Yuji Yasunaga

Expression of MicroRNA-146a in osteoarthritis cartilage.

OBJECTIVE A role of microRNA, which are approximately 22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). METHODS The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1beta (IL-1beta) was examined by quantitative RT-PCR. RESULTS All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0-5, grade II = moderate OA scored 6-10, and grade III = severe OA scored 11-14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1beta stimulation in human chondrocytes in vitro. CONCLUSION This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1beta. Thus, miR-146 might play a role in OA cartilage pathogenesis.

Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair

A basic experiment of peripheral nerve regeneration using neuronal progenitor cells embedded in collagen gel was performed in a rat sciatic nerve defect. First, when neuronal progenitor cells derived from the fetal rat hippocampus were cultured in atelocollagen-containing medium, neurospheres positive for anti-nestin antibody were confirmed after 8 days. These cells differentiated into astrocytes positive for anti-glial fibrillary acidic protein (GFAP) antibody, oligodendrocytes positive for anti-galactocerebroside (GalC) antibody and neurons positive for anti-neurofilament 200 (NF200) antibody, and they were capable of extending axons. They also differentiated into Schwann-like supportive cells positive for anti-s100 and anti-p75 antibody. Next, a 15-mm defect was prepared in the sciatic nerve of mature rats, and the nerve was bridged with a silicone tube filled with neuronal progenitor cells (1 x 10(5)) embedded in collagen gel. The transplanted neuronal progenitor cells were labeled in advance with 5-bromo-2-deoxyuridine (BrdU). When the regenerated tissue was examined 6 weeks and 10 weeks after grafting, the number and diameter of myelinated fibers were significantly increased compared with a control tube without neuronal progenitor cells. Action potentials were detected in the regenerated nerve. Also, cells positive for both anti-BrdU antibody and anti-S100 or anti-p75 antibody were observed in the regenerated tissue, and part of the grafted neural stem cells were considered to have differentiated into Schwann cell-like supportive cells. From these results neuronal progenitor cells derived from the fetal rat hippocampus are considered to retain their proliferative and differentiating abilities in collagen gel, and when transplanted to a site of peripheral nerve defect, part of them differentiate into supportive cells and they contributed to promotion of axonal regeneration.

Evaluation of systemic chemotherapy with magnetic liposomal doxorubicin and a dipole external electromagnet

The development of an active drug delivery system is an attractive approach to increase the targetability of anticancer agents. In the present study, we examined the efficiency of systemic chemotherapy with small magnetic liposomes containing doxorubicin (magnetic DOX liposomes) and an externally applied electromagnetic force in osteosarcoma‐bearing hamsters. Syrian male hamsters inoculated with osteosarcoma, OS515, in the limb were studied 7 days after inoculation. The efficiency of this system was evaluated by measuring the tissue distribution and tumor‐suppressing effects of DOX on primary tumor growth and lung metastases. A DC dipole electromagnet was used, and the hamsters tumor‐bearing limb was placed between 2 poles after the i.v. administration of liposomes. The dose of DOX and the magnetic field strength were fixed at 5 mg/kg and 0.4 T, respectively. Administration of magnetic DOX liposomes followed by 60 min application of magnetic field produced a 3‐ to 4‐fold higher maximum DOX concentration in the tumor. This newly designed systemic chemotherapy significantly suppressed primary tumor growth for at least 2 weeks, though other DOX treatments also suppressed compared to control. Histologic examination confirmed a greater antitumor effect of this systemic chemotherapy compared to standard methods. In addition, this approach significantly suppressed lung metastases measured at 3 weeks posttreatment. These results suggest that this systemic chemotherapy can effectively reduce primary tumor growth and suppress lung metastasis due to increased targeting of DOX. Such targeted drug delivery for anticancer agents would provide clinical advantages compared to current methods.

Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes.

Transplantation of neural progenitor cells (NPCs) has been reported recently to promote regeneration of the injured spinal cord. In the majority of these reports, cell transplantation was performed by local injection with a needle. However, direct injection might be too invasive for clinical use; therefore, the authors investigated a new method of delivering NPCs for the treatment of spinal cord injury. In this study, NPCs were obtained from E15 fetal hippocampus of transgenic rats expressing green fluorescent protein and 100,000 cells were transplanted intravenously into each animal 24h after contusion injury. It was found that the injected NPCs migrated to the lesion site widely and demonstrated nestin at an early phase after transplantation. These NPCs differentiated into neurons, astrocytes and oligodendrocytes, and survived at least for 56 days. These results indicated that intravenously injected neural stem cells migrated into the spinal cord lesion while preserving their potential as NPCs, and that this procedure is a potential method of delivering cells into the lesion for the treatment of spinal cord injury.

Effects of transforming growth factor-β 1 on the early stages of healing of the Achilles tendon in a rat model

We studied the effects of transforming growth factor-β 1 (TGF-β 1) on the genetic expression of procollagen type I and III and its effects on structural properties in the early stages of healing in rat Achilles tendon. The Achilles tendons in 90 rats were transsected and repaired immediately. TGF-β 1 dissolved in phosphate-buffered saline was injected locally at the repair site using two different doses, and outcomes in both groups were compared to that in the control group given phosphate-buffered saline only. Five animals in each group were killed at one, two, and four weeks postoperatively, and the healing tendon was evaluated. A dose-dependent increase in the expression of procollagen type I and III mRNA was found one week postoperatively. The failure load and stiffness of the healing tendon were increased by TGF-β 1 at two and four weeks.

The state of the articular cartilage at the time of surgery as an indication for rotational acetabular osteotomy

We have studied whether the state of the articular cartilage at the time of rotational acetabular osteotomy for dysplasia of the hip affects the outcome 2 to 5.5 years after surgery. Arthroscopy in 57 patients (59 joints) at the time of the operation showed grade-0 changes in seven, grade-1 in nine, grade-2 in 17, grade-3 in 14 and grade-4 in 12 joints, according to the classification of Outerbridge. There was radiological evidence of the progression of arthritis in four joints which were classified at arthroscopy as grade 4. Stepwise regression analysis showed that damage to acetabular or femoral articular cartilage significantly affected the progression of arthritis. We conclude that the short-term results of successful rotational acetabular osteotomy for dysplasia are affected by the state of the articular cartilage.

Transplantation of Meniscus Regenerated by Tissue Engineering With a Scaffold Derived From a Rat Meniscus and Mesenchymal Stromal Cells Derived From Rat Bone Marrow

The purpose of this study was to assess transplantation of regenerated menisci using scaffolds from normal allogeneic menisci and bone-marrow-derived mesenchymal stromal cells (BM-MSCs) of rats. We reported that scaffolds derived from normal menisci seeded with BM-MSCs in vitro could form meniscal tissues within 4 weeks. Then, we hypothesized that our tissues could be more beneficial than allogeneic menisci regarding early maturation and chondroprotective effect. Bone marrow was aspirated from enhanced green fluorescent protein transgenic rats. BM-MSCs were isolated and seeded onto scaffolds which were prepared from Sprague-Dawley rat menisci. After 4 weeks in coculture, the tissues were transplanted to the defect of menisci. Repopulation of BM-MSCs and expression of extracellular matrices were observed in the transplanted tissues at 4 weeks after surgery. At 8 weeks, articular cartilage in the cell-free group was more damaged compared to that in the cell-seeded group or the meniscectomy group.

Relationship Between Osteochondritis Dissecans of the Lateral Femoral Condyle and Lateral Menisci Types

The purpose of this study was to examine the relationship between osteochondritis dissecans (OCD) of the lateral femoral condyle and lateral menisci. From 1993 to 2002, 38 knees (28 patients) were diagnosed with OCD of the lateral femoral condyle. OCD locations were graded by the Cahill and Berg classification. The types of lateral menisci were classified by Watanabes classification. The relationship between OCD of the lateral femoral condyle and lateral menisci was examined. On the anterior-posterior view, 25 OCDs were located in zone 4 and 13 in zone 5. The types of lateral menisci were complete discoid in 19 knees, incomplete discoid in 15, and normal in 4. Ten of the 19 complete discoid menisci were damaged. Complete discoid menisci without tears were found in OCDs located in zone 4; incomplete discoid menisci were found in OCDs located in zone 5. The authors found a relationship between the type of OCD and the state of the lateral meniscus.

Multiinstitutional epidemiological study regarding osteoarthritis of the hip in Japan

Background. Osteoarthritis (OA) of the hip is a major disease that affects the healthy lifespan of a population. It is necessary to fully understand the patients’ conditions before a systematic treatment can be applied. However, a nationwide epidemiological study regarding hip OA has not yet been conducted in Japan. The present study examined the current status of patients with hip OA, including the disease etiology. Methods. This is a multiinstitutional study of new patients presenting with hip OA at the orthopedic outpatient clinics of 15 institutions in fi ve geographical areas of Japan. The collected data from each patient included the sex, age, treatment history for developmental dysplasia of the hip (DDH), the clinical score of the hip joints based on the Japanese Orthopaedic Association (JOA) scoring system, and the pelvic inclination according to anteroposterior radiographs. In addition, the etiology was determined from the following 17 options: primary OA, acetabular dysplasia, intragluteal dislocation, osteonecrosis, trauma, Perthes disease, slipped capital femoral epiphysis, infection, rheumatoid arthritis, ankylosing spondylitis, neuroarthropathy, endocrine diseases, metabolic diseases, hereditary bone diseases, synovial chondromatosis, generalized OA, and others. Results. There were a substantially larger number of female patients than male patients. This difference regarding sex was present in each generation. The mean age of the patients was 58 ± 14 years. The peak age at presentation was approximately 50 years. Most patients had no history of therapy for DDH. The older patients had lower gait and activities of daily living scores. The etiology was assessed to be acetabular dysplasia in most of the patients. A lower frequency of elderly patients demonstrated acetabular dysplasia. The patients who had a pelvic posterior inclination increased with increasing age. Conclusions. The patients with hip OA in Japan were unique in regard to age distribution, sexual heterogeneity, and disease

Bone-marrow-derived mononuclear cells with a porous hydroxyapatite scaffold for the treatment of osteonecrosis of the femoral head: A preliminary study

We have investigated the effectiveness of the transplantation of bone-marrow-derived mononuclear cells (BMMNCs) with interconnected porous calcium hydroxyapatite (IP-CHA) on early bone repair for osteonecrosis of the femoral head. We studied 22 patients (30 hips) who had osteonecrosis with a minimum follow-up of one year after implantation of BMMNCs. The mean age at surgery was 41 years (18 to 64) and the mean period of follow-up was 29 months (19 to 48). In a control group, cell-free IP-CHA was implanted into a further eight patients (9 hips) with osteonecrosis of the femoral head and the outcomes were compared. A reduction in the size of the osteonecrotic lesion was observed subsequent to hypertrophy of the bone in the transition zone in the BMMNC group. In three patients in the treatment group progression to extensive collapse was detected. In the control group subtle bone hypertrophy was observed, but severe collapse of the femoral head occurred in six of eight hips. In this limited study the implantation of BMMNCs and IP-CHA appears to confer benefit in the repair of osteonecrosis and in the prevention of collapse.