The Pavlik harness in the treatment of developmentally dislocated hips: results of Japanese multicenter studies in 1994 and 2008.

BACKGROUND: It has already been more than 50 years since the Pavlik harness was introduced in Japan, and today the Pavlik harness is widely recognized as the standard initial treatment modality for developmental dysplasia of the hip. We performed a multicenter nationwide questionnaire study concerning the results of Pavlik harness treatment twice in 1994 and 2008. METHODS: In 1994 and in 2008, we sent questionnaires to 12 institutes in Japan specializing mainly in pediatric orthopedics. We compare the results of these two studies and discuss differences in reduction rates, incidence of avascular necrosis in the femoral epiphysis and the percentage of joints with acceptable morphology (Severin grade I + II/total) at skeletal maturity. We statistically assessed these results to see whether there were changes in the treatment outcomes over this 14-year period. RESULTS: Reduction of the dislocated hips was obtained by the Pavlik harness in 80.2% (1990/2481 hips; 1994) and 81.9% (1248/1523 hips; 2008). The incidences of avascular necrosis of the proximal femoral epiphysis in the dysplastic hips were 14.3% (119/835 hips; 1994) and 11.5% (76/663 hips; 2008). The type of avascular necrosis in hips from the 2008 study was determined according to the classification of Kalamchi and MacEwen: 24/69 hips (34.8%) were classified as group I; 20/69 hips (29.0%) as group II; 11/69 hips (15.9%) as group Ill; 14/69 hips (20.3%) as group IV. The percentages of hips with acceptable outcomes at skeletal maturity discerned from Severin X-ray changes (grade I + II/total) were 72.3% (604/835 hips; 1994) and 77.7% (488/628 hips; 2008). CONCLUSION: Reduction rates and the incidence of avascular necrosis in 2008 were statistically similar to the results in 1994. The rate of acceptable outcome (Severin grade I + II/total) in 2008 was statistically higher than that of 1994.

Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells.

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.

Demography and productivity during the recovery time sequence of a wild edible bamboo after large-scale anthropogenic disturbance.

Anthropogenic disturbances in forest management practices can affect wild edible plants. Soil scarification is a large-scale disturbance that may cause long-term reduction in productivity of edible dwarf bamboo, Sasa kurilensis, in northern Japan. For their effective and sustainable use, we need to understand the recovery process after such disturbances. At 14 study sites in the Teshio Experimental Forest of Hokkaido University where soil scarification had been conducted between 2 and 44 years prior, the number and stem diameter of old and young (newly emerged, edible) culms was recorded. At sites that were within 11 years of soil scarification, the proportion of old culms (<11%) was lower than in the control area where soil scarification had never been conducted. At sites where more than 15 years had passed since soil scarification, the relative number of old culms was nearly equal to that in control area. Additionally, the number of young culms increased with an increasing number of old culms. These results suggest that recovery of productivity (in term of number) of edible culms may take a few decades. In contrast, the culm diameter of young culms increased linearly with time since soil scarification, but the 95% confidence interval in this relationship suggests that dwarf bamboo can produce thick edible culms soon after soil scarification. These findings will provide useful insights into how to obtain high quality bamboo culms following anthropogenic disturbances in future.

Cell-specific epigenetic regulation of ChM-I gene expression: crosstalk between DNA methylation and histone acetylation.

The expression of the chondromodulin-I (ChM-I) gene, a cartilage-specific gene, is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma (OS) cells. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by histone deacetylase 2 (HDAC2) in three ChM-I-negative OS cell lines. Treatment with an HDAC inhibitor induced the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and, surprisingly, the demethylation of the core promoter region. The demethylation was transient, and gradually replaced by methylation after a rapid recovery of histone deacetylaion. These results represent an example of the plasticity of differentiation being regulated by the cell-specific plasticity of epigenetic regulation.

Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.

Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion.

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A-positive cells were Ki67-negative and senescence associated beta-galactosidase-positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty-five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.

Response of a Wild Edible Plant to Human Disturbance: Harvesting Can Enhance the Subsequent Yield of Bamboo Shoots.

Wild edible plants, ecological foodstuffs obtained from forest ecosystems, grow in natural fields, and their productivity depends on their response to harvesting by humans. Addressing exactly how wild edible plants respond to harvesting is critical because this knowledge will provide insights into how to obtain effective and sustainable ecosystem services from these plants. We focused on bamboo shoots of Sasa kurilensis, a popular wild edible plant in Japan. We examined the effects of harvesting on bamboo shoot productivity by conducting an experimental manipulation of bamboo shoot harvesting. Twenty experimental plots were prepared in the Teshio Experimental Forest of Hokkaido University and were assigned into two groups: a harvest treatment, in which newly emerged edible bamboo shoots were harvested (n = 10); and a control treatment, in which bamboo shoots were maintained without harvesting (n = 10). In the first year of harvesting (2013), bamboo shoot productivities were examined twice; i.e., the productivity one day after harvesting and the subsequent post-harvest productivity (2-46 days after harvesting), and we observed no difference in productivity between treatments. This means that there was no difference in original bamboo shoot productivity between treatments, and that harvesting did not influence productivity in the initial year. In contrast, in the following year (2014), the number of bamboo shoots in the harvested plots was 2.4-fold greater than in the control plots. These results indicate that over-compensatory growth occurred in the harvested plots in the year following harvesting. Whereas previous research has emphasized the negative impact of harvesting, this study provides the first experimental evidence that harvesting can enhance the productivity of a wild edible plant. This suggests that exploiting compensatory growth, which really amounts to less of a decline in productivity, may be s a key for the effective use of wild edible plants.

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland.

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo-stellate cells in the anterior pituitary glands of 60-day-old Wistar-Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo-stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats.

Prostaglandin E2 receptor type 2-selective agonist prevents the degeneration of articular cartilage in rabbit knees with traumatic instability.

INTRODUCTION: Osteoarthritis (OA) is a common cause of disability in older adults. We have previously reported that an agonist for subtypes EP2 of the prostaglandin E2 receptor (an EP2 agonist) promotes the regeneration of chondral and osteochondral defects. The purpose of the current study is to analyze the effect of this agonist on articular cartilage in a model of traumatic degeneration. METHODS: The model of traumatic degeneration was established through transection of the anterior cruciate ligament and partial resection of the medial meniscus of the rabbits. Rabbits were divided into 5 groups; G-S (sham operation), G-C (no further treatment), G-0, G-80, and G-400 (single intra-articular administration of gelatin hydrogel containing 0, 80, and 400 µg of the specific EP2 agonist, ONO-8815Ly, respectively). Degeneration of the articular cartilage was evaluated at 2 or 12 weeks after the operation. RESULTS: ONO-8815Ly prevented cartilage degeneration at 2 weeks, which was associated with the inhibition of matrix metalloproteinase-13 (MMP-13) expression. The effect of ONO-8815Ly failed to last, and no effects were observed at 12 weeks after the operation. CONCLUSIONS: Stimulation of prostaglandin E2 (PGE2) via EP2 prevents degeneration of the articular cartilage during the early stages. With a system to deliver it long term, the EP2 agonist could be a new therapeutic tool for OA.

A novel method to isolate mesenchymal stem cells from bone marrow in a closed system using a device made by nonwoven fabric.

Bone marrow stromal cells (BMSCs) include cells with multidirectional differentiation potential described as mesenchymal stem cells. For clinical use, it is important to develop a way to isolate BMSCs from bone marrow in a closed system without centrifugation. After screening 200 biomaterials, we developed a device containing a nonwoven fabric filter composed of rayon and polyethylene. The filter selectively traps BMSCs among mononuclear cells in bone marrow based on affinity, not cell size. The cells are then recovered by the retrograde flow. Using canine and human bone marrow cells, the biological properties of BMSCs isolated by the device were compared with those obtained by conventional methods using centrifugation. The total number isolated by the device was larger, as was the number of CD106(+)/STRO-1(+) double-positive cells. The cells showed osteogenic, chondrogenic, and adipogenic differentiation potential in vitro. Finally, the direct transplantation of cells isolated by the device without in vitro cultivation accelerated bone regeneration in a canine model of osteonecrosis in vivo. The proposed method is rapid and efficient, does not require a biological clean area, and will be useful for the clinical application of mesenchymal stem cells in bone marrow.