Takashi Taniyama

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.

Repair of Osteochondral Defects in a Rabbit Model Using a Porous Hydroxyapatite Collagen Composite Impregnated With Bone Morphogenetic Protein-2

Articular cartilage has a limited capacity for spontaneous repair, and an effective method to repair damaged articular cartilage has not yet been established. The purpose of this study was to evaluate the effect of transplantation of porous hydroxyapatite collagen (HAp/Col) impregnated with bone morphogenetic protein-2 (BMP-2). To evaluate the characteristics of porous HAp/Col as a drug delivery carrier of recombinant human BMP-2 (rhBMP-2), the rhBMP-2 adsorption capacity and release kinetics of porous HAp/Col were analyzed. Porous HAp/Col impregnated with different amounts of rhBMP-2 (0, 5, and 25 μg) was implanted into osteochondral defects generated in the patellar groove of Japanese white rabbits to evaluate the effect on osteochondral defect regeneration. At 3, 6, 12, and 24 weeks after operation, samples were harvested and subjected to micro-computed tomography analysis and histological evaluation of articular cartilage and subchondral bone repair. The adsorption capacity was 329.4 μg of rhBMP-2 per cm(3) of porous HAp/Col. Although 36% of rhBMP-2 was released within 24 h, more than 50% of the rhBMP-2 was retained in the porous HAp/Col through the course of the experiment. Defects treated with 5 μg of rhBMP-2 showed the most extensive subchondral bone repair and the highest histological regeneration score, and differences against the untreated defect group were significant. The histological regeneration score of defects treated with 25 μg of rhBMP-2 increased up to 6 weeks after implantation, but then decreased. Porous HAp/Col, therefore, is an appropriate carrier for rhBMP-2. Implantation of porous HAp/Col impregnated with rhBMP-2 is effective for rigid subchondral bone repair, which is important for the repair of the smooth articular surface.

Modified K-line in magnetic resonance imaging predicts insufficient decompression of cervical laminoplasty.

Study Design. A retrospective single-center study. Objective. To clarify preoperative factors predicting unsatisfactory indirect decompression after laminoplasty in patients with cervical spondylotic myelopathy. Summary of Background Data. Many authors have shown that inadequate indirect decompression after laminoplasty can inhibit neural recovery and should be considered a complication. We previously demonstrated that residual anterior compression of the spinal cord (ACS) impaired recovery of upper extremity motor function. Although the K-line has been established as a predictive index indicating that laminoplasty is required in patients with ossification of the posterior longitudinal ligament, it remains unclear what preoperative factors can predict insufficient posterior cord decompression in patients with cervical spondylotic myelopathy. Methods. Forty-six consecutive patients who underwent laminoplasty for the treatment of cervical spondylotic myelopathy at our hospital were reviewed. A modified K-line was defined as the line connecting the midpoints of the spinal cord at C2 and C7 on a T1-weighted sagittal magnetic resonance image. We also determined the minimum interval between the tip of local kyphosis and a line connecting the midpoint of the cord at the level of the inferior endplates of C2 and C7 (INTmin) on the midsagittal image. Data analysis involved logistic regression and receiver operating characteristic curve analysis to select the most valuable index for predicting postoperative ACS. Results. Ten patients had ACS immediately after laminoplasty. Logistic regression analysis showed that INTmin was a significant predictive factor for the occurrence of postoperative ACS (odds ratio = 0.485; 95% confidence interval = 0.29–0.81; P = 0.02). Receiver operating characteristic curve analysis showed an area under the curve of 0.871. A cutoff of 4.0 mm had a sensitivity of 80% and a specificity of 80.6% for prediction of postoperative ACS. Conclusion. The parameter INTmin correlated with the occurrence of postoperative ACS. A cutoff point of 4.0 mm is most appropriate for alerting spine surgeons to a high likelihood of postoperative ACS.

Modified K-line in magnetic resonance imaging predicts clinical outcome in patients with nonlordotic alignment after laminoplasty for cervical spondylotic myelopathy.

Study Design. Retrospective single-center study. Objective. To investigate whether a preoperative index predicts clinical outcome after laminoplasty for cervical spondylotic myelopathy. Summary of Background Data. This is the first study using the modified K-line, which connects the midpoints of the spinal cord at the C2 and C7 levels on midsagittal magnetic resonance imaging, to assess the relationship between postoperative clinical outcome and anticipated degree of spinal cord shifting. Methods. Sixty-one consecutive patients who underwent laminoplasty for the treatment of cervical spondylotic myelopathy between 2000 and 2011 at our hospital were retrospectively reviewed. The interval between the preoperative mK-line and the anterior structure of the spinal canal at each segment of the C3 to C6 levels (INTn, n = 3–6) were measured on sagittal T1-weighted magnetic resonance imaging, and the sum of the INTn (INTsum) was then calculated. The degree of posterior cord shift was defined as follows: %Csum = &Sgr;Cn; Cn = (Bn−An) × 100/An (n = 3–6; An and Bn represent the preoperative and postoperative intervals between the midpoint of the spinal cord and the anterior impingement at each segment on sagittal T1-weighted magnetic resonance imaging, respectively). In addition, we defined INTmin as the minimum interval of the INTn in each patient. All patients were divided into lordotic and nonlordotic groups on the basis of lateral neutral radiography. The Japanese Orthopaedic Association (JOA) scoring system and recovery rate of the JOA score for cervical myelopathy was evaluated as clinical outcomes. Results. The recovery rate of the JOA score was 48.1%. The lordotic and nonlordotic groups contained 38 and 23 patients, respectively. Linear regression analysis revealed that INTmin was significantly correlated with the recovery rate of the patients in the nonlordotic group, whereas INTsum was not associated with recovery of the JOA score. Conclusion. We identified INTmin as a predictive factor for clinical outcomes in patients with nonlordotic alignment after laminoplasty. Level of Evidence: 4

Bone Defect Regeneration by a Combination of a β-Tricalcium Phosphate Scaffold and Bone Marrow Stromal Cells in a Non-Human Primate Model

Background: Reconstruction of large bone defects is a great challenge in orthopedic research. In the present study, we prepared composites of bone marrow-derived stromal cells (BMSCs) and β-tricalcium phosphate (β-TCP) with three novel aspects: proliferation of BMSCs with continuous dexamethasone treatment, cell loading under low pressure, and use of autologous plasma as the cell loading medium. The effectiveness of the resulting composite for large bone-defect reconstruction was tested in a non-human primate model, and the bone union capability of the regenerated bones was examined. Materials and Methods: Primary surgery: Bone defects (5 cm long) were created in the left femurs of nine cynomolgus monkeys with resection of the periosteum (five cases) or without resection (four cases), and porous β-TCP blocks were transplanted into the defects. Secondary surgery: Bone marrow aspirates harvested from seven of the nine monkeys were cultured with dexamethasone, and BMSCs were obtained. BMSCs were suspended in autologous plasma and introduced into a porous β-TCP block under low-pressure conditions. The BMSC/β-TCP composites were transplanted into bone defects created at the same sites as the primary surgery. Bone union evaluation: Five regenerated femurs were shortened by osteotomy surgery 8 to 15 months after transplantation of the β-TCP/BMSC composites, and bone union was evaluated radiographically. Results: After the primary surgery and treatment with β-TCP alone, one of the five periosteum-resected monkeys and two of the four periosteum-preserved monkeys exhibited successful bone reconstruction. In contrast, five of the seven cases treated with the β-TCP/MSC composite showed successful bone regeneration. In four of the five osteotomy cases, bone union was confirmed. Conclusion: We validated the effectiveness of a novel β-TCP/BMSC composite for large bone defect regeneration and confirmed the bone union capability of the regenerated bone.

Porous/dense Composite Hydroxyapatite for Anterior Cervical Discectomy and Fusion

Study Design. A prospective analysis Objective. Our aim was to investigate the efficacy of new synthetic porous/dense composite hydroxyapatite (HA) for use in anterior cervical discectomy and fusion (ACDF). Summary of Background Data. Iliac crest bone graft (ICBG) has been traditionally used as the “gold standard” for ACDF. The significant complication rate associated with harvesting tricortical ICBG, however, has encouraged development of alternative graft substitutes. Methods. The morphology of the porous/dense HA was observed by scanning electron microscopy (SEM), and the in vitro compressive strength of the composite HA was measured. From April 2005, 51 consecutive patients underwent 81 levels of ACDF using the composite HA with percutaneously harvested trephine bone chips. Clinical and radiological evaluation was performed during the postoperative hospital stay and at follow-up. Furthermore, the outcomes in ACDF using the composite HA were compared with those using tricortical ICBG. Results. The SEM images demonstrated 100- to 300-&mgr;m pores (approximately 40% of porosity) in the porous layers of the HA. The compressive strength of the composite HA was 203.1 ± 4.1 MPa. In the clinical study, the demographic data of the patients were similar in HA and ICBG groups. The fusion rates in HA group were comparable with those in ICBG group. The cervical lordosis was enhanced postoperatively in both groups and well preserved at 2-year follow-up without significant differences between the groups. The intraoperative blood loss in HA group was significantly lesser than that in ICBG group. Donor site complications were found in 29.2% of the patients in ICBG group, whereas no donor site morbidity was found in HA group. No major collapse or fragmentation of the composite HA was found. Conclusion. The hybrid graft of composite HA and percutaneously harvested trephine chips seemed promising as a graft substitute for ACDF. Level of Evidence: 4

After repeated division, bone marrow stromal cells express inhibitory factors with osteogenic capabilities, and EphA5 is a primary candidate

The differentiation capability of human bone marrow stromal cells (hBMSCs) is thought to deteriorate over multiple doubling processes. To clarify the deterioration mechanisms, the multilineage differentiation capabilities of short- and long-term passaged BMSCs were compared. Predictably, long-term passaged BMSCs showed reduced differentiation capacities compared to short-term passaged cells. Furthermore, a non-human primate heterotopic bone formation model demonstrated that long-term passaged BMSCs have bone formation capabilities but also exert inhibitory effects on bone formation. This finding indicated that long-term passaged BMSCs express higher levels of inhibitory factors than short-term passaged BMSCs do. Co-culture assays of short- and long-term passaged BMSCs suggested that the inhibitory signals required cell-cell contact and would therefore be expressed on the cell membrane. A microarray analysis of BMSCs identified ephrin type-A receptor 5 (EphA5) as an inhibitory factor candidate. Quantitative PCR revealed that among all members of the ephrin and Eph receptor families, only the expression of EphA5 was increased by BMSC proliferation. A gene knockdown analysis using siRNAs demonstrated that knockdown of EphA5 gene expression in long-term passaged BMSCs led to an increase in ALP mRNA expression. These results indicate that EphA5 may be a negative regulator of bone formation. A better understanding of the roles of the ephrin and Eph receptor families in hBMSCs may lead to alternative approaches for manipulating hBMSC fate. In addition, this avenue of discovery may provide new therapeutic targets and quality-control markers of the osteogenic differentiation capabilities of hBMSCs.

Dynamic changes in spinal cord compression by cervical ossification of the posterior longitudinal ligament evaluated by kinematic computed tomography myelography.

Study Design. A prospective clinical study. Objective. To investigate the dynamic causative factor in the pathogenesis of myelopathy in patients with cervical ossification of the posterior longitudinal ligament (OPLL) using kinematic computed tomography (CT) myelography. Summary of Background Data. Kinematic CT myelography is useful for dynamically evaluating the cervical spine with high-resolution images, particularly in bony compressive lesions. However, no studies have evaluated the dynamic factors in patients with OPLL using kinematic CT myelography. Methods. From 2008 to 2013, 51 consecutive patients with OPLL who presented with myelopathy were prospectively enrolled in this study. The patients were examined with kinematic (flexion-extension) CT myelography using a multidetector CT scanner. The range of motion at C2–C7 from flexion to extension was measured in the sagittal view. The segmental range of motion, anterior-posterior diameter and cross-sectional area (CSA) of the spinal cord were measured at the level where the spinal cord was most compressed by OPLL. Results. The neurological condition of the patients evaluated by Japanese Orthopaedic Association scores were 10.8 ± 2.4 points. The mean range of motion at C2–C7 and at the most compressed segment were 23.1 ± 11.7 and 7.0 ± 4.4°, respectively. Both the anterior-posterior diameter and the CSA at the most compressed levels were significantly decreased during neck extension compared with flexion. Interestingly, the anterior-posterior diameter and the CSA were decreased during neck flexion in 13.7% (7/51) of the patients. All 7 of these patients had massive OPLL with an occupying rate 60% or more. The dynamic change rate of CSA (flexion/extension) was significantly smaller in patients with an OPLL occupying rate 60% or more compared with patients with an occupying rate less than 60%. Conclusion. Although spinal cord compression was increased during neck extension in most of the patients, greater levels of compression could be placed on the spinal cord during neck flexion when the patients had OPLL with a high occupying rate. Level of Evidence: 4

Massive bone reconstruction with heat‐treated bone graft loaded autologous bone marrow‐derived stromal cells and β‐tricalcium phosphate composites in canine models

Bone marrow‐derived stromal cells (BMSCs) contain mesenchymal stem cells that are capable of forming various mesenchymal tissues. We hypothesized that BMSCs and β‐tricalcium phosphate (β‐TCP) composites would promote the remodeling of large‐sized autologous devitalized bone grafts; therefore, the aim of this study was to evaluate the effects of the composites on the remodeling of autologous devitalized bone grafts. Autologous BMSCs cultured in culture medium containing dexamethasone (10−7 M) were loaded into porous β‐TCP granules under low‐pressure. Theses BMSC/TCP composites were put into the bone marrow cavity of autologous heat‐treated bone (femoral diaphysis, 65‐mm long, 100°C, 30 min) and put back to the harvest site. In the contralateral side, β‐TCP without BMSC were used in the same manner as the opposite side as the control. Treatment with the BMSC/TCP composites resulted in a significant increase in thickness, bone mineral density, and matured bone volume of the cortical bone at the center of the graft compared to the control. Histological analysis showed matured regenerated bone in the BMSC loaded group. These results indicate that BMSC/TCP composites facilitated bone regeneration and maturation at the graft site of large‐sized devitalized bone. This method could potentially be applied for clinical use in the reconstruction of large bone defects such as those associated with bone tumors.

Spinal epidural abscess caused by Bacteroides fragilis group after dilation and curettage for incomplete abortion

Spinal epidural abscess (SEA) is a rare infection complicated in patients who have some risk factors such as injection-drug use, diabetes mellitus, and several illnesses. However, no case of SEA associated with abortion has been reported. Here we report a case of SEA in a 30-year-old woman after dilation and curettage for incomplete abortion. The diagnosis of SEA was done by MRI and pus was drained after the cervical discectomy. Bacteroides fragilis group was cultured from the aspirated pus sample. The patient responded to surgical drainage and antibiotics.