Naohisa Miyakoshi

In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with bone morphogenetic protein-4

This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation.

Development and evaluation of C-telopeptide enzyme-linked immunoassay for measurement of bone resorption in mouse serum

The mouse is increasingly being used as an animal model for the study of skeletal phenotypes in humans, mainly because of the ease of genetic manipulation. Biochemical markers of bone metabolism provide a valuable parameter for the assessment of skeletal metabolism. In the mouse model, assays for bone formation have been available for a long time; however, little is known about bone resorption markers. The present study describes the development of a serum C-telopeptide enzyme-linked immunoassay (ELISA), which measures degradation products of type I collagen that are generated by osteoclastic bone resorption. The C-telopeptide ELISA uses affinity-purified antibodies generated against human sequence DFSFLPQPPQEKAHDGGR. The epitope involves an amino acid sequence, which is identical in the mouse and human C-terminal peptide of type I collagen (alpha1 chain). Sensitivity of the ELISA used was <0.1 ng/mL. The average intra- (n = 10) and interassay (n = 8) coefficient of variation for two controls was <12%. The average dilution and spike recovery rates were 98% and 97%, respectively. Application of the ELISA to measure C-telopeptide in 3-4-week postovariectomized (ovx) C57BL/6J (B6) mice (n = 9 or 10) showed a 45% higher C-telopeptide concentration than the sham-operated mice. Treatment of ovx mice with estradiol (400 microg/kg body weight) or alendronate (1.0 mg/kg body weight) resulted in a 20%-50% decrease in C-telopeptide levels compared to the vehicle-treated ovx group. In addition, B6 mice fed a calcium-deficient diet (0.01% calcium) showed a 50% higher C-telopeptide concentration compared to the B6 mice receiving a normal diet (0.6% calcium). In conclusion, the C-telopeptide ELISA exhibited acceptable analytical performance and sufficient discriminatory power to show expected directional changes in the rate of bone resorption following ovariectomy, ovx plus estradiol or alendronate treatment, and administration of a calcium-deficient diet. Therefore, the ELISA developed in this study could be used for measuring bone resorption in the mouse model.

ACDF with a PEEK cage clinically provides a good outcome with minor donor site morbidity despite unsatisfactory radiological findings-A prospective cohort study of a PEEK cage in stand-alone usage-

Study Design A prospective cohort study was conducted on patients with anterior cervical decompression and fusion (ACDF) with a polyetheretherketone cage (PEEKc). Background Advantages of a PEEKc have been proposed in the study. However, benefits of using a PEEKc in ACDF are still controversial. Objective To investigate the advantages of a PEEKc in ACDF. Materials and Methods A total of 27 patients was enrolled in the study. The mean age of patients was 55±10 years (mean±standard deviation). The mean duration of symptoms was 17±21 months. Surgery was conducted at C3/4 in 1, C4/5 in 3, C5/6 in 11, C6/7 in 9, C7/T1 in 2, and C5/6/7 in 1 patient. The mean follow-up period was 2.1±1.3 years. Clinical outcomes were analyzed by the Japanese Orthopedic Association Scores (JOA scores) and its recovery rate. Perioperative complications were also investigated. Radiologically, studies were conducted on interbody lordotic angle (IBLA), interbody height (IBH), and bone fusion rates. Results The JOA score was 14.7±1.4 preoperatively and 16.3±1.3 at the final follow-up. A significant improvement was observed (p<0.05). The mean recovery rate of JOA scores was 74.0±25.0%. The preoperative IBLA was 0.5±6.1°. The mean IBLA at the final follow-up was 1.9±5.6°. The preoperative IBH was 34.2±3.5 mm. The mean IBH at the final follow-up was 34.3±3.5 mm. No significant improvement in IBLA and IBH was observed. A complete union rate at 1 year and 2.3 years (range, 2.0-6.0) after surgery was 29% (8/28 segments) and 61% (11/18 segments). No major complications were observed. Conclusions Despite an unsatisfactory bone union rate and no significant improvement in IBLA and IBH at the final follow-up, ACDF with a PEEKc clinically provided a stable outcome with less surgical invasion and minor donor-site morbidity.

Sacroiliac joint pain after multiple-segment lumbar fusion: a long-term observational study-Non-fused sacrum vs. fused sacrum

Introduction Sacroiliac joint pain (SIJP) after lumbar fusion surgery has recently gained attention as a source of low back pain after lumbar fusion. There are two risk factors for postoperative SIJP, i.e., fusion involving the sacrum and multiple-segment fusion. In this study, we examined whether SIJP could occur more frequently in patients with two risk factors (multiple-segment fusion to sacrum). Further, we examined SIJP after multiple-segment (≥3) lumbar fusion, focusing on the difference between floating fusion (non-fused sacrum) and fixed fusion (fused sacrum). Methods Ninety-one patients who underwent multiple-segment lumbar fusion were included. Patients without preoperative clinical SIJP were considered. Of these, 17 developed new-onset SIJP. We investigated postoperative SIJP development, duration from surgery to SIJP onset, and postoperative treatment outcomes of SIJP patients using Japanese Orthopaedic Association (JOA) scores. We compared the findings between floating fusion group and fixed fusion group. Results The incidence of SIJP was significantly higher with fixed fusion (32.1%) than with floating fusion (12.7%). The mean time of onset of sacroiliac joint pain was at 8.63 (2-13) months after surgery in the floating fusion group and 3.78 (1-10) months after surgery in the fixed fusion group, indicating that incidence occurred significantly earlier in the fixed fusion group. Our treatment outcome indicated that the mean JOA score significantly improved in the floating fusion group from 5.13 at the time of onset to 9.50 at the time of final follow-up; however, in the fixed fusion group, it improved from 5.78 at the time of onset to 7.33 at the time of final follow-up, indicating no significant improvement. Conclusions In multiple-segment lumbar fusion, fixed fusion (fused sacrum) has a very high risk of SIJP. In addition, the onset of SIJP in such cases may occur earlier. This aspect deserves consideration, given the difficulty of pain treatment.