590368

research-article2015

JDRXXX10.1177/0022034515590368Journal of Dental ResearchBone Regeneration of Blood-derived Stem Cells within Dental Implants

Research Reports: Biomaterials & Bioengineering

Bone Regeneration of Blood-derived Stem Cells within Dental Implants

Journal of Dental Research 1­–8 © International & American Associations for Dental Research 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0022034515590368 jdr.sagepub.com

R.C. Zheng1, Y.K. Park2, S.K. Kim1, J. Cho3, S.J. Heo1, J.Y. Koak1, S.J. Lee4, J.M. Park5, J.H. Lee6, and J.H. Kim1

Abstract Peripheral blood (PB) is known as a source of mesenchymal stem cells (MSCs), as is bone marrow (BM), and is acquired easily. However, it is difficult to have enough MSCs, and their osteogenic capacity with dental implantations is scarce. Therefore, we characterized peripheral blood mesenchymal stem cells (PBMSCs) cultured on a bone marrow–derived mesenchymal stem cell (BMMSC) natural extracellular matrix (ECM) and demonstrated the osteogenic capability in an experimental chamber implant surgery model in rabbits. We isolated PBMSCs from rabbits by culturing on a natural ECM-coated plate during primary culture. We characterized the PBMSCs using a fluorescence-activated cell scanner, cell proliferation assay, and multiple differentiation assay and compared them with BMMSCs. We also analyzed the osteogenic potential of PBMSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) by transplanting them into immunocompromised mice. Then, the mixture was applied to the canals. After 3 and 6 wk, we analyzed new bone (NB) formation inside the chambers using histological and histomorphometric analyses. The PBMSCs had a similar rate of BrdU-positive cells to BMMSCs, positively expressing CD90 but negative for CD14. The PBMSCs also showed osteogenic, adipogenic, and chondrogenic ability in vitro and osteogenic ability in vivo. Histological and histomorphometric results illustrated that the PBMSC and BMMSC groups showed higher NB than the HA/TCP and defect groups in the upper and lower chambers at 6 wk and in the upper canal at 3 wk; however, there was no difference in NB among all groups in the lower canal at 3 wk. The PBMSCs have characteristics and bone regeneration ability similar to BMMSCs both in vitro and in vivo. ECM was effective for obtaining PBMSCs. Therefore, PBMSCs are a promising source for bone regeneration for clinical use. Keywords: peripheral blood, mesenchymal stem cell, dental implantation, extracellular matrix, bone marrow stromal cells, bone remodeling

Introduction

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Mesenchymal stem cells (MSCs) are known as the stem cell source for stem cell–based bone regeneration (Jiang et al. 2002) and are generally isolated from not only bone marrow (BM) but also fat, cord blood, and even peripheral blood (PB). Bone marrow–derived mesenchymal stem cells (BMMSCs) have been considered a source for regenerating bone defects (Kuznetsov et al. 2001), but the method of BM aspiration from patients was aggressed to the donor sites (Bain 2005). PB has been noticed as a source of MSCs, which can be isolated with minimal invasiveness compared with BM (Koerner et al. 2006). PB includes progenitor cells that may differentiate into numerous types of cells depending on the specific microenvironment, for example, hematopoietic stem cells (HSCs) (Damon and Damon 2009), endothelial progenitor cells (EPCs) (Yoder Mervin 2012), MSCs (Zvaifler et al. 2000), osteoclast precursor cells (Costa-Rodrigues et al. 2010), hematopoietic osteoclast precursor cells (Muto et al. 2011), and circulating fibrocytes (Galligan and Fish 2013). The osteogenic differentiation capacity of the cells also has been reported (He et al. 2007). Under specific pathological conditions when MSCs can be involved in the tissue repair process, the numbers

Department of Prosthodontics & Dental Research Institute, Seoul National University Dental Hospital, School of Dentistry, Seoul National University, Seoul, South Korea 2 Department of Dental Research Institute, Brain Korea 21, Seoul National University, Seoul, South Korea 3 Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul, South Korea 4 Department of Orthodontics & Dental Research Institute, School of Dentistry, Seoul National University, Seoul, South Korea 5 Department of Prosthodontics, Seoul National University Gwanak Dental Hospital, Seoul, South Korea 6 Department of Prosthodontics, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, South Korea A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. Corresponding Authors: S.K. Kim, Department of Prosthodontics & Dental Research Institute, Seoul National University Dental Hospital, School of Dentistry, Seoul National University, 28 Yeongun-dong, Chongno-Gu, Seoul, 110-749, South Korea. Email: [email protected] J. Cho, Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul National University, 28 Yeongun-dong, Chongno-Gu, Seoul, 110-749, South Korea. Email: [email protected]

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of PBMSCs are increased (Roufosse et al. 2004; Rochefort et al. 2006; Bian et al. 2009) but are scarce under normal conditions. Since the lack of the isolated cell numbers from PB, it remains a problem for research and practical applications (Zvaifler et al. 2000). Recently, we successfully isolated and maintained MSCs from BM-suspended cells and showed their osteogenic capacity in vitro and in vivo (double-canaled implant). In this investigation, the natural extracellular matrix (ECM) from BMMSCs promoted the proliferation, self-renewal, and multipotential differentiation of the suspended BMMSCs (Zheng et al. 2014). The double-canaled implant used in this study is a well-­ established model to evaluate bone regeneration in different areas of the bone and presents an easy to locate defect in bone replacement, which helps to maintain mechanical stability and volume during the healing period (Lee et al. 2011). Previous studies reported that the double-canaled implant could be filled and serve as bone grafting material in addition to applying MSCs for bone regeneration evaluation (Lee et al. 2011; Zheng et al. 2014). Thus, we hypothesized that the BMMSC-derived ECM would be an important component that can improve the osteogenic capacity of PBMSCs like the suspended MSCs. Therefore, we acquired MSCs from the PB of rabbits by culturing BMMSC-derived ECM. Then, we identified the characteristics of PBMSCs in vitro and in vivo. Furthermore, we investigated the osteogenic capacity of PBMSCs on BMMSCderived ECM in the novel double-canal implant in rabbits. This study introduces that PBMSCs can be a useful supplement for helping bone regeneration after implant surgery.

Materials and Methods Acquisition and Characterization of PBMSCs from Rabbits New Zealand white rabbits and immunocompromised mice (NOD/SCID) were used and treated under the Guidelines and Regulations of Dentistry of Seoul National University (SNU: 130312-1). PBMSCs isolated from PB were obtained on ECMcoated culture plates, which were conducted according to a previous study (Zheng et al. 2014). BMMSCs isolated from BM were used as a control (Fig. 1A). The PBMSCs were characterized and compared with BMMSCs by flow cytometry analysis, a proliferation assay, and an in vitro differentiation assay and were evaluated for in vivo osteogenic potential by subcutaneous transplantation into immunocompromised mice. The detailed methods are described in the Appendix.

Bone Regeneration Capacity of PBMSCs in Double-canaled Implants Eighty double-canaled implants were prepared as described in our previous study (Zheng et al. 2014). The canals were not filled (defect group), filled with hydroxyapatite/tricalcium phosphate (HA/TCP group), or filled with BMMSCs (BMMSCs

group) or PBMSCs (PBMSCs group) mixed with HA/TCP and prepared to examine the ability of PBMSCs to regenerate bone. The implants were installed in rabbit tibias (upper canal to cortical area and lower canal to marrow area). After 3 and 6 wk, 20 rabbits were sacrificed and 80 implant sections were prepared for histological and histomorphometric analyses. The details are described in the Appendix.

Statistical Analysis Student’s t test and a multivariate analysis of variance were used. A P value of less than 0.05 was considered a significant difference. The details are described in the Appendix.

Results Proliferation and Flow Cytometry Analysis of PBMSCs PBMSCs were obtained based on their adherence to ECMcoated culture plates during 14 d of primary seeding. After 3 to 4 d of primary seeding, the attached cells were elongated from a round shape into a spindle shape. Over the next 5 to 14 d, the attached cells grew into a colony and showed a similar homogeneous fibroblast-like shape to the control BMMSCs, which were cultured on a plastic culture plate. The PBMSCs (Fig. 1B-b) showed BrdU incorporation similar to BMMSCs (Fig. 1B-a) following subculturing on a plastic culture plate. There was no statistical difference in BrdU-positive cells between BMMSCs (82.79% ± 0.09%) and PBMSCs (87.10% ± 0.06%) when these cells were subcultured on the plastic culture plate (P > 0.05) (Fig. 1B-c). Next, flow cytometry analysis indicated that BMMSCs did not readily express CD14 (Fig. 1C-a) but were positive for CD90 (Fig. 1C-c). These same findings were true for the PBMSCs (Fig. 1C-b, C-d). The positive cell expression of BMMSCs (CD14: 3.67% ± 0.54%, CD90: 47.09% ± 1.01%) was higher than PBMSCs (CD14: 0.85% ± 0.01%, CD90: 31.79% ± 1.72%) (P < 0.05) (Fig. 1C-e).

Differentiation Potential of the PBMSC In Vitro Assay Regarding osteogenic potential, PBMSC (Fig. 2A-b, d) morphology changed from a spindle shape to a more polygonal appearance after induction in an osteogenesis medium for 1 wk. After 1 to 3 wk, the PBMSCs formed nodules that became larger and more uniformly scattered, like those of the BMMSCs (Fig. 2A-a, c). In the study of osteogenesis, the bone nodule formation of PBMSCs (69.07% ± 7.53%) was similar to that of BMMSCs (72.76% ± 1.56%) (P > 0.05) (Fig. 2E). For adipogenic potential, the PBMSC nuclei became larger after incubation with an adipogenic medium over 7 to 10 d. The intracellular lipid droplets (Fig. 2B-b) appearing in the cytoplasm of PBMSCs grew larger and increased in number over 2 to 3 wk, as did BMMSCs (Fig. 2B-a). In adipogenic experiments, the percentage of Oil Red O–positive cells of PBMSCs (57.65% ±

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Figure 1.  Acquisition and characterization of PBMSCs. (A) Diagrammatic scheme for obtaining PBMSCs and BMMSCs. PB and BM were obtained from the marginal ear vein and bone marrow and isolated by density gradient centrifugation to collect PBMNCs and BMMNCs, respectively. Cell suspensions of the PBMNC pellets were transferred onto an ECM-coated culture plate, and the BMMNC pellets were suspended on a plastic culture plate. After 14 d of culturing, PBMSCs obtained by ECM-coated culture plate adhesion and BMMSCs obtained by plastic culture plate adhesion were used in this study. (B) The proliferation rate of the PBMSCs and the BMMSCs. Incorporated BrdU was stained in the BMMSCs (a) and the PBMSCs (b) at 400×. (c) There was no significant difference in the percentage of BrdU-positive nuclei cells relative to total cells between the BMMSCs and PBMSCs (P > 0.05). (C) Flow cytometry analysis of the cultured BMMSCs and PBMSCs. The PBMSCs (b) and BMMSCs (a) did not express the HSC marker CD14, but the PBMSCs (d) and BMMSCs (c) positively expressed the MSC marker CD90. The black histogram represents isotype-matched antibody control staining. (e) The analysis revealed that the BMMSCs showed higher expression of MSC (CD90) and HSC (CD14) surface markers than PBMSCs, *P < 0.05. (The graph shows mean ± SD.) BM, bone marrow; PB, peripheral blood; PBMNCs, peripheral blood mononuclear cells; BMMNCs, bone marrow mononuclear cells; MNCs, mononuclear cells; ECM, extracellular matrix; PBMSCs, peripheral blood mesenchymal stem cells; BMMSCs, bone marrow mesenchymal stem cells; BrdU, bromodeoxyuridine; CD14, cluster of differentiation 14; CD90, cluster of differentiation 90.

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Figure 2.  Differentiation potential of the PBMSCs in vitro and in vivo. (A) In an osteogenic induction culture, the PBMSCs (b, d) and the BMMSCs (a, c) formed calcium mineralization nodules that were stained by Alizarin Red S and von Kossa. (B) The small lipid droplets formed by the PBMSCs (b) and BMMSCs (a) in an adipogenic induction culture turned red upon Oil Red O staining. (C) After chondrogenic induction culture, the PBMSCs (b, d) and BMMSCs (a, c) were able to form a cartilage matrix, which is indicated by Safranin-O staining and Alcian blue staining. (D) Evidence of the osteogenic potential of the PBMSCs and the BMMSCs in vivo. The PBMSCs and BMMSCs were transplanted into immunocompromised mice using HA/TCP as a carrier. H&E staining indicated newly formed mineralized bone on the surface of the HA/TCP in the PBMSCs (b) and the BMMSCs (a). There was no significant difference in osteogenic (E) or adipogenic potential in vitro (F) or in vivo osteogenic potential (G) between the BMMSCs and the PBMSCs (P > 0.05). (The graph represents mean ± SD.) BMMSCs, bone marrow mesenchymal stem cells; PBMSCs, peripheral blood mesenchymal stem cells; HA/TCP, hydroxyapatite/tricalcium phosphate; H&E, hematoxylin and eosin.

3.07%) was similar to that of BMMSCs (57.93% ± 1.26%) (P > 0.05) (Fig. 2F). In a chondrogenic inductive culture, the PBMSCs (Fig. 2C-b, d) were capable of forming cartilage matrix, as were BMMSCs (Fig. 2C-a, c).

the PBMSCs (40.93% ± 3.87%) and the BMMSCs (43.01 ± 3.97%) was not significantly different (P > 0.05) (Fig. 2G).

Osteogenic Potential Ability of the In Vivo PBMSC Transplantation Assay

At lower magnifications, toluidine blue and von Kossa staining indicated the formation of mineralized tissues in the PBMSC group, and staining was similar to that of the original tibia bone tissues at 3 and 6 wk, as seen in the BMMSC group. In the upper canal at 3 wk, a larger amount of mineralized tissue was observed in the PBMSC (Fig. 3A-d, 3D-d) and BMMSC groups (Fig. 3A-c, D-c) than in the defect (Fig. 3A-a, D-a) and HA/TCP groups (Fig. 3A-b, D-b). Compared with the upper

Hematoxylin and eosin (H&E) staining showed that PBMSCs (Fig. 2D-b) regenerated mineralized bone structures around HA/TCP surfaces. The mineralized bone structures, which were also generated by BMMSCs (Fig. 2D-a), were similar to typical bone structures. The amount of bone formation between

Histological and Histomorphometric Analyses

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Figure 3.  Bone regeneration of PBMSCs in double-canaled implants at 3 wk. (A) Toluidine blue staining for histological view of the double-canaled implant at 12.5×. (a) Defect group: canals were not filled, (b) HA/TCP group: canals were filled with HA/TCP, (c) BMMSC group: canals were filled with BMMSCs mixed with HA/TCP, and (d) PBMSC group: canals were filled with PBMSC mixed with HA/TCP. (B) Toluidine blue staining for histological view of the double-canaled implant at 40×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. (C) Toluidine blue staining for histological view of the double-canaled implant at 200×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. New bone (NB), osteocytes (O), graft material (GM), and marrow space (MS) were observed. (D) von Kossa staining for histological view of the double-canaled implant, 12.5×. (a) Defect group, (b) HA/TCP group, (c) BMMSC group, and (d) PBMSC group. (E) von Kossa staining for histological view of the double-canaled implant, 40×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. (F) von Kossa staining for histological view of the double-canaled implant, 200×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. New bone (NB), osteocytes (O), graft material (GM), and marrow space (MS) were observed. (G) NB formation inside the upper and lower implant canals implanted into rabbit tibia for 3 wk. NB formation capacity of the PBMSC and the BMMSC groups was significantly higher than the HA/TCP and defect groups in the upper canal (*P < 0.0311); however, there was no significant difference among all groups in the lower canal. There was no significant difference of NB formation capacity between the PBMSC and the BMMSC groups in the upper and lower canals. Also, there was no significant difference of NB formation capacity between the defect group and HA/TCP group in the upper canal (P = 0.4111). (The graph bar represents means ± SD.) HA/TCP, hydroxyapatite/tricalcium phosphate; BMMSCs, bone marrow mesenchymal stem cells; PBMSCs, peripheral blood mesenchymal stem cells.

canal, less mineralized tissue was observed in all groups in the lower canal at 3 wk. In addition, at 6 wk, a larger amount of mineralized tissue was detected in the PBMSC (Fig. 4A-d, D-d) and BMMSC groups (Fig. 4A-c, D-c) than in the defect (Fig. 4A-a, D-a) and HA/TCP groups (Fig. 4A-b, D-b), both in the upper and lower canals. Compared with the results at 3 wk, more mineralized tissue was observed in all groups both in the upper and lower canals at 6 wk. The details of these observations at higher magnification are described below.

At 3 wk in the upper canal, the defect group (Fig. 3B-a, 3E-a) showed new bone (NB) mainly growing into the central part from the lateral side of the canal. At higher magnification (Fig. 3C-a, F-a), we further confirmed NB was located in the lateral side of the canal. The HA/TCP group showed mostly HA/TCP remnants, and fibrovascular tissue was observed. At higher magnification (Fig. 3C-b, F-b), a small amount of mineralized bone tissue near the lateral side of the canal and mostly fibrovascular tissue around HA/TCP in the central part of the

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Figure 4.  Bone regeneration of PBMSCs in double-canaled implant at 6 wk. (A) Toluidine blue staining for histological view of the double-canaled implant, 12.5×. (a) Defect group: canals were not filled, (b) HA/TCP group: canals were filled with HA/TCP, (c) BMMSC group: canals were filled with BMMSCs mixed with HA/TCP, and (d) PBMSC group: canals were filled with PBMSC mixed with HA/TCP. (B) Toluidine blue staining for histological view of the double-canaled implant, 40×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. (C) Toluidine blue staining for histological view of the double-canaled implant at 200×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. New bone (NB), osteocytes (O), graft material (GM), and marrow space (MS) were observed. (D) von Kossa staining for histological view of the double-canaled implant at 12.5×. (a) Defect group, (b) HA/TCP group, (c) BMMSC group, and (d) PBMSC group. (E) von Kossa staining for histological view of the double-canaled implant at 40×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. (F) von Kossa staining for histological view of the double-canaled implant at 200×. Columns, left to right: (a, e) defect group, (b, f) HA/TCP group, (c, g) BMMSC group, and (d, h) PBMSC group; rows, top to bottom: upper canal and lower canal. New bone (NB), osteocytes (O), graft material (GM), and marrow space (MS) were observed. (G) NB formation within the upper and lower canals implanted into the rabbit tibia after 6 wk. NB formation in the PBMSC and BMMSC groups was significantly higher than that in the HA/TCP and defect groups in the upper (*P < 0.0001) and lower canals (*P < 0.0255). The NB formation in the upper canal in the HA/TCP group was significantly greater than that in the defect group (*P = 0.0074). However, there was no significant difference in NB formation in the upper and lower canals between the PBMSC and the BMMSC groups. Also, there was no significant difference in NB formation in the lower canal between the defect and HA/TCP groups. (The graph represents mean ± SD.) HA/TCP, hydroxyapatite/tricalcium phosphate; BMMSCs, bone marrow mesenchymal stem cells; PBMSCs, peripheral blood mesenchymal stem cells.

canal were observed. The PBMSC (Fig. 3B-d, E-d) and BMMSC (Fig. 3B-c, E-c) groups showed mature mineralized woven bone detected in the central and lateral side of the canal. At higher magnification, mature mineralized woven bone regenerated by the PBMSC group (Fig. 3C-d, F-d) was mainly attached to HA/TCP, and a small amount of fibrous tissue was also observed, as seen in the BMMSC groups (Fig. 3C-c, F-c). At 3 wk in the lower canal, marrow spaces were primarily observed in the defect group (Fig. 3B-e, E-e), which was

further confirmed at higher magnification (Fig. 3C-e, F-e). In the HA/TCP group (Fig. 3B-f, E-f), mostly remnants of the HA/TCP scaffold and fatty marrow spaces were observed, while we observed fibrous tissue around HA/TCP at higher magnification (Fig. 3C-f, F-f). The PBMSC (Fig. 3B-h, E-h) and BMMSC (Fig. 3B-g, E-g) groups showed fibrovascular tissues with HA/TCP. A few samples of mineralized woven bone with osteogenic marker-positive cells regenerated by the PBMSC group (Fig. 3C-h, F-h) were observed at higher

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Bone Regeneration of Blood-derived Stem Cells within Dental Implants magnification, as was also seen in the BMMSC group (Fig. 3C-g, F-g). At 6 wk in the upper canal, more NB grew into the central part from the lateral side of the canal in the defect group compared with that at 3 wk (Fig. 4B-a, E-a). We also confirmed at higher magnification that a larger amount of NB was located in the central part of the canal (Fig. 4C-a, Fig. 4F-a) compared with that seen at 3 wk. In the HA/TCP group (Fig. 4B-b, E-b), mature mineralized woven bone with osteogenic marker-positive cells appeared around HA/TCP. Fewer gaps were present at the boneparticle interface at higher magnification (Fig. 4C-b, F-b) compared with those seen at 3 wk. In the PBMSC (Fig. 4B-d, E-d) and BMMSC (Fig. 4B-c, E-c) groups, more mature NB was detected in the central and lateral side of the canals at 6 wk than at 3 wk. Compared with the results at 3 wk, higher magnification analysis of the BMMSC (Fig. 4C-c, F-c) and PBMSC groups (Fig. 4C-d, F-d) showed mature NB with numerous rounded osteocytes and few fibrovascular tissues around HA/TCP. In addition, no gaps were present at the bone-particle interface. At 6 wk in the lower canal, mostly fatty marrow spaces were observed in the defect group (Fig. 4B-e, E-e), similar to the results at 3 wk. The same observation was detected at higher magnification (Fig. 4C-e, F-e). In the HA/TCP group (Fig. 4B-f, E-f), more newly formed woven bone with limited invasion of fibrovascular tissue was observed at 6 wk than at 3 wk. We also confirmed these observations at higher magnification (Fig. 4C-f, F-f). In the PBMSC (Fig. 4B-h, E-h) and BMMSC (Fig. 4B-g, E-g) groups, a small amount of mature bone was detected around HA/TCP; moreover, more NB with osteogenic marker-positive cells was observed at high magnification in the PBMSC (Fig. 4C-h, F-h) and BMMSC (Fig. 4C-g, F-g) groups compared with that seen at 3 wk. The histomorphometric analysis results showed no significant difference in bone regeneration capacity between the PBMSC and BMMSC groups in the upper and lower canals. Within the upper canals at 3 wk, the PBMSC (15.81% ±1.86%) and BMMSC groups (16.92% ± 3.58%) showed more NB than the HA/TCP (12.22% ± 3.40%) and defect groups (10.28% ± 1.71%) (P < 0.0311). However, there was no difference in NB among the defect (3.65% ± 1.34%), HA/TCP (4.82% ± 1.15%), BMMSC (4.92% ± 1.15%), and PBMSC groups (4.51% ± 0.89%) in the lower canal at 3 wk (Fig. 3G). Within the upper and lower canals at 6 wk, the PBMSC (upper canal: 36.30% ± 3.71%, lower canal: 7.56% ± 1.20%) and BMMSC groups (upper canal: 35.45% ± 5.74%, lower canal: 7.26% ± 1.10%) showed significantly more NB than did the HA/TCP (upper canal: 21.48% ± 4.87%, lower canal: 5.86% ± 1.03%) and defect (upper canal: 14.69% ± 2.61%, lower canal: 4.94% ± 0.77%) groups (upper, P < 0.0001; lower, P < 0.0255). However, there was no significant difference in NB between the PBMSC group and BMMSC group (Fig. 4G). Within the upper canal at 6 wk, the HA/TCP group showed more NB than the defect group; there was no significant difference in NB between the HA/TCP and defect groups within the upper canal at 3 wk or within the lower canal at 3 or 6 wk. Detailed percentages of NB, graft material (GM), and marrow space (MS) at 3 and 6 wk are described in Appendix Tables 1 and 2.

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Discussion In contrast to BM, the collection of PB as an MSC source is minimally invasive, is relatively simple, and does not lead to further complications. However, the isolation and expansion of PBMSCs are relatively not easy because of their limited cell number. The common method of isolating PBMSCs is by selecting MSCs via a simple medium after the first cell seeding on a plastic plate (Kim et al. 2006). In addition, magnetic bead–activated cell sorting has been used for selecting MSCs from PBMNCs (Lisa et al. 2000). However, it is known that the proliferative and differential capacity of PB-derived MSCs is limited. In this study, we obtained rabbit PBMSCs by culturing on BMMSC-derived ECM-coated culture plates. Our previous study reported that the ECM-coated culture plates were effective for obtaining suspended BMMSCs (Zheng et al. 2014). Similar results also showed that ECM facilitated the expansion of BMMSCs (Chen et al. 2007). Other researchers have suggested that ECM components might modulate the behavior and properties of MSCs (Lai et al. 2010; Prewitz et al. 2013). Therefore, further studies should clearly identify which components of the ECM control PBMSC binding efficiency. The cells showed similar capacities and similar proliferation and differentiation characteristics compared with BMMSCs. PBMSCs express CD90 but not CD14, like BMMSCs (Fig. 1). Although the expression level of CD90 is relatively lower in PBMMCs than in BMMSCs, the result indicates PBMSCs have the mesenchymal stem cell character. Thus, we suggest that BM may contain more primitive MSCs than PB, and the mixed degree of distinct cells may affect the expression of surface molecules. Similar observations related to the expression of PBMSCs were previously reported (Kassis et al. 2006). These results imply that PBMSCs differ from HSCs but have similar characteristics to BMMSCs. PBMSCs showed multipotent capacity as mesenchymal stem cells. We showed that the cells have osteogenic, adiposgenic, and chondrogenic differentiation potential as BMMSCs. Therefore, PBMSCs have stem cell potential similar to BMMSCs. We used the double-canaled dental implant for the application of MSCs and quantification of bone regeneration in cortical and marrow areas. Such implants have been shown to be the easiest way to detect bone regeneration according to the bone area (Lee et al. 2011). The result that bone regeneration was increased from 3 to 6 wk of observation is similar to that of the previous study (Cho et al. 2010). Histological results showed that NB was higher in cortical than in marrow areas at 3 and 6 wk. These observations were explained by a previous study, which showed that bone growth was more pronounced in cortical than in marrow areas in the rabbit tibia (Lee et al. 2009; Lu et al. 1998). In the same double-canaled implant model, only 17% and 5% NB in the upper and lower canals was reported in the HA/TCP group, respectively, while the BMMSC groups reported increases of 28% and 10% NB (Zheng et al. 2014). In the present study, NB area in the HA/TCP group was increased by 12% and 4.8% in the upper and lower canals, respectively, at 3 wk. In contrast, NB area in the BMMSC and PBMSC groups

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8

Journal of Dental Research 

was 17% and 15% in the upper canal and 4.9% and 4.5% in the lower canal at 3 wk, respectively. In contrast, it was 35% and 36% in the upper canal and 7.2% and 7.5% in the lower canal at 6 wk, respectively. It is possible that these results are due to BMMSCs and PBMSCs existing in the initial stage of osteogenic differentiation in this study at 3 wk. A previous study reported that woven bone was generally characterized by the orientation of fibrous collagen with osteocytes (Hardwick and Dahlin 1994). A similar observation noted an early oriented remodeling phase of woven bone in the osseointegration process after 2 wk (Terheyden et al. 2012); these observations were supported by histological results. In the present study, there may have been some limitations influencing bone regeneration. For example, the duration of survival of transplanted MSCs and the definitive role of PBMSCs in bony regeneration may have negatively affected regeneration. The ratio between cortical and marrow areas of the tibia, mechanical stability of the canal, and biological environment also affect bone regeneration. Within these limitations, our results indicate that PBMSCs remained at the graft and have a positive effect on bone regeneration. In conclusion, the present study suggests that PBMSCs, which are easily obtained, could be a promising source for bone regeneration in dental implant surgery.

Author Contributions S.K. Kim, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; R.C. Zheng, contributed to design, data acquisition, and analysis, drafted and critically revised the manuscript; Y.K. Park, contributed to conception and data analysis, drafted the manuscript; J. Cho, contributed to design, data analysis, and interpretation, drafted and critically revised the manuscript; S.J. Heo, contributed to data acquisition and analysis, drafted the manuscript; J.Y. Koak, contributed to conception, drafted the manuscript; J.H. Lee, contributed to data analysis and interpretation, critically revised the manuscript; S.J. Lee, contributed to design and data analysis, drafted and critically revised the manuscript; J.M. Park, J.H. Kim, contributed to data interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (no. 2011-0028067); by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120304); and by a grant 03-2013-0031 from the SNUDH Research Fund. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

References Bain B. 2005. Bone marrow biopsy morbidity: review of 2003. J Clin Pathol. 58(4):406–408. Bian ZY, Li G, Gan YK, Hao YQ, Xu WT, Tang T. 2009. Increased number of mesenchymal stem cell-like cells in peripheral blood of patients with bone sarcomas. Arch Med Res. 40(3):163–168.

Chen XD, Dusevich V, Feng JQ, Manolagas SC, Jilka RL. 2007. Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J Bone Miner Res. 22(12):1943–1956. Cho YJ, Heo SJ, Koak JY, Kim SK, Lee SJ, Lee JH. 2010. Promotion of osseointegration of anodized titanium implants with a 1α, 25-dihydroxyvitamin D3 submicron particle coating. Int J Oral Maxillofac Implants. 26(6):1225–1232. Costa-Rodrigues J, Teixeira CA, Sampaio P, Fernandes MH. 2010. Characterization of the osteoclastogenic potential of human osteoblastic and fibroblastic conditioned media. J Cell Biochem. 109(1):205–216. Damon LE, Damon LE. 2009. Mobilization of hematopoietic stem cells into the peripheral blood. Expert Rev Hematol. 2(6):717–733. Galligan CL, Fish EN. 2013. The role of circulating fibrocytes in inflammation and autoimmunity. J Leukoc Biol. 93(1):45–50. Hardwick R, Dahlin C. 1994. Healing pattern of bone regeneration in membrane-protected defects: a histologic study in the canine mandible. Int J Oral Maxillofac Implants. 9(1):13–29. He Q, Wan C, Li G. 2007. Concise review: multipotent mesenchymal stromal cells in blood. Stem Cells. 25(1):69–77. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, OrtizGonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, et al. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418(6893):41–49. Kassis I, Zangi L, Rivkin R, Levdansky L, Samuel S, Marx G, Gorodetsky R. 2006. Isolation of mesenchymal stem cells from G-CSF–mobilized human peripheral blood using fibrin microbeads. Bone Marrow Transplant. 37(10):967–976. Kim S, Honmou O, Kato K, Nonaka T, Houkin K, Hamada H, Kocsis JD. 2006. Neural differentiation potential of peripheral blood- and bone-marrow– derived precursor cells. Brain Res. 1123(1):27–33. Koerner J, Nesic D, Romero JD, Brehm W, Mainil-Varlet P, Grogan SP. 2006. Equine peripheral blood–derived progenitors in comparison to bone marrow–derived mesenchymal stem cells. Stem cells. 24(6):1613–1619. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. 2001. Circulating skeletal stem cells. J Cell Biol. 153(5):1133–1140. Lai Y, Sun Y, Skinner CM, Son EL, Lu Z, Tuan RS, Jilka RL, Ling J, Chen XD. 2010. Reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem Cells Dev. 19(7):1095–1107. Lee JE, Heo SJ, Koak JY, Kim SK, Han CH. 2011. Bone regeneration with rabbit bone marrow–derived mesenchymal stem cells and bone graft materials. Int J Oral Maxillofac Implants. 27(6):1389–1399. Lee JE, Heo SJ, Koak JY, Kim SK, Han CH, Lee SJ. 2009. Healing response of cortical and cancellous bone around titanium implants. Int J Oral Maxillofac Implants. 24(4):655–662. Lisa G, Barbara M, Dongmei W, Francis K, Fraser F, Mickie B. 2000. Identification of novel circulating human embryonic blood stem cells. Blood. 96(5):1740–1747. Lu J, Gallur A, Flautre B, Anselme K, Descamps M, Thierry B, Hardouin P. 1998. Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical, and medullar bone sites in rabbits. J Biomed Mater Res. 42(3):357–367. Muto A, Mizoguchi T, Udagawa N, Ito S, Kawahara I, Abiko Y, Arai A, Harada S, Kobayashi Y, Nakamichi Y, et al. 2011. Lineage-committed osteoclast precursors circulate in blood and settle down into bone. J Bone Miner Res. 26(12):2978–2990. Prewitz MC, Seib FP, von Bonin M, Friedrichs J, Stißel A, Niehage C, Müller K, Anastassiadis K, Waskow C, Hoflack B, et al. 2013. Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods. 10(8):788–794. Rochefort GY, Delorme B, Lopez A, Herault O, Bonnet P, Charbord P, Eder V, Domenech J. 2006. Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia. Stem Cells. 24(10):2202–2208. Roufosse CA, Direkze N, Otto W, Wright N. 2004. Circulating mesenchymal stem cells. Int J Biochem Cell Biol. 36(4):585–597. Terheyden H, Lang NP, Bierbaum S, Stadlinger B. 2012. Osseointegration— communication of cells. Clin Oral Implants Res. 23(10):1127–1135. Yoder Mervin C. 2012. Human endothelial progenitor cells. Cold Spring Harb Perspect Med. 2(7):1–14. Zheng RC, Park YK, Cho JJ, Kim SK, Heo SJ, Koak JY, Lee JH. 2014. Bone regeneration at dental implant sites with suspended stem cells. J Dent Res. 93(10):1005–1013. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN. 2000. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res. 2(6):477–488.

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Bone Regeneration of Blood-derived Stem Cells within Dental Implants.

Peripheral blood (PB) is known as a source of mesenchymal stem cells (MSCs), as is bone marrow (BM), and is acquired easily. However, it is difficult ...
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