Takumi Ikeda

Bone marrow-derived cells from the footprint infiltrate into the repaired rotator cuff

BACKGROUND Cells from the bone marrow are considered important during the rotator cuff repair process, but the kinetics of bone marrow-derived cells in this process is unknown. PURPOSE To analyze the kinetics of bone marrow cells during the rotator cuff repair process, to review whether or not they are histologically involved in rotator cuff healing, and to analyze the biomechanics of the repaired tissues. METHODS Bone marrow chimeric rats that express green fluorescent protein (GFP) only in bone marrow- and circulation-derived cells were created. Bilateral supraspinatus tendons were separated from the greater tuberosity of the humeral head to produce a rotator cuff transection model. Drilling into the bone marrow was performed in the greater tuberosity of the right humerus and the supraspinatus tendon was repaired (drilling group), while the supraspinatus tendon was repaired on the left shoulder without drilling (control group). We examined the histology of the rotator cuff, the ultimate force-to-failure, and the proportion of GFP-positive cells in the repaired rotator cuff at 2, 4 and 8 weeks after surgery. RESULTS Mesenchymal cells were observed in the repaired rotator cuff at 2 weeks in both groups. There were more GFP-positive cells in the drilling group than the control group at 2, 4 and 8 weeks. The ultimate force-to-failure was significantly higher in the drilling group than the control group at 4 and 8 weeks. CONCLUSION Bone marrow-derived cells passed through holes drilled in the humerus footprint, infiltrated the repaired rotator cuff and contributed to postsurgical rotator cuff healing.

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Is posterior spinal cord shifting by extensive posterior decompression clinically significant for multisegmental cervical spondylotic myelopathy

Study Design. Posterior cervical spinal cord shifting after selective single laminectomy associated with partial laminotomies was compared with that after bilateral open-door laminoplasty between the C3 and C7 levels in relation to the clinical results of each procedure. Objectives. To investigate the clinical significance of posterior spinal cord shifting after extensive cervical laminoplasty. Summary of Background Data. Current techniques used for cervical laminoplasty for multisegmental cervical spondylotic myelopathy (CSM) are consecutively performed between the C3 and C6 or C7 levels with expectation that the spinal cord will shift backward to keep it clear of anterior compression. However, the clinical significance of the posterior spinal cord shifting remains controversial, and there has been no report verifying it by comparing limited posterior decompression procedures with conventional extensive ones. Methods. Twenty-six patients with consecutive 2- to 3-level CSM who underwent selective laminoplasty (Group A) were enrolled in the study, and among 56 CSM patients who underwent bilateral open-door laminoplasty between the C3 and C7 levels, 25 who had consecutive 2- or 3- level stenosis identified by preoperative magnetic resonance imaging were used as controls (Group B). The recovery rate was calculated using preoperative and postoperative Japanese Orthopedic Association (JOA) scores for each patient, and for each patient’s magnetic resonance imaging, the postoperative cervical curvature index was obtained according to Ishihara’s method and the magnitude of postoperative backward shifting of the spinal cord was measured. Results. There was no significant difference between the subjects in Groups A and B with respect to the spinal curvature index, preoperative JOA scores, and recovery rate, but the magnitude of the postoperative posterior shifting of the spinal cord was greater for those in Group B than for those in Group A. There was no correlation between the recovery rate and posterior shifting of the spinal cord for each group, and no correlation was also found between the curvature index and posterior shifting of the spinal cord. Conclusions. The outcome of posterior decompression surgery for multisegmental CSM is not correlated with the magnitude of postoperative backward shifting of the spinal cord. Extensive and consecutive decompression performed in conventional cervical laminoplasties is therefore not always necessary for multisegmental CSM.

In vivo evaluation of rabbit sciatic nerve regeneration with diffusion tensor imaging (DTI): correlations with histology and behavior

Diffusion tensor imaging (DTI) is widely used in the study of the central nervous system. DTI represents a potential diagnostic tool for the peripheral nerve. However, more detailed information is needed for application of DTI in the clinical setting. In this study, peripheral degeneration and regeneration were evaluated using DTI-based analyses in a rabbit model. The changes in DTI parameters were compared to histological and functional changes after nerve injury. We used a high magnetic field (7.04T) MRI system. Japanese white male rabbits were used as the model of sciatic nerve crush injury. MR images were obtained before injury and at 2, 4, 6 and 8 weeks post-injury. The DTI parameters of fractional anisotropy (FA), axial diffusivity (λ||), and radial diffusivity (λ⊥) were calculated. Our results showed decreased FA and increased λ⊥ during the degenerative phase after sciatic nerve injury. In contrast, increased FA and decreased λ⊥ were observed during the regenerative phase. FA changes were correlated with axon number and with motor function recovery, assessed with the toe-spreading index. This study clearly demonstrates the validity of applying DTI parameters to the in vivo evaluation of peripheral nerve regeneration. Furthermore, results suggest that DTI can be a potent tool for predicting the extent of functional recovery after peripheral nerve injury.

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

Three-dimensional morphology and kinematics of the craniovertebral junction in rheumatoid arthritis.

Study Design. A case-series study. Objectives. To measure the 3-dimensional (3D) morphology and kinematics of the craniovertebral junction (CVJ) using a 3D computed tomography (CT) model; to reveal abnormal patterns and the relationships between pathology and kinematics. Summary of Background Data. Evaluations using radiography, 2-dimensional (2D) CT and magnetic resonance imaging have limitations because of the complex 3D structure of the CVJ. Methods. Twenty-four rheumatoid arthritis patients (21 females, 3 males) with cervical involvement underwent CT scanning of the cervical spine from the basilar process of the occipital bone to the first thoracic vertebra in neutral and flexed positions. The 3D morphology of the occipital condyle, atlas, and axis were classified based on the type of deformity observed. Periodontoid lesions (continuous bony lesions between the atlas and the odontoid process) were also noted. The 3D kinematics in the atlanto-occipital and atlantoaxial joints were evaluated using the volume merge method. Results. Deformities in the atlanto-occipital joints appeared more frequently than those in the atlantoaxial joints. The most common instability pattern was flexural rotation during flexion at the CVJ. The direction of translational motions during flexion was posterior in the atlanto-occipital joint and anterior and caudal in the atlantoaxial joint. Conclusion. The results suggest that bilateral occipital condyle deformation, unilateral and bilateral mass collapse, and periodontoid lesions may affect flexion/extension rotational instability in the atlantoaxial joint. In addition, unilateral occipital condyle deformation and atlantoaxial joint stability may affect sagittal translational instability to the posterior side in the atlanto-occipital joint. The noninvasive 3D CT imaging technique employed here would be useful for predicting the prognosis of patients with rheumatoid deformities at the CVJ.

The clinical usefulness of preoperative dynamic MRI to select decompression levels for cervical spondylotic myelopathy.

The study subjects included 54 patients with cervical spondylotic myelopathy who underwent a selective laminoplasty. The patients were divided into three groups according to the number of decompressed levels: two levels, three levels and four or five levels. The number of cord compressions at every intervertebral level was determined in the flexion, neutral, and extension position using a dynamic magnetic resonance imaging (MRI) scan in consideration of both static and dynamic compressions. For each group, the clinical outcomes were evaluated. Moreover, the patients were divided into two groups according to their age. Then, the appearance ratios of cord compression between the neutral and extension position were compared at each intervertebral level. The clinical outcomes were satisfactory. There were no statistical differences among the three groups, except for the age and operation time. The position of the neck influenced the number of cord compressions. The appearance ratios of cord compression, which were especially prominent at C2/3, C3/4 and C4/5, showed high scores in the aged. The preoperative dynamic MRI scan was clinically useful. In the aged, attention should be given to C2/3, C3/4 and C4/5.

Hepatocyte growth factor/c-met promotes proliferation, suppresses apoptosis, and improves matrix metabolism in rabbit nucleus pulposus cells in vitro

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti‐apoptotic and anti‐inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H2O2 or TNF‐α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase‐2 and matrix metalloproteinase‐3 and ‐9 by TNF‐α was significantly suppressed by HGF treatment. Expression of c‐Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF‐α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c‐Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities.

Effect of therapeutic insoles on the medial longitudinal arch in patients with flatfoot deformity: a three-dimensional loading computed tomography study.

BACKGROUND Insoles are frequently used in orthotic therapy as the standard conservative treatment for symptomatic flatfoot deformity to rebuild the arch and stabilize the foot. However, the effectiveness of therapeutic insoles remains unclear. In this study, we assessed the effectiveness of therapeutic insoles for flatfoot deformity using subject-based three-dimensional (3D) computed tomography (CT) models by evaluating the load responses of the bones in the medial longitudinal arch in vivo in 3D. METHODS We studied eight individuals (16 feet) with mild flatfoot deformity. CT scans were performed on both feet under non-loaded and full-body-loaded conditions, first with accessory insoles and then with therapeutic insoles under the same conditions. Three-dimensional CT models were constructed for the tibia and the tarsal and metatarsal bones of the medial longitudinal arch (i.e., first metatarsal bone, cuneiforms, navicular, talus, and calcaneus). The rotational angles between the tarsal bones were calculated under loading with accessory insoles or therapeutic insoles and compared. FINDINGS Compared with the accessory insoles, the therapeutic insoles significantly suppressed the eversion of the talocalcaneal joint. INTERPRETATION This is the first study to precisely verify the usefulness of therapeutic insoles (arch support and inner wedges) in vivo.

Effects of stress-shielding on the dynamic viscoelasticity and ordering of the collagen fibers in rabbit Achilles tendon.

We investigated the effects of stress‐shielding on both viscoelastic properties and microstructure of collagen fibers in the Achilles tendon by proton double‐quantum filtered (1H‐DQF) NMR spectroscopy. The right hind‐limbs of 20 Japanese white rabbits were immobilized for 4 weeks in a cast with the ankle in plantarflexion. Dynamic viscoelasticity of the Achilles tendons was measured using a viscoelastic spectrometer. Proton DQF NMR signals were analyzed to determine the residual dipolar coupling of bound water molecules in the Achilles tendons. Both the dynamic storage modulus (E′) and dynamic loss modulus (E″) decreased significantly in the Achilles tendons of the stress‐shielding group. The results of the 1H‐DQF NMR examination demonstrated significantly reduced residual dipolar coupling in the Achilles tendons of this same group. The disorientation of collagen fibers by stress‐shielding should contribute to degradation of the dynamic storage and loss moduli. The alterations of the collagen fiber orientation that contributed to the function of tendinous tissue can be evaluated by performing an analysis of 1H DQF NMR spectroscopy.