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Stromal cell-derived factor-1 accelerates bone regeneration through multiple regenerative mechanisms
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 31 (2019) 245–250
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Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Original Research
Stromal cell-derived factor-1 accelerates bone regeneration through multiple regenerative mechanisms
T
Yuji Andoa,b, Jun Ishikawaa,c, Masahito Fujioa, Yoshihiro Matsushitaa,c, Hirotaka Wakayamaa,b, ⁎ Hideharu Hibia, Akihito Yamamotod, a
Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Department of Oral and Maxillofacial Surgery, Chutoen General Medical Center 1-1 Shobugaike, Kakegawa, Shizuoka, 436-8555, Japan c Department of Oral and Maxillofacial Surgery, Kariya Toyota General Hospital, 5-15 Sumiyoshi-cho, Kariya, Aichi, 448-8505, Japan d Department of Tissue regeneration, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-5 Kuramoto-cho, Tokushima 770-8504, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteogenesis Stromal cell-derived factor-1 Endothelial progenitor cells Bone marrow stromal cells Cell recruitment
Objective: Stem cell transplantation has become a popular and promising therapeutic approach for many clinical conditions. Engrafted mesenchymal stem cells (MSCs) can promote bone regeneration in both humans and model animals; however, the differentiation and survival rates of the engrafted MSCs are reported to be poor. We hypothesized that MSCs promote bone regeneration primarily through paracrine mechanisms. SDF-1 is a factor secreted from MSCs that is known to be involved in cell-recruitment and angiogenesis. In this study, we examined the bone-regenerating activity of SDF-1 in the rat calvarial bone defect model. Methods: Collagen sponge containing SDF-1 was transplanted into the rat calvarial bone defect. The effects of treatment were examined both histological and microcomputed tomography analysis. The effects of SDF-1 on cellular migration and proliferationin vitro were assessed by transwell and WST-assay, respectively. Results: SDF-1 significantly enhanced the bone regeneration in rat calvarial bone defect model. SDF-1 promoted the recruitment of endogenous MSCs/osteogenic progenitors and promoted angiogenesisin vivo. Furthermore, SDF-1 directly enhanced the cell-migration activity of human bone marrow MSCs (hBMMSCs), human umbilical vein endothelial cells (HUVECs), and rat calvarial periosteum cells (rCPCs), without affecting their proliferative activities, in vitro. Conclusions: Our findings suggest that the local administration of SDF-1 enhances bone regeneration by inducing multiple endogenous tissue-regenerating activities.
1. Introduction In oral and maxillofacial surgery, autologous bone graft has been applied clinically to regenerate bone defects caused by tumors and trauma; however, damage of the donor site, resorption of the engrafted bone mass, and infection accompanying graft-surgery are major challenges associated with this procedure. Stem cell transplantation has drawn intense attention as an alternative cellular resource for bone regenerating therapy [1]. The engraftment of various types of adult mesenchymal stem cells (MSCs) and their derivatives into damaged areas promotes bone regeneration in both humans and model animals [2,3]. However, the engrafted MSCs exhibit poor differentiation and survival rates, suggesting that they promote bone regeneration primarily by activating endogenous tissue-repairing mechanisms [4,5]. We previously showed that the local administration of MSC-conditioned
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medium promotes tissue regeneration through the recruitment of endogenous osteogenic and vasculogenic progenitors, bone marrow MSCs (BMSCs)/periosteal cells (PEs) and bone marrow endothelial cells (BMECs)/endothelial progenitor cells (EPCs), respectively, to the injury [6]. The identification of the progenitor-recruiting factor derived from MSCs could lead to novel bone regenerating therapies based on the activation of endogenous tissue-repairing activities. We hypothesized that MSCs promote bone regeneration primarily through paracrine mechanisms. SDF-1 is a factor secreted from MSCs that is known to be involved in cell-recruitment and angiogenesis [7,8]. SDF-1, which belongs to the CXC subfamily of chemokines, binds to the G-protein-coupled seven-transmembrane receptors CXCR4 and CXCR7 [9,10]. Notably, SDF-1 is secreted from MSCs [7], and recombinant SDF-1 protein recruits both BMSCs and BM-ECs/EPCs [8]. Thus, SDF-1 is a promising candidate for a progenitor-recruiting factor from MSCs.
Corresponding author. E-mail address: [email protected] (A. Yamamoto).
https://doi.org/10.1016/j.ajoms.2019.02.005 Received 27 November 2017; Received in revised form 25 December 2018; Accepted 20 February 2019 Available online 08 March 2019 2212-5558/ © 2019 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd All rights reserved.
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 31 (2019) 245–250
Y. Ando, et al.
2.4. Cell culture
In this study, we administered SDF-1 locally into a rat calvarial bone defect model, and evaluated its tissue-regenerating activity.
Human bone marrow MSCs (hBMMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Walkersville (Basel, Switzerland) and from Health Science Research Resources Bank Japan, respectively. The hBMMSCs and HUVECs were cultured as recommended by the suppliers. Briefly, the cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS) (Lonza Walkersville) or EGM-2 (Endothelial Growth Medium; Lonza) at 37 °C in a 5% CO2 atmosphere and were subcultured every 6–7 days. Rat calvarial periosteum cells (rCPCs) were isolated as described previously [18]. Briefly, the rat calvarial periosteum was isolated and then digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37 °C. Single-cell suspensions were plated on culture dishes in DMEM supplemented with 10% FBS, then incubated at 37 °C in 5% CO2. rCPCs were plated at 3 × 104 cells/well into 24-well plates. Equal numbers of cells were incubated with osteogenic supplements (10% FBS, 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 100 nM dexamethasone; Sigma) in DMEM, and the medium was exchanged three times each week. After 14 days of induction culture, rCPCs were fixed and stained with Alizarin red-S to detect mineralized nodules formed in vitro, as described previously [19]. Briefly, the cells were fixed in 4% paraformaldehyde and incubated with Alizarin red-S (40 mM, pH 6.4; Sigma) for 10 min at room temperature. After washing the wells with distilled water, the plates were photographed. The Alizarin red dye was then extracted with 10% formic acid.
2. Materials and methods 2.1. Rat calvarial bone defect model All animal experiments were performed in accordance with the Guidelines for Animal Experimentation of the Nagoya University School of Medicine. Eight- to ten-week-old male SD rats (n = 24) were anesthetized by intraperitoneal injection of pentobarbital (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan) (20 mg/kg body weight). After shaving the skin, an incision was made in the skull, and the periosteum was opened to expose the surface of the calvarial bones. Two circular bone defects (full-thickness, 5 mm in diameter) were made in the calvarial bone using a trephine bur and were irrigated with saline to remove bone debris. The following experimental materials were then implanted into the defects. Atelocollagen Honeycomb sponge (Koken, Tokyo, Japan) was suspended in 20 μL of PBS, or of PBS containing 200 ng SDF1 (10 μg/mL in PBS). According to previous literature, we determined the range of the SDF-1 dose [11–13]. In preliminary analysis, as we observed a significant bone healing in 200 ng SDF-1 group, we chose the dose. We defined the following groups based on the implanted materials: (1) PBS: PBS/collagen; (2) SDF-1: SDF-1/collagen. Two circular bone defects received same treatment. The rats were sacrificed 2 or 6 weeks after implantation (n = 6 per group).
2.2. Radiographic analyses 2.5. Migration and proliferation studies
The calvarial bones were harvested at 6 weeks after surgery, fixed in 4% paraformaldehyde solution, and analyzed by microcomputed tomography (micro-CT) using a laboratory X-ray CT device (LATheta; Aloka Co., Tokyo, Japan). Images were compiled and analyzed to render 3D images using OsiriX imaging software (Ver.3.9; www.osirixviewer.com/). The area (mm2) of newly regenerated bone in the PBS and SDF-1 groups was compared [14–16].
The migration ability of hBMMSCs, HUVECs, or rCPCs in response to SDF-1 was assessed using transwell-membrane chambers (pore size 8 μm; BD Falcon; BD, Franklin Lakes, NJ) as previously described [20,21]. Briefly, 1 × 105 cells were seeded into the upper chamber in 700 μl serum-free DMEM, and 1400 μl of serum-free DMEM (control) or DMEM containing 10 ng/ml or 100 ng/ml SDF-1 (R&D Systems, Minneapolis, MN) was added to the lower chamber. To inhibit CXCR4 function, the cells were pre-incubated for 30 min at 37 ℃ with 5 μg/mL AMD3100 (Sigma) [22]. After 8 h of incubation in the transwell chamber, the membranes were fixed in a 4% paraformaldehyde solution and stained with hematoxylin. Non-migratory cells from the upper chamber were removed with cotton swabs. Cells that had migrated to the lower side of the membrane were counted in 10 randomly selected fields (size of analyzed field: 310 × 410 μm). As a positive control for migration, DMEM containing 10% FBS was added to the lower chamber. The cell proliferation rate was assessed using a cell-counting kit (WST-8 s; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions [23]. Cells were plated at a density of 2.0 × 104 cells/mL/ well in 12-well plates (Greiner Bio-One, Frickenhausen, Germany) in each medium. Before addition of SDF-1, we cultured cells in serum free medium for one hour. After a 24-h incubation in DMEM containing SDF-1 with/without 5 μg/mL AMD3100, each absorbance was measured in triplicate.
2.3. Histological analysis The calvarial bones were embedded in SCEM gel (8091140; Leica, Tokyo, Japan) and frozen in cooled isopentane. Non-decalcified calvarial bone sections were generated using Kawamoto's film method (8091098; Leica) [17]. Cryostat sections (5-μm thick) were stained with hematoxylin and eosin or subjected to immunohistological analysis. For immunostaining, the sections were fixed in 99.5% ethanol for 10 min at room temperature, blocked with 5% bovine serum albumin/PBS for 30 min, stained with primary antibodies in blocking buffer for 1 h, stained with secondary antibodies for 30 min, mounted with SCMM R3 (Leica), and examined with a BZ9000 fluorescence microscope (Keyence, Osaka, Japan). The following antibodies were used for immunostaining: mouse monoclonal antibody (mAb) against CD90 (Thy1.1) (MAB1406; Millipore, Darmstadt, Germany), rabbit polyclonal antibody against CD271 (NGF receptor) (AB1554; Millipore), rabbit mAb against alpha smooth muscle actin (αSMA) (04–1094; Millipore), and mouse mAb against Von Willebrand Factor (vWF) (F8/86; Dako, Glostrup, Denmark). Secondary antibodies were conjugated with Alexa Fluor 488 or 555 (Invitrogen, Waltham, MA). No cross reaction of mouse and rabbit second antibody against rat antigen was confirmed by staining with second Abs alone. Cell nuclei were labeled with 4′, 6diamidino-2-phenylindole (DAPI) (Invitrogen, Waltham, MA) and were counted in randomly selected fields (size of analyzed field: 310 × 410 μm). The microvessel content was assessed by measuring the number and density of vWF-positive cells around muscle fibers, using a fluorescence microscope (BZ9000; Keyence).
2.6. Quantification and statistical analysis Cells expressing a particular marker were counted under a fluorescence microscope (BZ9000, Keyence) and analyzed with ImageJ software. All experiments were conducted in triplicate and repeated at least twice. The SPSS statistical package was used for statistical analysis. Comparisons were made using Student’s t-test or Tukey’s post hoc test. Results were expressed as the mean ± standard deviation (SD: p < 0.05 was considered statistically significant). 246
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3. Results 3.1. SDF-1 enhances bone regeneration in a rat calvarial bone defect Five-millimeter bone defects were created in the left and right side of the calvarial bones of 8-10-week-old rats. This critically sized calvarial defect cannot heal spontaneously during the eight weeks after surgery [24]. Micro-CT analysis performed six weeks after the operation showed that little or no bone healing in the PBS-treated group, while the SDF-1-treated group exhibited clear healing (Fig. 1A). Quantitative analysis revealed that the area of newly regenerated bone in the SDF-1 group (57.9 ± 15.4%) was significantly larger than that in the PBS group (36.5 ± 9.6%) (Fig. 1B). Histological analysis showed that the bone defect in the SDF-1 group was covered with regenerated bone. In the PBS group some regenerated bone was observed, but most of the defective area was filled with connective tissue (Fig. 1C). These results demonstrated that the calvarial defect was healed by SDF-1 treatment (Fig. 1C and D). Notably, we found a significant bone tissue in the middle of the defects as well as the peripheral of it. These boneisland may be formed with cells recruited from calvalial periosteum. 3.2. SDF-1 accelerates endogenous rat BMSC recruitment and blood vessel formation CD90+/CD271+ rat bone marrow cells consist of multipotent BMSCs [25–27]. Immunohistological analysis showed that the recruitment of CD90+/CD271+ cells was increased in the SDF-1-treated compared with the PBS-treated group at two weeks post-operation (Fig. 2A). Quantitative analysis showed that the actual number of CD90+/CD271+ cells and the ratio of the CD90+/CD271+ cells to total DAPI + cells in the SDF-1 group was 1.76 times and 1.87 times that in the PBS group, respectively (Fig. 2B–D). We next examined whether SDF-1 promotes the formation of mature vessels composed of two types of cells, vWF+ endothelial cells and αSMA+ pericytes. Endothelial cells (ECs) line the vessel lumen as a continuous layer, while pericytes, which are of mesenchymal origin, constitute the outer layer of micro-vessels. In the SDF-1-treated group, there were many tubular micro-vessel structures, which were composed of an internal endothelial cell layer and an outer pericyte layer (Fig. 3A). Quantitative analysis showed that the number and density of vWF+/αSMA+ vessels were significantly increased in the SDF-1 treated group (Fig. 3B–D). 3.3. SDF-1 promotes the recruitment of osteogenic progenitors derived from calvarial periosteum rCPCs were isolated as described [18]. These cells exhibited a spindle or cobblestone shape (Fig. 4A). In an osteogenic differentiation medium, rCPCs formed many Alizarin Red S-positive condensed nodules (Fig. 4B and C), demonstrating that the rCPCs exhibited strong osteogenic activity. Cell migration analyses revealed that SDF-1 accelerated the migration of hBMMSCs, HUVECs, and rCPCs in a dose-dependent manner, and that this increased migration was abolished by AMD3100, a specific antagonist against CXCR4 (Fig. 5A–C). In contrast, SDF-1 treatment had little or no effect on the cell proliferation of these three types of cells (Fig. 6A–C).
Fig. 1. SDF-1 enhances bone regeneration in a rat calvarial bone defect. (A) X-ray and Micro-CT images at six weeks post-operation showed little or no bone healing in the PBS-treated group, while the SDF-1-treated group exhibited clear healing. (B) Quantitative analysis revealed that the area of newly regenerated bone in the SDF-1 group was significantly larger than that in the PBS group. Data represent mean ± SD; * p < 0.05; n = 6 per group. (C) A small amount of regenerated bone was observed in the PBS group, but most of the defective area was filled with connective tissue. In the SDF-1 group, regenerated bone covered the bone defect. Bar = 1 mm. (D) Increased magnification of the SDF-1-treated defect.
4. Discussion In this study, we examined the bone-regenerating activity of locally administered SDF-1 protein in the rat calvarial bone defect model. Our findings showed that SDF-1 significantly enhanced the bone regeneration, in association with an increased recruitment of endogenous MSCs and accelerated neo-vessel formation. We found that new bone tissue was formed in the middle of the bone defects in SDF-1-treated group. 247
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Fig. 2. SDF-1 accelerates endogenous rat BMSC recruitment. (A) Representative images showing immunohistochemical staining of the defect treated with PBS or SDF-1; samples were obtained at two weeks post-operation. White arrowhead indicates an MSC detected with CD90 (green) and CD271 (red) staining. Bar = 10 μm. (B–D) Quantification of CD90+ and CD271+ MSCs, DAPI+ cell counts, and CD90+ CD271+/DAPI+ percentages. Statistically significant differences between treatment results were calculated using unpaired t-tests. Data represent mean ± SD; * p < 0.05: n = 6 per group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 3. SDF-1 accelerates endogenous rat EC recruitment and blood-vessel formation. (A) Representative images showing immunohistochemical staining of the defect treated with PBS or SDF-1; samples were obtained at two weeks post-operation. ECs and vascular smooth muscle cells were detected with α-SMA (green) and vWF (red) staining, respectively. Mature blood vessels in the defect consisted of CD31positive endothelial cells and αSMA-positive pericytes; these vessels were increased in the SDF-1 group. Bar = 50 μm. (B–D) Quantification of vWF+ capillary structures, vWF+ α-SMA + vessel counts, and vWF+ α-SMA+ vessel density. Statistically significant differences between treatment results were calculated using unpaired t-tests. Data represent mean ± SD; * p < 0.05: n = 6 per group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 4. Rat calvarial periosteum cells (rCPCs). (A) rCPCs exhibited a spindle or cobblestone shape. Bar = 30 μm. (B) rCPCs cultured without osteogenic differentiation medium for 2 weeks were Alizarin Red S-negative. (C) rCPCs cultured in osteogenic differentiation medium for 2 weeks formed many Alizarin Red S-positive condensed nodules, demonstrating that these rCPCs had strong osteogenic activity (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Tissue regeneration requires the recruitment of endogenous ECs to the target site, where they promote the formation of new blood vessels that transport oxygen, nutrients, trophic factors, and various cells that are essential for tissue repair [28]. MSCs can differentiate into various tissue-specific cell types, including osteoblasts for bone regeneration, according to the disease conditions, and their trophic activities promote
CPCs and ECs from calvalial periosteum may be attributed to this boneregeneration. We further demonstrated that SDF-1 directly increased the cell-migration activity of the hBMMSCs, HUVECs, and rCPCs, without affecting their proliferation, in vitro. Taken together, our findings suggest that SDF-1 may provide significant benefits for bone regeneration therapy. 248
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Fig. 5. Effect of SDF-1 on cell migration. The migration response of hBMMSCs (A), HUVECs (B), or freshly isolated rat calvarial periosteal cells (rCPCs) (C) to SDF-1 was measured by a modified Boyden chamber migration assay (see Materials and Methods). Y-axis: migrated cells. An antagonist for SDF-1, AMD3100, suppressed the SDF-1-induced migration of each cell type. Triplicate wells were used for each treatment. FOV: field of view. Data shown represent mean ± SD of three independent experiments; * p < 0.05.
Fig. 6. Effect of SDF-1 on cell proliferation. The proliferation response of hBMMSCs (A), HUVECs (B), or freshly isolated rat calvarial periosteal cells (rCPCs) (C) to SDF-1 was measured by WST-8 assay (see Materials and Methods). Y-axis: absorbance. AMD3100, an antagonist for SDF-1, had little or no effect on cell proliferation. Triplicate wells were used for each treatment. Data shown represent mean ± SD of three independent experiments; * p < 0.05.
In calvarial bone regeneration, the periosteum plays an important role in supplying osteogenic and vasculogenic progenitors. The periosteum consists of two layers: an outer fibrous layer containing fibroblasts dispersed between collagen fibers, and an inner cambium layer containing skeletal progenitor cells and osteoblasts, and is highly vascularized and innervated [30]. In culture, periosteum-derived cells (PDCs) exhibit a fibroblast-like morphology. PDCs can be induced to differentiate along the osteogenic, chondrogenic, or adipogenic lineage in vitro by specific culture conditions [31–35]. PDCs are reported to be responsive to the chemokines CCL2 (monocyte chemoattractant protein-1, MCP-1), CCL25, CXCL8 (interleukin 8, IL-8), CXCL12 (SDF-1α), and CXCL13 [36]. Our study suggests that the local administration of SDF-1 recruited osteogenic and vasculogenic progenitors derived from PDCs to the bone defect and accelerated bone regeneration.
vascularization and suppress inflammatory responses [2,3]. In bone regeneration, ECs and MSCs coordinately establish tissue-regenerating conditions. Our results showed that SDF-1 promotes bone regeneration by enhancing the recruitment of both of these cell types. Furthermore, SDF-1 promoted the formation of mature micro-vessels, which were stabilized by their association with α-SMA-positive pericytes. We speculate that MSCs and ECs are recruited from tissues surrounding the defects, thereby SDF-1-induced bone regeneration could occur first in peripheral part of the defect, but not uniform manner. In previous reports, it has been shown that local administration of SDF-1 recruited labeled exogenous mesenchymal stem cells into the bone injury, mandibular distraction osteogenesis in rat as well as bone-fracture in mouse [11,29]. These previous reports support the notion that local administration of SDF-1 promote bone-regeneration through the recruitment of MSCs in the systemic circulation. Our in vitro cell-migration assay revealed that the efficacies of cell-recruitment activity of SDF-1 against hBMMSCs and rCPCs were similar, suggesting that most of bone-regenerating cells in SDF-1 treated bone defect derived from adjacent periosteum rather than systemic circulation. To clarify the mechanisms underlying the SDF-1 mediated bone-regeneration, it will be necessary to investigate the precise origin of cells recruited by SDF-1 action in future.
5. Conclusion SDF-1 significantly enhanced bone regeneration, in association with the recruitment of endogenous MSCs and the acceleration of neo-vessel formation. SDF-1 also directly activated the cell-migration activity of hBMMSCs, HUVECs, and rCPCs without affecting their proliferation in vitro. SDF-1 may provide significant benefits for bone-regeneration 249
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therapy. Ethical Approval The study was approved by the Ethics Committee of the Nagoya University School of Medicine.
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Conflict of interest There is no conflict of interest. Acknowledgements We thank Drs. Kiyoshi Sakai, Matsubara Kohki, Masashi Osugi, and the members of the Department of Oral and Maxillofacial Surgery for their encouragement to complete this study. We also thank the Division of Experimental Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine, for the housing of mice and for microscope maintenance. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, 15K15736. References [1] Bruder SP, Jaiswal N, Ricalton NS, Mosca JD, Kraus KH, Kadiyala S. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res 1998:S247–56. [2] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726–36. [3] Tolar J, Le Blanc K, Keating A, Blazar BR. Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells 2010;28:1446–55. [4] Kotobuki N, Katsube Y, Katou Y, Tadokoro M, Hirose M, Ohgushi H. In vivo survival and osteogenic differentiation of allogeneic rat bone marrow mesenchymal stem cells (MSCs). Cell Transplant 2008;17:705–12. [5] Zimmermann CE, Gierloff M, Hedderich J, Acil Y, Wiltfang J, Terheyden H. Survival of transplanted rat bone marrow-derived osteogenic stem cells in vivo. Tissue Eng Part A 2011;17:1147–56. [6] Ando Y, Matsubara K, Ishikawa J, Fujio M, Shohara R, Hibi H, et al. Stem cellconditioned medium accelerates distraction osteogenesis through multiple regenerative mechanisms. Bone 2014;61:82–90. [7] Ranganath SH, Levy O, Inamdar MS, Karp JM. Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease. Cell Stem Cell 2012;10:244–58. [8] Petit I, Jin D, Rafii S. The SDF-1-CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis. Trends Immunol 2007;28:299–307. [9] Salvucci O, Yao L, Villalba S, Sajewicz A, Pittaluga S, Tosato G. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1. Blood 2002;99:2703–11. [10] Strasser GA, Kaminker JS, Tessier-Lavigne M. Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 2010;115:5102–10. [11] Cao J, Wang L, Du ZJ Liu P, Zhang YB, Sui JF, Liu YP, et al. Recruitment of exogenous mesenchymal stem cells in mandibular distraction osteogenesis by the stromal cell-derived factor-1/chemokine receptor-4 pathway in rats. Br J Oral Maxillofac Surg 2013;51:937–41. [12] Fujio M, Yamamoto A, Ando Y, Shohara R, Kinoshita K, Kaneko T, et al. Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors. Bone 2011;49:693–700. [13] Chen G, Fang T, Qi Y, Yin X, Di T, Feng G, et al. Combined use of mesenchymal stromal cell sheet transplantation and local injection of SDF-1 for bone repair in a rat nonunion model. Cell Transplant 2016;25:1801–17. [14] Osugi M, Katagiri W, Yoshimi R, Inukai T, Hibi H, Ueda M. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects.
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Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Original Research
Stromal cell-derived factor-1 accelerates bone regeneration through multiple regenerative mechanisms
T
Yuji Andoa,b, Jun Ishikawaa,c, Masahito Fujioa, Yoshihiro Matsushitaa,c, Hirotaka Wakayamaa,b, ⁎ Hideharu Hibia, Akihito Yamamotod, a
Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan Department of Oral and Maxillofacial Surgery, Chutoen General Medical Center 1-1 Shobugaike, Kakegawa, Shizuoka, 436-8555, Japan c Department of Oral and Maxillofacial Surgery, Kariya Toyota General Hospital, 5-15 Sumiyoshi-cho, Kariya, Aichi, 448-8505, Japan d Department of Tissue regeneration, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-5 Kuramoto-cho, Tokushima 770-8504, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteogenesis Stromal cell-derived factor-1 Endothelial progenitor cells Bone marrow stromal cells Cell recruitment
Objective: Stem cell transplantation has become a popular and promising therapeutic approach for many clinical conditions. Engrafted mesenchymal stem cells (MSCs) can promote bone regeneration in both humans and model animals; however, the differentiation and survival rates of the engrafted MSCs are reported to be poor. We hypothesized that MSCs promote bone regeneration primarily through paracrine mechanisms. SDF-1 is a factor secreted from MSCs that is known to be involved in cell-recruitment and angiogenesis. In this study, we examined the bone-regenerating activity of SDF-1 in the rat calvarial bone defect model. Methods: Collagen sponge containing SDF-1 was transplanted into the rat calvarial bone defect. The effects of treatment were examined both histological and microcomputed tomography analysis. The effects of SDF-1 on cellular migration and proliferationin vitro were assessed by transwell and WST-assay, respectively. Results: SDF-1 significantly enhanced the bone regeneration in rat calvarial bone defect model. SDF-1 promoted the recruitment of endogenous MSCs/osteogenic progenitors and promoted angiogenesisin vivo. Furthermore, SDF-1 directly enhanced the cell-migration activity of human bone marrow MSCs (hBMMSCs), human umbilical vein endothelial cells (HUVECs), and rat calvarial periosteum cells (rCPCs), without affecting their proliferative activities, in vitro. Conclusions: Our findings suggest that the local administration of SDF-1 enhances bone regeneration by inducing multiple endogenous tissue-regenerating activities.
1. Introduction In oral and maxillofacial surgery, autologous bone graft has been applied clinically to regenerate bone defects caused by tumors and trauma; however, damage of the donor site, resorption of the engrafted bone mass, and infection accompanying graft-surgery are major challenges associated with this procedure. Stem cell transplantation has drawn intense attention as an alternative cellular resource for bone regenerating therapy [1]. The engraftment of various types of adult mesenchymal stem cells (MSCs) and their derivatives into damaged areas promotes bone regeneration in both humans and model animals [2,3]. However, the engrafted MSCs exhibit poor differentiation and survival rates, suggesting that they promote bone regeneration primarily by activating endogenous tissue-repairing mechanisms [4,5]. We previously showed that the local administration of MSC-conditioned
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medium promotes tissue regeneration through the recruitment of endogenous osteogenic and vasculogenic progenitors, bone marrow MSCs (BMSCs)/periosteal cells (PEs) and bone marrow endothelial cells (BMECs)/endothelial progenitor cells (EPCs), respectively, to the injury [6]. The identification of the progenitor-recruiting factor derived from MSCs could lead to novel bone regenerating therapies based on the activation of endogenous tissue-repairing activities. We hypothesized that MSCs promote bone regeneration primarily through paracrine mechanisms. SDF-1 is a factor secreted from MSCs that is known to be involved in cell-recruitment and angiogenesis [7,8]. SDF-1, which belongs to the CXC subfamily of chemokines, binds to the G-protein-coupled seven-transmembrane receptors CXCR4 and CXCR7 [9,10]. Notably, SDF-1 is secreted from MSCs [7], and recombinant SDF-1 protein recruits both BMSCs and BM-ECs/EPCs [8]. Thus, SDF-1 is a promising candidate for a progenitor-recruiting factor from MSCs.
Corresponding author. E-mail address: [email protected] (A. Yamamoto).
https://doi.org/10.1016/j.ajoms.2019.02.005 Received 27 November 2017; Received in revised form 25 December 2018; Accepted 20 February 2019 Available online 08 March 2019 2212-5558/ © 2019 Asian AOMS, ASOMP, JSOP, JSOMS, JSOM, and JAMI. Published by Elsevier Ltd All rights reserved.
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2.4. Cell culture
In this study, we administered SDF-1 locally into a rat calvarial bone defect model, and evaluated its tissue-regenerating activity.
Human bone marrow MSCs (hBMMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Walkersville (Basel, Switzerland) and from Health Science Research Resources Bank Japan, respectively. The hBMMSCs and HUVECs were cultured as recommended by the suppliers. Briefly, the cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS) (Lonza Walkersville) or EGM-2 (Endothelial Growth Medium; Lonza) at 37 °C in a 5% CO2 atmosphere and were subcultured every 6–7 days. Rat calvarial periosteum cells (rCPCs) were isolated as described previously [18]. Briefly, the rat calvarial periosteum was isolated and then digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37 °C. Single-cell suspensions were plated on culture dishes in DMEM supplemented with 10% FBS, then incubated at 37 °C in 5% CO2. rCPCs were plated at 3 × 104 cells/well into 24-well plates. Equal numbers of cells were incubated with osteogenic supplements (10% FBS, 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 100 nM dexamethasone; Sigma) in DMEM, and the medium was exchanged three times each week. After 14 days of induction culture, rCPCs were fixed and stained with Alizarin red-S to detect mineralized nodules formed in vitro, as described previously [19]. Briefly, the cells were fixed in 4% paraformaldehyde and incubated with Alizarin red-S (40 mM, pH 6.4; Sigma) for 10 min at room temperature. After washing the wells with distilled water, the plates were photographed. The Alizarin red dye was then extracted with 10% formic acid.
2. Materials and methods 2.1. Rat calvarial bone defect model All animal experiments were performed in accordance with the Guidelines for Animal Experimentation of the Nagoya University School of Medicine. Eight- to ten-week-old male SD rats (n = 24) were anesthetized by intraperitoneal injection of pentobarbital (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan) (20 mg/kg body weight). After shaving the skin, an incision was made in the skull, and the periosteum was opened to expose the surface of the calvarial bones. Two circular bone defects (full-thickness, 5 mm in diameter) were made in the calvarial bone using a trephine bur and were irrigated with saline to remove bone debris. The following experimental materials were then implanted into the defects. Atelocollagen Honeycomb sponge (Koken, Tokyo, Japan) was suspended in 20 μL of PBS, or of PBS containing 200 ng SDF1 (10 μg/mL in PBS). According to previous literature, we determined the range of the SDF-1 dose [11–13]. In preliminary analysis, as we observed a significant bone healing in 200 ng SDF-1 group, we chose the dose. We defined the following groups based on the implanted materials: (1) PBS: PBS/collagen; (2) SDF-1: SDF-1/collagen. Two circular bone defects received same treatment. The rats were sacrificed 2 or 6 weeks after implantation (n = 6 per group).
2.2. Radiographic analyses 2.5. Migration and proliferation studies
The calvarial bones were harvested at 6 weeks after surgery, fixed in 4% paraformaldehyde solution, and analyzed by microcomputed tomography (micro-CT) using a laboratory X-ray CT device (LATheta; Aloka Co., Tokyo, Japan). Images were compiled and analyzed to render 3D images using OsiriX imaging software (Ver.3.9; www.osirixviewer.com/). The area (mm2) of newly regenerated bone in the PBS and SDF-1 groups was compared [14–16].
The migration ability of hBMMSCs, HUVECs, or rCPCs in response to SDF-1 was assessed using transwell-membrane chambers (pore size 8 μm; BD Falcon; BD, Franklin Lakes, NJ) as previously described [20,21]. Briefly, 1 × 105 cells were seeded into the upper chamber in 700 μl serum-free DMEM, and 1400 μl of serum-free DMEM (control) or DMEM containing 10 ng/ml or 100 ng/ml SDF-1 (R&D Systems, Minneapolis, MN) was added to the lower chamber. To inhibit CXCR4 function, the cells were pre-incubated for 30 min at 37 ℃ with 5 μg/mL AMD3100 (Sigma) [22]. After 8 h of incubation in the transwell chamber, the membranes were fixed in a 4% paraformaldehyde solution and stained with hematoxylin. Non-migratory cells from the upper chamber were removed with cotton swabs. Cells that had migrated to the lower side of the membrane were counted in 10 randomly selected fields (size of analyzed field: 310 × 410 μm). As a positive control for migration, DMEM containing 10% FBS was added to the lower chamber. The cell proliferation rate was assessed using a cell-counting kit (WST-8 s; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions [23]. Cells were plated at a density of 2.0 × 104 cells/mL/ well in 12-well plates (Greiner Bio-One, Frickenhausen, Germany) in each medium. Before addition of SDF-1, we cultured cells in serum free medium for one hour. After a 24-h incubation in DMEM containing SDF-1 with/without 5 μg/mL AMD3100, each absorbance was measured in triplicate.
2.3. Histological analysis The calvarial bones were embedded in SCEM gel (8091140; Leica, Tokyo, Japan) and frozen in cooled isopentane. Non-decalcified calvarial bone sections were generated using Kawamoto's film method (8091098; Leica) [17]. Cryostat sections (5-μm thick) were stained with hematoxylin and eosin or subjected to immunohistological analysis. For immunostaining, the sections were fixed in 99.5% ethanol for 10 min at room temperature, blocked with 5% bovine serum albumin/PBS for 30 min, stained with primary antibodies in blocking buffer for 1 h, stained with secondary antibodies for 30 min, mounted with SCMM R3 (Leica), and examined with a BZ9000 fluorescence microscope (Keyence, Osaka, Japan). The following antibodies were used for immunostaining: mouse monoclonal antibody (mAb) against CD90 (Thy1.1) (MAB1406; Millipore, Darmstadt, Germany), rabbit polyclonal antibody against CD271 (NGF receptor) (AB1554; Millipore), rabbit mAb against alpha smooth muscle actin (αSMA) (04–1094; Millipore), and mouse mAb against Von Willebrand Factor (vWF) (F8/86; Dako, Glostrup, Denmark). Secondary antibodies were conjugated with Alexa Fluor 488 or 555 (Invitrogen, Waltham, MA). No cross reaction of mouse and rabbit second antibody against rat antigen was confirmed by staining with second Abs alone. Cell nuclei were labeled with 4′, 6diamidino-2-phenylindole (DAPI) (Invitrogen, Waltham, MA) and were counted in randomly selected fields (size of analyzed field: 310 × 410 μm). The microvessel content was assessed by measuring the number and density of vWF-positive cells around muscle fibers, using a fluorescence microscope (BZ9000; Keyence).
2.6. Quantification and statistical analysis Cells expressing a particular marker were counted under a fluorescence microscope (BZ9000, Keyence) and analyzed with ImageJ software. All experiments were conducted in triplicate and repeated at least twice. The SPSS statistical package was used for statistical analysis. Comparisons were made using Student’s t-test or Tukey’s post hoc test. Results were expressed as the mean ± standard deviation (SD: p < 0.05 was considered statistically significant). 246
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3. Results 3.1. SDF-1 enhances bone regeneration in a rat calvarial bone defect Five-millimeter bone defects were created in the left and right side of the calvarial bones of 8-10-week-old rats. This critically sized calvarial defect cannot heal spontaneously during the eight weeks after surgery [24]. Micro-CT analysis performed six weeks after the operation showed that little or no bone healing in the PBS-treated group, while the SDF-1-treated group exhibited clear healing (Fig. 1A). Quantitative analysis revealed that the area of newly regenerated bone in the SDF-1 group (57.9 ± 15.4%) was significantly larger than that in the PBS group (36.5 ± 9.6%) (Fig. 1B). Histological analysis showed that the bone defect in the SDF-1 group was covered with regenerated bone. In the PBS group some regenerated bone was observed, but most of the defective area was filled with connective tissue (Fig. 1C). These results demonstrated that the calvarial defect was healed by SDF-1 treatment (Fig. 1C and D). Notably, we found a significant bone tissue in the middle of the defects as well as the peripheral of it. These boneisland may be formed with cells recruited from calvalial periosteum. 3.2. SDF-1 accelerates endogenous rat BMSC recruitment and blood vessel formation CD90+/CD271+ rat bone marrow cells consist of multipotent BMSCs [25–27]. Immunohistological analysis showed that the recruitment of CD90+/CD271+ cells was increased in the SDF-1-treated compared with the PBS-treated group at two weeks post-operation (Fig. 2A). Quantitative analysis showed that the actual number of CD90+/CD271+ cells and the ratio of the CD90+/CD271+ cells to total DAPI + cells in the SDF-1 group was 1.76 times and 1.87 times that in the PBS group, respectively (Fig. 2B–D). We next examined whether SDF-1 promotes the formation of mature vessels composed of two types of cells, vWF+ endothelial cells and αSMA+ pericytes. Endothelial cells (ECs) line the vessel lumen as a continuous layer, while pericytes, which are of mesenchymal origin, constitute the outer layer of micro-vessels. In the SDF-1-treated group, there were many tubular micro-vessel structures, which were composed of an internal endothelial cell layer and an outer pericyte layer (Fig. 3A). Quantitative analysis showed that the number and density of vWF+/αSMA+ vessels were significantly increased in the SDF-1 treated group (Fig. 3B–D). 3.3. SDF-1 promotes the recruitment of osteogenic progenitors derived from calvarial periosteum rCPCs were isolated as described [18]. These cells exhibited a spindle or cobblestone shape (Fig. 4A). In an osteogenic differentiation medium, rCPCs formed many Alizarin Red S-positive condensed nodules (Fig. 4B and C), demonstrating that the rCPCs exhibited strong osteogenic activity. Cell migration analyses revealed that SDF-1 accelerated the migration of hBMMSCs, HUVECs, and rCPCs in a dose-dependent manner, and that this increased migration was abolished by AMD3100, a specific antagonist against CXCR4 (Fig. 5A–C). In contrast, SDF-1 treatment had little or no effect on the cell proliferation of these three types of cells (Fig. 6A–C).
Fig. 1. SDF-1 enhances bone regeneration in a rat calvarial bone defect. (A) X-ray and Micro-CT images at six weeks post-operation showed little or no bone healing in the PBS-treated group, while the SDF-1-treated group exhibited clear healing. (B) Quantitative analysis revealed that the area of newly regenerated bone in the SDF-1 group was significantly larger than that in the PBS group. Data represent mean ± SD; * p < 0.05; n = 6 per group. (C) A small amount of regenerated bone was observed in the PBS group, but most of the defective area was filled with connective tissue. In the SDF-1 group, regenerated bone covered the bone defect. Bar = 1 mm. (D) Increased magnification of the SDF-1-treated defect.
4. Discussion In this study, we examined the bone-regenerating activity of locally administered SDF-1 protein in the rat calvarial bone defect model. Our findings showed that SDF-1 significantly enhanced the bone regeneration, in association with an increased recruitment of endogenous MSCs and accelerated neo-vessel formation. We found that new bone tissue was formed in the middle of the bone defects in SDF-1-treated group. 247
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Fig. 2. SDF-1 accelerates endogenous rat BMSC recruitment. (A) Representative images showing immunohistochemical staining of the defect treated with PBS or SDF-1; samples were obtained at two weeks post-operation. White arrowhead indicates an MSC detected with CD90 (green) and CD271 (red) staining. Bar = 10 μm. (B–D) Quantification of CD90+ and CD271+ MSCs, DAPI+ cell counts, and CD90+ CD271+/DAPI+ percentages. Statistically significant differences between treatment results were calculated using unpaired t-tests. Data represent mean ± SD; * p < 0.05: n = 6 per group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 3. SDF-1 accelerates endogenous rat EC recruitment and blood-vessel formation. (A) Representative images showing immunohistochemical staining of the defect treated with PBS or SDF-1; samples were obtained at two weeks post-operation. ECs and vascular smooth muscle cells were detected with α-SMA (green) and vWF (red) staining, respectively. Mature blood vessels in the defect consisted of CD31positive endothelial cells and αSMA-positive pericytes; these vessels were increased in the SDF-1 group. Bar = 50 μm. (B–D) Quantification of vWF+ capillary structures, vWF+ α-SMA + vessel counts, and vWF+ α-SMA+ vessel density. Statistically significant differences between treatment results were calculated using unpaired t-tests. Data represent mean ± SD; * p < 0.05: n = 6 per group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 4. Rat calvarial periosteum cells (rCPCs). (A) rCPCs exhibited a spindle or cobblestone shape. Bar = 30 μm. (B) rCPCs cultured without osteogenic differentiation medium for 2 weeks were Alizarin Red S-negative. (C) rCPCs cultured in osteogenic differentiation medium for 2 weeks formed many Alizarin Red S-positive condensed nodules, demonstrating that these rCPCs had strong osteogenic activity (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Tissue regeneration requires the recruitment of endogenous ECs to the target site, where they promote the formation of new blood vessels that transport oxygen, nutrients, trophic factors, and various cells that are essential for tissue repair [28]. MSCs can differentiate into various tissue-specific cell types, including osteoblasts for bone regeneration, according to the disease conditions, and their trophic activities promote
CPCs and ECs from calvalial periosteum may be attributed to this boneregeneration. We further demonstrated that SDF-1 directly increased the cell-migration activity of the hBMMSCs, HUVECs, and rCPCs, without affecting their proliferation, in vitro. Taken together, our findings suggest that SDF-1 may provide significant benefits for bone regeneration therapy. 248
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Fig. 5. Effect of SDF-1 on cell migration. The migration response of hBMMSCs (A), HUVECs (B), or freshly isolated rat calvarial periosteal cells (rCPCs) (C) to SDF-1 was measured by a modified Boyden chamber migration assay (see Materials and Methods). Y-axis: migrated cells. An antagonist for SDF-1, AMD3100, suppressed the SDF-1-induced migration of each cell type. Triplicate wells were used for each treatment. FOV: field of view. Data shown represent mean ± SD of three independent experiments; * p < 0.05.
Fig. 6. Effect of SDF-1 on cell proliferation. The proliferation response of hBMMSCs (A), HUVECs (B), or freshly isolated rat calvarial periosteal cells (rCPCs) (C) to SDF-1 was measured by WST-8 assay (see Materials and Methods). Y-axis: absorbance. AMD3100, an antagonist for SDF-1, had little or no effect on cell proliferation. Triplicate wells were used for each treatment. Data shown represent mean ± SD of three independent experiments; * p < 0.05.
In calvarial bone regeneration, the periosteum plays an important role in supplying osteogenic and vasculogenic progenitors. The periosteum consists of two layers: an outer fibrous layer containing fibroblasts dispersed between collagen fibers, and an inner cambium layer containing skeletal progenitor cells and osteoblasts, and is highly vascularized and innervated [30]. In culture, periosteum-derived cells (PDCs) exhibit a fibroblast-like morphology. PDCs can be induced to differentiate along the osteogenic, chondrogenic, or adipogenic lineage in vitro by specific culture conditions [31–35]. PDCs are reported to be responsive to the chemokines CCL2 (monocyte chemoattractant protein-1, MCP-1), CCL25, CXCL8 (interleukin 8, IL-8), CXCL12 (SDF-1α), and CXCL13 [36]. Our study suggests that the local administration of SDF-1 recruited osteogenic and vasculogenic progenitors derived from PDCs to the bone defect and accelerated bone regeneration.
vascularization and suppress inflammatory responses [2,3]. In bone regeneration, ECs and MSCs coordinately establish tissue-regenerating conditions. Our results showed that SDF-1 promotes bone regeneration by enhancing the recruitment of both of these cell types. Furthermore, SDF-1 promoted the formation of mature micro-vessels, which were stabilized by their association with α-SMA-positive pericytes. We speculate that MSCs and ECs are recruited from tissues surrounding the defects, thereby SDF-1-induced bone regeneration could occur first in peripheral part of the defect, but not uniform manner. In previous reports, it has been shown that local administration of SDF-1 recruited labeled exogenous mesenchymal stem cells into the bone injury, mandibular distraction osteogenesis in rat as well as bone-fracture in mouse [11,29]. These previous reports support the notion that local administration of SDF-1 promote bone-regeneration through the recruitment of MSCs in the systemic circulation. Our in vitro cell-migration assay revealed that the efficacies of cell-recruitment activity of SDF-1 against hBMMSCs and rCPCs were similar, suggesting that most of bone-regenerating cells in SDF-1 treated bone defect derived from adjacent periosteum rather than systemic circulation. To clarify the mechanisms underlying the SDF-1 mediated bone-regeneration, it will be necessary to investigate the precise origin of cells recruited by SDF-1 action in future.
5. Conclusion SDF-1 significantly enhanced bone regeneration, in association with the recruitment of endogenous MSCs and the acceleration of neo-vessel formation. SDF-1 also directly activated the cell-migration activity of hBMMSCs, HUVECs, and rCPCs without affecting their proliferation in vitro. SDF-1 may provide significant benefits for bone-regeneration 249
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therapy. Ethical Approval The study was approved by the Ethics Committee of the Nagoya University School of Medicine.
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