Our results suggest strongly that CXM, the intact trimeric NC1 domain of type X collagen, escapes degradation in the skeletal growth plate and can be detected in blood, where its concentration reflects overall growth plate activity in the body and correlates with velocity of skeletal growth. Hence, this degradation by-product of skeletal growth behaves as a real-time marker for linear skeletal growth velocity and has many potential clinical applications.

Analysis of paired serum, plasma, and DBS samples showed that CXM concentrations were similar across sample types, although plasma and DBS readings tended to be slightly higher on average than serum values (table S1). Differences in marker concentrations have been shown in matched biological sample types, so this result is not surprising ( 29 ). The DBS determinations were on average closer to those of the plasma samples rather than serum, suggesting that plasma may be the preferred choice of blood specimens for this assay in the future, but more definitive studies will be needed to resolve this question. Notably, the overall inter- and intra-assay variations of plasma and serum samples were comparable, but DBS samples varied more, perhaps reflecting variability in sampling technique and DBS handling.

Our mouse studies suggest that the CXM marker in vivo half-life is relatively short (~30 min). In contrast, the marker is very stable in vitro, in isolated serum, plasma, and DBS samples. For example, CXM displays <10% degradation in serum for 18 hours at 37°C (fig. S4), can undergo multiple freeze thaws, and resists degradation at temperatures above freezing point. The ability of the marker to resist proteolysis likely reflects its compact molecular configuration ( 17 ). CXM’s resistance to serum proteases and low urinary excretion suggests that another clearance pathway is involved. Trimeric adiponectin, a circulating hormone that is both genetically closely related to type X collagen and structurally similar to CXM, is rapidly cleared by the liver with a very similar half-life ( 28 ), suggesting that CXM may be removed through a similar mechanism, although the mechanism was not investigated here and will require further study.

Type X collagen has two proposed collagenase cleavage sites in its helical domain ( Fig. 1 ). The ~50-kDa size of the predominant fragment detected by Western blot suggests that CXM is the product of the carboxy collagenase cleavage plus additional cleavage events that trim the fragment to smaller sizes. The mouse Cxm appears to undergo cleavages similar to the human CXM. Detection of distinct bands slightly larger and smaller than the predominant 50-kDa human CXM band combined with the mass spectrometry results implies that there are favored cleavage sites at the N-terminal end of the C-terminal collagenase cleavage fragment. Our attempts to identify the cleavage sites by N-terminal sequencing have been unsuccessful to date.

The synthesis of type X collagen is normally restricted to the hypertrophic zone of the skeletal growth plate, where it is secreted into cartilage matrix during the latter stages of endochondral ossification in all growing bones. This matrix serves as a template for bone growth during which it is degraded as growth proceeds until growth stops after adolescence. The interface between the hypertrophic zone and newly formed bone—ossification front—is highly enriched in extracellular proteolytic enzymes engaged in degrading and removing hypertrophic cartilage matrix as the ossification front expands and the bone lengthens. The enzymes known to have collagenase activity, which are thereby candidates for type X collagen degradation, include matrix metalloproteinase 13 (MMP13) secreted from terminally differentiated hypertrophic chondrocytes, MMP9 secreted from osteochondroclasts, and proteases released from vascular cell precursors that invade the cartilage template from the bone marrow ( 27 ).

Clinical relevance

It is well established that growth velocity is highest in young infants, drops substantially over the first 2 to 3 years, remains relatively low during childhood, increases modestly during the pubertal growth spurt, and drops to zero after the spurt is complete. The scatterplot of our cross-sectional serum data from healthy infants and children shows a similar trend (Fig. 6A). Our numbers are preliminary in that they represent the first attempt to relate CXM to established human growth data, but they provide strong support for our hypothesis that the marker levels reflect skeletal growth velocity.

We must emphasize that CXM presumably represents a real-time readout of growth plate activity that corresponds to instantaneous skeletal growth velocity at the time of sampling in contrast to average growth velocity calculated from measuring incremental growth over several months, typically 6 months or more. Hence, no comparable marker exists for CXM validation. If growth were a slow, steady, and constant process, then one would expect the real-time and average velocities to be very similar. However, if growth varies from day to day or even by time of day, as our preliminary data suggest, then the two might not agree. Similarly, CXM does not necessarily predict length or height, both of which reflect accumulated growth in contrast to CXM, which measures growth rate at a single point of time. Despite these caveats, both mouse Cxm and human CXM values correlate with velocities calculated from measured interim growth, suggesting that variability must not be too great.

The correlation of CXM to growth velocity in human subjects was higher using a nonlinear power curve [adjusted R2 (weighted) = 0.88, P < 0.001] rather than a linear best fit (Pearson’s r = 0.66, P < 0.001) that was used with the mouse data. Figure 6 included some participants with more than one data observation. The relaxation of the assumption of independence might lead to narrower sample variability and risk modest inflation of the association of CXM and growth velocity. With a larger data set, it may be found that a linear fit is more appropriate for plotting growth velocity versus CXM concentration; however, the strong correlation from our data set demonstrates that CXM has the potential to provide estimates of growth velocity with narrow margins of error. Further investigation can clarify whether the relationship of CXM to growth velocity is linear or curvilinear and whether that varies within certain ranges of growth velocity. Accordingly, CXM appears to be an informative, real-time indicator of skeletal growth velocity that has considerable potential benefit for the clinical management of skeletal growth and its disorders.

We anticipate that CXM-based estimates of height velocity will be compared to conventional stadiometer-based height velocity determinations. Accordingly, we must emphasize that they measure different parameters of growth, instantaneous growth velocity versus growth velocity averaged over 6 to 12 months, respectively. Consequently, they have different clinical applications and different utilities. For example, stadiometer-based methods will be most useful for cross-sectional, long-term studies. In contrast, we predict that CXM measurements will be most useful for assessing responses of individual children to interventions that affect growth in days to a few weeks. The difference is analogous clinically to the difference between measuring serum glucose and hemoglobin A1c in diabetic patients. The former measures glucose concentration at the time of sampling; the latter is an indicator of glucose metabolism over ~3 months (30). Both are used in the management of diabetes but for different purposes; the utility of one marker does not diminish the utility of the other.

The most obvious practical application of the CXM marker is for monitoring the growth response of poorly growing infants and children to interventions designed to improve growth. Examples include growth hormone and C-type natriuretic peptide derivative therapies for infants and children with growth hormone deficiency and achondroplasia, respectively. Compared to cross-sectional studies, the infant or child serves as his/her own control in this setting, minimizing person-to-person variation. It is likely that treatments that directly or indirectly improve growth begin to act on the bone growth machinery within days or a few weeks at the least and that resulting changes in growth velocity could be detected by measuring CXM within this time frame assuming that baseline concentrations were determined. Information about how an infant/child responds to treatment a month after initiation would be a substantial advantage over the current practice of waiting 6 months or more for growth velocity information. Being able to detect responses to therapeutic interventions in a much shorter time frame would greatly facilitate the adjustment and comparison of therapeutic interventions in these instances. It would also provide a new tool to investigate in depth how the skeleton responds to growth-promoting interventions. Similarly, CXM testing may facilitate the assessment and comparison of the efficacy of programmatic interventions developed to alleviate malnutrition and other chronic diseases that negatively affect growth in resource-restricted regions of the world.

Our testing of healthy diabetic children suggests that CXM exhibits diurnal variation with values highest in the morning, which would be consistent with the notion that diurnal factors, such as growth hormone, drive bone growth (31). Alternatively, diurnal variation of CXM could simply reflect loading (rising from bedtime horizontal position to daytime upright stature forces CXM from the growth plate into subchondral blood vessels). Further investigation is needed to resolve the nature of apparent diurnal variation; however, in the interim, it seems prudent to sample blood in the morning if CXM is being used to monitor growth of a child over time. Similarly, additional investigation will likely refine the optimal conditions for measuring and interpreting CXM values as is typical after the initial identification of a new diagnostic marker.

Lampl and colleagues (32) have proposed that growth occurs in an episodic or saltatory fashion in short-duration, high-velocity spurts separated by periods of little or no growth. Others have argued against this model (33). Our data showing ~20% average variation in day-to-day and week-to-week CXM levels seem less than one would expect for the marked changes in growth predicted by the saltatory growth model. If confirmed by further investigation, then our preliminary observations would be most compatible with small to modest variation in growth velocity from day to day. CXM could serve as a valuable tool to investigate short-term variations in bone growth and their relationship to conventional parameters of growth.

Many of the growth plates that contribute to blood CXM values may not contribute to skeletal length or height, so one might argue that linking it to linear growth may not represent a perfect correlation. However, we believe that the largest and most active growth plates in the body, namely, those in the proximal and distal femurs and tibias, as well as the less active growth plates of the vertebral bodies, are likely to contribute most of the measurable CXM. Moreover, the correlations that we detect for CXM versus length/height velocity and remarkable similarities of plotting CXM versus age to curves that plot clinically determined growth velocity to age argue that CXM is a useful indicator of linear bone growth.

The CXM marker has potential applications beyond those directly related to bone growth. For example, the management of idiopathic scoliosis frequently involves bracing and surgical fusion of the spine (34). In both cases, the timing of intervention depends on the timing of the pubertal growth spurt; bracing takes advantage of the spurt, whereas surgical fusion is done after the spurt is finished. Frequent CXM testing could be used to guide the timing of both interventions.

Long bone fractures heal through endochondral ossification during which type X collagen–containing fracture callus is degraded and replaced by bone much like that which occurs in the growth plate, although the rate is influenced by other factors such as fracture severity, site, and stabilization (23). The data shown in Fig. 7 are clearly preliminary, but they show that CXM concentrations increase temporarily during the time frame when fractures would be expected to heal. They also lend evidence to the fact that our assay is sensitive enough to detect small changes over baseline CXM levels in adult subjects. Furthermore, these data support the concept that CXM is an indicator of endochondral ossification.

Articular chondrocytes often terminally differentiate (hypertrophy) in osteoarthritis (OA), raising the possibility that type X collagen could be used as a marker of OA activity (35). Low levels of type X collagen have been detected in sera from adults with severe OA (36). The reported concentrations (24 to 128 pg/ml) are about three orders of magnitude lower than those that we detect in growing infants but within the detectable limits of our assay. The epitope for the assay developed by these investigators maps to the NC1 domain of type X collagen. It is possible that the mAb reported in this publication detects the same NC1 fragment reported here, although no biochemical studies were done to characterize the antibody target.

Type X collagen has been linked to cancer in two publications. In one case, it was detected by ELISA in sera of adult patients with colon cancer (37). The authors speculated that Runx2, a known transcriptional regulator of COL10A1 expression, is responsible for type X collagen production in the tumors. The second report detected expression of COL10A1 mRNA by microarray analysis in diverse cancer types but not in normal tissues (38). Immunostaining of breast cancer tissues localized it to blood vessels, suggesting that its expression is associated with vascular invasion of tumors. These reports raise the possibility that CXM could also be used as a marker for cancer detection in adults, but this will require further investigation.