Periodontitis is a world-wide infectious disease that destroys the tooth-supporting attachment apparatus, which consists of alveolar bone, cementum, and periodontal ligament. Recent studies have reported numerous associations between periodontitis and systemic diseases, such as cardiovascular disease (de Oliveira et al., 2010) and diabetes mellitus (Lalla and Papapanou, 2011), as well as a higher risk of preterm low birth-weight babies (Offenbacher et al., 1996). Furthermore, researches have recently shown that Bisphosphonate-Related Osteonecrosis of the Jaws (BRONJ) is also associated with severe periodontitis (Vescovi et al., 2011). Therefore, periodontal treatment may not only contribute to oral hygiene but also improvement of systemic conditions (Seymour et al., 2007). Conventional treatments, such as scaling, root-planing, and surgical cleaning, have been performed to remove the bacteria and contaminated tissue. However, these procedures frequently result in the formation of a weak attachment, a condition termed “long junctional epithelium (LJE)” (Caton et al., 1980), wherein the patients tend to present with a recurrence of disease without maintenance therapies (Axelsson and Lindhe, 1981). To overcome this problem, various regenerative therapies, such as guided tissue regeneration (GTR) and enamel matrix derivative, have been introduced in clinical practice. The use of cell-occlusive membranes for GTR is regarded as the first generation of periodontal regeneration, whereas the development and use of growth factors and endogenous regenerative technology for periodontal regeneration is regarded as the second generation of periodontal regeneration (Ishikawa et al., 2009). However, the outcomes of these studies were limited and associated with poor clinical predictability (Esposito et al., 2009). Therefore, stem cell-based approaches for periodontal regeneration have been studied and translated into clinical settings as the third generation. In this chapter, we would like to describe the principles of “Cell Sheet Engineering” and its application of clinical settings, featuring our recent translational research for periodontal regeneration.
2. “Cell Sheet Engineering (CSE)”
The cell delivery for periodontal regeneration is usually performed with the combination use of cells and scaffolds, although the location and the differentiation of transplanted is difficult to control. In contrast to approaches that utilize scaffolds, we have developed an alternative technology for cell transplantation using temperature responsive culture dishes, which we call “Cell Sheet Engineering”.
2.1. Intelligent surface of
N-isopropylacrylamide (PIPAAm) and fabrication of cell sheets
A: Cells can attach and proliferate on grafted surface of the temperature responsive polymer (poly (N-isopropylacrylamide: PIPAAm) at 37 °C, wherein PIPAAm is extensively dehydrated and compact. B: At temperatures below 32 °C, cells with extracellular matrix proteins spontaneously detach from the temperature responsive culture dishes, wherein PIPAAm is fully hydrated with an extended-chain conformation. A simple temperature change can control cell attachment/detachment without any damages. Modified and reprint from Iwata et al., 2013.
The application of this technology has enabled the retrieval of confluently cultured cells, such as keratinocytes (Yamato et al., 2001), corneal epithelial cell sheets (Nishida et al., 2004a), and oral mucosal epithelial cells (Ohki et al., 2006) in the form of a “cell sheet”. The epithelial cell sheets are multi-layered and preserve the integrity of proteins such as E-cadherin and laminin 5 that are typically destroyed in the process of enzymatic treatments (Yamato et al., 2001). In addition, recent studies revealed that epithelial cell sheets can be fabricated using temperature responsive culture inserts without feeder layers (Murakami et al., 2006a; b), thereby eliminating exclude xenogeneic factors for animal-free cell transplantation (Takagi et al., 2011).
To fabricate thick tissues, cell sheets can be stacked in layers because they can connect to one another very quickly. A study demonstrated that bilayer cardiomyocyte sheets were completely coupled 46 ± 3 min (mean ± SEM) after the initial layering (Haraguchi et al., 2006), suggesting that multi-layered cell sheets can communicate and become synchronized as functional tissues. Based on this study, multi-layered transplantation was performed (Shimizu et al., 2006b). When more than three cardiomyocyte sheets were layered and transplanted into the subcutaneous space in rats, the appearance of fibrosis and disordered vasculature indicated the presence of fibrotic areas within the transplanted laminar structures. Although the rapid establishment of microvascular networks occurred within the engineered tissues, this formation of new vessels did not rescue the tissues when the thickness was above 80 µm. Using a multiple-step transplantation protocol at 1 or 2 day intervals resulted in rapid neovascularization of the engineered myocardial tissues with a thickness of more than 1 mm (Shimizu et al., 2006b), and these results led us to fabricate prevascularized cell sheets (Sekine et al., 2011). Recent studies demonstrate that the combination of different types of cells, for example an endothelial cell sheet sandwiched with other types of cell sheets, can lead to pre-vascularization
2.2. Cell sheet transplantation in animal models
From the beginning of the 21st century, various types of cells have been extracted, cultured in temperature responsive dishes, and fabricated as cell sheets. Transplantation has been performed, and the efficacy of these cell sheets was evaluated in most of the studies.
2.2.1. Corneal regeneration
Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and loss of vision. To recruit limbal stem cells, a novel cell-sheet manipulation technology that takes advantage of temperature responsive culture surfaces was developed (Nishida et al., 2004a). The results reveal that multi-layered corneal epithelial cell sheets were successfully fabricated and that their characteristics were similar to those of native tissues. Transplantation of these cell sheets induced corneal surface reconstruction in rabbits. For patients who suffer from unilateral limbal stem deficiency, corneal epithelial cell sheets can be cultured from autologous limbal stem cells. When the objective is to repair the bilateral corneal stem cell deficiency, autologous oral mucosal epithelial cells are utilized to create oral mucosal epithelial cell sheets. The cell sheets contain both cell-to-cell junctions and extracellular matrix proteins, and can be transplanted without the use of any carrier substrates or sutures. Therefore, oral mucosal epithelial sheets were examined as an alternative cell source to expand the possibilities of autologous transplantation. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface and restored transparency. Four weeks after the transplantation, epithelial stratification was similar to that of normal corneal epithelia, although the keratin expression profile retained characteristics of the oral mucosal epithelium.
2.2.2. Cardiac regeneration
To enhance the function of cardiac tissue, neonatal rat cardiomyocyte sheets were fabricated and examined (Shimizu et al., 2002). When 4 sheets were layered, spontaneous beating of the engineered constructs was observed. When they were transplanted subcutaneously, heart tissue-like structures and neovascularization within the contractile tissues were observed. The long-term survival of pulsatile cardiac grafts was confirmed for more than one year in rats (Shimizu et al., 2006a). Another study was performed to create thick tissue in rats (Shimizu et al., 2006b). However, the thickness limit for the layered cell sheets of subcutaneous tissue was ~80 μm (3 layers). To overcome this limitation, several transplantations of triple-layer grafts were performed, resulting in an approximately 1 mm-thick myocardium with a well-organized microvascular network. Other types of cell sheets were also examined to improve cardiac function. Adipose-derived mesenchymal stem cells in mice (Miyahara et al., 2006) and skeletal myoblasts in dogs, rats, and hamsters (Hata et al., 2006; Hoashi et al., 2009; Kondoh et al., 2006) were transplanted as cell sheets, demonstrating the efficacy of the method for cardiac repair.
2.2.3. Cartilage regeneration
Chondrocyte sheets applicable to cartilage regeneration were prepared using cell sheet manufacturing technique that takes advantage of temperature responsive culture dishes. The layered chondrocyte sheets were able to maintain the phenotype of cartilage and could be attached to sites that exhibited cartilage damage. The cell sheets act as a barrier for preventing the loss of proteoglycan from these sites and for protection against catabolic factors in the joints of rabbits (Kaneshiro et al., 2006).
2.2.4. Esophageal regeneration
With the recent development of endoscopic submucosal dissection (ESD), large esophageal cancers can be removed using a single procedure. However, complications, such as postoperative inflammation and stenosis, frequently occur after an aggressive ESD procedure, which can considerably affect the quality of life of the patient. Therefore, a novel treatment combining ESD and the endoscopic transplantation of tissue-engineered cell sheets created using autologous oral mucosal epithelial cells, was examined in a canine model (Ohki et al., 2006). The results confirm the efficacy of the novel combination of the endoscopic approach with the potential treatment of esophageal cancers that can effectively enhance wound healing and possibly prevent postoperative esophageal stenosis.
2.2.5. Hepatocyte regeneration
To address the demand for therapeutic benefits for patients suffering from liver disease, the development of new therapeutic applications is crucial. Therefore, hepatic tissue sheets transplanted into the subcutaneous space of mice have been investigated, resulting in the efficient engraftment of the surrounding cells, as well as the formation of a two-dimensional hepatic tissues network, which was stable for more than 200 days (Ohashi et al., 2007). The engineered hepatic cell sheets also showed several characteristics of liver-specific functionality, and the use of bilayered sheets enhanced these characteristics.
2.2.6. Fibroblast sheet transplantation for sealing air leaks
In thoracic surgery, the development of postoperative air leaks is the most common cause of prolonged hospitalization. To seal the lung leakage, use of autologous fibroblast sheets on the defects was demonstrated to be an effective treatment for permanently sealing air leaks in a dynamic fashion in rats (Kanzaki et al., 2007). Using roughly the same procedures, pleural defects were also closed by fibroblast sheets in pigs (Kanzaki et al., 2008).
2.2.7. Mesothelial cells for the prevention of post-operative adhesions
Post-operative adhesions often cause severe complications such as bowel obstruction and abdominopelvic pain. The use of mesothelial cell sheets was investigated to prevent post-operative adhesions in a canine model (Asano et al., 2006). Mesothelial cells were harvested from tunica vaginalis (Asano et al., 2005) and cell sheets were fabricated on a fibrin gel. The results demonstrated that mesothelial cell sheets are effective for preventing post-operative adhesion formation.
2.2.8. Retinal Pigment Epithelial (RPE) cell regeneration
The retinal pigment epithelium (RPE) plays an important role in maintaining the health of the neural retina. RPE cell sheets were fabricated as a monolayer structure with intact cell-to-cell junctions, similar to that of native RPE (Kubota et al., 2006). In the transplantation study, RPE cell sheets attached to the host tissues in the subretinal space were more effective than the use of injected isolated cell suspensions in rabbits (Yaji et al., 2009).
2.2.9. Urothelial regeneration
Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured
2.2.10. Islet regeneration
To establish a novel approach for diabetes mellitus, pancreatic islet cell sheets were fabricated and transplanted in rats (Shimizu et al., 2009). Laminin-5 was coated on temperature responsive dishes to enhance the initial cell attachment, and the presence of specific molecules, such as insulin and glucagon, was also observed in the recipient site.
2.2.11. Thyroid regeneration
For hormonal deficiencies caused by endocrine organ diseases, continuous oral hormone administration is indispensable to supplement the shortage of hormones. To verify the cytotherapeutic approach, cells from rat thyroid were spread on temperature responsive culture dishes, and cell sheets were created (Arauchi et al., 2009). Rats were exposed to total thyroidectomy as hypothyroidism models and received the thyroid cell sheet transplantation 1 week after the total thyroidectomy. The transplantation of the thyroid cell sheets was able to restore the thyroid function 1 week after the cell sheet transplantation and the improvement was observed long after the surgery.
2.3. Cell sheet transplantation in human clinical trials
In Japan, 6 clinical trials using cell sheet engineering technology have been started or have already been completed.
2.3.1. Corneal reconstruction
The first clinical trial of the cell sheet engineering technology involved a corneal reconstruction using autologous mucosal epithelial cells, and the results were published in 2004 (Nishida et al., 2004b). Oral mucosal tissue was harvested from 4 patients with bilateral total corneal stem-cell deficiencies. Subsequently, cells were cultured for two weeks using a mitomycin C-treated 3T3 feeder layer and transplanted directly into the denuded corneal surfaces without sutures. The results demonstrated that complete re-epithelialization of the corneal surfaces occurred, and the vision of all patients was restored. Recently, autologous oral mucosal epithelial cell sheets cultured with UpCell-Insert technology (CellSeed, Tokyo, Japan) without the feeder layer were transplanted into 25 patients for the treatment of corneal limbal epithelial deficiency in France The safety of the products was established during the 360-day follow-up, and the results confirmed its efficacy for reconstructing the ocular surface. (Burillon et al., 2012).
2.3.2. Endoscopic treatment of esophageal ulceration
Using a canine model (Ohki et al., 2006), autologous oral mucosal epithelial cell sheets were fabricated using the UpCell-Insert technology. After performing the esophageal endoscopic submucosal dissection to remove superficial esophageal neoplasms, cell sheets were transplanted, resulting in the complete prevention of stricture formation in patients with partial circumferential resection (Ohki et al., 2009; Ohki et al., 2012).
2.3.3. Improvements in ischemic cardiomyopathy
Autologous myoblast cells from a patient’s thigh were fabricated as cell sheets, and these cell sheets were transplanted into end-stage dilated cardiomyopathy patients in need of left ventricular assist systems (Sawa et al., 2012). The myoblastic cell sheets were transplanted into the affected part of the heart in the patients. The first patient was successfully treated and discharged from the hospital without requiring a ventricular assisting device.
2.3.4. Cartilage regeneration
A clinical trial for cartilage regeneration began in 2011 at Tokai University, Japan. In this study, autologous chondrocytes and synoviocytes were co-cultured with the UpCell-Insert technology. After a period of cultivation, co-cultured cell sheets were combined into three layers and transplanted into the cartilage defects of patients.
2.3.5. Nasal mucosa epithelial cell sheet transplantation to the middle ear bone for preventing hearing loss
A clinical trial for preventing hearing loss began in 2014 at The Jikei University, Japan. Autologous nasal mucosa epithelial cell sheets were transplanted to the surface of bone of the middle ear, and inhibit such as the hyperplasy of granulation tissue and bone, and the progression of fibroblast within middle ear cavity, which induce hearing loss after the surgery of otitis media.
3. Periodontal regeneration
Our laboratory started to introduce cell sheet engineering for periodontal regeneration since sometime after 2000. A key event in periodontal regeneration involves the formation of periodontal ligament and cementum complex (MacNeil and Somerman, 1999), which is a thin surface structure that anchors the tooth to the alveolar socket. Several studies have demonstrated that the cell sheet engineering approach can deliver functional cells in the form of a thin layered sheet, wherein the extracellular matrices, cell-cell junctions, and cell-matrix interactions are well-preserved (Kumashiro et al., 2010). Thus, we have attempted to regenerate this periodontal attachment apparatus based on the technology of “cell sheet engineering” (Yang et al., 2007).
3.1. Small animal studies
Human PDL (hPDL) cell sheets were successfully created using temperature responsive dishes, and the characteristics of hPDL cell sheets were investigated (Hasegawa et al., 2005). In this study, explant culture methods were utilized for the primary culture of hPDL cells. The hPDL cell sheets cultured with ascorbic acid were recovered from the culture dishes as a contiguous sheet accompanied by abundant extracellular matrix components, including type I collagen, integrin β1 and fibronectin. Then, hPDL cell sheets were transplanted as cell pellets into a mesial dehiscence model in athymic rats. Four weeks after surgery, newly formed immature fibers with obliquely anchored dentin surfaces were observed in all the experimental sites, whereas no such findings were observed in any control sites (Figure 2).These results suggest that this procedure based upon the principles of cell sheet engineering can be applied to periodontal regeneration.
A: Nontransplanted control site. B: hPDL transplanted experimental site. Regeneration of periodontal ligament-like structure was observed only in the experimental site. Azan staining. Modified and reprint from Hasegawa et al., 2005.
Next, the optimal culture condition was examined. Because the osteoinductive medium, which contains 50 µg/ml of ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone, enhanced both osteoblastic/cementoblastic and the periodontal differentiation of PDL cells
3.2. Large animal studies
Based on the successful results from small animal studies, we next utilized canine periodontal defect models. Dog PDL (dPDL) cells were extracted using collagenase/dispase digestion. Four individual dPDL cells were successfully isolated and expanded
Next, we evaluated the safety and efficacy of PDL cell sheets in a one-wall infrabony defect model (Tsumanuma et al., 2011), which is considered to be a severe defect model (Kim et al., 2004). In this study, we also compared the differences in the periodontal healing of various cell sources. PDL cells, bone marrow derived mesenchymal stem cells, and alveolar periosteal cells were obtained from each animal, three-layered canine cell sheets were transplanted in an autologous manner, and bone defects were filled with porous β-TCP with 3% type I collagen gelto stabilize the graft shape. Eight weeks after transplantation, significantly more periodontal regeneration was observed in the newly formed cementum and well-oriented PDL fibers more in the PDL cell sheets group than in the other groups. These results indicate that PDL cell sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration.
3.3. Optimization of human PDL cells
To protect human rights as subjects in clinical trials, the protocol of cytotherapy should be designed based on Good Clinical Practice (GCP) and Good Manufacturing Practice (GMP). Culturing hPDL cells from a single tooth is essential in performing our clinical trial. However, appropriate method for the extraction and expansion of hPDL cells are still not well understood. Thus, we determined the optimal method of isolation and expansion of hPDL cells and then examined their gene expression levels and differentiation potentials, and eventually validated the common characteristics of hPDL cells from 41 samples (Iwata et al., 2010). The hPDL cells were successfully extracted with collagenase/dispase, and then clonal proliferation was performed. Typically, 10 to 100 colonies were observed for a few days after the initial spreading. hPDL cells exhibit the ability to be highly proliferative when cultured at a low cell density. The cells were subcultured for 3 to 4 days, reaching one million cells in 2 weeks. Then, cells were spread on temperature responsive dishes to create a cell sheet in the presence of the osteoinductive medium. Cell sheets were harvested 2 weeks after spreading because the mRNA expression of osteogenic marker genes was strong after that period of time. Quality assurance tests were performed on at least 7 samples, and then the standard phenotypes of hPDL cell sheets were determined.
According to the GCP and GMP guidelines, hPDL cell sheets were created from three healthy volunteer donors at the GMP-grade Cell Processing Center (CPC) in our university (Washio et al., 2010). GMP-grade reagents and certified materials were used for culturing the hPDL cells. The safety and efficacy of “the product (hPDL cell sheets in this case)” was validated for a clinical trials. Prior to performing the cell culture, autologous serum was prepared from the donors. The hPDL cells were cultured under xeno-free conditions, and cell sheets were fabricated using the temperature responsive dishes. Culture sterility was confirmed using conventional tests. Safety was evaluated using the following tests: 1) the soft-agar colony-formation assay, 2) transplantation into nude mice, and 3) the karyotype test (Yoshida et al., 2012). The efficacy of the cell sheets was verified by transplantation with a dentin block into SCID mice. All of these tests revealed that hPDL cell sheets created at the CPC were safe and exhibited the ability to regenerate periodontal tissues. Another set of three hPDL cell sheets from healthy volunteer donors were created at the CPC to optimize the procedures.
3.4. The clinical trial
After approval on the 5th of January 2011, our clinical trial called “Periodontal regeneration with autologous periodontal ligament cell sheets” was initiated to treat patients presenting with the following ailments: 1) infrabony defects with a probing depth of more than 4 mm after the initial therapy, 2) radiographic evidence of infrabony defects, and 3) a redundant tooth that contains healthy periodontal tissue as a cell source. All patients provided written informed consent according to the GCP. Exclusion criteria included the following: 1) relevant medical conditions contraindicating surgical interventions (e.g., diabetes mellitus, cardiovascular, kidney, liver, or lung disease, or compromised immune system), 2) pregnancy or lactation, and 3) heavy tobacco smoking (more than 11 cigarettes a day). The primary outcome of this trial is to evaluate the safety and efficacy of autologous transplantation of periodontal ligament cell sheets. As of the end of May in 2014, 10 cases of autologous PDL cell sheets were transplanted, and the healing process took place uneventfully.
The applications of cell sheet engineering for regenerative medicine are mentioned. Various types of cells have been examined and most of them improved the functions of recipients, suggesting that cell sheet engineering can be an alternative strategy for the therapy of tissue engineering. The implementation of robotic systems that allow the safe mass production of sterile cell sheets automatically, as well as further collaboration between researchers and medical professionals will make “cell sheet engineering” the leading edge solution for regenerative medicine (Elloumi-Hannachi et al., 2010).
This study was supported by Creation of innovation centers for advanced interdisciplinary research areas Program in the Project for Developing Innovation Systems “Cell Sheet Tissue Engineering Center (CSTEC)” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Teruo Okano is a director of the board of Cell Seed, a stake holder of Cell Seed, and an inventor of cell sheet-related patent. Masayuki Yamato had been a science consultant of Cell Seed until 2011, stake holder and an inventor of cell sheet-related patent. Tokyo Women's Medical University is receiving research fund from CellSeed Inc.
Arauchi A, Shimizu T, Yamato M, Obara T, Okano T (2009). Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models. Tissue Eng Part A15(12) :3943-3949.
Asakawa N, Shimizu T, Tsuda Y, Sekiya S, Sasagawa T, Yamato M et al.(2010). Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials31(14) :3903-3909.
Asano T, Takazawa R, Yamato M, Kageyama Y, Kihara K, Okano T (2005). Novel and simple method for isolating autologous mesothelial cells from the tunica vaginalis. BJU Int96(9) :1409-1413.
Asano T, Takazawa R, Yamato M, Takagi R, Iimura Y, Masuda H et al.(2006). Transplantation of an autologous mesothelial cell sheet prepared from tunica vaginalis prevents post-operative adhesions in a canine model. Tissue Eng12(9) :2629-2637.
Axelsson P, Lindhe J (1981). The significance of maintenance care in the treatment of periodontal disease. Journal of Clinical Periodontology8(4) :281-294.
Burillon C, Huot L, Justin V, Nataf S, Chapuis F, Decullier E et al.(2012). Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Investigative ophthalmology & visual science53(3) :1325-1331.
Caton J, Nyman S, Zander H (1980). Histometric evaluation of periodontal surgery. II. Connective tissue attachment levels after four regenerative procedures. J Clin Periodontol7(3) :224-231.
de Oliveira C, Watt R, Hamer M (2010). Toothbrushing, inflammation, and risk of cardiovascular disease: results from Scottish Health Survey. Bmj340(c2451.
Elloumi-Hannachi I, Yamato M, Okano T (2010). Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. Journal of Internal Medicine267(1) :54-70.
Esposito M, Grusovin MG, Papanikolaou N, Coulthard P, Worthington HV (2009). Enamel matrix derivative (Emdogain(R)) for periodontal tissue regeneration in intrabony defects. Cochrane database of systematic reviews4) :CD003875.
Flores MG, Hasegawa M, Yamato M, Takagi R, Okano T, Ishikawa I (2008a). Cementum-periodontal ligament complex regeneration using the cell sheet technique. J Periodontal Res43(3) :364-371.
Flores MG, Yashiro R, Washio K, Yamato M, Okano T, Ishikawa I (2008b). Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats. J Clin Periodontol35(12) :1066-1072.
Haraguchi Y, Shimizu T, Yamato M, Kikuchi A, Okano T (2006). Electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation. Biomaterials27(27) :4765-4774.
Haraguchi Y, Shimizu T, Sasagawa T, Sekine H, Sakaguchi K, Kikuchi T et al.(2012). Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc7(5) :850-858.
Hasegawa M, Yamato M, Kikuchi A, Okano T, Ishikawa I (2005). Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng11(3-4) :469-478.
Hata H, Matsumiya G, Miyagawa S, Kondoh H, Kawaguchi N, Matsuura N et al.(2006). Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-induced canine heart failure model. J Thorac Cardiovasc Surg132(4) :918-924.
Hoashi T, Matsumiya G, Miyagawa S, Ichikawa H, Ueno T, Ono M et al.(2009). Skeletal myoblast sheet transplantation improves the diastolic function of a pressure-overloaded right heart. J Thorac Cardiovasc Surg138(2) :460-467.
Ishikawa I, Iwata T, Washio K, Okano T, Nagasawa T, Iwasaki K et al.(2009). Cell sheet engineering and other novel cell-based approaches to periodontal regeneration. Periodontol 200051(220-238.
Iwata T, Yamato M, Tsuchioka H, Takagi R, Mukobata S, Washio K et al.(2009). Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials30(14) :2716-2723.
Iwata T, Yamato M, Zhang Z, Mukobata S, Washio K, Ando T et al.(2010). Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use. Journal of Clinical Periodontology37(12) :1088-1099.
Iwata T, Washio K, Yoshida T, Ishikawa I, Ando T, Yamato M et al.(2013). Cell sheet engineering and its application for periodontal regeneration. J Tissue Eng Regen Med.
Kaneshiro N, Sato M, Ishihara M, Mitani G, Sakai H, Mochida J (2006). Bioengineered chondrocyte sheets may be potentially useful for the treatment of partial thickness defects of articular cartilage. Biochem Biophys Res Commun349(2) :723-731.
Kanzaki M, Yamato M, Yang J, Sekine H, Kohno C, Takagi R et al.(2007). Dynamic sealing of lung air leaks by the transplantation of tissue engineered cell sheets. Biomaterials28(29) :4294-4302.
Kanzaki M, Yamato M, Yang J, Sekine H, Takagi R, Isaka T et al.(2008). Functional closure of visceral pleural defects by autologous tissue engineered cell sheets. Eur J Cardiothorac Surg34(4) :864-869.
Kim CS, Choi SH, Chai JK, Cho KS, Moon IS, Wikesjo UM et al.(2004). Periodontal repair in surgically created intrabony defects in dogs: influence of the number of bone walls on healing response. Journal of Periodontology75(2) :229-235.
Kondoh H, Sawa Y, Miyagawa S, Sakakida-Kitagawa S, Memon IA, Kawaguchi N et al.(2006). Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters. Cardiovasc Res69(2) :466-475.
Kubota A, Nishida K, Yamato M, Yang J, Kikuchi A, Okano T et al.(2006). Transplantable retinal pigment epithelial cell sheets for tissue engineering. Biomaterials27(19) :3639-3644.
Kumashiro Y, Yamato M, Okano T (2010). Cell attachment-detachment control on temperature-responsive thin surfaces for novel tissue engineering. Ann Biomed Eng38(6) :1977-1988.
Lalla E, Papapanou PN (2011). Diabetes mellitus and periodontitis: a tale of two common interrelated diseases. Nat Rev Endocrinol7(12) :738-748.
MacNeil RL, Somerman MJ (1999). Development and regeneration of the periodontium: parallels and contrasts. Periodontology 200019(1) :8-20.
Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H et al.(2006). Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med12(4) :459-465.
Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J et al.(2006a). The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets. Biomaterials27(32) :5518-5523.
Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J et al.(2006b). Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. J Artif Organs9(3) :185-191.
Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H et al.(2004a). Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation77(3) :379-385.
Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E et al.(2004b). Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med351(12) :1187-1196.
Offenbacher S, Katz V, Fertik G, Collins J, Boyd D, Maynor G et al.(1996). Periodontal infection as a possible risk factor for preterm low birth weight. Journal of Periodontology67(10 Suppl) :1103-1113.
Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M et al.(2007). Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med13(7) :880-885.
Ohki T, Yamato M, Murakami D, Takagi R, Yang J, Namiki H et al.(2006). Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut55(12) :1704-1710.
Ohki T, Yamato M, Ota M, Murakami D, Takagi R, Kondo M et al.(2009). Endoscopic Transplantation of Human Oral Mucosal Epithelial Cell Sheets-World's First Case of Regenerative Medicine Applied to Endoscopic Treatment. Gastrointestinal Endoscopy69(5) :AB253-AB254.
Ohki T, Yamato M, Ota M, Takagi R, Murakami D, Kondo M et al.(2012). Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets. Gastroenterology143(3) :582-588 e581-582.
Okano T, Yamada N, Okuhara M, Sakai H, Sakurai Y (1995). Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces. Biomaterials16(4) :297-303.
Pirraco RP, Obokata H, Iwata T, Marques AP, Tsuneda S, Yamato M et al.(2011). Development of osteogenic cell sheets for bone tissue engineering applications. Tissue engineering Part A17(11-12) :1507-1515.
Sasagawa T, Shimizu T, Sekiya S, Haraguchi Y, Yamato M, Sawa Y et al.(2009). Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials.
Sawa Y, Miyagawa S, Sakaguchi T, Fujita T, Matsuyama A, Saito A et al.(2012). Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case. Surg Today42(2) :181-184.
Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M et al.(2011). Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue engineering Part A17(23-24) :2973-2980.
Seymour GJ, Ford PJ, Cullinan MP, Leishman S, Yamazaki K (2007). Relationship between periodontal infections and systemic disease. Clin Microbiol Infect13 Suppl 4(3-10.
Shimizu H, Ohashi K, Utoh R, Ise K, Gotoh M, Yamato M et al.(2009). Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus. Biomaterials30(30) :5943-5949.
Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K et al.(2002). Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res90(3) :e40.
Shimizu T, Sekine H, Isoi Y, Yamato M, Kikuchi A, Okano T (2006a). Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets. Tissue Eng12(3) :499-507.
Shimizu T, Sekine H, Yang J, Isoi Y, Yamato M, Kikuchi A et al.(2006b). Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J20(6) :708-710.
Shiroyanagi Y, Yamato M, Yamazaki Y, Toma H, Okano T (2003). Transplantable urothelial cell sheets harvested noninvasively from temperature-responsive culture surfaces by reducing temperature. Tissue Eng9(5) :1005-1012.
Shiroyanagi Y, Yamato M, Yamazaki Y, Toma H, Okano T (2004). Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps. BJU Int93(7) :1069-1075.
Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T et al.(2011). Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. Journal of tissue engineering and regenerative medicine5(4) :e63-73.
Tsuda Y, Shimizu T, Yamato M, Kikuchi A, Sasagawa T, Sekiya S et al.(2007). Cellular control of tissue architectures using a three-dimensional tissue fabrication technique. Biomaterials28(33) :4939-4946.
Tsumanuma Y, Iwata T, Washio K, Yoshida T, Yamada A, Takagi R et al.(2011). Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model. Biomaterials32(25) :5819-5825.
Vescovi P, Campisi G, Fusco V, Mergoni G, Manfredi M, Merigo E et al.(2011). Surgery-triggered and non surgery-triggered Bisphosphonate-related Osteonecrosis of the Jaws (BRONJ): A retrospective analysis of 567 cases in an Italian multicenter study. Oral Oncol47(3) :191-194.
Washio K, Iwata T, Mizutani M, Ando T, Yamato M, Okano T et al.(2010). Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study. Cell and Tissue Research341(3) :397-404.
Yaji N, Yamato M, Yang J, Okano T, Hori S (2009). Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model. Biomaterials30(5) :797-803.
Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y (1990). Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Die Makromolekulare Chemie, Rapid Communications11(11) :571-576.
Yamato M, Utsumi M, Kushida A, Konno C, Kikuchi A, Okano T (2001). Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng7(4) :473-480.
Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M et al. (2007). Reconstruction of functional tissues with cell sheet engineering. Biomaterials 28(34):5033-5043.
Yoshida T, Washio K, Iwata T, Okano T, Ishikawa I (2012). Current status and future development of cell transplantation therapy for periodontal tissue regeneration. Int J Dent 2012(307024.