Insulin receptor substrate-1 in osteoblast is indispensable for maintaining bone turnover

Insulin receptor substrates (IRS-1 and -2) are essential for intracellular signaling by insulin and IGF-I, anabolic regulators of bone metabolism. Mice lacking the IRS-1 gene IRS-1^–/– showed severe osteopenia with low bone turnover. IRS-1 was expressed in osteoblasts, but not in osteoclasts, of wild-type (WT) mice. IRS-1^–/– osteoblasts treated with insulin or IGF-I failed to induce tyrosine phosphorylation of cellular proteins, and they showed reduced proliferation and differentiation. Osteoclastogenesis in the coculture of hemopoietic cells and osteoblasts depended on IRS-1 expression in osteoblasts and could not be rescued by IRS-1 expression in hemopoietic cells in the presence of not only IGF-I but also 1,25(OH)₂D₃. In addition, osteoclast differentiation factor (RANKL/ODF) was not induced by these factors in IRS-1^–/– osteoblasts. We conclude that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation, differentiation, and support of osteoclastogenesis, resulting in low-turnover osteopenia. Osteoblastic IRS-1 is essential for maintaining bone turnover, because it mediates signaling by IGF-I and insulin and, we propose, also by other factors, such as 1,25(OH)₂D₃.

Insulin and IGF-I induce a wide variety of growth and metabolic responses and play important roles in the anabolic regulation of bone metabolism (1–4). Patients with insulin deficiency as exemplified by insulin-dependent diabetes mellitus (IDDM) are reported to be associated with decreased bone mass: osteoporosis (5–7). Patients with Laron syndrome caused by IGF-I deficiency also exhibit osteoporosis (8). However, the cellular and molecular mechanism underlying the bone loss by the deficit of insulin and IGF-I signaling has not yet been clarified.

Insulin and IGF-I initiate cellular responses by binding to and activating their endogenous tyrosine kinase receptors (9, 10). The major substrates of both insulin and IGF-I receptor tyrosine kinases are known to be closely related high-molecular-weight proteins, insulin receptor substrate-1 and -2 (IRS-1 and –2), which become rapidly phosphorylated on multiple tyrosine residues after ligand stimulation (11). These phosphorylated substrates bind to proteins containing Src homology-2 domains, and these intermediate signals stimulate a variety of different downstream biological effects (11–13).

We and others generated mice lacking the IRS-1 gene (IRS-1^–/– mice) by homologous recombination (14, 15). Our previous report has shown that the growth of IRS-1^–/– mice was retarded after 15.5 embryonic days and that the body weight was about 30% less than that of wild-type (WT) littermates at the age of 3, 8, and 15 weeks (14). In addition, IRS-1^–/– mice developed resistance to the glucose-lowering effects of insulin and IGF-I. The serum level of insulin was higher whereas that of IGF-I was normal in IRS-1^–/– mice (14). Except for these changes, IRS-1^–/– mice exhibited normal appearance and no abnormalities in major organs such as brain, heart, liver, spleen, and kidney. The survival rate was identical between WT and IRS-1^–/– mice, and both male and female IRS-1^–/– mice were fertile (14).

In this study, we found severe osteopenia in IRS-1^–/– mice and investigated the cellular and molecular mechanism underlying the abnormality of bone metabolism using in vivo morphological analyses and ex vivo cell culture systems. Here we demonstrate the significant similarity between the pathophysiology of osteopenia in IRS-1^–/– mice and that in low-turnover osteoporosis in humans. IRS-1^–/– mice could be a new laboratory animal model for low-turnover osteoporosis and could give us a new clue to understanding the mechanism of bone turnover regulation.

In vivo skeletal findings and blood chemistries. Because the lengths of femora and tibiae were about 20% shorter in IRS-1^–/– than those of WT littermates at 8 weeks of age (Figure 1a), IRS-1 was suggested to be involved in the regulation of skeletal growth. Significant difference of the bone shape could not be detected between WT and IRS-1^–/– mice. However, IRS-1^–/– mice showed severe osteopenia in these long bones by x-ray and three-dimensional computed tomographic (CT) analyses (Figure 1a). This finding was also observed in vertebral bodies (data not shown). BMD of these bones was decreased by more than 30% in IRS-1^–/– mice at 4, 8, 12, and 16 weeks of age compared with those in WT littermates (Figure 1b, left). To investigate the distribution of BMD of long bones at 8 weeks, the femora and tibiae were analyzed by dividing them longitudinally into 20 equal regions, and BMD of each fraction was measured (Figure 1b, right). BMD of each fraction was decreased to a similar extent in IRS-1^–/– mice, suggesting that both trabecular and cortex bones were equally affected. Histological analyses in the proximal tibiae of 8-week-old IRS-1^–/– mice revealed the decreases in trabecular bones, osteoid surface, and TRAP-positive osteoclasts (Figure 2). Histomorphometric measurements supported these histological observations (Table 1). Trabecular and cortex thickness was decreased by 51% and 45%, respectively. Parameters for both bone formation (Ob.S/BS and BFR/BS) and resorption (Oc.N/B.Pm, Oc.S/BS, and ES/BS) were also significantly lower in IRS-1^–/– mice. The decrease in bone formation parameters (approximately 70–85% reduction) exceeded the decrease in bone resorption parameters (approximately 50–60% reduction), indicating a state of low-turnover osteopenia that is a characteristic feature of senile osteoporosis in aged humans (16).

Figure 1

Radiological findings of the long bones of WT and IRS-1^–/– mice. (a) Plain x-ray images of femora (left) and tibiae (middle) and three-dimensional CT images of distal femora (right) of representative 8-week-old WT and IRS-1^–/– mice that are littermates generated from the intercross between heterozygous mice. (b) BMD of the femora and tibiae of WT and IRS-1^–/– littermates generated from 3 pairs of heterozygous mice. Left: BMD of the whole femora and tibiae at 4, 8, 12, and 16 weeks of age. Significantly different from WT mice, *P < 0.01. Right: BMD of each of 20 equal longitudinal divisions of the femora and tibiae of 8-week-old mice. Data are expressed as mean (symbols) ± SEM (error bars) for 8 bones/group for WT and IRS-1^–/– mice.

Figure 2

Histological features of the proximal metaphysis of 8-week-old WT and IRS-1^–/– mice. In Villanueva-Goldner staining for representative WT (a) and IRS-1^–/– (b) littermates (originally ×20), mineralized bone is stained green and unmineralized osteoid is stained red. In TRAP staining for WT (c) and IRS-1^–/– (d) littermates (originally ×100), TRAP-positive osteoclasts are stained red. Histomorphometric parameters are shown in Table 1.

Table 1

Histomorphometry of trabecular and cortex bones of tibiae

The thickness of the growth plate at the proximal tibiae was moderately decreased in IRS-1^–/– mice as compared with WT littermates (60.7 ± 4.8 and 77.5 ± 4.0 μm, mean ± SEM of 7 mice each, respectively). Although chondrocytes of various differentiation stages were arranged regularly in a cartilaginous column, the number of columns and that of chondrocytes per column were decreased by approximately 20% and 30%, respectively, in the IRS-1^–/– growth plate cartilage. Because growth plate chondrocytes express IGF-I receptors and are responsive to IGF-I (17), these abnormalities may be due to the lack of action of IGF-I in IRS-1^–/– mice.

The serum insulin level was significantly higher in IRS-1^–/– mice than in WT littermates both before (0.48 ± 0.06 vs. 0.25 ± 0.03 ng/mL, mean ± SEM of 5 mice each) and after (0.52 ± 0.06 vs. 0.31 ± 0.04 ng/mL) glucose load at 8 weeks of age as reported previously (14). Serum IGF-I and IGF-II levels were similar between IRS-1^–/– and WT littermates (129.07 ± 7.37 vs. 131.89 ± 12.62 ng/mL and 9.60 ± 0.46 vs. 9.22 ± 1.16 ng/mL, respectively) at 8 weeks of age. The serum IGF-I level did not change significantly over the age of 8–16 weeks in either type of mice. No sex differences were apparent for any of these in vivo skeletal findings and blood chemistries.

Expression and signal transduction of IRS-1 in bone cells. Because IRS-1 and -2 were reported to be differentially expressed in the variety of cells and tissues having different biological roles (18–22), we investigated the expressions of IRS-1 and -2 in cells of osteoblastic and osteoclastic lineages by RT-PCR. Both substrates were expressed in WT osteoblasts from neonatal calvariae, whereas only IRS-2 was detected in IRS-1^–/– osteoblasts (Figure 3a). Immunoblotting using an anti–IRS-1 antibody showed that IRS-1 protein was expressed in WT osteoblasts (Figure 3b, lanes 7–9), but not in IRS-1^–/– osteoblasts (lanes 10–12). Moreover, immunoblotting experiments using an anti-phosphotyrosine antibody of total cellular extracts (lanes 1–3) or immunoprecipitates with an anti–IRS-1 antibody (lanes 13–15) revealed that IGF-I and insulin induced tyrosine phosphorylation of cellular proteins including IRS-1 (arrowhead) in WT osteoblasts. However, in IRS-1^–/– osteoblasts neither phosphorylation of cellular proteins (lanes 4–6) nor IRS-1 (lanes 16–18) induced by IGF-I or insulin was observed, indicating that IRS-1 is essential for signal transduction of IGF-I and insulin in osteoblasts.

Figure 3

The expression of IRS-1 and -2 and the phosphorylation of cellular proteins in cell cultures. (a) Messenger RNA expression patterns of IRS-1 and IRS-2 in osteoblasts and osteoclasts from WT and IRS-1^–/– littermates and mouse osteoclast progenitor C7 cells at various differentiation stages were determined by semiquantitative RT-PCR within an exponential phase of the amplification. Total RNA was extracted from cultured osteoblasts from neonatal calvariae, osteoclasts formed in the coculture of marrow cells and osteoblasts, and C7 cells cultured for 2, 4, and 6 days in the presence of RANKL/ODF and M-CSF. IRS-1 expression was seen only in osteoblasts but not in cells of osteoclastic lineage, even when the amount of template cDNA or the number of amplification cycles was increased. (b) Induction of tyrosine phosphorylation of cellular proteins and IRS-1 in osteoblasts from WT and IRS-1^–/– mice by IGF-I and insulin. Osteoblasts from neonatal calvariae of WT (lanes 1–3, 7–9, 13–15) and IRS-1^–/– (lanes 4–6, 10–12, 16–18) littermates were treated with vehicle (lanes 1, 4, 7, 10, 13, and 16), IGF-I (lanes 2, 5, 8, 11, 14, and 17) or insulin (lanes 3, 6, 9, 12, 15, and 18) for 2 minutes, and extracted cellular proteins were subjected to SDS-PAGE. Lanes 1–6: immunoblotting with an anti-phosphotyrosine antibody; lanes 7–12: immunoblotting with an anti–IRS-1 antibody; lanes 13–18: immunoblotting with an anti–IRS-1 antibody for the osteoblast extracts immunoprecipitated with an anti-phosphotyrosine antibody. Arrowheads indicate 185-kD protein (IRS-1).

On the contrary, IRS-1 expression was not detected in mature osteoclasts formed and isolated from cocultured marrow cells and osteoblasts not only from IRS-1^–/– mice but also from WT mice (Figure 3a). Because osteoclast precursor cells are difficult to isolate in high purity, we used immortal mouse macrophage cell line C7 cells, which are known to differentiate into osteoclasts after 5–6 days of culture in the presence of soluble RANKL/ODF and M-CSF (23, 24). IRS-1 expression also could not be detected in C7 cells cultured for 2, 4, and 6 days, including various differentiation stages of osteoclastic cells. In contrast, IRS-2 was abundantly expressed in isolated mature osteoclasts and C7 cells of various differentiation stages.

Analyses of cultured osteoblasts and osteoclasts. To learn the cellular mechanism underlying the abnormalities in the bone of IRS-1^–/– mice, cultured osteoblasts and osteoclasts were examined. Cultured osteoblasts from IRS-1^–/– mice showed significantly reduced proliferation determined by [³H]-TdR uptake as compared with WT osteoblasts (Figure 4a) in the control culture. In osteoblasts from WT mice, IGF-I stimulated not only cell proliferation but also cell differentiation determined by ALP activity (Figure 4, a and b) and insulin-stimulated proliferation (Figure 4a). There was no stimulation of proliferation or differentiation by IGF-I or insulin in IRS-1^–/– osteoblast cultures. On the other hand, the stimulation of cell proliferation by FGF-2 and the stimulation of cell differentiation by BMP-2 was similar in both WT and IRS-1^–/– osteoblasts, indicating that impaired proliferation and differentiation in cultured osteoblasts from IRS-1^–/– mice were specific to IGF-I and insulin.

Figure 4

Cultures of osteoblasts from WT and IRS-1^–/– mice. (a) Cell proliferation of cultured osteoblasts. Osteoblasts from neonatal calvariae of WT and IRS-1^–/– littermates were cultured with or without IGF-I (10 nM), insulin (100 nM), or FGF-2 (1 nM) for 24 hours, and the cell proliferation was determined by [³H]-TdR incorporation into DNA. (b) ALP activity of osteoblasts from WT or IRS-1^–/– neonatal calvariae cultured with or without IGF-I (10 nM), insulin (100 nM), or BMP-2 (30 ng/mL) at 14 days of culture. Data are expressed as mean (bars) ± SEM (error bars) for 8 wells/group. Significantly different from WT cultures, ^AP < 0.05, ^BP < 0.01.

Osteoclasts are known to be derived from hemopoietic cells and to require the cell-cell interaction with osteoblasts/stromal cells for differentiation (25). To investigate osteoclast formation, we measured the number of TRAP-positive multinucleated osteoclasts formed in the coculture system of bone marrow cells and primary osteoblasts. Osteoclastogenesis in the coculture induced not only by IGF-I but also by 1,25(OH)₂D₃ or their combination was decreased when both cells were derived from IRS-1^–/– mice as compared with when both cells were derived from WT mice (Figure 5a). Other conventional bone resorptive factors, PGE₂, PTH, and IL-11, also induced osteoclastogenesis in the coculture; however, this stimulation was not distinguishable between the cultures of WT:WT and IRS-1^–/–:IRS-1^–/– cells. These results were reproducible when spleen cells were used instead of bone marrow cells as a source of hemopoietic cells (data not shown). Recently, the TNF family molecule RANKL/ODF (also known as TRANCE and OPGL) was identified as a key membrane-associated molecule expressed on osteoblasts/stromal cells inducing osteoclastogenesis (24, 26). Hence, we examined the regulation of RANKL/ODF expression in osteoblasts from WT and IRS-1–/– mice by these resorptive factors. The regulation of RANKL/ODF expression and of osteoclastogenesis by these factors showed good correlation: RANKL/ODF induction by 1,25(OH)₂D₃ and/or IGF-I was decreased in IRS-1–/– osteoblasts as compared with WT osteoblasts, whereas the induction by PGE₂, PTH, and IL-11 was identical in WT and IRS-1–/– osteoblasts (Figure 5a).

Figure 5

Osteoclastogenesis in the coculture of bone marrow cells and osteoblasts. (a) The number of TRAP-positive multinucleated osteoclasts formed in the coculture of marrow cells and osteoblasts both from WT or IRS-1^–/– littermates for 8 days in the presence or absence of 1,25(OH)₂D₃ (VD₃, 10 nM), IGF-I (10 nM), PGE₂ (100 nM), PTH (10 nM), and IL-11 (10 ng/mL) was counted. Data are expressed as mean (bars) ± SEM (error bars) for 8 wells/group. Significantly different from WT cultures, ^AP < 0.05, ^BP < 0.01. Steady-state mRNA levels of RANKL/ODF in cultured osteoblasts from neonatal WT and IRS-1^–/– littermates were determined by Northern blot analysis after 24 hours of culture with or without the factors above. (b) The number of osteoclasts formed in the coculture of marrow cells and osteoblasts from WT and IRS-1^–/– littermates in the presence of 1,25(OH)₂D₃ (10 nM) (top) and the pit area resorbed by osteoclasts for an additional 48 hours of coculture on a dentine slice (bottom). Data are expressed as mean (bars) ± SEM (error bars) for 12 wells/group. Significantly different from WT:WT culture, ^AP < 0.01.

In the presence of 1,25(OH)₂D₃, impaired osteoclastogenesis in the coculture of IRS-1–/– cells was restored by replacing osteoblasts with WT osteoblasts, but not by replacing marrow cells with WT marrow cells (Figure 5b, top). This result was also reproducible when spleen cells were used as a source of hemopoietic cells (data not shown). When osteoclasts formed in the coculture were isolated and further cultured on a dentine slice for two days, the area of resorption pits correlated with the number of osteoclasts formed (Figure 5b, bottom). In addition, individual osteoclasts from IRS-1–/– mice formed resorption pits approximately equal in area to those formed by WT mice. When the same number of osteoclasts were seeded, the total pit area resorbed by IRS-1–/– osteoclasts was 108.2 ± 9.7% (mean ± SEM) of that resorbed by WT osteoclasts. Survival curves showed no difference between the half-lives of WT and IRS-1–/– osteoclasts (12.1 hours and 12.9 hours, respectively). These results strongly suggest that the decrease in bone resorption in IRS-1–/– mice is caused by a defect in osteoblasts to support osteoclast differentiation, but is not due to intrinsic abnormalities of osteoclast progenitors or reduced activity/survival of mature osteoclasts.

We have shown in this study by radiological and histological analyses of bone phenotypes of IRS-1–/– mice that IRS-1 is essential to maintain bone mass and bone turnover. As a mechanism of this abnormality of IRS-1–/– bones, ex vivo cell culture experiments showed that the stimulatory effects of IGF-I and insulin on proliferation and differentiation seen in WT osteoblasts that abundantly express IRS-1 were abrogated in IRS-1–/– osteoblasts. Moreover, the differentiation of osteoclasts that do not express IRS-1 was also decreased in IRS-1–/– mice owing to the impairment of the ability of osteoblasts to support osteoclastogenesis.

Because signal transduction by IGF-I and insulin was markedly diminished in IRS-1–/– osteoblasts, the severe osteopenia in IRS-1–/– mice may largely be due to the loss of IGF-I and insulin signaling. IGF-I is a potent autocrine/paracrine factor for osteoblast proliferation and differentiation (3, 4). Actions of other anabolic agents such as PTH and prostaglandin on bone formation are also reported to be mediated by IGF-I production by osteoblasts (27–29). A relationship between BMD and IGF-I is suggested by a study showing a correlation of serum and skeletal IGF-I levels with BMD between 2 healthy inbred strains of mice (30). In humans, accumulating evidence has suggested a positive correlation between the serum IGF-I level and BMD in postmenopausal women (31, 32). The anabolic effect of insulin on bone formation may be related to its ability to stimulate cell proliferation (1, 2). Streptozotocin-induced diabetic animals with impaired pancreatic insulin production lose bone rapidly, and this loss is offset by insulin replacement (33). Insulin deficiency as exemplified by IDDM patients is also associated with decreased bone mass (5–7). However, the change of bone mass in non–insulin-dependent diabetes mellitus (NIDDM) patients is controversial (5–7, 34–36). Although previous reports demonstrated a decrease in bone mass in NIDDM patients (5, 35), accumulating evidence has shown that obese NIDDM patients have normal or even increased bone mass (6, 7, 34, 36). This might be due to the increased physical stress on the skeleton or to hyperinsulinemia in NIDDM as a result of the osteoanabolic effect of insulin. Whether or not the response of bone cells to insulin is affected in NIDDM patients similarly to those of cells regulating glucose metabolism such as muscle, liver, and adipose tissues is a point to be elucidated. Although the IRS-1–/– mouse is considered to be a model resembling NIDDM patients with hyperinsulinemia/insulin resistance in whom IRS-1 function might be impaired (37), this model is unlikely to define mechanisms of abnormalities of bone metabolism in these patients. The IRS-1–/– mouse may serve as a model of low-turnover osteopenia associated with insulin and IGF-I resistance.

The decrease in IRS-1–/– osteoblast proliferation was seen not only in the stimulated culture by IGF-I or insulin but also in the control culture, although not as strongly in the latter (Figure 4a). This possibly may be due to the blockage of signaling of endogenous IGF-I acting as an autocrine/paracrine factor in the culture. The concentrations of IGF-I in the culture medium were 0.52 ± 0.10 and 0.49 ± 0.09 nM (mean ± SEM) in the control WT and IRS-1–/– cultures, respectively. In addition, serum IGF-I levels were similar between WT and IRS-1–/– mice over the age of 8–16 weeks, suggesting the absence of systemic compensation for impaired IGF-I bioactivity. Hence, the decreased bone formation in vivo in IRS-1–/– mice is likely to be due to the deficit of anabolic signaling of endogenous IGF-I produced by osteoblasts acting as an autocrine/paracrine factor. IGF-I may also mediate the effects of other anabolic factors (27–29), but stimulation of osteoblast proliferation by FGF-2 and stimulation of differentiation by BMP-2 were not reduced in IRS-1–/– osteoblast cultures, indicating that the anabolic effects of these factors are not mediated by IGF-I/IRS-1.

The decrease in 1,25(OH)₂D₃- or IGF-I–stimulated osteoclast formation from IRS-1–/– hemopoietic cells showed good correspondence with decreased RANKL/ODF mRNA expression in IRS-1–/– osteoblasts and was reversed by WT osteoblasts. In addition, there was no reduction in IRS-1–/– osteoclast activity or survival. We conclude, therefore, that the decrease in osteoclastogenesis in IRS-1–/– mice was caused by the decreased ability of IRS-1–/– osteoblasts to support osteoclast differentiation. The impairment of RANKL/ODF induction in IRS-1–/– osteoblasts stimulated by 1,25(OH)₂D₃ suggests some cross-talk mechanism between the signaling pathways of IRS-1 and 1,25(OH)₂D₃. The fact that mRNA and protein levels of vitamin D receptor (VDR) determined by RT-PCR and Western blotting, respectively, were identical between WT and IRS-1–/– osteoblasts (data not shown) suggests the convergence of these signaling pathways after 1,25(OH)₂D₃ binds to VDR. Because the main signaling pathways downstream of IRS-1 are known to be those of mitogen-activated protein (MAP) kinase and phosphatidylinositol 3 (PI3) kinase (11), it is possible that either pathway may be connected with the VDR signaling. In fact, several cross-talk mechanisms of signal transduction pathways between MAP kinase and nuclear receptors have been identified recently (38). Estrogen receptor, another nuclear receptor that is essential to regulate osteoblast function, is known to be activated through phosphorylation by MAP kinase (39–41). Regarding VDR, its heterodimer partner, RXR, has been reported to be phosphorylated by MAP kinase activation; however, the phosphorylation of RXR is inhibitory on 1,25(OH)₂D₃ signal transduction (42). Hence, the signaling pathways of VDR and IRS-1 might rather converge on some molecules in PI3 kinase pathways. Although these cross-talk mechanisms have not been investigated in osteoblasts, further studies will elucidate the detailed mechanisms of cross-talk of membrane and nuclear-receptor signaling such as IRS-1 and VDR.

Despite the fact that both IRS-1 and -2 are ubiquitously expressed, these expression patterns are different among cells and tissues (18–22). Most hemopoietic cells express IRS-2 but not IRS-1 (21), and cells of osteoclastic lineage showed an identical expression pattern in this study. IGF-I is known to stimulate osteoclastic bone resorption not only through indirect action but also through direct action on osteoclast progenitors or mature osteoclasts (43–46). The receptor for insulin has also been identified on mature osteoclasts and regulates osteoclast function (47). Osteoclastogenesis from osteoclast progenitors was impaired but not abrogated by IRS-1 deficiency, and the activity and survival of mature osteoclasts were not affected. Hence, IRS-2 may play an important role in the direct action of IGF-I and insulin on osteoclastic cells and contribute to the regulation of bone turnover in a different way from IRS-1. This question is currently being addressed in our laboratory.

From this study, it is concluded that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation and differentiation, as well as support of osteoclastogenesis, resulting in low-turnover osteopenia. We therefore propose that osteoblastic IRS-1 plays a critical role in maintaining bone turnover, not only because it is essential for IGF-I and insulin signaling, but also because it may be involved in signal transduction for other factors such as 1,25(OH)₂D₃. Further clinical studies will reveal the contribution of IRS-1 to the pathophysiology of several bone disorders such as osteoporosis.

We thank K. Hoshi, I. Nakamura, C.C. Pilbeam, and L.G. Raisz for suggestions and critical review, and the hard tissue research team at Kureha Chemical Co., Ltd. for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture (11470301), a Grant-in-Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports, and Culture, the Japan Orthopaedics and Traumatology Foundation (0103), a Bristol-Myers Squibb/Zimmer Unrestricted Research Grant, and the Uehara Memorial Foundation.

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