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- MATERIALS AND METHODS
The objective of this study was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a rat model. A segmental bone defect (5 mm) was created in the femur and fixed with a mini-plate. There were two groups: EPC-treated (N = 28) and control (N = 28). Seven animals were sacrificed from each group at 1, 2, 3, and 10 weeks postoperatively. Healing of the defect was evaluated with radiographic, histological, and quantitative micro-computed tomography (micro-CT) scans. Radiographically, mean scores of the EPC and control groups were, respectively, 1.16–0.61 (p < 0.05) at 1 week, 2.53–1.54 (p < 0.05) at 2 weeks, and 4.58–2.35 at 3 weeks (p < 0.05). At 10 weeks, all the animals in the EPC-treated group had complete union (7/7), but in the control group none achieved union (0/7). Histological evaluation revealed that specimens from EPC-treated animals had abundant new bone and vessel formation compared to that in controls. Micro-CT assessment of the samples from the animals sacrificed at 10 weeks (N = 14) showed significantly improved parameters of bone volume (36.58–10.57, p = 0.000), bone volume density (0.26–0.17, p = 0.000), model index −2.22–2.79, p = 0.000), trabecular number (1.28–0.91, p = 0.063), trabecular thickness (0.21–0.15, p = 0.001), trabecular spacing (0.63–1.07, p = 0.022), bone surface (353.75–152.08, p = 0.000), and bone surface to bone volume ratio (9.54–14.24, p = 0.004) for the EPC group compared to control, respectively. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental defect model in rat femur diaphysis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1007–1014, 2010
Bone healing constitutes a unique regenerative process which requires coordinated coupling between osteogenesis and angiogenesis. Although regeneration of the bone can be completed without any scarring, a significant percentage of fractures fail to heal adequately.1, 2 Segmental bone defects after severe trauma, infection, and surgical removal of tumors remain a major clinical problem to be addressed. Vascular in-growth at the fracture site has a cardinal role in the healing process and regeneration of the bone postfracture.3 This observation has provided the rationale for the investigations showing the positive effects of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) on fracture healing.4, 5
Endothelial progenitor cells (EPCs), first described by Asahara et al.6 in 1997, are bone marrow-derived cells with the ability to differentiate into endothelial cells and to participate in the establishment of neovasculature mainly by indirect, paracrine mechanisms. EPCs have been shown to express various endothelial surface markers such as CD34, VEGFR2, and CD133, and to home to sites of ischemia. These findings constituted the basis for the use of ex vivo expanded EPCs as a novel therapeutic option to augment neovascularization in animal models of myocardial infarction and limb ischemia.7 Studies in myocardial infarction models demonstrated that infusion of ex vivo expanded EPCs significantly improved blood flow and cardiac function, and reduced left ventricular scarring.8 Likewise, ex vivo expanded EPCs improved the neovascularization in rat models with hind limb ischemia.9 It was described previously that EPCs mobilized by signals from the bone regeneration sites may contribute to neovascularization and, thus, new bone formation in fracture healing.10 However, effects of local treatment with ex vivo expanded EPCs on healing of a segmental bone defect have not been reported to date.
The purpose of this investigation was to evaluate the effects of the local use of ex vivo expanded EPCs on the stimulation of angiogenesis and the promotion of bone healing at a fracture site in a rat femur osteotomy model.
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- MATERIALS AND METHODS
Endothelial progenitor cells have been shown by many researchers to be effective in cell-based therapies to improve vascularization for a variety of therapeutic applications.17, 18 To our knowledge, this is the first study reporting the effects of local EPC therapy on healing of a segmental bone defect. Our results demonstrated that local use of EPCs in a defect model in a rat femur significantly improved callus formation and healing of the osteotomy gap.
In our study design, we primarily aimed to evaluate the effects of local use of EPCs at a segmental bone defect, and compare the difference that EPC therapy could make versus a control group. Therefore, the size of the osteotomy gap (5 mm) we created, although smaller than those reported in similar studies, was not considered as a limitation.19 In addition, we subdivided both the EPC and the control groups into 1-, 2-, 3-, and 10-week groups in order to be able to monitor the effects of EPC therapy both during early and later stages of fracture healing, and the differences between the two groups at these time points postfracture. Our analysis revealed the remarkable effects of EPCs in enhancing bone regeneration even at earlier time points, such as 2 and 3 weeks after the surgery. Findings at 10 weeks after the surgery also showed an impressive difference between the two groups. Complete fracture union was observed in all the animals in the EPC group, whereas in the control group, none of the animals had bridging callus formation. These results encourage us to speculate that local EPC therapy may become an effective cell-based treatment modality to promote bone healing in the near future.
An important question concerning postfracture local EPC therapy relates to the possible mechanisms that contribute to enhanced bone regeneration. Previous reports have demonstrated EPCs' potential to differentiate into endothelial cells and participate in neovascularization.20–22 Moreover, it was reported that there is a high plasticity among cells of the myeloid lineage and, under specific growth conditions, these cells may be possibly differentiating into cells of another lineage with distinct functional properties.23 Supporting this, Matsumoto et al.24 recently highlighted multilineage differentiation of CD34-positive cells into endothelial cells and also osteoblasts to develop a favorable environment for fracture healing via vasculogenesis and osteogenesis. In our study, we did not correlate the amount of new vessel formation at the osteotomy gap, particularly during the early phase of fracture healing, with the significant differences in the amount of new bone formation during the same postoperative time periods. However, we comment that transplanted EPCs also had osteogenic effects along with vasculogenesis in the treatment group. Further investigation is required to determine the relative proportions of locally transplanted EPCs that differentiate into endothelial cells for neovascularization or into osteoblasts for osteogenesis, and promote the tremendous bone regeneration potential we observed in our study. It would also raise the possibility that EPCs may be acting by paracrine mechanisms releasing chemokines and growth factors that may enhance bone regeneration.
Several animal studies have previously suggested that transplantation of culture expanded bone marrow-derived mesenchymal stem cells (MSCs) has value in bone-healing applications.25 We did not compare EPC therapy with other local cell-based therapies such as MSCs, and this may appear to be a potential limitation of this study that warrants discussion. However, since there were no previous studies in the literature reporting the effects of local EPC therapy on bone regeneration in a segmental defect model, our primary objective was to demonstrate the value of EPCs as a new cell-based treatment modality to promote bone healing. Hence, future studies focusing on the local use of EPCs postfracture, and comparing EPCs with other cell-based treatment modalities, will be important to fully realize the potential of these cells.
Another point which requires discussion is the use of gel-foam as a scaffold to transfer the EPCs to the osteotomy site. Gel-foam provides a mechanical matrix that facilitates clotting and offers a framework for deposition of the cellular elements of blood.26 Given the importance of hematoma formation postfracture, we speculate that gel-foam did not have any inhibitory effect on fracture healing; on the contrary, it possibly provided a superior environment at the osteotomy site. In our study design, all the animals in both groups were treated with gel-foam pieces of same size to minimize any additional influence on bone regeneration at the fracture site.
We transferred the EPCs within gel-foam and 0.3 ml media including growth factors, as opposed to the control group where gel-foam was implanted with 0.3 ml saline. Our aim was to keep the cells viable from the time of collection until transplantation to the animals, and this may appear as a limitation of our study. In a separate experiment to determine the effect of the media and growth factors, we compared a group of animals treated with gel-foam and 0.3 ml media including growth factors with control animals where gel-foam was implanted with 0.3 ml saline. There was no difference between these two groups with regard to bone healing during a period of 10 weeks postoperatively. Hence, we state that the superior healing effect we observed in the group treated with EPCs should be attributed solely to the use of EPCs.
In conclusion, local use of ex-vivo expanded endothelial progenitor cells significantly enhanced bone healing in a segmental defect model in rat femur diaphysis, compared with that in control animals. On the basis of these results, local use of EPCs should be further investigated as a promising cell-based therapy to promote bone regeneration at a fracture site.