**2. Research projects**

This chapter was edited by collecting all the achievement performed in the laboratory of oral and maxillofacial surgery and it brings together the specific experiences of the scientific community in these experiences of our scientific community in this field as well as the clinical experiences of the most renowned experts in the fields from all over Nagoya University. The editors are especially proud of bringing together the leading biologists and material scientists together with dentist, plastic surgeons and surgeons of all specialities from all department of the medical school of Nagoya University. Taken together, this unique collection of worldwide

© 2013 Ueda; licensee InTech. This is an open access article 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. © 2013 Ueda; licensee InTech. This is a paper 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.

expert achievement and experiences represents the current spectrum of possibilities in tissue engineered substitution.

### **2.1. Bone regeneration with self assembling peptide nanofiber scaffolds in tissue engineering for osseointegration of dental implants**

The aim of this study was to evaluate the correlation between the osseointegration of dental implants and tissue‐engineered bone using a nanofiber scaffold, *PuraMatrix* (PM). The first molar and all premolars in the mandible region of dogs were extracted, and three bone defects were prepared with a trephine bar on both sides of the mandible after 4 weeks. The experi‐ mental groups were as follows: 1) PM, 2) PM and dog mesenchymal stem cells (MSCs), 3) PM, dog MSCs, and platelet‐rich plasma (PRP), and 4) a control (defect only). Implants were placed with in the prepared areas 8 weeks later, and assessed by histological and histomorphometric analyses (bone‐implant contact (BIC)). The BICs for groups 1, 2, 3, and 4 were 40.77%, 50.35%, 55.64% and 30.57%, respectively. The findings indicate that PM may be useful as a scaffold for bone regeneration around dental implants.

**Figure 1.** Molecular structure of PM. Molecular models of amiphiphilic self-complementary peptide have 16 amino acids with an alternating polar and nanopolar pattern. A= alanine; R= arginine; D= aspartic acid; + and – refer to the positively and negatively charged residues, respectively (From Kohgo et al. [1]. Reprinted with permission from Quin‐ tessence Publishing Co, Inc, Chicago).

**Figure 3.** Photographs of histologic sections, as seen on light microscopy, 8 weeks after implant placement. (a to c) In the control group, the buccal and lingual walls were not sufficiently for dental implants. (d to f) In the PM group, slight bone regeneration in the lingual wall was observed. (g to i) However, slightly more could be seen in the PM/dog MSCs group. (j to l) On the other hand, the amount of regenerated bone was greatest in the PM/dog MSCs/PRP group (a, d, g and j, magnification tion rat, e, h, and k, magnification ×200; c, f, i, and l, maginification ×200) (From Kohgo et al. [1].

Tissue Engineering and Regenerative Medicine 125

BIC = bone-to-implant contact; SD = standard deviation; PM = PuraMatrix; dMSCs = dog mesenchymal stem cells; PRP = Platelet-rich plasma \* P < 0.01 \*\*P < 0.05 (From Kohgo et al. 2011. Reprinted with permission from Quintessence

Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

Publishing Co, Inc, Chicago).

**Table 1.** Results of BIC (mean ±SD)†

**Figure 2.** PM is an injectable scaffold and shows good plasticity. Bar = 5 mm. (From Kohgo et al. [1]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 3.** Photographs of histologic sections, as seen on light microscopy, 8 weeks after implant placement. (a to c) In the control group, the buccal and lingual walls were not sufficiently for dental implants. (d to f) In the PM group, slight bone regeneration in the lingual wall was observed. (g to i) However, slightly more could be seen in the PM/dog MSCs group. (j to l) On the other hand, the amount of regenerated bone was greatest in the PM/dog MSCs/PRP group (a, d, g and j, magnification tion rat, e, h, and k, magnification ×200; c, f, i, and l, maginification ×200) (From Kohgo et al. [1]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).


BIC = bone-to-implant contact; SD = standard deviation; PM = PuraMatrix; dMSCs = dog mesenchymal stem cells; PRP = Platelet-rich plasma \* P < 0.01 \*\*P < 0.05 (From Kohgo et al. 2011. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Table 1.** Results of BIC (mean ±SD)†

expert achievement and experiences represents the current spectrum of possibilities in tissue

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

The aim of this study was to evaluate the correlation between the osseointegration of dental implants and tissue‐engineered bone using a nanofiber scaffold, *PuraMatrix* (PM). The first molar and all premolars in the mandible region of dogs were extracted, and three bone defects were prepared with a trephine bar on both sides of the mandible after 4 weeks. The experi‐ mental groups were as follows: 1) PM, 2) PM and dog mesenchymal stem cells (MSCs), 3) PM, dog MSCs, and platelet‐rich plasma (PRP), and 4) a control (defect only). Implants were placed with in the prepared areas 8 weeks later, and assessed by histological and histomorphometric analyses (bone‐implant contact (BIC)). The BICs for groups 1, 2, 3, and 4 were 40.77%, 50.35%, 55.64% and 30.57%, respectively. The findings indicate that PM may be useful as a scaffold for

**Figure 1.** Molecular structure of PM. Molecular models of amiphiphilic self-complementary peptide have 16 amino acids with an alternating polar and nanopolar pattern. A= alanine; R= arginine; D= aspartic acid; + and – refer to the positively and negatively charged residues, respectively (From Kohgo et al. [1]. Reprinted with permission from Quin‐

**Figure 2.** PM is an injectable scaffold and shows good plasticity. Bar = 5 mm. (From Kohgo et al. [1]. Reprinted with

**2.1. Bone regeneration with self assembling peptide nanofiber scaffolds in tissue**

**engineering for osseointegration of dental implants**

bone regeneration around dental implants.

tessence Publishing Co, Inc, Chicago).

permission from Quintessence Publishing Co, Inc, Chicago).

engineered substitution.

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The results suggest that tissue‐engineered bone can integrate well around dental implants. PM is a 3D structure may have the potential to be a scaffold applicable in bone tissue engineering.

### **2.2. Self‐assembling peptide nanofiber scaffolds, platelet rich plasma, and mesenchymal stem cells for injectable bone regeneration with tissue engineering**

The ideal biomaterial scaffolds, particularly in cranio‐maxillofacial, plastic, or orthopedic fields with complicated bone‐defect shapes, should have excellent plasticity for fitting into complex defect shapes and the rate of absorption needs to be fast in order to avoid infection. Moreover, these materials and their internal structures may not provide a particularly favorable environment for cell survival and bone regeneration, and internal, microscale environment of these materials needs to serve as an extracellular matrix (ECM). ECM is a dynamic organized nanocomposite that not only provides mechanical support for embedded cells but also interacts with cells and promotes and regulates cellular functions such as adhesion, migration, proliferation, and differentiation and is consequently involved in three‐ dimensional morphogenesis. This study considered the use of the matrix material PM, which is synthesized by chemical peptide methods and has similarities to the fibers and pore sizes found in the ECM. We investigated a capability of PM as a scaffold for bone regeneration in combination with dog MSCs and/or PRP using tissue engineering and regenerative medicine technology. First, teeth were extracted from an adult hybrid dog's mandible region. After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. The listed graft materials were implanted into these defects: 1) control (defect only), 2) PM, 3) PM/ PRP, 4) PM/dog MSCs, and 5) PM/dog MSCs/PRP. At 2, 4, and 8 weeks after implantation, each sample was collected from the graft area with a trephine bar and assessed by histological and histomorphometric analyses.

From histological evaluation, It was observed that the bone regenerated by PM/dog MSCs/PRP was excellent quality, and it was found that mature bone had been formed. Histometrically, at 8 weeks newly formed bone areas comprised 12.39 ± 1.29% (control), 25.28 ± 3.92% (PM), 27.72 ± 3.15% (PM/PRP), 50.07 ± 3.97% (PM/dog MSCs), and 58.43 ± 5.06% (PM/ dog MSCs/PRP). The PM/dog MSCs and PM/dog MSCs/PRP groups showed a significant increase at all weeks compared with the control, PM, or PM/PRP. These results showed that MSCs might keep their own potential and promote new bone regeneration in the three‐ dimensional structure by PM scaffolds. Taken together, it is suggested that PM might be useful as a scaffold of bone regeneration in cell therapy, and these results might lead to an effective treatment method for bone defects. In the future this tissue‐engineered bone grafting material could be used as a minimally invasive method instead of traditional grafting procedures.

### **2.3. Effects of self‐assembling peptide hydrogel scaffold on bone regeneration with recombinant human bone morphogenetic protein‐2**

Various biomaterials have been tested as scaffolds for bone regeneration, such as beta‐ tricalcium phosphate, hydroxyapatite, and polymers. However, a scaffold has still not been found that has the characteristics of biologic safety, absorbability, cell interaction, and bone inductivity. A self‐assembling peptide hydrogel scaffold is made of artificial synthetic

**Figure 4.** Schema of the experimental protocol (From Yoshimi et al. [2, 3]. Reprinted with permission).

Tissue Engineering and Regenerative Medicine 127

The results suggest that tissue‐engineered bone can integrate well around dental implants. PM is a 3D structure may have the potential to be a scaffold applicable in bone tissue engineering.

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**2.2. Self‐assembling peptide nanofiber scaffolds, platelet rich plasma, and mesenchymal**

The ideal biomaterial scaffolds, particularly in cranio‐maxillofacial, plastic, or orthopedic fields with complicated bone‐defect shapes, should have excellent plasticity for fitting into complex defect shapes and the rate of absorption needs to be fast in order to avoid infection. Moreover, these materials and their internal structures may not provide a particularly favorable environment for cell survival and bone regeneration, and internal, microscale environment of these materials needs to serve as an extracellular matrix (ECM). ECM is a dynamic organized nanocomposite that not only provides mechanical support for embedded cells but also interacts with cells and promotes and regulates cellular functions such as adhesion, migration, proliferation, and differentiation and is consequently involved in three‐ dimensional morphogenesis. This study considered the use of the matrix material PM, which is synthesized by chemical peptide methods and has similarities to the fibers and pore sizes found in the ECM. We investigated a capability of PM as a scaffold for bone regeneration in combination with dog MSCs and/or PRP using tissue engineering and regenerative medicine technology. First, teeth were extracted from an adult hybrid dog's mandible region. After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. The listed graft materials were implanted into these defects: 1) control (defect only), 2) PM, 3) PM/ PRP, 4) PM/dog MSCs, and 5) PM/dog MSCs/PRP. At 2, 4, and 8 weeks after implantation, each sample was collected from the graft area with a trephine bar and assessed by histological

From histological evaluation, It was observed that the bone regenerated by PM/dog MSCs/PRP was excellent quality, and it was found that mature bone had been formed. Histometrically, at 8 weeks newly formed bone areas comprised 12.39 ± 1.29% (control), 25.28 ± 3.92% (PM), 27.72 ± 3.15% (PM/PRP), 50.07 ± 3.97% (PM/dog MSCs), and 58.43 ± 5.06% (PM/ dog MSCs/PRP). The PM/dog MSCs and PM/dog MSCs/PRP groups showed a significant increase at all weeks compared with the control, PM, or PM/PRP. These results showed that MSCs might keep their own potential and promote new bone regeneration in the three‐ dimensional structure by PM scaffolds. Taken together, it is suggested that PM might be useful as a scaffold of bone regeneration in cell therapy, and these results might lead to an effective treatment method for bone defects. In the future this tissue‐engineered bone grafting material could be used as a minimally invasive method instead of traditional grafting procedures.

**2.3. Effects of self‐assembling peptide hydrogel scaffold on bone regeneration with**

Various biomaterials have been tested as scaffolds for bone regeneration, such as beta‐ tricalcium phosphate, hydroxyapatite, and polymers. However, a scaffold has still not been found that has the characteristics of biologic safety, absorbability, cell interaction, and bone inductivity. A self‐assembling peptide hydrogel scaffold is made of artificial synthetic

**recombinant human bone morphogenetic protein‐2**

**stem cells for injectable bone regeneration with tissue engineering**

and histomorphometric analyses.

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126

**Figure 4.** Schema of the experimental protocol (From Yoshimi et al. [2, 3]. Reprinted with permission).

**Figure 6.** Photographs of surgical procedure. In the left and right parietal and frontal bones, 4 circular slits were pre‐ pared. Five holes were prepared in outer cortical bone inside this circle (a). Apical end of the 4 titanium cylinders was pressed into each slit, and primary fixation was obtained (b). Four titanium cylinders were filled with respective materi‐ als (c). The top of the cylinders was closed with a titanium lid (d) (From Ikeno et al. [4]. Reprinted with permission from

Tissue Engineering and Regenerative Medicine 129

**Figure 7.** Higher magnification of a histological section Bar = 100μm. NB, newly formed bone; CT, connective tissue; BV, blood vessel; OB, osteoblast-like cells (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publish‐

New bone formation seemed to occur from the calvarial bone through the perforations in the outer cortical bone; newly formed bone was also observed in all groups. Under higher magnification, newly formed bone, including cells and some blood vessels, was observed in the connective tissue and the edges of bone were lined with osteoblast‐like cells in the cylinder (Figure 8). Regenerated tissue in the sample treated with PM/rhBMP‐2 was observed in about

Histomorphometric analysis showed thatregenerated tissue in the cylinder with PM/rhBMP‐2 was significantly increased compared to the empty control (Fig. 8). The mean area values of regenerated tissue in the cylinders were 35.80% ± 10.35% (control), 47.94% ± 5.65% (rhBMP‐2), 48.94% ± 11.33% (PM), and 58.06% ± 14.84% (PM/rhBMP‐2). The mean area values of newly formed bone in the cylinders were 9.39% ± 4.34% (control), 14.03% ± 2.25% (rhBMP‐2), 13.99% ± 2.15% (PM), and 16.61% ± 3.79% (PM/rhBMP‐2). NeitherrhBMP‐2 nor PM alone significantly

enhanced bone regeneration compared to the empty control cylinder.

Quintessence Publishing Co, Inc, Chicago).

ing Co, Inc, Chicago).

two thirds of the cylinders (Figure 9).

**Figure 5.** Histological evaluation of control, PM, PM/PRP, PM/dog MSCs, and PM/dog MSCs/PRP implantations at each time point (center figure:lower magnification, right figure: higher magnification). Sections of representative im‐ plants are shown from the respective group. The sections were stained with hematoxylin and eosin. Original magnifi‐ cation, ×25 for all photographs. (A) 2 weeks Control group, (B) 4 weeks Control group, (C) 8 weeks Control group, (D) 2 weeks PM group, (E) 4 weeks PM group, (F) 8 weeks PM group, (G) 2 weeks PM/PRP group, (H) 4 weeks PM/PRP group, (I) 8 weeks PM/PRP group, (J) 2 weeks PM/dog MSCs group, (K) 4 weeks PM/dog MSCs group, (L) 8 weeks PM/dog MSCs group, (M) 2 weeks PM/dog MSCs/PRP group, (N) 4 weeks PM/dog MSCs/PRP group, and (O) 8 weeks PM/dog MSCs/PRP group (From Yoshimi et al. [4, 5]. Reprinted with permission).

materials featuring biologic safety and absorbability. PM is expected to be a candidate as a scaffold for bone regeneration. Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) has exhibited high osteogenic activity in experimental studies. The objective of this pilot study was to histologically evaluate bone regeneration using a self‐assembling peptide hydrogel scaffold with rhBMP‐2 on the bone augmentation in a rabbit calvaria model (Figure 6).

**Figure 6.** Photographs of surgical procedure. In the left and right parietal and frontal bones, 4 circular slits were pre‐ pared. Five holes were prepared in outer cortical bone inside this circle (a). Apical end of the 4 titanium cylinders was pressed into each slit, and primary fixation was obtained (b). Four titanium cylinders were filled with respective materi‐ als (c). The top of the cylinders was closed with a titanium lid (d) (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 7.** Higher magnification of a histological section Bar = 100μm. NB, newly formed bone; CT, connective tissue; BV, blood vessel; OB, osteoblast-like cells (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publish‐ ing Co, Inc, Chicago).

New bone formation seemed to occur from the calvarial bone through the perforations in the outer cortical bone; newly formed bone was also observed in all groups. Under higher magnification, newly formed bone, including cells and some blood vessels, was observed in the connective tissue and the edges of bone were lined with osteoblast‐like cells in the cylinder (Figure 8). Regenerated tissue in the sample treated with PM/rhBMP‐2 was observed in about two thirds of the cylinders (Figure 9).

Histomorphometric analysis showed thatregenerated tissue in the cylinder with PM/rhBMP‐2 was significantly increased compared to the empty control (Fig. 8). The mean area values of regenerated tissue in the cylinders were 35.80% ± 10.35% (control), 47.94% ± 5.65% (rhBMP‐2), 48.94% ± 11.33% (PM), and 58.06% ± 14.84% (PM/rhBMP‐2). The mean area values of newly formed bone in the cylinders were 9.39% ± 4.34% (control), 14.03% ± 2.25% (rhBMP‐2), 13.99% ± 2.15% (PM), and 16.61% ± 3.79% (PM/rhBMP‐2). NeitherrhBMP‐2 nor PM alone significantly enhanced bone regeneration compared to the empty control cylinder.

materials featuring biologic safety and absorbability. PM is expected to be a candidate as a scaffold for bone regeneration. Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) has exhibited high osteogenic activity in experimental studies. The objective of this pilot study was to histologically evaluate bone regeneration using a self‐assembling peptide hydrogel scaffold with rhBMP‐2 on the bone augmentation in a rabbit calvaria model (Figure 6).

PM/dog MSCs/PRP group (From Yoshimi et al. [4, 5]. Reprinted with permission).

**Figure 5.** Histological evaluation of control, PM, PM/PRP, PM/dog MSCs, and PM/dog MSCs/PRP implantations at each time point (center figure:lower magnification, right figure: higher magnification). Sections of representative im‐ plants are shown from the respective group. The sections were stained with hematoxylin and eosin. Original magnifi‐ cation, ×25 for all photographs. (A) 2 weeks Control group, (B) 4 weeks Control group, (C) 8 weeks Control group, (D) 2 weeks PM group, (E) 4 weeks PM group, (F) 8 weeks PM group, (G) 2 weeks PM/PRP group, (H) 4 weeks PM/PRP group, (I) 8 weeks PM/PRP group, (J) 2 weeks PM/dog MSCs group, (K) 4 weeks PM/dog MSCs group, (L) 8 weeks PM/dog MSCs group, (M) 2 weeks PM/dog MSCs/PRP group, (N) 4 weeks PM/dog MSCs/PRP group, and (O) 8 weeks

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

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killed, and non‐decalcified section was obtained. BIC and bone area (BA) around the implants were assessed with cortical bone and cancellous bone. Furthermore, bone density (BD) was evaluated in a 500mm wide zone of cancellous bone lateral to the implants (Figure10). At 28 and 56 days after implantation, no significant difference was found between the OVX and SHAM groups for BIC and BA in cortical bone. BIC, BA, and BD with cancellous bone was lower in OVX group than in SHAM group. However, BIC and BA tended to improve by the

Tissue Engineering and Regenerative Medicine 131

**Figure 10.** Histomorphological observation and morphological evaluations (From Tateishi et al. [5, 6]. Reprinted with

variance of implant surface (Figure 10.11.12.13).

permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 8.** Histological sections of cylinders with empty control (a), rhBMP-2 (b), PM (c), and PM/rhBMP-2 (d) (toluidine blue stain) (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 9.** Regenerated tissue area measured by image analysis. The percentage of area of regenerated tissue (left), maximum height of newly-formed bone (middle), and area of newly-formed bone (right) in the cylinder with each group was displayed. Bar = SD. Analysis of variance (ANOVA) (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

PM, a synthetic self‐assembling peptide, used in combination with recombinant human bone morphogenetic protein‐2 significantly enhanced bone regeneration in a bone augmentation model in rabbits. PM promises to be an alternative synthetic material as a useful carrier for recombinant human bone morphogenetic protein for bone regeneration.

#### **2.4. A study of bone healing around the titanium screw implants in osteoporosis: Can sandblasted surface contribute to the implant stability?**

Dental implants are widely performed as a prosthetic treatment in edentulous patients. However, in the aged patients, there are various systemic risk factors such a osteoporosis. The aim of this study was to investigate whether estrogen deficiency interrupts bone healing around titanium implants and to evaluate whether bone healing around implants under the condition of estrogen deficiency is affected by implant surface variance. Forty eight female SD rats were divided into two groups: ovariectomized rats (OVX; n=24) and sham operated rats (SHAM; n=24). Each group was further divided into two groups: a machine polished implants placed group and a sandblasted implants placed group. Both implants were placed in the rat left femur centrifugal site 84 days after OVX or sham surgery. After 28, 56 days, the rats were killed, and non‐decalcified section was obtained. BIC and bone area (BA) around the implants were assessed with cortical bone and cancellous bone. Furthermore, bone density (BD) was evaluated in a 500mm wide zone of cancellous bone lateral to the implants (Figure10). At 28 and 56 days after implantation, no significant difference was found between the OVX and SHAM groups for BIC and BA in cortical bone. BIC, BA, and BD with cancellous bone was lower in OVX group than in SHAM group. However, BIC and BA tended to improve by the variance of implant surface (Figure 10.11.12.13).

**Figure 8.** Histological sections of cylinders with empty control (a), rhBMP-2 (b), PM (c), and PM/rhBMP-2 (d) (toluidine

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 9.** Regenerated tissue area measured by image analysis. The percentage of area of regenerated tissue (left), maximum height of newly-formed bone (middle), and area of newly-formed bone (right) in the cylinder with each group was displayed. Bar = SD. Analysis of variance (ANOVA) (From Ikeno et al. [4]. Reprinted with permission from

PM, a synthetic self‐assembling peptide, used in combination with recombinant human bone morphogenetic protein‐2 significantly enhanced bone regeneration in a bone augmentation model in rabbits. PM promises to be an alternative synthetic material as a useful carrier for

**2.4. A study of bone healing around the titanium screw implants in osteoporosis: Can**

Dental implants are widely performed as a prosthetic treatment in edentulous patients. However, in the aged patients, there are various systemic risk factors such a osteoporosis. The aim of this study was to investigate whether estrogen deficiency interrupts bone healing around titanium implants and to evaluate whether bone healing around implants under the condition of estrogen deficiency is affected by implant surface variance. Forty eight female SD rats were divided into two groups: ovariectomized rats (OVX; n=24) and sham operated rats (SHAM; n=24). Each group was further divided into two groups: a machine polished implants placed group and a sandblasted implants placed group. Both implants were placed in the rat left femur centrifugal site 84 days after OVX or sham surgery. After 28, 56 days, the rats were

recombinant human bone morphogenetic protein for bone regeneration.

**sandblasted surface contribute to the implant stability?**

Quintessence Publishing Co, Inc, Chicago).

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130

blue stain) (From Ikeno et al. [4]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 10.** Histomorphological observation and morphological evaluations (From Tateishi et al. [5, 6]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

Estrogen deficiency affected bone healing and bone density around the titanium implants, especially in cancellous bone, but sandblasted surface property has the possibility to improve osseointegration. However, the positive effect by rough surface property is limited on the

Tissue Engineering and Regenerative Medicine 133

**2.5. Osteogenic induction of bone marrow‐derived stromal cells on simvastatin‐releasing,**

Tissue engineering is an effective approach forthe treatment of bone defects. Statins have been demonstrated topromote osteoblastic differentiation of bone marrow‐derived MSCs. Electro‐ spun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano‐ to microscale topography and high porosity similar to the natural ECM. The aim of this study was to investigate the feasibility of a simvastatin‐ releasing, biodegradable, nano‐ to microscale fiber scaffold (SRBFS) for bone tissue engineer‐ ing with MSCs. Simvastatin was released from SRBFS slowly (Figure 14). MSCs were observed to spread actively and rigidly adhere to SRBFS. MSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture (Figure 15). Furthermore, osteoclastogenesis was suppressed by SRBFS *in vitro* (Figure 16a‐c). The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell‐scaffold construct into an ectopic site on the murine back (Figure 17a‐c). These results suggest that SRBFS promoted osteoblastic differentiation of MSCs *in vitro* and *in vivo*, and demonstrate feasibility as a bone engineering

**Figure 14.** Cumulative release of SRBFS in vitro (From Wadagaki et al. [7, 8]. Reprinted with permission).

implant surface.

scaffold.

**biodegradable, nano‐ to microscale fiber scaffolds**

**Figure 11.** Bone to implant contact (BIC) at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted with per‐ mission from Quintessence Publishing Co, Inc, Chicago).

**Figure 12.** Bone area (BA) inside the implant thread at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 13.** Bone density (BD) around the implants at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

Estrogen deficiency affected bone healing and bone density around the titanium implants, especially in cancellous bone, but sandblasted surface property has the possibility to improve osseointegration. However, the positive effect by rough surface property is limited on the implant surface.

#### **2.5. Osteogenic induction of bone marrow‐derived stromal cells on simvastatin‐releasing, biodegradable, nano‐ to microscale fiber scaffolds**

Tissue engineering is an effective approach forthe treatment of bone defects. Statins have been demonstrated topromote osteoblastic differentiation of bone marrow‐derived MSCs. Electro‐ spun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano‐ to microscale topography and high porosity similar to the natural ECM. The aim of this study was to investigate the feasibility of a simvastatin‐ releasing, biodegradable, nano‐ to microscale fiber scaffold (SRBFS) for bone tissue engineer‐ ing with MSCs. Simvastatin was released from SRBFS slowly (Figure 14). MSCs were observed to spread actively and rigidly adhere to SRBFS. MSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture (Figure 15). Furthermore, osteoclastogenesis was suppressed by SRBFS *in vitro* (Figure 16a‐c). The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell‐scaffold construct into an ectopic site on the murine back (Figure 17a‐c). These results suggest that SRBFS promoted osteoblastic differentiation of MSCs *in vitro* and *in vivo*, and demonstrate feasibility as a bone engineering scaffold.

**Figure 11.** Bone to implant contact (BIC) at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted with per‐

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 12.** Bone area (BA) inside the implant thread at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted

**Figure 13.** Bone density (BD) around the implants at 28 days after implantation (From Tateishi et al. [5, 6]. Reprinted

mission from Quintessence Publishing Co, Inc, Chicago).

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132

with permission from Quintessence Publishing Co, Inc, Chicago).

with permission from Quintessence Publishing Co, Inc, Chicago).

**Figure 14.** Cumulative release of SRBFS in vitro (From Wadagaki et al. [7, 8]. Reprinted with permission).

**Figure 15.** ALP activity of MSCs on SRBFS and BFS measured on days 7 and 14 (From Wadagaki et al. [7, 8]. Reprinted with permission).

**Figure 17.** (a) Bone formation area analyzed by H&E staining 12 weeks after implantation. (b) Percent BSP (bone sialo protein) -positive cells as determined by immunohistochemical staining with BSP 12 weeks after implantation. (c) Ac‐ cumulated calcium content per sample 12 weeks after implantation (From Wadagaki et al. [7, 8]. Reprinted with per‐

Tissue Engineering and Regenerative Medicine 135

**2.6. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat**

Recently tissue engineering has become available as a treatment procedure for bone augmen‐ tation. However, this procedure has several problems such as an expensive cost for capital investment and cell culture, complicated safety and quality management of cell handling and invasiveness of cell collection for patients. On the other hand, it was reported that the stem cells secreted many growth factors and chemokines during their cultivation and that could affect on the cellular characteristics and behavior. This study investigated the effect of stem cell cultured conditioned media on bone regeneration (Fig.18). Cultured conditioned media from human bone‐marrow‐derived mesenchymal stem cells (MSC‐CM) enhanced the migra‐ tion, proliferation and expression of osteogenetic marker genes, such as *osteocalcin* and *Runx2,* of rat MSCs *in vitro*. MSC‐CM included cytokines such as insulin‐like‐growth factor (IGF)‐1

*In vivo*, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC‐CM/ agarose (MSC‐CM), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using micro‐computed tomography (micro‐CT) and histological analysis. Micro‐CT analysis indicated that the MSC‐CM group had a greater area of newly regenerated bone compared with the other groups (P < 0.05) (Figure 19) and histological analysis at 8 weeks

mission).

**calvarial bone defects**

and vascular endothelial growth factor (VEGF).

**Figure 16.** Simvastatin released from SRBFS inhibits RANKL-induced osteoclastogenesis 5 days after stimulation. Cells were cultured for 5 days with (a) SRBFS and (b) BFS after RANKL treatment and stained for TRAP expression. Bar = 500 μm. (c) The total number of TRAP-positive multinucleated osteoclasts (i.e., those containing three nuclei) per well were counted (From Wadagaki et al. [7, 8]. Reprinted with permission).

**Figure 17.** (a) Bone formation area analyzed by H&E staining 12 weeks after implantation. (b) Percent BSP (bone sialo protein) -positive cells as determined by immunohistochemical staining with BSP 12 weeks after implantation. (c) Ac‐ cumulated calcium content per sample 12 weeks after implantation (From Wadagaki et al. [7, 8]. Reprinted with per‐ mission).

**Figure 15.** ALP activity of MSCs on SRBFS and BFS measured on days 7 and 14 (From Wadagaki et al. [7, 8]. Reprinted

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 16.** Simvastatin released from SRBFS inhibits RANKL-induced osteoclastogenesis 5 days after stimulation. Cells were cultured for 5 days with (a) SRBFS and (b) BFS after RANKL treatment and stained for TRAP expression. Bar = 500 μm. (c) The total number of TRAP-positive multinucleated osteoclasts (i.e., those containing three nuclei) per well were

counted (From Wadagaki et al. [7, 8]. Reprinted with permission).

with permission).

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#### **2.6. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects**

Recently tissue engineering has become available as a treatment procedure for bone augmen‐ tation. However, this procedure has several problems such as an expensive cost for capital investment and cell culture, complicated safety and quality management of cell handling and invasiveness of cell collection for patients. On the other hand, it was reported that the stem cells secreted many growth factors and chemokines during their cultivation and that could affect on the cellular characteristics and behavior. This study investigated the effect of stem cell cultured conditioned media on bone regeneration (Fig.18). Cultured conditioned media from human bone‐marrow‐derived mesenchymal stem cells (MSC‐CM) enhanced the migra‐ tion, proliferation and expression of osteogenetic marker genes, such as *osteocalcin* and *Runx2,* of rat MSCs *in vitro*. MSC‐CM included cytokines such as insulin‐like‐growth factor (IGF)‐1 and vascular endothelial growth factor (VEGF).

*In vivo*, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC‐CM/ agarose (MSC‐CM), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using micro‐computed tomography (micro‐CT) and histological analysis. Micro‐CT analysis indicated that the MSC‐CM group had a greater area of newly regenerated bone compared with the other groups (P < 0.05) (Figure 19) and histological analysis at 8 weeks

indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the effects of MSC‐CM were stronger than those of the MSCs group.

*In vivo* imaging also showed that migration of injected rMSCs to the bone defect in the MSC‐

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These results demonstrated that MSC‐CM can regenerate bone through mobilization of endogenous stem cells. The use of stem cell cultured conditioned media for bone regeneration will be a unique conceptthat utilizes paracline factors of stem cells without celltransplantation.

**2.7. Recovery of neo‐callus formation in the H‐DO gap by the angiogenic activities of SDF‐1**

Distraction osteogenesis (DO) is a unique therapy that induces skeletal tissue regeneration without stem/progenitor cell transplantation. Although the self‐regeneration property of DO provides many clinical benefits, the long treatment period required is a major drawback. A high‐speed DO mouse model (H‐DO), in which the distraction was done two times faster than in control DO (C‐DO) mice, failed to generate new bone callus in the DO gap. We found that this was caused by the unsuccessful recruitment of bone marrow endothelial cells (BM‐ECs)/ endothelial progenitor cells (EPCs) into the gap. We then tested the ability of a local application of stromal cell‐derived factor‐1 (SDF‐1), a major chemo‐attractant for BM‐ECs/EPCs, to accelerate the bone regeneration in H‐DO. Our data showed that, in H‐DO, SDF‐1 induced callus formation in the gap through the recruitment of BM‐ECs/EPCs, the maturation of neo‐ blood vessels, and increased blood flow. These results indicate that the active recruitment of endogenous BM‐ECs/EPCs may provide a substantial clinical benefit for shortening the

CM group was greater than in the other groups (Figure 20).

**Figure 20.** In vivo imaging of infected rat MSCs migration to implants.

treatment period of DO.

**Figure 18.** Outline of the experimental protocol.

**Figure 19.** Micro-CT analysis of bone regeneration following implantation of MSC-CM or controls into a bone defect.

*In vivo* imaging also showed that migration of injected rMSCs to the bone defect in the MSC‐ CM group was greater than in the other groups (Figure 20).

**Figure 20.** In vivo imaging of infected rat MSCs migration to implants.

indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 19.** Micro-CT analysis of bone regeneration following implantation of MSC-CM or controls into a bone defect.

effects of MSC‐CM were stronger than those of the MSCs group.

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**Figure 18.** Outline of the experimental protocol.

These results demonstrated that MSC‐CM can regenerate bone through mobilization of endogenous stem cells. The use of stem cell cultured conditioned media for bone regeneration will be a unique conceptthat utilizes paracline factors of stem cells without celltransplantation.

#### **2.7. Recovery of neo‐callus formation in the H‐DO gap by the angiogenic activities of SDF‐1**

Distraction osteogenesis (DO) is a unique therapy that induces skeletal tissue regeneration without stem/progenitor cell transplantation. Although the self‐regeneration property of DO provides many clinical benefits, the long treatment period required is a major drawback. A high‐speed DO mouse model (H‐DO), in which the distraction was done two times faster than in control DO (C‐DO) mice, failed to generate new bone callus in the DO gap. We found that this was caused by the unsuccessful recruitment of bone marrow endothelial cells (BM‐ECs)/ endothelial progenitor cells (EPCs) into the gap. We then tested the ability of a local application of stromal cell‐derived factor‐1 (SDF‐1), a major chemo‐attractant for BM‐ECs/EPCs, to accelerate the bone regeneration in H‐DO. Our data showed that, in H‐DO, SDF‐1 induced callus formation in the gap through the recruitment of BM‐ECs/EPCs, the maturation of neo‐ blood vessels, and increased blood flow. These results indicate that the active recruitment of endogenous BM‐ECs/EPCs may provide a substantial clinical benefit for shortening the treatment period of DO.

**Figure 21.** Traction speed affects new bone callus formation in mouse DO models.(A, B) Surgical procedure for the mouse DO model. An anterior longitudinal incision was made on the right leg of the animal. Needles were inserted through the skin into the proximal and distal metaphysis of the tibia (A). Subsequently, sets of needles were fixed to the custom-made fixator with acrylic resin (B). After polymerization of the resin, osteotomy was carried out at the mid‐ dle of the diaphysis (arrow). (C) Distraction protocols and experimental design. After a 5-day latency period, distrac‐ tion was started at a rate of 0.2 mm/12 h (C-DO) or 0.4 mm/12 h (H-DO). The lengthening was continued for 8 days in the C-DO and 4 days in the H-DO model, resulting in a length increase of 3.2 mm. Black arrowheads indicate the time points of sacrifice. White arrowheads indicate the time points for injecting 200 ng SDF-1 protein (+SDF-1). LA, latency period; AD, active distraction period; CO, consolidation period. (D–I) Representative micrographs of sections displaying the DO gap stained with Hematoxylin–Eosin (HE) (D, F and H) and Alcian Blue–Fast red (E, G and I) (n = 8). The left and right of each figure correspond to the end of the distal and proximal bone fragment, respectively. Neo-callus forma‐ tion was evident within the C-DO gap at the end of the consolidation period (D). A little cartilage was observed in the periosteal but not in the endosteal region (E). The H-DO gap was filled with fibrotic tissues and periosteum-derived cartilages (F, G). The local administration SDF-1 rescued the callus formation in the H-DO gap (H, I). Bar = 300 μm (D–I). (From Fujio et al. [9]. Reprinted with permission).

**Figure 22.** Contribution of BM-ECs/EPCs to DO healing. Mice were sacrificed at the middle of the active distraction period in each group: day 9 and day 7, respectively, for the C-DO and H-DO group. (A, B, and C) The recruitment of BM-ECs/EPCs was evaluated by immunofluorescence staining for CD31 (red). CD31+ BM-ECs/EPCs accumulated in the C-DO (A), but not in the H-DO gap (B). Local administration of SDF- 1 rescued the recruitment of BM-ECs/EPCs to the H-DO gap (C) (n = 8). (D) Boxed area in (C) is shown in higher-magnification micrographs, in which the CD31 signals are seen together with Sca-1 (green). Note that SDF-1 treatment increased the number of CD31+Sca-1 cells in the gap. (E) CD31-single-positive and CD31+Sca-1+ cells were counted by Image J software. The number of CD31+Sca-1+ BM-ECs/EPCs in the C-DO gap was significantly higher than that in intact bone marrow, whereas that in the H-DO gap was significantly lower. SDF-1 treatment rescued the number of BM-ECs/EPCs in the H-DO gap. The dotted line represents native bone. Data represent the mean ± SD. \*\*P > 0.01 and \*P > 0.05. Intact: intact bone marrow. Bar = 100 μm (A, B,

Tissue Engineering and Regenerative Medicine 139

**2.8. Effect of GDF‐5 and BMP‐2 on the expression of tendo/ligamentogenesis‐related**

The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament‐derived cells was investigated with specialreference to tendo/ ligamen‐ togenesis‐related markers.The results from this study showed that both GDF‐5 and BMP‐2 affect the differentiation of PDL‐derived cells in vitro. However, the effect on those differential ligament‐makers was not identical and the underlying mechanisms might be complex.This study focused on the tendo‐/ligamentogenesis related markers and confirmed the effect of those factors not only on crude PDL‐derived cells but also on STRO‐1+ and STRO‐1‐ PDL‐ derived cells. The results from our study showed some potential beneficial effect of GDF‐5 on periodontal tissue regeneration. However, the underlying mechanisms appear to be compli‐ cated, and the overall benefit of the clinical application of the factor requires further analyses.

C) and 50 μm (D) (From Fujio et al. [9]. Reprinted with permission).

**markers in human PDL‐derived cells**

We tested whether the local administration of SDF‐1, a chemo‐ attractant for BM‐ECs/EPCs, would rescue the disrupted callus formation and integration of BM‐ECs/EPCs in the H‐DO gap. A collagen gel matrix containing 200 ng of SDF‐1 protein was injected into the H‐DO gap every other day (Figure 21C). We found that the high levels of SDF‐1 rescued the callus formation and increased the number of BM‐ECs/EPCs in the H‐DO gap (Figures 21H, I and 22C). Although about 80% of the CD31+ cells co‐expressed Sca‐1 in the C‐DO gap, in the H‐ DO gap treated with SDF‐1, 50% of the CD31+ cells were negative for Sca‐1, suggesting that some of the BM‐ECs/EPCs recruited by SDF‐1 had already differentiated into mature endo‐ thelial cells (Figure 22E).

In summary, our study demonstrated that locally administered SDF‐1 promotes the recruit‐ ment of endogenous BM‐ECs/EPCs and neo callus formation in the DO gap. We propose that the regulation of endogenous stem/progenitor cell trafficking is a powerful therapeutic strategy in skeletal regeneration.

**Figure 21.** Traction speed affects new bone callus formation in mouse DO models.(A, B) Surgical procedure for the mouse DO model. An anterior longitudinal incision was made on the right leg of the animal. Needles were inserted through the skin into the proximal and distal metaphysis of the tibia (A). Subsequently, sets of needles were fixed to the custom-made fixator with acrylic resin (B). After polymerization of the resin, osteotomy was carried out at the mid‐ dle of the diaphysis (arrow). (C) Distraction protocols and experimental design. After a 5-day latency period, distrac‐ tion was started at a rate of 0.2 mm/12 h (C-DO) or 0.4 mm/12 h (H-DO). The lengthening was continued for 8 days in the C-DO and 4 days in the H-DO model, resulting in a length increase of 3.2 mm. Black arrowheads indicate the time points of sacrifice. White arrowheads indicate the time points for injecting 200 ng SDF-1 protein (+SDF-1). LA, latency period; AD, active distraction period; CO, consolidation period. (D–I) Representative micrographs of sections displaying the DO gap stained with Hematoxylin–Eosin (HE) (D, F and H) and Alcian Blue–Fast red (E, G and I) (n = 8). The left and right of each figure correspond to the end of the distal and proximal bone fragment, respectively. Neo-callus forma‐ tion was evident within the C-DO gap at the end of the consolidation period (D). A little cartilage was observed in the periosteal but not in the endosteal region (E). The H-DO gap was filled with fibrotic tissues and periosteum-derived cartilages (F, G). The local administration SDF-1 rescued the callus formation in the H-DO gap (H, I). Bar = 300 μm (D–I).

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

We tested whether the local administration of SDF‐1, a chemo‐ attractant for BM‐ECs/EPCs, would rescue the disrupted callus formation and integration of BM‐ECs/EPCs in the H‐DO gap. A collagen gel matrix containing 200 ng of SDF‐1 protein was injected into the H‐DO gap every other day (Figure 21C). We found that the high levels of SDF‐1 rescued the callus formation and increased the number of BM‐ECs/EPCs in the H‐DO gap (Figures 21H, I and 22C). Although about 80% of the CD31+ cells co‐expressed Sca‐1 in the C‐DO gap, in the H‐ DO gap treated with SDF‐1, 50% of the CD31+ cells were negative for Sca‐1, suggesting that some of the BM‐ECs/EPCs recruited by SDF‐1 had already differentiated into mature endo‐

In summary, our study demonstrated that locally administered SDF‐1 promotes the recruit‐ ment of endogenous BM‐ECs/EPCs and neo callus formation in the DO gap. We propose that the regulation of endogenous stem/progenitor cell trafficking is a powerful therapeutic

(From Fujio et al. [9]. Reprinted with permission).

thelial cells (Figure 22E).

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strategy in skeletal regeneration.

**Figure 22.** Contribution of BM-ECs/EPCs to DO healing. Mice were sacrificed at the middle of the active distraction period in each group: day 9 and day 7, respectively, for the C-DO and H-DO group. (A, B, and C) The recruitment of BM-ECs/EPCs was evaluated by immunofluorescence staining for CD31 (red). CD31+ BM-ECs/EPCs accumulated in the C-DO (A), but not in the H-DO gap (B). Local administration of SDF- 1 rescued the recruitment of BM-ECs/EPCs to the H-DO gap (C) (n = 8). (D) Boxed area in (C) is shown in higher-magnification micrographs, in which the CD31 signals are seen together with Sca-1 (green). Note that SDF-1 treatment increased the number of CD31+Sca-1 cells in the gap. (E) CD31-single-positive and CD31+Sca-1+ cells were counted by Image J software. The number of CD31+Sca-1+ BM-ECs/EPCs in the C-DO gap was significantly higher than that in intact bone marrow, whereas that in the H-DO gap was significantly lower. SDF-1 treatment rescued the number of BM-ECs/EPCs in the H-DO gap. The dotted line represents native bone. Data represent the mean ± SD. \*\*P > 0.01 and \*P > 0.05. Intact: intact bone marrow. Bar = 100 μm (A, B, C) and 50 μm (D) (From Fujio et al. [9]. Reprinted with permission).

## **2.8. Effect of GDF‐5 and BMP‐2 on the expression of tendo/ligamentogenesis‐related markers in human PDL‐derived cells**

The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament‐derived cells was investigated with specialreference to tendo/ ligamen‐ togenesis‐related markers.The results from this study showed that both GDF‐5 and BMP‐2 affect the differentiation of PDL‐derived cells in vitro. However, the effect on those differential ligament‐makers was not identical and the underlying mechanisms might be complex.This study focused on the tendo‐/ligamentogenesis related markers and confirmed the effect of those factors not only on crude PDL‐derived cells but also on STRO‐1+ and STRO‐1‐ PDL‐ derived cells. The results from our study showed some potential beneficial effect of GDF‐5 on periodontal tissue regeneration. However, the underlying mechanisms appear to be compli‐ cated, and the overall benefit of the clinical application of the factor requires further analyses.

**Figure 23.** ALP activity of crude PDL-derived cells in passage 2. ALP activity of the Dex group was significantly greater compared with control, BMP-2 and GDF-5 groups. \*P < 0.05. Values are the mean ± standard deviation of five experi‐ ments (From Inoue, M. et al. [10]. Reprinted with permission).

**Figure 25.** Western blot analyses of scleraxis crude PDL-derived cells (a) and STRO-1-PDL-derived cells (b) at passage 2. Expression of scleraxis was detected in all samples. An experiment representative of five similar studies is shown. (c) GDF-5 treated crude PDL-derived cells had significantly higher scleraxis expression than the other groups. (d) There was a similar tendency to crude PDL-derived cells, but no significant difference in STRO-1- PDL-derived cells among all groups. Values are the mean ± standard deviation of five experiments (From Inoue, M. et al. [10]. Reprinted with per‐

Tissue Engineering and Regenerative Medicine 141

**Figure 26.** The results from western blot analyses of scleraxis in crude PDL-derived cells,STRO-1+ and STRO-1- PDL-de‐ rived cells at passage 2. All samples were treated with rmGDF-5. (a) The expression of scleraxis proteins was detected in all groups for 7 days. An experiment representative of six similar studies is shown. (b) Reduced scleraxis expression in STRO-1+PDL-derived cells was statistically significant compared to crude P2 and STRO-1- PDL-derived cells. Values are the

mean ± standard deviation of six experiments (From Inoue, M. et al. [10]. Reprinted with permission).

mission).

**Figure 24.** Quantitative RT-PCR analysis for scleraxis (a) and tenomodulin (b) gene expression of crude PDL-derived cells in passage 2. Cells were cultured with culture medium with or without BMP-2, GDF-5 for 1, 3 and 7 days. There were no significant differences among control, BMP-2 and GDF-5 groups on any time points. Values are the mean ± standard deviation of five experiments (From Inoue, M. et al. [10]. Reprinted with permission).

**Figure 25.** Western blot analyses of scleraxis crude PDL-derived cells (a) and STRO-1-PDL-derived cells (b) at passage 2. Expression of scleraxis was detected in all samples. An experiment representative of five similar studies is shown. (c) GDF-5 treated crude PDL-derived cells had significantly higher scleraxis expression than the other groups. (d) There was a similar tendency to crude PDL-derived cells, but no significant difference in STRO-1- PDL-derived cells among all groups. Values are the mean ± standard deviation of five experiments (From Inoue, M. et al. [10]. Reprinted with per‐ mission).

**Figure 23.** ALP activity of crude PDL-derived cells in passage 2. ALP activity of the Dex group was significantly greater compared with control, BMP-2 and GDF-5 groups. \*P < 0.05. Values are the mean ± standard deviation of five experi‐

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 24.** Quantitative RT-PCR analysis for scleraxis (a) and tenomodulin (b) gene expression of crude PDL-derived cells in passage 2. Cells were cultured with culture medium with or without BMP-2, GDF-5 for 1, 3 and 7 days. There were no significant differences among control, BMP-2 and GDF-5 groups on any time points. Values are the mean ±

standard deviation of five experiments (From Inoue, M. et al. [10]. Reprinted with permission).

ments (From Inoue, M. et al. [10]. Reprinted with permission).

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**Figure 26.** The results from western blot analyses of scleraxis in crude PDL-derived cells,STRO-1+ and STRO-1- PDL-de‐ rived cells at passage 2. All samples were treated with rmGDF-5. (a) The expression of scleraxis proteins was detected in all groups for 7 days. An experiment representative of six similar studies is shown. (b) Reduced scleraxis expression in STRO-1+PDL-derived cells was statistically significant compared to crude P2 and STRO-1- PDL-derived cells. Values are the mean ± standard deviation of six experiments (From Inoue, M. et al. [10]. Reprinted with permission).

#### **2.9.TransientTWEAK overexpression leads to a general salivary epithelial cell proliferation**

Tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro‐apoptotic, pro‐angiogenic and pro‐inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested *in vitro*, and then administered to male Balb/c mice via cannulation of Whartonʹs duct. TWEAK expression *in vivo* was monitored as protein secreted into saliva and serum by enzyme‐linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. AdhTWEAK administration led to a dose‐dependent, transient TWEAK protein expression (Figure 27), detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3 (Figure 28, 29). TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

**Figure 27.** Effect of the AdhTWEAK dose administered on the detection of hTWEAK in murine saliva and serum (From Sugito et al. [11]. Reprinted with permission).

**Figure 28.** Detection of proliferating cell nuclear antigen (PCNA) staining in submandibular glands of mice following AdhTWEAK administration. AdhTWEAK (109 particles per gland), or saline, was delivered to both submandibular glands (n = 4 mice per group) and PCNA staining performed on gland sections as described in Materials and methods to evaluate cell proliferation. Brown staining represents PCNA-positive nuclei. Sections are counterstained with hema‐ toxylin. (a) Day 0 after saline administration; (b) Day 1 after AdhTWEAK administration; (c) Day 2 after-AdhTWEAK administration; (d) Day 3 after AdhTWEAK administration. Bar = 20 μm (From Sugito et al. [11]. Reprinted with permis‐

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sion).

**2.9.TransientTWEAK overexpression leads to a general salivary epithelial cell proliferation**

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro‐apoptotic, pro‐angiogenic and pro‐inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested *in vitro*, and then administered to male Balb/c mice via cannulation of Whartonʹs duct. TWEAK expression *in vivo* was monitored as protein secreted into saliva and serum by enzyme‐linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. AdhTWEAK administration led to a dose‐dependent, transient TWEAK protein expression (Figure 27), detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on approximately days 2 and 3 (Figure 28, 29). TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation

**Figure 27.** Effect of the AdhTWEAK dose administered on the detection of hTWEAK in murine saliva and serum (From

Sugito et al. [11]. Reprinted with permission).

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of epithelial cells vs a selective stimulation of a salivary progenitor cell.

**Figure 28.** Detection of proliferating cell nuclear antigen (PCNA) staining in submandibular glands of mice following AdhTWEAK administration. AdhTWEAK (109 particles per gland), or saline, was delivered to both submandibular glands (n = 4 mice per group) and PCNA staining performed on gland sections as described in Materials and methods to evaluate cell proliferation. Brown staining represents PCNA-positive nuclei. Sections are counterstained with hema‐ toxylin. (a) Day 0 after saline administration; (b) Day 1 after AdhTWEAK administration; (c) Day 2 after-AdhTWEAK administration; (d) Day 3 after AdhTWEAK administration. Bar = 20 μm (From Sugito et al. [11]. Reprinted with permis‐ sion).

**Figure 29.** Quantification of AdTWEAK-induced salivary epithelial cell proliferation (From Sugito et al. [11]. Reprinted with permission).

### **2.10. Injectable soft‐tissue augmentation by tissue engineering and regenerative medicine with human mesenchymal stem cells, platelet‐rich plasma, and hyaluronic acid scaffolds**

The restoration of soft tissue by adequate implant material is needed in case of functional and aesthetic impairments from loss of soft connective tissue. The implants vary, and repeated injections are generally required for varying degrees of material resorption. Recently it was reported that autologous cell injection was useful to improve soft tissue. The aim of this study was to evaluate the possibility of soft‐tissue augmentation adopting tissue engineering and regenerative medicine (TERM) technology for a longer duration of injected implants. TERM is the combination and reorganization of three types of injection materials to regenerate organs or tissues: 1) living cells, including cultured human MSCs or human fibroblasts (Fibro); 2) scaffolds of hyaluronic acid (HA); and 3) growth factors of PRP. The experimental combina‐ tions were as follows: HA, HA/Fibro, HA/MSCs, HA/PRP, HA/PRP/Fibro and HA/PRP/MSCs. These were intradermally injected into immunodeficient rats and evaluated by histological analysis, the percentage of original volume and the maintenance volume (Figure 30).

The percentage of original volume values at 14 days showed significant differences between groups with and without PRP upon comparison (Table 2). As for the maintenance volume values, HA/PRP/Fibro, and HA/PRP/MSCs from 7 to 14 days were higher than others (Figure 31). HA/PRP/MSCs groups maintained the shape and dimensions of the injected implant, indicating that the injected cells produced type I collagen (Figure 32). The findings suggest that a soft tissue‐engineered procedure with MSCs may be useful for longer‐lasting soft‐tissue augmentation.

**Figure 30.** Schema of experimental protocol (a) and dermal mound formed after sample injection and the measure‐

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ment diagram (b) (From Okabe et al. [12]. Reprinted with permission).

**Figure 29.** Quantification of AdTWEAK-induced salivary epithelial cell proliferation (From Sugito et al. [11]. Reprinted

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**2.10. Injectable soft‐tissue augmentation by tissue engineering and regenerative medicine with human mesenchymal stem cells, platelet‐rich plasma, and hyaluronic acid scaffolds**

The restoration of soft tissue by adequate implant material is needed in case of functional and aesthetic impairments from loss of soft connective tissue. The implants vary, and repeated injections are generally required for varying degrees of material resorption. Recently it was reported that autologous cell injection was useful to improve soft tissue. The aim of this study was to evaluate the possibility of soft‐tissue augmentation adopting tissue engineering and regenerative medicine (TERM) technology for a longer duration of injected implants. TERM is the combination and reorganization of three types of injection materials to regenerate organs or tissues: 1) living cells, including cultured human MSCs or human fibroblasts (Fibro); 2) scaffolds of hyaluronic acid (HA); and 3) growth factors of PRP. The experimental combina‐ tions were as follows: HA, HA/Fibro, HA/MSCs, HA/PRP, HA/PRP/Fibro and HA/PRP/MSCs. These were intradermally injected into immunodeficient rats and evaluated by histological

analysis, the percentage of original volume and the maintenance volume (Figure 30).

The percentage of original volume values at 14 days showed significant differences between groups with and without PRP upon comparison (Table 2). As for the maintenance volume values, HA/PRP/Fibro, and HA/PRP/MSCs from 7 to 14 days were higher than others (Figure 31). HA/PRP/MSCs groups maintained the shape and dimensions of the injected implant, indicating that the injected cells produced type I collagen (Figure 32). The findings suggest that a soft tissue‐engineered procedure with MSCs may be useful for longer‐lasting soft‐tissue

with permission).

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augmentation.

**Figure 30.** Schema of experimental protocol (a) and dermal mound formed after sample injection and the measure‐ ment diagram (b) (From Okabe et al. [12]. Reprinted with permission).

> place of surgical operation and medical treatment of wound healing. It is well known that human dental pulp cell shows the property that resembled MSCs. In this research, we examined the benefit of stem cells from human exfoliated deciduous teeth (SHEDs) in wound

Tissue Engineering and Regenerative Medicine 147

**Figure 33.** Schema of experimental protocol (From Nishino et al. [13]. Reprinted with permission).

**Figure 34.** Wound measurement (From Nishino et al. [13]. Reprinted with permission).

was used to visualize the nuclei (blue fluorescence) (Figure 35).

SHEDs and MSCs significantly accelerated wound closure compared with Fibro and control treatment (Figure 34). At day 7 and 14, the evaluation by fluorescence microscope showed that PKH 26 positive cells (Fibro, MSCs, SHEDs) were surrounded by hyaluronic acid binding protein production of hyaluronic acid. The plasma membrane of transplanted cells showed red fluorescence by PKH 26. Hyaluronic acid was visualized with green fluorescence. DAPI

healing (Figure 33).

**Figure 31.** The percentage of maintenance volume values (%) in HA/PRP, HA/PRP/Fibro and HA/PRP/MSCs. Bar = SD. \*P < 0.05 (From Okabe et al. [12]. Reprinted with permission).

**Figure 32.** Distribution of type I collagen produced by MSCs (a) or Fibro (b) in the HA/PRP gels. Bar = 50 μm (From Okabe et al. [12]. Reprinted with permission).

#### **2.11. Potentiality of new cell therapy for skin regeneration in wound healing**

The process of wound healing depends upon a variety of interactions between cells and the extracellular matrix. There is hyaluronic acid in one of the extracellular matrix. It is well known that hyaluronic acid not only supports tissue architecture as a passive structural component of the matrix in various connective tissues but is also involved in dynamic cellular processes during wound healing. Recently, cell therapies which is a low aggression is paid attention in place of surgical operation and medical treatment of wound healing. It is well known that human dental pulp cell shows the property that resembled MSCs. In this research, we examined the benefit of stem cells from human exfoliated deciduous teeth (SHEDs) in wound healing (Figure 33).

**Figure 33.** Schema of experimental protocol (From Nishino et al. [13]. Reprinted with permission).

**Figure 31.** The percentage of maintenance volume values (%) in HA/PRP, HA/PRP/Fibro and HA/PRP/MSCs. Bar = SD.

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 32.** Distribution of type I collagen produced by MSCs (a) or Fibro (b) in the HA/PRP gels. Bar = 50 μm (From

The process of wound healing depends upon a variety of interactions between cells and the extracellular matrix. There is hyaluronic acid in one of the extracellular matrix. It is well known that hyaluronic acid not only supports tissue architecture as a passive structural component of the matrix in various connective tissues but is also involved in dynamic cellular processes during wound healing. Recently, cell therapies which is a low aggression is paid attention in

**2.11. Potentiality of new cell therapy for skin regeneration in wound healing**

\*P < 0.05 (From Okabe et al. [12]. Reprinted with permission).

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Okabe et al. [12]. Reprinted with permission).

**Figure 34.** Wound measurement (From Nishino et al. [13]. Reprinted with permission).

SHEDs and MSCs significantly accelerated wound closure compared with Fibro and control treatment (Figure 34). At day 7 and 14, the evaluation by fluorescence microscope showed that PKH 26 positive cells (Fibro, MSCs, SHEDs) were surrounded by hyaluronic acid binding protein production of hyaluronic acid. The plasma membrane of transplanted cells showed red fluorescence by PKH 26. Hyaluronic acid was visualized with green fluorescence. DAPI was used to visualize the nuclei (blue fluorescence) (Figure 35).

skeletal regeneration and wound healing is valuable for the treatment of neonatal congenital abnormality (Figure 36). Umbilical cords are routinely discarded as medical wastes in clinic. We have succeeded in isolating stem cells from the Wharton's Jelly in umbilical cords (so called umbilical cord matrix stem cells: UCMSCs). UCMSCs exhibited multipotential differentiation activities. In this research, we focused on the isolation and identification of MSCs from the Wharton's jelly of umbilaical cord.The effect oflocal injection ofUCMSCs on cutenaous wound

Tissue Engineering and Regenerative Medicine 149

**Figure 36.** New concept of treatment of cleft lip & plate (From Shohara et al. [14, 15]. Reprinted with permission).

We have succeeded in isolating stem cells from umbilical cords (Figure 37A, B). Proliferation of UCMSCs was significantly greater than that of bone marrow stromal cells. UCMSCs proliferated much faster than did bone marrow stromal cells. UCMSCs exhibited multipoten‐ tial differentiation activities toward osteogenic, adipogenic. We found that UCMSCs shared most of their immunophenotype with bone marrow stromal cells, including positivity for CD90, CD73, CD105, but negativity for CD11b, CD34(endothelial progenitor cell marker), CD45(hematopoietic markers), and HLA‐DR. It shows that UCMSCs expressed high levels of mesenchymal stem cell markers. To investigate the wound repair activity of UCMSCs, we transplanted them in mouse excisional wound splinting model and the acceleration of the wound closure was evaluated (Figure 38). We found that the wound receiving the UCMSCs exhibit significantly faster healing compared with PBS‐injected control (Figure 39A). After 14 days from operation, the closed wound area was 99.72 ± 0.17 % in the UCMSCs‐group, while that in the control was 82.13 ± 5.85 % (Figure 39B). Histological analysis of wounds on day 14

healing was examined using excisional wound model in mice.

**Figure 35.** Histological evaluation (From Nishino et al. [13]. Reprinted with permission).

**Table 2.** Hyaluronic Acid Contents at Day 7 and 14 (From Nishino et al. [13]. Reprinted with permission).

Quantitate of hyaluronic acid was determined by measuring with ELISA. Significantly increased amounts of hyaluronic acid in wounded tissues were observed at day 7 and 14 in MSCs, SHEDs, and Fibro as compared with control (P < 0.05). This study demonstrated that deciduous teeth, considered as medical waste, would be novel therapeutic approaches in the treatment of wounds and new stem cell source for wound healing.

#### **2.12. Umbilical cord Wharton's Jelly: A new potential cell source of mesenchymal stem cells for wound healing**

Neonatal congenital disease, such as cleft and lip palate, involves soft tissue defect as well as skeletal abnormality. Thus the development of the therapeutic approaches accelerating both skeletal regeneration and wound healing is valuable for the treatment of neonatal congenital abnormality (Figure 36). Umbilical cords are routinely discarded as medical wastes in clinic. We have succeeded in isolating stem cells from the Wharton's Jelly in umbilical cords (so called umbilical cord matrix stem cells: UCMSCs). UCMSCs exhibited multipotential differentiation activities. In this research, we focused on the isolation and identification of MSCs from the Wharton's jelly of umbilaical cord.The effect oflocal injection ofUCMSCs on cutenaous wound healing was examined using excisional wound model in mice.

**Figure 35.** Histological evaluation (From Nishino et al. [13]. Reprinted with permission).

**Table 2.** Hyaluronic Acid Contents at Day 7 and 14 (From Nishino et al. [13]. Reprinted with permission).

treatment of wounds and new stem cell source for wound healing.

**for wound healing**

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Quantitate of hyaluronic acid was determined by measuring with ELISA. Significantly increased amounts of hyaluronic acid in wounded tissues were observed at day 7 and 14 in MSCs, SHEDs, and Fibro as compared with control (P < 0.05). This study demonstrated that deciduous teeth, considered as medical waste, would be novel therapeutic approaches in the

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**2.12. Umbilical cord Wharton's Jelly: A new potential cell source of mesenchymal stem cells**

Neonatal congenital disease, such as cleft and lip palate, involves soft tissue defect as well as skeletal abnormality. Thus the development of the therapeutic approaches accelerating both

**Figure 36.** New concept of treatment of cleft lip & plate (From Shohara et al. [14, 15]. Reprinted with permission).

We have succeeded in isolating stem cells from umbilical cords (Figure 37A, B). Proliferation of UCMSCs was significantly greater than that of bone marrow stromal cells. UCMSCs proliferated much faster than did bone marrow stromal cells. UCMSCs exhibited multipoten‐ tial differentiation activities toward osteogenic, adipogenic. We found that UCMSCs shared most of their immunophenotype with bone marrow stromal cells, including positivity for CD90, CD73, CD105, but negativity for CD11b, CD34(endothelial progenitor cell marker), CD45(hematopoietic markers), and HLA‐DR. It shows that UCMSCs expressed high levels of mesenchymal stem cell markers. To investigate the wound repair activity of UCMSCs, we transplanted them in mouse excisional wound splinting model and the acceleration of the wound closure was evaluated (Figure 38). We found that the wound receiving the UCMSCs exhibit significantly faster healing compared with PBS‐injected control (Figure 39A). After 14 days from operation, the closed wound area was 99.72 ± 0.17 % in the UCMSCs‐group, while that in the control was 82.13 ± 5.85 % (Figure 39B). Histological analysis of wounds on day 14 indicated that granulation tissue of UCMSCs‐group appeared to be thicker and larger as compared with the untreated group. Thus, these results demonstrated that the engrafted UCMSCs accelerated wound healing process.

accelerated cutenaous wound healing process. This stem cells can be isolated without invasive surgical procedures, providing unique cellresources forregenerative medicine. Together with the distinct advantages of UCMSCs, such as accessibility, painless procedures to donors, possible source for autologous cell therapy and lower risk of viral contamination, we suggest

Tissue Engineering and Regenerative Medicine 151

Recently, it has been revealed that bone marrow‐derived MSCs accelerate skin wound healing, and it is attracting attention as a new cell therapy. However, MSCs are isolated from bone marrow from patient, and collection of the bone marrow is considerably invasive. In addition, there is a problem that proliferative capacity of MSCs decreases with aging. Thus, application ofMSCs for elderly patients andfresh cases wasdifficult because ittakes a long time to cultivate the cells. On the other hand, it has been known that MSCs secrete many growth factors. We presumed that growth factors secreted by MSCs play a main role in wound healing effect, and

**Figure 40.** Scheme of the experiment(From Tamari et al. [16-18]. Reprinted with permission from Quintessence Pub‐

We observed wound healing process macroscopically and histologically using an excisional wound splinting mouse model, and examined expression level of hyaluronic acid related to the wound healing process to evaluate wound‐healing effect of MSCs, MSC‐CM, and control (PBS) (Figure 39). The MSCs and MSC‐CM groups accelerated wound healing as compared with the control group. The area of wound in the MSCs and MSC‐CM groups at 5 days and later indicated statistically significant difference as compared with the control group.(Figure

that UCMSCs should be considered a promising cell resource for cell therapy.

**2.13. Acceleration of wound healing with stem cell–derived growth factors**

examined effect of MSC‐CM on wound healing.

lishing Co, Inc, Chicago).

**Figure 37.** (A) Morphology and cross sectional image of umbilical cord. (B) UCMSCs exhibited fibroblastic morphology with bipolar spindles shape (From Shohara et al. [14, 15]. Reprinted with permission).

**Figure 38.** 6 mm full-thickness excicional wound splinted with silicone plate (From Shohara et al. [14, 15]. Reprinted with permission).

**Figure 39.** (A) Representative photograph of the wound at day 0, 7, 14 after UCMSCs transplantation. (B) Measure‐ ment of wound closure at different time points (From Shohara et al. [14, 15]. Reprinted with permission).

In summary, the present study describes the isolation and primary characterization of stem cells from medical wastes, such as umbilical cord. Furthermore, local injection of UCMSCs accelerated cutenaous wound healing process. This stem cells can be isolated without invasive surgical procedures, providing unique cellresources forregenerative medicine. Together with the distinct advantages of UCMSCs, such as accessibility, painless procedures to donors, possible source for autologous cell therapy and lower risk of viral contamination, we suggest that UCMSCs should be considered a promising cell resource for cell therapy.

#### **2.13. Acceleration of wound healing with stem cell–derived growth factors**

indicated that granulation tissue of UCMSCs‐group appeared to be thicker and larger as compared with the untreated group. Thus, these results demonstrated that the engrafted

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 37.** (A) Morphology and cross sectional image of umbilical cord. (B) UCMSCs exhibited fibroblastic morphology

**Figure 38.** 6 mm full-thickness excicional wound splinted with silicone plate (From Shohara et al. [14, 15]. Reprinted

**Figure 39.** (A) Representative photograph of the wound at day 0, 7, 14 after UCMSCs transplantation. (B) Measure‐

In summary, the present study describes the isolation and primary characterization of stem cells from medical wastes, such as umbilical cord. Furthermore, local injection of UCMSCs

ment of wound closure at different time points (From Shohara et al. [14, 15]. Reprinted with permission).

with bipolar spindles shape (From Shohara et al. [14, 15]. Reprinted with permission).

UCMSCs accelerated wound healing process.

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with permission).

Recently, it has been revealed that bone marrow‐derived MSCs accelerate skin wound healing, and it is attracting attention as a new cell therapy. However, MSCs are isolated from bone marrow from patient, and collection of the bone marrow is considerably invasive. In addition, there is a problem that proliferative capacity of MSCs decreases with aging. Thus, application ofMSCs for elderly patients andfresh cases wasdifficult because ittakes a long time to cultivate the cells. On the other hand, it has been known that MSCs secrete many growth factors. We presumed that growth factors secreted by MSCs play a main role in wound healing effect, and examined effect of MSC‐CM on wound healing.

**Figure 40.** Scheme of the experiment(From Tamari et al. [16-18]. Reprinted with permission from Quintessence Pub‐ lishing Co, Inc, Chicago).

We observed wound healing process macroscopically and histologically using an excisional wound splinting mouse model, and examined expression level of hyaluronic acid related to the wound healing process to evaluate wound‐healing effect of MSCs, MSC‐CM, and control (PBS) (Figure 39). The MSCs and MSC‐CM groups accelerated wound healing as compared with the control group. The area of wound in the MSCs and MSC‐CM groups at 5 days and later indicated statistically significant difference as compared with the control group.(Figure

40, 41) At 7 days after administration of MSCs or MSC‐CM, epithelialization was accelerated, thick connective tissue was formed in the skin defective area, and the area of wound was reduced in the MSCs and MSC‐CM groups as compared with the control group. Hyaluronic acid was expressed in the marginal part of the wound significantly. At 14 days after operation, infiltration of inflammatory cells was decreased as compared with 7 days, and the wound was closed in the MSCs and MSC‐CM groups, while defective part of epithelium was observed in the control group. Expression of hyaluronic acid was decreased from 7 days. At 7 and 14 days after operation, expression levels of hyaluronic acid were 1512.0 ± 84.10 ng/mg and 683.7±56.4 ng/mg in control group, 2450.3 ± 225.7 ng/mg and 1690.2 ± 170.2 ng/mg in MSCs group, and 2360.6 ± 230.0 ng/mg and 1570.1 ± 142.5 ng/mg in MSC‐CM group. At 7 and 14 days, the MSCs and MSC‐CM groups expressed significantly high level of hyaluronic acid as compared with the control group (P < 0.05). The expression level of hyaluronic acid was lower at 14 days than that at 7 days in all three groups.

These experimental results indicated that both MSCs and MSC‐CM groups have wound healing acceleration effect as compared with the control group. The wound healing accelera‐ tion effects of the MSC‐CM group and the MSCs group were equivalent. Accordingly, it is suggested that the MSC‐CM contains growth factor derived from stem cells and is able to provide wound healing acceleration effect equivalent to stem cell transplantation, and may

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**Figure 42.** Time-dependent changes in the area of wound area determined with image analysis software. area of actual wound/area of original wound ×100. Test for significant difference (ANOVA). MSC-CM group vs. control group, \*P < 0.05. \*\*P < 0.01 (From Tamari et al. [16-18]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

**2.14. Human dental pulp‐derived stem cells promote locomotor recovery after complete**

Spinal cord injury resulted in severe functional disability. In worldwide, 2.5 million people live with spinal cord injury More than 200,000 new injuries reported each year. Currently no curative therapy is available. Recent study has demonstrated that transplantation of stem cells in injured spinal cord would support functional recovery. The cellular resources of stem cell are Human embryonic stem cell, Induced pluripotent stem cells, Human embryonic neural stem cell, and adult mesenchymal stem cells from bone marrow. These stem cells have been shown to be valuable cellular resources for treatment of animal model of SCI. However, to use

**transection of the rat spinal cord by multiple neuro‐regenerative mechanisms**

become new therapeutic method for wound healing in the future.

**Figure 41.** Macroscopic findings at 7 and 14 days after injection. Left panels (A, C, and E) at 7 days after administra‐ tion; Right panels (B, D, and F) at 14 days after administration. (A and B) control group; (C and D) MSCs group; (E and F) MSC-CM group. Bar = 3 mm (From Tamari et al. [16-18]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

These experimental results indicated that both MSCs and MSC‐CM groups have wound healing acceleration effect as compared with the control group. The wound healing accelera‐ tion effects of the MSC‐CM group and the MSCs group were equivalent. Accordingly, it is suggested that the MSC‐CM contains growth factor derived from stem cells and is able to provide wound healing acceleration effect equivalent to stem cell transplantation, and may become new therapeutic method for wound healing in the future.

40, 41) At 7 days after administration of MSCs or MSC‐CM, epithelialization was accelerated, thick connective tissue was formed in the skin defective area, and the area of wound was reduced in the MSCs and MSC‐CM groups as compared with the control group. Hyaluronic acid was expressed in the marginal part of the wound significantly. At 14 days after operation, infiltration of inflammatory cells was decreased as compared with 7 days, and the wound was closed in the MSCs and MSC‐CM groups, while defective part of epithelium was observed in the control group. Expression of hyaluronic acid was decreased from 7 days. At 7 and 14 days after operation, expression levels of hyaluronic acid were 1512.0 ± 84.10 ng/mg and 683.7±56.4 ng/mg in control group, 2450.3 ± 225.7 ng/mg and 1690.2 ± 170.2 ng/mg in MSCs group, and 2360.6 ± 230.0 ng/mg and 1570.1 ± 142.5 ng/mg in MSC‐CM group. At 7 and 14 days, the MSCs and MSC‐CM groups expressed significantly high level of hyaluronic acid as compared with the control group (P < 0.05). The expression level of hyaluronic acid was lower at 14 days than

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

**Figure 41.** Macroscopic findings at 7 and 14 days after injection. Left panels (A, C, and E) at 7 days after administra‐ tion; Right panels (B, D, and F) at 14 days after administration. (A and B) control group; (C and D) MSCs group; (E and F) MSC-CM group. Bar = 3 mm (From Tamari et al. [16-18]. Reprinted with permission from Quintessence Publishing

that at 7 days in all three groups.

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Co, Inc, Chicago).

**Figure 42.** Time-dependent changes in the area of wound area determined with image analysis software. area of actual wound/area of original wound ×100. Test for significant difference (ANOVA). MSC-CM group vs. control group, \*P < 0.05. \*\*P < 0.01 (From Tamari et al. [16-18]. Reprinted with permission from Quintessence Publishing Co, Inc, Chicago).

#### **2.14. Human dental pulp‐derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro‐regenerative mechanisms**

Spinal cord injury resulted in severe functional disability. In worldwide, 2.5 million people live with spinal cord injury More than 200,000 new injuries reported each year. Currently no curative therapy is available. Recent study has demonstrated that transplantation of stem cells in injured spinal cord would support functional recovery. The cellular resources of stem cell are Human embryonic stem cell, Induced pluripotent stem cells, Human embryonic neural stem cell, and adult mesenchymal stem cells from bone marrow. These stem cells have been shown to be valuable cellular resources for treatment of animal model of SCI. However, to use

them for patient, there are significant ethnical, safety and invasive problems. Thus, an ideal stem cellresource for SCI treatment is still an elusive subject. Here we have examined a neuro‐ regenerative activity of tooth derived stem cells in a rat model of SCI. Merits of teeth derived stem cells for SCI treatment are 1, They can be easily isolated from medical wast such as wisdom teeth and milk teeth. 2, AS they are originated from Neural crest,they can be a valuable cellular resources for treatment of neuro‐degenerative disease. 3, as they are autologous cellular resources, must be safe for cell therapy.

more than 3 and 5 times, respectively. These results demonstrate that SHED and DPSC, stem cells derived dental pulps, are valuable cellular resources for neuro‐regeneration therapy. SHEDs or DPSCs were transplanted into the transected spinal cord as described in the Materials and Methods. At 8 weeks post‐engraftment, BBB scoring suggested a recovery of hindlimb locomotor function both in SHEDs or DPSCs‐transplanted rats (n = 11 for SHEDs and n=10 for DPSCs) in comparison with vehicle controls (n = 10) analysis revealed a higher frequency of locomotion recovery (BBB score > 7 for SHEDs and > 6 for DPSCs) in SHEDs or DPSCs vs. vehicle controls. Thus, both SHED and DPSC promote functional recovery of completely transected spinal cord. As axonal myelination promotes functional recovery of injured spinal cord, we studied whether transplanted SHEDs preserved myelinated area in injured spinal cord. In the PBS‐treated control group, the transverse area of the lesion site exhibited no or little staining of Fluoro‐myelin. In SHEDs‐transplanted, however, myelin positive area covered 15% and 80% of entire spinal cord, at epicenter and 5mm caudal lesion site, respectively, showing that transplanted SHEDs play significant role in preservation of

Tissue Engineering and Regenerative Medicine 155

Our study revealed that engrafted SHEDs exhibited three major therapeutic benefits for recovery after SCI, including inhibition of the SCI‐induced apoptosis of neurons, astrocytes, and oligodendrocytes, which promoted the preservation of neural fibers and myelin sheaths, regeneration of the transected axon through the direct inhibition of multiple AGI signals, such as chondroitin sulfate proteoglycans and MAG, by paracrine mechanisms, and replacement of lost or damaged oligodendrocytes after SCI through specific differentiation into mature oligodendrocytes under the extreme conditions of SCI. To our knowledge, the neuro‐regen‐ erative activities and are unique to tooth‐derived stem cells, and are not exhibited by any other previously described stem cells. Thus, our data demonstrate that tooth‐derived stem cells may provide significant therapeutic benefits for treating the acute phase of SCI through both cell‐ autonomous and paracrine/trophic regenerative activities. We demonstrated multifaceted neuro‐regenerative activities of tooth‐derived stem cells that fulfill many requirements for functionalrecovery after SCI. In addition to theirremarkable neuro‐regenerative activities, we did not observe the malignant transformation of engrafted SHEDs 8 weeks after their implan‐ tation (data not shown). Furthermore, SHEDs and DPSCs can be obtained from exfoliated deciduous and impacted adult wisdom teeth without adverse health effects. Thus, there are few ethical concerns regarding their clinical use. We propose that tooth‐derived stem cells may

**2.15. Growth factors derived from dental pulp stem cells: A new potential clinical benefits**

There is no effective treatment for SCI. In the acute phase of SCI, damaged neurons are going to die within a day after the injury by inflammatory reaction. Until a weak later, multiple axon growth inhibitors (AGIs) are produced by the astroglial scar and degenerated myelin sur‐ rounding the injured CNS (Figure 44). We have been reported the clinical benefits of the engrafted human DPSCs (hDPSCs) and stem cells from human SHEDs in the treatment of acute phase rat SCI model. Recently, growth factors secreted from stem cells were important to stem

be an excellent and practical cellular resource for the treatment of SCI.

axonal myelination in injured spinal cord.

**for CNS regeneration therapy**

**Figure 43.** Merits of teeth derived stem cells for SCI treatment.

Flow cytometry analysis showed that SHEDs and DPSCs expressed a set of mesechymal stem cell marker, CD90, CD73, CD105 and CD90, but not endothelial/hematopoietic marker, CD54, CD34, CD45, CD11b/c or HLA‐DR, as describe previously. Majority of these stem cells uniquely co‐expressed several neural linage markers, including Nestin and Doublecortin (neural stem cell marker), GFAP (astrocyte marker), CNPase (immature oligodendrocyte maeker) and A2B5 (oligodendrocyte precursor marker) altogether. Next we have examined expression of neurotropic factors by real‐time PCR. Comparing with skin‐derived fibroblasts SHEDs expressed NT‐3 (Neurotrophin‐3) and BDNF (Brain Derived Neurotrophic Factor), more than 3 and 5 times, respectively. These results demonstrate that SHED and DPSC, stem cells derived dental pulps, are valuable cellular resources for neuro‐regeneration therapy. SHEDs or DPSCs were transplanted into the transected spinal cord as described in the Materials and Methods. At 8 weeks post‐engraftment, BBB scoring suggested a recovery of hindlimb locomotor function both in SHEDs or DPSCs‐transplanted rats (n = 11 for SHEDs and n=10 for DPSCs) in comparison with vehicle controls (n = 10) analysis revealed a higher frequency of locomotion recovery (BBB score > 7 for SHEDs and > 6 for DPSCs) in SHEDs or DPSCs vs. vehicle controls. Thus, both SHED and DPSC promote functional recovery of completely transected spinal cord. As axonal myelination promotes functional recovery of injured spinal cord, we studied whether transplanted SHEDs preserved myelinated area in injured spinal cord. In the PBS‐treated control group, the transverse area of the lesion site exhibited no or little staining of Fluoro‐myelin. In SHEDs‐transplanted, however, myelin positive area covered 15% and 80% of entire spinal cord, at epicenter and 5mm caudal lesion site, respectively, showing that transplanted SHEDs play significant role in preservation of axonal myelination in injured spinal cord.

them for patient, there are significant ethnical, safety and invasive problems. Thus, an ideal stem cellresource for SCI treatment is still an elusive subject. Here we have examined a neuro‐ regenerative activity of tooth derived stem cells in a rat model of SCI. Merits of teeth derived stem cells for SCI treatment are 1, They can be easily isolated from medical wast such as wisdom teeth and milk teeth. 2, AS they are originated from Neural crest,they can be a valuable cellular resources for treatment of neuro‐degenerative disease. 3, as they are autologous

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Flow cytometry analysis showed that SHEDs and DPSCs expressed a set of mesechymal stem cell marker, CD90, CD73, CD105 and CD90, but not endothelial/hematopoietic marker, CD54, CD34, CD45, CD11b/c or HLA‐DR, as describe previously. Majority of these stem cells uniquely co‐expressed several neural linage markers, including Nestin and Doublecortin (neural stem cell marker), GFAP (astrocyte marker), CNPase (immature oligodendrocyte maeker) and A2B5 (oligodendrocyte precursor marker) altogether. Next we have examined expression of neurotropic factors by real‐time PCR. Comparing with skin‐derived fibroblasts SHEDs expressed NT‐3 (Neurotrophin‐3) and BDNF (Brain Derived Neurotrophic Factor),

cellular resources, must be safe for cell therapy.

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154

**Figure 43.** Merits of teeth derived stem cells for SCI treatment.

Our study revealed that engrafted SHEDs exhibited three major therapeutic benefits for recovery after SCI, including inhibition of the SCI‐induced apoptosis of neurons, astrocytes, and oligodendrocytes, which promoted the preservation of neural fibers and myelin sheaths, regeneration of the transected axon through the direct inhibition of multiple AGI signals, such as chondroitin sulfate proteoglycans and MAG, by paracrine mechanisms, and replacement of lost or damaged oligodendrocytes after SCI through specific differentiation into mature oligodendrocytes under the extreme conditions of SCI. To our knowledge, the neuro‐regen‐ erative activities and are unique to tooth‐derived stem cells, and are not exhibited by any other previously described stem cells. Thus, our data demonstrate that tooth‐derived stem cells may provide significant therapeutic benefits for treating the acute phase of SCI through both cell‐ autonomous and paracrine/trophic regenerative activities. We demonstrated multifaceted neuro‐regenerative activities of tooth‐derived stem cells that fulfill many requirements for functionalrecovery after SCI. In addition to theirremarkable neuro‐regenerative activities, we did not observe the malignant transformation of engrafted SHEDs 8 weeks after their implan‐ tation (data not shown). Furthermore, SHEDs and DPSCs can be obtained from exfoliated deciduous and impacted adult wisdom teeth without adverse health effects. Thus, there are few ethical concerns regarding their clinical use. We propose that tooth‐derived stem cells may be an excellent and practical cellular resource for the treatment of SCI.

#### **2.15. Growth factors derived from dental pulp stem cells: A new potential clinical benefits for CNS regeneration therapy**

There is no effective treatment for SCI. In the acute phase of SCI, damaged neurons are going to die within a day after the injury by inflammatory reaction. Until a weak later, multiple axon growth inhibitors (AGIs) are produced by the astroglial scar and degenerated myelin sur‐ rounding the injured CNS (Figure 44). We have been reported the clinical benefits of the engrafted human DPSCs (hDPSCs) and stem cells from human SHEDs in the treatment of acute phase rat SCI model. Recently, growth factors secreted from stem cells were important to stem cell therapy. So we focused on the paracrine effect of growth factors derived from SHEDs. Here, we show that local administration of a serum‐free conditioned media of SHEDs (SHED‐ CM) into the rat SCI resulted in remarkable recovery of hindlimb locomotor functions. We found that this effect is associated with several paracrine effect of SHED‐CM.

glutamate and NO level in microglia culture medium reduced only SHED‐CM groups. Damaged neurons and glial cells are going to die within a day after injury, and expand cell death in one week. These cell death leads to degeneration of myelin and chronic atrophy of spinal cord. So we tried TUNEL staining. Compared to the control groups, SHED‐CM groups were down regulated TUNEL positive cells significantly at 24hour and 1week after SCI. And we stained myelin by fluoromyelin at eight weeks after SCI. At control groups, spinal cord was going to atrophy from epicenter, but on the other handSHED‐CMgroups were maintained myelin and kept form of spinal cord from epicenter. Until a week after SCI, multiple AGIs are produced by astroglial scar and degenerated myelin surrounding injury site. These AGIs accelerate the neuronal apoptosis and inhibit axonal regrowth. We got primary cerebellum granule neurons (CGNs) and tried neurite outgrowth assay on AGIs coating dish. And we foundthat CGNs only SHEDs andDPSC‐CMculture groups inhibitedAGIs activity andstrong promoted their neurite. So we stained by NFM and 5‐HT eight weeks after SCI. And we found that neuronal axon extended beyond the injury epicenter to caudal. Our studies demonstrate potential clinical benefits of SHED‐CM for the treatment of the acute phase of SCI, providing a novel, safe and effective neuro‐regenerative therapy that protects patient's CNS from the

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traumatic and ischemic CNS injury.

**Figure 45.** Multifaceted treatment strategy.

**Figure 44.** Acute phase of SCI.

We examined the effects of the treatment with SHED‐CM, MSC‐CM or Fibro‐CM on the functional recovery after contusion SCI. CMs were continuously delivered intrathecally by infusion pump into the SCI epicenter. The level of recovery of hindlimb locomotion was evaluated using the Basso, Beattie, Bresnahan locomotor rating scale (BBB scale). After the recovery period, the rats that had received SHED‐CM were able to support their weight through the plantar surface of the paw and step with fore‐hindlimb coordinated manner In contrast, although the MSC‐CM or Fibro‐CM treated rats move 3 joints of hindlimb, they were not able to walk with weight support. These results demonstrate that SHED‐CM, but not MSC‐ CM and Fibro‐CM, provides significant therapeutic benefit forthe treatment of the acute phase of SCI. In the acute phase of SCI, much inflammatory cytokines are released. These inflam‐ matory cytokines activate microglia and leads to expansion of inflammatory reaction. We got rat spinal cord mRNA after SCI, and analyzed expression of inflammatory and anti‐inflam‐ matory cytokines. We found that inflammatory cytokines expression down regulated MSC‐ CM, Fibro‐CM, and SHED‐CM groups rather than control groups at three time points. On the other hand,importantly, anti‐inflammatory cytokines expression up regulated only SHED‐CM groups. We got mouse primary microglia mRNA after LPS stimulation and analyzed the expression. Similarly *in vivo* results, inflammatory cytokines expression down regulated MSC‐ CM, Fibro‐CM, and SHED‐CM culture groups rather than control groups. But,uniquely, glutamate and NO level in microglia culture medium reduced only SHED‐CM groups. Damaged neurons and glial cells are going to die within a day after injury, and expand cell death in one week. These cell death leads to degeneration of myelin and chronic atrophy of spinal cord. So we tried TUNEL staining. Compared to the control groups, SHED‐CM groups were down regulated TUNEL positive cells significantly at 24hour and 1week after SCI. And we stained myelin by fluoromyelin at eight weeks after SCI. At control groups, spinal cord was going to atrophy from epicenter, but on the other handSHED‐CMgroups were maintained myelin and kept form of spinal cord from epicenter. Until a week after SCI, multiple AGIs are produced by astroglial scar and degenerated myelin surrounding injury site. These AGIs accelerate the neuronal apoptosis and inhibit axonal regrowth. We got primary cerebellum granule neurons (CGNs) and tried neurite outgrowth assay on AGIs coating dish. And we foundthat CGNs only SHEDs andDPSC‐CMculture groups inhibitedAGIs activity andstrong promoted their neurite. So we stained by NFM and 5‐HT eight weeks after SCI. And we found that neuronal axon extended beyond the injury epicenter to caudal. Our studies demonstrate potential clinical benefits of SHED‐CM for the treatment of the acute phase of SCI, providing a novel, safe and effective neuro‐regenerative therapy that protects patient's CNS from the traumatic and ischemic CNS injury.

**Figure 45.** Multifaceted treatment strategy.

cell therapy. So we focused on the paracrine effect of growth factors derived from SHEDs. Here, we show that local administration of a serum‐free conditioned media of SHEDs (SHED‐ CM) into the rat SCI resulted in remarkable recovery of hindlimb locomotor functions. We

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

We examined the effects of the treatment with SHED‐CM, MSC‐CM or Fibro‐CM on the functional recovery after contusion SCI. CMs were continuously delivered intrathecally by infusion pump into the SCI epicenter. The level of recovery of hindlimb locomotion was evaluated using the Basso, Beattie, Bresnahan locomotor rating scale (BBB scale). After the recovery period, the rats that had received SHED‐CM were able to support their weight through the plantar surface of the paw and step with fore‐hindlimb coordinated manner In contrast, although the MSC‐CM or Fibro‐CM treated rats move 3 joints of hindlimb, they were not able to walk with weight support. These results demonstrate that SHED‐CM, but not MSC‐ CM and Fibro‐CM, provides significant therapeutic benefit forthe treatment of the acute phase of SCI. In the acute phase of SCI, much inflammatory cytokines are released. These inflam‐ matory cytokines activate microglia and leads to expansion of inflammatory reaction. We got rat spinal cord mRNA after SCI, and analyzed expression of inflammatory and anti‐inflam‐ matory cytokines. We found that inflammatory cytokines expression down regulated MSC‐ CM, Fibro‐CM, and SHED‐CM groups rather than control groups at three time points. On the other hand,importantly, anti‐inflammatory cytokines expression up regulated only SHED‐CM groups. We got mouse primary microglia mRNA after LPS stimulation and analyzed the expression. Similarly *in vivo* results, inflammatory cytokines expression down regulated MSC‐ CM, Fibro‐CM, and SHED‐CM culture groups rather than control groups. But,uniquely,

found that this effect is associated with several paracrine effect of SHED‐CM.

**Figure 44.** Acute phase of SCI.

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> almost the same high score for motor function in the early stages as shown in Figure 47 right. Differences in the score appeared gradually between the two groups during the middle stage. On day 15, progressive improvement in motor disability in the SHED‐CM group became significant compared to the PBS group. As shown in Figure 47 left, there was a significant decrease in infarct volume on day 16 in the SHED‐CM group compared to the PBS group. These results suggest that SHED‐CM promoted regeneration. Recently, we reported the characteristics of SHEDs compared with DPSCs and MSCs. The results indicated that SHEDs possessed high proliferation ability and were enriched with extracellular matrix, suggesting it may be a useful source for stem cell‐based therapy. In addition, using micro array analysis, we showed that SHEDs had higher expression levels of several growth factors, such as fibroblast growth factor, transforming growth factor, connective tissue growth factor, nerve growth factor, and bone morphogenetic protein. Taken together, these findings indicate SHEDs are a more potentially useful source of stem cells for celltherapy than DPSCs andMSCs.

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However, cell therapy is always associated with problems, such as canceration, immune rejection, and ethical issues. Therefore, it is necessary to find alternative treatments to cell therapy. Studies in recent years have resulted in the recognition of a paracrine function in factors, and have suggested that stem cell transplantation may also be regarded as "cell‐based" cytokine therapy. Accordingly, we investigated two steps for developing a new treatment for cerebral ischemia. In the first step, we used SHED‐CM as a new source fortreatment of cerebral ischemia. We have reported previously in a rat model of stroke that needle administration of DPSCs induced recovery of motor disability and reduction in infarct volume, proliferation of their presumptive progeny in SVZ, migration to the infarct, and differentiation into the appropriate neurons. As several growth factors involved in neural regeneration are secreted from DPSCs, we hypothesized in the current study that SHED‐CM may improve recovery of motor disability and reduce infarct volume. In the second step, we investigated intranasal administration of SHED‐CM. Using this administration, therapeutic molecules traverse the BBB through the olfactory pathway and the less‐studied trigeminal neural pathway. An important advantage of intranasal administration is that it is less invasive with the factors

**Figure 47.** Evaluation of motor function and Reduction in infarct volume.

being delivered directly to the brain.

### **2.16. SHED‐CM rnhances recovery of focal cerebral ischemia in rats**

Regenerative therapy using stem cells is a promising approach for treatment of stroke. Recently, we reported that DPSCs ameliorated ischemic tissue injury in rat brain and acceler‐ ated functional recovery after middle cerebral artery occlusion (MCAO). In this study, we investigated the effects of SHED‐CM after pMCAO (permanent middle cerebral artery occlusion). Adult male Sprague‐Dawley rats were subjected to pMCAO. SHED‐CM was then administered intranasally and motor function and infarct volume evaluated. SHEDs were cultured in DMEM serum‐free medium. Conditioned medium of SHEDs was collected after 48 h of culture and centrifuged at 1500 rpm for 5 min. The supernatant was recentrifuged at 3000 rpm for 3 min followed by collection of the second supernatant, named SHED‐CM. Seventy‐two hours after pMCAO, the rats were anesthetized again with 1.5% isoflurane in a mixture of 70% N2O and 30% O2. A total of 100 μL of SHED‐CM was administered to rat via the olfactory pathway using a Hamilton microsyringe (Figure 46). The SHED‐CM preparation was administered in 10 μL at a time, with an interval of 2 min between each administration. Intranasal administration was performed everyday from days 3 to 15.

**Figure 46.** Intranasal administration.

The rats were blindly examined on days 1, 3, 6, 9, 12, and 15 using a standardized motor disability scale with slight modifications. The cryosections obtained from samples on day 16 were stained with hematoxylin and eosin. Image J was used to determine each infarct area in 12 coronal sections at 1.00‐mm intervals. The entire infarction area was covered by these 12 coronal sections. Regional infarct volumes were calculated by summing the infarct areas and multiplying these areas by the distance between sections (1.00 mm). The two groups displayed almost the same high score for motor function in the early stages as shown in Figure 47 right. Differences in the score appeared gradually between the two groups during the middle stage. On day 15, progressive improvement in motor disability in the SHED‐CM group became significant compared to the PBS group. As shown in Figure 47 left, there was a significant decrease in infarct volume on day 16 in the SHED‐CM group compared to the PBS group. These results suggest that SHED‐CM promoted regeneration. Recently, we reported the characteristics of SHEDs compared with DPSCs and MSCs. The results indicated that SHEDs possessed high proliferation ability and were enriched with extracellular matrix, suggesting it may be a useful source for stem cell‐based therapy. In addition, using micro array analysis, we showed that SHEDs had higher expression levels of several growth factors, such as fibroblast growth factor, transforming growth factor, connective tissue growth factor, nerve growth factor, and bone morphogenetic protein. Taken together, these findings indicate SHEDs are a more potentially useful source of stem cells for celltherapy than DPSCs andMSCs.

**Figure 47.** Evaluation of motor function and Reduction in infarct volume.

**2.16. SHED‐CM rnhances recovery of focal cerebral ischemia in rats**

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Intranasal administration was performed everyday from days 3 to 15.

**Figure 46.** Intranasal administration.

Regenerative therapy using stem cells is a promising approach for treatment of stroke. Recently, we reported that DPSCs ameliorated ischemic tissue injury in rat brain and acceler‐ ated functional recovery after middle cerebral artery occlusion (MCAO). In this study, we investigated the effects of SHED‐CM after pMCAO (permanent middle cerebral artery occlusion). Adult male Sprague‐Dawley rats were subjected to pMCAO. SHED‐CM was then administered intranasally and motor function and infarct volume evaluated. SHEDs were cultured in DMEM serum‐free medium. Conditioned medium of SHEDs was collected after 48 h of culture and centrifuged at 1500 rpm for 5 min. The supernatant was recentrifuged at 3000 rpm for 3 min followed by collection of the second supernatant, named SHED‐CM. Seventy‐two hours after pMCAO, the rats were anesthetized again with 1.5% isoflurane in a mixture of 70% N2O and 30% O2. A total of 100 μL of SHED‐CM was administered to rat via the olfactory pathway using a Hamilton microsyringe (Figure 46). The SHED‐CM preparation was administered in 10 μL at a time, with an interval of 2 min between each administration.

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The rats were blindly examined on days 1, 3, 6, 9, 12, and 15 using a standardized motor disability scale with slight modifications. The cryosections obtained from samples on day 16 were stained with hematoxylin and eosin. Image J was used to determine each infarct area in 12 coronal sections at 1.00‐mm intervals. The entire infarction area was covered by these 12 coronal sections. Regional infarct volumes were calculated by summing the infarct areas and multiplying these areas by the distance between sections (1.00 mm). The two groups displayed

However, cell therapy is always associated with problems, such as canceration, immune rejection, and ethical issues. Therefore, it is necessary to find alternative treatments to cell therapy. Studies in recent years have resulted in the recognition of a paracrine function in factors, and have suggested that stem cell transplantation may also be regarded as "cell‐based" cytokine therapy. Accordingly, we investigated two steps for developing a new treatment for cerebral ischemia. In the first step, we used SHED‐CM as a new source fortreatment of cerebral ischemia. We have reported previously in a rat model of stroke that needle administration of DPSCs induced recovery of motor disability and reduction in infarct volume, proliferation of their presumptive progeny in SVZ, migration to the infarct, and differentiation into the appropriate neurons. As several growth factors involved in neural regeneration are secreted from DPSCs, we hypothesized in the current study that SHED‐CM may improve recovery of motor disability and reduce infarct volume. In the second step, we investigated intranasal administration of SHED‐CM. Using this administration, therapeutic molecules traverse the BBB through the olfactory pathway and the less‐studied trigeminal neural pathway. An important advantage of intranasal administration is that it is less invasive with the factors being delivered directly to the brain.

Our results suggested that SHED‐CM including some growth factors may produce effects in stroke model. The success of the two steps investigated suggest it may be possible to use a shortcut for clinical application. Administration of SHED‐CM resolves the ethical issues involved with cell therapies, as SHED‐CM is not a cell but rather a conjugate of many growth factors. As SHED‐CM can be stocked, it is possible to use it for acute stages of stroke, either alone or with ready‐made treatment, such as recombinant tissue plasminogen activator, anticoagulation, and antiplatelet therapy. This study suggested that intranasal administration of SHED‐CM may help recovery in acute stroke patients in the future. In conclusion, regen‐ eration therapy using SHED‐CM is a very safe method with no associated problems and is therefore a potential candidate for innovative treatment of cerebral ischemia.

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