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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.

Study Design Surgeon survey. Objective To analyze multimodal intraoperative monitoring (MIOM) for different combinations of methods based on the collected data and determine the best combination. Methods A questionnaire was sent to 72 training institutions to analyze and compile data about monitoring that had been conducted during the preceding 5 years to obtain data on the following: (1) types of monitoring; (2) names and number of diseases; (3) conditions of anesthesia; (4) condition of stimulation, the monitored muscle and its number; (5) complications; and (6) preoperative and postoperative manual muscle testing, presence of dysesthesia, and the duration of postoperative motor deficit. Sensitivity and specificity, false-positive rates, and false-negative rates were examined for each type of monitoring, along with the relationship between each type of monitoring and the period of postoperative motor deficit. Results Comparison of the various combinations showed transcranial electrical stimulation motor evoked potential (TcMEP) + cord evoked potential after stimulation to the brain (Br-SCEP) combination had the highest sensitivity (90%). The TcMEP + somatosensory evoked potential (SSEP) and TcMEP + spinal cord evoked potential after stimulation to the spinal cord (Sp-SCEP) combinations each had a sensitivity of 80%, exhibiting little difference between their sensitivity and that obtained when TcMEP alone was used. Meanwhile, the sensitivity was as low as 50% with Br-SCEP + Sp-SCEP (i.e., the cases where TcMEP was not included). Conclusions The best multimodality combination for intraoperative spinal cord monitoring is TcMEP + Br-SCEP, which had the highest sensitivity (90%), the lowest false-positive rate (6.1%), and the lowest false-negative rate (0.2%).

There have been no prospective studies comparing anterior surgery and posterior method in terms of long-term outcomes. The purposes of this study is to clarify whether there is any difference in long-term clinical and radiologic outcomes of anterior decompression with fusion (ADF) and laminoplasty (LAMP) for the treatment of cervical spondylotic myelopathy (CSM).Ninety-five patients were prospectively treated with ADF or LAMP for CSM in our hospital from 1996 through 2003. On alternate years, patients were enrolled to receive ADF (1997, 1999, 2001, and 2003: ADF group, n = 45) or LAMP (1996, 1998, 2000, and 2002: LAMP group, n = 50). We excluded 19 patients who died during follow-up, and 25 who were lost to follow-up. Clinical outcomes were evaluated by the recovery rate of the Japanese Orthopaedic Association (JOA) score between the two groups. Sagittal alignment of the C2-7 lordotic angle and range of motion (ROM) in flexion and extension on plain X-ray were measured.Mean age at the time of surgery was 58.3 years in the ADF group and 57.9 years in the LAMP group. Mean preoperative JOA score was 10.0 and 10.5, respectively. Mean recovery rate of the JOA score at 3-5 years postoperatively was significantly higher in the ADF group (p 0.05). Reoperation was required in 1 patient for pseudarthrosis and in 1 patient for recurrence of myelopathy in the ADF group; no patient in the LAMP group underwent a second surgery. There was a significant difference in maintenance of the lordotic angle in the ADF group compared with the LAMP group (p 0.05), but not in ROM.Both ADF and LAMP provided similar good outcomes at 10-year time-point whereas ADF could achieve more satisfactory outcomes and better sagittal alignment at the middle-term. However, the incidence of reoperation and complication in the ADF group were higher than those in the LAMP group.A prospective comparative study (not randomized).

Gene therapy for neuropathic pain requires efficient gene delivery to both central and peripheral nervous systems. We previously showed that an adenoassociated virus serotype 9 (AAV9) vector expressing short-hairpin RNA (shRNA) could suppress target molecule expression in the dorsal root ganglia (DRG) and spinal cord upon intrathecal injection. To evaluate the therapeutic potential of this approach, we constructed an AAV9 vector encoding shRNA against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, but its role in chronic neuropathic pain remains unclear. We intrathecally injected it into the subarachnoid space at the upper lumbar spine of mice 3 weeks after spared nerve injury (SNI). Delivered shTRPV1 effectively suppressed mRNA and protein expression of TRPV1 in the DRG and spinal cord, and it attenuated nerve injury-induced thermal allodynia 10-28 days after treatment. Our study provides important evidence for the contribution of TRPV1 to thermal hypersensitivity in neuropathic pain and thus establishes intrathecal AAV9-mediated gene delivery as an investigative and potentially therapeutic platform for the nervous system.

Object Although multimodal intraoperative spinal cord monitoring provides greater accuracy, transcranial electrical stimulation motor evoked potential (TcMEP) monitoring became the gold standard for intraoperative spinal cord monitoring. However, there is no definite alarm point for TcMEPs because a multicenter study is lacking. Thus, based on their experience with 48 true-positive cases (that is, a decrease in potentials followed by a new neurological motor deficit postoperatively) encountered between 2007 and 2009, the authors set a 70% decrease in amplitude as the alarm point for TcMEPs. Methods A total of 959 cases of spinal deformity, spinal cord tumor, and ossification of the posterior longitudinal ligament (OPLL) treated between 2010 and 2012 are included in this prospective multicenter study (18 institutions). These institutions are part of the Japanese Society for Spine Surgery and Related Research monitoring working group and the study group on spinal ligament ossification. The authors prospectively analyzed TcMEP variability and pre- and postoperative motor deficits. A 70% decrease in amplitude was designated as the alarm point. Results There were only 2 false-negative cases, which occurred during surgery for intramedullary spinal cord tumors. This new alarm criterion provided high sensitivity (95%) and specificity (91%) for intraoperative spinal cord monitoring and favorable accuracy, except in cases of intramedullary spinal cord tumor. Conclusions This study is the first prospective multicenter study to investigate the alarm point of TcMEPs. The authors recommend the designation of an alarm point of a 70% decrease in amplitude for routine spinal cord monitoring, particularly during surgery for spinal deformity, OPLL, and extramedullary spinal cord tumor.

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.

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a(-/-) mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a(-/-) mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1(-/-) and Sema3aosx(-/-) mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin(-/-) and Sema3anestin(-/-) mice) had low bone mass, similar to Sema3a(-/-) mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin(-/-) mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin(-/-) mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a(-/-) mice, such as olfactory development, were identified in Sema3asynasin(-/-) mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a(-/-) mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Cervical sagittal balance has received increased attention as an important determinant of radiological and clinical outcomes. However, no prospective studies have compared the impact of cervical sagittal balance between anterior and posterior surgeries. We previously conducted a prospective study comparing anterior decompression with fusion (ADF) and laminoplasty (LAMP) for degenerative cervical myelopathy (DCM) and reported; however, analysis of cervical alignment within the concept of sagittal balance has yet to be performed, because that concept has recently been proposed. This study aimed to review this prospective cohort, specifically focusing on cervical sagittal balance. We prospectively performed ADF or LAMP for DCM patients based on the year of enrollment: ADF was performed in odd-numbered years and LAMP in even-numbered years. Cervical lateral X-ray images taken in the neutral standing position were evaluated preoperatively and at a 1-year follow-up. The radiographic measurements included the following: (1) CL (cervical lordosis: C2–7 lordotic angle), (2) CGH (center of gravity of the head)-C7 SVA (sagittal vertical axis), and (3) C7 slope. The clinical results were evaluated using the Japanese Orthopedic Association scoring system for cervical myelopathy (C-JOA score). We analyzed the data for 66 patients (ADF n = 28, LAMP n = 38). While the CL and CGH-C7 SVA in the ADF were unchanged after the operation, those in the LAMP group worsened, especially in patients with preoperative cervical sagittal imbalance. The C7 slopes were not affected by the operation in either group. The postoperative decreases in the CL in the LAMP group correlated with the preoperative CGH-C7 SVA (r = 0.618, P

Study design Retrospective case-control study. Objective The purpose of this study was to examine the factors of postoperative paralysis in patients who have undergone thoracic ossification of posterior longitudinal ligament (OPLL) surgery. Summary of background data A higher percentage of thoracic OPLL patients experience postoperative aggravation of paralysis than cervical OPLL patients, including patients that presented great difficulties in treatment. However, there were a few reports to prevent paralysis thoracic OPLL. Methods The 156 patients who had received thoracic OPLL surgery were selected as the subjects of this study. The items for review were the duration of disease; the preoperative muscle strength (Muscle Manual Testing); OPLL levels (T1/2-4/5: high, T5/6-8/9: middle, and T9/10-11/12: low); the spinal canal occupancy ratio; the ratio of yellow ligament ossification as a complication; the ratio of transcranial-motor evoked potential (Tc-MEP) derivation; the preoperative/postoperative kyphotic angles in the thoracic vertebrae; the correction angle of kyphosis; the duration of surgery; and the amount of bleeding. The subjects were divided into two groups based on the absence or presence of postoperative paralysis to determine the factors of postoperative paralysis. Results Twenty-three patients (14.7%) exhibited postoperative paralysis. Multivariate analysis identified factors associated with postoperative paralysis: the duration of disease (odds ratio, OR = 3.3); the correction angle of kyphosis (OR = 2.4); and the ratio of Tc-MEP derivation (OR = 2.2). Conclusion The risk factors of postoperative paralysis are a short duration of disease and a small correction angle of kyphosis. In addition, ratios of Tc-MEP derivation below 50% may anticipate paralysis. Level of evidence 4.

This study was performed to investigate the effects of the co-administration of proton pump inhibitor (PPI) on the efficacy of bisphosphonate (BP) treatment for osteoporosis. A total of 180 women with low bone mineral density were randomly divided into four groups, one in which sodium risedronate was administered with sodium rabeprazole and one in which only risedronate was administered (BP + PPI and BP groups, respectively). The biomarkers were measured at the baseline and every 3 months, inlcuding: N-terminal telopeptide of type I collagen corrected for creatinine, bone-specific alkaline phosphatase (BAP), parathyroid hormone, bone mineral density (BMD) of the lumbar spine and physical parameters evaluated according to the SF-36v2™ Health Survey. Statistical comparisons of these parameters were performed after 9 months. Data were available for a total of 137 patients (62 in the BP group and 75 in the BP + PPI group). The Δ % value of increase in BMD and improvement of physical functioning in the BP + PPI group were significantly larger, and its decrease in BAP in the BP + PPI group was significantly smaller than that in the BP group. It is expected that risedronate administration in combination with a PPI may be more effective not only for treating osteoporosis but also improving physical fitness than treatment with risedronate alone.

Flap necrosis remains a major complication of reconstructive surgery. To improve skin flap survival, various treatments with vasodilators, antiplatelet drugs, or the local administration of growth factors have been performed. However, the sufficient prevention of skin necrosis is not well established. Platelet-rich plasma has been used as an autologous factor and includes various growth factors. The authors evaluated whether or not platelet-rich plasma can improve skin flap survival in an experimental rat model.Cranially based dorsal cutaneous flaps were elevated in 48 rats. The animals received subcutaneous injections of either platelet-rich plasma (100 μl) or platelet-poor plasma (100 μl). The rats were divided into three groups: the platelet-rich plasma group (n = 16), the platelet-poor plasma group (n = 16), and the nontreatment group (n = 16). Flap survival was measured and histologic specimens were collected on day 7. Real-time polymerase chain reaction specimens were collected after 8 hours, 24 hours, 3 days, and 7 days.Platelet-rich plasma significantly improved flap survival rates (61.2 percent) compared with the platelet-poor plasma treatment (35.8 percent) and nontreatment groups (28.0 percent). A histologic analysis showed that significantly fewer inflammatory cells and an increased blood vessel density were observed in the platelet-rich plasma rats versus the platelet-poor plasma or nontreatment rats. In addition, platelet-rich plasma treatment significantly increased the mRNA levels of vascular endothelial growth factor and platelet-derived growth factor.Platelet-rich plasma modulates the genes involved in angiogenesis and improves skin flap survival.

Gene therapy for neurological diseases requires efficient gene delivery to target tissues in the central and peripheral nervous systems. Although adeno-associated virus is one of the most promising vectors for clinical use against neurological diseases, it is difficult to get it across the blood-brain barrier. A clinically practical approach to using a vector based on adeno-associated virus to decrease the expression of a specific gene in both the central and the peripheral nervous system has yet to be established. Here, we analyzed whether upper lumbar intrathecal administration of a therapeutic vector incorporating adeno-associated virus and short-hairpin RNA against superoxide dismutase-1 bypassed the blood-brain barrier to target the spinal cord and dorsal root ganglia. The therapeutic vector effectively suppressed mRNA and protein expression of endogenous superoxide dismutase-1 in the lumbar spinal cord and dorsal root ganglia. Moreover, neither neurological side effects nor toxicity due to the incorporated short-hairpin RNA occurred after the injection. We propose that this approach could be developed into novel therapies for motor neuron diseases and chronic pain conditions, such as complex regional pain syndrome, through silencing of the genes responsible for pathologies in the spinal cord and dorsal root ganglia.

STUDY DESIGN A retrospective single-center study. OBJECTIVE To investigate how functional recovery is influenced by anterior compression of the spinal cord (ACS) and instability at the level of ACS after laminoplasty in patients with cervical spondylotic myelopathy. SUMMARY OF BACKGROUND DATA There have been many reports that patients whose spinal cord cannot be decompressed sufficiently after laminoplasty are likely to show unsatisfactory neurologic outcomes. Notably, postoperative ACS is well known to cause problems. Clinically, however, it remains unknown how functional recovery is inhibited by postoperative ACS. METHODS Sixty-four consecutive patients who underwent expansive laminoplasty for the treatment of myelopathy at our hospital between 1998 and 2005 were reviewed. All 64 patients were available for follow-up. The average follow-up period was 97 months (60-156 months). Patients were divided into 2 groups: the ACS(+) group comprised 16 patients who had ACS 3 years postoperatively, and the ACS(-) group comprised 48 patients with no ACS. Clinical outcome was compared in terms of the Japanese Orthopaedic Association score (mean total score, mean score of each item, and recovery rates). RESULTS Demographics were similar between the 2 groups. Mean Japanese Orthopaedic Association score at final follow-up was 12.1 points (recovery rate 34.0%) in the ACS(+) group and 13.8 points (recovery rate 56.6%) in the ACS(-) group, and there was a significant difference in recovery rate between the groups (P < 0.05). Notably, a significant difference was found between the 2 groups in improvement of upper extremity motor function (P < 0.05). In addition, we found that not only the presence of ACS but also postoperative hypermobility of the intervertebral segment with ACS influenced clinical outcome negatively. CONCLUSION These results demonstrate that ACS after laminoplasty could be a risk factor for clinical outcome and might prevent improvement in upper extremity motor function in patients with myelopathy.

It is known that Rho family small GTPases activate a number of signal transduction pathways involved in cell cycle progression, gene expression, and cell survival. These small G proteins play an important role in neuronal survival and axon regeneration in neural injury. In this study, we tested whether the activity of RhoA or Rac1 regulates neurite extension in dorsal root ganglia (DRGs) in vitro and nerve regeneration in injured sciatic nerves. Regeneration of neurites from explanted DRGs was accelerated by combined suppression of RhoA and Rac1 activity using adenoviruses expressing dominant negative (DN) forms of both RhoA and Rac1 (Ad-Rho/RacDN) in vitro. Rat sciatic nerves were cut and Ad-Rho/RacDN was injected into the proximal stumps. After bridge grafting with chitosan mesh tubes, muscle evoked potentials induced by transcranial electrical stimulation were recorded eight weeks postoperatively. The terminal latencies were shorter in the Ad-Rho/RacDN group than in the control group. Histological analysis revealed extensive regrowth of neurofilament-positive and myelinated axons within the tubes in the group that received Ad-Rho/RacDN. These findings suggest that combined regulation of RhoA and Rac1 using DN adenoviral transgenic methods has the potential to modify injured peripheral nerve tissues directly.

Background We previously reported the usefulness of neuromagnetic recordings for the diagnosis of disorders in peripheral nerves or the spinal cord. However, there have been no reports on incomplete conduction block of the spinal cord, which is clinically common in conditions such as cervical myelopathy. Here, we estimated the usefulness of measuring spinal cord evoked magnetic fi elds for evaluating incomplete conduction block. Methods Incomplete conduction block models of the spinal cord of the rabbit were established using a Fogarty balloon catheter that was inserted into the epidural space of the cervical spine. Electrical stimuli were applied to the lower thoracic spinal cord with an epidural catheter electrode. Spinal cord evoked potentials were recorded using epidural electrodes. Spinal cord evoked magnetic fi elds were recorded over the skin surface of the neck using a biomagnetometer. Results The decrease in the conduction velocity and amplitude at the compression site could be detected by spinal cord evoked potentials from the epidural space, confi rming the spinal cord lesion. The waveforms of the magnetic fi elds showed a biphasic confi guration. The distribution of magnetic fi elds showed a characteristic quadrupolar pattern propagating from caudal to cranial. After compression, the amplitude and the conduction velocity of the magnetic fi elds decreased, and the distribution of magnetic fi elds were attenuated and decelerated near the compression site especially in the trailing magnetic fi elds. Diagnosis of the incomplete conduction block was thus possible. Conclusions We report the fi rst measurement of the spinal cord evoked magnetic fi eld in the intact spinal cord from the skin surface and that it can be applied to incomplete conduction block of the injured spinal cord. The use of a biomagnetometer is promising as a less-invasive method for clinically evaluating spinal cord function.

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1(f/f) mice and alpha1(II) Runx1(f/f) mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and alpha1(II) DKO mice, respectively]. Prx1 Runx1(f/f) mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, alpha1(II) Runx1(f/f) mice and alpha1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of alpha1(II) collagen (Col2a1 - Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of alpha1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

Trabecular bone fragments can be percutaneously harvested from the ilium using methods that are similar in invasiveness to aspiration of bone marrow. In this study, we investigated the use of the trabecular bone as a cell source for bone tissue engineering. Trabecular bone-derived progenitor cells (TB cells) were isolated with a simple method in which trabecular fragments were first cultured as explants, and then the cells were released by trypsin digestion and advanced to a monolayer culture. The properties of TB cells prepared in this procedure were compared with bone marrow-derived progenitor cells (BM cells). A large number of TB cells could be obtained with less variation among donors, compared with BM cells. In multiple harvests of donor tissue through the same aspiration hole at the cortex, TB cells could be more consistently obtained in primary culture. The proliferative potential of BM and TB cells was similar in serial subculture. TB cells showed a higher alkaline phosphatase expression in the surface marker analysis and greater in vitro osteogenic abilities than BM cells after the initial 14 days of culture. In in vivo bone formation studies, TB cells also showed a higher osteogenic potential than BM cells. The results of this study suggest that TB cells can be considered an attractive source for clinical bone regeneration.

Articular cartilage has a limited capacity for self-renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF-2 to repair large osteochondral defects in a rabbit model. Ninety-six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 microl of FGF-2 at a concentration of 10 or 100 microg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 microl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline-like appearance. These findings suggest that porous HAp/Col with FGF-2 augments the cartilage repair process.

To potentiate the bone formation capability of bone marrow stromal cell (BMSC)/beta-tricalcium phosphate (beta-TCP) constructs, we devised an autologous plasma-based construct. We tested its effectiveness and investigated the effects of its components on a monkey ectopic bone formation model. The autologous plasma (platelet-rich plasma, PRP, or platelet-poor plasma, PPP)/BMSC/beta-TCP construct (R group or P group) showed significantly more bone formation at 3 and 6 weeks after implantation than a conventional BMSC/beta-TCP construct using a culture medium (M group). There was no significant difference between the P and R groups. Moreover, the P group constructs with a 10-fold lower cell concentration yielded equivalent bone formation to the M group at 5 weeks after implantation. To elucidate the effect of fibrin and serum contained in the plasma, five constructs were prepared using the following cell vehicles: autologous serum + fibrinogen (0, 1, 4, or 16 mg/mL) or phosphate-buffered saline + fibrinogen (4 mg/mL). The serum + fibrinogen (4 mg/mL, physiological concentration of monkeys) construct showed the most abundant bone formation at 3 weeks after implantation, though at 5 weeks no statistical difference existed among the groups. Autologous plasma efficiently promoted osteogenesis of BMSCs/porous beta-TCP constructs, and both fibrin and serum proved to play significant roles in the mechanism.

Background. Simultaneous bilateral total hip arthroplasty (THA) can offer a potential benefit of greater postoperative hip motion without the negative influence of contralateral hip disabilities, compared to two-stage THA. However, postoperative changes in hip motion after simultaneous bilateral THA have rarely been reported. The purpose of this study was to clarify the efficacy of simultaneous procedures on postoperative hip motion and functional recovery. Methods. We retrospectively compared hip motion in 27 patients treated with simultaneous bilateral THA to those in 11 patients with two-stage bilateral THA, 35 patients with unilateral THA for unilateral disease, and 15 patients with unilateral THA for bilateral disease. We also evaluated the clinical manifestations according to the Japanese orthopaedic Association (JoA) hip scores and compared the outcomes among the groups. All of the THA surgeries were primarily performed through a posterolateral approach using cementless prostheses. The diagnosis at surgery was dysplastic osteoarthritis in all patients, and the patients were followed up for at least 4 years. Results. The postoperative improvement of motion in hip flexion was significantly greater in patients treated with simultaneous procedures compared to patients with two-stage THA and unilateral THA for bilateral disease. The differences in improvement of motion in hip abduction were less marked than the improvement in hip flexion among the groups. Although there were no significant differences in pain scores among the groups, the values for activity of daily living were significantly greater in patients treated with simultaneous bilateral THA and patients with unilateral THA for unilateral disease than in patients with unilateral and two-stage bilateral THA for bilateral disease. Conclusions. The simultaneous procedure was considered to be more effective in patients with bilateral hip osteoarthritis and demonstrated a substantial improvement in hip motion and functional recovery after THA.

Recent advances in tissue engineering techniques have allowed porous biomaterials to be combined with osteogenic cells for effective bone regeneration. We developed a simple low-pressure cell-loading method using only syringes and stopcocks, and examined the effect of this method on osteogenesis when applied to the combination of highly porous β-tricalcium phosphate (β-TCP) and fresh autologous bone marrow. Both block and granule β-TCP scaffolds were used to prepare implants in three different ways: without bone marrow as a control, with bone marrow that was allowed to penetrate spontaneously under atmospheric pressure (AP group), and with bone marrow that was seeded under low pressure (ULP group). These implants were transplanted into rabbit intramuscular sites, and the samples were examined biologically and histologically. The penetration efficiency of the block implants after marrow introduction was significantly higher in the ULP group than in the AP group. In the transplanted block samples, alkaline phosphatase activity was significantly higher in the ULP group at 2 weeks after implantation, and significantly more newly formed bone was observed in the ULP group at both 5 and 10 weeks compared with the AP group. Similar results were observed even in the experiment using β-TCP granules, which are smaller than the blocks and frequently used clinically. Because of its convenience and safety, this low-pressure method might be a novel, effective treatment to promote osteogenesis with bone marrow in clinical bone reconstruction surgeries. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1–7, 2009

Objective We have previously reported that the measurement of spinal cord evoked magnetic fields (SCEFs) could be a helpful method for evaluating spinal cord function or detecting conduction blocks in the spinal cord. However, there have been no reports about segmental-SCEFs as a complex of axonal and synaptic activities in the spinal cord. The purpose of this study is to record and evaluate segmental-SCEFs. Methods The segmental-SCEFs were measured over the lumbar dural tubes of adult rabbits using our SQUID system following sciatic nerve stimulation; spinal cord evoked potentials (SCEPs) were also measured to compare the results. Results SCEPs showed conductive sharp waves following gentle waves, suggesting action potentials and synaptic potentials, respectively. The isomagnetic field maps of SCEFs showed a quadrupolar pattern propagating from the caudal to the cranial region within a short latency time, and after the conductive magnetic fields passed, stationary dipolar fields appeared and were sustained at some vertebral levels. Conclusions The quadrupolar magnetic fields were estimated to be generated from conducting action potentials, and the dipolar fields were thought to be caused by synaptic activities. Significance Through the measurement of segmental-SCEFs, the conductive neural and synaptic activities in the spinal cord can be visualized and distinguished. This is the first report to record and visualize the sequence of events ranging from the axonal activities of peripheral nerves and the spinal tract to the synaptic activities in the spinal cord.

The causes of early degeneration and calcification of the nucleus pulposus in the chondrodystrophoid dog are poorly understood, and the underlying molecular mechanism of this process has not yet been clearly defined. Type X collagen is one of the key molecules in endochondral bone growth and development, especially matrix calcification. The relationship between type X collagen and disc degeneration and calcification in chondrodystrophoid dogs has not yet been studied. We analyzed the expression of type X collagen in degeneration and calcification of the intervertebral disc in chondrodystrophoid dogs, using type X collagen immunohistochemistry. Control intervertebral discs were collected from five dogs (4 female, 1 male, average age 1.3 years, beagle breed). Degenerated intervertebral discs were surgically removed from 11 canine patients with intervertebral disc extrusion (1 female, 10 male, average age 5.1 years, dachshund breed) in Nippon Veterinary and Animal Science University. All extruded disc samples showed hypertrophic changes and clustering of cells, typical features observed in the degenerated nucleus pulposus. The relative expression of type X collagen in the degenerated nucleus pulposus (84.3 +/- 11.0%) was significantly increased compared to the control nucleus pulposus (5.4 +/- 5.4%). Our findings suggest that type X collagen might contribute to the development of degeneration or calcification in the nucleus pulposus of the chondrodystrophoid dog.

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu-/-) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system-mediated inhibition of bone formation was abolished in Nmu-/- mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50–760 mmHg, 1–10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous β-tricalcium phosphate (β-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/β-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into β-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

The effects of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on bone marrow stromal cells (MSCs) with respect to proliferation, osteogenic differentiation, and bone formation capability were investigated. MSCs derived from rats were cultured in medium containing mixtures of PRP and PPP. Fibrinogen was eliminated prior to the experiment. The DNA content and alkaline phosphatase (ALP) activity were measured. PRP stimulated cell proliferation and inhibited osteoblastic differentiation. To examine the effects of fibrin in plasma, MSCs were cultured in PRP or PPP fibrin gels formed both on a cell culture insert installed in a culture well and on the bottom surface of the same culture well. The ALP activities of the MSCs in both of the gels were higher than those on the surface of the culture wells. The MSCs cultured on the PPP gel showed the highest ALP activity. The effects of PRP and PPP used as scaffolds for bone formation were also investigated. MSCs were suspended in PRP or PPP, introduced into porous β-tricalcium phosphate blocks, and then implanted into subcutaneous sites. Subsequently, bone formation was quantified. Further in vivo studies found that implants prepared using PPP had a greater osteoinductive capability than implants prepared with PRP.

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

ED-71, a novel analog of 1alpha,25-(OH)2 D3, increases bone mass to a greater extent than alfacalcidol, an 1alpha,25-(OH)2 D3 prodrug. In this study, we used a murine bone marrow ablation model to compare the effect of ED-71 on bone formation and resorption in vivo with that of 1alpha,25-(OH)2 D3. We discovered that bone matrix remodeling occurring within the first week after bone marrow ablation was enhanced by a single injection of ED-71, but not by 1alpha,25-(OH)2 D3. This enhancement was associated with an increase in bone surface. Trabecular bone resorption occurring from 1 to 2 weeks after the procedure was suppressed by a single injection of ED-71, but not 1alpha,25-(OH)2 D3, with treated mice exhibiting a reduction in osteoclast numbers, despite increases in osteoblast surface. As seen with the single injection, daily administration of ED-71 also enhanced bone modeling. Bone marrow osteoblast differentiation was also augmented by ED-71 pretreatment. Furthermore, ED-71 treatment immediately after bone marrow ablation enhanced angiogenesis within the bone marrow cavity via enhancement of VEGF(120) expression. In this paper, we clearly demonstrate that ED-71 is an orally administered small molecular weight compound with an anabolic effect on bone metabolism.

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at -20 degrees C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140 degrees C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 microm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.

Hydroxyapatite (HAp) coatings are applied to orthopedic and dental implants made of titanium (Ti) and its alloys in order to increase their bioactivities and to offset the mechanical weakness of HAp. We examined the in vivo effects of electrical polarization on the bone bonding of HAp-coated Ti. Polarized samples with a negatively or positively charged HAp-coated surface (N- or P-surface, respectively) were randomly implanted in the femora and tibiae of canines. As controls, nonpolarized HAp-coated Ti substrates with 0-surfaces were implanted. Direct bonding between the newly formed bone and HAp-coated Ti was observed with the O-, N-, and P-surfaces. The results of a pullout test were consistent with the amount of newly formed bone bonded directly to the surface of HAp-coated Ti. Electrically polarized HAp-coated Ti substrates, especially those with N-surfaces, exposed to recipient bone enhance bone bonding and could enable earlier weight-bearing loads after operations.

Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBPzeta) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBPbeta and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPbeta, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPbeta and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.

Transplantation surgeries of autologous bone require a second surgery with inherent risks. To avoid these risks, we developed a multi porous implant of hydroxyapatite/collagen composite with desirable biophysical properties (flexibility, elasticity and compression resistance) for use with OP-1 as a graft implant. In this study, we tested the efficacy of this multi porous implant as OP-1 carrier using rabbit posterolateral lumbar fusion model (PLF). PLFs were performed in the following 4 groups of 8 New Zealand white rabbits each: autograft, HAp/Col alone, HAp/Col plus 0.3 mg OP-1, and HAp/Col plus 1.2 mg OP-1. At 5 weeks, fusion masses were analyzed by radiographic and biomechanical tests. Implants consisting of HAp/Col plus OP-1 were more effective than autologous bone in promoting spinal fusion. Low dose and high dose OP-1 were equally effective.

In the field of bone tissue engineering using cells combined with scaffolds, it is important to efficiently load cells into porous scaffolds. We devised a novel cell-loading method into porous beta-tricalcium phosphate (β-TCP) blocks. In this study, we compared this method with two conventional cell-loading methods in terms of cell-loading efficiency and in vivo bone formation capability. Bone marrow stromal cells (BMSCs) were obtained from the femurs of Fisher rats. After about 10 days of culture, BMSCs were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using three loading methods: a soaking method, a post low-pressure method and a pre low-pressure method (the novel method). These β-TCP cubes were cross-sectioned and stained with toluidine blue and cell-counted. Cell-loading efficiency was significantly higher when using the novel methods. For the study of in vivo bone formation capability, nearly confluent BMSCs were exposed in an osteogenic medium supplemented with 10-7 M dexamethasone, 50µg/ml L-ascorbic acid phosphate and 10mM β-glycerophosphate for 4 days. These osteogenic cells were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using the three cell-loading methods. Immediately, these β-TCP cubes were implanted at subcutaneous sites in the backs of 7-week-old male Fisher rats and harvested at postoperative 3 and 6 weeks. After cross-sectioning, these sections were stained with hematoxylin and eosin, and the new bone formation area was quantified. Consistent with cell-loading efficiency, in vivo bone formation capability was significantly higher in the novel method at postoperative 6 weeks. We showed the usefulness of the novel cell-loading method in bone tissue engineering.

Hydroxyapatite/hyaluronic acid (HAp/HyA) and hydroxyapatite/chondroitin sulfate (HAp/ChS) microparticles, which show the high adsorption ability of proteins, high biocompatibility and osteoconductivity, are potential scaffolds for a time-controlled BMP release. The present study evaluated the biocompatibility and osteoconductivity of the composites after injection into bone defect. Drilled bone holes were made at tibia and femur of Japanese white rabbits, and HAp/HyA or HAp/ChS was implanted into each bone hole using an injection syringe. After 2 and 4 weeks of implantation, rabbits were sacrificed and histological observations were conducted with HE, TRAP, and ALP staining. Histological observations revealed that HAp/ChS has superior biocompatibility compared with HAp/HyA, and 20% HAp/ChS promotes bone formation as well as osteoblast activities compared with lower ratios of HAp/ChS.

The porous hydroxyapatite/collagen (HAp/Col = 80/20 in weight ratio) composite with unique three-dimensional pore structures was fabricated by the control of ice crystal growth using the liquid N 2 as a cooling medium and subsequent freeze-dry process, and the pore structure and mechanical properties were evaluated by SEM and compression tests. The porous composite had the unidirectionally interconnected micropores along the ice growth direction with a spindle-shaped cross-section (distributed from 40.1 × 11.0 to 110 × 21.8 μm in size). When the container was set on a cooling stage at − 30 °C, the length of the long-axes (distributed from 75.2 to 134.3 μm) reached the range that is necessary to bone tissue penetration. The defined pore structure improved its mechanical resistance against the compression and gave anisotropic compression properties against the pore directions. The porous HAp/Col composite with flexibility and shape recover properties will be a promised biomaterial for bone regeneration and other scaffold for tissue engineering.

This study investigated the effects of the three-dimensional (3-D) pore structure of a porous hydroxyapatite/collagen (HAp/Col) composite on their mechanical properties and in vivo tissue ingrowth. The unique 3-D pore structure, comprising unidirectionally interconnected pores, was fabricated by the unidirectional growth of ice crystals by using a cooling stage and a subsequent freeze-drying process. The unidirectional pores had a spindle-shaped cross section, and their size gradually increased from the bottom to the upper face. The porous composite showed an elastic property and anisotropic compressive strength for the pore directions. While the strength and modulus parallel to the pore axis were 1.3- and twofold higher than those of the porous composite with spherical pores formed randomly, the strength and modulus perpendicular to the pore axis showed the lowest values. The subcutaneous implantations revealed that when compared with the random pores, the unidirectional pores promote the ingrowth of the surrounding tissues into the pores.

Background In the autogenous tendon for anterior cruciate ligament reconstruction, intrinsic fibroblasts are necrotized immediately after surgery, and repopulation and revascularization occur. Vascular endothelial growth factor is considered to be a potent mediator of angiogenesis. Hypothesis An application of vascular endothelial growth factor significantly enhances angiogenesis in the in situ frozen anterior cruciate ligament, and the application significantly affects mechanical properties of the in situ frozen anterior cruciate ligament. Study Design Controlled laboratory study. Methods Right anterior cruciate ligaments from 66 rabbits underwent the freeze-thaw treatment, and animals were then divided into 3 groups. Group I served as a freeze-thaw but otherwise untreated control. In group II, 0.2 mL phosphate-buffered saline alone was applied. In group III, 30 µ g vascular endothelial growth factor was applied. The groups were compared on the basis of histologic revascularization examinations using the Chalkley score, an indicator of the microvessel density, and mechanical evaluations, which included the anterior-posterior translation of the tibia relative to the femur during ± 10 N of anterior-posterior load and the mechanical properties of the anteromedial bundle of the anterior cruciate ligament. Results Group III's Chalkley score was significantly greater than that of groups I and II. The tensile strength and the tangent modulus of anterior cruciate ligaments in groups I, II, and III were significantly lower than those of a normal anterior cruciate ligament, although there were no significant differences among groups I, II, and III. Conclusion Vascular endothelial growth factor, as administered in this study, significantly promoted angiogenesis in the devitalized anterior cruciate ligament with in situ freeze-thaw treatment, but it did not affect the mechanical properties of the in situ frozen-thawed anterior cruciate ligament in the rabbit model. Clinical Relevance An application of the recombinant anterior cruciate ligament is a potential future strategy to enhance revascularization of the autograft in anterior cruciate ligament reconstruction.

Summary Mutations of PQBP-1 (polyglutamine binding protein-1) have been shown recently to cause human mental retardation accompanied by microcephaly at a high frequency. As a first step towards understanding the molecular basis of this developmental anomaly, we analysed developmental expression of PQBP-1 by in situ hybridization, immunohistochemsitry and Western blot analysis. Although it had been shown by Northern blot analysis that PQBP-1 mRNA is expressed in multiple organs in adult mice, our present results revealed that PQBP-1 mRNA and protein are dominantly expressed in the central nervous system (CNS) in embryos and in newborn mice. The mean expression level of PQBP-1 reaches a peak around birth and is down-regulated in adulthood. Furthermore, the expression pattern in the CNS changes remarkably following birth. PQBP-1 mRNA in the cerebral cortex is high in embryos but it rapidly decreases after birth. PQBP-1 mRNA increases in external and internal granular cell layers of the cerebellum from postnatal day 1 (P1) to P5. In addition, expression in the subventricular zone, where neurogenesis occurs, was high from P5 to adulthood. Collectively, these findings suggest that PQBP-1 might be involved in neuronal proliferation and/or maturation. These ideas may be relevant to the insufficient growth of brain structure reported in PQBP-1-linked human mental retardation.

Recently, MMP-7 and MMP-3 have been found to play a crucial role in the natural resorption process of herniated discs. We therefore examined the role of these recombinant human matrix metalloproteinases (rh MMPs) in the treatment of herniated discs.(a) Surgical samples of herniated disc were cultured in the presence or absence of rh MMPs, and wet weight was measured 24h later. (b) The rh MMPs were administered into normal rabbit intervertebral discs, and after 1 week spine samples were stained with Safranin O. (c) The rh MMPs were administered into canine herniated discs in vivo. Myelography and MRI were performed prior to and 1 week after administration. Spine samples were examined histologically. Whole disc tissue was collected, total protein was extracted, and Western blot analysis was performed.(a) Proteoglycan degradation was found in MMP-7, MMP-3, and chymopapain-treated samples. MMP-7 and chymopapain-treated samples displayed a significant loss in wet weight (p0.01). (b) Normal disc tissues after administration of rh MMP-7, MMP-3, and chymopapain showed an extensive loss of Safranin O staining. (c) The rh MMP-7-treated discs had a marked decrease in protruded herniation by MRI. Herniated discs after administration of MMP-7 and chymopapain showed a significant decrease in protruded mass 7 days after administration compared with saline-treated discs when evaluated by myelography (p0.01). The rh MMP-7-treated discs displayed a clear loss of Safranin O staining in the nucleus pulposus. Proteoglycan expression was barely detectable in disc tissues after MMP-7 administration, whereas obvious expression was obtained in saline-treated or untreated disc tissues.Exposure to rh MMP-7 resulted in promising proteoglycan loss in human surgical samples, normal rabbit intervertebral discs, and natural canine herniated discs. Administration of rh MMP-7 may facilitate the resorption process of herniated discs.

The correlation between the insertion torque of a lumbar pedicle screw and the mechanical stability of the screw in the bone has been mentioned in in vitro studies. The purpose of this study was to confirm the factors affecting the insertion torque of such screws in vivo. Also, the contribution of insertion torque to the initial stability of the fusion area was to be analyzed in vivo. A series of 23 cases representing 50 lumbar vertebrae were included in this study, in which we examined bone mineral density using quantitative computed tomography (CT) prior to operation. Two screw shapes were utilized, with the insertion torque for each screw measured at two points in time. The correlation between insertion torque and mineral density was investigated. Screw positions were confirmed on postoperative CT scans, and the effect of the screw thread cutting into the cortex bone was investigated. Radiographic changes at three points during a period of 3 months were also measured, and we then evaluated the interrelations between these changes and insertion torque. Furthermore, the relation between insertion torque and instability at 3 months was investigated. Correlations of insertion torque and bone mineral density depended on screw shape. There was no correlation found with mineral density in the case of cylindrical screws. Insertion torque was not affected by the screw thread cutting into the cortex of bone. As for postoperative alignment changes, no definitive trends could be ascertained, and no interrelations with torque and alignment changes were observed. There is a possibility that insertion torque was related to early-stage stability, but no statistical relation could be determined.

Recently it has been thought that neuromagnetic recording is expected to be a helpful alternative examination, since it can detect neural conduction by non-invasive skin surface recording. In this study, we recorded three-dimensional spinal cord evoked magnetic fields (SCEFs) in the cervical spinal cord of rabbits, and evaluated their usefulness in the examination of spinal cord function. The rabbits were anesthetized and put on a respirator. Electrical stimuli (10 Hz square wave pulses, 0.03 ms in duration, 8–10 mA in intensity) were applied to the lower thoracic spinal cord with an epidural electrode. SCEFs were recorded at 64–96 measurement points on the skin surface over the cervical spine. In addition, spinal cord evoked potentials (SCEPs) were recorded at the skin surface. The recordings were done before and after the disconnection of the cervical spinal cord. Three-dimensional SCEFs were recorded in all animals and the conducting neuronal activity was visualized by magnetic contour map. The three components of the SCEFs showed characteristic patterns, respectively. The positions of action currents were estimated by dipole localization. Although further studies are necessary, three-dimensional recording of SCEFs could be a helpful method to evaluate the function of the spinal cord.

Segmental-SCEFs of rabbits after sciatic nerve stimulation were detected by neuromagnetic recordings, and conductive neural activities and synaptic activities in the spinal cord were able to be visualized. The measurement of segmental-SCEFs could be a helpful method to evaluate the function of the spinal cord.