Association of α₂-HS Glycoprotein with Neurogenic Heterotopic Ossification in Patients with Spinal Cord Injury

Abstract

Background

Neurogenic heterotopic ossification (NHO) is the abnormal formation of mature lamellar bone where bone does not normally exist, usually in the soft tissues [1]. It is one of most common complications in patients with central nervous system disorders such as spinal cord injury (SCI). Severe heterotopic ossification might limit the degree of joint movement, and even cause ankylosis, reducing the ability to perform activities of daily living and affecting the recovery of patients [2,3]. There is no effective treatment for neurogenic heterotopic ossification, and the early detection of NHO is crucial for early treatment and good prognosis. Risk factors such as edema, prolonged swelling and demographic factors (e.g., age and sex) have been reported to be associated with increased risk of NHO [2,4–6]. Spasticity, pressure ulcers, and urinary tract infections have also been suggested to be risk factors for NHO formation [7]. Although many risk factors have been identified for the formation of NHO, most do not have clear biological basis and the association with NHO is relatively weak.

The α₂-HS glycoprotein, also referred to as human fetuin A (Fetuin A of α2 Heremans-Schmid glycoprotein, AHSG), is a plasma glycoprotein belonging to the fetuin family [8]. It serves as an extracellular calcium-regulatory protein, which inhibits Ca×PO4 precipitation [9–12]. The α₂-HS glycoprotein is abundant in the extracellular space which is responsible for over half of the precipitation inhibitory effect of serum [8,11]. It can also markedly inhibit ectopic calcification [13]. To search for risk factors with a relatively clear biological basis, we hypothesized that α₂-HS glycoprotein downregulation might be associated with the occurrence of neurogenic heterotopic ossification. Therefore, we analyzed the α₂-HS glycoprotein level in SCI patients with or without hip heterotopic ossification in this study.

Material and Methods

Study design

From January 2011 to January 2012, 75 patients (67 male) with paraplegia caused by spinal cord injury were treated in the Rehabilitation Department of the Third Hospital of Hebei Medical University (Figure 1). The inclusion criteria were: age 16 to 65 years; strong awareness and compliance of rehabilitation; course of disease ranging from 0.5 to 12 months; no history of hip injury, bone and joint infection, and bone tumor; and no history of pelvis or femur fractures. Patients were excluded with the following diseases which may influence the level of α₂-HS glycoprotein: acute myocardial infarction (AMI), acute leukemia, chronic myeloid leukemia, myeloma cells and degeneration of bone marrow fibrosis, rheumatoid arthritis, lymphoma, alcohol hepatitis, liver cirrhosis, fatty liver, systemic lupus erythematosus, and Crohn’s disease. The study was approved by the Ethics Committee of the Third Hospital of Hebei Medical University. Informed consent was obtained from all patients.

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Figure 1

The flow chart of the retrospective study.

The mean age of the patients was 36.2 years, ranging from 16 to 58 years. The paraplegias were caused by traffic injury (18 patients), injury from a high fall (31 patients), crushing (11 patients), and other reasons (15 patients). The patients were divided into 2 groups according to the occurrence of heterotopic ossification based on the results of high-frequency ultrasound on the bilateral hip joint (Figure 2): Group A (patients with heterotopic ossification after spinal cord injury, 28 cases) and Group B (patients without heterotopic ossification after SCI, 47 cases).

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Figure 2

Representatives of sonographic findings of neurogenic heterotopic ossification. (A) Abnormal muscle structure: the anteromedial muscle group of left thigh showed structure derangement, and there was low and no echo area with irregular shape and island shape enhanced echo area (area indicated by dotted line in white and green). (B) Calcification: There were multiple strip or block enhanced echo areas (indicated by dotted line in white and green) with posterior acoustic shadowing in the anteromedial muscle group of left thigh.

Furthermore, the 28 cases in group A were divided into a mature group (20 cases) and an immature group (8 cases) according to the degree of maturity of neurogenic heterotopic ossification based on the results of high-frequency ultrasound (Figure 3). The ultrasonographic manifestations of mature NHO showed patchy hyperechoic lesion site, uneven surface, the rear sound shadow, boundary less clear, and unsmooth edge. Reactive thickening of the periosteum may be found adjacent to cortical bone, and sparse blood flow signals on color Doppler flow imaging (CDFI). The sonographic findings of immature NHO showed inhomogeneous and hypoechoic mass at the lesion, and the boundary was clear. The involved muscles can be localized, enlarged, and sometimes interrupted by muscle fibers. CDFI showed that the blood flow signals in the damaged muscle tissues increased more than that of the surrounding normal muscle tissues. With the extension of the disease, the signal of blood flow decreased gradually.

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Figure 3

Representatives of sonographic findings related to the 2 stages of neurogenic heterotopic ossification. (A) Mature type: There were irregular lamellar and block enhanced echo areas with posterior acoustic shadowing in front of neck of femur in left thigh (dotted line in white). (B) Immature type: The thickened muscle and uneven low-level echo lump in front of neck of femur in left thigh (area indicated by dotted line in white and green).

At the first visit, the sensory plane, the plane of motion, the muscle strength, the reflex, the joint activity, and the ADL (activities of daily living) ability were evaluated for all patients. We also instructed patients and their family members to do physically active and passive activities in order to avoid contracture and joint mobility limitation due to joint disuse. The movements should be gentle to avoid joint soft tissue injury, hyperemia, hemorrhage, edema, and even organization which might induce or aggravate ectopic ossification.

Results

Discussion

We studied a group of 75 patients with paraplegia caused by spinal cord injury, with or without the presence of NHO. We found that the levels of α₂-HS glycoprotein, CRP, serum calcium, d-dimer, and BMP in SCI patients with NHO were all significantly different from those in SCI patients without NHO. A significant decrease of α₂-HS glycoprotein in SCI patients with NHO was found, as compared with SCI patients without NHO. The degree of maturity of NHO did not influence the level of α₂-HS glycoprotein. Multivariate liner regression analysis showed that the level of serum α2-HS glycoprotein was correlated with CRP and spasticity. Our findings suggest that the decreased level of α2-HS glycoprotein may be related to the formation of neurogenic heterotopic ossification in patients with spinal cord injury, and a decreased level of α₂-HS glycoprotein might be a risk factor for NHO in patients with SCI.

After spinal cord injury, the humoral factors, nerve immune function, metabolism function, and the local microenvironment all change, but the exact mechanism by which these factors induce the formation of NHO is still not clear. Chalmers et al. proposed that the formation of heterotopic ossification must have the following 3 conditions: osteogenic induction, osteogenic precursor cells, and an environment which allow the osteogenesis [15]. According to Kaplan et al., 4 elements are required for heterotopic ossification: 1) local edema around the joint after SCI (initial events); 2) local infiltration of inflammatory cells and release of cytokines (efferent signal); 3) migration of mesenchymal stem cells, accumulation of osteoblasts, production of tropocollagen which aggregated into collagen, and new bone formation (stem cells with osteogenic potential); and 4) local abnormal changes in nutritional status after SCI, local circulation dynamic changes in the tissues, decreased PO2, microvascular dysfunction, and independent nerve dysfunction, providing a suitable environment for the growth of ectopic bone tissue organization (a suitable environment). Among the above elements, the signal factors are most important for elaborating the metabolic effects on NHO after SCI. This was why we chose the regulatory protein α₂-HS glycoprotein to study the effect of heterotopic ossification after spinal cord injury.

The α₂-HS glycoprotein has been considered to be the major calcification inhibitor in the extracellular space [9,11,12]. Jahnen-Dechent et al. found a significant inhibition of calcification in mice knocked-out of α₂-HS glycoprotein, and severe micro-calcification was found in soft tissue and blood vessels [12]. In vitro experiments have also confirmed that the α₂-HS glycoprotein dose-dependently inhibits calcification of bovine vascular smooth muscle [16,17]. Although there are many reports in this field, less is known about the effect of alpha 2-HS glycoprotein on heterotopic ossification, especially in SCI patients with NHO. In our study, we found that the serum level of α₂-HS glycoprotein was significantly lower in SCI patients with NHO as compared with SCI patients without NHO. This indicates that the decrease of serum level of α₂-HS glycoprotein may be related to the formation of NHO. It should be noted that we found no significant difference in different degrees of maturity of neurogenic heterotopic ossification, which suggests that the level of α₂-HS glycoprotein may be not associated with the maturity degree of neurogenic heterotopic ossification. However, due to the limited size of our sample, further studies with larger sample sizes are needed to verify these results.

Wittenberg et al. [18] reported that the development of NHO is related to the site of injury, regardless of the severity and occurrence of SCI, which means that the incidence rate NHO in cervical and thoracic spinal cord injury patients is higher as compared with other types of injury with low paraplegia plane. However, NHO mostly occurs in severe SCI patients (ASIA A neurological classification and ASIA B neurological classification). In patients with only loss of sensory function, none developed NHO in our group of cases. Our results suggest the formation of NHO is significantly correlated with the severity of SCI, but is not associated with the location of SCI. Bravo-Payno et al. [6] reported that the incidence rate of NHO in patients with spasm was significantly higher than in those without spasm. However, we found that the formation of NHO and degree of maturity of NHO were not correlated with increased muscle tension (spasm) after injury. This result may be caused by the relatively small sample size in our study, and further studies enrolling more patients are needed.

Deep venous thrombosis (DVT) is a common complication of SCI, with clinical features of lower-limb swelling and edema. Serum D-dimer is a breakdown product of fibrin, which serves as a useful diagnostic marker for DVT [19–22]. It has been reported that the incidence rate of DVT was 6.55% in acute SCI patients, and the incidence rate of NHO was 8%, with a correlation between the DVT and NHO [23]. Perkash et al. emphasized that NHO can cause a secondary hypercoagulation state in patients with spinal cord injury, and thus lead to the occurrence of DVT [24]. In agreement with these findings, in our study, 11 of the 28 (39.3%) SCI patients with NHO had DVT, and the level of D-dimer was significantly higher these patients than in SCI patients without NHO.

C-reactive protein (CRP), a nonspecific biomarker of inflammation, is upregulated in acute trauma or infection [25]. It is a positive acute-phase reactant. Unlike CRP, α₂-HS glycoprotein is a negative reactant. Both variables represent the same biological event, and thus are considered to be inflammation-related variables [26]. It has been reported that α₂-HS glycoprotein is an alternative to CRP in the Würzburg dialysis cohort, and is inversely correlated with CRP concentrations [26]. Our results showed that the level of CRP was positively correlated with the occurrence of NHO after spinal cord injury, which accords with published data [27].

Conclusions

In conclusion, we found that decreased level of α₂-HS glycoprotein may be related to the formation of neurogenic heterotopic ossification in patients with spinal cord injury, and the level of α₂-HS glycoprotein was not influenced by the degree of maturity of NHO. Our results suggest that decreased levels of α₂-HS glycoprotein might be a risk factor for NHO in patients with SCI, which may have potential benefits for patients with SCI though early monitoring and diagnosis of NHO. However, the sample size in this study is relatively small. Further well-designed clinical studies with larger sample sizes and long-term follow-up are needed to elucidate the actual role of α₂-HS glycoprotein in NHO.

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