Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by infiltrates of lymphocytes in the salivary glands. In this disease, the immune system attacks the salivary glands, particularly the acinar cells. This causes an irreversible damage that is accompanied with a progressive loss of fluid-secreting acinar cells, and leads to a considerable decrease of saliva secretion. The challenge of successful cell-based therapy for autoimmune diseases is not only to regenerate the tissue but also to prevent it from the same autoimmune attack that was responsible for its destruction at the first place (Kodama et al., 2003). Following the abovementioned concept, we initially copied a therapy proposed by Faustman and colleagues to reverse end-stage diabetes in Non-obese diabetic (NOD) mouse, but for the treatment of SSlike disease (Tran et al., 2007). NOD mice develop SS-like disease and a progressive loss of saliva secretion. This therapy had two components. First, it was an injection of complete Freund's adjuvant (CFA) to induce endogenous TNF-α to exterminate autoreactive T lym‐ phocytes. The second component was the transplantation of MHC class I-matched normal spleen cells. As a result, our group showed that injections of CFA combined with spleen cells restored salivary function in NOD mice with SS-like disease (Tran et al., 2007). However for future clinical consideration, spleen cells are not easily obtained from patients. Therefore our group modified the Faustman's two-component therapy by using BMDCs (Khalili et al., 2010). The spleen and bone marrow are closely related organs, and both are among the first sites of hematopoiesis during gestation. We injected CFA and MHC class I-matched normal BMDCs in 7-week-old NOD mice, which had not yet developed SS. At 52 weeks posttreatment, we found that all NOD mice receiving BMDCs and CFA had a recovery of saliva secretion and were protected from SS and diabetes. Although a very small number of donor BMDCs had differentiated into salivary epithelial cells (<0.1%), including acinar cells was observed, the size of focus score (the number of foci, infiltrated lymphocytes) was bigger in the non-treated mice (Figure 2).

(Modified from Khalili et al., 2010)

of-concept of the paracrine effect of (BMDCs) cell-based therapy, our group recently demon‐ strated that the injection of bone marrow cell-extracts (a bioactive lysate of BMDCs) could be advantageously used to repair the radiogenic dysfunction of salivary glands rather than BMDCs transplantation (Tran et al., 2013). Although the mechanisms of regeneration are not well understood at the present, these findings provided a promising result for future clinical

**Figure 1.** Salivary flow rate recovery after BMDCs transplantation at 8, 16, 24 weeks post-irradiation. SFR were higher

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

in BMDC-transplanted mice (18Gy+BMDC) when compared to non-transplanted ones (18Gy).

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by infiltrates of lymphocytes in the salivary glands. In this disease, the immune system attacks the salivary glands, particularly the acinar cells. This causes an irreversible damage that is accompanied with a progressive loss of fluid-secreting acinar cells, and leads to a considerable decrease of saliva secretion. The challenge of successful cell-based therapy for autoimmune diseases is not only to regenerate the tissue but also to prevent it from the same autoimmune attack that was responsible for its destruction at the first place (Kodama et al., 2003). Following the abovementioned concept, we initially copied a therapy proposed by Faustman and colleagues to reverse end-stage diabetes in Non-obese diabetic (NOD) mouse, but for the treatment of SSlike disease (Tran et al., 2007). NOD mice develop SS-like disease and a progressive loss of saliva secretion. This therapy had two components. First, it was an injection of complete Freund's adjuvant (CFA) to induce endogenous TNF-α to exterminate autoreactive T lym‐ phocytes. The second component was the transplantation of MHC class I-matched normal

trials of cell-based therapy using BMDCs or MSCs.

**2.3. Behaviors of donor BMDCs in SS model**

(Modified from Sumita et al., 2011)

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**Figure 2.** Focus score change after BMDCs transplantation. The size of the focus score (black arrows) was bigger in non-treated mice (Non-treated NOD) when compared to BMDC-treated mice (BMDC-treated NOD).

This study suggests that a combined immuno and cell-based therapy can permanently prevent SS and restore the salivary function in NOD mice through mechanisms involving the paracrine and immunomodulatory effects. Moreover, we analyzed whether this BMDC-treatment would be effective in restoring salivary gland function if the treatment was given at a late phase of SS (20-week old NOD) when minimal saliva was secreted. In consequence, saliva secretion improved and was at best 50 % of pre-symptomatic levels. The treatment also decreased TNFα and TGF-β1 levels while increasing EGF and regulatory T cells. This study provided that the cell-based therapy using BMDCs holds a promising effect, even when given in an advanced phase of SS. Likewise, we have also confirmed MSCs derived from bone marrow display similar effectiveness (Khalili et al., 2012). The combined use of CFA and MSCs was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands. Thus, cell-based therapy must be recognized as one of the promising options for future clinical trials. Recently, our group has also shown that the injection of bone marrow cell extracts (a

bioactive lysate of BMDCs) could restore the function of damaged salivary gland in NOD mice (Misuno et al., 2014).

osteogenic cells. For this purpose, various parameters including cell separation procedures, culture media, type and origin of serum, cell seeding density, timing of passaging, the number of passages, content and concentrations of reagents for induction and the period of induction should be optimized. Similarly, scaffold material and shape should be optimized depending on the purpose. To establish a standard operating protocol for the preparation of cells for clinical bone tissue engineering, we have investigated optimal cell culture and induction procedures using human BMSCs and granular type β-tricalcium phosphate (β-TCP) as a scaffold. The results showed that the passage number, seeding density and the period of induction significantly affected the osteogenic ability of BMSCs (Agata et al., 2010). In particular, human BMSCs lose their *in vivo* bone forming ability very rapidly after passaging and no bone formation was observed with cells after the fourth cell passage (Fig. 3) (Agata et al., 2010). The results of those basic studies should be considered in the establishment of an

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**Figure 3.** Effect of passage numbers on BMSC's ability for ectopic bone formation. Upper panels show ectopic bone formation on the back of nude mice with tissue-engineered bone using human BMSCs at passage 1 (a) and passage 5 (b). The success of ectopic bone formation quickly decrease after cell passage and no bone formation was observed after passage 4 (c). Note that the bone forming ability was quickly lost during passage. Adapted from Kagami et al.,

optimal cell culture/induction protocol.

2014 and modified from original figure in Agata et al., 2010.

### **3. Cell-based therapy to atrophic alveolar bone (bone tissue engineering)**

Atrophic alveolar bone is one of the major obstacles for dental implant therapy because dental implant placement requires the presence of a minimal amount of bone. For patients with severe bone atrophy, functional recovery with conventional dentures is difficult and most of the patients suffer from masticatory disturbance. A combination of autologous bone graft and dental implant installation is a realistic option for those patients with severe alveolar bone atrophy. However, the procedure is accompanied by swelling and pain of the donor site and causes morbidity. Bioartificial bone substitutes have been frequently used as an alternative, although the artificial materials cannot induce bone regeneration and the application is limited. Accordingly, tissue engineering and regenerative medicine of bone tissue is now receiving significant attention (Kagami et al., 2014).

#### **3.1. Scientific bases for bone tissue engineering**

Osteogenic ability is the function to generate bone after transplantation. Before the develop‐ ment of tissue-engineered bone, only vascular bone grafts were known to have this function. Tissue-engineered bone is designed to contain living osteogenic cells and thus possesses osteogenic function. The characteristic features of tissue-engineered bone is based on the presence of osteogenic cells.

As a source for osteogenic cells, pluripotent mesenchymal stromal cells from bone marrow aspirates have been widely used. Initially, this group of cells was defined as adherent fibro‐ blast-like cells in bone marrow. Since the culture contains cells which can generate single cellderived colonies, those cells were referred to as colony forming units-fibroblasts (CFUs-F). Eventually, CFUs-F were proved to have high proliferating potential and even single-colony derived cells were shown capable of forming bone (Friedenstein et al., 1966; Friedenstein et al. 1970; Friedenstein et al., 1987). Subsequently, the capability of CFUs-F to differentiate to various mesenchymal tissues has been reported. Then, these cells were re-named as"mesen‐ chymal stem cells" (MSCs) (Caplan, 1991). However, not all cells are multipotent; MSCs are also designated as "bone marrow stromal cells (BMSCs)", which is a relatively widely accepted nomenclature (Prockop, 2009). Although BMSCs can be obtained from a patient's own bone marrow aspirate, the percentage of BMSCs in total bone marrow cells is likely less than 0.01% (Pittenger et al., 1999). Accordingly, cell cultivation is required to obtain a sufficient number of cells for clinical bone tissue engineering. For example, bone marrow from a single rat can provide a sufficient number of osteogenic cells for only two or three bone-forming transplants. In contrast, after *in vitro* expansion of cells from the same volume of bone marrow, more than 50 bone-forming transplants are possible (Yoshikawa et al., 1996).

The quality of cells is an important factor for tissue engineering as it could affect the treatment outcome. Accordingly, BMSCs should be appropriately processed, cultured and induced into osteogenic cells. For this purpose, various parameters including cell separation procedures, culture media, type and origin of serum, cell seeding density, timing of passaging, the number of passages, content and concentrations of reagents for induction and the period of induction should be optimized. Similarly, scaffold material and shape should be optimized depending on the purpose. To establish a standard operating protocol for the preparation of cells for clinical bone tissue engineering, we have investigated optimal cell culture and induction procedures using human BMSCs and granular type β-tricalcium phosphate (β-TCP) as a scaffold. The results showed that the passage number, seeding density and the period of induction significantly affected the osteogenic ability of BMSCs (Agata et al., 2010). In particular, human BMSCs lose their *in vivo* bone forming ability very rapidly after passaging and no bone formation was observed with cells after the fourth cell passage (Fig. 3) (Agata et al., 2010). The results of those basic studies should be considered in the establishment of an optimal cell culture/induction protocol.

bioactive lysate of BMDCs) could restore the function of damaged salivary gland in NOD mice

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

**3. Cell-based therapy to atrophic alveolar bone (bone tissue engineering)**

Atrophic alveolar bone is one of the major obstacles for dental implant therapy because dental implant placement requires the presence of a minimal amount of bone. For patients with severe bone atrophy, functional recovery with conventional dentures is difficult and most of the patients suffer from masticatory disturbance. A combination of autologous bone graft and dental implant installation is a realistic option for those patients with severe alveolar bone atrophy. However, the procedure is accompanied by swelling and pain of the donor site and causes morbidity. Bioartificial bone substitutes have been frequently used as an alternative, although the artificial materials cannot induce bone regeneration and the application is limited. Accordingly, tissue engineering and regenerative medicine of bone tissue is now receiving

Osteogenic ability is the function to generate bone after transplantation. Before the develop‐ ment of tissue-engineered bone, only vascular bone grafts were known to have this function. Tissue-engineered bone is designed to contain living osteogenic cells and thus possesses osteogenic function. The characteristic features of tissue-engineered bone is based on the

As a source for osteogenic cells, pluripotent mesenchymal stromal cells from bone marrow aspirates have been widely used. Initially, this group of cells was defined as adherent fibro‐ blast-like cells in bone marrow. Since the culture contains cells which can generate single cellderived colonies, those cells were referred to as colony forming units-fibroblasts (CFUs-F). Eventually, CFUs-F were proved to have high proliferating potential and even single-colony derived cells were shown capable of forming bone (Friedenstein et al., 1966; Friedenstein et al. 1970; Friedenstein et al., 1987). Subsequently, the capability of CFUs-F to differentiate to various mesenchymal tissues has been reported. Then, these cells were re-named as"mesen‐ chymal stem cells" (MSCs) (Caplan, 1991). However, not all cells are multipotent; MSCs are also designated as "bone marrow stromal cells (BMSCs)", which is a relatively widely accepted nomenclature (Prockop, 2009). Although BMSCs can be obtained from a patient's own bone marrow aspirate, the percentage of BMSCs in total bone marrow cells is likely less than 0.01% (Pittenger et al., 1999). Accordingly, cell cultivation is required to obtain a sufficient number of cells for clinical bone tissue engineering. For example, bone marrow from a single rat can provide a sufficient number of osteogenic cells for only two or three bone-forming transplants. In contrast, after *in vitro* expansion of cells from the same volume of bone marrow, more than

The quality of cells is an important factor for tissue engineering as it could affect the treatment outcome. Accordingly, BMSCs should be appropriately processed, cultured and induced into

(Misuno et al., 2014).

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86

significant attention (Kagami et al., 2014).

presence of osteogenic cells.

**3.1. Scientific bases for bone tissue engineering**

50 bone-forming transplants are possible (Yoshikawa et al., 1996).

**Figure 3.** Effect of passage numbers on BMSC's ability for ectopic bone formation. Upper panels show ectopic bone formation on the back of nude mice with tissue-engineered bone using human BMSCs at passage 1 (a) and passage 5 (b). The success of ectopic bone formation quickly decrease after cell passage and no bone formation was observed after passage 4 (c). Note that the bone forming ability was quickly lost during passage. Adapted from Kagami et al., 2014 and modified from original figure in Agata et al., 2010.

In terms of the safety of bone tissue engineering, BMSCs have been used clinically for more than ten years and no major side effect has been reported (Quatro et al., 2001). Accordingly, bone tissue engineering using BMSCs is considered relatively safe. However, genetic instabil‐ ity has been reported in mouse BMSCs, changes that might lead to carcinogenesis (reviewed by Prockop, 2009). Quest for the usage of more potent stem cells might be accompanied by a higher risk of genetic alteration, an issue that should be kept in mind in therapeutic applica‐ tions of stem cells.

#### **3.2. Clinical bone tissue engineering in dentistry and maxillofacial surgery**

Tissue-engineered bone was first applied for long-bone defects (Quatro et al., 2001). In Quatro's study, autologous BMSCs were cultured with hydroxyapatite blocks and transplanted to the defective site. This study was the first to prove the clinical feasibility of bone tissue engineering.

In the field of dentistry, results from a first clinical study were reported in 2004. MSCs were mixed with platelet rich plasma as a scaffold and transplanted to the site of atrophic alveolar bone at the time of dental implant installation (Yamada et al., 2004). They reported that bone regeneration was observed in all cases. More recently, another clinical study using BMSCs was reported (Meijer et al., 2008). BMSCs were cultured and seeded on hydroxyapatite granules. Then, the cells were induced into osteogenic cells for one week and transplanted. In this study, the subjects included severe atrophy cases in which dental implant could not be installed at the time of cell transplantation (two steps approach). Bone formation was observed in three cases. However, in two cases where the atrophy was severe, newly formed bone was only observed in the area adjacent to the native bone, which might imply osteoconduction rather than bone regeneration by the transplanted cells. Accordingly, the efficacy of clinical alveolar bone tissue engineering for severe atrophy cases was not established in this study.

We have conducted a clinical study of bone tissue engineering for severe atrophy of alveolar bone. BMSCs were harvested from each patient's bone marrow, expanded *in vitro*, induced into osteogenic cells and transplanted together with β-TCP granules (Fig. 4). The results following two-and five-year observations proved that bone regeneration was successful and no side effects or related complications have been observed. On the other hand, one of the important findings through this clinical study was the presence of individual variations in cell growth, differentiation and levels of bone regeneration (Asahina et al., manuscript in prepa‐ ration). This problem is not limited to bone tissue engineering and might be an important issue for the all therapies using autologous cells. Development of novel technologies to reduce individual variation is a current research target.

engineering will provide more options. Accordingly, the selection of an adequate technique

**Figure 4.** The procedure for clinical study of alveolar bone regeneration at The Research Hospital, The Institute of

Potential Cell-Based Therapies for Irreversibly Damaged Salivary Glands...

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89

When the tissue defect is relatively large and the condition of the recipient site is not optimal, the use of more invasive technique such as autologous bone grafting might be acceptable. However, future bone regeneration therapy will probably avoid surgical removal of healthy bone in favour of less invasive procedures. Bone tissue engineering is one of the most prom‐ ising approaches and is expected to replace cases of autologous bone grafting. Currently, the best applications and most significant limitations of bone tissue engineering are not clear. Welldesigned clinical studies should be performed to answer these important questions, and these

In the near future, a cell-based therapy will be accepted as a useful or even an essential treatment option for various diseases in dentistry and maxillofacial surgery. Based on previous and ongoing studies, radiation-damaged salivary glands and atrophic alveolar bone are the

clinical trials will contribute to the wide acceptance of bone tissue engineering.

Medical Science, The University of Tokyo. Modified from original figure in Kagami et al., 2014.

for each case is of importance for practitioners.

**4. Conclusion**

#### **3.3. Future prospect of bone tissue engineering in dentistry and maxillofacial surgery**

Currently, various techniques for bone regeneration/reconstruction are available, including microvascular-free flaps, autologous block bone transplantation, allogeneic or xenogeneic bone substitutes or purely artificial bone substitutes. Furthermore, recent development of additional strategies such as destruction osteogenesis, growth factor administration and tissue Potential Cell-Based Therapies for Irreversibly Damaged Salivary Glands... http://dx.doi.org/10.5772/58913 89

**Figure 4.** The procedure for clinical study of alveolar bone regeneration at The Research Hospital, The Institute of Medical Science, The University of Tokyo. Modified from original figure in Kagami et al., 2014.

engineering will provide more options. Accordingly, the selection of an adequate technique for each case is of importance for practitioners.

When the tissue defect is relatively large and the condition of the recipient site is not optimal, the use of more invasive technique such as autologous bone grafting might be acceptable. However, future bone regeneration therapy will probably avoid surgical removal of healthy bone in favour of less invasive procedures. Bone tissue engineering is one of the most prom‐ ising approaches and is expected to replace cases of autologous bone grafting. Currently, the best applications and most significant limitations of bone tissue engineering are not clear. Welldesigned clinical studies should be performed to answer these important questions, and these clinical trials will contribute to the wide acceptance of bone tissue engineering.

#### **4. Conclusion**

In terms of the safety of bone tissue engineering, BMSCs have been used clinically for more than ten years and no major side effect has been reported (Quatro et al., 2001). Accordingly, bone tissue engineering using BMSCs is considered relatively safe. However, genetic instabil‐ ity has been reported in mouse BMSCs, changes that might lead to carcinogenesis (reviewed by Prockop, 2009). Quest for the usage of more potent stem cells might be accompanied by a higher risk of genetic alteration, an issue that should be kept in mind in therapeutic applica‐

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

Tissue-engineered bone was first applied for long-bone defects (Quatro et al., 2001). In Quatro's study, autologous BMSCs were cultured with hydroxyapatite blocks and transplanted to the defective site. This study was the first to prove the clinical feasibility of bone tissue engineering.

In the field of dentistry, results from a first clinical study were reported in 2004. MSCs were mixed with platelet rich plasma as a scaffold and transplanted to the site of atrophic alveolar bone at the time of dental implant installation (Yamada et al., 2004). They reported that bone regeneration was observed in all cases. More recently, another clinical study using BMSCs was reported (Meijer et al., 2008). BMSCs were cultured and seeded on hydroxyapatite granules. Then, the cells were induced into osteogenic cells for one week and transplanted. In this study, the subjects included severe atrophy cases in which dental implant could not be installed at the time of cell transplantation (two steps approach). Bone formation was observed in three cases. However, in two cases where the atrophy was severe, newly formed bone was only observed in the area adjacent to the native bone, which might imply osteoconduction rather than bone regeneration by the transplanted cells. Accordingly, the efficacy of clinical alveolar

bone tissue engineering for severe atrophy cases was not established in this study.

individual variation is a current research target.

We have conducted a clinical study of bone tissue engineering for severe atrophy of alveolar bone. BMSCs were harvested from each patient's bone marrow, expanded *in vitro*, induced into osteogenic cells and transplanted together with β-TCP granules (Fig. 4). The results following two-and five-year observations proved that bone regeneration was successful and no side effects or related complications have been observed. On the other hand, one of the important findings through this clinical study was the presence of individual variations in cell growth, differentiation and levels of bone regeneration (Asahina et al., manuscript in prepa‐ ration). This problem is not limited to bone tissue engineering and might be an important issue for the all therapies using autologous cells. Development of novel technologies to reduce

**3.3. Future prospect of bone tissue engineering in dentistry and maxillofacial surgery**

Currently, various techniques for bone regeneration/reconstruction are available, including microvascular-free flaps, autologous block bone transplantation, allogeneic or xenogeneic bone substitutes or purely artificial bone substitutes. Furthermore, recent development of additional strategies such as destruction osteogenesis, growth factor administration and tissue

**3.2. Clinical bone tissue engineering in dentistry and maxillofacial surgery**

tions of stem cells.

Medicine

88

In the near future, a cell-based therapy will be accepted as a useful or even an essential treatment option for various diseases in dentistry and maxillofacial surgery. Based on previous and ongoing studies, radiation-damaged salivary glands and atrophic alveolar bone are the

most realistic targets. Despite rapid progress in studies of cell-based therapies, this novel technology has yet to gain acceptance in ordinary practice. Since cell-based therapy is still expensive, determination of adequate treatment targets should be an important research goal to facilitate its widespread use. Furthermore, it is essential to reduce treatment costs without sacrificing safety. Development of automated cell culture systems and efficient safety tests are the key technologies to achieve this goal.

[2] Atkinson, J.B., Mahoney, F.J., Schwartz, I.R. & Hesch, J.A. (1959). *Therapy of acute leu‐ kemia by whole-body irradiation and bone marrow transplantation from an identical normal*

Potential Cell-Based Therapies for Irreversibly Damaged Salivary Glands...

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91

[3] Baum BJ, Tran SD. Synergy between genetic and tissue engineering – creating an arti‐

[4] Baum, B.J., Alevizos, I., Zheng, C., Cotrim, A.P., Liu, S., McCullagh, L., Goldsmith, C.M., Burbelo, P.D., Citrin, D.E., Mitchell, J.B., Nottingham, L.K., Rudy, S.F., Van Waes, C., Whatley, M.A., Brahim, J.S., Chiorini, J.A., Danielides, S., Turner, R.J., Pa‐ tronas, N.J., Chen, C.C., Nikolov, N.P. & Illei, G.G. (2012). *Early responses to adenovi‐ ral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.* Proc Natl Acad Sci USA, Vol. 109, No. 47, (November 2012), pp. 19403-19407, ISSN:

[5] Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O. & Peterson, L. (1994), *Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.* N Engl J Med, Vol. 331, No. 14, (October 1994), pp. 889-895, ISSN 0021-9738 (Print).

[6] Caplan, A.I. (1991), *Mesenchymal stem cells.* J Orthop Res, (September 1991), Vol. 9,

[7] Friedenstein, A.J., Piatetzky-Chapiro, I.I. & Petrakova, K.V. (1966), *Osteogenesis in transplants of bone marrow cells.* J Embryol Exp Morph, (December 1966), Vol.16, No. 3,

[8] Friedenstein, A.J., Chailakhjan, R.K. & Lalykina, K.S. (1970). *The development of fibro‐ blast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells.* Cell Tissue

[9] Friedenstein, A.J., Challakhyan, R.K., Gerasimov, U.V. (1987). *Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers.* Cell Tissue Kinet,

[10] Gallico, G. G. 3rd, O'Connor, N. E., Compton, C. C., Kehinde, O. & Green, H. (1984). Permanent Coverage of Large Burn Wounds with Autologous Cultured Human Epi‐ thelium. *N Engl J Med,* Vol. 311, No. 7, (August 1984), pp. 448-451, ISSN 0028-4793.

[11] Kagami, H., Wang, S. & Hai, B. (2008). *Restoring the function of salivary glands.* Oral

[12] Kagami, H., Agata, H., Inoue, M., Asahina, I., Tojo, A., Yamashita, N. & Imai, K. (2014). *The Use of Bone Marrow Stromal Cells (Bone Marrow-Derived Multipotent Mesen‐ chymal Stromal Cells) for Alveolar Bone Tissue Engineering: Basic Science to Clinical Trans‐*

[13] Khalili, S., Liu, Y., Sumita, Y., Maria, O.M., Blank, D., Key, S., Mezey, E. & Tran, S.D. (2010). *Bone marrow cells are a source of undifferentiated cells to prevent Sjogren's syn‐*

Dis. Vol. 14, No. 1, (January 2008), pp. 15-24, ISSN 1354-523X (Print).

*lation.* In press, (2014), ISSN 1937-3368 (Print).

Kinet (October 1970), Vol. 3, No. 4, pp.393–403, ISSN:0008-8730 (Print).

(May 1987), Vol. 20, No. 3, pp. 263–272, ISSN:0008-8730 (Print).

*twin.* Blood. Vol. 14, No. 3, (March 1959), pp. 228-234, ISSN 0006-4971 (Print).

ficial salivary gland. *Periodontology2000*. 2006;41:218-223

No. 5, pp. 641–650, ISSN:0736-0266 (Print).

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0027-8424 (Print).

### **Acknowledgements**

This work was supported in part by "Health Labour Sciences Research Grants" from the Ministry of Health, Labor and Welfare of Japan,"Grant-in-Aid for Scientific Research (B)", "Grant-in-Aid for challenging Exploratory Research" from Japan Society for the Promotion of Science (JSPS) and "Translational Research Network Program" from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

#### **Author details**

Yoshinori Sumita1 , Izumi Asahina1 , Simon D. Tran2 , Hideki Agata1 , Minoru Inoue3,4, Arinobu Tojo3 and Hideaki Kagami3,4

1 Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

2 Laboratory of Craniofacial Tissue Engineering, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

3 Tissue Engineering Research Group, Division of Molecular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

4 Department of Oral and Maxillofacial Surgery, Matsumoto Dental University, Shiojiri, Japan

#### **References**

[1] Agata, H., Asahina, I., Watanabe, N., Ishii, Y., Kubo, N., Ohshima, S., Yamazaki, M., Tojo, A. &Kagami, H. (2010). *Characteristic change and loss of in vivo osteogenic abilities of human bone marrow stromal cells during passage.* Tissue Eng Part A, Vol.16, No.2,(Oc‐ tober 2010), pp. 663–673, ISSN 1937-3341 (Print).

[2] Atkinson, J.B., Mahoney, F.J., Schwartz, I.R. & Hesch, J.A. (1959). *Therapy of acute leu‐ kemia by whole-body irradiation and bone marrow transplantation from an identical normal twin.* Blood. Vol. 14, No. 3, (March 1959), pp. 228-234, ISSN 0006-4971 (Print).

most realistic targets. Despite rapid progress in studies of cell-based therapies, this novel technology has yet to gain acceptance in ordinary practice. Since cell-based therapy is still expensive, determination of adequate treatment targets should be an important research goal to facilitate its widespread use. Furthermore, it is essential to reduce treatment costs without sacrificing safety. Development of automated cell culture systems and efficient safety tests are

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

This work was supported in part by "Health Labour Sciences Research Grants" from the Ministry of Health, Labor and Welfare of Japan,"Grant-in-Aid for Scientific Research (B)", "Grant-in-Aid for challenging Exploratory Research" from Japan Society for the Promotion of Science (JSPS) and "Translational Research Network Program" from Ministry of Education,

, Simon D. Tran2

3 Tissue Engineering Research Group, Division of Molecular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo,

4 Department of Oral and Maxillofacial Surgery, Matsumoto Dental University, Shiojiri,

[1] Agata, H., Asahina, I., Watanabe, N., Ishii, Y., Kubo, N., Ohshima, S., Yamazaki, M., Tojo, A. &Kagami, H. (2010). *Characteristic change and loss of in vivo osteogenic abilities of human bone marrow stromal cells during passage.* Tissue Eng Part A, Vol.16, No.2,(Oc‐

1 Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate

2 Laboratory of Craniofacial Tissue Engineering, Faculty of Dentistry, McGill University,

, Hideki Agata1

, Minoru Inoue3,4,

the key technologies to achieve this goal.

Culture, Sports, Science and Technology (MEXT) of Japan.

, Izumi Asahina1

School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

tober 2010), pp. 663–673, ISSN 1937-3341 (Print).

and Hideaki Kagami3,4

**Acknowledgements**

Medicine

90

**Author details**

Yoshinori Sumita1

Montreal, Quebec, Canada

Arinobu Tojo3

Japan

Japan

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[29] Sumita, Y., Liu, Y., Khalili, S., Maria, O.M., Xia, D., Key, S., Cotrim, A.P., Mezey, E. & Tran, S.D. (2011). *Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation.* Int J Biochem Cell Biol, (January 2011), Vol. 43, No. 1, pp.

[30] Thomas, E.D., Lochte, H.L. Jr., Cannon, J.H., Sahler, O.D., Ferrebee, J.W. (1959). *Su‐ pralethal whole body irradiation and isologous marrow transplantation in man.* J Clin In‐ vest. (October 1959), Vol. 38, No. 10 (Pt 1-2), pp. 1709-1716, ISSN 0021-9738 (Print). [31] Tran, S.D., Wang, J., Bandyopadhyay, B.C., Redman, R.S., Dutra, A., Pak, E., Swaim, W.D., Gerstenhaber, J.A., Bryant, J.M., Zheng, C., Goldsmith, C.M., Kok, M.R., Well‐ ner, R.B. & Baum, B.J. (2005). *Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.* Tissue Eng, (January-February 2005),

[32] Tran, S.D., Kodama, S., Lodde, B.M., Szalayova, I., Key, S., Khalili, S., Faustman, D.L. & Mezey, E. (2007). *Reversal of Sjogren's-like syndrome in non-obese diabetic mice.* Ann

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[34] Tran, S.D., Redman, R.S., Barrett, A.J., Pavletic, S.Z., Key, S., Liu, Y., Carpenter, A., Nguyen, H.M., Sumita, Y., Baum, B.J., Pillemer, S.R. & Mezey, E. (2011). *Microchimer‐*

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[24] Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. & Marshak, D.R. (1999). *Multilineage po‐ tential of adult human mesenchymal stem cells.* Science (April 1999), Vol. 284, No. 5411, pp. 143–147, ISSN:0036-8075 (Print).

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

[15] Khalili, S., Faustman DL, Liu, Y., Sumita, Y., Blank, D., Peterson, A., Kodama, S. & Tran, S.D. (2014). *Treatment for salivary gland hypofunction at both initial and advanced stages of Sjogren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period.* Cytotherapy, (March 2014), Vol. 6, No. 3, pp.

[16] Kodama, S., Kuhtreiber, W., Fujimura, S., Dale, E.A. & Faustman, D.L. (2003). *Islet re‐ generation during the reversal of autoimmune diabetes in NOD mice.* Science, (November

[17] Kojima, T., Knemaru, S.-I., Hirano, S., Tateya, I., Ohno, S., Nakamura, T. & Ito, J. (2011). *Regeneration of radiation damaged salivary glands with adipose-derived stromal cells.* Laryngoscope, (September 2011), Vol. 121, No. 9, pp. 1864-1869, ISSN:0023-852X

[18] Lim, J.Y., Yi, T., Choi, J.S., Jang, Y.H., Lee, S., Kim, H.J., Song, S.U. & Kim, Y.M. (2013). *Intraglandular transplantation of bone marrow-derived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage.* Oral Oncol, (February

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[22] Misuno, K., Tran, S.D., Khalili, S., Huang, J., Liu, Y. & Hu, S. (2014). *Quantitative anal‐ ysis of protein and gene expression in salivary glands of Sjorgren's-like disease NOD mice treated by bone marrow soup.* PLoS One, (January 2014), Vol. 9, No. 1, pp. e87158, ISSN:

[23] Ogawa, M., Oshima, M., Imamura, A., Sekine, Y., Ishida, K., Yamashita, K., Nakaji‐ ma, K., Hirayama, M., Tachikawa, T. & Tsuji, T. (2013). *Functional salivary gland regen‐*

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> *ism in salivary glands after blood- and marrow-derived stem cell transplantation.* Biol Blood Marrow Transplant, (March, 2011), Vol. 17, No. 3, pp. 429-433, ISSN:1083-8791 (Print).

**Chapter 8**

**Bone and Periodontal Tissue**

**from Bone Marrow Derived**

**Mesenchymal Stem Cells**

Wataru Katagiri

**1. Introduction**

**Regeneration by the Secretomes**

Additional information is available at the end of the chapter

(Kawasaki et al., 1998; Perri et al., 2007, Vaidya et al., 2007).

studies of bone and periodontal regenerative medicine.

Alveolar bone regeneration with grafting is often carried out prior to placement of dental implants. Several graft materials have been used including autogenous bone, xenogeneic bone, and synthetic bone substitutes. Autogenous bone grafts have been used for a long time with good predictability and are considered the "gold standard" because of their osteoinductive and osteoconductive properties and immunogenic compatibility. However, autogenous bone must be harvested from a donor site of the patient and is associated with higher morbidity (Arrington et al., 1996; Joshi et al., 2004). Xenogeneic bone and synthetic bone substitutes such as deproteinized bovine bone, hydroxyapatite, and calcium triphosphate are often used clinically as osteoconductive scaffolds, but they provide limited osteoinductivity and a potential risk of infection and extrusion (Damien et al., 1991). Osteoinductive growth factors such as bone morphogenic protein (BMP)-2 have been used with these osteoconductive materials to promote bone regeneration (Herford & Boyne, 2008). However, recent studies have indicated unexpected effects on bone regeneration including induction of a severe inflammatory response, because of the higher dose with clinical application of BMP-2

Recently, the concept of tissue engineering and regenerative medicine has been widely accepted (Langer & Vacanti, 1993), and many clinical studies have been performed including

> © 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.

**Chapter 8**

**Bone and Periodontal Tissue Regeneration by the Secretomes from Bone Marrow Derived Mesenchymal Stem Cells**

Wataru Katagiri

*ism in salivary glands after blood- and marrow-derived stem cell transplantation.* Biol Blood Marrow Transplant, (March, 2011), Vol. 17, No. 3, pp. 429-433, ISSN:1083-8791

[35] Tran, S.D., Liu, Y., Xia, D., Maria, O.M., Khalili, S., Wang, R.W., Quan, V.H., Hu, S. & Seuntjens, J. (2013). *Paracrine effects of bone marrow soup restore organ function, regenera‐ tion, and repair in salivary glands damaged by irradiation.* PLoS One, (April 2013), Vol. 8,

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

[36] Yamada, Y., Ueda, M., Hibi, H. & Nagasaka, T. (2004). *Translational research for injecta‐ ble tissue-engineered bone regeneration using mesenchymal stem cells and platelet-rich plas‐ ma: from basic research to clinical case study.* Cell Transplant, Vol. 13, No. 4, pp. 343–355,

[37] Yoshikawa, T., Ohgushi, H. & Tamai, S. (1996). *Immediate bone forming capability of pre‐ fabricated osteogenic hydroxyapatite.* J Biomed Mater Res (November 1996), Vol. 32, No.

[38] Xu, J., Xu, J., Wang, D., Liu, D., Fan, Z., Zhang, H., Liu, O., Ding, G., Gao, R., Zhang, C., Ding, Y., Bromberg, J.S., Chen, W., Sun, L. & Wang, S. (2012). *Allogeneic mesenchy‐ mal stem cell treatment alleviates experimental and clinical Sjogren's syndrome.* Blood. (Oc‐

tober 2011), Vol. 120, No. 15, pp. 3142-3151, ISSN:0006-4971 (Print).

No 4, pp. e61632, ISSN:1932-6203 (Electronic).

3, pp. 481–492, ISSN:0021-9304 (Print).

(Print).

Medicine

94

ISSN:0963-6897 (Print).

Additional information is available at the end of the chapter

#### **1. Introduction**

Alveolar bone regeneration with grafting is often carried out prior to placement of dental implants. Several graft materials have been used including autogenous bone, xenogeneic bone, and synthetic bone substitutes. Autogenous bone grafts have been used for a long time with good predictability and are considered the "gold standard" because of their osteoinductive and osteoconductive properties and immunogenic compatibility. However, autogenous bone must be harvested from a donor site of the patient and is associated with higher morbidity (Arrington et al., 1996; Joshi et al., 2004). Xenogeneic bone and synthetic bone substitutes such as deproteinized bovine bone, hydroxyapatite, and calcium triphosphate are often used clinically as osteoconductive scaffolds, but they provide limited osteoinductivity and a potential risk of infection and extrusion (Damien et al., 1991). Osteoinductive growth factors such as bone morphogenic protein (BMP)-2 have been used with these osteoconductive materials to promote bone regeneration (Herford & Boyne, 2008). However, recent studies have indicated unexpected effects on bone regeneration including induction of a severe inflammatory response, because of the higher dose with clinical application of BMP-2 (Kawasaki et al., 1998; Perri et al., 2007, Vaidya et al., 2007).

Recently, the concept of tissue engineering and regenerative medicine has been widely accepted (Langer & Vacanti, 1993), and many clinical studies have been performed including studies of bone and periodontal regenerative medicine.

© 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.

We previously developed a technique whereby autogenous human mesenchymal stem cells (hMSCs) from the patient's bone marrow are combined with platelet-rich plasma for use as an alternative to such materials with predictable good prognosis (Yamada et al., 2004, 2013). However, clinical use of stem cells requires highly qualified safety investigation and quality management of cell handling, and is very expensive. These limitations currently impede the widespread use of stem cells for alveolar bone regeneration therapy. Moreover, recent studies have revealed that the implanted cells do not survive long (Ide et al., 2010; Perri et al., 2007; Toma et al., 2009). As an alternative, the effects of the secretomes, the various factors secreted into the medium, from stem cells on tissue repair and regeneration have attracted much attention (Baglio et al., 2012; Chen et al., 2008; Ciapetti et al., 2012).

**Factors Concentration (pg/mL)** IGF-1 1515.6±211.83 VEGF 465.84±108.81 TGF-β1 339.82±14.41 HGF 20.32±7.89 PDGF-BB N.D BMP-2 N.D FGF-2 N.D SDF-1 N.D

Bone and Periodontal Tissue Regeneration by the… 97

The concentrations of the cytokines IGF-1, VEGF, TGF-β1, HGF, FGF-2, PDGF-BB, BMP-2, and SDF-1α in MSC-CM were quantified with ELISA. Cytokines were not detected in DMEM-0% and DMEM-30%. However, MSC-CM contained IGF-1, VEGF, TGF-β1, and HGF at concen‐ trations of 1386 ± 465, 468.5 ± 109, 339.8 ± 14.4, and 20.3 ± 7.8 pg/ml, respectively. The other

The migratory properties of rMSCs and rPDLCs were examined using the CytoSelect Wound Healing Assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cell suspension was added to the well with a plastic insert in place. The insert was removed from the well after a monolayer of cells had formed, creating a wound gap of 0.9 mm. After washing, cells were incubated at 37 °C for 48 h in MSC-CM with 30% FBS or serum-free DMEM. The extent of wound closure was determined with a light microscope (CK40; Olympus, Tokyo, Japan) at ×40 magnification. The percentage of rMSCs in the wound area of DMEM (−) was 9.28 ± 4.41. There were 70.9 ± 6.8% rMSCs in the wound area of positive control (30% FBS). MSC-CM exerted significant effects (p < 0.05) and closed the wound to 43.4

The percentage of rPDLCs in the wound area of DMEM (−) was 2.36 ± 2.32%, with 48.01 ± 6.28% in 30% FBS and 17.98 ± 4.14% in MSC-CM. Thus, MSC-CM increased rMSC migration more than four-fold and rPDLC migration more than seven-fold compared with that in DMEM (−). These differences were statistically significant (p < 0.05), indicating that MSC-CM enhanced

The level of cellular fill within the wound area in response to MSC-CM was compared with the wound-fill response in the presence of 30% FBS or serum-free DMEM as control after 48 h (a). The migration of rMSCs and rPDLSCs cultured in MSC-CM was enhanced compared with

**Table 1.** The Levels of Cytokines Present in MSC-CM

factors assayed were not detected in MSC-CM (Table 1).

rMSC and rPDLC migration and proliferation (Fig. 1b).

**2.3. MSC-CM enhances migration and proliferation of MSCs and PDLCs**

**2.2. Cytokines present in MSC-CM**

± 10.6% rMSCs (Fig. 1a).

We have reported the effects of the secretomes in the conditioned medium from bone marrowderived mesenchymal stem cells (MSC-CM) on bone and periodontal tissue regeneration *in vitro* and *in vivo*. MSC-CM contains several cytokines such as insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β1. MSC-CM enhances cell proliferation, mobilization, angiogenesis, and expression of osteogenic markers such as *alkaline phosphatase*, *collagen type I*, and *Runx2* genes (Katagiri et al., 2013). MSC-CM also recruits endogenous stem cells to the grafted site and shows early bone and perio‐ dontal regeneration in rat calvarial bone defects and dog periodontal bone defects (Inukai et al., 2013; Osugi et al., 2012). Furthermore, the concentrations of cytokines contained in MSC-CM are relatively low such that use of MSC-CM does not induce the severe histological inflammatory responses that are observed with the clinical use of recombinant human BMP-2 (Katagiri et al., 2013).

In this section, our *in vitro* and *in vivo* studies about the effects of MSC-CM on bone and periodontal tissue regeneration were summarized and introduced.

### **2. Biological effects of MSC-CM in bore and periodontal tissue regeneration**

#### **2.1. Preparation of MSC-CM**

The hMSCs were purchased from Lonza and cultured in MSC basal medium (Lonza) with MSC growth medium (SingleQuots, Lonza). Rat MSCs (rMSCs) were isolated from the femora of 7-week-old Wistar/ST rats (Japan SLC) and expanded and cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS). Cells of the second to fourth passages were used in the experiments in this study. Cells were maintained at 37°C in 5% carbon dioxide/95% air. The hMSCs were cultured in a culture dish (100 × 20 mm). When hMSCs reached 70% to 80% confluence, the medium was refreshed with 10 mL of serum-free DMEM containing antibiotics (100 units/mL penicillin G, 100 µg/mL streptomycin, and 0.25µg/ mL amphotericinB; Gibco). The culture media (CM) was collected after 48 hours of incubation. This media was defined as hMSC culture-conditioned media (MSC-CM) and stored at 4°C or –80°C before being used for subsequent experiments.

**Table 1.** The Levels of Cytokines Present in MSC-CM

#### **2.2. Cytokines present in MSC-CM**

We previously developed a technique whereby autogenous human mesenchymal stem cells (hMSCs) from the patient's bone marrow are combined with platelet-rich plasma for use as an alternative to such materials with predictable good prognosis (Yamada et al., 2004, 2013). However, clinical use of stem cells requires highly qualified safety investigation and quality management of cell handling, and is very expensive. These limitations currently impede the widespread use of stem cells for alveolar bone regeneration therapy. Moreover, recent studies have revealed that the implanted cells do not survive long (Ide et al., 2010; Perri et al., 2007; Toma et al., 2009). As an alternative, the effects of the secretomes, the various factors secreted into the medium, from stem cells on tissue repair and regeneration have attracted much

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

We have reported the effects of the secretomes in the conditioned medium from bone marrowderived mesenchymal stem cells (MSC-CM) on bone and periodontal tissue regeneration *in vitro* and *in vivo*. MSC-CM contains several cytokines such as insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β1. MSC-CM enhances cell proliferation, mobilization, angiogenesis, and expression of osteogenic markers such as *alkaline phosphatase*, *collagen type I*, and *Runx2* genes (Katagiri et al., 2013). MSC-CM also recruits endogenous stem cells to the grafted site and shows early bone and perio‐ dontal regeneration in rat calvarial bone defects and dog periodontal bone defects (Inukai et al., 2013; Osugi et al., 2012). Furthermore, the concentrations of cytokines contained in MSC-CM are relatively low such that use of MSC-CM does not induce the severe histological inflammatory responses that are observed with the clinical use of recombinant human BMP-2

In this section, our *in vitro* and *in vivo* studies about the effects of MSC-CM on bone and

The hMSCs were purchased from Lonza and cultured in MSC basal medium (Lonza) with MSC growth medium (SingleQuots, Lonza). Rat MSCs (rMSCs) were isolated from the femora of 7-week-old Wistar/ST rats (Japan SLC) and expanded and cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS). Cells of the second to fourth passages were used in the experiments in this study. Cells were maintained at 37°C in 5% carbon dioxide/95% air. The hMSCs were cultured in a culture dish (100 × 20 mm). When hMSCs reached 70% to 80% confluence, the medium was refreshed with 10 mL of serum-free DMEM containing antibiotics (100 units/mL penicillin G, 100 µg/mL streptomycin, and 0.25µg/ mL amphotericinB; Gibco). The culture media (CM) was collected after 48 hours of incubation. This media was defined as hMSC culture-conditioned media (MSC-CM) and stored at 4°C or

attention (Baglio et al., 2012; Chen et al., 2008; Ciapetti et al., 2012).

periodontal tissue regeneration were summarized and introduced.

–80°C before being used for subsequent experiments.

**2. Biological effects of MSC-CM in bore and periodontal tissue**

(Katagiri et al., 2013).

Medicine

96

**regeneration**

**2.1. Preparation of MSC-CM**

The concentrations of the cytokines IGF-1, VEGF, TGF-β1, HGF, FGF-2, PDGF-BB, BMP-2, and SDF-1α in MSC-CM were quantified with ELISA. Cytokines were not detected in DMEM-0% and DMEM-30%. However, MSC-CM contained IGF-1, VEGF, TGF-β1, and HGF at concen‐ trations of 1386 ± 465, 468.5 ± 109, 339.8 ± 14.4, and 20.3 ± 7.8 pg/ml, respectively. The other factors assayed were not detected in MSC-CM (Table 1).

#### **2.3. MSC-CM enhances migration and proliferation of MSCs and PDLCs**

The migratory properties of rMSCs and rPDLCs were examined using the CytoSelect Wound Healing Assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cell suspension was added to the well with a plastic insert in place. The insert was removed from the well after a monolayer of cells had formed, creating a wound gap of 0.9 mm. After washing, cells were incubated at 37 °C for 48 h in MSC-CM with 30% FBS or serum-free DMEM. The extent of wound closure was determined with a light microscope (CK40; Olympus, Tokyo, Japan) at ×40 magnification. The percentage of rMSCs in the wound area of DMEM (−) was 9.28 ± 4.41. There were 70.9 ± 6.8% rMSCs in the wound area of positive control (30% FBS). MSC-CM exerted significant effects (p < 0.05) and closed the wound to 43.4 ± 10.6% rMSCs (Fig. 1a).

The percentage of rPDLCs in the wound area of DMEM (−) was 2.36 ± 2.32%, with 48.01 ± 6.28% in 30% FBS and 17.98 ± 4.14% in MSC-CM. Thus, MSC-CM increased rMSC migration more than four-fold and rPDLC migration more than seven-fold compared with that in DMEM (−). These differences were statistically significant (p < 0.05), indicating that MSC-CM enhanced rMSC and rPDLC migration and proliferation (Fig. 1b).

The level of cellular fill within the wound area in response to MSC-CM was compared with the wound-fill response in the presence of 30% FBS or serum-free DMEM as control after 48 h (a). The migration of rMSCs and rPDLSCs cultured in MSC-CM was enhanced compared with

> tail and abdominal region at 1, 24, and 48 h, and at 1 week after injection. We confirmed that there were no signals at the defect area in the cranium at any time point. In the PBS group, fluorescent signals were observed in the tail and in the abdominal area at 24 and 48h after injection, and very low signals were observed in the breast and cranial area after 1 week. In the MSC-CM group, a moderate increase in signal intensity was observed in the area of the tail and the abdominal region during the first 24h after injection. At 48h after injection, signal intensity in the MSC-CM-implanted area of the parietal bone started to increase. The maximum

Bone and Periodontal Tissue Regeneration by the… 99

In vivo imaging analysis shows that DiR-labeled rMSCs that were injected into the caudal vein just after implantation of the materials into the calvarial bone defects started to mi-grate immediately after injection. At 24 h, 48 h, 72h and 1 week after injection, the signal of the fluorescent-labeled rMSCs was only detected in the tail and abdominal region in the control PBS group. In the MSC-CM group, a moderate increase in signal intensity was observed in the abdominal region during the first 24 h after injection. Forty-eight hours after injection, the MSC-CM-implanted area of calvarial bone started to increase in signal intensity, and, 1 week after injection, the MSC-CM implanted area, as well as the implanted bone cavity, showed the

fluorescent signal in the implanted area was observed 1 week after injection (Fig. 2).

highest fluorescent signal of the experimental groups.

**Figure 2.** In vivo imaging of injected rMSC migration to implants.

in EM (Fig. 3).

**2.5. MSC-CM enhanced osteogenic and angiogenic marker gene expression**

The levels of expression of the *ALP*, *OCN*, *Runx2*, *VEGF-A*, *ANG-1,* and *ANG-2* genes were significantly upregulated in rMSCs cultured with MSC-CM compared with rMSCs cultured

**Figure 1.** MSC-CM promoted migration and proliferation of rMSCs and rPDLCs in the wound-healing assay.

rMSCs and rPDLSCs cultured in DMEM (−). MSC-CM increased wound area more than 4-fold compared to that in DMEM(-) (b, \*p < 0.05).

#### **2.4. MSC-CM enhanced MSC migration in rat calvarial bone defect model**

rMSCs were harvested and cultured as described previously and were labeled with the lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR; Molecu‐ lar Probes, Eugene, OR) for all imaging ex-periments (Kalchenko et al., 2006). This fluorophore is excited at 750 nm and has an emission peak at 782 nm. The cells were incubated with DiR (3 x106 cells in 10 mL PBS containing 3.5 mg/mL dye and 0.5% ethanol) for 30 min at 37°C. The cells were then washed twice with PBS and injected intravenously into the caudal vein of Wistar/ST rats with the bone defect de-scribed previously, which had been implanted with vari-ous implant materials just before the injection of rMSCs. The rats were anesthetized by intraperitoneal injection of Somnopentyl® prior to injection. Xenogen's IVIS® 200 Series Imaging System (Xenogen, Alameda, CA) was used to monitor DiR-labeled rMSC localization within live, as well as sacrificed, animals. Imaging was performed at 1, 24, and 48h, and at 1 week after injection of DiR-labeled cells. Migration of rMSCs to the implants in vivo was analyzed in rats of the different implantation groups in which DiR-labeled rMSCs were injected into the caudal vein. Although it is not possible to detect DiR-labeled cells at a great depth with the imaging system used, this system can detect DiR-labeled cells that accumulate on the calvarial bone. The fluorescent signal in all groups increased immediately after injection. In the control Defect group, the fluorescent signal of the labeled rMSCs was only detected in the tail and abdominal region at 1, 24, and 48 h, and at 1 week after injection. We confirmed that there were no signals at the defect area in the cranium at any time point. In the PBS group, fluorescent signals were observed in the tail and in the abdominal area at 24 and 48h after injection, and very low signals were observed in the breast and cranial area after 1 week. In the MSC-CM group, a moderate increase in signal intensity was observed in the area of the tail and the abdominal region during the first 24h after injection. At 48h after injection, signal intensity in the MSC-CM-implanted area of the parietal bone started to increase. The maximum fluorescent signal in the implanted area was observed 1 week after injection (Fig. 2).

In vivo imaging analysis shows that DiR-labeled rMSCs that were injected into the caudal vein just after implantation of the materials into the calvarial bone defects started to mi-grate immediately after injection. At 24 h, 48 h, 72h and 1 week after injection, the signal of the fluorescent-labeled rMSCs was only detected in the tail and abdominal region in the control PBS group. In the MSC-CM group, a moderate increase in signal intensity was observed in the abdominal region during the first 24 h after injection. Forty-eight hours after injection, the MSC-CM-implanted area of calvarial bone started to increase in signal intensity, and, 1 week after injection, the MSC-CM implanted area, as well as the implanted bone cavity, showed the highest fluorescent signal of the experimental groups.

**Figure 2.** In vivo imaging of injected rMSC migration to implants.

rMSCs and rPDLSCs cultured in DMEM (−). MSC-CM increased wound area more than 4-fold

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

**Figure 1.** MSC-CM promoted migration and proliferation of rMSCs and rPDLCs in the wound-healing assay.

rMSCs were harvested and cultured as described previously and were labeled with the lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR; Molecu‐ lar Probes, Eugene, OR) for all imaging ex-periments (Kalchenko et al., 2006). This fluorophore is excited at 750 nm and has an emission peak at 782 nm. The cells were incubated with DiR

 cells in 10 mL PBS containing 3.5 mg/mL dye and 0.5% ethanol) for 30 min at 37°C. The cells were then washed twice with PBS and injected intravenously into the caudal vein of Wistar/ST rats with the bone defect de-scribed previously, which had been implanted with vari-ous implant materials just before the injection of rMSCs. The rats were anesthetized by intraperitoneal injection of Somnopentyl® prior to injection. Xenogen's IVIS® 200 Series Imaging System (Xenogen, Alameda, CA) was used to monitor DiR-labeled rMSC localization within live, as well as sacrificed, animals. Imaging was performed at 1, 24, and 48h, and at 1 week after injection of DiR-labeled cells. Migration of rMSCs to the implants in vivo was analyzed in rats of the different implantation groups in which DiR-labeled rMSCs were injected into the caudal vein. Although it is not possible to detect DiR-labeled cells at a great depth with the imaging system used, this system can detect DiR-labeled cells that accumulate on the calvarial bone. The fluorescent signal in all groups increased immediately after injection. In the control Defect group, the fluorescent signal of the labeled rMSCs was only detected in the

**2.4. MSC-CM enhanced MSC migration in rat calvarial bone defect model**

compared to that in DMEM(-) (b, \*p < 0.05).

(3 x106

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#### **2.5. MSC-CM enhanced osteogenic and angiogenic marker gene expression**

The levels of expression of the *ALP*, *OCN*, *Runx2*, *VEGF-A*, *ANG-1,* and *ANG-2* genes were significantly upregulated in rMSCs cultured with MSC-CM compared with rMSCs cultured in EM (Fig. 3).

The explants were decalci-fied with K-CX solution (FALMA) and dehydrated using a graded series of ethanols, cleared with xylene, and embedded in paraffin. The specimens were cut in a sagittal direction to make 3-µm-thick histologic sections and stained with hematoxylin-eosin. Histologic analysis was performed using a light microscope. MSC-CM/Terudermis or control implants were placed into rat calvarial bone defects (Fig 4a). The area of newly regenerated bone was determined as a percentage of the total graft area at 2 and 4 weeks post-implantation using micro-CT (Fig 4b). After 2 weeks, the mean area of newly regenerated bone in the MSC-CM defects (81.50% ± 2.7%) was significantly increased compared to that of the unfilled defects (8.63% ± 1.78%) and the PBS-treated sites (60.63% ± 5.8%). After 4 weeks, the defect areas were almost completely filled by newly regenerated bone in the MSC-CM (93.07% ± 6.6%) and PBS (84.04% ± 4.9%) groups (Fig 4b). Moreover, histologic analysis also showed well-regenerated bone in the MSC-CM group compared with the PBS groups (Fig 5). At 2 weeks, the bone defect was almost covered with newly regenerated bone in the MSC-CM group, in contrast to the PBS group, where the defect was covered with a large amount of connective tissue. At 4 weeks, newly regenerated bone was partially noticeable within the defect of the PBS group, but in the MSC-CM group, the defect was almost completely replaced by mature bone tissue (Fig 5).

Bone and Periodontal Tissue Regeneration by the… 101

**Figure 4.** (a) Bone defect 5mm in diameter was prepared in each rat calvaria. (b) Micro-CT analysis of bone regenera‐

tion.

**Figure 3.** MSC-CM enhanced osteogenic and angiogenic marker gene expression.

The mRNA expression levels of *ALP*, *OCN*, *Runx2*, *VEGF-A*, *ANG-1,* and *ANG-2* were deter‐ mined relative to the level of *Glyceraldehyde 3-phosphate dehydrogenase*(*GAPDH*) mRNA in each sample and were quantified and standardized. The level of expression of the each gene was significantly upregulated in rMSCs cultured with MSC-CM compared with rMSCs cultured in DMEM (\*p < 0.05).

#### **2.6. MSC-CM enhanced bone regeneration, migration of endogeneous MSCs and angiogenesis in rat calvarial bone defect model**

Twenty-four 10-week-old male Wistar/ST rats were anesthetized by intraperitoneal injection of pento-barbital (Somnopentyl, Kyoritsu Seiyaku) (20 mg/kg body weight). Two circular fullthickness (through-and-through) bone defects (5 mm in diameter) were made in the calvarial bone using a trephine bur and were irrigated with saline to remove the bone debris. The experimental materials were then implanted into the defects. Atelocollagen (Terudermis, Olympus Terumo Biomaterials), which was cut into the desired form, was suspended in MSC-CM or PBS. Three groups were de-fined: *(1)* MSC-CM group: MSC-CM/Terudermis; *(2)* PBS group: PBS/Terudermis; and *(3)* defect group: unfilled defect.

Rats were sacrificed at 2 or 4 weeks after transplantation (n=4 per group). The surgical sites were dissected, fixed in 10% formalin, and subjected to micro-computed tomography (micro-CT) analysis using a laboratory x-ray CT device (LATheta, Hitachi Aloka). Images were compiled and analyzed to render three-dimensional images using OsiriX imaging software (version 3.9). Then the area (mm2 ) of newly regenerated bone was compared between groups. The explants were decalci-fied with K-CX solution (FALMA) and dehydrated using a graded series of ethanols, cleared with xylene, and embedded in paraffin. The specimens were cut in a sagittal direction to make 3-µm-thick histologic sections and stained with hematoxylin-eosin. Histologic analysis was performed using a light microscope. MSC-CM/Terudermis or control implants were placed into rat calvarial bone defects (Fig 4a). The area of newly regenerated bone was determined as a percentage of the total graft area at 2 and 4 weeks post-implantation using micro-CT (Fig 4b). After 2 weeks, the mean area of newly regenerated bone in the MSC-CM defects (81.50% ± 2.7%) was significantly increased compared to that of the unfilled defects (8.63% ± 1.78%) and the PBS-treated sites (60.63% ± 5.8%). After 4 weeks, the defect areas were almost completely filled by newly regenerated bone in the MSC-CM (93.07% ± 6.6%) and PBS (84.04% ± 4.9%) groups (Fig 4b). Moreover, histologic analysis also showed well-regenerated bone in the MSC-CM group compared with the PBS groups (Fig 5). At 2 weeks, the bone defect was almost covered with newly regenerated bone in the MSC-CM group, in contrast to the PBS group, where the defect was covered with a large amount of connective tissue. At 4 weeks, newly regenerated bone was partially noticeable within the defect of the PBS group, but in the MSC-CM group, the defect was almost completely replaced by mature bone tissue (Fig 5).

**Figure 3.** MSC-CM enhanced osteogenic and angiogenic marker gene expression.

**angiogenesis in rat calvarial bone defect model**

group: PBS/Terudermis; and *(3)* defect group: unfilled defect.

in DMEM (\*p < 0.05).

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(version 3.9). Then the area (mm2

The mRNA expression levels of *ALP*, *OCN*, *Runx2*, *VEGF-A*, *ANG-1,* and *ANG-2* were deter‐ mined relative to the level of *Glyceraldehyde 3-phosphate dehydrogenase*(*GAPDH*) mRNA in each sample and were quantified and standardized. The level of expression of the each gene was significantly upregulated in rMSCs cultured with MSC-CM compared with rMSCs cultured

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

Twenty-four 10-week-old male Wistar/ST rats were anesthetized by intraperitoneal injection of pento-barbital (Somnopentyl, Kyoritsu Seiyaku) (20 mg/kg body weight). Two circular fullthickness (through-and-through) bone defects (5 mm in diameter) were made in the calvarial bone using a trephine bur and were irrigated with saline to remove the bone debris. The experimental materials were then implanted into the defects. Atelocollagen (Terudermis, Olympus Terumo Biomaterials), which was cut into the desired form, was suspended in MSC-CM or PBS. Three groups were de-fined: *(1)* MSC-CM group: MSC-CM/Terudermis; *(2)* PBS

Rats were sacrificed at 2 or 4 weeks after transplantation (n=4 per group). The surgical sites were dissected, fixed in 10% formalin, and subjected to micro-computed tomography (micro-CT) analysis using a laboratory x-ray CT device (LATheta, Hitachi Aloka). Images were compiled and analyzed to render three-dimensional images using OsiriX imaging software

) of newly regenerated bone was compared between groups.

**2.6. MSC-CM enhanced bone regeneration, migration of endogeneous MSCs and**

**Figure 4.** (a) Bone defect 5mm in diameter was prepared in each rat calvaria. (b) Micro-CT analysis of bone regenera‐ tion.

Images of micro-CT indicated that the newly regenerated bone was seen at an early stage of bone regeneration and almost covered the defect at 4 weeks. (c) Percent bone regeneration by area within defects as measured by micro-CT at 2 and 4 weeks. MSC-CM increased the bone regeneration significantly compared with other groups at 2 and 4 weeks (\*p<0.01, #p<0.05).

Furethermore, we performed the immunohistochemical staining against CD31 and CD105 antibody to confirm the endogeneous MSC migration and angiogenesis during the bone regeneration by MSC-CM. In the MSC-CM group, numerous CD31-or CD105--positive cells were seen the MSC-CM implanted area. In contrast, there were fewer CD31-or CD105-positive cells in both the PBS and Defect groups (Fig 6). These results indicated MSC-CM enhanced the endogeneous MSC migration and angiogenesis.

Newly regenerated bone in the defect, PBS and MSC-CM groups 2 or 4 weeks after implanta‐ tion was evaluated. In the defect group, majority of the defect was filled with connectivetissue and the infiltrations of inflammatory cells were seen both at 2 and 4 weeks. In the MSC-CM group, newly regenerated bone had begun to cover the defect at 2 weeks and ossification had progressed gradually. In the PBS group, newly regenerated bone and partial connective tissue had covered the defect at 4 weeks.

**2.7. MSC−CM enhanced periodontal tissue regeneration in dog periodontal defect model**

Bone and Periodontal Tissue Regeneration by the… 103

**Figure 6.** Immunohistochemical analysis of newly generated bone area 2 weeks after implantation.

All animal experiments were approved by the Nagoya University animal experiment com‐ mittee. After a period of acclimatization of 30 days, five hybrid dogs were operated on under general anesthesia by intravenous injection of pentobarbital (Somnopentyl®) (20mg/kg body weight), and under local anesthesia with 2% lidocaine (with 1:80,000 epinephrine, ORA® Inj. Dental Cartridge; Showa Yakuhin Kako, Tokyo, Japan). Before the experimental surgery, the mandibular first and third or fourth premolars were extracted, and the extraction sites were allowed to heal for 8 weeks. For the experimental surgery, buccal and lingual mucoperiosteal flaps were elevated, and critical-size, box-type, one-wall intrabony defects (width, 4mm; height, 5 mm) were created at the distal aspect of the second, and the mesial aspect of the fourth premolars in the right and left jaw quadrants (Kim, 2005). Following root planing to remove the root cementum, a reference notch indicating a 5-mm distance from the cement-enamel junction to the bottom of the defect was made with a burr into the root surface at the base of the defects. With no differences in bone regeneration in the various grafted areas in terms of bone healing, two defects were created and implanted with two materials at random sites. An absorbable atelo-collagen sponge (TERUPLUG®; OLYMPUS TERUMO BIOMATERIALS, Tokyo, Japan) was used as a scaffold and contained 300µl MSC-CM or PBS. The dogs with defects were randomly divided into three groups (n=6 each) and implanted with graft materials: MSC-CM plus scaffold, PBS plus scaffold, or unfilled defect. The mucogingival flaps were advanced, adapted, and completely closed. Post-surgical management involved antibi‐ otics (Azithromycin, 250 mg; Pfizer, Tokyo, Japan) daily for 3 days, a soft diet, and topical application of 2% chlorhexidine (Hibitane concentrate; Dainippon Sumitomo Pharma, Osaka, Japan) twice a week. After 4 weeks, the dogs were given general anesthesia and sacrificed by

Standardized radiographic images of the defect sites were obtained with an X-ray apparatus (Dent navi Hands; Yoshida Co., Ltd., Tokyo, Japan) and dental X-ray films (BW-100; Hanshin Technical Laboratory, Nishinomiya, Japan) immediately, and 4 weeks after, transplantation.

exsanguination after injection of heparin sodium (400 U/kg).

**Figure 5.** Histological analysis of newly regenerated bone.

Two weeks after implantation into carvalial defects, tissue specimens were analyzed using immunohistostaining for: CD31 (RED), a marker for rat endothelial cells; CD105 (GREEN), a marker for rat stem cells. Cell nuclei were labeled with DAPI (blue). In MSC-CM group, both CD105 and CD31 positive cells were more prominent than those of the control PBS group.

**Figure 6.** Immunohistochemical analysis of newly generated bone area 2 weeks after implantation.

Images of micro-CT indicated that the newly regenerated bone was seen at an early stage of bone regeneration and almost covered the defect at 4 weeks. (c) Percent bone regeneration by area within defects as measured by micro-CT at 2 and 4 weeks. MSC-CM increased the bone regeneration significantly compared with other groups at 2 and 4 weeks (\*p<0.01, #p<0.05).

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

Furethermore, we performed the immunohistochemical staining against CD31 and CD105 antibody to confirm the endogeneous MSC migration and angiogenesis during the bone regeneration by MSC-CM. In the MSC-CM group, numerous CD31-or CD105--positive cells were seen the MSC-CM implanted area. In contrast, there were fewer CD31-or CD105-positive cells in both the PBS and Defect groups (Fig 6). These results indicated MSC-CM enhanced the

Newly regenerated bone in the defect, PBS and MSC-CM groups 2 or 4 weeks after implanta‐ tion was evaluated. In the defect group, majority of the defect was filled with connectivetissue and the infiltrations of inflammatory cells were seen both at 2 and 4 weeks. In the MSC-CM group, newly regenerated bone had begun to cover the defect at 2 weeks and ossification had progressed gradually. In the PBS group, newly regenerated bone and partial connective tissue

Two weeks after implantation into carvalial defects, tissue specimens were analyzed using immunohistostaining for: CD31 (RED), a marker for rat endothelial cells; CD105 (GREEN), a marker for rat stem cells. Cell nuclei were labeled with DAPI (blue). In MSC-CM group, both CD105 and CD31 positive cells were more prominent than those of the control PBS group.

endogeneous MSC migration and angiogenesis.

**Figure 5.** Histological analysis of newly regenerated bone.

had covered the defect at 4 weeks.

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#### **2.7. MSC−CM enhanced periodontal tissue regeneration in dog periodontal defect model**

All animal experiments were approved by the Nagoya University animal experiment com‐ mittee. After a period of acclimatization of 30 days, five hybrid dogs were operated on under general anesthesia by intravenous injection of pentobarbital (Somnopentyl®) (20mg/kg body weight), and under local anesthesia with 2% lidocaine (with 1:80,000 epinephrine, ORA® Inj. Dental Cartridge; Showa Yakuhin Kako, Tokyo, Japan). Before the experimental surgery, the mandibular first and third or fourth premolars were extracted, and the extraction sites were allowed to heal for 8 weeks. For the experimental surgery, buccal and lingual mucoperiosteal flaps were elevated, and critical-size, box-type, one-wall intrabony defects (width, 4mm; height, 5 mm) were created at the distal aspect of the second, and the mesial aspect of the fourth premolars in the right and left jaw quadrants (Kim, 2005). Following root planing to remove the root cementum, a reference notch indicating a 5-mm distance from the cement-enamel junction to the bottom of the defect was made with a burr into the root surface at the base of the defects. With no differences in bone regeneration in the various grafted areas in terms of bone healing, two defects were created and implanted with two materials at random sites. An absorbable atelo-collagen sponge (TERUPLUG®; OLYMPUS TERUMO BIOMATERIALS, Tokyo, Japan) was used as a scaffold and contained 300µl MSC-CM or PBS. The dogs with defects were randomly divided into three groups (n=6 each) and implanted with graft materials: MSC-CM plus scaffold, PBS plus scaffold, or unfilled defect. The mucogingival flaps were advanced, adapted, and completely closed. Post-surgical management involved antibi‐ otics (Azithromycin, 250 mg; Pfizer, Tokyo, Japan) daily for 3 days, a soft diet, and topical application of 2% chlorhexidine (Hibitane concentrate; Dainippon Sumitomo Pharma, Osaka, Japan) twice a week. After 4 weeks, the dogs were given general anesthesia and sacrificed by exsanguination after injection of heparin sodium (400 U/kg).

Standardized radiographic images of the defect sites were obtained with an X-ray apparatus (Dent navi Hands; Yoshida Co., Ltd., Tokyo, Japan) and dental X-ray films (BW-100; Hanshin Technical Laboratory, Nishinomiya, Japan) immediately, and 4 weeks after, transplantation.

Dental X-ray films were placed parallel to the tooth axis, and radiographic images of the defect site were ta-ken in the buccolingual direction. The defect sites were dissected and fixed in 10% neutral-buffered formalin (Wako, Japan) 4 weeks after transplantation. The specimens were decalcified in Plank-Rychro solution (Wako) for 8 weeks, routinely processed into 5µm-thick paraffin-embedded sections, stained with hematoxylin and eosin, and observed under a light microscope (Olympus). Histometric parameters were quantified using a computer-based image analysis system (Image J 1.44; National Institutes of Health). The following parameters were analyzed:

**1.** Cementum regeneration height: distance from the root surface notch to the coronal

extension of newly formed cementum on the root surface.

**2.** Bone regeneration height: distance from the root surface notch to the coronal

extension of newly formed bone along the root surface.

**3.** Bone regeneration area: area of new alveolar bone formed coronally from the apical

extension of the root surface notch.

Clinical healing was generally uneventful. The results from the histometric analysis are shown in Fig. 8. The cementum regeneration height, the bone regeneration height, and the bone regeneration area of the MSC-CM group were 3.01 ± 0.16 mm, 3.19 ± 0.51 mm, and 4.89 ± 1.08 mm2 , respectively. A large amount of new lamellar and woven bone formation was observed in the MSC-CM group. Thick-layered and cellular cementum on the root surface was also frequently observed in the MSC-CM group.

White arrows: osteoblasts. Black arrows: newly regenerated cellular cementum. Photomicrographs: hematoxylin and

**Figure 8.** (A, E and I) overview of periodontal defect site. Original magnification x12.5, scale bar=1 mm. (B, C, F, G, J and K) Higher magnification of the boxed areas. Original magnification x100, scale bar=50 lm. (D, H and L) Radio‐ graphic images were taken at 0 (left) and 4 weeks (right) after surgery. Histometric analysis of periodontal regenera‐

From the results of these studies, it was suggested that MSC-CM contributes to upregulation of several processes of bone and periodontal tissue regeneration through the angiogenesis and mobilization of endogenous MSCs, and thus enhanced bone and periodontal tissue regener‐ ation. Using MSC-CM for bone and periodontal tissue regeneration may be effective because several cytokines, including MSC-CM, contribute several processes to the complex system of bone and periodontal tissue regeneration. If the MSC-CM treatment protocol is to be estab‐ lished for bone and periodontal tissue regeneration, it is essential that effective and therapeutic doses of MSC-CM as well as the safety of the therapy should be carefully established. Further

in dog one-wall intrabony defects (means ± SD in mm or

Bone and Periodontal Tissue Regeneration by the… 105

eosin staining.

mm2) (p < 0.05).

**3. Conclusion**

tion following surgical implantation of MSC-CM/TERUPLUG®

investigation regarding these matters is now in progress.

On the other hand, less newly regenerated bone and cementum compared to the MSC-CM group was observed in the PBS group. Dense collagen fibers were observed frequently in the PBS group. Newly regenerated bone and cementum were not apparent in the defect group. Furthermore, there was minimal inflammatory cell infiltration in the MSC-CM group com‐ pared to the other groups.

**Figure 7.** Representative photomicrographs and radiographic images from sites receiving experimental materials.

White arrows: osteoblasts. Black arrows: newly regenerated cellular cementum. Photomicrographs: hematoxylin and eosin staining.

**Figure 8.** (A, E and I) overview of periodontal defect site. Original magnification x12.5, scale bar=1 mm. (B, C, F, G, J and K) Higher magnification of the boxed areas. Original magnification x100, scale bar=50 lm. (D, H and L) Radio‐ graphic images were taken at 0 (left) and 4 weeks (right) after surgery. Histometric analysis of periodontal regenera‐ tion following surgical implantation of MSC-CM/TERUPLUG® in dog one-wall intrabony defects (means ± SD in mm or mm2) (p < 0.05).

#### **3. Conclusion**

Dental X-ray films were placed parallel to the tooth axis, and radiographic images of the defect site were ta-ken in the buccolingual direction. The defect sites were dissected and fixed in 10% neutral-buffered formalin (Wako, Japan) 4 weeks after transplantation. The specimens were decalcified in Plank-Rychro solution (Wako) for 8 weeks, routinely processed into 5µm-thick paraffin-embedded sections, stained with hematoxylin and eosin, and observed under a light microscope (Olympus). Histometric parameters were quantified using a computer-based image analysis system (Image J 1.44; National Institutes of Health). The following parameters

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

**1.** Cementum regeneration height: distance from the root surface notch to the coronal

**3.** Bone regeneration area: area of new alveolar bone formed coronally from the apical

Clinical healing was generally uneventful. The results from the histometric analysis are shown in Fig. 8. The cementum regeneration height, the bone regeneration height, and the bone regeneration area of the MSC-CM group were 3.01 ± 0.16 mm, 3.19 ± 0.51 mm, and 4.89 ± 1.08

On the other hand, less newly regenerated bone and cementum compared to the MSC-CM group was observed in the PBS group. Dense collagen fibers were observed frequently in the PBS group. Newly regenerated bone and cementum were not apparent in the defect group. Furthermore, there was minimal inflammatory cell infiltration in the MSC-CM group com‐

**Figure 7.** Representative photomicrographs and radiographic images from sites receiving experimental materials.

, respectively. A large amount of new lamellar and woven bone formation was observed in the MSC-CM group. Thick-layered and cellular cementum on the root surface was also

**2.** Bone regeneration height: distance from the root surface notch to the coronal

extension of newly formed cementum on the root surface.

extension of newly formed bone along the root surface.

extension of the root surface notch.

pared to the other groups.

frequently observed in the MSC-CM group.

were analyzed:

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mm2

From the results of these studies, it was suggested that MSC-CM contributes to upregulation of several processes of bone and periodontal tissue regeneration through the angiogenesis and mobilization of endogenous MSCs, and thus enhanced bone and periodontal tissue regener‐ ation. Using MSC-CM for bone and periodontal tissue regeneration may be effective because several cytokines, including MSC-CM, contribute several processes to the complex system of bone and periodontal tissue regeneration. If the MSC-CM treatment protocol is to be estab‐ lished for bone and periodontal tissue regeneration, it is essential that effective and therapeutic doses of MSC-CM as well as the safety of the therapy should be carefully established. Further investigation regarding these matters is now in progress.

hema-topoietic cell homing. *J Biomed Opt Vol.* 11, No.5, (January 2007), pp.50507,

Bone and Periodontal Tissue Regeneration by the… 107

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4, (March 2014), pp.1009-1016, ISSN 1942-4434.

#### **Author details**

Wataru Katagiri

Nagoya University Graduate School of Medicine, Japan

#### **References**

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[11] Katagiri W, Osugi M, Kawai T & Ueda M. (2013). Novel cell-free regenerative medi‐ cine of bone using stem cell derived factors. *Int J Oral Maxillofac Implants* Vol. 28, No. 4, (March 2014), pp.1009-1016, ISSN 1942-4434.

**Author details**

Medicine

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Wataru Katagiri

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**Chapter 9**

**Whole Tooth Regeneration Using a Bioengineered**

The tooth, which is an ectodermal organ whose development is regulated by reciprocal epithelial-mesenchymal interactions (Jussila et al., 2013), contributes to oral functions associ‐ ated with mastication and enunciation, which are important aspects of general health and quality of life (Proffit et al., 2004). Teeth have a three-dimensional multicellular structure composed of characteristic hard tissues, *e.g.,* enamel, dentin, cementum and alveolar bone. Teeth also have soft connective tissues, such as pulp and periodontal ligaments, which contain nerve fibres and blood vessels that are important for maintaining tooth homeostasis (Avery, 2002). Dental caries, periodontal disease and trauma, which have high prevalence rates in dental disorders, cause fundamental problems for oral function and are associated with oral and general health issues (Proffit et al., 2004). To restore occlusal function after tooth loss, conventional dental treatments based on replacing teeth with artificial materials, such as fixed or removable dentures, have been established. Dental implants, which are able to stand alone in the jawbone without invading the adjacent teeth, have been used for the rehabilitation of tooth loss. Although these artificial therapies are widely applied to treat dental disorders, recent advances in tissue regeneration have been made that enhance the functions of the biological tooth, allow for underlying tooth movement through bone remodelling and aid the ability to perceive noxious stimuli (Huang et al., 2009). Substantial advances in the develop‐ ment of regenerative therapies have been driven by our understanding of embryonic devel‐ opment, stem cell biology and tissue engineering technologies (Yelick & Vacanti, 2006). Currently, an important concept in regenerative therapy is the transplantation of tissuederived stem cells or *in vitro*-manipulated induced pluripotent stem (iPS) cells (Volponi et al., 2010). These therapies are attractive therapeutic concepts that have the potential to repair damaged tissues and restore the partial loss of organ function (Korbling & Estrov, 2003). In

> © 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.

**Tooth**

Masamitsu Oshima and Takashi Tsuji

http://dx.doi.org/10.5772/58908

**1. Introduction**

Additional information is available at the end of the chapter

## **Whole Tooth Regeneration Using a Bioengineered Tooth**

Masamitsu Oshima and Takashi Tsuji

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58908

#### **1. Introduction**

The tooth, which is an ectodermal organ whose development is regulated by reciprocal epithelial-mesenchymal interactions (Jussila et al., 2013), contributes to oral functions associ‐ ated with mastication and enunciation, which are important aspects of general health and quality of life (Proffit et al., 2004). Teeth have a three-dimensional multicellular structure composed of characteristic hard tissues, *e.g.,* enamel, dentin, cementum and alveolar bone. Teeth also have soft connective tissues, such as pulp and periodontal ligaments, which contain nerve fibres and blood vessels that are important for maintaining tooth homeostasis (Avery, 2002). Dental caries, periodontal disease and trauma, which have high prevalence rates in dental disorders, cause fundamental problems for oral function and are associated with oral and general health issues (Proffit et al., 2004). To restore occlusal function after tooth loss, conventional dental treatments based on replacing teeth with artificial materials, such as fixed or removable dentures, have been established. Dental implants, which are able to stand alone in the jawbone without invading the adjacent teeth, have been used for the rehabilitation of tooth loss. Although these artificial therapies are widely applied to treat dental disorders, recent advances in tissue regeneration have been made that enhance the functions of the biological tooth, allow for underlying tooth movement through bone remodelling and aid the ability to perceive noxious stimuli (Huang et al., 2009). Substantial advances in the develop‐ ment of regenerative therapies have been driven by our understanding of embryonic devel‐ opment, stem cell biology and tissue engineering technologies (Yelick & Vacanti, 2006). Currently, an important concept in regenerative therapy is the transplantation of tissuederived stem cells or *in vitro*-manipulated induced pluripotent stem (iPS) cells (Volponi et al., 2010). These therapies are attractive therapeutic concepts that have the potential to repair damaged tissues and restore the partial loss of organ function (Korbling & Estrov, 2003). In

© 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. The mechanisms of tooth development**

periodontal ligaments and alveolar bone (Avery, 2002).

tooth structures (Honda et al., 2007; Yelick & Vacanti, 2006).

**regeneration**

recipient's jaw (Fig. 2, lower).

Ectodermal organs, such as the teeth, hair and salivary glands, arise from their respective organ germs through reciprocal epithelial-mesenchymal interactions in the developing embryo. These interactions, which involve various signalling molecules and transcription factors, are the principal mechanism regulating organogenesis (Jussila et al., 2013). In tooth germ devel‐ opment, the dental lamina first thickens (lamina stage). This stage is followed by epithelial thickening (placode stage) at the future location of the tooth and subsequent epithelial budding to the underlying neural crest-derived ecto-mesenchyme. Tooth germ formation is initiated on embryonic days (EDs) 10-11 in mice by epithelial signals that include fibroblast growth factor (FGF) 8, bone morphogenetic protein (BMP) 4, sonic hedgehog (Shh), tumour necrosis factor (TNF) and Wnt10b. These signals induce the expression of several transcription factors in the dental mesenchyme that condense around the developing epithelial bud (bud stage) (Jussila et al., 2013; O'connell et al., 2013). At ED13.5-14.5, the first enamel knot, which acts as a signalling centre to orchestrate tooth development by controlling the gene expression of various signalling molecules and transcription factors, is formed in the dental epithelium (cap stage). At ED16-18, the epithelial and mesenchymal cells in the tooth germ terminally differ‐ entiate into the tooth-tissue progenitor cells, such as ameloblasts, odontoblasts, and dental follicle cells (bell stage). Ameloblasts and odontoblasts accumulate the enamel and dentin matrix, respectively, at the boundary surface between the epithelium and mesenchyme, while dental follicle cells differentiate into the periodontal tissues, which include the cementum,

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111

**3. A novel three-dimensional cell manipulation method for whole tooth**

One current biological approach for the regeneration of three-dimensional organs is based on recapitulating organogenesis by mimicking the reciprocal epithelial-mesenchymal interac‐ tions that occur in the developing embryo, thereby developing fully functional bioengineered organs from a bioengineered organ germ generated from immature stem cells via threedimensional cell manipulation *in vitro*. For tooth regeneration, one proposed concept has been to transplant a bioengineered tooth germ into the recipient jaw and allowing it to develop into a functional mature tooth *in situ* (Fig. 2, upper). It is also expected that it will be possible to transplant a bioengineered tooth unit that includes mature tooth, periodontal ligament and alveolar bone, which will achieve biological engraftment through bone integration with the

To realise whole tooth replacement, the first critical issue is to develop a three-dimensional cell manipulation method using completely dissociated epithelial and mesenchymal cells *in vitro*. Previously, it has been reported that using a polyglycolic acid and poly-L-lactate-coglycolide copolymer (PLA/PLGA) or a collagen sponge as a tooth-shaped scaffold and seeding them with epithelial and mesenchymal cells isolated from tooth buds could generate small

dental medicine, tooth tissue-derived stem cells and the cytokine network that regulates tooth development have been well characterised at the molecular level (Jussila et al., 2013). These advances can be applied to the repair of dental pulp and periodontal tissues, including the alveolar bone (Egusa et al., 2012, 2013). Organ replacement regenerative therapy, which involves constructing a fully functional bioengineered organ using three-dimensional cell manipulation *in vitro*, holds great promise for the replacement of dysfunctional organs following disease, injury or aging. Tooth regenerative therapy would also involve the replace‐ ment of a lost or damaged tooth with a bioengineered tooth, constructed with stem cells, that has the capacity to become a functional unit comprising the whole tooth and periodontal tissue (Yen & Sharpe, 2008). It is anticipated that tooth replacement therapy will be established in the near future as a novel biological treatment for the functional recovery of lost teeth to satisfy both aesthetic and physiological requirements (Fig. 1). Over the past three decades, many approaches for replacing missing teeth have been studied, including three-dimensional bioengineered teeth and tooth germ generation using biodegradable materials and cell aggregation methods (Volponi et al., 2010). Recently, studies have reported tooth replacement by transplantation of fully functioning bioengineered teeth having the correct tooth structure, masticatory performance, proper responsiveness to mechanical stress and neural function after transplantation into the region of tooth loss (Ikeda et al., 2009; Nakao et al., 2007; Oshima et al., 2011). In this chapter, we describe novel technologies for whole tooth replacement that have the potential to provide functional recovery and could someday replace current dental treatments based on artificial materials.

**Figure 1.** Concepts of tooth regenerative therapy

Recent approaches for tooth regenerative therapy have included tissue repair and whole tooth replacement. Tooth regenerative therapy and stem cell transplantation therapies are regarded as attractive approaches for repairing tissue that has been damaged by dental caries or periodontal disease. The transplantation of dental stem cells has been examined for the treatment of dental caries, pulp injury and periodontal disease.

#### **2. The mechanisms of tooth development**

dental medicine, tooth tissue-derived stem cells and the cytokine network that regulates tooth development have been well characterised at the molecular level (Jussila et al., 2013). These advances can be applied to the repair of dental pulp and periodontal tissues, including the alveolar bone (Egusa et al., 2012, 2013). Organ replacement regenerative therapy, which involves constructing a fully functional bioengineered organ using three-dimensional cell manipulation *in vitro*, holds great promise for the replacement of dysfunctional organs following disease, injury or aging. Tooth regenerative therapy would also involve the replace‐ ment of a lost or damaged tooth with a bioengineered tooth, constructed with stem cells, that has the capacity to become a functional unit comprising the whole tooth and periodontal tissue (Yen & Sharpe, 2008). It is anticipated that tooth replacement therapy will be established in the near future as a novel biological treatment for the functional recovery of lost teeth to satisfy both aesthetic and physiological requirements (Fig. 1). Over the past three decades, many approaches for replacing missing teeth have been studied, including three-dimensional bioengineered teeth and tooth germ generation using biodegradable materials and cell aggregation methods (Volponi et al., 2010). Recently, studies have reported tooth replacement by transplantation of fully functioning bioengineered teeth having the correct tooth structure, masticatory performance, proper responsiveness to mechanical stress and neural function after transplantation into the region of tooth loss (Ikeda et al., 2009; Nakao et al., 2007; Oshima et al., 2011). In this chapter, we describe novel technologies for whole tooth replacement that have the potential to provide functional recovery and could someday replace current dental

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

Recent approaches for tooth regenerative therapy have included tissue repair and whole tooth replacement. Tooth regenerative therapy and stem cell transplantation therapies are regarded as attractive approaches for repairing tissue that has been damaged by dental caries or periodontal disease. The transplantation of dental stem cells has been examined for the

treatments based on artificial materials.

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110

**Figure 1.** Concepts of tooth regenerative therapy

treatment of dental caries, pulp injury and periodontal disease.

Ectodermal organs, such as the teeth, hair and salivary glands, arise from their respective organ germs through reciprocal epithelial-mesenchymal interactions in the developing embryo. These interactions, which involve various signalling molecules and transcription factors, are the principal mechanism regulating organogenesis (Jussila et al., 2013). In tooth germ devel‐ opment, the dental lamina first thickens (lamina stage). This stage is followed by epithelial thickening (placode stage) at the future location of the tooth and subsequent epithelial budding to the underlying neural crest-derived ecto-mesenchyme. Tooth germ formation is initiated on embryonic days (EDs) 10-11 in mice by epithelial signals that include fibroblast growth factor (FGF) 8, bone morphogenetic protein (BMP) 4, sonic hedgehog (Shh), tumour necrosis factor (TNF) and Wnt10b. These signals induce the expression of several transcription factors in the dental mesenchyme that condense around the developing epithelial bud (bud stage) (Jussila et al., 2013; O'connell et al., 2013). At ED13.5-14.5, the first enamel knot, which acts as a signalling centre to orchestrate tooth development by controlling the gene expression of various signalling molecules and transcription factors, is formed in the dental epithelium (cap stage). At ED16-18, the epithelial and mesenchymal cells in the tooth germ terminally differ‐ entiate into the tooth-tissue progenitor cells, such as ameloblasts, odontoblasts, and dental follicle cells (bell stage). Ameloblasts and odontoblasts accumulate the enamel and dentin matrix, respectively, at the boundary surface between the epithelium and mesenchyme, while dental follicle cells differentiate into the periodontal tissues, which include the cementum, periodontal ligaments and alveolar bone (Avery, 2002).

### **3. A novel three-dimensional cell manipulation method for whole tooth regeneration**

One current biological approach for the regeneration of three-dimensional organs is based on recapitulating organogenesis by mimicking the reciprocal epithelial-mesenchymal interac‐ tions that occur in the developing embryo, thereby developing fully functional bioengineered organs from a bioengineered organ germ generated from immature stem cells via threedimensional cell manipulation *in vitro*. For tooth regeneration, one proposed concept has been to transplant a bioengineered tooth germ into the recipient jaw and allowing it to develop into a functional mature tooth *in situ* (Fig. 2, upper). It is also expected that it will be possible to transplant a bioengineered tooth unit that includes mature tooth, periodontal ligament and alveolar bone, which will achieve biological engraftment through bone integration with the recipient's jaw (Fig. 2, lower).

To realise whole tooth replacement, the first critical issue is to develop a three-dimensional cell manipulation method using completely dissociated epithelial and mesenchymal cells *in vitro*. Previously, it has been reported that using a polyglycolic acid and poly-L-lactate-coglycolide copolymer (PLA/PLGA) or a collagen sponge as a tooth-shaped scaffold and seeding them with epithelial and mesenchymal cells isolated from tooth buds could generate small tooth structures (Honda et al., 2007; Yelick & Vacanti, 2006).

> bioengineered tooth unit comprising a mature tooth, periodontal ligament and alveolar bone after transplantation into the subrenal capsule (Fig. 3B). These technologies have the potential to be adapted for successful functional tooth replacement *in vivo* and are expected to represent

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113

**Figure 3.** The organ germ method: three-dimensional cell processing A) Dissociated mesenchymal cells at a high den‐ sity are injected into the centre of a collagen drop. Dissociated tooth germ-derived epithelial cells are subsequently injected into the drop adjacent to the mesenchymal cell aggregate (upper). Within 1 day of organ culture, bioengi‐ neered tooth germ formation with appropriate compartmentalisation between epithelial and mesenchymal cells and cell-to-cell compaction was observed (lower). B) By transplanting a bioengineered tooth germ into a subrenal capsule for 30 days (left panel), a bioengineered tooth unit comprising a mature tooth with the correct structural components such as enamel (E), dentin (D), periodontal ligament (PDL) and alveolar bone (AB) can be produced (right panel).

Oral functions such as mastication, pronunciation, and facial aesthetics have an important influence on quality of life because they facilitate both oral communication and nutritional intake. These functions are achieved with the teeth, masticatory muscles and the temporo‐ mandibular joint, under control of the central nervous system. For the realisation of tooth replacement regenerative therapy, a regenerated tooth developing from bioengineered germ tissue or a transplanted bioengineered mature tooth unit must be capable of properly engraft‐ ing into the lost tooth region in an adult oral environment and acquiring full functionality, including sufficient masticatory performance, biochemical cooperation with periodontal tissues and afferent responsiveness to noxious stimulations in the maxillofacial region (Proffit

**4. Functional tooth replacement therapy**

et al., 2004).

a substantial advance in bioengineered organ replacement regenerative therapy.

**Figure 2.** Strategies for whole tooth replacement via regenerative therapies. Functioning teeth can now be regenerat‐ ed *in vivo* by transplanting bioengineered tooth germ generated from epithelial and mesenchymal cells via the organ germ method, or bioengineered tooth units with periodontal ligament and alveolar bone developed from bioengi‐ neered tooth germ.

In addition, the cell aggregation method, which aims to reconstitute a bioengineered organ germ, has been applied for the transplantation of cell aggregates constructed from dental epithelial and mesenchymal cells, and it has been reported that this approach can generate appropriate tooth formation (Hu et al., 2006). It has also been reported that mixed cell aggre‐ gates of tooth germ-derived epithelial and mesenchymal cells can develop into a tooth with the correct structure, following epithelial cell sorting and subsequent self-organisation of the epithelial and mesenchymal cells (Song et al., 2006). However, these approaches suffer from critical limitations, including a low frequency of tooth formation and irregularity of the resulting tooth tissue structures, for example with enamel-dentin complex formation and the arrangements of the ameloblast/odontoblast cell lineages.

To achieve precise replication of the processes in organogenesis, an *in vitro* three-dimensional novel cell manipulation method designated as the bioengineered organ germ method has been developed (Nakao et al., 2007). This innovative method is based on compartmentalisation of the epithelial and mesenchymal cells at a high-cell density in a type I collagen gel (Fig. 3A). Bioengineered tooth germ created by this technique, which could allow for large-scale organ development, mimics the multicellular assembly underlying epithelial-mesenchymal interac‐ tions during natural tooth development. This bioengineered tooth germ generates a correct tooth structure after transplantation in an organ culture *in vitro* as well as following placement into a subrenal capsule *in vivo*. The bioengineered tooth germ generated by this method was also found to develop in the oral cavity to form the proper tooth structure (Nakao et al., 2007). Furthermore, this unique technology can successfully generate a size-controlled bioengineered tooth unit comprising a mature tooth, periodontal ligament and alveolar bone after transplantation into the subrenal capsule (Fig. 3B). These technologies have the potential to be adapted for successful functional tooth replacement *in vivo* and are expected to represent a substantial advance in bioengineered organ replacement regenerative therapy.

**Figure 3.** The organ germ method: three-dimensional cell processing A) Dissociated mesenchymal cells at a high den‐ sity are injected into the centre of a collagen drop. Dissociated tooth germ-derived epithelial cells are subsequently injected into the drop adjacent to the mesenchymal cell aggregate (upper). Within 1 day of organ culture, bioengi‐ neered tooth germ formation with appropriate compartmentalisation between epithelial and mesenchymal cells and cell-to-cell compaction was observed (lower). B) By transplanting a bioengineered tooth germ into a subrenal capsule for 30 days (left panel), a bioengineered tooth unit comprising a mature tooth with the correct structural components such as enamel (E), dentin (D), periodontal ligament (PDL) and alveolar bone (AB) can be produced (right panel).

#### **4. Functional tooth replacement therapy**

**Figure 2.** Strategies for whole tooth replacement via regenerative therapies. Functioning teeth can now be regenerat‐ ed *in vivo* by transplanting bioengineered tooth germ generated from epithelial and mesenchymal cells via the organ germ method, or bioengineered tooth units with periodontal ligament and alveolar bone developed from bioengi‐

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

In addition, the cell aggregation method, which aims to reconstitute a bioengineered organ germ, has been applied for the transplantation of cell aggregates constructed from dental epithelial and mesenchymal cells, and it has been reported that this approach can generate appropriate tooth formation (Hu et al., 2006). It has also been reported that mixed cell aggre‐ gates of tooth germ-derived epithelial and mesenchymal cells can develop into a tooth with the correct structure, following epithelial cell sorting and subsequent self-organisation of the epithelial and mesenchymal cells (Song et al., 2006). However, these approaches suffer from critical limitations, including a low frequency of tooth formation and irregularity of the resulting tooth tissue structures, for example with enamel-dentin complex formation and the

To achieve precise replication of the processes in organogenesis, an *in vitro* three-dimensional novel cell manipulation method designated as the bioengineered organ germ method has been developed (Nakao et al., 2007). This innovative method is based on compartmentalisation of the epithelial and mesenchymal cells at a high-cell density in a type I collagen gel (Fig. 3A). Bioengineered tooth germ created by this technique, which could allow for large-scale organ development, mimics the multicellular assembly underlying epithelial-mesenchymal interac‐ tions during natural tooth development. This bioengineered tooth germ generates a correct tooth structure after transplantation in an organ culture *in vitro* as well as following placement into a subrenal capsule *in vivo*. The bioengineered tooth germ generated by this method was also found to develop in the oral cavity to form the proper tooth structure (Nakao et al., 2007). Furthermore, this unique technology can successfully generate a size-controlled

arrangements of the ameloblast/odontoblast cell lineages.

neered tooth germ.

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Oral functions such as mastication, pronunciation, and facial aesthetics have an important influence on quality of life because they facilitate both oral communication and nutritional intake. These functions are achieved with the teeth, masticatory muscles and the temporo‐ mandibular joint, under control of the central nervous system. For the realisation of tooth replacement regenerative therapy, a regenerated tooth developing from bioengineered germ tissue or a transplanted bioengineered mature tooth unit must be capable of properly engraft‐ ing into the lost tooth region in an adult oral environment and acquiring full functionality, including sufficient masticatory performance, biochemical cooperation with periodontal tissues and afferent responsiveness to noxious stimulations in the maxillofacial region (Proffit et al., 2004).

#### **4.1. Transplantation of bioengineered tooth germ or a bioengineered mature tooth unit as a tooth replacement therapy**

The critical issue dictating the success of tooth regenerative therapy via the transplantation of bioengineered tooth germ tissue into the lost tooth region is whether the germ can erupt and occlude properly with the opposing tooth in an adult oral environment. It has previously been demonstrated that transplanted natural tooth germ erupts in a murine toothless diastema region (Ohazama et al., 2004). We have also reported that a bioengineered tooth germ can develop the correct tooth structure in an oral cavity and successfully erupt 37 days after transplantation (Ikeda et al., 2009). The bioengineered tooth subsequently reached the occlusal plane and achieved occlusion with the opposing tooth from 49 days onwards (Fig. 4A, B). In the case of a transplanted bioengineered mature tooth unit comprising mature tooth, perio‐ dontal ligament and alveolar bone, the most critical consideration is whether that unit can be engrafted into the tooth loss region through bone integration, which involves natural bone remodelling in the recipient. A bioengineered tooth unit transplanted at a position reaching the occlusal plane with the opposing upper first molar was successfully engrafted after 40 days and thereafter maintained the periodontal ligament originating from the bioengineered tooth unit through successful bone integration (Fig. 4C) (Oshima et al., 2011). The enamel and dentin hardness of the bioengineered tooth components were in the normal range when analysed by the Knoop hardness test (Ikeda et al., 2009; Oshima et al., 2011). These approaches demonstrate the potential to successfully recover masticatory performance and natural tooth tissue through state-of-the-art bioengineering technology.

**4.2. Biological response of bioengineered teeth to mechanical stress**

bone (Ikeda et al., 2009; Oshima et al., 2011).

cooperation with the maxillofacial region.

**4.3. Perceptive neuronal potential of bioengineered teeth**

Biological oral functions require cooperation between teeth and the maxillofacial region through the connection of periodontal ligaments (Dawson, 2006). Tooth loss and periodon‐ tal disease cause fundamental problems for oral function, including mastication, as well as associated health issues. The periodontal ligament plays an essential role in the pathogen‐ ic and physiological tooth response to extreme mechanical forces from bone remodelling accompanied by orthodontic tooth movement (Proffit et al., 2004). Studies on autologous tooth transplantation have indicated that healthy periodontal tissue remaining on the tooth root can successfully restore physiological tooth function, including bone remodelling, and effectively prevent ankylosis. In contrast, the absence of a periodontal ligament in osseointegrated dental implants is associated with deficiencies in essential tooth functions and in the natural structural relationship between the tooth root and alveolar bone (Dawson, 2006). The periodontal ligament of bioengineered teeth that erupted following the transplan‐ tation of bioengineered tooth germ and mature tooth units achieved functional tooth movement comparable with that of natural teeth. Bioengineered teeth also successfully underwent bone remodelling in response to mechanical stress via the proper localisation of osteoclasts and osteoblasts, indicating that a bioengineered tooth can reproduce critical tooth functions by restoring and re-establishing cooperation with the surrounding jaw‐

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The peripheral nervous system is established by the growth of axons that navigate and establish connections with developing target organs during embryogenesis (Guyton & Hall, 2000). The perceptive potential for noxious stimulation, including mechanical stress and pain, is important for proper organ function (Guyton & Hall, 2000). Additionally, it is believed that the recovery of the nervous system, which requires the re-entry of nerve fibres following organ transplantation, is critical for reconstituting organ function. Teeth are a peripheral organ for sensory and sympathetic nerves, both of which play important roles in tooth function and protection (Dawson, 2006). It is anticipated that tooth regenerative therapies will be able to recover the neuronal ability related to the perception of mechani‐ cal forces that are lacking in implant patients. Importantly, sensory and sympathetic nerve fibres innervate both the pulp and periodontal ligament of a bioengineered tooth follow‐ ing its eruption (Ikeda et al., 2009). Thus, these bioengineered teeth possess appropriate perceptive potential for nociceptive pain stimulations, such as pulp injury and orthodon‐ tic treatment, and can properly transduce these events to the central nervous system through c-Fos immunoreactive neurons (Ikeda et al., 2009; Oshima et al., 2011). In this way, bioengineered teeth can indeed restore the perceptive potential for noxious stimuli in

**Figure 4.** Regeneration of a bioengineered tooth in an adult oral environment A) A transplanted bioengineered tooth germ erupted and reached the occlusal plane with the opposing lower first molar 49 days after transplantation. B) GFP-labelled bioengineered tooth (right panel) erupted in the oral environment of adult mice. C) A bioengineered tooth unit was engrafted by bone integration and reached the occlusal plane with the opposing upper first molar at 40 days post transplantation.

#### **4.2. Biological response of bioengineered teeth to mechanical stress**

**4.1. Transplantation of bioengineered tooth germ or a bioengineered mature tooth unit as**

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

The critical issue dictating the success of tooth regenerative therapy via the transplantation of bioengineered tooth germ tissue into the lost tooth region is whether the germ can erupt and occlude properly with the opposing tooth in an adult oral environment. It has previously been demonstrated that transplanted natural tooth germ erupts in a murine toothless diastema region (Ohazama et al., 2004). We have also reported that a bioengineered tooth germ can develop the correct tooth structure in an oral cavity and successfully erupt 37 days after transplantation (Ikeda et al., 2009). The bioengineered tooth subsequently reached the occlusal plane and achieved occlusion with the opposing tooth from 49 days onwards (Fig. 4A, B). In the case of a transplanted bioengineered mature tooth unit comprising mature tooth, perio‐ dontal ligament and alveolar bone, the most critical consideration is whether that unit can be engrafted into the tooth loss region through bone integration, which involves natural bone remodelling in the recipient. A bioengineered tooth unit transplanted at a position reaching the occlusal plane with the opposing upper first molar was successfully engrafted after 40 days and thereafter maintained the periodontal ligament originating from the bioengineered tooth unit through successful bone integration (Fig. 4C) (Oshima et al., 2011). The enamel and dentin hardness of the bioengineered tooth components were in the normal range when analysed by the Knoop hardness test (Ikeda et al., 2009; Oshima et al., 2011). These approaches demonstrate the potential to successfully recover masticatory performance and natural tooth tissue through

**Figure 4.** Regeneration of a bioengineered tooth in an adult oral environment A) A transplanted bioengineered tooth germ erupted and reached the occlusal plane with the opposing lower first molar 49 days after transplantation. B) GFP-labelled bioengineered tooth (right panel) erupted in the oral environment of adult mice. C) A bioengineered tooth unit was engrafted by bone integration and reached the occlusal plane with the opposing upper first molar at

**a tooth replacement therapy**

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114

state-of-the-art bioengineering technology.

40 days post transplantation.