SCIENTIFIC FOUNDATION

Self-Assembling Peptide Nanofiber Scaffolds, Platelet-Rich Plasma, and Mesenchymal Stem Cells for Injectable Bone Regeneration With Tissue Engineering
Ryoko Yoshimi, DDS,* Yoichi Yamada, DDS, PhD, Kenji Ito, DDS, PhD, Sayaka Nakamura, DDS, PhD,* Akihiro Abe, MD, PhD, Tetsuro Nagasaka, MD, PhD,|| Kazuto Okabe, DDS,* Tomoyuki Kohgo, DDS,* Shunsuke Baba, DDS, PhD, and Minoru Ueda, DDS, PhD*
Abstract: The purpose of this study was to investigate a capability of PuraMatrix (PM), which is a self-assembling peptide nanomaterial, as a scaffold for bone regeneration in combination with dog mesenchymal stem cells (dMSCs) and/or platelet-rich plasma (PRP) using tissue engineering and regenerative technology. Initially, teeth were extracted from an adult hybrid dogs mandible region. After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. The following graft materials were implanted into these defects: (1) control (defect only), (2) PM, (3) PM/PRP, (4) PM/dMSCs, and (5) PM/dMSCs/PRP. From scanning electron microscope images, PM had a three-dimensional nanostructure, and dMSCs attached on the surface of PM. At 2, 4, and 8 weeks after implantation, each sample was collected from the graft area with a trephine bar and assessed by histologic and histomorphometric analyses. It was observed that the bone regenerated by PM/dMSCs/PRP was of excellent quality, and mature bone had been formed. Histometrically, at 8 weeks, newly formed bone areas comprised 12.39 T 1.29% (control), 25.28 T 3.92% (PM), 27.72 T 3.15% (PM/PRP), 50.07 T 3.97% (PM/dMSCs), and 58.43 T 5.06% (PM/dMSCs/PRP). The PM/dMSCs and PM/dMSCs/PRP groups showed a signicant increase at all weeks compared with the control, PM, or PM/PRP (P G 0.05 at 2, 4, and 8 weeks, analysis of variance). 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. Key Words: Tissue engineering and regenerative medicine, bone regeneration, PuraMatrix (PM), mesenchymal stem cells (MSCs), platelet-rich plasma (PRP) (J Craniofac Surg 2009;20: 1523Y1530) t present, the gold standard of clinical treatment of bone defects from tumor resection, congenital malformation, trauma, or periodontitis is autogenous bone grafting. However, preferred autogenous material is limited in supply, is associated with attendant donor site morbidity, and is occasionally not suitable for the proposed reconstruction because of poor tissue quality or difculty in shaping the graft. A previous approach to this problem focused on the development of various articial materials, including hydroxyapatite, tricalcium phosphate, anorganic porous bovine-derived bone mineral scaffolds, and so on, and it was thought that these materials might be used instead of the autogenous bone.1Y6 However, most of these articial materials did not induce adequate bone formation and were difcult to t into complicated bone defects, had increased susceptibility to infection, had an uncertain long-term interaction with the hosts physiology, and were often animal-derived.7,8 The ideal biomaterial scaffolds, particularly in craniomaxillofacial, plastic, or orthopedic elds with complicated bone defect shapes, should have excellent plasticity for tting into complex defect shapes, and the rate of absorption needs to be fast to avoid infection. On the other hand, in the clinical use of animal-derived materials, there are some problems, such as, they may not have uniform quality, foreign substances may contaminate the material, and animal infection may occur. Furthermore, there is also currently an insufcient amount of information available on the essential properties of scaffolds for bone regeneration. Moreover, these materials and their internal structures may not provide a particularly favorable environment for cell survival and bone regeneration, and the internal microscale environment of these materials needs to serve as an extracellular matrix (ECM). The 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.9 Recent studies have shown that the promotion of

From the *Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine; Center for Genetic and Regenerative Medicine and Department of Clinical Cell Therapy and Tissue Engineering, Nagoya University School of Medicine; and Departments of Hematology and ||Clinical Pathology, Nagoya University Graduate School of Medicine, Nagoya; and Department of Regenerative Medicine, Institute of Biomedical Research and Innovation, Kobe, Japan. Received November 22, 2008. Accepted for publication March 17, 2009. Address correspondence and reprint requests to Yoichi Yamada, DDS, PhD, Center for Genetic and Regenerative Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan; E-mail: yyamada@med.nagoya-u.ac.jp This work was support in part by the Japan Society for the Promotion of Science (KAKENHI 16390583), and the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI 19791505). Copyright * 2009 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0b013e3181b09b7e

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multidimensional cell-cell interactions by biomaterials was crucial for proper cellular differentiation and for subsequent tissue formation.10 This study considered the use of the matrix material PuraMatrix (PM; 3D-Matrix, Ltd, Tokyo, Japan), which is synthesized by chemical

peptide methods and has similarities to the bers and pore sizes found in the ECM. PuraMatrix is a synthetic biologic material formed through the assembly of ionic self-complementary peptides consisting of a 16Yamino acid sequence (Ac-RADARADARADARADACONH2), which is called RADA16-I composed of alanine (A), arginine (R), and aspartic acid (D), and has greater than 99.5% water content (standard amino acids content, 1% wt/vol)11 and good uid characteristics. The peptides have regular repeating units of positively charged residues (arginine) and negatively charged residues (aspartic acid) separated by hydrophobic residues (alanine) and contain 50% charged residues and are characterized by their periodic repeats of alternating ionic hydrophilic and uncharged hydrophobic amino acids.12 The peptides interact individually by self-complementary and amphiphilic property and self-assembly. Thus, PM possesses good plasticity, absorption, and biocompatible properties and is devoid of animal-derived pathogens and antigens.13 The selfassembled three-dimensional microenvironment structure is also similar to natural ECM, and PM may be useable as a scaffold because of these nanoscale morphologic features that may help to control cell behavior.12Y16 Recently, in clinical applications, treatments based on tissue engineering and regenerative medicine technology are expected to be of benet from the viewpoint of reduced hospital days, donor site morbidity, and immune reactions.17,18 The concept is to regenerate tissues by transplanting cells with scaffolds made from natural or synthetic materials and appropriate signal molecules, and bone regeneration is regarded as an application of this concept. The selected approach involved the application of mesenchymal stem cells (MSCs) as the cells and platelet-rich plasma (PRP) as the source of signal molecules. Mesenchymal stem cells are easy to harvest by bone marrow aspiration and can be cultured in the laboratory. They are multipotent and show active proliferation.19 Moreover, PRP is collected by centrifugation of peripheral blood, and it is known that growth factors in PRP include platelet-derived growth factor, transforming growth factor A, vascular endothelial growth factor, and epithelial growth factor.20,21 These growth factors are released from the degranulation of platelets and act as the key factors in tissue repair. Taken together, the current study investigated effective bone regeneration using tissue engineering and regenerative medicine technology with a self-assembling peptide hydrogel as the graft material.

MATERIALS AND METHODS Animal Model

All animal experiments were performed in strict accordance with protocols approved by the institutional animal care committee. After a period of acclimatization, adult hybrid dogs with a mean age of 2 years were operated on under general anesthesia. The rst molar and all premolars in the mandible region were extracted and were then allowed to heal for 1 month. In each side of mandible, 3 bone defects were prepared using a 10-mm-diameter trephine bar. After preparing the defects, the following graft materials were implanted: PM; PM and PRP (PM/PRP); PM and dog MSCs (PM/dMSCs); PM, dMSCs, and PRP (PM/dMSCs/PRP); and control (defect only). The selection of treatments and treatment sites was done at random. The resultant osteogenesis was examined by histologic and histomorphometric analyses.

FIGURE 1. Images of PM and PM/dMSCs. A, Molecular model of the peptide RADA16-I included in PM. A indicates alanine; D, aspartic acid; R, arginine. j and + refer to the positively and negatively charged residues. B, Photograph of PM at normal temperature. C and D, Scanning electron microscope images of PM. Two photographs are shown at different levels of magnication (5000 [C] and 70,000 [D]). At lower magnication (C), the PM surface appears like a sheet. At higher magnication (D), peptides composed of interwoven nanobers that are approximately 10 to 20 nm in diameter are visible. E and F, Dog MSCs are sandwiched in PM sheets at lower magnication (E). Higher magnication reveals dMSCs attached to the PM surface (F). A scale bar on each image is included: 10 Km (C, E) and 100 nm (D, F).

MSCs Isolation and Cultivation

Dog MSCs from iliac aspirates were cultured in conditioned medium consisting of low-glucose Dulbeccos modied * 2009 Mutaz B. Habal, MD

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Eagle medium with growth supplements (50 mL of fetal bovine serum, 10 mL of 200-mmol/L L-glutamine, and 0.5 mL of penicillin-streptomycin mixture containing 25 U of penicillin and 25 Kg of streptomycin [Lonza, Inc, Walkersville, MD]) at 37-C in a humidied atmosphere containing 95% air and 5% carbon dioxide. After a sufcient number of cells were obtained, dMSCs were induced to form osteogenic cells for 7 days. The osteogenic induction medium included conditioned medium and 3 supplements (0.1 KL of dexamethasone, 10-mmol/L sodium Aglycerophosphate, and L-ascorbic acid 2-phosphate [Sigma Chemical Co, St Louis, MO]). The differentiated dMSCs were trypsinized and collected for implantation.

Platelet-Rich Plasma Preparation and Injection of PM With dMSCs and/or PRP

Approximately 50 mL of whole blood was withdrawn from each dog into a centrifuge tube containing 10 mL of culture medium with 250 U/mL of preservative-free heparin. The blood was rst centrifuged (Himac CT; Hitachi Koki, Hitachi, Japan) for 10 minutes at 1100 rpm. Subsequently, the yellow plasma containing the buffy coat with platelets and leukocytes was taken up into a neutral monovette using a long cannula. A second centrifugation at 2500 rpm for 10 minutes was performed to combine the platelets into a single pellet, and the plasma supernatant, which was platelet-poor plasma containing relatively few cells, was removed. The resulting pellet of

FIGURE 2. Schema of the experimental protocol. * 2009 Mutaz B. Habal, MD

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FIGURE 3. Macroscopic and radiographic observations of bone regeneration. A, Bone defects were prepared with a trephine bar with a diameter of 10 mm. Control were defects only. BYE, The defects were lled by PM (B), PM/PRP (C), PM/dMSCs (D), and PM/dMSCs/PRP (E). F, At 8 weeks, bone regeneration in the control (defect only) was not complete. GYJ, After 8 weeks, new bone regeneration was observed with PM (G), PM/PRP (H), PM/dMSCs (I), and PM/dMSCs/PRP (J). Newly formed bone in the PM/dMSCs/PRP group lled the bone defects almost completely (J). KYP, Radiographs of a bone defect before implantation (K) and with each material at 8 weeks, namely, control (L), PM (M), PM/PRP (N), PM/dMSCs (O), and PM/dMSCs/PRP (P). platelets, termed the buffy coat/plasma fraction, was resuspended in 5 mL of residual plasma and used for the platelet gel. Platelet-rich plasma (PRP) was stored at room temperature in a conventional shaker until use. Five thousand units of bovine thrombin in powdered form were dissolved in 5 mL of 10% calcium chloride in a separate sterile cup. Next, 4 samples were prepared and mixed in syringes, PM/dMSCs/PRP (372 KL of PM, 186 KL of 10% sucrose, 62 KL of phosphate-buffered saline [PBS], 1.0 107 dMSCs, 372 KL of PRP, 62 KL of thrombin/calcium chloride, and air), PM/dMSCs (500 KL of PM, 250 KL of 10% sucrose, 250 KL of PBS, and 1.0 107 dMSCs), PM/PRP (372 KL of PM, 186 KL of 10% sucrose, 62 KL of PBS, 62 KL of thrombin/calcium chloride, 372 KL of PRP, and air), and PM (500 KL of PM, 250 KL of 10% sucrose, and 250 KL of PBS). In this study, the gel was injected into the bone defect eld using a needle attached to a 2.5-mL syringe. Samples were analyzed at 2, 4, and 8 weeks after injection. cally for 30 minutes and mixed with same volume of 10% sucrose. Dog MSCs were subcultured in PM for 5 days as a sample of PM/ dMSCs and were removed from the medium before observation. The samples were xed in 2% glutaraldehyde/PBS for 30 minutes and rinsed with PBS and exposed to 1% osmium tetroxide solution for 30 minutes. Next, the samples were treated with tannic acid for 30 minutes and exposed to 1% osmium tetroxide solution for 30 minutes again. All samples were dehydrated using a graded series of ethanol solutions (70%, 80%, 90%, 95%, and 100%), treated with butyl alcohol, and then freeze-dried with liquid nitrogen. Each sample was mounted, splutter-coated with osmium, and examined using a scanning electron microscope (SEM; Hitachi S800; Hitachi High-Technologies Co, Tokyo, Japan).

Histologic and Histomorphometric Analyses

Each implantation site was collected as a cylindrical sample with a 2-mm-diameter trephine bar above the native bone at 2, 4, and 8 weeks after implantation, and the section was assessed histologically. Specimens were xed in 10% buffered formalin, decalcied (K-CX; Falma Co, Tokyo, Japan), and stained with hematoxylin and * 2009 Mutaz B. Habal, MD

Scanning Electron Microscopy In Vitro

For scanning electron microscopy, 2 samples, PM and PM/ dMSCs, were prepared in vitro. PuraMatrix was treated ultrasoni-

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eosin. Each image of the specimens was duplicated and digitized with 8-bit density values. The images were transferred to a microcomputer for analysis (Scion Image, version 4.0.3; Scion Corporation, Frederick, MD). The volume of newly formed bone in the augmented area without native bone was quantied using this computer-based image analysis system, and then the area was calculated as the percentage of regenerated bone. Statistical differences among the volumes in different implants were evaluated by the Tukey-Kramer test after 1-way analysis of variance (ANOVA). A value of P G 0.05 was considered to be statistically signicant.

RESULTS Nanobers Hydrogel Peptide (PM) and dMSCs

The self-assembling peptide RADA16-I (Fig. 1A) was gellike in appearance (Fig. 1B). The SEM images of the peptide hydrogel at low magnication showed a smooth surface (Fig. 1C). At high magnication, SEM revealed a brous structure consisting of interwoven laments with diameters of 10 to 20 nm and porous enclosures of approximately 50 to 200 nm in diameter (Fig. 1D). After 5 days, dMSCs cultured in PM were encapsulated and attached to the surface of PM (Fig. 1E and F).

In Vivo Macroscopic Findings, Radiographic Assessment of PM, PM/PRP, PM/dMSCs, and PM/dMSCs/PRP Implants Compared With the Control
Dog MSCs were used for the implants at 1.0 107 cells. The mean platelet count of PRP was 890,000 with a range of 830,000 to 964,000. These values showed that the concentration was 289% greater than the baseline platelet count. The experimental protocol and design are shown in Figures 2 and 3. PuraMatrix, PM/PRP, PM/ dMSCs, and PM/dMSCs/PRP were implanted into the dogs mandible defects. Macroscopic ndings showed that these scaffolds had almost completely disappeared without infection after implantation and that bone regeneration with PM/dMSCs/PRP almost reached the natural marginal bone level. In addition, the radiopaque area of bone regeneration was supported by radiographs at 8 weeks compared with the control (Fig. 3).

Histologic Assessment of PM, PM/PRP, PM/dMSCs, and PM/dMSCs/PRP Implants Compared With the Control
Implanted and nonimplanted control regions were collected after 2, 4, and 8 weeks for histologic examination. The cortical continuity was never restored, and the cavities were invaded by vascular brous tissue in defects lled with control, PM, and PM/ PRP (Figs. 4 and 5). On the other hand, cavities lled with PM/ dMSCs and PM/dMSCs/PRP resulted in new bone formation even after 2 weeks, which was manifested in a tubular pattern and by abundant vascularization after 4 weeks. This pattern was indicative of normal bone macrostructure with well-differentiated marrow cavity and cortices compared with cavities lled with control, PM, and PM/PRP (Figs. 4 and 5). After 8 weeks, lamellar bone was observed in the PM/dMSCs/PRP group. On the other hand, lamellar bone was still not observed in the PM/dMSCs group after 8 weeks. The control, PM, and PM/PRP groups did not exhibit appreciable bone formation.

Histomorphometric Analysis
The bone-regenerating ability of all implants was assessed by measuring the cortical and medullary bone surface areas without * 2009 Mutaz B. Habal, MD

FIGURE 4. Histologic evaluation of control, PM, PM/PRP, PM/dMSCs, and PM/dMSCs/PRP implantations at each time point (lower magnication). Sections of representative implants are shown from the respective group. The sections were stained with hematoxylin and eosin. Original magnication, 25 for all photographs: at 2 (A), 4 (B), and 8 (C) weeks for the control group; at 2 (D), 4 (E), and 8 (F) weeks for the PM group; at 2 (G), 4 (H), and 8 (I) weeks for the PM/PRP group; at 2 (J), 4 (K), and 8 (L) weeks for the PM/dMSCs group; and at 2 (M), 4 (N), and 8 (O) weeks for the PM/dMSCs/PRP group.

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TABLE 1. Bone Volume in Each Grafted Material

Histomorphometric analysis of control, PM, PM/PRP PM/dMSCs, and PM/ , dMSCs/PRP groups at 2, 4, and 8 weeks. The values are the mean T SE. The PM/dMSCs and PM/dMSCs/PRP groups showed a signicant increase compared with control, PM, and PM/PRP groups (*P G 0.05 by paired Tukey-Kramer test after 1-way ANOVA).

native bone by image analysis (Table 1). Adding PM or PM/PRP to the cavity did not signicantly increase the cortical or medullary bone surface area compared with the control at 2, 4, and 8 weeks. In contrast, the PM/dMSCs and PM/dMSCs/PRP groups showed a signicant increase in the surface area at all 3 time points compared with the control, PM, or PM/PRP groups (P G 0.05 at 2, 4, and 8 weeks, ANOVA). In addition, there was no signicant difference in newly formed bone between PM/dMSCs and PM/dMSCs/PRP over time.

DISCUSSION
Tissue-engineered bone regeneration has been developed as a promising treatment concept, and a variety of bone-substitute materials have been examined. Materials such as A-tricalcium phosphate, hydroxyapatite, and polymers have been used as scaffolds for bone regeneration. However, these materials lack the plasticity to t into the varied shapes of bone defects and have a drawback that infection can be initiated by the retardation of absorption. In many native tissues, it is well known that ECM plays a crucial role in controlling cell behavior and is composed of a basement membrane and an interstitial network of chemically and physically cross-linked proteins and glycosaminoglycans, and all of these features are in the nanometer size range. This microstructure supports cell adhesion, migration, proliferation, differentiation, and the three-dimensional organization of the tissue.9 It is thought that scaffolds with natural ECM-like structures have similar biologic effects on tissue repair at graft sites. Therefore, the novel hydrated material, PM, which is composed of amino acids and has an ECMlike structure, could be applied in tissue engineering. It is thought that ideal scaffolds should have good plasticity, high absorption ratios, and three-dimensional structures similar to natural ECM. PuraMatrix exists in a gel state, because of its water content of more than 99.5%, and has good uidity, stickiness, and plasticity * 2009 Mutaz B. Habal, MD

FIGURE 5. Histologic evaluation of control, PM, PM/PRP, PM/dMSCs, and PM/dMSCs/PRP groups at each time point (higher magnication). Sections of representative implants are shown from each group. The sections were stained with hematoxylin and eosin. Original magnication, 100 for all photographs: at 2 (A), 4 (B), and 8 (C) weeks for the control group; at 2 (D), 4 (E), and 8 (F) weeks for the PM group; at 2 (G), 4 (H), and 8 (I) weeks for the PM/PRP group; at 2 (J), 4 (K), and 8 (L) weeks for the PM/dMSCs group; and at 2 (M), 4 (N), and 8 (O) weeks for the PM/dMSCs/PRP group.

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characteristics (Fig. 1B), and it was easy to t, keep its shape in the bone defects, and form its shape concomitantly with implantation (Fig. 3BYE). In addition, in the specimens of PM only, there were no residues after 2 weeks. This result shows that PM has a high absorption ratio. The high absorption ratio is required for graft materials in tissue regeneration, and this is an advantageous characteristic of PM. In addition to amino acids and water, RADA16-I is present and combined with the amino acids in PM (Fig. 1A). RADA16-I is a self-assembling ionic self-complementary peptide and constructs the nanometer-scale brous structure by either exposing to physiological concentrations of salt solutions or changing to neutral pH. This process occurs as a result of weak noncovalent bonds, hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, and watermediated hydrogen bonds.10,22 Moreover, PM forms a complex three-dimensional microenvironment with nanobers of 7- to 10-nmand 50- to 200-nm-sized nanopores, closely mimicking the morphologic characteristics of natural ECM (Fig. 1D). In SEM images, it was clear that the nanobers constructed a porous structure, and dMSCs were encapsulated and attached to the PM structure (Fig. 1E and F). These results suggest that the complex formed from PM peptides is benecial for the attachment and support of cells. This study demonstrated that the PM structure had advantages to control cell behavior in a similar manner to native ECM. On the other hand, the MSCs are multipotent, show active proliferation, and differentiate to osteoblasts in osteogenic induction medium.19 In previous studies, MSCs cultured with osteogenic induction medium for 7 days showed a higher expression of alkaline phosphatase than those cultured normal medium in vitro.23,24 In addition, human and rabbit predifferentiated MSCs in vitro showed more extensive in vivo bone formation on transplantation than nondifferentiated MSCs.25,26 And previous studies have not demonstrated sufcient efcacy of marrow cells when freshly aspirated cells were implanted.25 These investigators hypothesized that the number of implanted cells might be too low to achieve bone regeneration and that it might be necessary to differentiate the MSCs into osteoblasts using osteoinductive factors before implantation.27 According to their reports, we used MSCs cultured in an osteogenic induction medium before implanting because it is the in vivo bone regeneration efcacy of MSCs that is to be greatly enhanced by the osteogenic differentiation before transplantation using PM.23,24,28 Moreover, the number of cells needed was achieved by expanding it ex vivo. Indeed, we have clinically achieved good results.29,30 In histologic analysis and macroscopic ndings, PM/dMSCs and PM/dMSCs/PRP showed better bone regeneration than the control (defect only), PM, and PM/PRP groups did (Figs. 3Y5). Histometrically, at 8 weeks, the newly formed bone areas were 12.39 T 1.29% (control), 25.28 T 3.92% (PM), 27.72 T 3.15% (PM/PRP), 50.07 T 3.97% (PM/dMSCs), and 58.43 T 5.06% (PM/dMSCs/PRP). Furthermore, the PM/dMSCs and PM/dMSCs/PRP groups showed better bone volume than the control, PM, and PM/PRP groups in the analysis at each time point (Table 1). These results suggested that the activity of dMSCs was maintained by the nanobrous threedimensional structure of PM peptides. In addition, MSCs would be able to survive and retain the potential to differentiate into various mesenchymal phenotypes in vivo and, in particular, would be able to participate in the process of osteogenesis in PM.25,26 It also might be that stimulation of reactive host bone formation by the implanted MSCs occurs and that stimulatory signals and the PRP are transiently presented to this site by the MSCs. On the other hand, PRP alone did not promote bone regeneration in PM. The macroscopic ndings showed that PM/ dMSCs/PRP generated larger amounts of mature bone than PM/ dMSCs, and the regenerated bone approached the level of the native marginal bone (Fig. 3I and J). Newly formed bone was observed in * 2009 Mutaz B. Habal, MD

the histologic analysis after 2 weeks, and mature lamellar bone areas were observed at 8 weeks in the PM/dMSCs/PRP group (Fig. 5O). But only premature bone areas were observed with PM/dMSCs at 8 weeks (Fig. 5L). From these results, the new bone made in the PM/ dMSCs/PRP group might have matured more rapidly than that in the PM/dMSCs group. In the previous study, it was conrmed that bone regeneration with MSCs was facilitated by PRP.29,31,32 This result also might implicate that the acceleration of bone formation may be due to PRP, which contains cytokines and growth factors, and PM may be clinically helpful for regeneration for bone defects at earlier stages. It is concluded that tissue-engineered bone from PM with MSCs and PRP might be useful in a clinical setting. In summary, these results indicate that PM with dMSCs and PRP have excellent osteogenic characteristics and support the potential of PM in the repair of bone defects. In the future, this tissue-engineered bone grafting material can be used as a minimally invasive method instead of traditional grafting procedures.

ACKNOWLEDGMENTS
This authors thank Drs Shuguang Zhang of the Center for Biomedical Engineering at Massachusetts Institute of Technology and Hideharu Hibi, Wataru Katagiri, and members of the Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, for their generous assistance and contributions to this study. This authors also thank ArBlast Co, Ltd, Kobe, Japan, and 3D-Matrix Co, Ltd, Tokyo, Japan, for their help.

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