheet. Fibroblasts from the submucosal layers grew more quickly in culture than did keratinocytes from the mucosal layer. Bacterial and fungal contamination was not found in any of the cultures from all samples. The in vitro culture was also successfully performed from biopsy samples harvested from the buccal mucosa of SD rats. The viability and colony-forming ability of the cells were similar to those of the human samples. Three or more cell sheets of 5-cm2 size from each sample were generated within 2–3 weeks from the time point of mucosal sampling. The three-dimensional cell sheets were flexible enough to be easily stretched up to double their size without tearing or injury. The sheets were also easily detached, using fine-tip forceps and without the need for an enzymatic detachment solution, for transfer to other dishes and to cut for grafting (Fig. 2). Histological examination of the mucosal cell sheets revealed their similarity to the histological characteristics of the oral mucosa (Fig. 3). The mucosal layer of the cell sheet included 2–4 layers of cuboidal-shaped nucleated epithelial cells interspersed with elongated cells. The submucosal layer of the cell sheet included fibroblasts and fibrin embedded in the scaffold, similar to the oral submucosal structures of the extracellular matrix and fibroblasts, except for the interspersed vessels. However, the cell sheet had no evidence of the rete ridges between epithelial and
subepithelial layers observed in normal oral mucosa. The cultured keratinocytes and the epithelial layers of the cell sheet were enriched with p63-positive cells. In addition, immunohistochemical analyses showed that most cells in epithelial layers of cell sheets were positive for cytokeratin 5/6 and AE1/AE2 pancytokeratin, and were negative for ki67 (data not shown). Promotion of oral wound healing using the in vitro-engineered mucosal cell sheet The deep wounds made in the ventral tongue of SD rats were covered with (or without, as the wound control) mucosal cell sheets. Gross photographs taken regularly after the surgery showed earlier closure of the wound defects in the mucosal cell sheet group than that in the wound control group (P < 0.05, Figs. 4 and 5). The wound control had substantial inflammatory reactions and sloughs covering the surgical defect on the ventral tongue surface during the early stage after wounding. In addition, the anterior to posterior length of the tongue was shortened, probably by the devascularization and mutilation of the tongue tip of the injured rats. On the other hand, the viable mucosal cell sheet covering the surgical defect in the cell sheet group promoted wound healing and prevented further injury of the mucosa-deficient tongue. Reepithelialization was completed within 3 weeks in both the wound control and cell sheet groups, but was more rapid in the latter group (P < 0.05, Fig. 5). Microscopic evidence of accelerated wounding healing by mucosal cell sheet grafting Microscopic examinations of the surgical wounds according to postsurgical days supported the observed gross findings and differences between the wound control and cell sheet groups (Fig. 6). Sloughs were found in the denuded mucosal surface of the wound control rats, whereas the surgical defect was covered with the mucosal sheet in the cell sheet group. Inflammatory reactions and muscular disruption of the injured submucosa were also more observed in the wound control than the cell sheet group during the early stage of wound healing (P < 0.05). At the late stage, the mucosal and submucosal compositions of the cell sheet group appeared to be more similar to those of the unwounded normal rat tongue, whereas those of the wound control rats were more thickened and fibrotic (P < 0.05). The characteristics of the healed mucosa and submucosa were evaluated at postsurgical days 14 and 28 and compared between the two experimental groups (Fig. 7). In both groups, the mucosa and submucosa were thickened, and although collagen deposition
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Fig. 2. Photographs showing the in vitro-engineered mucosal cell sheet. (A) Air-lifting culture of the three-dimensional mucosal cell sheet. (B) The mucosal cell sheet in an insert wall. (C) The ease in detaching, transferring, and handling a cell sheet prior to transplantation.
Fig. 3. Microscopic examination of an autologous mucosal cell sheet produced from human and rat oral mucosae. Hematoxylin and eosin stains of the human oral mucosa (A) and in vitro-engineered three-dimensional mucosal cell sheet (B). P63 immunohistochemistry of the mucosal cell sheet (C).
had increased during the early stage of wound healing, this lessened during the later stage. Compared with the cell sheet group, the healed mucosa and submucosa were thicker and collagen was more deposited in the wound control group (P < 0.05). The finally healed mucosa and submucosa compositions of the cell sheet group appeared to be similar to those of the normal ventral rat tongue. Microscopic muscular disruption was found in the ventral tongue of the wound control rats but not in that of the cell sheet group.
Discussion This study showed that an in vitro-engineered oral mucosal equivalent can be produced by use of a completely autologous mucosa and plasma. Epithelial keratinocytes and lamina propria fibroblasts obtained from oral biopsy grew well in the air-liquid interface inserts without a 3T3 feeder layer. The mucosal cell sheet showed similarity to the histological characteristics of the oral
mucosa, with high p63 positivity. The engineered sheets were easy to handle and transfer, and showed flexibility to stretch without tearing. In the in vivo rat model mimicking the surgical extirpation of tongue cancer, the cell sheet promoted oral tongue wound healing with re-epithelialization, and showed microscopic evidence of accelerated wound healing with minimal fibrosis. The cell sheethealed tongues appeared have similar histology to that of a normal tongue. Therefore, our study suggests that our newly developed three-dimensional mucosal cell sheet can be used to restore the mucosal lining or soft tissue defects in the oral cavity. In vitro-tissue engineering has been used to restore tissue or organ defects with or without biodegradable scaffolds [14]. Cell transfer without scaffolds is widely used for a variety of human diseases, but many cells are lost soon after transplantation [15]. Accordingly, tissue engineering methods have been developed to overcome this problem, using biodegradable scaffolds to support tissue formation. In vitro-engineered cell sheets have been made using collagen glue [11], the acellular dermis [7], and the amniotic
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Fig. 4. Comparison of macroscopic wound healing between the wound control and mucosal cell sheet groups. Gross photographs taken at days 0, 3, 7, and 21 after deep wounding of the ventral tongue in the wound control (upper panels) and cell sheet groups (lower panels). *Asterisks indicate the well-adapted and well-survived mucosal cell sheet grafts at the early stage of wound healing.
Fig. 5. Comparison of wound sizes between the wound control and mucosal cell sheet groups. The wound size was measured regularly after deep wounding of the ventral tongue in Sprague-Dawley rats and is represented relative to that of a surgical defect at day 0. *P < 0.05 between the wound control (ctr) and cell sheet groups.
membrane [10], etc. as scaffolds to support the cells. Cell sheet engineering using a temperature-responsive polymer without biodegradable scaffolds has also been introduced [16]. Our in vitro-cultured cell sheets contain autologous fibrin and fibroblasts as well as keratinocyte layers. Autologous fibrin glue can be easily obtained from an abundant source of blood, ensuring safe biocompatibility and avoiding immunorejection.
Fibrin glue, as the first of the natural biomaterials, with autologous transplantation of cultured keratinocytes has been applied to tissue engineering for the closure of chronic wounds and burns [17]. Covering oral surgical wounds with only an autologous fibrin glue without cell components helped to relieve postoperative pain, and prevented viral infections and allergic reactions in patients undergoing partial glossectomy [18]. However, the absence of fibroblasts in the collagen scaffold might result in poor epithelial organization, as previously noted [19]. Another study reported that the death of epithelial cells seeded on an acellular dermal scaffold was due to the absence of fibroblasts [20]. Fibroblasts incorporated in fibrin glue promoted keratinocyte growth in vitro, whereas fibroblasts incorporated within a subepithelial substratum promoted mucosal epithelial maturation and organization in vivo [21]. Therefore, fibroblasts appear to be fundamental for preserving the coherence of the three-dimensional structure, by producing collagen fibers and thereby replacing the fibrin matrix [11,22]. Our in vitro-cultured mucosal cell sheet has the advantages of flexibility to stretch up to double its size, and of ease of handling and transfer to other dishes or to the defect site. The cell sheet can be detached manually using fine-point forceps, without the need for enzymatic detachment solutions. The transfer to other locations was also easy, without the need for carriers for placing the sheet, such as vaseline gauze [23]. Furthermore, our autologous cell sheet adapted well and immediately on the oral mucosal defect. The time from mucosal biopsy to production of the mucosal cell sheet was relatively fast, with completion within 2–3 weeks, which is acceptable in the clinical setting. For patients undergoing surgery for removal of an oral lesion, the biopsy for a small piece of normal tissue can be done simultaneously with the biopsy for the pathological lesion. The in vitro-cultured mucosal cell sheet can be used to cover the tissue or mucosal lining defect after the surgical extirpation of pathological oral lesions. Another advantage of our
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Fig. 6. Comparison of microscopic wound healing between the wound control and mucosal cell sheet groups. Microscopic photographs of tongues stained with hematoxylin and eosin after deep wounding of the ventral tongue in the wound control (ctr, A–C) and cell sheet groups (E and F). Asterisks indicate sloughs covering the unhealed wounds at the early stage (arrows). Open arrows indicate re-epithelialization of the ventral tongue wound (C, E, and F) and the ventral side of the normal tongue (D) in SpragueDawley rats. The bars indicate 500 lm.
autologous mucosal cell sheet is its simple and cheap production, because most of the essential compositions can be obtained from the same patient. Moreover, our cell sheet has histological similarity to the normal oral mucosa and an enrichment of p63-positive cells as an oral stem cell marker [24], giving it high regenerative potential to improve wound healing and restore defects. The present study examined the in vivo efficacy of in vitrocultured autologous mucosal cell sheets in an oral tongue wound model mimicking surgical treatment cases for tongue cancer. After transplantation, the survival and status of grafts are critical to the repair of intraoral defects. The survival of grafts has relied mainly on nutrient diffusion from the wound bed at the early stage of transplantation [25]. The fibroblasts in cultured grafts and wounds might help to adapt the keratinocyte growth and maturation in a defective oral mucosal lining [26]. Cultured fibroblasts provide cytokines and growth factors that stimulate early wound healing [27]. Inflammation at wounds is an important determinant of the wound healing process. An excessively activated inflammatory response might hinder the regeneration process and eventually lead to hypertrophic scar contraction and fibrosis [28]. The present study showed that the autologous mucosal sheet graft adapted well to, and survived in, the surgical tongue wound. The quick recovery of the mucosal layer can provide a barrier to infection from pathogens and maintain water homeostasis within the mucosa. The grafts also prevent excessive inflammatory responses and granulation formation in the wounds, eventually leading to a more natural wound healing process. These findings might be related to proper microvascular formation and less fibrosis in the wounds. At a later stage, this also contributes to the natural healing of the wounded oral mucosa, mimicking the histological characteristics of the normal tongue. However, our present findings will require further validations in other animal models with wounding
in different anatomical sites, and in human cases with surgical extirpation of pathological oral lesions. There is the potential risk of grossly normal but microscopically/genetically abnormal oral mucosa of oral cancer patients that is used for the production of tissue-engineered mucosal cell sheet. This can be solved by the use of other mucosa or low lip mucosa that involves relatively low risk of co-existing mucosal abnormality if the patients had no lip cancers. The site may be the advantages of easy accessibility and minimal morbidity for sampling. The mucosa cell sheets engineered from the nasal cavity may be also used, which will be better for lining surface defects in the upper respiratory tract or other sites requiring ciliated epithelium. The other potential advantage of our mucosal cell sheets is the use of completely autologous sources of cells and matrices, which needs acceptable costs for its production as well as avoids potential post-transplantation immunorejection. However, future studies will be required to ensure no acceleration of residual cancer growth or potential side effects for long-term follow-ups in the surgical animal models with oral cancers and clinical patients.
Conclusions The present study examined the potential efficacy of mucosal equivalents that were in vitro-engineered using completely autologous mucosae and plasma. The mucosal cell sheets proved to be highly efficient at wound healing, fast and simple to produce, highly flexible, and easy to handle and transfer. Autologous cell sheets transplanted into the deep wounds of the rat ventral tongue promoted oral wound healing, with earlier re-epithelialization and less submucosal fibrosis than that seen in the wound control. The cell sheet-healed tongue had similar histology to that of a normal tongue. Our engineered mucosal cell sheets thus have potential
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Fig. 7. Characteristics of the healed mucosa and submucosa. (A–C) Masson’s trichrome staining of the wound control (ctr, A) and mucosal cell sheet groups (B) on postsurgical day 28, and a normal tongue (NT, C). (D and E) Thickness of the epithelial and subepithelial layers on days 14 and 28. (F) Quantification of the collagen intensity in the image. *P < 0.05.
applicability for the rapid healing of oral mucosal lining and soft tissue defects, without scarring, adhesion, and functional deficits. Conflict of interest statement The authors declare no conflicts of interest. Funding This study was supported by the grants (No. HI15C2920 and HI14C23050000) from the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health & Welfare, Seoul, Republic of Korea (J.L. Roh). References [1] Kelm JM, Fussenegger M. Scaffold-free cell delivery for use in regenerative medicine. Adv Drug Deliv Rev 2010;62:753–64. [2] Takagi R, Yamato M, Kanai N, Murakami D, Kondo M, Ishii T, et al. Cell sheet technology for regeneration of esophageal mucosa. World J Gastroenterol 2012;18:5145–50. [3] Bates D, Kampa P. Cell-based regenerative approaches to the treatment of oral soft tissue defects. Int J Oral Maxillofac Implants 2013;28:e424–31. [4] O’Connor NE, Mulliken JB, Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75–8. [5] Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting of burn patients with allografts of cultured epidermal cells. Lancet 1983;2:428–30. [6] Oshima H, Inoue H, Matsuzaki K, Tanabe M, Kumagai N. Permanent restoration of human skin treated with cultured epithelium grafting–wound healing by stem cell based tissue engineering. Hum Cell 2002;15:118–28.
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