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.

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.

Therapeutic strategy of third-generation autologous chondrocyte implantation for osteoarthritis.

BACKGROUND: Autologous chondrocyte implantation (ACI) is considered a promising choice for the treatment of cartilage defects. However, the application of ACI to osteoarthritic patients is, in general, contraindicated. The purpose of this study is to evaluate the efficiency of three-dimensionally structured ACI (3D-ACI; CaReS) in a rat model of knee osteoarthritis (OA). METHODS: OA-like degenerative changes in the articular cartilage were created by transecting the anterior cruciate ligament (ACLT) in athymic nude rats. Two weeks later, CaReS was transplanted at the cartilage injury sites created by micro-drilling in the patella groove (Chondrocyte-implanted (CI) group: CaReS collagen with human chondrocytes; Collagen group: CaReS collagen without cells; and Sham group: sham operation; n = 15/group). RESULTS: Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis demonstrated the expression of human-specific type 2 collagen and Sry-type high-mobility-group box 9 (SOX9) in the CI group-not in the other groups-throughout the study period. Double immunohistochemistry for human-specific type 2 collagen and human leukocyte antigen-abacavir (HLA-ABC) at week 4 showed positive staining in the CI group only. Macroscopic assessment showed better repair at the cartilage defect sites in the CI group, compared to the other groups. Histological assessment with toluidine blue staining showed that the thickness of the articular cartilage and semi-quantitative histological scores were higher in the CI group than in the other groups up to week 20. CONCLUSIONS: We demonstrate, for the first time, that 3D-ACI is effective in repairing cartilage defects in a rat model of ACLT-induced OA.

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.