**Meet the editor**

Hideharu Hibi received his DDS and PhD degrees from Tokyo Medical and Dental University in 1987 and 1991, respectively. He served as an associate professor at the Center for Genetic and Regenerative Medicine in Nagoya University School of Medicine from 2004 to 2008. Since 2009, he has been an associate professor at the Department of Oral and Maxillofacial Surgery in Nagoya University Graduate School of Medicine. His academic interests include translational research on bone tissue engineering for maxillomandibular reconstruction.

Minoru Ueda received his DDS degree from Tokyo Medical and Dental University in 1978 and his PhD degree in medical science from Nagoya University in 1982. He was appointed professor and chair of the Clinical Department of Oral and Maxillofacial Surgery at Nagoya University School of Medicine in 1994. Recently, he served as a visiting professor of stem cell engineering at the Institute of Medical Science, University of Tokyo, Japan, in 2003 and Korea University Ansan Hospital, Korea, in 2011. He was appointed as chairman of the scientific advisory board at Japan Tissue Engineering, Inc., which, in 1999, became the first tissue engineering company to commercialize cultured skin. He received the "2004 President Award" from the Science Council of Japan for his contribution to the development of tissue-engineered skin and cartilage. His former titles include president of the Japanese Forum for Regenerative Dentistry, Japanese Tissue Engineering Society, and Asian Tissue Engineering Society, and vice-president of the Tissue Engineering International & Regenerative Medicine Society.

Contents

**Preface VII**

Yoshie

Hideto Saijo

**Regeneration 55**

**Cord Injury 67**

and Minoru Ueda

Chapter 1 **Cell Sheet Engineering for Periodontal Regeneration 1**

Chapter 2 **The Cell-Multilayered Periosteal Sheet — A Promising**

Misako Nakashima and Koichiro Iohara

Chapter 5 **Feasibility of Molecularly Targeted Therapy for Tooth**

Katsu Takahashi, Honoka Kiso, Kazuyuki Saito, Yumiko Togo, Hiroko Tsukamoto, Boyen Huang and Kazuhisa Bessho

Akihito Yamamoto, Kohki Matsubara, Kiyoshi Sakai, Fumiya Kano

Yoshinori Sumita, Izumi Asahina, Simon D. Tran, Hideki Agata,

Chapter 6 **Multifaceted Neuro-Regenerative Activities of Human Dental Pulp Stem Cells for Functional Recovery after Spinal**

Chapter 7 **Potential Cell-Based Therapies for Irreversibly Damaged Salivary Glands and Atrophic Alveolar Bone 81**

Minoru Inoue, Arinobu Tojo and Hideaki Kagami

Chapter 4 **Regenerative Medicine in the Oral and Maxillofacial Region 47**

Tomohiro Ando, Masayuki Yamato and Teruo Okano

Chapter 3 **Pulp Regeneration by Harnessing Dental Pulp Stem Cells 37**

**Osteogenic and Osteoinductive Grafting Material 19**

Takanori Iwata, Kaoru Washio, Toshiyuki Yoshida, Isao Ishikawa,

Tomoyuki Kawase, Kazuhiro Okuda, Masaki Nagata and Hiromasa

### Contents

#### **Preface XI**

Preface

duction.

sequent implantation *in vivo.*

in not only dentistry and also medicine.

This book has been edited through collection of all the achievement performed by the main members of The Forum of Tissue Engineering in Dentistry which was established in 2000 and it brought together the specific experiences of the scientific community as well as the clinical experiences of the most renowned experts in the field of all over dentistry. The edi‐ tors are especially proud of bringing together the leading biologists and dentists of all spe‐ cialties. Taken together, this unique collection of world-wide expert achievement and experiences represents the current spectrum of possibilities in tissue engineered substitution

Historically, tissue engineering, which aims at regenerating new tissues, as well as substitut‐ ing lost organs by making use of autogenic or allogeneic cells in combination with biomate‐ rials, is an emerging biomedical engineering field. There are several driving forces that presently make tissue engineering very challenging and important: 1) the limitations in bio‐ logical functions of current artificial tissues and organs made from man-made materials alone, 2) the shortage of donor tissues and organs for organ transplantation, 3) recent re‐ markable advances in regeneration mechanisms made by molecular biologists, as well as 4) achievements in modern biotechnology for large-scale tissue culture and growth factor pro‐

The idea of tissue engineering is not quite as new as it seems. The Nobel Laureate Alexis Carrel performed seminal work in the early 1900s that paved the way for today's tissue en‐ gineers. Carrel even caught the imagination of the pilot Charles Lindbergh. After his historic first solo flight across the Atlantic Ocean, Lindbergh worked with Carrel at the Rockefeller Institute in New York, with the goal of maintaining viable tissue and organs *in vitro* for sub‐

The earliest attempts at engineering tissue were carried out in skin by Bell, Yannas, and Green at the Massachusetts Institute of Technology in the late 1970s, early 1980s.Dr. Iannas Yannas, collaborated in studies both in the laboratory and in humans to generate a tissueengineered skin substitute using a collagen matrix to support the growth of dermal fibro‐ blasts. Dr. Eugine Bell later transferred sheets of keratinocytes with fibroblasts referring to them as contracted collagen gels. From a more clinical aspect, one of the most exciting ad‐ vances in culturing skin epithelium was carried out by Dr. Howard Green and it has been the ability to use these cells in treating patients with burns and other skin disorders. The technique of culturing human skin epithelium in defined media and the exciting possibility to multiply epithelial cells up to ten thousand fold the amount of the original skin sample has been available for more than three decades by now. Many scientific pioneers in the field of biology, surgery and other disciplines have added their efforts with enormous creativity

### Preface

Chapter 8 **Bone and Periodontal Tissue Regeneration by the Secretomes**

Chapter 9 **Whole Tooth Regeneration Using a Bioengineered Tooth 109**

Masamitsu Oshima and Takashi Tsuji

Wataru Katagiri

**VI** Contents

**from Bone Marrow Derived Mesenchymal Stem Cells 95**

This book has been edited through collection of all the achievement performed by the main members of The Forum of Tissue Engineering in Dentistry which was established in 2000 and it brought together the specific experiences of the scientific community as well as the clinical experiences of the most renowned experts in the field of all over dentistry. The edi‐ tors are especially proud of bringing together the leading biologists and dentists of all spe‐ cialties. Taken together, this unique collection of world-wide expert achievement and experiences represents the current spectrum of possibilities in tissue engineered substitution in not only dentistry and also medicine.

Historically, tissue engineering, which aims at regenerating new tissues, as well as substitut‐ ing lost organs by making use of autogenic or allogeneic cells in combination with biomate‐ rials, is an emerging biomedical engineering field. There are several driving forces that presently make tissue engineering very challenging and important: 1) the limitations in bio‐ logical functions of current artificial tissues and organs made from man-made materials alone, 2) the shortage of donor tissues and organs for organ transplantation, 3) recent re‐ markable advances in regeneration mechanisms made by molecular biologists, as well as 4) achievements in modern biotechnology for large-scale tissue culture and growth factor pro‐ duction.

The idea of tissue engineering is not quite as new as it seems. The Nobel Laureate Alexis Carrel performed seminal work in the early 1900s that paved the way for today's tissue en‐ gineers. Carrel even caught the imagination of the pilot Charles Lindbergh. After his historic first solo flight across the Atlantic Ocean, Lindbergh worked with Carrel at the Rockefeller Institute in New York, with the goal of maintaining viable tissue and organs *in vitro* for sub‐ sequent implantation *in vivo.*

The earliest attempts at engineering tissue were carried out in skin by Bell,Yannas, and Green at the Massachusetts Institute of Technology in the late 1970s, early 1980s.Dr. Iannas Yannas, collaborated in studies both in the laboratory and in humans to generate a tissueengineered skin substitute using a collagen matrix to support the growth of dermal fibro‐ blasts. Dr. Eugine Bell later transferred sheets of keratinocytes with fibroblasts referring to them as contracted collagen gels. From a more clinical aspect, one of the most exciting ad‐ vances in culturing skin epithelium was carried out by Dr. Howard Green and it has been the ability to use these cells in treating patients with burns and other skin disorders. The technique of culturing human skin epithelium in defined media and the exciting possibility to multiply epithelial cells up to ten thousand fold the amount of the original skin sample has been available for more than three decades by now. Many scientific pioneers in the field of biology, surgery and other disciplines have added their efforts with enormous creativity

and ambition to further improve this method. The introduction of this technique into burn treatment in the early eighties of the past century was followed by initial enthusiasm about the prospect of saving the lives of many extensively burned patients. Our early success in Nagoya experiences have started by following the Green's artificial skin. Because he kindly provided the 3T3-J2 cell line and it supported our research works.

tokines change the environment dramatically from sever condition chemically to the adequate condition for tissue repair. Then Mother Nature's repair cells, which are pro‐ gramed to mature and repair tissue damage of a certain magnitude, are activated. Therefore I can conclude that the main player for the tissue regeneration is not stem cells but the cyto‐ kines produced by the stem cell. From these speculations we have tried several experiments using the established model animals such as diabetes, hepatitis, stroke, spinal cord injury and so on. The results of the experimental studies were excellent and showed that stem cell based cytokines have almost the same ability as the stem cell for the regeneration in several organs. I believe that we establish a novel approach for tissue and organ regeneration by

**Minoru Ueda**

Japan

Preface IX

Department of Oral & Maxillofacial Surgery, Nagoya University Graduate School of Medicine,

using stem cell based cytokines.

Tissue engineering was catapulted to the forefront of public awareness with the airing of a BBC broadcast on the potential of tissue-engineered cartilage using images of the now-infa‐ mous "mouse with the human ear", fondly referred to as auriculosaurus, from the laborato‐ ry of Dr. Charles Vacanti at University of Massachusetts Medical Center. This has become known as tissue engineering, which were all based on the same premise, that new functional replacement tissue could be generated from living cells seeded onto appropriately config‐ ured scaffoldings. In the example of cartilage, viable chondrocytes were seeded onto porous polymer fibers and configured in the shape of the desired tissue. Other potential applica‐ tions of tissue engineering include the replacement of worn and poorly functioning tissues as exemplified by replacement of small caliber arteries, veins, bone and cartilage; replace‐ ment of the bladder, muscle and nerve tube; and restoration of cells to produce necessary enzymes, hormones, and other bioactive secretory products, such as salivary gland.

In spite of significant scientific progress in tissue engineering, there are few examples of hu‐ man application. Two potential explanations for this may be 1) problems associated with "scale up" and 2) cell death associated with implantation. Large numbers of cells are needed to generate relatively small volumes of tissues. These problems are always associated with the human application of tissue engineering concept. Fortunately, dentistry is the advanta‐ geous field because the volume of the newly formed tissue to be needed is relatively small compared with other fields of medicine. However, to ultimately be effective in humans, tis‐ sue engineering must generate relatively large volume of tissue, starting with the very few cells. Cell implantation and its associated vascular disruption result in a relatively hypoxic environment and cell death. The potential for different cell types to be expanded *in vitro* and survive a relatively hostile environment at the time of implantation is now being explored. To be effective, cells should be easily procured, be effectively expanded *in vitro* , survive the initial implantation, be accepted as self, function normally, and not become malignant.

Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) may have very similar potentials and risk to develop into the different cellular elements necessary for effec‐ tive tissue regeneration. ES cells have been postulated to retain a greater ability to produce healthier tissue despite its ethical issues. At the point in time, there is little evidence that iPS cells can be consistently driven to form only the cell type needed for the tissue to be engi‐ neered without any risk of carcinogenicity. Being derived from autogenic cells, iPS cells have the associated problem of malignancy.

It is my belief that some forms of tissue-specific adult stem cells are the most hopeful cell at this moment for clinical use because it may represent Mother Nature's repair cells. Such cells are potentially present within all of the tissue of the body and may remain dormant until they are activated in response to tissue injury. However the chemical environment such as a low oxygen and a poor nutrition at the site of any injury is very hostile. When adequate numbers of cells transplanted into the damaged tissue, the adult stem cell can't survive in this environment. The important thing is the cytokines produced by the trans‐ planted stem cell. They can recruit stem cells from the distant site of whole body. These cy‐ tokines change the environment dramatically from sever condition chemically to the adequate condition for tissue repair. Then Mother Nature's repair cells, which are pro‐ gramed to mature and repair tissue damage of a certain magnitude, are activated. Therefore I can conclude that the main player for the tissue regeneration is not stem cells but the cyto‐ kines produced by the stem cell. From these speculations we have tried several experiments using the established model animals such as diabetes, hepatitis, stroke, spinal cord injury and so on. The results of the experimental studies were excellent and showed that stem cell based cytokines have almost the same ability as the stem cell for the regeneration in several organs. I believe that we establish a novel approach for tissue and organ regeneration by using stem cell based cytokines.

and ambition to further improve this method. The introduction of this technique into burn treatment in the early eighties of the past century was followed by initial enthusiasm about the prospect of saving the lives of many extensively burned patients. Our early success in Nagoya experiences have started by following the Green's artificial skin. Because he kindly

Tissue engineering was catapulted to the forefront of public awareness with the airing of a BBC broadcast on the potential of tissue-engineered cartilage using images of the now-infa‐ mous "mouse with the human ear", fondly referred to as auriculosaurus, from the laborato‐ ry of Dr. Charles Vacanti at University of Massachusetts Medical Center. This has become known as tissue engineering, which were all based on the same premise, that new functional replacement tissue could be generated from living cells seeded onto appropriately config‐ ured scaffoldings. In the example of cartilage, viable chondrocytes were seeded onto porous polymer fibers and configured in the shape of the desired tissue. Other potential applica‐ tions of tissue engineering include the replacement of worn and poorly functioning tissues as exemplified by replacement of small caliber arteries, veins, bone and cartilage; replace‐ ment of the bladder, muscle and nerve tube; and restoration of cells to produce necessary

enzymes, hormones, and other bioactive secretory products, such as salivary gland.

In spite of significant scientific progress in tissue engineering, there are few examples of hu‐ man application. Two potential explanations for this may be 1) problems associated with "scale up" and 2) cell death associated with implantation. Large numbers of cells are needed to generate relatively small volumes of tissues. These problems are always associated with the human application of tissue engineering concept. Fortunately, dentistry is the advanta‐ geous field because the volume of the newly formed tissue to be needed is relatively small compared with other fields of medicine. However, to ultimately be effective in humans, tis‐ sue engineering must generate relatively large volume of tissue, starting with the very few cells. Cell implantation and its associated vascular disruption result in a relatively hypoxic environment and cell death. The potential for different cell types to be expanded *in vitro* and survive a relatively hostile environment at the time of implantation is now being explored. To be effective, cells should be easily procured, be effectively expanded *in vitro* , survive the initial implantation, be accepted as self, function normally, and not become malignant.

Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) may have very similar potentials and risk to develop into the different cellular elements necessary for effec‐ tive tissue regeneration. ES cells have been postulated to retain a greater ability to produce healthier tissue despite its ethical issues. At the point in time, there is little evidence that iPS cells can be consistently driven to form only the cell type needed for the tissue to be engi‐ neered without any risk of carcinogenicity. Being derived from autogenic cells, iPS cells

It is my belief that some forms of tissue-specific adult stem cells are the most hopeful cell at this moment for clinical use because it may represent Mother Nature's repair cells. Such cells are potentially present within all of the tissue of the body and may remain dormant until they are activated in response to tissue injury. However the chemical environment such as a low oxygen and a poor nutrition at the site of any injury is very hostile. When adequate numbers of cells transplanted into the damaged tissue, the adult stem cell can't survive in this environment. The important thing is the cytokines produced by the trans‐ planted stem cell. They can recruit stem cells from the distant site of whole body. These cy‐

have the associated problem of malignancy.

provided the 3T3-J2 cell line and it supported our research works.

VIII Preface

**Minoru Ueda**

Department of Oral & Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Japan

**Chapter 1**

**Cell Sheet Engineering for Periodontal Regeneration**

Periodontitis is a world-wide infectious disease that destroys the tooth-supporting attach‐ ment apparatus, which consists of alveolar bone, cementum, and periodontal ligament. Re‐ cent 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 (Offen‐ bacher et al., 1996). Furthermore, researches have recently shown that Bisphosphonate-Re‐ lated Osteonecrosis of the Jaws (BRONJ) is also associated with severe periodontitis (Vescovi et al., 2011). Therefore, periodontal treatment may not only contribute to oral hy‐ giene but also improvement of systemic conditions (Seymour et al., 2007). Conventional treatments, such as scaling, root-planing, and surgical cleaning, have been performed to re‐ move 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 with‐ out maintenance therapies (Axelsson and Lindhe, 1981). To overcome this problem, various regenerative therapies, such as guided tissue regeneration (GTR) and enamel matrix deriva‐ tive, 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 regener‐ ation 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 clini‐ cal predictability (Esposito et al., 2009). Therefore, stem cell-based approaches for periodon‐ tal regeneration have been studied and translated into clinical settings as the third

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Takanori Iwata, Kaoru Washio, Toshiyuki Yoshida,

Isao Ishikawa, Tomohiro Ando,

**1. Introduction**

Masayuki Yamato and Teruo Okano

Additional information is available at the end of the chapter

### **Cell Sheet Engineering for Periodontal Regeneration**

Takanori Iwata, Kaoru Washio, Toshiyuki Yoshida, Isao Ishikawa, Tomohiro Ando, Masayuki Yamato and Teruo Okano

Additional information is available at the end of the chapter

#### **1. Introduction**

Periodontitis is a world-wide infectious disease that destroys the tooth-supporting attach‐ ment apparatus, which consists of alveolar bone, cementum, and periodontal ligament. Re‐ cent 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 (Offen‐ bacher et al., 1996). Furthermore, researches have recently shown that Bisphosphonate-Re‐ lated Osteonecrosis of the Jaws (BRONJ) is also associated with severe periodontitis (Vescovi et al., 2011). Therefore, periodontal treatment may not only contribute to oral hy‐ giene but also improvement of systemic conditions (Seymour et al., 2007). Conventional treatments, such as scaling, root-planing, and surgical cleaning, have been performed to re‐ move 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 with‐ out maintenance therapies (Axelsson and Lindhe, 1981). To overcome this problem, various regenerative therapies, such as guided tissue regeneration (GTR) and enamel matrix deriva‐ tive, 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 regener‐ ation 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 clini‐ cal predictability (Esposito et al., 2009). Therefore, stem cell-based approaches for periodon‐ tal regeneration have been studied and translated into clinical settings as the third

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

were limited and associated with poor clinical predictability (Esposito et al., 2009). Therefore, stem cell-New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative Medicine 2

generation. In this chapter, we would like to describe the principles of "Cell Sheet Engineer‐ ing" and its application of clinical settings, featuring our recent translational research for pe‐ riodontal regeneration. 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.

based approaches for periodontal regeneration have been studied and translated into clinical settings as

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-cad‐ herin and laminin 5 that are typically destroyed in the process of enzymatic treatments (Ya‐ mato 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 transplanta‐

Cell Sheet Engineering for Periodontal Regeneration 3

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 com‐ pletely 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 (Shimi‐ zu 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 (Se‐ kine 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 *in vitro*, which may allow the graft to survive and function (Haraguchi et al., 2012; Pirraco et al., 2011). Furthermore, the three-dimensional manipulation of fibroblast cell sheets and micro-patterned endothelial cells with a gelatin-coated stacking manipulator produced microvascular-like networks within a 5-day *in vitro* culture (Tsuda et al., 2007). Non-patterned endothelial cell sheets and other types of cell sheets with a fibrin gel manipu‐ lator can also produce pre-vascular networks both *in vitro* (Asakawa et al., 2010) and *in vivo*

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

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

performed, and the efficacy of these cell sheets was evaluated in most of the studies.

tion (Takagi et al., 2011).

(Sasagawa et al., 2009).

*2.2.1. Corneal regeneration*

**2.2. Cell sheet transplantation in animal models**

#### **2. "Cell Sheet Engineering (CSE)" 2. "Cell Sheet Engineering (CSE)"**

**¶(14pt)**

**¶(6pt)**

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 dif‐ ficult to control. In contrast to approaches that utilize scaffolds, we have developed an alter‐ native technology for cell transplantation using temperature responsive culture dishes, which we call "Cell Sheet Engineering". 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". **¶(14pt)**

#### **2.1. Intelligent surface of** *N***-isopropylacrylamide (PIPAAm) and fabrication of cell sheets 2.1 Intelligent Surface of** *N***-isopropylacrylamide (PIPAAm) and Fabrication of Cell Sheets**

Poly(*N*-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been widely utilized for novel biomedical applications. We have developed a PIPAAm-grafted surface as a smart biointerface wherein cell attachment/detachment can be easily controlled by simply changing the temperature (Okano et al., 1995; Yamada et al., 1990). This surface is slightly hydrophobic under cell culture conditions of 37 °C, but readily becomes hydrated and hydrophilic below its lower critical solution temperature (LCST) of 32 °C. Cells can ad‐ here, spread, and proliferate similarly to that on ungrafted tissue culture grade polystyrene surfaces at 37 °C (Figure 1A), and cells detach from the surface by reducing temperature be‐ low LCST, making it possible to harvest the cells from the culture surfaces without the use of proteolytic enzymes (Figure 1B). Poly(*N*-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been widely utilized for novel biomedical applications. We have developed a PIPAAm-grafted surface as a smart biointerface wherein cell attachment/detachment can be easily controlled by simply changing the temperature (Okano et al., 1995; Yamada et al., 1990). This surface is slightly hydrophobic under cell culture conditions of 37 °C, but readily becomes hydrated and hydrophilic below its lower critical solution temperature (LCST) of 32 °C. Cells can adhere, spread, and proliferate similarly to that on ungrafted tissue culture grade polystyrene surfaces at 37 °C (Figure 1A), and cells detach from the surface by reducing temperature below LCST, making it possible to harvest the cells from the culture surfaces without the use of proteolytic enzymes (Figure 1B). ¶(9pt)

¶(4pt) **Figure 1.** The principle of "Cell Sheet Engineering".

Fig. 1. The principle of "Cell Sheet Engineering". A: Cells can attach and proliferate on grafted surface of the temperature responsive polymer (poly (Nisopropylacrylamide: PIPAAm) at 37 °C, wherein PIPAAm is extensively dehydrated and compact. B: At 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 dehy‐ drated 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 con‐ trol 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-cad‐ herin and laminin 5 that are typically destroyed in the process of enzymatic treatments (Ya‐ mato 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 transplanta‐ tion (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 com‐ pletely 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 (Shimi‐ zu 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 (Se‐ kine 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 *in vitro*, which may allow the graft to survive and function (Haraguchi et al., 2012; Pirraco et al., 2011). Furthermore, the three-dimensional manipulation of fibroblast cell sheets and micro-patterned endothelial cells with a gelatin-coated stacking manipulator produced microvascular-like networks within a 5-day *in vitro* culture (Tsuda et al., 2007). Non-patterned endothelial cell sheets and other types of cell sheets with a fibrin gel manipu‐ lator can also produce pre-vascular networks both *in vitro* (Asakawa et al., 2010) and *in vivo* (Sasagawa et al., 2009).

#### **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*

generation. In this chapter, we would like to describe the principles of "Cell Sheet Engineer‐ ing" and its application of clinical settings, featuring our recent translational research for pe‐

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

were limited and associated with poor clinical predictability (Esposito et al., 2009). Therefore, stem cellbased 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

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 dif‐ ficult to control. In contrast to approaches that utilize scaffolds, we have developed an alter‐ native technology for cell transplantation using temperature responsive culture dishes,

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

Poly(*N*-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been widely utilized for novel biomedical applications. We have developed a PIPAAm-grafted surface as a smart biointerface wherein cell attachment/detachment can be easily controlled by simply changing the temperature (Okano et al., 1995; Yamada et al., 1990). This surface is slightly hydrophobic under cell culture conditions of 37 °C, but readily becomes hydrated and hydrophilic below its lower critical solution temperature (LCST) of 32 °C. Cells can adhere, spread, and proliferate similarly to that on ungrafted tissue culture grade polystyrene surfaces at 37 °C (Figure 1A), and cells detach from the surface by reducing temperature below LCST, making it possible to harvest the cells from the culture surfaces

A: Cells can attach and proliferate on grafted surface of the temperature responsive polymer (poly (Nisopropylacrylamide: PIPAAm) at 37 °C, wherein PIPAAm is extensively dehydrated and compact. B: At

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 dehy‐ drated 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 con‐ trol cell attachment/detachment without any damages. Modified and reprint from Iwata et

**2.1. Intelligent surface of** *N***-isopropylacrylamide (PIPAAm) and fabrication of cell sheets** Poly(*N*-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been widely utilized for novel biomedical applications. We have developed a PIPAAm-grafted surface as a smart biointerface wherein cell attachment/detachment can be easily controlled by simply changing the temperature (Okano et al., 1995; Yamada et al., 1990). This surface is slightly hydrophobic under cell culture conditions of 37 °C, but readily becomes hydrated and hydrophilic below its lower critical solution temperature (LCST) of 32 °C. Cells can ad‐ here, spread, and proliferate similarly to that on ungrafted tissue culture grade polystyrene surfaces at 37 °C (Figure 1A), and cells detach from the surface by reducing temperature be‐ low LCST, making it possible to harvest the cells from the culture surfaces without the use

**2.1 Intelligent Surface of** *N***-isopropylacrylamide (PIPAAm) and Fabrication of Cell Sheets**

using temperature responsive culture dishes, which we call "Cell Sheet Engineering".

riodontal regeneration.

Medicine

2

**¶(6pt)**

regeneration. **¶(14pt)**

**2. "Cell Sheet Engineering (CSE)"**

**2. "Cell Sheet Engineering (CSE)"**

which we call "Cell Sheet Engineering".

**¶(14pt)**

of proteolytic enzymes (Figure 1B).

¶(9pt)

¶(4pt)

al., 2013.

**Figure 1.** The principle of "Cell Sheet Engineering".

without the use of proteolytic enzymes (Figure 1B).

Fig. 1. The principle of "Cell Sheet Engineering".

A B

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

> 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 heal‐

Cell Sheet Engineering for Periodontal Regeneration 5

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 ef‐ ficient 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 functional‐

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, pleu‐

Post-operative adhesions often cause severe complications such as bowel obstruction and abdominopelvic pain. The use of mesothelial cell sheets was investigated to prevent postoperative 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

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 cellto-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

Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contig‐

ing and possibly prevent postoperative esophageal stenosis.

ity, and the use of bilayered sheets enhanced these characteristics.

*2.2.7. Mesothelial cells for the prevention of post-operative adhesions*

ral defects were also closed by fibroblast sheets in pigs (Kanzaki et al., 2008).

than the use of injected isolated cell suspensions in rabbits (Yaji et al., 2009).

*2.2.6. Fibroblast sheet transplantation for sealing air leaks*

*2.2.8. Retinal Pigment Epithelial (RPE) cell regeneration*

*2.2.5. Hepatocyte regeneration*

adhesion formation.

*2.2.9. Urothelial regeneration*

fabricated and that their characteristics were similar to those of native tissues. Transplanta‐ tion 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 ma‐ trix 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 ex‐ pand 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 ob‐ served. 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 subcu‐ taneous 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 myocardi‐ um with a well-organized microvascular network. Other types of cell sheets were also exam‐ ined 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 man‐ ufacturing 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 prevent‐ ing 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 postop‐ erative 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 heal‐ ing and possibly prevent postoperative esophageal stenosis.

#### *2.2.5. Hepatocyte regeneration*

fabricated and that their characteristics were similar to those of native tissues. Transplanta‐ tion 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 ma‐ trix 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 ex‐ pand 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

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

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 ob‐ served. 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 subcu‐ taneous 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 myocardi‐ um with a well-organized microvascular network. Other types of cell sheets were also exam‐ ined 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

Chondrocyte sheets applicable to cartilage regeneration were prepared using cell sheet man‐ ufacturing 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 prevent‐ ing the loss of proteoglycan from these sites and for protection against catabolic factors in

With the recent development of endoscopic submucosal dissection (ESD), large esophageal cancers can be removed using a single procedure. However, complications, such as postop‐ erative inflammation and stenosis, frequently occur after an aggressive ESD procedure,

mucosal epithelium.

Medicine

4

*2.2.2. Cardiac regeneration*

efficacy of the method for cardiac repair.

the joints of rabbits (Kaneshiro et al., 2006).

*2.2.3. Cartilage regeneration*

*2.2.4. Esophageal 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 ef‐ ficient 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 functional‐ ity, 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, pleu‐ ral 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 postoperative 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 cellto-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, contig‐

uous urothelial cell sheets cultured *in vitro* should eliminate these complications. Canine ur‐ othelial cell sheets were grown and their structures were shown to be appropriate (Shiroyanagi et al., 2003). Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suturing or fixation, and generated a multi-layered uro‐ thelium *in vivo* (Shiroyanagi et al., 2004). The novel intact cell-sheet grafting method rapidly produced native-like epithelium *in vivo*.

*2.3.2. Endoscopic treatment of esophageal ulceration*

*2.3.3. Improvements in ischemic cardiomyopathy*

*2.3.4. Cartilage regeneration*

*loss*

of otitis media.

**3. Periodontal regeneration**

engineering" (Yang et al., 2007).

circumferential resection (Ohki et al., 2009; Ohki et al., 2012).

layers and transplanted into the cartilage defects of patients.

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 trans‐ planted, resulting in the complete prevention of stricture formation in patients with partial

Cell Sheet Engineering for Periodontal Regeneration 7

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

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

*2.3.5. Nasal mucosa epithelial cell sheet transplantation to the middle ear bone for preventing hearing*

A clinical trial for preventing hearing loss began in 2014 at The Jikei University, Japan. Au‐ tologous 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 pro‐ gression of fibroblast within middle ear cavity, which induce hearing loss after the surgery

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 pe‐ riodontal 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 demon‐ strated 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 in‐ teractions are well-preserved (Kumashiro et al., 2010). Thus, we have attempted to regener‐ ate this periodontal attachment apparatus based on the technology of "cell sheet

and discharged from the hospital without requiring a ventricular assisting device.

#### *2.2.10. Islet regeneration*

To establish a novel approach for diabetes mellitus, pancreatic islet cell sheets were fabricat‐ ed and transplanted in rats (Shimizu et al., 2009). Laminin-5 was coated on temperature re‐ sponsive 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 ad‐ ministration is indispensable to supplement the shortage of hormones. To verify the cyto‐ therapeutic 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 thyroi‐ dectomy 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 reconstruc‐ tion using autologous mucosal epithelial cells, and the results were published in 2004 (Nish‐ ida 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 mito‐ mycin C-treated 3T3 feeder layer and transplanted directly into the denuded corneal surfa‐ ces 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 cor‐ neal 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 trans‐ planted, 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*

uous urothelial cell sheets cultured *in vitro* should eliminate these complications. Canine ur‐ othelial cell sheets were grown and their structures were shown to be appropriate (Shiroyanagi et al., 2003). Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suturing or fixation, and generated a multi-layered uro‐ thelium *in vivo* (Shiroyanagi et al., 2004). The novel intact cell-sheet grafting method rapidly

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

To establish a novel approach for diabetes mellitus, pancreatic islet cell sheets were fabricat‐ ed and transplanted in rats (Shimizu et al., 2009). Laminin-5 was coated on temperature re‐ sponsive dishes to enhance the initial cell attachment, and the presence of specific

For hormonal deficiencies caused by endocrine organ diseases, continuous oral hormone ad‐ ministration is indispensable to supplement the shortage of hormones. To verify the cyto‐ therapeutic 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 thyroi‐ dectomy 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

In Japan, 6 clinical trials using cell sheet engineering technology have been started or have

The first clinical trial of the cell sheet engineering technology involved a corneal reconstruc‐ tion using autologous mucosal epithelial cells, and the results were published in 2004 (Nish‐ ida 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 mito‐ mycin C-treated 3T3 feeder layer and transplanted directly into the denuded corneal surfa‐ ces 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 cor‐ neal 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

molecules, such as insulin and glucagon, was also observed in the recipient site.

produced native-like epithelium *in vivo*.

*2.2.10. Islet regeneration*

Medicine

6

*2.2.11. Thyroid regeneration*

was observed long after the surgery.

already been completed.

*2.3.1. Corneal reconstruction*

surface. (Burillon et al., 2012).

**2.3. Cell sheet transplantation in human clinical trials**

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. Au‐ tologous 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 pro‐ gression 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 pe‐ riodontal 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 demon‐ strated 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 in‐ teractions are well-preserved (Kumashiro et al., 2010). Thus, we have attempted to regener‐ ate this periodontal attachment apparatus based on the technology of "cell sheet engineering" (Yang et al., 2007).

> dicularly into the newly formed cementum-like tissue, and this orientation resembled the native Sharpey's fibers. In addition, the regenerative potential of hPDL cell sheets cultured with the osteoinductive medium the was confirmed, when hPDL cell sheets were trans‐ planted onto the root surface of periodontal defects in athymic rat mandibles (Flores et al., 2008b). The results indicate that most of the specimens in the experimental group exhibited a newly-formed cementum and a new attachment of collagen fibers to the cementum layer. No clear cementum layer was observed in the control group (in the absence of osteoinduc‐ tive supplements). As shown in these experiments, hPDL cells cultured with osteoinductive

Cell Sheet Engineering for Periodontal Regeneration 9

medium could contribute to the simultaneous regeneration of cementum and PDL.

allow for the regeneration of periodontal tissues composed of hard and soft tissues.

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 perios‐ teal 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 pe‐

Based on the successful results from small animal studies, we next utilized canine periodon‐ tal defect models. Dog PDL (dPDL) cells were extracted using collagenase/dispase digestion. Four individual dPDL cells were successfully isolated and expanded *ex vivo*. Cells were cul‐ tured in a standard medium with osteoinductive supplements for 5 days, because longer cultivation induced spontaneous detachment of cell sheets from the UpCell Surfaces. Threelayered dPDL cell sheets were fabiricated with woven polyglycolic acid (PGA) for cell sheet transfer. This PGA product has a number of advantages, including: 1) cell sheets can be easi‐ ly peeled from temperature responsive dishes, because cell sheets can be attached to the fi‐ bers of the woven PGA, 2) the shrinkage of cell sheets can be prevented, 3) easy stacking of multi-layered cell sheets can be achived in a short period of time (see the video attached to the manuscript (Iwata et al., 2009)), 4) easy adjustment of different sizes of cell sheets can be used to cover any defect shape by simply trimming the cell sheets, 5) the ability to make contact on hard tissues and curved surfaces, and 6) the transplant is visible to the operators. dPDL cell sheets were transplanted into the surface of dental roots containing three-wall pe‐ riodontal defects in an autologous manner, and bone defects were filled with porous betatricalcium phosphate (β-TCP). Cell sheet transplantation regenerated both new bone and cementum connecting with the well-oriented collagen fibers, while only limited bone regen‐ eration was observed in the control group where cell sheet transplantation was not per‐ formed. These results suggest that PDL cells have multiple differentiation properties that

**3.2. Large animal studies**

riodontal regeneration.

#### **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 engineer‐ ing can be applied to periodontal regeneration.

¶(4pt) **Figure 2.** PDL regeneration at 4 weeks postsurgery.

Fig. 2. PDL regeneration at 4 weeks postsurgery. 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. A: Nontransplanted control site. B: hPDL transplanted experimental site. Regeneration of periodontal ligament-like structure was observed only in the experimental site. Azan stain‐ ing. Modified and reprint from Hasegawa et al., 2005.

¶(6pt) 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 *in vitro*, we compared hPDL cell sheets cultured in the absence and presence of these osteoinductive supplements in a xenogeneic transplantation model (Flores et al., 2008a). Three layered hPDL cell sheets were constructed with fibrin gel and transplanted with a human dentin block into the back of a subcutaneous athymic rat. The constructs were excised for histological investigation 6 weeks after the transplantation. The three-layered hPDL cell sheets-dentin block constructs induced a new cementum-like hard tissue on the surface of the 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 dexame‐ thasone, enhanced both osteoblastic/cementoblastic and the periodontal differentiation of PDL cells *in vitro*, we compared hPDL cell sheets cultured in the absence and presence of these osteoinductive supplements in a xenogeneic transplantation model (Flores et al., 2008a). Three layered hPDL cell sheets were constructed with fibrin gel and transplanted with a human dentin block into the back of a subcutaneous athymic rat. The constructs were excised for histological investigation 6 weeks after the transplantation. The three-layered hPDL cell sheets-dentin block constructs induced a new cementum-like hard tissue on the surface of the dentin in more than 60% of the samples. Collagen fibers were inserted perpen‐

dentin in more than 60% of the samples. Collagen fibers were inserted perpendicularly into the newly formed cementum-like tissue, and this orientation resembled the native Sharpey's fibers. In addition, the regenerative potential of hPDL cell sheets cultured with the osteoinductive medium the was confirmed, when hPDL cell sheets were transplanted onto the root surface of periodontal defects in athymic rat mandibles (Flores et al., 2008b). The results indicate that most of the specimens in the experimental group exhibited a newly-formed cementum and a new attachment of collagen fibers to the cementum layer. No clear cementum layer was observed in the control group (in the absence of osteoinductive supplements). As shown in these experiments, hPDL cells cultured with osteoinductive medium could contribute to the

simultaneous regeneration of cementum and PDL.

dicularly into the newly formed cementum-like tissue, and this orientation resembled the native Sharpey's fibers. In addition, the regenerative potential of hPDL cell sheets cultured with the osteoinductive medium the was confirmed, when hPDL cell sheets were trans‐ planted onto the root surface of periodontal defects in athymic rat mandibles (Flores et al., 2008b). The results indicate that most of the specimens in the experimental group exhibited a newly-formed cementum and a new attachment of collagen fibers to the cementum layer. No clear cementum layer was observed in the control group (in the absence of osteoinduc‐ tive supplements). As shown in these experiments, hPDL cells cultured with osteoinductive medium could contribute to the simultaneous regeneration of cementum and PDL.

#### **3.2. Large animal studies**

**3.1. Small animal studies**

Medicine

8

¶(4pt)

¶(6pt)

Fig. 2. PDL regeneration at 4 weeks postsurgery.

simultaneous regeneration of cementum and PDL.

from Hasegawa et al., 2005.

ing. Modified and reprint from Hasegawa et al., 2005.

**Figure 2.** PDL regeneration at 4 weeks postsurgery.

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

A: Nontransplanted control site. B: hPDL transplanted experimental site. Regeneration of periodontal ligament-like structure was observed only in the experimental site. Azan stain‐

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 dexame‐ thasone, enhanced both osteoblastic/cementoblastic and the periodontal differentiation of PDL cells *in vitro*, we compared hPDL cell sheets cultured in the absence and presence of these osteoinductive supplements in a xenogeneic transplantation model (Flores et al., 2008a). Three layered hPDL cell sheets were constructed with fibrin gel and transplanted with a human dentin block into the back of a subcutaneous athymic rat. The constructs were excised for histological investigation 6 weeks after the transplantation. The three-layered hPDL cell sheets-dentin block constructs induced a new cementum-like hard tissue on the surface of the dentin in more than 60% of the samples. Collagen fibers were inserted perpen‐

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 *in vitro*, we compared hPDL cell sheets cultured in the absence and presence of these osteoinductive supplements in a xenogeneic transplantation model (Flores et al., 2008a). Three layered hPDL cell sheets were constructed with fibrin gel and transplanted with a human dentin block into the back of a subcutaneous athymic rat. The constructs were excised for histological investigation 6 weeks after the transplantation. The three-layered hPDL cell sheets-dentin block constructs induced a new cementum-like hard tissue on the surface of the dentin in more than 60% of the samples. Collagen fibers were inserted perpendicularly into the newly formed cementum-like tissue, and this orientation resembled the native Sharpey's fibers. In addition, the regenerative potential of hPDL cell sheets cultured with the osteoinductive medium the was confirmed, when hPDL cell sheets were transplanted onto the root surface of periodontal defects in athymic rat mandibles (Flores et al., 2008b). The results indicate that most of the specimens in the experimental group exhibited a newly-formed cementum and a new attachment of collagen fibers to the cementum layer. No clear cementum layer was observed in the control group (in the absence of osteoinductive supplements). As shown in these experiments, hPDL cells cultured with osteoinductive medium could contribute to the

ing can be applied to periodontal regeneration.

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 engineer‐

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

A B

Based on the successful results from small animal studies, we next utilized canine periodon‐ tal defect models. Dog PDL (dPDL) cells were extracted using collagenase/dispase digestion. Four individual dPDL cells were successfully isolated and expanded *ex vivo*. Cells were cul‐ tured in a standard medium with osteoinductive supplements for 5 days, because longer cultivation induced spontaneous detachment of cell sheets from the UpCell Surfaces. Threelayered dPDL cell sheets were fabiricated with woven polyglycolic acid (PGA) for cell sheet transfer. This PGA product has a number of advantages, including: 1) cell sheets can be easi‐ ly peeled from temperature responsive dishes, because cell sheets can be attached to the fi‐ bers of the woven PGA, 2) the shrinkage of cell sheets can be prevented, 3) easy stacking of multi-layered cell sheets can be achived in a short period of time (see the video attached to the manuscript (Iwata et al., 2009)), 4) easy adjustment of different sizes of cell sheets can be used to cover any defect shape by simply trimming the cell sheets, 5) the ability to make contact on hard tissues and curved surfaces, and 6) the transplant is visible to the operators. dPDL cell sheets were transplanted into the surface of dental roots containing three-wall pe‐ riodontal defects in an autologous manner, and bone defects were filled with porous betatricalcium phosphate (β-TCP). Cell sheet transplantation regenerated both new bone and cementum connecting with the well-oriented collagen fibers, while only limited bone regen‐ eration was observed in the control group where cell sheet transplantation was not per‐ formed. These results suggest that PDL cells have multiple differentiation properties that allow for the regeneration of periodontal tissues composed of hard and soft tissues.

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 perios‐ teal 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 pe‐ riodontal regeneration.

> come of this trial is to evaluate the safety and efficacy of autologous transplantation of perio‐ dontal ligament cell sheets. As of the end of May in 2014, 10 cases of autologous PDL cell

> 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 re‐

> 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.

, Toshiyuki Yoshida1

2 Department of Oral and Maxillofacial Surgery, Tokyo Women's Medical University, Japan

[1] Arauchi A, Shimizu T, Yamato M, Obara T, Okano T (2009). Tissue-engineered thy‐ roid cell sheet rescued hypothyroidism in rat models after receiving total thyroidec‐ tomy comparing with nontransplantation models. *Tissue Eng Part A* 15(12)*:*3943-3949.

, Isao Ishikawa1

, Tomohiro Ando2

Cell Sheet Engineering for Periodontal Regeneration 11

,

sheets were transplanted, and the healing process took place uneventfully.

generative medicine (Elloumi-Hannachi et al., 2010).

**4. Conclusion**

**Acknowledgements**

**Author details**

Masayuki Yamato1

**References**

Takanori Iwata1,2, Kaoru Washio1

and Teruo Okano1

1 Institute of Advanced Biomedical Engineering and Science, Japan

#### **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. Howev‐ er, appropriate method for the extraction and expansion of hPDL cells are still not well un‐ derstood. Thus, we determined the optimal method of isolation and expansion of hPDL cells and then examined their gene expression levels and differentiation potentials, and eventual‐ ly 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 prolif‐ eration 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 pre‐ pared 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 con‐ firmed using conventional tests. Safety was evaluated using the following tests: 1) the softagar 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) rele‐ vant medical conditions contraindicating surgical interventions (e.g., diabetes mellitus, car‐ diovascular, 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 out‐ come of this trial is to evaluate the safety and efficacy of autologous transplantation of perio‐ dontal 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.

#### **4. Conclusion**

**3.3. Optimization of human PDL cells**

Medicine

10

phenotypes of hPDL cell sheets were determined.

the procedures.

**3.4. The clinical trial**

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. Howev‐ er, appropriate method for the extraction and expansion of hPDL cells are still not well un‐ derstood. Thus, we determined the optimal method of isolation and expansion of hPDL cells and then examined their gene expression levels and differentiation potentials, and eventual‐ ly 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 prolif‐ eration 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

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

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 pre‐ pared 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 con‐ firmed using conventional tests. Safety was evaluated using the following tests: 1) the softagar 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

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) rele‐ vant medical conditions contraindicating surgical interventions (e.g., diabetes mellitus, car‐ diovascular, 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 out‐ 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 re‐ generative medicine (Elloumi-Hannachi et al., 2010).

#### **Acknowledgements**

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.

#### **Author details**

Takanori Iwata1,2, Kaoru Washio1 , Toshiyuki Yoshida1 , Isao Ishikawa1 , Tomohiro Ando2 , Masayuki Yamato1 and Teruo Okano1

#### **References**

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Cell Sheet Engineering for Periodontal Regeneration 13

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New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

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[54] Tsuda Y, Shimizu T, Yamato M, Kikuchi A, Sasagawa T, Sekiya S *et al.* (2007). Cellu‐ lar control of tissue architectures using a three-dimensional tissue fabrication techni‐

[55] 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

[56] Vescovi P, Campisi G, Fusco V, Mergoni G, Manfredi M, Merigo E *et al.* (2011). Sur‐ gery-triggered and non surgery-triggered Bisphosphonate-related Osteonecrosis of the Jaws (BRONJ): A retrospective analysis of 567 cases in an Italian multicenter

[57] Washio K, Iwata T, Mizutani M, Ando T, Yamato M, Okano T *et al.* (2010). Assess‐ ment of cell sheets derived from human periodontal ligament cells: a pre-clinical

[58] Yaji N, Yamato M, Yang J, Okano T, Hori S (2009). Transplantation of tissue-engi‐ neered retinal pigment epithelial cell sheets in a rabbit model. *Biomaterials* 30(5)*:*

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[60] Yamato M, Utsumi M, Kushida A, Konno C, Kikuchi A, Okano T (2001). Thermo-re‐ sponsive culture dishes allow the intact harvest of multilayered keratinocyte sheets

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**Chapter 2**

**The Cell-Multilayered Periosteal Sheet — A Promising**

The present is an exciting time in the field of regenerative biology in dental medicine. Many basic and clinical studies have been performed to improve periodontal therapy and rehabili‐ tation therapy for morphological and functional recovery of the maxillofacial bones. Many materials, e.g., hydroxyapatite, GTR membrane, enamel matrix derivative, and platelet-rich plasma, have been applied to osseous defects to achieve significant outcomes. In contrast, cell therapy, as an advanced tissue-engineering technology, using bone marrow stem cells, periosteal cells, or periodontal ligament cells have just been started in several university

The periosteum has been recognized as a promising source of immature osteogenic progenitor cells. Osteogenic cells are important components as a promoter of bone regenerative therapy because their bioactivities are crucial to reconstituting an active site of controlled bone formation. We demonstrated that implantation of cultured periosteal sheets into nude mice could induce the formation of bone-like tissue [Kawase *et al*., 2009]. In addition, we established regenerative surgery with autologous cultured periosteal sheets in combination with plateletrich plasma (PRP) and hydroxyapatite (HA) in periodontitis patients (Fig. 1) [Yamamiya *et al*., 2008, Okuda *et al*.,2013]. Furthermore, we reported remarkable bone remodeling in reduced alveolar bone from autologous bone grafting with cultured periosteal sheets [Nagata *et al*.,

In this chapter, we will introduce the characteristics of periosteal sheets and clinical outcome by cell-based therapy in patients with periodontitis and alveolar jaw reduction. In addition,

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

future perspectives on regenerative therapy will be described.

**Osteogenic and Osteoinductive Grafting Material**

Tomoyuki Kawase, Kazuhiro Okuda, Masaki Nagata and Hiromasa Yoshie

http://dx.doi.org/10.5772/58902

hospitals and institutes in Japan.

**1. Introduction**

2012].

Additional information is available at the end of the chapter

## **The Cell-Multilayered Periosteal Sheet — A Promising Osteogenic and Osteoinductive Grafting Material**

Tomoyuki Kawase, Kazuhiro Okuda, Masaki Nagata and Hiromasa Yoshie

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58902

#### **1. Introduction**

The present is an exciting time in the field of regenerative biology in dental medicine. Many basic and clinical studies have been performed to improve periodontal therapy and rehabili‐ tation therapy for morphological and functional recovery of the maxillofacial bones. Many materials, e.g., hydroxyapatite, GTR membrane, enamel matrix derivative, and platelet-rich plasma, have been applied to osseous defects to achieve significant outcomes. In contrast, cell therapy, as an advanced tissue-engineering technology, using bone marrow stem cells, periosteal cells, or periodontal ligament cells have just been started in several university hospitals and institutes in Japan.

The periosteum has been recognized as a promising source of immature osteogenic progenitor cells. Osteogenic cells are important components as a promoter of bone regenerative therapy because their bioactivities are crucial to reconstituting an active site of controlled bone formation. We demonstrated that implantation of cultured periosteal sheets into nude mice could induce the formation of bone-like tissue [Kawase *et al*., 2009]. In addition, we established regenerative surgery with autologous cultured periosteal sheets in combination with plateletrich plasma (PRP) and hydroxyapatite (HA) in periodontitis patients (Fig. 1) [Yamamiya *et al*., 2008, Okuda *et al*.,2013]. Furthermore, we reported remarkable bone remodeling in reduced alveolar bone from autologous bone grafting with cultured periosteal sheets [Nagata *et al*., 2012].

In this chapter, we will introduce the characteristics of periosteal sheets and clinical outcome by cell-based therapy in patients with periodontitis and alveolar jaw reduction. In addition, future perspectives on regenerative therapy will be described.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**2.1. Tissue-like thickness**

Dispersed periosteal cells (living)

**Table 1.** The apparent stiffness of periosteal sheets.

vitro under our static culture expansion conditions.

**Figure 2.** Effects of culture media on the morphology of periosteal sheets.

Saggital paraffin sections were stained with hematoxylin & eosin.

Because of the cell-multilayered structure, as shown in Figure 2 (Left), the periosteal sheet is substantially thicker than a monolayer cell sheet [Kawase *et al*., 2009]. In addition, the thickness of the periosteal sheet can be enhanced by many types of commercially available stem-cell culture media [Uematsu *et al*., 2013a, 2013b] (Fig. 2 Right). However, intact periosteum tissue is hardly excised for expansion culture, and the native periosteum-like structure, dual-layers composed of the cambium and fibrous layers, could not be newly formed or regenerated in

The Cell-Multilayered Periosteal Sheet — A Promising Osteogenic ...

http://dx.doi.org/10.5772/58902

21

Periosteal sheets were expanded with the conventional growth medium (Medium 199 supplemented with 10% FBS) or a commercial stem-cell medium (MesenCult) for 35 days.

Interestingly, as far as we could attempt and achieve, similar cell-multilayered structures can barely be formed by similar expansion cultures of other connective tissues, such as epidermal and periodontal ligament tissue [Kawase *et al*., Unpublished observations]. It is also impossible to form similar structures using dispersed single periosteal cell populations in conventional two-dimensional (2D) culture systems. However, using appropriate scaffolding materials, we developed a 3D culture system for preparation of similar periosteal sheets [Kawase *et al*., 2014].

**Material Region Stiffness n** Periosteal sheet central 5.59 ± 3.24 kPa 76 Periosteal sheet peripheral 4.35 ± 2.69 kPa 63 Dispersed periosteal cells 14.3 ± 9.42 kPa 118

Tissue culture polystyrene 2.34 ± 1.54 GPa 79

Stiffness was evaluated by the nanoindentation technique using an atomic force microscopy.

9.04 ± 9.20 kPa 54

**Figure 1.** Cultured periosteal sheet application to infrabony defects in patients with periodontitis

#### **2. Biological characteristics of the cultured periosteal sheet**

Our periosteal sheets are prepared using static explant cultures of small pieces of alveolar periosteum tissue. The number of cells can be expanded at least by 1000 times by the end of the 6-week preparation, and the resulting periosteal sheet is mechanically tough enough to be picked up by forceps [Kawase *et al*., 2009]. In our experience preparing more than 2000 periosteal sheets, we confirmed that the growth is not significantly dependent either on age or sex. The successful preparation is rather dependent on the cell density in excised periosteum tissue segments. It is also important to handle the excised periosteum tissue in a timely manner, especially in the initial preparation of the "cut and paste" onto culture dishes because this procedure includes a time-sensitive semi-drying process for facilitating tissue segment attachment onto the plastic surface.

We developed this grafting material as a substitute for crushed autologous bone, a gold standard osteogenic grafting material. Therefore, the periosteal sheets are expected to express osteogenic and osteoinductive capabilities besides (or rather than) the osteoconductive capability displayed by most recent bone filler made of bioactive ceramics.

In our basic studies using in vitro cell culture systems and in vivo animal implantation systems, we have demonstrated several remarkable characteristics of the periosteal sheet, which are summarized below:

**Tissue-like thickness:** The periosteal sheet displays a unique structure that is composed of cell-multilayers and abundantly deposited extracellular matrices (ECM).

**Osteogenicity:** Periosteal cells within the periosteal sheet differentiate to osteogenic cells slowly but spontaneously with time of expansion using a conventional medium.

**Osteoinduction:** The periosteal sheet produces the major growth factors involved in bone metabolism.

#### **2.1. Tissue-like thickness**

**Figure 1.** Cultured periosteal sheet application to infrabony defects in patients with periodontitis

**2. Biological characteristics of the cultured periosteal sheet**

capability displayed by most recent bone filler made of bioactive ceramics.

cell-multilayers and abundantly deposited extracellular matrices (ECM).

slowly but spontaneously with time of expansion using a conventional medium.

attachment onto the plastic surface.

summarized below:

Medicine

20

metabolism.

Our periosteal sheets are prepared using static explant cultures of small pieces of alveolar periosteum tissue. The number of cells can be expanded at least by 1000 times by the end of the 6-week preparation, and the resulting periosteal sheet is mechanically tough enough to be picked up by forceps [Kawase *et al*., 2009]. In our experience preparing more than 2000 periosteal sheets, we confirmed that the growth is not significantly dependent either on age or sex. The successful preparation is rather dependent on the cell density in excised periosteum tissue segments. It is also important to handle the excised periosteum tissue in a timely manner, especially in the initial preparation of the "cut and paste" onto culture dishes because this procedure includes a time-sensitive semi-drying process for facilitating tissue segment

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

We developed this grafting material as a substitute for crushed autologous bone, a gold standard osteogenic grafting material. Therefore, the periosteal sheets are expected to express osteogenic and osteoinductive capabilities besides (or rather than) the osteoconductive

In our basic studies using in vitro cell culture systems and in vivo animal implantation systems, we have demonstrated several remarkable characteristics of the periosteal sheet, which are

**Tissue-like thickness:** The periosteal sheet displays a unique structure that is composed of

**Osteogenicity:** Periosteal cells within the periosteal sheet differentiate to osteogenic cells

**Osteoinduction:** The periosteal sheet produces the major growth factors involved in bone

Because of the cell-multilayered structure, as shown in Figure 2 (Left), the periosteal sheet is substantially thicker than a monolayer cell sheet [Kawase *et al*., 2009]. In addition, the thickness of the periosteal sheet can be enhanced by many types of commercially available stem-cell culture media [Uematsu *et al*., 2013a, 2013b] (Fig. 2 Right). However, intact periosteum tissue is hardly excised for expansion culture, and the native periosteum-like structure, dual-layers composed of the cambium and fibrous layers, could not be newly formed or regenerated in vitro under our static culture expansion conditions.

**Figure 2.** Effects of culture media on the morphology of periosteal sheets.

Periosteal sheets were expanded with the conventional growth medium (Medium 199 supplemented with 10% FBS) or a commercial stem-cell medium (MesenCult) for 35 days. Saggital paraffin sections were stained with hematoxylin & eosin.

Interestingly, as far as we could attempt and achieve, similar cell-multilayered structures can barely be formed by similar expansion cultures of other connective tissues, such as epidermal and periodontal ligament tissue [Kawase *et al*., Unpublished observations]. It is also impossible to form similar structures using dispersed single periosteal cell populations in conventional two-dimensional (2D) culture systems. However, using appropriate scaffolding materials, we developed a 3D culture system for preparation of similar periosteal sheets [Kawase *et al*., 2014].

Stiffness was evaluated by the nanoindentation technique using an atomic force microscopy.

**Table 1.** The apparent stiffness of periosteal sheets.

On the other hand, from a biomechanical point of view, ECM-enriched structures function as a soft scaffolding material for proliferating periosteal cells. As shown in Table 1, the stiffness of a conventional plastic dish is several GPa, whereas that of a light-fixed periosteal sheet is 4-6 kPa [Horimizu *et al*., 2013]. Because stiffer materials are in general more suitable for cell mitosis and proliferation [Vincent *et al*., 2013], it can be predicted that conventional culture dishes provide more preferable places for periosteal cells to proliferate. This is most likely a major reason why dispersed periosteal cells grow faster than cells contained in a periosteal sheet. It has also been reported that the optical stiffness for the osteoblastic differentiation is 25-40 kPa, and softer material surfaces maintain cells in the osteoblast lineage at immature stages [Engler *et al*., 2006]. Therefore, it is presumable that a thicker periosteal sheet could be suitable for the expansion of immature osteoprogenitor cells without inducing the osteoblastic differentiation.

#### **2.2. Osteogenicity**

As a promising osteogenic grafting material, a periosteal sheet should produce mineral deposits in vitro and form bone-like tissue in vivo. In conventional growth medium, Medium 199 supplemented with 10% fetal bovine serum (FBS), periosteal cells gradually and increas‐ ingly express alkaline phosphatase (ALP) with expansion time [Kawase *et al*., 2009]. The addition of chemical agents involved in osteoblastic differentiation, such as dexamethasone, β-glycerophosphate, and ascorbate, immediately increases ALP activity and simultaneously mineral deposit formation [Kawase *et al*., 2009, 2010, 2011].

**Phenotype Genes**

**Table 2.** Effects of the osteoinduction on the growth factor production by the periosteal sheet.

**Changes Growth factors**

moderate IL-6 MCP-1, TPO

moderate GRO-α, IL-8, IL-13, IGFBP-2 substantial GRO, IL-5, IL-7, IL-10, MCP-3

Osteoclast TRAP, Cathepsin K, Osteoclast-like cell cDNA

Osteoblast MSX2, ALP, BMP4, Bone gla protein, IGF2

Osteoblast RUNX2, SatB2, Osteoprotegerin, BMP1, TGFβ1, integrin α5β1, ICAM1

Growth factors released by the control and the osteoinduced periosteal sheets to the low-serum culture medium were

The Cell-Multilayered Periosteal Sheet — A Promising Osteogenic ...

http://dx.doi.org/10.5772/58902

23

The human periosteal sheets, which were implanted into the subcutaneous tissue of nude mice, were retrieved along with the surrounding mouse tissue. Mouse-specific mRNAs expressed in the surrounding tissue were evaluated by an

We should again emphasize that the periosteal sheet is characterized by its unique structure. Dispersed periosteal cells and other cells in the osteoblast lineage expanded in 2D culture systems are capable of producing growth factors involved in bone metabolism and forming mineral deposits as well as the periosteal sheet. However, these cells require appropriate scaffolding materials for implantation. In addition, a 3D culture system without using appropriate scaffolding materials, such as accumulation of cell sheets and large-scale spheroid

**Table 3.** Global DNA microarray analyses of gene expression in the surrounding mouse connective tissue.

Upregulation (> twofolds)

control > differentiated

control < differentiated

Downregulation (> twofolds)

**2.4. Conclusion**

Blood vessel Tie1

Stem cell SOX2

Osteoclast CSF1

Agilent Mouse Genome (4 x 44K) microarray.

Chondrocyte SOX9, COL2

semi-quantitatively evaluated using an antibody array.

When implanted into the subcutaneous tissue of nude mice, these periosteal sheets, regardless of their differentiation stages, are capable of forming osteoid and bone-like tissue more potently than dispersed periosteal cells combined with porous hydroxyapatite blocks [Kawase *et al*., 2009, Uematsu *et al*., 2013a]. In addition, several periosteal cells, especially those embed‐ ded in dense collagen matrices, become osteocyte-like cells. These data suggest the "osteoge‐ nicity" of the periosteal sheet.

#### **2.3. Osteoinduction**

We have detected major growth factors, such as several interleukins, FGF-2 (fibroblast growth factor-2), MCP-1 (monocyte chemotactic protein-1), TPO (thrombopoeitin), IGFBP-2 (insulinlike growth factor binding protein-2), GRO (growth-related oncogene) and VEGF (vascular endothelial growth factor) in conditioned media of the periosteal sheet [Kawase *et al*., 2009, Kobayashi *et al*., Manuscript in submission]. The osteogenic induction using chemical agents drastically changed the profile of cytokine release, as shown in Table 2.

Following implantation of periosteal sheets into the subcutaneous tissue of nude mice, global DNA microarray analyses demonstrated that the periosteal sheet influences the surrounding host tissue and induces osteoclastic phenotypes (Table 3). We also observed that the osteo‐ blastic phenotypes could be induced by the implanted periosteal sheet. In addition, the osteoinduced periosteal sheet is potently capable of inducing angiogenesis and osteoclastic formation. Therefore, these data suggest "osteoinduction" by the periosteal sheet.

Growth factors released by the control and the osteoinduced periosteal sheets to the low-serum culture medium were semi-quantitatively evaluated using an antibody array.

**Table 2.** Effects of the osteoinduction on the growth factor production by the periosteal sheet.

The human periosteal sheets, which were implanted into the subcutaneous tissue of nude mice, were retrieved along with the surrounding mouse tissue. Mouse-specific mRNAs expressed in the surrounding tissue were evaluated by an Agilent Mouse Genome (4 x 44K) microarray.

**Table 3.** Global DNA microarray analyses of gene expression in the surrounding mouse connective tissue.

#### **2.4. Conclusion**

On the other hand, from a biomechanical point of view, ECM-enriched structures function as a soft scaffolding material for proliferating periosteal cells. As shown in Table 1, the stiffness of a conventional plastic dish is several GPa, whereas that of a light-fixed periosteal sheet is 4-6 kPa [Horimizu *et al*., 2013]. Because stiffer materials are in general more suitable for cell mitosis and proliferation [Vincent *et al*., 2013], it can be predicted that conventional culture dishes provide more preferable places for periosteal cells to proliferate. This is most likely a major reason why dispersed periosteal cells grow faster than cells contained in a periosteal sheet. It has also been reported that the optical stiffness for the osteoblastic differentiation is 25-40 kPa, and softer material surfaces maintain cells in the osteoblast lineage at immature stages [Engler *et al*., 2006]. Therefore, it is presumable that a thicker periosteal sheet could be suitable for the expansion of immature osteoprogenitor cells without inducing the osteoblastic

New Trends in Tissue Engineering and Regenerative Medicine - Official Book of the Japanese Society for Regenerative

As a promising osteogenic grafting material, a periosteal sheet should produce mineral deposits in vitro and form bone-like tissue in vivo. In conventional growth medium, Medium 199 supplemented with 10% fetal bovine serum (FBS), periosteal cells gradually and increas‐ ingly express alkaline phosphatase (ALP) with expansion time [Kawase *et al*., 2009]. The addition of chemical agents involved in osteoblastic differentiation, such as dexamethasone, β-glycerophosphate, and ascorbate, immediately increases ALP activity and simultaneously