Ichiro Torigoe

Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor-2 (FGF-2)

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 µl of FGF‐2 at a concentration of 10 or 100 µg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 µl 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.

Middle-Term Results of a Prospective Comparative Study of Anterior Decompression With Fusion and Posterior Decompression With Laminoplasty for the Treatment of Cervical Spondylotic Myelopathy

Study Design. A clinical prospective study. Objective. To assess whether clinical and radiologic outcomes differ between anterior decompression and fusion (ADF) and laminoplasty (LAMP) in the treatment of cervical spondylotic myelopathy (CSM). Summary of Background Data. No reports to date have accurately and prospectively compared middle-term clinical outcomes after anterior and posterior decompression for CSM. Methods. We prospectively performed LAMP (n = 50) in 1996, 1998, 2000, and 2002, and ADF (n = 45) in 1997, 1999, 2001, and 2003. The Japanese Orthopedic Association (JOA) score, recovery rate, and each item of the JOA score were evaluated. For radiographic evaluation, the lordotic angle and range of motion (ROM) at C2–C7 and residual anterior compression to the spinal cord (ACS) after LAMP on magnetic resonance imaging were investigated. Results. Eighty-six patients (ADF n = 39; LAMP n = 47) could be followed for more than 5 years (follow-up rate 91.5%). Demographics were similar between the two groups. The mean JOA score and recovery rate in the ADF group were superior to those in the LAMP group from 2-year data collected after surgery. However, LAMP was safer and less invasive than ADF with respect to physical status and complications in the perioperative period. For individual items of the JOA score, the ADF group showed significantly more improvement of upper extremity motor function than the LAMP group (P < 0.05). There was a significant difference in maintenance of the lordotic angle in the ADF group compared with the LAMP group despite no difference in ROM. The LAMP group was divided into two subgroups: (1) LAMP(+) (n = 16) comprising patients who had ACS at 2 years after surgery, and (2) LAMP(–) (n = 31) comprising patients without ACS. Recovery rate differed significantly between the LAMP(+) and LAMP(−) groups despite there being no difference between the LAMP(−) and ADF groups. Conclusion. The recovery rate of the JOA score in the ADF group was better than that in the LAMP group. The clinical outcomes after LAMP could be influenced by ACS.

Fresh bone marrow introduction into porous scaffolds using a simple low-pressure loading method for effective osteogenesis in a rabbit model.

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.

Cervical Sagittal Imbalance is a Predictor of Kyphotic Deformity After Laminoplasty in Cervical Spondylotic Myelopathy Patients Without Preoperative Kyphotic Alignment.

Study Design. A retrospective cohort study. Objective. The aim of this study is to investigate the preoperative factors for postlaminoplasty kyphotic deformity in cervical spondylotic myelopathy (CSM) patients without preoperative kyphotic alignment focused on the cervical sagittal balance. Summary of Background Data. After laminoplasty (LAMP), appropriate decompression may be obtained when cervical lordosis is maintained to allow the posterior shift of the spinal cord. Therefore, LAMP is not suitable for patients with preoperative cervical kyphosis. However, we sometimes encounter patients who developed postoperative kyphosis despite normal preoperative alignment. The risk factors of postlaminoplasty kyphotic deformity for the patients without preoperative kyphotic alignment are not well known. Methods. A total of 174 consecutive patients who received a double-door LAMP for CSM without preoperative kyphotic alignment and completed a 1-year follow-up were enrolled. Cervical lateral X-ray images obtained in the standing position were measured at the preoperative stage and during a 1-year follow-up visit. The radiographic measurements included the following: (1) C2-7 lordotic angle (C2-7 angle), (2) C2-7 range of motion (C2-7 ROM), (3) CGH (center of gravity of the head)-C7 SVA, and (4) C7 slope. The clinical results were evaluated using the Japanese Orthopedic Association score system for cervical myelopathy (C-JOA score). Results. Postoperative kyphotic deformity was observed in 9 patients (5.2%). The recovery rates of the C-JOA scores at the 1-year follow-up period in the kyphotic deformity (+) group were inferior to those of the kyphotic deformity (−) group. The CGH-C7 SVA and advanced age were detected as preoperative risk factors using multivariate analysis. Cutoff values for predicting postlaminoplasty kyphotic deformity were a CGH-C7 SVA = 42 mm and an age of 75 years. Conclusion. Cervical sagittal imbalance and advanced age were the preoperative risk factors for kyphotic deformity after LAMP for CSM in patients without preoperative cervical kyphotic alignment. Level of Evidence: 4

Bone Regeneration with Autologous Plasma, Bone Marrow Stromal Cells, and Porous β-Tricalcium Phosphate in Nonhuman Primates

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.

Novel cell seeding system into a porous scaffold using a modified low-pressure method to enhance cell seeding efficiency and bone formation.

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.

Effects of gamma-ray irradiation on mechanical properties, osteoconductivity, and absorption of porous hydroxyapatite/collagen.

In this study, the effects of gamma-ray irradiation on the mechanical properties, absorbability, and osteoconductivity of porous hydroxyapatite/collagen (HAp/Col) were investigated. Porous HAp/Col was exposed to 16, 25, 35, or 50 kGy of gamma-ray irradiation. The compressive elastic modulus showed irradiation dose-dependence, with a particularly pronounced decrease in the 50-kGy treatment group. An in vitro enzymatic digestion test showed that gamma-ray irradiation of porous HAp/Col resulted in accelerated degradation by collagenase. For in vivo studies, porous HAp/Col was transplanted into the back muscles or bone defects in the femoral condyle of rats. Specimens were obtained at 2, 4, and 8 weeks postoperatively. Absorption of the implants in the muscle was time- and irradiation dose-dependent, with notable absorption for the 35- and 50-kGy groups at 2 weeks. At the skeletal sites, porous HAp/Col demonstrated high osteoconductivity in all irradiation treatment groups. Interestingly, not only implant absorption but also bone formation was irradiation dose-dependent at early time points.

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.

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.

145. Alarm point of muscle evoked potentials after brain stimulation in intraoperative spinal cord monitoring

Mechanisms of the dementia have gradually elucidated in aging society. Recently, there are many researches with MRI, SPECT, and PET focused on the mild cognitive impairments (MCI), for the establishment of the early diagnosis of MCI. However, it is a pivotal node to assess clinically the higher brain functions non-invasively and repetitively. We clarified the cognitive functions from normal subjects and subjects suffering from MCI with a whole head magnetoencephalography (MEG). Our studies showed the significantly decreased and delayed activities of the hippocampus of each hemisphere and the left posterior inferior frontal (PIF) area in the subjects with MCI, compared with the normal subjects. Both results were more significant in the subjects with Alzheimer’s disease than MCI. Disorders of the cognitive processing in the left PIF area proposing the association with social communication suggest early cognitive impairments with social cognitive impairments. Based on these results, MEG is a useful method for early diagnosis of MCI as non-invasively neurophysiological clarification of the cognitive brain processing, and this method would produce precious information for clinical examination and basic brain researches of the cognitive disorders.