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Spinal cord injury (SCI) results in profound bone loss due to muscle paralysis and the inability to ambulate. Sclerostin, a Wnt signaling pathway antagonist produced by osteocytes, is a potent inhibitor of bone formation. Short-term studies in rodent models have demonstrated increased sclerostin in response to mechanical unloading that is reversed with reloading. These studies suggest that complete spinal cord injury, a condition resulting in mechanical unloading of the paralyzed lower extremities, will be associated with high sclerostin levels. We assessed the relationship between circulating sclerostin and bone density in 39 subjects with chronic SCI and 10 without SCI. We found that greater total limb bone mineral content was significantly associated with greater circulating levels of sclerostin. Sclerostin levels were reduced, not elevated, in subjects with SCI who use a wheelchair compared to those with SCI who walk regularly. Similarly, sclerostin levels were lower in subjects with SCI who use a wheelchair compared to persons without SCI who walk regularly. These findings suggest that circulating sclerostin is a biomarker of osteoporosis severity, not a mediator of ongoing bone loss, in long-term, chronic paraplegia. This is in contrast to the acute sclerostin-mediated bone loss demonstrated in animal models of mechanical unloading where high sclerostin levels suppress bone formation. As these data indicate important differences in the relationship between mechanical unloading, sclerostin, and bone in chronic SCI compared to short-term rodent models, it is likely that sclerostin is not a good therapeutic target to treat chronic SCI-induced osteoporosis.
SCI causes rapid bone loss that increases the risk of low-impact fractures. More than 50% of people with complete SCI will fracture at some point following their injury. SCI-induced osteoporosis is unique in that the distal femur and proximal tibia are the most frequently fractured bones, and fractures at these locations often requires prolonged hospitalization to treat (1). Once these fractures occur, medical complications are common. These include pressure ulcer formation, increased pain, spasticity, fracture non-union, and lower limb amputation. Despite these serious complications, there are multiple barriers to osteoporosis diagnosis, wide variations in treatment practices, and no standard of care for the prevention or treatment of SCI-induced osteoporosis (1–4). Part of this clinical void is due to a poor understanding of the underlying causes leading to altered bone metabolism following neurological injury.
Recently, a molecular mechanism involving the Wnt signaling pathway has been described to explain the response of bone to mechanical unloading (5–9). This discovery has radically transformed our understanding of the cellular and molecular mechanisms responsible for the adaptation of bone to unloading (6;7). According to this model, mechanical unloading causes up-regulation of sclerostin, a Wnt signaling antagonist produced almost exclusively by osteocytes. This represents the first cellular response to unloading (10). Sclerostin, in turn, can selectively inhibit the canonical Wnt/β-catenin signaling pathway, thus suppressing the activity of osteoblasts as well as the viability of osteoblasts and osteocytes, ultimately resulting in bone loss.
In humans, mechanical unloading of bone occurs in diseases that cause muscle paralysis or the inability to walk. Very few studies address the impact of mechanical unloading on human bone loss. Postmenopausal women have significantly higher serum sclerostin levels than premenopausal women (11), but postmenopausal osteoporosis is not necessarily associated with the inability to walk. Stroke may cause paralysis, and sclerostin levels are increased in stroke patients with mobility impairments who were studied a mean of 10 months after stroke (12). These studies demonstrate an association between circulating levels of sclerostin and circulating markers of bone turnover. The association between sclerostin and bone loss is expected to be strongest in human disease conditions associated with muscle paralysis and the inability to walk, such as SCI. However, there are no reports in the literature that assess the relationship between sclerostin and changes in bone density in response to paralysis in humans. In this study we sought to determine the relationship between circulating sclerostin levels and bone mass in chronic SCI (more than 2 years after injury).
We studied a convenience sample of 39 subjects with chronic SCI recruited from veterans and individuals in the community previously participating in a larger epidemiological study assessing health at our VA facility (13;14). Subjects with SCI were eligible if they were 22 years of age or older, 2 years or more after injury, and had no other neurological condition (multiple sclerosis, stroke, past polio). We also studied 10 subjects without SCI who were recruited from VA primary care clinics. Subjects without SCI were eligible for recruitment if they did not require an ambulatory aid, had no neurological conditions preventing independent walking, and did not have a history of osteoporosis. The study was approved by our institutional review boards, and all study subjects gave informed consent.
Motor level and completeness of injury was confirmed by physical exam. Injury completeness was reported according to the American Spinal Injury Association Impairment Scale (AIS) as previously described (15). Participants were classified as AIS A or B (motor complete, no motor function below the neurological level of injury); AIS C (motor incomplete, motor function preserved below the neurological level and more than half the key muscles below the neurological level are not strong enough to overcome gravity); or AIS D (motor incomplete, motor function preserved below the neurological level, and more than half the key muscles below the neurological level strong enough to overcome gravity). Injury severity was then classified in 3 categories: motor complete SCI (AIS A/B), motor incomplete SCI (AIS C or D), or no SCI.
Bone mineral density (BMD) was determined by Dual X-ray Absorptiometry (DXA) scan using a 5th generation GE iDXA densitometer. Fractures are most common at the knee (distal femur or proximal tibia) after SCI. Therefore, scans were also performed at both SCI-specific (proximal tibia, distal femur) and standard skeletal sites (radius) as previously described (3). Unless there was a previous fracture or instrumentation, the non-dominant lower extremity and radius were scanned. For the distal femur, the proximal edge of the region of interest (ROI) was set at 20% of the femur length (measured from the lateral femoral condyle), and the distal edge was set at the visible intersection between the patella and the femur, excluding the patella from the ROI. For the proximal tibia, the proximal edge was set at the most proximal point of contact between the tibia and fibular head sites, avoiding regions of overlap between the fibula and the tibia. Customized research software supplied by General Electric was used to determine BMD at the knee. As a standard procedure, a quality assurance phantom supplied by the manufacturer was measured at least every 2 days to confirm accuracy of the densitometer.
Plasma samples were drawn into an EDTA tube and immediately delivered to the core blood research laboratory at our facility. The samples were centrifuged for 15 min at 2600 rpm (1459 × g) at 4°C and stored at −80°C until batch analysis. Sclerostin was measured by quantitative sandwich enzyme immunoassay (ELISA, Alpco Diagnostics, Salem, NH). The assay possesses a sensitivity of 8.9 pmol/L and a run-to-run imprecision at sclerostin concentrations of 13 and 35 pmol/L of 5.0 and 6.0%, respectively. Osteocalcin was measured as an indicator of bone formation by electrochemiluminescence immunoassay on a 2010 Elecsys autoanalyzer (Roche Diagnostics, Indianapolis, IN). The lowest detection limit of this assay is 0.50 ng/mL, and the day-to-day imprecision values at concentrations of 7.04, 25.5 and 78.1 ng/mL are 1.6%, 1.4%, and 1.5%, respectively. C-telopeptide was measured as an indicator of bone resorption by electrochemiluminescence immunoassay on a 2010 Elecsys autoanalyzer (Roche Diagnostics, Indianapolis, IN). The lowest detection limit of this assay is 0.07 ng/mL, and the day-to-day imprecision values at concentrations of 0.08, 0.39, and 3.59 ng/mL are 4.7%, 4.3%, and 1.6%, respectively.
We considered socio-demographic factors, medication use, and various health behaviors reported at the time of DXA scan. Participants were weighed and supine length measured for the calculation of body mass index (BMI). In subjects with severe joint contractures, length was self-reported (n=6). Usual mobility mode (more than 50% of the time) was considered in the following 4 categories: motorized wheelchair, hand-propelled wheelchair, walk with aid (crutch, cane, or similar aid), or walk without assistance. Usual mobility mode also was defined as wheelchair use/no wheelchair use. Smokers were defined as smoking 20 or more packs of cigarettes or using 12 ounces of tobacco or more in a lifetime, or smoking 1 or more cigarettes per day for at least 1 year. Current smokers reported cigarette use within 1 month of testing.
All analyses were performed using SAS 9.2 (SAS Institute, Inc., Cary, NC). Since the distribution of sclerostin was skewed, natural log-transformation was used to normalize the distribution of the outcome and stabilize the variance. General linear models (PROC GLM) were applied to assess associations between selected covariates with sclerostin and BMD. Separate regression models adjusting for age were used for each sclerostin subgroup analysis, with the exception of the SCI (no wheelchair) and No SCI group. Due to the limited sample size in this subgroup, age-adjusted sclerostin values and coefficient for age were obtained using a multivariate model that included all observations and terms for age and subgroup. Tukey correction was used to adjust for multiple comparisons when appropriate. T-tests were used to compare BMD values in subgroup analyses in Table 3.
All participants were male and a majority were white (See Table 1). Ages ranged from 30 to 78, with a mean of 55.8 ± 12.1 years. For those with SCI, the duration of injury ranged from 4.1 to 42.6 years, with a mean of 22.4 ± 11.2 years. Of the subjects with SCI, 29 subjects had motor complete SCI, 10 had motor incomplete SCI of which the majority (9/10) were AIS D SCI. Three people with SCI reported using a bisphosphonate. BMD could not be determined in one subject with complete SCI due to knee contractures preventing correct scan positioning. BMD at the radius was available in 40 persons as scanning at this site was introduced after the study began.
We examined the association between sclerostin and injury severity, injury duration, wheelchair use, age, BMI, smoking history, and markers of bone turnover (c-telopeptide and osteocalcin). One subject with no SCI had a sclerostin value of >250 pmol/L (beyond the detection limits of the assay). This value was considered to be an outlier and was excluded from subsequent sclerostin analyses. There was no significant association between sclerostin levels and duration of injury, BMI, smoking history (current, former, or never), or markers of bone turnover (data not shown, p=0.16–0.83). When considering subjects with SCI, sclerostin levels increased significantly with age (p=0.0007, Figure 1). Among people with SCI, age-adjusted sclerostin levels were significantly lower in wheelchair users compared to those who walk (p=0.05, Table 2). Similarly, age-adjusted sclerostin levels were significantly lower in wheelchair users with SCI compared to subjects without SCI (p=0.03, Table 2). There was no significant difference in sclerostin levels in subjects with SCI who walk compared to subjects without SCI (p=0.99, Table 2).
There was no significant association between age and BMD at the distal femur (p=0.06), proximal tibia (p=0.09), or radius (p=0.81). When considering subjects with SCI, BMD was significantly lower at the distal femur (p=0.0001, Table 3) and proximal tibia (p<0.0001, Table 3) in wheelchair users compared to those who walk. Likewise, BMD was significantly lower at these skeletal sites in wheelchair users with SCI compared to subjects with no SCI (p<0.0001, Table 3). There was no significant difference in BMD between subjects with SCI who walk and subjects without SCI at the distal femur (p=0.23, table 3) and proximal tibia (p=0.09, table 3). There was no significant difference in radius BMD based on ability to walk in SCI or when comparing subjects with SCI who walk compared to subjects without SCI (p=0.84–0.99, Table 3). Including age in the multivariate models did not change these associations (data not shown).
In subjects with SCI, sclerostin levels were positively associated with BMD at both the distal femur (Figure 2, R2=0.11, p=0.04) and proximal tibia (R2=0.22, p=0.003), but not the radius (R2=0.007, p=0.65). These relationships did not change when excluding the 3 subjects taking a bisphosphonate.
In this study we examined the relationship between circulating sclerostin levels and bone in subjects with chronic SCI (more than 2 years post-injury). We found that sclerostin levels increase significantly with age. These results are in agreement with a recent report that circulating sclerostin levels increase with age, and are positively associated with bone density in elderly men and women (16). When considering the SCI subjects only, age-adjusted sclerostin levels were significantly reduced in people using a wheelchair compared to those who walk. Similarly, age-adjusted sclerostin levels were significantly reduced in people with SCI who use a wheelchair compared to people without SCI. Subjects with SCI who use a wheelchair had lower bone density at sublesional skeletal sites (distal femur and proximal tibia) compared to subjects with SCI who walk. We found no difference in age-adjusted sclerostin or bone density when comparing subjects with SCI who walk to subjects without SCI. Because there are few subjects in each of these 2 groups, these findings should be confirmed in a larger study. However, this suggests that, from a bone perspective, people with SCI who walk are similar to people without SCI.
For people with SCI, sclerostin levels were significantly associated with bone mineral density at the knee (distal femur, proximal tibia) but not the radius. For the majority of subjects with SCI in this study, the knee but not the radius, was subjected to partial or total mechanical unloading due to paralysis. This suggests that in the case of complete SCI with associated severe osteoporosis, local reductions in sclerostin production in osteoporotic sublesional bones can be meaningfully detected in circulating sclerostin assays. It is possible that in less severe SCI (such as incomplete SCI with preservation of ambulation) sclerostin levels are changed regionally to a lesser degree than seen in complete SCI, yet when assessed systemically these changes are not detected. This should be explored further in animal models where local levels of sclerostin within the bone microenvironment can be compared across injury severities.
We report lower, not higher, sclerostin levels associated with SCI and wheelchair use. We speculate that our findings are due to the fact that we studied chronic SCI. Prior studies have attempted to characterize bone loss following SCI (17–21). Based on these reports, bone loss is thought to occur in 2 phases: 1) rapid, acute bone loss that plateaus somewhere between 18–24 months post-injury and 2) chronic, ongoing bone loss that is more gradual in nature. In the chronic phase of SCI, the majority of bone already has been resorbed and it is likely that ongoing bone loss is more gradual. Based on a rodent model of SCI-induced osteoporosis (22), nearly all of the trabeculae in the sublesional long bones have been resorbed after injury with extensive cortical thinning. The osteocytes that produce sclerostin are found in trabecular and cortical bone. Therefore, dramatic sublesional bone loss would result in fewer sclerostin-producing cells and a lower basal level of sclerostin. Our findings of reduced sclerostin levels may be reflective of the severity of bone loss that occurs in chronic SCI. This suggests that unloading leads to elevated sclerostin levels during the acute phase, which inhibits bone formation by suppressing osteoblastic differentiation and/or function (23–24). If inhibition of bone formation proceeds long term, without reintroduction of mechanical loading, extreme bone loss occurs. In severe osteoporosis, fewer bone cells exist to produce sclerostin, and sclerostin levels fall to levels lower than those seen under normal conditions. These findings suggest that, in chronic SCI, circulating sclerostin is a biomarker of osteoporosis severity, not a mediator of ongoing bone loss.
Sclerostin, encoded by the sost gene, is a potent inhibitor of bone growth (5;25;26). Several elegant animal studies have shown sclerostin levels are inversely proportional to bone mass (5;7) and that production of sclerostin by osteocytes is dramatically reduced by mechanical loading in rodents (6;8;9). The currently accepted paradigm for the Wnt signaling pathway states that Wnt binds to a co-receptor complex involving Frizzled receptor and low-density lipoprotein receptor-related protein (LRP)-5 or LRP-6, both present on osteoblasts. This binding stabilizes cytoplasmic β-catenin and causes it to translocate to the nucleus. Translocation of β-catenin, in turn, activates the transcription of genes that promote osteoblast proliferation, differentiation, and function, ultimately resulting in new bone formation. Several antagonists have been described that can inhibit this signaling pathway. For instance, molecules like secreted frizzled-related proteins, Wif (Wnt inhibitor factor), and Cerberus can bind Wnt and functionally block the pathway. Dickkopfs (Dkk1) and sclerostin, on the other hand, inhibit the Wnt pathway by preventing the formation of the Wnt- Frizzled-LRP5 complex either by promoting the internalization of the LRP5/6 co-receptor (Dkk1) or by competitive binding to LRP5 (sclerostin) (27;28). These studies have established the central role of Wnt signaling antagonists in the pathogenesis of disuse osteoporosis and provide a basis for the regulation of bone responses to unloading via enhanced or reduced Wnt signaling due to mechanical stimulation or unloading, respectively.
Animal models clearly demonstrate elevated sclerostin levels in response to mechanical unloading that is reversed with reloading. These are short term studies that are thought to mimic the acute phase of SCI-induced bone loss. There are no animal studies reported in the literature that are long-term or mimic chronic SCI. Based on these reports and our results in chronic SCI, we propose a conceptual model of sclerostin mediated bone loss following SCI (Figure 3). Mechanical unloading (paralysis) in acute SCI subjects causes greater sclerostin levels. This increase contributes to decreased bone formation and subsequent bone loss during the acute phase of SCI. The ability to walk (mechanical loading) modulates the response of bone to paralysis by causing a smaller increase in sclerostin levels, thereby partially protecting from bone loss. In the chronic phase, bone wasting results in lower sclerostin levels than those observed in the able bodied. This effect is due to the reduction of sclerostin-producing osteocytes in the osteoporotic bone. In the chronic phase, similar to the acute phase, the ability to walk partially protects from bone loss.
Our novel finding of decreased sclerostin levels in extreme osteoporosis strongly supports a relatively narrow therapeutic window for targeting this pathway in disuse osteoporosis. Based on the literature supporting emerging therapeutic trials (29;30), treatment with an anti-sclerostin antibody would be ineffective in chronic SCI when sclerostin levels already are suppressed. This suggests that the sclerostin pathway should be targeted immediately after SCI, before excessive bone loss occurs. Anti-sclerostin antibodies are being tested in post-menopausal osteoporosis, and may be effective in acute-SCI. We found that wheelchair use in chronic SCI is associated with low sclerostin levels and low BMD at SCI- specific sites. This suggests that physical therapy programs that reintroduce mechanical loading soon after SCI may effectively reduce or block sclerostin-mediated bone loss. These findings have important implications for rehabilitation following acute SCI.
The effect of bisphosphonate therapy on the relationship between sclerostin and bone is also unknown. In this study only 3 subjects were actively taking a bisphosphonate. Despite bisphosphonate treatment, each subject had very low bone density at the distal femur (0.42–0.73 grams/cm2) compared to the mean BMD (0.97 grams/cm2) at the distal femur in the group with no SCI. This suggests that this therapy was started to treat severe osteoporosis, not prevent bone loss. This also suggests that in the context of severe osteoporosis, as seen in our subjects with chronic SCI, bisphosphonate therapy has little impact on the relationship between sclerostin and bone. In fact, when excluding the 3 subjects taking a bisphosphonate, the relationships between bone density and sclerostin did not change. These observations need to be confirmed in a larger study and also assessed in the acute phase of SCI.
The optimal time frame for targeting sclerostin in disuse osteoporosis, either with medication or with physical therapy, is currently unknown. Future studies to measure sclerostin in acute SCI are needed to clarify pathogenesis and prior to the consideration of anti-sclerostin antibodies to treat bone loss in the acute phase of SCI.
We thank Sam Davis, clinical research coordinator and technician, Boston VA Healthcare System, for assisting with bone density scans; Renee Mikorski and Heather Colburn, research assistants, Boston VA Healthcare System, for collection of anthropometric data; and C.W. Wolff, research coordinator, Spaulding Rehabilitation Hospital, for editorial assistance.
This study received support from: the National Institute of Child Health and Human Development [R21HD057030 and R21HD057030-02S1], the National Institute of Arthritis and Musculoskeletal and Skin Diseases [1R01AR059270-01], the Office of Research and Development, Rehabilitation Research and Development [Merit Review Grant B6618R], and the Massachusetts Veterans Epidemiology Research and Information Center, Cooperative Studies Program, Department of Veterans Affairs
All authors have no conflicts of interest.
Authors’ roles: Study design: LM, RB, DG, EG. Study conduct: AL, VD, SS, CT. Data collection: SD, VD. Data analysis: LM, EG, SS, DG. Data interpretation: LM, RB, EG, AL, DG. Drafting manuscript: LM, RB, EG, AL, SS. Revising manuscript content: LM, VD, CT, RB, EG, DG, AL, SS. Approving final version of manuscript: LM, SS, VD, CT, AL, DG, EG, RB. RB, EG, SS, DG, and LM take responsibility for the integrity of the data analysis.