Bayan Aghdasi

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis through Smoothened Binding

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2‐10B4 mouse marrow stromal cells. Oxy133‐induced activation of an 8X‐Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133‐induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X‐ray after 4 weeks and confirmed with manual assessment, micro‐CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein‐2 (rhBMP‐2). Unlike rhBMP‐2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development.

Prevalence and motion characteristics of degenerative cervical spondylolisthesis in the symptomatic adult.

Study Design. Retrospective analysis of kinetic magnetic resonance images. Objective. To define the prevalence of degenerative cervical spondylolisthesis in symptomatic patients and to analyze the motion characteristics and influence on the spinal canal at the affected level. Summary of Background Data. When compared with lumbar spondylolisthesis, there are few studies evaluating cervical spondylolisthesis, and the prevalence and motion characteristics of cervical spondylolisthesis are not well defined. Methods. Four hundred sixty-eight symptomatic patients underwent upright cervical kinetic magnetic resonance images in neutral, flexion, and extension positions. Segmental displacement and intervertebral angles were measured in 3 positions using computer analysis software. Spondylolisthesis was defined as the vertebral displacement more than 2 mm, and graded based on the magnitude into 2 groups at each level: grade 1 (2–3 mm), grade 2 (>3 mm). Instability was defined as segmental translational motion exceeding 3 mm. Results. Grade 1 and 2 spondylolisthesis at a minimum of 1 level were observed with a prevalence of 16.4% and 3.4% of all patients, respectively. The most affected levels were C4–C5 (6.2%) and C5–C6 (6.0%) followed by C3–C4 (3.6%) and C6–C7 (3.0%). Translational motion was greater in levels with grade 1 as compared with segments without spondylolisthesis, but there was no difference in angular motion between the 3 groups. Translational instability was observed with a prevalence of 16.7% in grade 2, 4.3% in grade 1, and 3.4% in segments without spondylolisthesis. Space available for the cord at the affected level was decreased and spinal cord compression grade was higher in grade 1 and grade 2 as compared with levels without spondylolisthesis. Conclusion. Cervical spondylolisthesis of at least 2 mm was observed in 20% of patients and was most common at C4–C5 and C5–C6. The presence of spondylolisthesis was associated with increased translational motion and decreased segmental spinal canal diameter. Level of Evidence: N/A

The Effect of Corticosteroid Administration on Soft-tissue Inflammation Associated With rhbmp-2 Use in a Rodent Model of Inflammation

Study Design. In vivo rodent model. Objective. Investigate the effect of systemic corticosteroid administration on soft-tissue inflammation after local delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2). Summary of Background Data. Corticosteroid use in cases of soft-tissue inflammation associated with the use of rhBMP-2 has been reported in clinical studies, but the effectiveness of its use and appropriate timing remain unclear. Methods. Absorbable collagen sponges were implanted with control or rhBMP-2 into the lumbar region of rats subcutaneously and intramuscularly. Four groups were studied: group I, control sponge only; group 2, BMP-2 sponge only; group III, BMP-2 sponge and preoperative intraperitoneal methylprednisolone (MPSS); group IV, BMP-2 sponge with MPSS given on day 2. Using magnetic resonance imaging, inflammation was assessed in terms of soft tissue edema volume at 0, 2, 4, and 7 days. Rats were sacrificed after 7 days for gross and histological analysis. Results. The peak mean intramuscular inflammatory volume occurred on day 2 in all groups. Group II (BMP-2 without MPSS) had a significantly higher peak mean inflammatory volume (405.46 mm3) on day 2 than that of groups I (266 mm3), III (278 mm3), and IV (291 mm3) (P = 0.001). No significant difference in intramuscular soft-tissue inflammation was observed between the control group and the groups receiving MPSS on day 0 or day 2 at any time point. No differences in the area of inflammatory cell infiltrate surrounding the sponge was observed histologically, after sacrificing them, in groups treated with BMP-2. Conclusion. Systemic MPSS administration reduced soft tissue edema associated with rhBMP-2 as measured by magnetic resonance imaging, but no effect was observed on the histological area of inflammation. Further studies are required to elucidate if there is any benefit to the use of corticosteroid administration in reducing the area of inflammation associated with the use of rhBMP-2.

Effect of cervical kyphotic deformity type on the motion characteristics and dynamic spinal cord compression.

Study Design. Retrospective analysis of kinematic magnetic resonance images. Objective. To provide baseline data on the segmental angular and translational motion of the degenerated cervical spine by subtype of kyphotic cervical deformity and to elucidate the relationship between motion and degree of spinal cord compression. Summary of Background Data. Kyphotic deformities of the cervical spine are relatively common and are classified as either global or focal. Nevertheless, the effects of kyphotic subtype on cervical segmental motion and degree of spinal cord compression are unknown. Methods. A total of 1171 symptomatic patients (618 females, 553 males) underwent cervical kinematic magnetic resonance imaging in the neutral, flexion, and extension positions. Cervical spines demonstrating kyphosis were included and classified into 3 groups: (1) “global kyphotic deformity” (C-type) (n = 54); (2) “sigmoid deformity” (S-type) with kyphotic upper and lordotic lower cervical segments (n = 29); and (3) “reverse sigmoid deformity” (R-type) with lordotic upper and kyphotic lower cervical segments (n = 39). Translational motion, angular motion, and degree of spinal cord compression were evaluated for each cervical level along with the changes associated with flexion and extension. Results. In the C- and R-types, angular motion with extension was increased in the upper cervical spine, where there was kyphosis; when compared with the S-type, in which there was lordosis in the upper segments. The results were opposite for flexion angular motion. R-type displayed more translational motion at C3–C4 and C5–C6. Degree of static spinal cord compression of R-type was higher than the others at C3–C4. The dynamic spinal cord compression increased in extension more than flexion in all subtypes. Conclusion. Cervical spine studies that aim to investigate kyphotic deformities should make efforts to discern the different subtypes of kyphotic deformities to more accurately characterize and study the effects that the sagittal alignment has on the kinematics of the spine and the degree of spinal cord compression. Level of Evidence: 3

Migration Inhibitory Factor Enhances Inflammation via CD74 in Cartilage End Plates with Modic Type 1 Changes on MRI

BackgroundType 1 Modic changes are characterized by edema, vascularization, and inflammation, which lead to intervertebral disc degeneration. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine closely related to the inflammatory cytokines detected in degenerative intervertebral disc tissues. However, the existence and role of MIF and its receptor CD74 in intervertebral disc degeneration have not been elucidated.Questions/purposesWe asked whether (1) MIF and its receptor CD74 are expressed in cartilage end plates with Type 1 Modic changes, (2) MIF is associated with cartilage end plate degeneration, (3) the MIF antagonist (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) suppresses MIF-induced inflammatory cytokine release, and (4) inflammatory cytokines are released by cartilage end plate chondrocytes via CD74 by activating the CD74 antibody (CD74Ab).MethodsWe examined MIF and CD74 expression by human cartilage end plate chondrocytes and tissues with Type 1 Modic changes from eight patients using immunocytofluorescence and immunohistochemistry. MIF production by the chondrocytes was assessed by ELISA and PCR. We compared cytokine release by chondrocytes treated with MIF in the presence or absence of exogenous ISO-1 by ELISA. Cytokine release by chondrocytes after treatment with CD74Ab was determined by ELISA.ResultsMIF was expressed in degenerated human cartilage end plate tissues and chondrocytes. Lipopolysaccharide and tumor necrosis factor α (TNF-α) upregulated MIF expression and increased MIF secretion in chondrocytes in a dose-dependent manner. MIF increased the secretion of IL-6, IL-8, and prostaglandin E2 (PGE2) in a dose-dependent manner. ISO-1 reduced the secretion of IL-6, IL-8, and PGE2. CD74Ab activated CD74 and induced release of inflammatory cytokines.ConclusionsChondrocytes in cartilage end plate with Type 1 Modic changes express MIF and its receptor CD74. MIF might promote the inflammatory response through CD74. MIF-induced cytokine release appears to be suppressed by ISO-1, and CD74Ab could induce cytokine release.Clinical RelevanceThe MIF/CD74 pathway may represent a crucial target for treating disc degeneration since inhibiting the function of MIF with its antagonist ISO-1 can reduce MIF-induced inflammation and exert potent therapeutic effects.

Growth-Factor Nanocapsules That Enable Tunable Controlled Release for Bone Regeneration.

Growth factors are of great potential in regenerative medicine. However, their clinical applications are largely limited by the short in vivo half-lives and the narrow therapeutic window. Thus, a robust controlled release system remains an unmet medical need for growth-factor-based therapies. In this research, a nanoscale controlled release system (degradable protein nanocapsule) is established via in situ polymerization on growth factor. The release rate can be finely tuned by engineering the surface polymer composition. Improved therapeutic outcomes can be achieved with growth factor nanocapsules, as illustrated in spinal cord fusion mediated by bone morphogenetic protein-2 nanocapsules.

Dynamic changes of the ligamentum flavum in the cervical spine assessed with kinetic magnetic resonance imaging.

The purpose of this article is to quantify changes in thickness of the ligamentum flavum (LF) associated with motion of the cervical spine and to compare the thickness of the LF at each cervical level using kinetic magnetic resonance imaging (kMRI). Two hundred fifty-seven symptomatic patients (129 men; 128 women) underwent kMRI in neutral, flexion, and extension positions. Midsagittal images were digitally marked and electronically analyzed by spine surgeons. Thickness of LF in the cervical region from C2–3 to C7–T1 was measured in all three positions. LF at C7–T1 was significantly thicker than C2–3 to C6–7 in neutral, flexion, and extension positions (p < 0.05). LF was significantly thicker in extension than in flexion at C3–4 to C6–7. LF thickness increases with extension and decreases with flexion. LF is uniquely thick at C6–7 and at C7–T1 in the extension position, which may predispose these levels to cord compression syndromes and associated neuropathies.

BMP-2 adverse reactions treated with human dose equivalent dexamethasone in a rodent model of soft-tissue inflammation.

Study Design. Basic science rodent model of bone morphogenetic protein-2 (BMP-2) soft-tissue inflammation. Objective. This study investigated the anti-inflammatory effect of human dose equivalent (HDE) dexamethasone (DM) for treatment of BMP-2-related soft-tissue inflammation in a rodent model and suggests an appropriate dose for utilization in the clinical practice of spine surgeons. Summary of Background Data. BMP-2 is frequently used in spinal surgery to augment fusion. Yet, side effects of soft-tissue inflammation have been observed. DM decreases proinflammatory cytokine production and cellular immune response. However, the anti-inflammatory effects of HDE DM in a rodent model of BMP-2-associated soft-tissue inflammation have not been reported. Methods. Five, 10, and 15 mg of HDE DM were administered 3 times perioperatively to rodent cohorts receiving BMP-2 paraspinal implants and compared against BMP-2 only positive controls and phosphate buffer negative controls (n = 6 subjects per group). Histopathology, magnetic resonance imaging, and gross dissection were used as measures of cellular, edematous, and exudative inflammatory response. Serial killings were made on day 2 and day 7 postoperatively. Results. Magnetic resonance imaging volume rendering demonstrated inflammatory edema decreased by 49% from 605.4 mm3 to 304.03 mm3 in subjects treated with 5, 10, or 15 mg of HDE DM (P < 0.05). Histopathological analysis demonstrated inflammatory cross-sectional area decreased 28.8% from 1.84 mm2 to 1.31 mm2 in subjects treated with 5, 10 or 15 mg of HDE DM (P < 0.05). Immune cellular infiltration depth decreased 38.5% from 0.26 mm to 0.16 in subjects treated with 15 mg of HDE DM (P < 0.05). Gross anatomical inflammatory exudates were prevented in 100% of subjects treated with 10 or 15 mg of HDE DM (P < 0.05). Conclusion. Low-dose DM administration is effective in controlling the cellular inflammation and edema resulting from BMP-2. Ten or 15 mg of DM might be considered by spine surgeons for controlling the inflammation and edema seen in spine surgery with BMP-2.

The Compensatory Relationship of Upper and Subaxial Cervical Motion in the Presence of Cervical Spondylosis.

Study Design:This study was an in vivo kinematic magnetic resonance imaging analysis of cervical spinal motion in human subjects. Objective:The objective of the study was to identify associations between disk degeneration in the subaxial cervical spine and upper cervical spinal motion in patients with general age-related cervical spondylosis. Summary of Background Data:The kinematic relationship between the occipital-atlantoaxial complex and subaxial cervical spine in patients with cervical spondylosis and decreased cervical motion is not well understood. Methods:A total of 446 symptomatic patients who had neck pain with and without neurogenic symptoms were included in this study. Kinematic magnetic resonance imaging was performed with dynamic motion of the cervical spine in upright, weight-bearing neutral, flexion, and extension positions. Intervertebral disk degeneration for each segment from C2–3 to C7–T1 and sagittal angular motion between flexion and extension for each segment from Oc–C1 to C7–T1 was evaluated. Depending on the amount of sagittal subaxial angular motion, the patients were classified into 3 groups by sagittal angular motion using cutoff points based on tertile (<36-degree group: 149 cases; 36–47-degree group: 148 cases; and >47-degree group: 149 cases). Results:A significant correlation was found between subaxial angular motion and intervertebral disk degeneration, indicating that the subaxial motion decreases according to the degree of disk degeneration. Mean angular motion of the occipital-atlantoaxial complex, especially of Oc–C1, was significantly higher in the <36-degree and 36–47-degree group than in the >47-degree group, whereas no significant difference was found at C1–C2. Conclusions:Our study demonstrates that decreased subaxial cervical spinal motion is associated with intervertebral disk degeneration in a symptomatic population. This decrease in mobility at the subaxial cervical spine is compensated for by an increase in angular mobility of the upper cervical spine at the occipital-atlantoaxial complex, especially at Oc–C1.

The Evaluation and Observation of “Hidden” Hypertrophy of Cervical Ligamentum Flavum, Cervical Canal, and Related Factors Using Kinetic Magnetic Resonance Imaging

Study Design Retrospective cohort study. Objective The objective was to measure the change of flavum ligament diameter during positional changes of the cervical spine using kinetic magnetic resonance imaging (MRI) and to examine the correlational diameter changes of the flavum ligament, disk bulging, and the spinal canal from extension to flexion positions. Methods One hundred eight-nine patients underwent kinetic MRI in neutral, extension, and flexion positions. The diameters of cervical ligamentum flavum, disk bulging, and cervical spinal canal and the disk degeneration grade and Cobb angles were measured from C2–C3 to C7–T1. Results In all, 1,134 cervical spinal segments from 189 patients were included. There was a 0.26 ± 0.85-mm average increase in the diameter of the ligamentum flavum from flexion to extension, and 62.70% of the segments had increased ligamentum flavum diameter from flexion to extension. For all segments of the 189 patients, the cervical spinal canal diameters had an average decrease at the disk level of 0.56 ± 1.21 mm from flexion to extension. For all segments with cervical spinal canal narrowing ≥1 mm from flexion to extension view, the ligamentum flavum diameters at C3–C4 to C5–C6 had significant increases compared with patients with spinal canal narrowing < 1 mm (p < 0.05). For patients with ligamentum flavum hypertrophy of ≥1 mm from the flexion to extension view, the cervical spinal canal diameters at C2–C3, C4–C5, and C5–C6 had significant decreases compared with patients with ligamentum flavum hypertrophy of <1 mm (p < 0.05). Conclusion The “hidden” hypertrophy of ligamentum flavum was significant at C4–C5 and C5–C6 and significantly contributes to the stenosis of cervical spinal canal in the extension position.