Yanlin Tan

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.

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.

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 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.

Analysis of Relationship between Paraspinal Muscle Fatty Degeneration and Cervical Spine Motion Using Kinetic Magnetic Resonance Imaging

The alignment and mobility of the cervical spine is influenced by factors related to the vertebral bodies, intervertebral discs, ligaments, facet joints, and muscles. Few reports have described the role played by the paraspinal muscles in cervical spine mobility. In this study, we investigate the relationship between fatty degeneration of the paraspinal muscles and cervical motion as assessed with kinetic magnetic resonance imaging (kMRI). One hundred eighty-eight symptomatic patients underwent cervical kMRI in neutral, flexion, and extension positions. We quantified cervical paraspinal muscle fatty infiltration and measured angular variation and translational motion at each cervical level, and the global Cobb angle. Cervical paraspinal muscle fatty degeneration demonstrated a pattern in which C3 and C7 had significantly more fatty infiltration than C4, C5, and C6. Additionally, when the normal group was compared with the fatty degeneration group with respect to angular variation, translational motion, and Cobb angle, no significant differences were found except in angular variation at the C3–C4 level. In conclusion, we found a significantly larger quantity of fatty degeneration in the paraspinal muscles at C3 and C7 than the middle cervical levels. Also, we demonstrate that fatty degeneration does not significantly affect cervical lordotic alignment or mobility characteristics.

Dynamic Evaluation of the Cervical Spine and the Spinal Cord of Symptomatic Patients Using a Kinetic Magnetic Resonance Imaging Technique.

Purpose: The purpose of this study was to examine the movement of the spinal cord and its relationship to the spinal canal in patients with mild spondylosis using kinetic magnetic resonance imaging (kMRI). Methods: Weight-bearing, multiposition kMRI was performed on symptomatic patients through a full range of flexion-extension. A total of 52 study patients were selected based on the C2–C7 Cobb angle of sagittal alignment: lordotic (from 30 to 45 degrees). We evaluated dynamic changes in different parameters from flexion-extension: spinal canal diameter (CD), spinal cord diameter (SCD), space available for the cord, anterior space available for the cord (ASAC), posterior space available for the cord (PSAC), average distance between the anterior canal and the cord (d-value), and global angle for the spinal canal and cord. Results: The CD tended to decrease from flexion to extension from C3/C4 to C6/C7, however, there were no significant differences at the proximal and distal levels, C2/C3 and C7/T1. There were no significant differences of SCD between different postures. The SCD tended to decrease from C2/C3 to C7/T1. The ASAC followed the same pattern as CD-values. The ASAC was narrowest at C4/C5 and C5/C6. The PSAC tended to increase from C2/C3 to C7/T1. The spinal cord shifted anteriorly with extension and posteriorly with flexion. In addition, the spinal cord maintained its curve with the movement. Conclusions: The kinematics of spinal cord motion may be associated with the pathogenesis of cervical spondylotic myelopathy. However, the spinal cord maintains its curve with position changes. Consequently, different motions of the cervical spine may affect spinal cord migration and cause changes in ASAC and PSAC.

The effect of bone morphogenetic protein-2 injection at different time points on intervertebral disk degeneration in a rat tail model.

Study Design: Prospective in vivo rat tail model of disk degeneration comparing the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) injection over various time points and grades of degeneration. Objective: To evaluate the effect of timing and disk grade on rhBMP-2 injection in a rat tail model of disk degeneration. Summary of Background Data: rhBMP-2 stimulates the proliferation of intervertebral disk cells and the secretion of extracellular matrix. However, few in vivo studies have demonstrated whether rhBMP-2 also improves disk degeneration and the severity of disk degeneration beyond which disks cannot be recovered by rhBMP-2 treatment. Methods: Two coccygeal disks of each rodent subject were punctured percutaneously using an 18 G needle. At 4 weeks after the puncture, disks demonstrating induced degeneration were divided into 3 groups. Groups 1, 2, and 3 were treated with 7.5 &mgr;g rhBMP-2 or phosphate buffered saline by injection into the disk at 4, 6, and 8 weeks postpuncture, respectively. Plain radiographs and magnetic resonance images (MRIs) were obtained on the day of puncture and every 2 weeks thereafter until sacrifice. At 6 weeks after injection, each group was killed and examined with histologic and immunohistochemical analysis. Results: According to MRI disk grade evaluation of the degenerative disk, rhBMP-2 significantly improved degeneration grade in group 1 at 2 weeks after injection. According to radiographic disk height index, groups 1 and 2 showed a trend toward improvement at 2 weeks after rhBMP-2 injection. Chondrogenic differentiation was noted on immunohistochemical staining of many disks treated with rhBMP-2. Conclusions: rhBMP-2 injection of degenerated disks at 4 weeks postpuncture induced a transient improvement in disk grade on MRI and stimulated chondrogenic differentiation. These data suggest rhBMP-2 as a potential therapy for degenerative disk disease.