SDF-1/CXCR4 axis in Tie2-lineage cells including endothelial progenitor cells contributes to bone fracture healing.

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal-derived-factor 1 (SDF-1). SDF-1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF-1/CXCR4 pathway in Tie2-lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2-Cre(ER) CXCR4 knockout mice (CXCR4(-/-) ) and wild-type mice (WT). We report here that in vitro, EPCs derived from of CXCR4(-/-) mouse bone marrow demonstrated severe reduction of migration activity and EPC colony-forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4(-/-) group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4(-/-) group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4(-/-) group. Especially, CXCR4(-/-) group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real-time RT-PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE-cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4(-/-) group. In the gain-of-function study, the fracture in the SDF-1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF-1 injected CXCR4(-/-) group. We demonstrated that an EPC SDF-1/CXCR4 axis plays an important role in bone fracture healing using Tie2-Cre(ER) CXCR4 conditional knockout mice.

Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion Following Bone Fracture.

We recently demonstrated that the local transplantation of human peripheral blood (PB) CD34(+) cells, an endothelial/hematopoietic progenitor cell-rich population, contributes to fracture repair via vasculogenesis/angiogenesis and osteogenesis. Human PB mononuclear cells (MNCs) are also considered a potential cell fraction for neovascularization. We have previously shown the feasibility of human PB MNCs to enhance fracture healing. However, there is no report directly comparing the efficacy for fracture repair between CD34(+) cells and MNCs. In addition, an unhealing fracture model, which does not accurately resemble a clinical setting, was used in our previous studies. To overcome these issues, we compared the capacity of human granulocyte colony-stimulating factor-mobilized PB (GM-PB) CD34(+) cells and human GM-PB MNCs in a nonunion model, which more closely resembles a clinical setting. First, the effect of local transplantation of 1 × 10(5) GM-PB CD34(+) cells (CD34(+) group), 1 × 10(7) GM-PB MNCs (containing approximately 1 × 10(5) GM-PB CD34(+) cells) (MNC group), and phosphate-buffered saline (PBS) (PBS group) on nonunion healing was compared. Similar augmentation of blood flow recovery at perinonunion sites was observed in the CD34(+) and MNC groups. Meanwhile, a superior effect on nonunion repair was revealed by radiological, histological, and functional assessment in the CD34(+) group compared with the other groups. Moreover, through in vivo and in vitro experiments, excessive inflammation induced by GM-PB MNCs was confirmed and believed to be one of the mechanisms underlying this potency difference. These results strongly suggest that local transplantation of GM-PB CD34(+) cells is a practical and effective strategy for treatment of nonunion after fracture.

A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization.

Lnk, an intracellular adapter protein, is expressed in hematopoietic cell lineages, which has recently been proved as an essential inhibitory signaling molecule for stem cell self-renewal in the stem cell factor-c-Kit signaling pathway with enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. Moreover, the therapeutic potential of hematopoietic stem/endothelial progenitor cells (EPCs) for fracture healing has been demonstrated with mechanistic insight into vasculogenesis/angiogenesis and osteogenesis enhancement in the fracture sites. We report here, Lnk siRNA-transfected endothelial commitment of c-kit+/Sca-1+/lineage- subpopulations of bone marrow cells have high EPC colony-forming capacity exhibiting endothelial markers, VE-Cad, VEGF and Ang-1. Lnk siRNA-transfected osteoblasts also show highly osteoblastic capacity. In vivo, locally transfected Lnk siRNA could successfully downregulate the expression of Lnk at the fracture site up to 1 week, and radiological and histological examination showed extremely accelerated fracture healing in Lnk siRNA-transfected mice. Moreover, Lnk siRNA-transfected mice exhibited sufficient therapeutic outcomes with intrinstic enhancement of angiogenesis and osteogenesis, specifically, the mice demonstrated better blood flow recovery in the sites of fracture. In our series of experiments, we clarified that a negatively regulated Lnk system contributed to a favorable circumstance for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. These findings suggest that downregulation of Lnk system may have the clinical potential for faster fracture healing, which contributes to the reduction of delayed unions or non-unions.

Therapeutic superiority for cartilage repair by CD271-positive marrow stromal cell transplantation.

Recent reports indicated that human isolated CD271+ bone marrow mesenchymal stromal cells (BM-MSCs) have a greater expansion and potential for multipotent differentiation including chondrogenesis than classical plastic adherent (PA) BM-MSCs in vitro. Therefore, we set up a hypothesis that CD271+ MSCs may have a greater chondrogenic potential than PA-MSCs in vitro and in vivo. We investigated the superiority of CD271+ MSCs on chondrogenesis using in vitro expansion and pellet culture system and in vivo rat model of cartilage defect when compared to PA-MSCs. In the in vitro study, CD271+ MSCs showed higher expansion potential and produced larger pellets with higher expressions of chondrogenic genes when compared to the control groups. During the culture, CD271 expression decreased, which resulted in decreased chondrogenesis. In the in vivo study, immunohistochemical staining demonstrated differentiated human chondrocytes identified as double-stained cells with human-specific collagen type 2 and human leukocyte antigen-ABC in CD271+ and PA groups. The number of double-stained cells was significantly higher in the CD271+ group than PA group. Real-time RT-PCR analysis of tissue RNA isolated from the chondral defect site for human-specific chondrogenic markers demonstrated a significantly higher expression in CD271+ group than PA group. Macroscopic examination of chondral defect sites at week 8 revealed glossy white and well-integrated repaired tissues in the CD271+ and PA groups, but not in the PBS group. The average histological score in the CD271+ group was significantly greater than in the other groups. Apoptosis analysis at the cell transplanted site with TUNEL staining showed that the CD271+ group had significantly fewer apoptotic chondrocytes compared with the PA group. These results indicate that CD271+ MSCs have a greater chondrogenic potential than PA-MSCs in both in vitro and in vivo conditions.

Local transplantation of ex vivo expanded bone marrow-derived CD34-positive cells accelerates fracture healing.

Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

Therapeutic effect of local administration of low-dose simvastatin-conjugated gelatin hydrogel for fracture healing.

Several reports have shown the therapeutic effect of statins on bone formation and neovascularization. However, the effect of the systemic administration of statins is limited due to its metabolism in the liver and clearance in the digestive system. In addition, high-dose administration may cause adverse side effects. To avoid low-efficacy/frequent side effects of high-dose statin treatment, we utilized biodegradable gelatin hydrogel as a drug delivery system of statin for fracture healing. A femoral fracture was created in rats with periosteum cauterization leading to nonunion at 8 weeks postfracture. Rats received local administration of either simvastatin-conjugated gelatin hydrogel (ST-Gel group) or gelatin hydrogel alone (Gel group). Approximately 70% of animals in the ST-Gel group achieved fracture union radiographically and histologically, while only 7% of animals achieved fracture healing in the Gel group. Functional bone healing was also significantly greater with increased angiogenesis- and osteogenesis-related growth factor expressions in periosteal granulation tissue in the ST-Gel group than in the Gel group. Simvastatin locally applied with gelatin hydrogel to fracture sites at a dose similar to that used in clinical settings successfully induced fracture union in a rat unhealing bone fracture model via its effect on both angiogenesis and osteogenesis.

Local transplantation of granulocyte colony-stimulating factor-mobilized human peripheral blood mononuclear cells for unhealing bone fractures.

We previously reported the therapeutic potential of human peripheral blood (hPB) CD34(+) cells for bone fracture healing via vasculogenesis/angiogenesis and osteogenesis. Transplantation of not only hPB CD34(+) cells but also hPB total mononuclear cells (MNCs) has shown their therapeutic efficiency for enhancing ischemic neovascularization. Compared with transplantation of purified hPB CD34(+) cells, transplantation of hPB MNCs is more attractive due to its simple method of cell isolation and inexpensive cost performance in the clinical setting. Thus, in this report, we attempted to test a hypothesis that granulocyte colony-stimulating factor-mobilized (GM) hPB MNC transplantation could also contribute to fracture healing via vasculogenesis/angiogenesis and osteogenesis. Nude rats with unhealing fractures received local administration of the following materials with atelocollagen: 1 × 10(7) GM hPB MNCs (Hi group), 1 × 10(6) GM hPB MNCs (Lo group), or PBS (PBS group). Immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated human cell-derived vasculogenesis and osteogenesis in the Hi and Lo groups, but not in the PBS group at week 1. Intrinsic angiogenesis and osteogenesis assessed by rat capillary, osteoblast density, and real-time RT-PCR analysis was significantly enhanced in the Hi group compared to the other groups. Blood flow assessment by laser doppler perfusion imaging showed a significantly higher blood flow ratio at week 1 in the Hi group compared with the other groups. Morphological fracture healing was radiographically and histologically confirmed in about 30% of animals in the Hi group at week 8, whereas all animals in the other groups resulted in nonunion. Local transplantation of GM hPB MNCs contributes to fracture healing via vasculogenesis/angiogenesis and osteogenesis.

Synergistic effect of adipose-derived stem cell therapy and bone marrow progenitor recruitment in ischemic heart.

Human multipotent adipose-derived stem cells (hMADSCs) have recently been isolated featuring extensive expansion capacity ex vivo. We tested the hypothesis that hMADSC transplantation might contribute to cardiac functional recovery by its direct or indirect effect on myocardial infarction (MI). Nude rats were either transplanted with hMADSCs or PBS (control) in ischemic myocardium immediately following MI. Echocardiographical assessment of cardiac function after MI with hMADSCs showed significant improvement of each parameter compared to that with PBS. Histological analysis also showed significantly reduced infarct size and increased capillary density in peri-infarct myocardium by hMADSC treatment. However, remarkable transdifferentiation of hMADSCs into cardiac or vascular lineage cells was not observed. Despite the less transdifferentiation capacity, hMADSCs produced robust multiple pro-angiogenic growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1α (SDF-1α). Specifically, hMADSC-derived SDF-1α had a crucial role for cooperative angiogenesis, with the paracrine effect of hMADSCs and Tie2-positive bone marrow (BM) progenitor recruitment in ischemic myocardium. hMADSCs exhibit a therapeutic effect on cardiac preservation following MI, with the production of VEGF, bFGF, and SDF-1α showing paracrine effects and endogenous BM stem/progenitor recruitment to ischemic myocardium rather than its direct contribution to tissue regeneration.

Local transplantation of G-CSF-mobilized CD34(+) cells in a patient with tibial nonunion: a case report.

Although implantation of crude bone marrow cells has been applied in a small number of patients for fracture healing, transplantation of peripheral blood CD34(+) cells, the hematopoietic/endothelial progenitor cell-enriched population, in patients with fracture has never been reported. Here, we report the first case of tibial nonunion receiving autologous, granulocyte colony stimulating factor mobilized CD34(+) cells accompanied with autologous bone grafting. No serious adverse event occurred, and the novel therapy performed 9 months after the primary operation resulted in bone union 3 months later without any symptoms including pain and gait disturbance.

Lnk-dependent axis of SCF-cKit signal for osteogenesis in bone fracture healing.

The therapeutic potential of hematopoietic stem cells/endothelial progenitor cells (HSCs/EPCs) for fracture healing has been demonstrated with evidence for enhanced vasculogenesis/angiogenesis and osteogenesis at the site of fracture. The adaptor protein Lnk has recently been identified as an essential inhibitor of stem cell factor (SCF)-cKit signaling during stem cell self-renewal, and Lnk-deficient mice demonstrate enhanced hematopoietic reconstitution. In this study, we investigated whether the loss of Lnk signaling enhances the regenerative response during fracture healing. Radiological and histological examination showed accelerated fracture healing and remodeling in Lnk-deficient mice compared with wild-type mice. Molecular, physiological, and morphological approaches showed that vasculogenesis/angiogenesis and osteogenesis were promoted in Lnk-deficient mice by the mobilization and recruitment of HSCs/EPCs via activation of the SCF-cKit signaling pathway in the perifracture zone, which established a favorable environment for bone healing and remodeling. In addition, osteoblasts (OBs) from Lnk-deficient mice had a greater potential for terminal differentiation in response to SCF-cKit signaling in vitro. These findings suggest that inhibition of Lnk may have therapeutic potential by promoting an environment conducive to vasculogenesis/angiogenesis and osteogenesis and by facilitating OB terminal differentiation, leading to enhanced fracture healing.

Local transplantation of human multipotent adipose-derived stem cells accelerates fracture healing via enhanced osteogenesis and angiogenesis.

Adipose tissue is one of the promising sources of multipotent stem cells in human. Human multipotent adipose-derived stem (hMADS) cells have recently been isolated and showed differentiation potential into multiple mesenchymal lineages in vitro and in vivo. On the basis of these evidences, we examined the therapeutic efficacy of hMADS cells for fracture healing in an immunodeficient rat femur non-union fracture model. Local transplantation of hMADS cells radiographically and histologically promoted fracture healing with significant improvement of biomechanical function at the fracture sites compared with local transplantation of human fibroblasts (hFB) or PBS administration. Histological capillary density and physiological blood flow by laser Doppler perfusion imaging were significantly greater in hMADS group than hFB and PBS groups. Expressions of intrinsic (rat) bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-1 in peri-fracture tissue were upregulated in hMADS group than other groups. In addition, presence of BMP-2 or VEGF activated the proliferation and migration of hMADS cells in vitro. These results indicate that hMADS cells stimulate the interaction between the transplanted cells and the resident cells stronger than other cells, and they promote fracture healing more effectively. Furthermore, immunohistochemistry for human-specific antibodies revealed direct differentiation of hMADS cells into osteoblasts or endothelial cells in newly formed callus or vasculature, respectively. RT-PCR for human-specific primers for osteogenic/endothelial markers also disclosed osteogenic and vasculogenic plasticity of the transplanted hMADS cells at the early stage of fracture healing. The present results suggest that transplantation of hMADS cells may become a useful strategy for cell-based bone regeneration in the future clinical setting.

A palmar fracture-dislocation of the proximal interphalangeal joint of the middle finger caused by bowling: a case report.

During bowling, a twenty year old man could not pull out his middle finger from the ball in release and injured his finger. X-ray revealed a palmar fracture- dislocation of the PIP joint. We manipulated the PIP joint, but a gap remained at the fracture site on the X-ray after reduction. Surgical treatment was performed with a screw. Postoperatively, the middle finger was fixed with a splint for two weeks, and then active range of motion exercises were started. One year after the operation, the fracture had healed with a congruous joint surface, and the patient had full range of motion in the middle finger with no difficulties in activities of daily living. The etiology of a palmar fracture-dislocation of the PIP joint is still controversial, but we suggested the mechanism of the fracture-dislocation was caused by a shearing force to the middle phalangeal base from a dorsal direction. The main cause of the current injury was the poor fit between the middle finger and the hole of the bowling ball. Bowling is a popular and safe sport, but we should be aware of unexpected hand injuries related to bowling which may occur, especially in players at a recreational level. Key pointsWe presented a palmar fracture-dislocation of the PIP joint in a middle finger that occured while bowling.We discussed the mechanism and suggested the main cause of the injury was the poor fit between the middle finger and the hole of the bowling ball.We advised that while bowling is recognized as a safe sport, due to its popularity we should be aware of unexpected hand injuries which may occur, especially in players at a recreational level.

Circulating endothelial/skeletal progenitor cells for bone regeneration and healing.

An emerging strategy in the regeneration and repair of bone is to use stem cells, including bone marrow mesenchymal stem cells, which are the most investigated and reliable source for tissue engineering, as well as circulating skeletal stem/progenitor cells, which are receiving abundant attention in regenerative medicine due to their ease of isolation and high osteogenic potential. Because failures in fracture healing are largely due to poor vascularization among many environmental factors, we highlight the first proof-of-principle experiments that elucidated the collaborative multi-lineage differentiation of circulating CD34 positive cells - a cell-enriched population of endothelial/hematopoietic progenitor cells - into not only endothelial cells but also osteoblasts. These cells develop a favorable environment for fracture healing via vasculogenesis/angiogenesis and osteogenesis, ultimately leading to functional recovery from fracture. This review will also highlight current concepts of circulating stem/progenitor cell-based therapy and their potential application for bone repair.

[Vascular regeneration].

Tissue regeneration by using stem/progenitor cells has been recognized as a maintenance or recovery system of many organs in adult. The isolation of endothelial progenitor cells (EPCs) derived from the peripheral blood (PB) was one of the amazing discovery for the recognition of "neovessel formation" in adult occurring as physiological and pathological responses. These findings that EPCs home to sites of neovascularization and differentiate into endothelial cells (ECs) in situ is consistent with "vasculogenesis", a critical paradigm well described for embryonic neovascularization, but proposed recently in adults in whom a reservoir of stem or progenitor cells contributes to vascular organogenesis. On the basis of the regenerative potency, these stem/progenitor cells are expected as a key factor of therapeutic applications for the ischemic diseases.

Local delivery of granulocyte colony stimulating factor-mobilized CD34-positive progenitor cells using bioscaffold for modality of unhealing bone fracture.

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM-PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM-PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen-ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 10(5) (Hi), 10(4) (Mid), or 10(3) (Lo) doses of GM-PB CD34+ cells or phosphate-buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM-PB CD34+ cells, enhancement of the intrinsic angio-osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM-PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing. Disclosure of potential conflicts of interest is found at the end of this article.

Administrations of peripheral blood CD34-positive cells contribute to medial collateral ligament healing via vasculogenesis.

Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to contribute to neoangiogenesis; however, the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD34+ cells with atelocollagen (CD34+ group), GM-PB mononuclear cells (MNCs) with atelocollagen (MNC group), or atelocollagen alone (control group) was locally transplanted after the creation of medial collateral ligament injury in immunodeficient rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining at the injury site demonstrated that molecular and histological expression of human-specific markers for endothelial cells was higher in the CD34+ group compared with the other groups at week 1. Endogenous effect, assessed by capillary density and mRNA expression of vascular endothelial growth factor, was significantly higher in CD34+ cell group than the other groups. In addition to the observation that, as assessed by real-time RT-PCR, gene expression of ligament-specific marker was significantly higher in the CD34+ group than in the other groups, ligament healing assessed by macroscopic, histological, and biomechanical examination was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data strongly suggest that local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.

Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing.

We recently reported that systemic administration of peripheral blood (PB) CD34+ cells, an endothelial progenitor cell (EPC)-enriched population, contributed to fracture healing via vasculogenesis/angiogenesis. However, pathophysiological role of EPCs in fracture healing process has not been fully clarified. Therefore, we investigated the hypothesis whether mobilization and incorporation of bone marrow (BM)-derived EPCs may play a pivotal role in appropriate fracture healing. Serial examinations of Laser doppler perfusion imaging and histological capillary density revealed that neovascularization activity at the fracture site peaked at day 7 post-fracture, the early phase of endochondral ossifification. Fluorescence-activated cell sorting (FACS) analysis demonstrated that the frequency of BM cKit+Sca1+Lineage- (Lin-) cells and PB Sca1+Lin- cells, which are EPC-enriched fractions, significantly increased post-fracture. The Sca1+ EPC-derived vasuculogenesis at the fracture site was confirmed by double immunohistochemistry for CD31 and Sca1. BM transplantation from transgenic donors expressing LacZ transcriptionally regulated by endothelial cell-specific Tie-2 promoter into wild type also provided direct evidence that EPCs contributing to enhanced neovascularization at the fracture site were specifically derived from BM. Animal model of systemic administration of PB Sca1+Lin- Green Fluorescent Protein (GFP)+ cells further confirmed incorporation of the mobilized EPCs into the fracture site for fracture healing. These findings indicate that fracture may induce mobilization of EPCs from BM to PB and recruitment of the mobilized EPCs into fracture sites, thereby augment neovascularization during the process of bone healing. EPCs may play an essential role in fracture healing by promoting a favorable environment through neovascularization in damaged skeletal tissue.