**3. Research projects**

The following research projects are introduced in the present chapter: 1) clinical research on tissue‐engineered osteogenic material (TEOM), 2)regulation of cell signaling pathways, 3) cell transplantation for improving bone quality, and 4) TEOM for promoting bone formation.

### **3.1. Clinical research on TEOM**

TEOM, ie, injectable tissue‐engineered bone, is a gel comprising *in vitro* expanded autologous bone marrow stromal cells (BMSCs) and platelet‐rich plasma (PRP). Alpha granules in the platelets release various growth factors and the plasma forms a fibrin network, which serves as cell scaffold. Translational researches on TEOM have produced positive data and TEOM is considered a promising material for bone formation [1‐4]. Yamada et al. reported a clinical study on injectable tissue‐engineered bone for maxillary sinus floor augmentation and simultaneous placement of dental implants (Figure 2). Their study included 16 sinus augmen‐ tations in 12 patients whose alveolar crest bone height was 2 to 10 mm. All 41 dental implants installed were clinically stable at the time of second‐stage surgery. Radiography at 2 years after implant installation showed increased mineralized tissue (mean, 8.8 ± 1.6 mm) compared with the preoperative bone height, and neither adverse effects norremarkable bone absorption were seen in the 2‐ to 6‐year follow‐up periods. These results support the feasibility of using injectable tissue‐engineered bone for successful bone formation and dental implants.

**Figure 2.** Treatment procedures with tissue-engineered bone A: Preoperative view. B: Magnified view of dental im‐ plants placed into the area of maxillary sinus floor augmentation and dental implant thread exposure. C: Tissue-engi‐ neered bone injection. D: Magnified view during second-stage surgery at 6 months after implant placement. The exposed thread is surrounded by newly formed bone (white arrow). The green arrow indicates the line of bone biop‐ sy. E: Provisional prosthesis. F: Micrograph of the regenerated bone area (×4); right-upper panel (×20). G: Orthopanto‐ mogram taken immediately after first-stage surgery. Line of substratum of the maxillary sinus mucosa (arrow). H: Orthopantomogram taken at 6 months after first-stage surgery. Line of maxillary sinus mucosa after sinus augmenta‐ tion (arrow). The left lower panel is a computed tomography (CT) image indicating radiodensity. (From [1]. Reprinted

Tissue Engineering and Regenerative Medicine for Bone Regeneration 323

with permission).

transplantation and osteogenic chemical induction are preferred for small tissue deficiencies, while bioartificial mineral construct implantation is considered suitable for large deficiencies.

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

The following research projects are introduced in the present chapter: 1) clinical research on tissue‐engineered osteogenic material (TEOM), 2)regulation of cell signaling pathways, 3) cell transplantation for improving bone quality, and 4) TEOM for promoting bone formation.

TEOM, ie, injectable tissue‐engineered bone, is a gel comprising *in vitro* expanded autologous bone marrow stromal cells (BMSCs) and platelet‐rich plasma (PRP). Alpha granules in the platelets release various growth factors and the plasma forms a fibrin network, which serves as cell scaffold. Translational researches on TEOM have produced positive data and TEOM is considered a promising material for bone formation [1‐4]. Yamada et al. reported a clinical study on injectable tissue‐engineered bone for maxillary sinus floor augmentation and simultaneous placement of dental implants (Figure 2). Their study included 16 sinus augmen‐ tations in 12 patients whose alveolar crest bone height was 2 to 10 mm. All 41 dental implants installed were clinically stable at the time of second‐stage surgery. Radiography at 2 years after implant installation showed increased mineralized tissue (mean, 8.8 ± 1.6 mm) compared with the preoperative bone height, and neither adverse effects norremarkable bone absorption were seen in the 2‐ to 6‐year follow‐up periods. These results support the feasibility of using

injectable tissue‐engineered bone for successful bone formation and dental implants.

**Figure 1.** Elements required for tissue regeneration

**3. Research projects**

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322

**3.1. Clinical research on TEOM**

**Figure 2.** Treatment procedures with tissue-engineered bone A: Preoperative view. B: Magnified view of dental im‐ plants placed into the area of maxillary sinus floor augmentation and dental implant thread exposure. C: Tissue-engi‐ neered bone injection. D: Magnified view during second-stage surgery at 6 months after implant placement. The exposed thread is surrounded by newly formed bone (white arrow). The green arrow indicates the line of bone biop‐ sy. E: Provisional prosthesis. F: Micrograph of the regenerated bone area (×4); right-upper panel (×20). G: Orthopanto‐ mogram taken immediately after first-stage surgery. Line of substratum of the maxillary sinus mucosa (arrow). H: Orthopantomogram taken at 6 months after first-stage surgery. Line of maxillary sinus mucosa after sinus augmenta‐ tion (arrow). The left lower panel is a computed tomography (CT) image indicating radiodensity. (From [1]. Reprinted with permission).

**3.3. Cell transplantation for improving bone quality**

with implants.

Low bone quality is a risk factor for successful osseointegration, a prerequisite for dental implants, and typically occurs in osteoporosis. Osteoporosis is a systemic skeletal disease leading to fragile bones with decreased microstructures due to the postmenopausal decrease in estrogen secretion. Okamoto et al. investigated whether BMSCs can promote bone healing around titanium implants in rat osteoporosis models [6]. Sprague‐Dawley rats were divided into 3 groups: the first group in which the ovaries were removed (OVX group), the second group in which a sham surgery was performed (SHAM group), and the third group in which BMSCs were transplanted to an OVX group (OVX‐BMSCs group) (Figure 4). In the OVX‐ BMSCs group, 1 × 105 BMSCs were transplanted into the femur with implant. Each value of the bone‐to‐implant contact and the bone area of each cortical bone and cancellous bone were obtained histomorphometrically. Bone density was measured over 500 μm distally and proximally from the implants. Each ratio of bone‐to‐implant contact, bone area, and bone density in the OVX‐BMSCs group was significantly higher than those of the OVX group as compared to the cancellous bone. BMSC transplantation therapy improved local bone healing in the cancellous bone surrounding implants and also significantly improved bone binding

Tissue Engineering and Regenerative Medicine for Bone Regeneration 325

**Figure 4.** Experimental design (left) and histological evaluation of the distal femur at 28 and 56 days after implanta‐ tion (right) A-F: Micrographs of the distal femur at 28 days after implantation. A and D: SHAM; B and E: OVX (ovaries had been removed); C and F: OVX-BMSCs (ovaries had been removed and bone marrow stromal cells had been trans‐ planted), (toluidine blue stain, A-C ×1.25, bar = 1.1 mm; D-F ×10, bar = 200 µm). In the cortical bone area, most of the implant surface was in direct contact with newly generated bones, and the thread was widely filled with bone tissues in all groups. No remarkable difference was observed among the groups. Slightly more osteogenesis was observed in the OVX-BMSCs group than in the OVX group. The osteogenetic area was remarkably smaller in the OVX group than in the SHAM group outside the thread. In addition, osteogenesis was observed at a higher rate in the OVX-BMSCs group than in the OVX group outside the thread. G-L: Micrographs of the distal femur at 56 days after implantation. G and J: SHAM; H and K: OVX; I and L: OVX-BMSCs, (toluidine blue stain, G-I ×1.25, bar = 1.1 mm; J-L ×10, bar = 200 µm). In the cortical bone area, the area on the implant surface that was in contact with newly generated bones was greater than that observed at 28 days, and the amount of bone tissue inside the thread had increased in all groups. In the cancellous bone area, the amount of bone that was in contact with the implant surface had increased in all groups. There was no remarkable difference between the SHAM group and the OVX-BMSCs group. In the OVX-BMSCs group,

trabecular structures were observed, presenting as web-like pattern. (From [6]. Reprinted with permission).

#### **3.2. Regulation of cell signaling pathways**

Improvements in cell culture efficiency may contribute to a shorter treatment time and decrease its cost. Katagiri et al. examined the cultivation process for human mesenchymal stem cells (MSCs) by regulating the Wnt signaling pathway [5]. Wnt signaling was activated and inhibited with LiCl and secreted frizzled‐related protein‐3 (sFRP‐3), respectively. Human MSCs were examined for proliferation (cell counting and BrdU assays), osteogenic differen‐ tiation (alizarin red staining), and osteogenic gene expression on days 7 and 14 after induction (reverse‐transcription polymerase chain reaction [RT‐PCR] and quantitative real‐time RT‐PCR analyses). Cell counting and BrdU assays showed higher proliferation rate of LiCl‐treated MSCs than of untreated MSCs. Alizarin red staining showed that sFRP‐3‐treated MSCs mineralized earlier(on day 7) than untreated MSCs (Figure 3). Both RT‐PCR analyses (on days 7 and 14) demonstrated that sFRP‐3‐treated MSCs expressed higher levels of the alkaline phosphatase gene than untreated MSCs. These results suggest that regulation of the Wnt signaling pathway contributes to cell proliferation andosteogenicdifferentiation ofMSCs.This study implies that effective and efficient bone regeneration technologies can be developed by regulating the Wnt signaling pathway.

**Figure 3.** Alizarin red staining of sFRP-3-treated human mesenchymal stem cells (MSCs) during osteogenic differentia‐ tion A: During osteogenic induction of MSCs, more mineralization occurred in MSCs treated with secreted frizzledrelated protein-3 (sFRP-3) than in untreated MSCs on days 7 and 14. B: Phase-contrast macroscopic views (×40). (From [5]. Reprinted with permission).

#### **3.3. Cell transplantation for improving bone quality**

**3.2. Regulation of cell signaling pathways**

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324

regulating the Wnt signaling pathway.

[5]. Reprinted with permission).

Improvements in cell culture efficiency may contribute to a shorter treatment time and decrease its cost. Katagiri et al. examined the cultivation process for human mesenchymal stem cells (MSCs) by regulating the Wnt signaling pathway [5]. Wnt signaling was activated and inhibited with LiCl and secreted frizzled‐related protein‐3 (sFRP‐3), respectively. Human MSCs were examined for proliferation (cell counting and BrdU assays), osteogenic differen‐ tiation (alizarin red staining), and osteogenic gene expression on days 7 and 14 after induction (reverse‐transcription polymerase chain reaction [RT‐PCR] and quantitative real‐time RT‐PCR analyses). Cell counting and BrdU assays showed higher proliferation rate of LiCl‐treated MSCs than of untreated MSCs. Alizarin red staining showed that sFRP‐3‐treated MSCs mineralized earlier(on day 7) than untreated MSCs (Figure 3). Both RT‐PCR analyses (on days 7 and 14) demonstrated that sFRP‐3‐treated MSCs expressed higher levels of the alkaline phosphatase gene than untreated MSCs. These results suggest that regulation of the Wnt signaling pathway contributes to cell proliferation andosteogenicdifferentiation ofMSCs.This study implies that effective and efficient bone regeneration technologies can be developed by

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

**Figure 3.** Alizarin red staining of sFRP-3-treated human mesenchymal stem cells (MSCs) during osteogenic differentia‐ tion A: During osteogenic induction of MSCs, more mineralization occurred in MSCs treated with secreted frizzledrelated protein-3 (sFRP-3) than in untreated MSCs on days 7 and 14. B: Phase-contrast macroscopic views (×40). (From Low bone quality is a risk factor for successful osseointegration, a prerequisite for dental implants, and typically occurs in osteoporosis. Osteoporosis is a systemic skeletal disease leading to fragile bones with decreased microstructures due to the postmenopausal decrease in estrogen secretion. Okamoto et al. investigated whether BMSCs can promote bone healing around titanium implants in rat osteoporosis models [6]. Sprague‐Dawley rats were divided into 3 groups: the first group in which the ovaries were removed (OVX group), the second group in which a sham surgery was performed (SHAM group), and the third group in which BMSCs were transplanted to an OVX group (OVX‐BMSCs group) (Figure 4). In the OVX‐ BMSCs group, 1 × 105 BMSCs were transplanted into the femur with implant. Each value of the bone‐to‐implant contact and the bone area of each cortical bone and cancellous bone were obtained histomorphometrically. Bone density was measured over 500 μm distally and proximally from the implants. Each ratio of bone‐to‐implant contact, bone area, and bone density in the OVX‐BMSCs group was significantly higher than those of the OVX group as compared to the cancellous bone. BMSC transplantation therapy improved local bone healing in the cancellous bone surrounding implants and also significantly improved bone binding with implants.

**Figure 4.** Experimental design (left) and histological evaluation of the distal femur at 28 and 56 days after implanta‐ tion (right) A-F: Micrographs of the distal femur at 28 days after implantation. A and D: SHAM; B and E: OVX (ovaries had been removed); C and F: OVX-BMSCs (ovaries had been removed and bone marrow stromal cells had been trans‐ planted), (toluidine blue stain, A-C ×1.25, bar = 1.1 mm; D-F ×10, bar = 200 µm). In the cortical bone area, most of the implant surface was in direct contact with newly generated bones, and the thread was widely filled with bone tissues in all groups. No remarkable difference was observed among the groups. Slightly more osteogenesis was observed in the OVX-BMSCs group than in the OVX group. The osteogenetic area was remarkably smaller in the OVX group than in the SHAM group outside the thread. In addition, osteogenesis was observed at a higher rate in the OVX-BMSCs group than in the OVX group outside the thread. G-L: Micrographs of the distal femur at 56 days after implantation. G and J: SHAM; H and K: OVX; I and L: OVX-BMSCs, (toluidine blue stain, G-I ×1.25, bar = 1.1 mm; J-L ×10, bar = 200 µm). In the cortical bone area, the area on the implant surface that was in contact with newly generated bones was greater than that observed at 28 days, and the amount of bone tissue inside the thread had increased in all groups. In the cancellous bone area, the amount of bone that was in contact with the implant surface had increased in all groups. There was no remarkable difference between the SHAM group and the OVX-BMSCs group. In the OVX-BMSCs group, trabecular structures were observed, presenting as web-like pattern. (From [6]. Reprinted with permission).

**3.4. TEOM for promoting bone formation**

compensate for insufficient distraction gaps.

solution. (From [9]. Reprinted with permission).

than 10 cm [7,8]. (Figure 5).

Distraction osteogenesis (DO) is considered *in vivo* tissue engineering and is a technique that provides autologous and predictable bone formation. Hibi & Ueda developed an internal device with which they were able to reconstruct a mandibular segmental bony defect of more

Tissue Engineering and Regenerative Medicine for Bone Regeneration 327

DO is a useful technique for reconstructing bony defects without performing grafting proce‐ dures; however, it requires a long treatment time that includes latent, lengthening, and consolidation periods. To shorten these periods, attempts of applying tissue engineering technologies for DO have been made [9, 10]. Kinoshita et al. investigated whether locally injected TEOM can promote bone regeneration in a rabbit high‐rate DO model (Figure 6). Bilateral osteotomies were performed in the maxilla; distraction devices were activated at a rate of 2.0 mm once daily for 4 days after a 5‐day latency period. Twelve rabbits were divided into 2 groups. At the end of distraction, the experimental group ofrabbits received an injection of TEOM into the distracted tissue on one side, whereas saline solution was injected into the distracted tissue on the contralateral side (as internal control). An additional control group received an injection of PRP or saline solution into the distracted tissue in the same way as the experimental group. The distraction regenerates were assessed by radiological and histomor‐ phometric analyses. The radiodensity of the distraction gap injected with TEOM was signifi‐ cantly higherthan that injected with PRP or saline solution at 2, 3, and 4 weeks postdistraction. The histomorphometric analysis also showed that both new bone zone and bony content in the distraction gap injected with TEOM were significantly increased when compared with PRP or saline solution. These results demonstrated that the distraction gap injected with TEOM showed significant new bone formation. Therefore, injections of TEOM may be able to

**Figure 6.** Experimental design (left) and radiographic view of the distracted maxilla (right) A: Two weeks after tissueengineered osteogenic material (TEOM) injection. B: Four weeks after TEOM injection. C: Two weeks after injection of saline solution. D: Four weeks after saline solution injection. E: Two weeks after platelet-rich plasma (PRP) injection. F: Four weeks after PRP injection. G: Two weeks after injection of saline solution. H: Four weeks after injection of saline

**Figure 5.** Two-step transport distraction osteogenesis (DO) for the posterior body and ramus of the mandible A: Man‐ dible with recurrent ameloblastoma. Computed tomography (CT) revealed a multilocular radiolucent area in the ra‐ mus and posterior body of the mandible. B: Custom-made distraction device with an artificial condyle on the patient's stereolithographic model. Locking plates and screws enabled supraperiosteal fixation of the transport segment. C: Su‐ praperiosteal fixation of the distraction device. A short buccal periosteal flap of the transport segment was reposi‐ tioned and sutured to cover the vertical osteotomy line. The transport segment was fixed with locking plates and screws supraperiosteally. D: Regenerated posterior mandibular body. Eight months after distraction, CT showed that the newly formed 50-mm-long bone in the distraction gap (arrowheads) progressed to continuous buccal and lingual cortical surfaces. E: Secondary vertical DO for reconstructing the ascending ramus. A short buccal periosteal flap of the secondary transport segment was repositioned and sutured to cover the horizontal osteotomy line. The transport seg‐ ment was fixed with another distraction device positioned supraperiosteally. F: Another transport segment. Immedi‐ ately after surgery, CT showed fixation of the secondary transport segment with the distraction device. G: Regenerated ascending ramus. Nine months after the secondary distraction, CT showed that newly formed bone in the distraction gap progressed to continuous buccal and lingual cortical surfaces of the ramus. H: Device removal and mandibular reconstruction. CT showed that the regenerated ramus was connected to the preserved condylar segment with a lag screw and plate. I: Regenerated mandibular body containing implants. CT showed 3 screw-type implants installed in the right second premolar and molar regions with a buccal veneer bone graft fixed with 2 screws. J: Recon‐ structed mandible and osseointegrated implants for occlusal function. CT showed that the mandibular angle was ad‐ justed with osteotomy and screw fixation in the angle region. K: Provisional implant-supported prosthesis on the reconstructed mandible. The ridge supporting the implants was surrounded by the attached mucosa, the buccal part of which originated from the palatal mucosa. (From [8]. Reprinted with permission).

#### **3.4. TEOM for promoting bone formation**

**Figure 5.** Two-step transport distraction osteogenesis (DO) for the posterior body and ramus of the mandible A: Man‐ dible with recurrent ameloblastoma. Computed tomography (CT) revealed a multilocular radiolucent area in the ra‐ mus and posterior body of the mandible. B: Custom-made distraction device with an artificial condyle on the patient's stereolithographic model. Locking plates and screws enabled supraperiosteal fixation of the transport segment. C: Su‐ praperiosteal fixation of the distraction device. A short buccal periosteal flap of the transport segment was reposi‐ tioned and sutured to cover the vertical osteotomy line. The transport segment was fixed with locking plates and screws supraperiosteally. D: Regenerated posterior mandibular body. Eight months after distraction, CT showed that the newly formed 50-mm-long bone in the distraction gap (arrowheads) progressed to continuous buccal and lingual cortical surfaces. E: Secondary vertical DO for reconstructing the ascending ramus. A short buccal periosteal flap of the secondary transport segment was repositioned and sutured to cover the horizontal osteotomy line. The transport seg‐ ment was fixed with another distraction device positioned supraperiosteally. F: Another transport segment. Immedi‐ ately after surgery, CT showed fixation of the secondary transport segment with the distraction device. G: Regenerated ascending ramus. Nine months after the secondary distraction, CT showed that newly formed bone in the distraction gap progressed to continuous buccal and lingual cortical surfaces of the ramus. H: Device removal and mandibular reconstruction. CT showed that the regenerated ramus was connected to the preserved condylar segment with a lag screw and plate. I: Regenerated mandibular body containing implants. CT showed 3 screw-type implants installed in the right second premolar and molar regions with a buccal veneer bone graft fixed with 2 screws. J: Recon‐ structed mandible and osseointegrated implants for occlusal function. CT showed that the mandibular angle was ad‐ justed with osteotomy and screw fixation in the angle region. K: Provisional implant-supported prosthesis on the reconstructed mandible. The ridge supporting the implants was surrounded by the attached mucosa, the buccal part

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

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of which originated from the palatal mucosa. (From [8]. Reprinted with permission).

Distraction osteogenesis (DO) is considered *in vivo* tissue engineering and is a technique that provides autologous and predictable bone formation. Hibi & Ueda developed an internal device with which they were able to reconstruct a mandibular segmental bony defect of more than 10 cm [7,8]. (Figure 5).

DO is a useful technique for reconstructing bony defects without performing grafting proce‐ dures; however, it requires a long treatment time that includes latent, lengthening, and consolidation periods. To shorten these periods, attempts of applying tissue engineering technologies for DO have been made [9, 10]. Kinoshita et al. investigated whether locally injected TEOM can promote bone regeneration in a rabbit high‐rate DO model (Figure 6). Bilateral osteotomies were performed in the maxilla; distraction devices were activated at a rate of 2.0 mm once daily for 4 days after a 5‐day latency period. Twelve rabbits were divided into 2 groups. At the end of distraction, the experimental group ofrabbits received an injection of TEOM into the distracted tissue on one side, whereas saline solution was injected into the distracted tissue on the contralateral side (as internal control). An additional control group received an injection of PRP or saline solution into the distracted tissue in the same way as the experimental group. The distraction regenerates were assessed by radiological and histomor‐ phometric analyses. The radiodensity of the distraction gap injected with TEOM was signifi‐ cantly higherthan that injected with PRP or saline solution at 2, 3, and 4 weeks postdistraction. The histomorphometric analysis also showed that both new bone zone and bony content in the distraction gap injected with TEOM were significantly increased when compared with PRP or saline solution. These results demonstrated that the distraction gap injected with TEOM showed significant new bone formation. Therefore, injections of TEOM may be able to compensate for insufficient distraction gaps.

**Figure 6.** Experimental design (left) and radiographic view of the distracted maxilla (right) A: Two weeks after tissueengineered osteogenic material (TEOM) injection. B: Four weeks after TEOM injection. C: Two weeks after injection of saline solution. D: Four weeks after saline solution injection. E: Two weeks after platelet-rich plasma (PRP) injection. F: Four weeks after PRP injection. G: Two weeks after injection of saline solution. H: Four weeks after injection of saline solution. (From [9]. Reprinted with permission).
