Postoperative Changes of Spinopelvic Sagittal Parameters After Cervical Laminoplasty for Cervical Spondylotic Myelopathy.

STUDY DESIGN: A retrospective analysis of prospectively collected data. OBJECTIVE: To investigate postoperative changes of spinopelvic sagittal parameters after laminoplasty for cervical spondylotic myelopathy (CSM) accompanying postoperative cervical kyphotic deformity or cervical regional sagittal imbalance. SUMMARY OF BACKGROUND DATA: To the best of our knowledge, no study has been reported concerning postoperative changes of spinopelvic sagittal parameters accompanying postoperative deterioration of cervical sagittal alignment or balance after cervical laminoplasty. METHODS: Forty-five CSM patients without preoperative cervical kyphosis who underwent laminoplasty were included. None of the 45 patients had a medical history of previous spine surgery, hip joint surgery, or knee joint surgery. The patients were divided into 2 groups (kyphosis and lordosis groups) according to postoperative C2-7 angle, and they were also divided into 2 other groups (imbalance and balance groups) according to postoperative C1-7 sagittal vertical axis. Postoperative changes (Δ) of T1 slope (T1S), thoracic kyphosis, thoracolumbar kyphosis (TLK), lumbar lordosis (LL), Pelvic tilt, and C7 sagittal vertical axis were measured comparing lateral radiographs of the whole spine in the standing position taken at 1 year postoperatively with those before surgery. RESULTS: Both T1S and TLK significantly decreased after cervical laminoplasty in the kyphosis group compared with the lordosis group. On the other hand, both T1S and TLK increased significantly, and LL significantly decreased after surgery in the imbalance group compared with the balance group. CONCLUSIONS: At 1 year after laminoplasty for CSM, both T1S and TLK significantly decreased accompanying postoperative cervical kyphotic deformity as a compensatory action for postoperative cervical kyphosis to maintain the global sagittal balance of the spine, whereas both T1S and TLK increased significantly, and LL significantly decreased accompanying postoperative cervical reginal sagittal imbalance which resulted in postoperative forward inclination of the whole spine.

Responsiveness of the Zurich Claudication Questionnaire, the Oswestry Disability Index, the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire, the 8-Item Short Form Health Survey, and the Euroqol 5 dimensions 5 level in the assessment of patients with lumbar spinal stenosis.

PURPOSE: To assess the responsiveness of the Zurich Claudication Questionnaire (ZCQ), the Oswestry Disability Index, the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire, the visual analog scale (VAS), the 8-Item Short Form Health Survey (SF-8), and the EuroQol 5 dimensions 5 level as methods of assessing outcomes of surgery for lumbar spinal stenosis. METHODS: We analyzed 218 patients who had undergone lumbar surgery for spinal stenosis and completed one year of follow-up. The internal responsiveness of each questionnaire and any domains was assessed by the effect size and standardized response mean. External responsiveness was assessed by the Spearman rank correlation coefficient and the receiver operating characteristics (ROC) curve. RESULTS: The most responsive assessments were "symptom severity" and "physical function" on the ZCQ, "walking ability" on the JOABPEQ, "leg pain" on the VAS, and "social function" on the JOABPEQ. The moderately responsive assessments were the physical component summary on the SF-8, the ODI, the EQ5D-5L, "low back pain" on the JOABPEQ, and "leg numbness" on the VAS. The least responsive assessments were "low back pain" on the VAS, "mental health" and "lumbar function" on the JOABPEQ, and the mental component summary on the SF-8. CONCLUSIONS: Because of its high responsiveness, "symptom severity" on the ZCQ is recommended as a primary tool for assessing outcome when designing prospective studies for lumbar spinal stenosis.

Early Fusion Status After Posterior Lumbar Interbody Fusion With Cortical Bone Trajectory Screw Fixation or Traditional Trajectory Screw Fixation: A Comparison Between the Titanium-coated Polyetheretherketone Cage and the Same Shape Polyetheretherketone Cage.

STUDY DESIGN: A retrospective analysis of prospectively collected data. OBJECTIVE: The aim was to examine whether posterior lumbar interbody fusion with cortical bone trajectory screw fixation (CBT-PLIF) is inferior to PLIF using traditional trajectory screw fixation (TT-PLIF) regarding early fusion status and whether the titanium-coated polyetheretherketone (PEEK) cage (TP cage) improves early fusion status compared with the same shape pure PEEK cage (P cage). SUMMARY OF BACKGROUND DATA: We recently reported that the fusion rate was relatively lower after CBT-PLIF than after TT-PLIF, although no significant difference was found and that compared with the carbon PEEK cage, the TP cage significantly reduced the incidence of vertebral endplate cysts (cyst signs) and slightly improved the early fusion rate at 1 year after CBT-PLIF. MATERIALS AND METHODS: The subjects were 37 patients undergoing TT-PLIF using P cages (TT-P group), 24 patients undergoing CBT-PLIF using P cages (CBT-P group), 32 patients undergoing TT-PLIF using TP cages (TT-TP group), and 20 patients undergoing CBT-PLIF using TP cages (CBT-TP group). On multiplanar reconstruction computed tomography at 6 months after surgery, cyst signs were evaluated and classified into diffuse or local cysts. Early fusion status was assessed using both dynamic plain radiographs and multiplanar reconstruction computed tomography at 1-year postoperatively. RESULTS: The incidence of the diffuse cyst was 27.0% in the TT-P group, 29.2% in the CBT-P group, 25.0% in the TT-TP group and 25.0% in the CBT-TP group (P>0.05). The early fusion rate was 75.7% in the TT-P group, 75.0% in the CBT-P group, 71.9% in the TT-TP group and 75.0% in the CBT-TP group (P>0.05). CONCLUSIONS: These results indicate that CBT-PLIF resulted in the equivalent early fusion status to TT-PLIF and that compared with the same shape P cage, the TP cage did not improve early fusion status after both TT-PLIF and CBT-PLIF.

Surgical outcomes after posterior lumbar interbody fusion using traditional trajectory screw fixation or cortical bone trajectory screw fixation: A comparative study between the polyetheretherketone cage and the same shape titanium-coated polyetheretherketone cage.

OBJECTIVE: To investigate whether or not the fusion rate after posterior lumbar interbody fusion with cortical bone trajectory screw fixation (CBT-PLIF) is lower than after PLIF using traditional trajectory screw fixation (TT-PLIF) and whether or not the titanium-coated polyetheretherketone (PEEK) cage (TiP cage) improves fusion status compared to the same shape uncoated PEEK cage (P cage). METHODS: The subjects were 37 patients undergoing TT-PLIF using P cages (P-TT group), 24 patients undergoing CBT-PLIF using P cages (P-CBT group), 32 patients undergoing TT-PLIF using TiP cages (TiP-TT group), and 20 patients undergoing CBT-PLIF using TiP cages (TiP-CBT group). All patients from the 4 groups underwent our unified PLIF procedure (total facetectomy, subtotal discectomy, and the same bone graft technique using the same shape cages) except for the screw trajectories and the surface materials of the cages. Clinical symptoms were assessed using the Japanese Orthopedic Association (JOA) score before surgery and at 2-year postoperatively. None of age at the time of surgery, gender, fused segment and preoperative JOA score showed significant differences among the 4 groups. On multiplanar reconstruction computed tomography (MPR-CT) at 6months after surgery, vertebral end plate cysts were evaluated and classified into local or diffuse cysts. Fusion status was assessed using both dynamic plain radiographs and MPR-CT at postoperative 2-year. RESULTS: Neither the mean JOA score nor the mean recovery rate of the JOA score at 2-year after surgery showed significant differences among the 4 groups. The incidence of the diffuse cyst (a known predictor of non-union) was 27.0% in the P-TT group, 29.2% in the P-CBT group, 25.0% in the TiP-TT group and 25.0% in the TiP-CBT group (P > 0.05). The fusion rate was 89.2% in the P-TT group, 91.7% in the P-CBT group, 90.6% in the TiP-TT group and 90.0% in the TiP-CBT group (P > 0.05). CONCLUSIONS: After our unified PLIF procedure except for the screw trajectories and the surface materials of the cages, CBT-PLIF resulted in the equivalent fusion rate to TT-PLIF and the TiP cage did not lead to the improved fusion rate compared to the same shape P cage.

Epidemiology of symptomatic ossification of the posterior longitudinal ligament: a nationwide registry survey.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is radiographically detectable in 3-6% of Asian individuals, although not all detectable OPLL cases lead to myelopathy. To date, it is unknown how many patients suffer from neurological symptoms due to OPLL. The purpose of this study was to investigate the epidemiology of symptomatic OPLL using Japan's national registry database. METHODS: We examined the registry data of patients with OPLL who held a certificate of medical subsidy from the Japanese Ministry of Health, Labor and Welfare. The study period was from January 1, 2011 to December 31, 2012. RESULTS: Registry data revealed that the incidence and the period prevalence of symptomatic OPLL were 0.005% (5 per 100,000 population) and 0.027% (27 per 100,000 population), respectively. OPLL occurred twice as often in men as in women. The peak age for onset of symptoms was 60-69 years. The mean Japanese Orthopedic Association (JOA) score was 9 points. Ninety percent of OPLL patients underwent surgery, and 90% of these surgeries were performed with a posterior approach. The most common indication for surgery was a JOA score of 11 points. CONCLUSIONS: According to registry data, the prevalence of symptomatic OPLL was less than one-hundredth of that of radiographically detected OPLL. This indicates that most cases of radiographically detectable OPLL may be asymptomatic.

Does Atherosclerosis Have Negative Impacts on Early Adjacent Segment Degeneration After Posterior Lumbar Interbody Fusion?

STUDY DESIGN: Retrospective study. OBJECTIVE: To examine whether atherosclerosis has negative impacts on early adjacent segment degeneration (ASD) after posterior lumbar interbody fusion using traditional trajectory pedicle screw fixation (TT-PLIF). METHODS: The subjects were 77 patients who underwent single-level TT-PLIF for degenerative lumbar spondylolisthesis. Using dynamic lateral radiographs of the lumbar spine before surgery and at 3 years postoperatively, early radiological ASD (R-ASD) was examined. Early symptomatic ASD (S-ASD) was diagnosed when neurologic symptoms deteriorated during postoperative 3-year follow-up and the responsible lesions adjacent to the fused segment were also confirmed on magnetic resonance imaging. According to the scoring system by Kauppila et al, the abdominal aortic calcification score (AAC score: a surrogate marker of systemic atherosclerosis) was assessed using preoperative lateral radiographs of the lumbar spine. RESULTS: The incidence of early R-ASD was 41.6% at the suprajacent segment and 8.3% at the subjacent segment, respectively. Patients with R-ASD had significantly higher AAC score than those without R-ASD. The incidence of early S-ASD was 3.9% at the suprajacent segment and 1.4% at the subjacent segment, respectively. Patients with S-ASD had higher AAC score than those without S-ASD, although there was no significant difference. CONCLUSIONS: At 3 years after surgery, the advanced AAC had significantly negative impacts on early R-ASD after TT-PLIF. This result indicates that impaired blood flow due to atherosclerosis can aggravate degenerative changes at the adjacent segments of the lumbar spine after PLIF.

Two-level Posterior Lumbar Interbody Fusion at the Lumbosacral Segment has a High Risk of Pseudarthrosis and Poor Clinical Outcomes: Comparison Between the Lumbar and Lumbosacral Segments.

STUDY DESIGN: This was a retrospective study. OBJECTIVES: The purposes of this study were to investigate the fusion rate and clinical outcomes of 2-level posterior lumbar interbody fusion (PLIF). SUMMARY OF BACKGROUND DATA: PLIF provides favorable clinical outcomes and a high fusion rate. However, most extant studies have been limited to the results of single-level PLIF. Clinical outcomes and fusion rate of 2-level PLIF are unknown. MATERIALS AND METHODS: In total, 73 patients who underwent 2-level PLIF below L3 between 2008 and 2016 (follow-up period >2 y) were included. Patients were divided into the 2 groups on the basis of surgical level. The lumbar group included 48 patients who underwent L3/4/5 PLIF, and the lumbosacral group included 25 patients who underwent L4/5/S PLIF. Fusion rate and clinical outcomes were compared. The Japanese Orthopedic Association Back Pain Evaluation Questionnaire (JOABPEQ) and a visual analog scale were used for evaluation. RESULTS: Fusion rate was significantly lower in the lumbosacral group (lumbar 96% vs. lumbosacral 64%; P<0.001). Eight of 9 cases of pseudarthrosis occurred at the lumbosacral segment. Improvement in the mental health domain of the JOAPEQ was significantly lower in the lumbosacral group (lumbar 16 vs. lumbosacral 10; P=0.02). The VAS data showed that improvements in the following variables were significantly lower in the lumbosacral group than in the lumbar group: pain in low back (lumbar -38 vs. lumbosacral -23; P=0.004), pain in buttocks or lower leg (lumbar -48 vs. lumbosacral -29; P=0.04), and numbness in buttocks or lower leg (lumbar -44 vs. lumbosacral -33; P=0.04). CONCLUSIONS: Two-level PLIF at the lumbosacral segment demonstrated a significantly lower fusion rate and poorer clinical outcomes than that at the lumbar-only segments. Some reinforcement for the sacral anchor is recommended to improve fusion rate, even for short fusion like 2-level PLIF, if the lumbosacral segment is included. LEVEL OF EVIDENCE: Level III.

Early cephalad adjacent segment degeneration after posterior lumbar interbody fusion: a comparative study between cortical bone trajectory screw fixation and traditional trajectory screw fixation.

OBJECTIVE: Cortical bone trajectory (CBT) screw insertion through a caudomedial starting point provides advantages in limiting dissection of the superior facet joints and reducing muscle dissection and the risk of superior-segment facet violation by the screw. These advantages of the cephalad CBT screw can result in lower rates of early cephalad adjacent segment degeneration (ASD) after posterior lumbar interbody fusion (PLIF) with CBT screw fixation (CBT-PLIF) than those after PLIF using traditional trajectory screw fixation (TT-PLIF). Here, the authors investigated early cephalad ASD after CBT-PLIF and compared these results with those after TT-PLIF. METHODS: The medical records of all patients who had undergone single-level CBT-PLIF or single-level TT-PLIF for degenerative lumbar spondylolisthesis (DLS) and with at least 3 years of postsurgical follow-up were retrospectively reviewed. At 3 years postoperatively, early cephalad radiological ASD changes (R-ASD) such as narrowing of disc height (> 3 mm), anterior or posterior slippage (> 3 mm), and posterior opening (> 5°) were examined using lateral radiographs of the lumbar spine. Early cephalad symptomatic adjacent segment disease (S-ASD) was diagnosed when clinical symptoms such as leg pain deteriorated during postoperative follow-up and the responsible lesion suprajacent to the fused segment was confirmed on MRI. RESULTS: One hundred two patients underwent single-level CBT-PLIF for DLS and were followed up for at least 3 years (CBT group). As a control group, age- and sex-matched patients (77) underwent single-level TT-PLIF for DLS and were followed up for at least 3 years (TT group). The total incidence of early cephalad R-ASD was 12.7% in the CBT group and 41.6% in the TT group (p < 0.0001). The incidence of narrowing of disc height, anterior slippage, and posterior slippage was significantly lower in the CBT group (5.9%, 2.0%, and 4.9%) than in the TT group (16.9%, 13.0%, and 14.3%; p < 0.05). Early cephalad S-ASD developed in 1 patient (1.0%) in the CBT group and 3 patients (3.9%) in the TT group; although the incidence was lower in the CBT group than in the TT group, no significant difference was found between the two groups. CONCLUSIONS: CBT-PLIF, as compared with TT-PLIF, significantly reduced the incidence of early cephalad R-ASD. One of the main reasons may be that cephalad CBT screws reduced the risk of proximal facet violation by the screw, which reportedly can increase biomechanical stress and lead to destabilization at the suprajacent segment to the fused segment.

Abdominal Aortic Calcification Is a Significant Poor Prognostic Factor for Clinical Outcomes After Decompressive Laminotomy for Lumbar Spinal Canal Stenosis.

STUDY DESIGN: Retrospective study. OBJECTIVE: To examine whether the presence of chronic kidney disease (CKD) or advanced abdominal aortic calcification (AAC) negatively affects clinical outcomes after decompression surgery for lumbar spinal canal stenosis (LSCS). METHODS: The subjects comprised 143 patients who underwent decompressive laminotomy for LSCS and were followed for ≥2 years. Fifty-five patients had CKD (Stage 3-4). Clinical outcome was assessed using the Japanese Orthopaedic Association (JOA) score before surgery and at 2-year postoperatively. According to the scoring system by Kauppila et al, the AAC score (a surrogate marker of systemic atherosclerosis) was assessed using preoperative lateral radiographs of the lumbar spine. RESULTS: Patient age had weak but significantly negative correlations with both the preoperative JOA score and the JOA score at 2 years after surgery, but did not have a significant correlation with the recovery rate of the JOA score at 2 years postoperatively. The JOA score before surgery, the JOA score at 2-year follow-up, and the recovery rate of the JOA score were slightly lower in the CKD patients than in those without CKD, although there were no significant differences between the 2 groups. On the contrary, the AAC score had a weak but significantly negative correlation with the preoperative JOA score, and had relatively strong and significantly negative correlations with both the JOA score at 2 years after surgery and the recovery rate of the JOA score. CONCLUSIONS: At 2 years after surgery, advanced AAC was a significant poor prognostic factor for clinical outcomes of decompression surgery for LSCS.

Synthesis of α-Methylene-γ-lactone Structure by Cyclization of ω-Formylallylsilane in Water.

Surfactant-type protonic acid-promoted intramolecular cyclization of functionalized allylsilanes was studied in water for the synthesis of α-methylene-γ-lactone compounds. ω-Formyl-ß-(acetoxymethyl)allylsilane afforded carbocyclic compounds in good yields, while the cyclization product was not obtained from the corresponding ß-ethoxycarbonyl derivative. It was found that (Z)-ß-(acetoxymethyl)allylsilane predominantly afforded the cis-product, while (E)-ß-(acetoxymethyl)allylsilane afforded both cis- and trans-products at a ratio of almost 1 : 1. The stereoselectivity of the cyclization reaction was almost the same as a protonic acid-promoted reaction in CH2Cl2 and was explained by an interaction between the C(Si)-C(alkene) bond and the carbonyl moiety. The cyclization products were converted to α-methylene-γ-lactone compounds.

Preoperative retrolisthesis as a predictive risk factor of reoperation due to delayed-onset symptomatic foraminal stenosis after central decompression for lumbar canal stenosis without fusion.

BACKGROUND CONTEXT: For patients diagnosed with lumbar central canal stenosis with asymptomatic foraminal stenosis (FS), surgeons occasionally only decompress central stenosis and preserve asymptomatic FS. These surgeries have the potential risk of converting preoperative asymptomatic FS into symptomatic FS postoperatively by accelerating spinal degeneration, which requires reoperation. However, little is known about delayed-onset symptomatic FS postoperatively. PURPOSE: This study aimed to evaluate the rate of reoperation for delayed-onset symptomatic FS after lumbar central canal decompression in patients with preoperative asymptomatic FS, and determine the predictive risk factors of those reoperations. STUDY DESIGN: This study is a retrospective cohort study. PATIENT SAMPLE: Two hundred eight consecutive patients undergoing posterior central decompression for lumbar canal stenosis between January 2009 and June 2014 were included in this study. OUTCOME MEASURES: The number of patients who had preoperative FS and the reoperation rate for delayed-onset symptomatic FS at the index levels were the outcome measures. METHODS: Patients were divided into two groups with and without preoperative asymptomatic FS at the decompressed levels. The baseline characteristics and revision rates for delayed-onset symptomatic FS were compared between the two groups. Predictive risk factors for such reoperations were determined using multivariate logistic regression and receiver operating characteristics analyses. RESULTS: Preoperatively, 118 patients (56.7%) had asymptomatic FS. Of those, 18 patients (15.3%) underwent reoperation for delayed-onset symptomatic FS at a mean of 1.9 years after the initial surgery. Posterior slip in neutral position and posterior extension-neutral translation were significant risk factors for reoperation due to FS. The optimal cutoff values of posterior slip in neutral position and posterior extension-neutral translation for predicting the occurrence of such reoperations were both 1 mm; 66.7% of patients who met both of these cutoff values had undergone reoperation. CONCLUSIONS: This study demonstrated that 15.3% of patients with preoperative asymptomatic FS underwent reoperation for delayed-onset symptomatic FS at the index levels at a mean of 1.9 years after central decompression, and preoperative retrolisthesis was a predictive risk factor for such a reoperation. These findings are valuable for establishing standards of appropriate treatment strategies in patients with lumbar central canal stenosis with asymptomatic FS.

Proximal Junctional Failure After Long-Segment Instrumentation for Degenerative Lumbar Kyphosis With Ankylosing Spinal Disorder.

STUDY DESIGN: Case report. OBJECTIVE: We report a case of proximal junctional failure at the ankylosed, but not the mobile, junction after segmental instrumented fusion for degenerative lumbar kyphosis with ankylosing spinal disorder. SUMMARY OF BACKGROUND DATA: Proximal junctional failure (PJF) and proximal junctional kyphosis (PJK) are important complications that occur subsequent to long-segment instrumentation for correction of adult spinal deformity. Thus far, most studies have focused on the mobile junction as a site at which PJK/PJF can occur, and little is known about the relationship between PJK/PJF and ankylosing spinal disorders such as diffuse idiopathic skeletal hyperostosis. METHODS: The patient was an 82-year-old female with degenerative lumbar kyphosis. She had abnormal confluent hyperostosis in the anterior longitudinal ligaments from Th5 to Th10. The patient was treated operatively with spinal instrumented fusion from Th10 to the sacrum. RESULTS: Four weeks subsequent to initial surgery, the patient developed progressive lower extremity paresis caused by the uppermost instrumented vertebrae fracture (Th10) and adjacent subluxation (Th9). Extension of fusion to Th5 with decompression at Th9-Th10 was performed. However, the patient showed no improvement in neurological function. CONCLUSION: PJF can occur at the ankylosing site above the uppermost instrumented vertebrae after long-segment instrumentation for adult spinal deformity. PJF in the ankylosed spine may cause severe fracture instability and cord deficit. The ankylosed spine should be integrated into the objective determination of materials contributing to the appropriate selection of fusion levels. LEVEL OF EVIDENCE: 3.

Immediate Reduction of a Retro-odontoid Synovial Cyst Following Lateral Atlantoaxial Joint Puncture and Arthrography: A Case Report.

STUDY DESIGN: Case report. OBJECTIVE: We report on a case with a retro-odontoid synovial cyst, and the immediate reduction of the cyst was confirmed after lateral atlantoaxial joint puncture and arthrography. SUMMARY OF BACKGROUND DATA: Retro-odontoid synovial cysts are rare diseases located posteriorly to a dense axis. Because most reports have focused on surgical treatment, only a few have examined nonsurgical treatment. However, several months are required after nonsurgical treatment until cyst regression. METHODS: A 52-year-old female presented with atlantoaxial instability. She complained of neck pain and numbness in her hands. Magnetic resonance imaging revealed a retro-odontoid synovial cyst. Lateral atlantoaxial joint puncture and arthrography were performed. RESULTS: Two days after treatment, the patient showed significant improvement in the numbness of her hands, and a follow-up magnetic resonance imaging revealed an immediate reduction in the cyst. During a 4.5-year follow-up period, no recurrence of the clinical symptoms occurred. CONCLUSION: Lateral atlantoaxial joint puncture may immediately reduce retro-odontoid synovial cysts, and the lateral atlantoaxial joint has a communication channel with the retro-odontoid synovial cyst via the atlantodental joint. Once disappearance of the cyst is confirmed, an acceptable long-term outcome can be achieved with nonsurgical treatment even in cases with atlantoaxial instability. LEVEL OF EVIDENCE: N/A.

SIK3 is essential for chondrocyte hypertrophy during skeletal development in mice.

Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.

Conditional deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, increasing volume of remodeling bone in mice.

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast-specific manner using a knock-in Cre mouse line to the cathepsin K locus (Ctsk(Cre/+);Bmpr1a(flox/flox), designated as Bmpr1a(ΔOc/ΔOc)). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre-dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1a(ΔOc/ΔOc) with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1a(ΔOc/ΔOc) mice. Expression of bone-resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5-, 8-, and 12-week-old Bmpr1a(ΔOc/ΔOc) mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone-formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1a(ΔOc/ΔOc) tibias at 8 weeks of age. For comparison, we also induced an osteoblast-specific deletion of Bmpr1a using Col1a1-Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling.

Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways.

During endochondral bone formation, Sox9 expression starts in mesenchymal progenitors, continues in the round and flat chondrocyte stages at high levels, and ceases just prior to the hypertrophic chondrocyte stage. Sox9 is important in mesenchymal progenitors for their differentiation into chondrocytes, but its functions post-differentiation have not been determined. To investigate Sox9 function in chondrocytes, we deleted mouse Sox9 at two different steps after chondrocyte differentiation. Sox9 inactivation in round chondrocytes resulted in a loss of Col2a1 expression and in apoptosis. Sox9 inactivation in flat chondrocytes caused immediate terminal maturation without hypertrophy and with excessive apoptosis. Inactivation of Sox9 in the last few cell layers resulted in the absence of Col10a1 expression, suggesting that continued expression of Sox9 just prior to hypertrophy is necessary for chondrocyte hypertrophy. SOX9 knockdown also caused apoptosis of human chondrosarcoma SW1353 cells. These phenotypes were associated with reduced Akt phosphorylation. Forced phosphorylation of Akt by Pten inactivation partially restored Col10a1 expression and cell survival in Sox9(floxdel/floxdel) mouse chondrocytes, suggesting that phosphorylated Akt mediates chondrocyte survival and hypertrophy induced by Sox9. When the molecular mechanism of Sox9-induced Akt phosphorylation was examined, we found that expression of the PI3K subunit Pik3ca (p110α) was decreased in Sox9(floxdel/floxdel) mouse chondrocytes. Sox9 binds to the promoter and enhances the transcriptional activities of Pik3ca. Thus, continued expression of Sox9 in differentiated chondrocytes is essential for subsequent hypertrophy and sustains chondrocyte-specific survival mechanisms by binding to the Pik3ca promoter, inducing Akt phosphorylation.

Insulation of the ubiquitous Rxrb promoter from the cartilage-specific adjacent gene, Col11a2.

The retinoid X receptor beta gene (Rxrb) is located just upstream of the alpha2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF.