Skeletal injuries remain among the most prevalent clinical problems, especially in the aging society. This problem is compounded in patients with rheumatoid arthritis (RA), who tend to have lower bone mineral density due to the combination of their disease-induced generalized osteopenia (1) and the consequences of steroid-induced osteoporosis (2). Although simple fractures are often treated effectively, osteoporotic hip fractures are associated with 24% mortality (3). The structural bone loss that occurs in compound fractures and periprosthetic osteolysis also exemplifies the serious clinical problems requiring massive structural bone reconstruction. In reconstructive surgery in such cases, autologous bone grafting is the gold standard due to the abilities of the live donor tissue, which serves as both the biologic scaffold and the source of osteogenesis via its mesenchymal stem cells (MSCs) (4).
Due to morbidity issues in terms of donor sites and the limited availability of bone autografts, allogeneic bone graft transplantation has become the standard of care. However, because the allograft bone is dead and void of osteogenic and osteoinductive properties (5), the long-term clinical results are poor. A recent study showed a 56.6% survival rate for structural allografts at 216 months (6). In order to overcome these limitations, it is of great importance to develop novel biologic strategies, which first requires elucidation of the molecular signals responsible for successful bone repair.
MSCs are pluripotent cells that differentiate into multiple cell lineages and can promote structural and functional repairs in many organs including bones, making MSCs an attractive candidate for cell-based bone regeneration (7). Many experimental and clinical studies have attempted to regenerate bones with MSCs, but the results present several notable shortcomings, such as vulnerability to infection, uncertainty regarding the differentiation capability in specific in vivo situations, the high cost of ex vivo cell handling, concern regarding the limited number of cells that can actually contribute to bone formation, and possibly even malignant transformation of the cells during ex vivo cell expansion (8–10). During the course of organ regeneration, however, it has been demonstrated that both local MSCs derived from the injured tissue and circulating MSCs collaborate in the healing of damaged organs. Circulating MSCs “sense” a tissue injury, migrate to the sites of damage, and undergo tissue-specific differentiation (11). However, the mechanisms responsible for MSC migration to the site of bone injury have not yet been revealed.
Stromal cell–derived factor 1 (SDF-1)/pre–B cell growth-stimulating factor belongs to the CXC subfamily of chemokines such as CXCL12, which was initially identified as a bone marrow stromal cell–derived factor (12) and as a bone marrow stromal cell–derived pre–B cell stimulatory factor (13). SDF-1 plays many important roles through activation of a G protein–coupled receptor, CXCR4 (13–15), and the interaction of SDF-1/CXCR4 and hematopoietic stem cells (HSCs) has been extensively reported. In bone marrow, endothelial cells and stromal cells express SDF-1, which not only acts as a chemoattractant for HSCs to a bone marrow niche but also supports their survival and proliferation (16, 17). Furthermore, during the last decade, accumulating data have supported an emerging hypothesis that SDF-1/CXCR4 also plays a pivotal role in the biologic and physiologic functions of MSCs (18, 19). SDF-1 is up-regulated at sites of injury and serves as a potent chemoattractant to recruit circulating or residing CXCR4-expressing MSCs, which are necessary for tissue-specific organ repair or the regeneration of the liver (20), heart (21, 22), brain (23), kidney (24), and skin (25). Moreover, the local delivery of SDF-1 into injured tissue promotes the recruitment of circulating mesenchymal stromal and progenitor cells to lesions in the heart (21, 22) and brain (23). However, the involvement of the SDF-1/CXCR4 axis on MSCs in bone repair has not been elucidated.
In this study, we attempted to investigate the hypothesis that SDF-1 plays an important role in endochondral bone repair. Using mouse segmental bone graft models, both live and dead, we demonstrated that SDF-1 recruits MSCs to bone repair sites during the early phase of bone repair. Our results not only lead to further understanding of the physiologic mechanisms of bone regeneration but also suggest new strategies for the therapeutic use of SDF-1 to promote successful bone healing.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we demonstrated that SDF-1 recruits MSCs during endochondral bone repair. We compared gene expression levels during the healing of live bone grafts and dead bone grafts and observed increased expression of SDF1 during the acute phase in the live graft model, whereas no remarkable increase was detected around dead bone. This differential increase in SDF1 expression suggests that this molecule might be a key regulator involved in successful bone repair. We next investigated the in vitro and in vivo chemotactic potency of SDF-1 and observed that SDF-1 promotes the migration of MSCs in vitro in a dose-dependent manner. We also verified that the BrdU-labeled mouse BMSCs injected intravenously were recruited to the live bone graft lesion, and, in addition, that this migration was inhibited by treatment with TF14016, an antagonist for CXCR4. These results strongly favor the notion that SDF-1 is an essential molecule for the migration of MSCs to sites of bone repair in vivo. Indeed, the bone grafts from CXCR4+/− mice showed the capability of restoring decreased bone formation in SDF-1+/− mice, but not vice versa.
Although the role of circulating MSCs in bone healing remains controversial, a recent study formally demonstrated the participation of circulating osteogenic connective tissue progenitor cells in a parabiotic mouse model of fracture healing (34). Moreover, another interesting study demonstrated that circulating bone marrow–derived osteoblast progenitor cells (MOPCs) are recruited to the bone-forming site via the SDF-1/CXCR4 axis in a bone-forming model of bone morphogenetic protein 2–induced ectopic bone formation (35). The effects of SDF-1 on MSCs in normal bone repair, however, have not yet been proven.
In this study, we demonstrated the migration of intravenously transplanted MSCs to the site of bone repair in a live bone graft model. The mobilization of BrdU-positive cells was observed around the graft bone, and the number of migrated cells was decreased by TF14016. These data support the existence of circulating MSCs and involvement of the SDF-1/CXCR4 axis in the migration of cells to the sites of bone repair. In fact, 2 recent studies demonstrated the expression of CXCR4 at the surface of CD45-negative MOPCs and human bone marrow–derived stromal stem cells (35, 36), and the MSCs used in the present study were derived from bone marrow and were negative for CD45. The expression of CXCR4 in MSCs at the mRNA level was confirmed by reverse transcription–PCR. Regarding the in vivo migration of BrdU-positive cells, a counter argument could be that SDF-1 merely recruited macrophages that phagocyted the transplanted BrdU-positive cells. However, 25.2% of total chondrocytes in the endochondral callus were BrdU positive, demonstrating chondrogenic differentiation of the migrated cells.
These observations support the notion that the migrated cells were not hematopoietic but mesenchymal cells and, what is more, actually participate in endochondral bone formation. To our knowledge, this study is the first to show an in vivo chemotactic function of SDF-1 on MSCs and the subsequent commitment of these cells to endochondral bone repair. Surprisingly, the percentage of BrdU-positive cells was higher than expected. This may be because the migrated cells proliferated and differentiated more rapidly than the resident cells, but this possibility remains to be elucidated.
One of the noteworthy findings is that blocking of SDF-1 or CXCR4 is directly connected to the decreased volume of newly formed bone. The loss-of-function studies revealed that anti–SDF-1 neutralizing antibody remarkably decreased the area of new bone formation on day 14 (Figure 4), and that treatment with TF14016 not only inhibited the migration of BrdU-labeled mouse BMSCs (Figure 3B) but also decreased the area of new bone formation on day 7 (Figure 4). Moreover, compared with that in wild-type mice, bone formation was significantly reduced in both SDF-1+/− and CXCR4+/− mice (Figures 5A and B). A clinical study showed that the volume of bone formation is related to the number of progenitor cells that were transplanted for the treatment of human fracture nonunion (9). Moreover, another recent study, using the same murine bone graft model, demonstrated statistically significant correlations between the graft and callus volume and the ultimate torque and torsional rigidity of the site of bone repair (37). Taken together, the results from the present study indicate that the SDF-1/CXCR4 axis plays a key role in the recruitment of MSCs to sites of bone healing and promotes successful endochondral bone regeneration.
The location and type of cells expressing SDF-1 during bone healing represent an intriguing and crucial question. To answer this, we performed gene expression and immunohistochemical analysis and observed that the periosteum of the long bones indeed expressed SDF1 mRNA, and that the expression of SDF-1 protein was highly increased in the periosteum of the live grafts, while no remarkable increase was observed either in the periosteum of the host bone or in that of the dead grafts (Figures 1B and C). It was previously reported that SDF-1 is expressed at the endosteum and the growth plate of normal long bones in adults (32), but a recent study showed that SDF-1 is expressed at the periosteum during embryonic endochondral bone development, and that expression is substantially reduced after birth (38). Combined with these reports, our results may lead to an interesting hypothesis that, in endochondral bone repair, progenitor cells in the periosteum regain the embryonic state and recruit MSCs through the up-regulation of SDF-1 expression, which is consistent with a well-accepted hypothesis that fracture healing recapitulates normal bone growth.
Another important issue is regulation of the expression of SDF-1 in bone repair. Increased expression of SDF1 mRNA was observed on days 2 and 3 in our models, although other studies had demonstrated an increase in SDF-1 expression within 24 hours after injury (22, 24, 25). The discrepancy in the peak point of SDF-1 expression may relate to the types of injuries. Many of the reported animal models are vascular injury types, in which oxygen tension changes rapidly (21–24). Because SDF-1 is reportedly regulated by a hypoxia-specific transcriptional factor, hypoxia-inducible factor 1, the expression of SDF-1 may increase rapidly after the blood supply is stopped in those models (25). However, any trophic vasculature was not apparently affected in our live bone graft model (33). Furthermore, it can be expected that infiltration from the neighboring bone marrow retains the blood supply to the graft bone to some extent. These unique conditions in our model may bring about the gradual hypoxic change of the injured lesion, resulting in the relatively delayed increase in SDF1 expression. Indeed, SDF1 was not up-regulated during the acute phase of the murine rib fracture model, in which the blood supply can be preserved well enough, and proper endochondral bone repair was observed (data not shown). The regulatory mechanism of SDF-1 expression in bone repair should be pursued further in future studies.
Finally, to prove the functional role of SDF-1 in MSC migration, we created an exchanging–bone graft model between heterozygous SDF-1 mice and CXCR4 mice. SDF-1−/− and CXCR4−/− mice die perinatally, with severe abnormalities affecting many organs including the hematopoietic system, cardiovascular system, and brain, although without major skeletal deformities. In contrast, SDF-1+/− and CXCR4+/− mice have been reported to grow up and appear normal in terms of skeletal development, similar to wild-type mice (13, 39). We observed that a CXCR4+/− mouse–derived bone graft restored impaired bone formation in SDF-1+/− mice. In contrast, an SDF-1+/− mouse–derived bone graft was not able to restore the potency of bone formation in CXCR4+/− mice. The immunohistochemical study showed SDF-1 being expressed in the periosteum of the graft bone, not host bone (Figure 1B), and these data strongly support the hypothesis that SDF-1 expressed from the graft bone, not from the host bone, can recruit MSCs to the sites of bone healing and plays a critical role in skeletal bone repair.
In conclusion, this study demonstrates, for the first time, that the SDF-1/CXCR4 axis plays a crucial role in the migration of MSCs and contributes to endochondral bone repair. SDF-1 is highly expressed in the periosteum of the live bone grafts during the acute phase of healing after surgery. SDF-1 recruits MSCs toward the graft lesion and allows them to participate in proper bone repair. Blockage of this axis inhibits cell migration and results in decreased callus formation. Moreover, CXCR4+/− mouse–derived bone grafts expressing SDF-1 restored impaired bone formation in SDF-1+/− mice, but not vice versa. Further understanding of the function of SDF-1 and the mechanism of endochondral bone repair is likely to lead to the development of a new, less-invasive strategy for the therapeutic use of SDF-1 to achieve successful bone repair.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Ito had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Kitaori, Ito, Schwarz.
Acquisiton of data. Kitaori, Ito, Tsutsumi, Oishi, Nakano, Fujii, Nagasawa.
Analysis and interpretation of data. Kitaori, Ito, Nakamura.
Manuscript preparation. Kitaori, Ito, Schwarz.
Statistical analysis. Kitaori.
Discussion. Kitaori, Ito, Schwarz, Yoshitomi, Nagasawa, Nakamura.