Natsuo Yasui

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel, were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II collagen. These chondrocytes were autoradiographically proven to have originated from the transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement over the results reported in previous studies on chondrocyte transplantation without collagen gel. By contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one of the most promising methods for the restoration of articular cartilage.

Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2αA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2αA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2αA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2αA. In the present study, we determined the direct regulation of PEBP2αA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2αA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2αA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2αA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2αA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2αA to MC3T3/E1 cells expressing endogenous PEBP2αA, ETS1 and opn. These findings suggest that PEBP2αA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

Mechanical Tension-Stress Induces Expression of Bone Morphogenetic Protein (BMP)-2 and BMP-4, but Not BMP-6, BMP-7, and GDF-5 mRNA, During Distraction Osteogenesis

Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats, and the effect of mechanical tension‐stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and Northern blot analysis using probes of BMP‐2, BMP‐4, BMP‐6, BMP‐7, and growth/differentiation factor (GDF)‐5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0.25 mm/12 h using a small external fixator. The signals of the above BMPs mRNA were not detected in the intact rat bone but they were induced after osteotomy except those for BMP‐7. By 4 days after osteotomy, BMP‐2 and BMP‐4 mRNAs were detected in chondrogenic precursor cells in the subperiosteal immature callus. BMP‐6 and GDF‐5 mRNA were detected in more differentiated cells in chondroid bone. By 7 days after osteotomy, cartilaginous external callus and bony endosteal callus were formed. Meanwhile, the signals of BMP‐2 and BMP‐4 mRNAs declined to preoperative levels, whereas the signals of BMP‐6 and GDF‐5 mRNAs were rather elevated. As distraction was started, the callus elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP‐2 and BMP‐4 mRNAs was markedly induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension‐stress at the fibrous interzone. BMP‐6 and GDF‐5 mRNAs were detected exclusively in chondrogenic cells at both ends of the fibrous interzone, where endochondral ossification occurred. But neither mRNA was detected in terminally differentiated hypertrophic chondrocytes. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. There was no new cartilage formation in the advanced stage of distraction. The signals of BMP‐6 and GDF‐5 mRNA declined by this stage, while those of BMP‐2 and BMP‐4 were maintained at high level for as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. BMP‐2, BMP‐4, BMP‐6, nor GDF‐5 was expressed at this stage. The signals of BMP‐7 were not detected throughout the experiment. The present results suggest that excellent and uninterrupted bone formation during distraction osteogenesis owes to enhanced expression of BMP‐2 and BMP‐4 genes by mechanical tension‐stress. Abundant gene products of BMP‐2 and BMP‐4 could induce in situ bone formation by paracrine and autocrine mechanisms.

Skeletal muscle gene expression in space-flown rats

Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of ~26,000 gastrocnemius muscle genes in space‐flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail‐suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight‐specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria‐anchoring proteins, A‐kinase anchoring protein, and cytoplasmic dynein, and 2) up‐regulated expression of ubiquitin ligase genes, MuRF‐1, Cbl‐b, and Siah‐1A, which are rate‐limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress‐inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.

Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures.

Chondrocytes isolated from sterna of 13-day-old chick embryos were cultured in vitro within collagen gels for six weeks. Under these culture conditions, chondrocytes assumed a rounded morphology, accumulated metachromatic matrix, and took on the cytological characteristics of in vivo cartilage cells. Most of the glycosaminoglycans synthesized were deposited within the collagen gels, and the rate of glycosaminoglycan synthesis was maintained over five weeks. Throughout the study, the major collagen synthesized was Type II. This cell-culture system, using collagen gels, provides chondrocytes with a suitable environment in which to accumulate synthesized matrix macromolecules and preserve their differentiated phenotype in vitro.

Expression of Bone Matrix Proteins mRNA During Distraction Osteogenesis

Distraction osteogenesis is a recently advanced principle of bone lengthening in which a bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. Appropriate mechanical tension‐stress is believed not to break the callus but rather to stimulate osteogenesis. To study the molecular features of this process, the expression and localization of the mRNAs encoding osteopontin (OPN), osteocalcin (OC), matrix Gla protein (MGP), osteonectin (ON), and collagen type I and II during distraction osteogenesis were examined by in situ hybridization and Northern blot analysis. The process can be divided into three distinct phases: the lag phase for 7 days between osteotomy and the beginning of distraction, the distraction phase for 21 days, and the consolidation phase for several weeks. The histologic and molecular events taking place during the lag phase were similar to those observed in fracture healing. The osteotomy site was surrounded by external callus consisting of hyaline cartilage. As distraction started at the rate of 0.25 mm/12 h, the cartilaginous callus was elongated, deformed, and eventually separated into proximal and distal segments. The chondrocytes were stretched along the tension vector and became fibroblast‐like in shape. Although morphologically these cells were distinguishable from osteogenic cells, they expressed OPN, OC, and alkaline phosphatase mRNAs. As distraction advanced, the cartilaginous callus was progressively replaced by bony callus by endochondral ossification and thereafter new bone was formed directly by intramembranous ossification. OPN mRNA was detected in preosteoblasts and osteoblasts at the boundary between fibrous tissue and new bone. ON, MGP, and OC mRNAs appeared early in the differentiation stage. The variety of cell types expressing mRNA encoding bone matrix proteins in distraction osteogenesis was much greater than that detected in the embryonic bone formation and fracture healing process. Moreover, the levels of OPN, ON, MGP, and OC mRNA expression markedly increased during the distraction phase. These results suggested that mechanical tension‐stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins.

Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

ABSTRACT Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine608-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.

MRI signal changes of the pedicle as an indicator for early diagnosis of spondylolysis in children and adolescents : A clinical and biomechanical study

Study Design. Clinical review of pediatric patients with lumbar spondylolysis and biomechanical analysis using finite-element lumbar spine model. Objectives. To evaluate the usefulness of the signal changes observed on MR images of the pedicle for the early diagnosis of spondylolysis, and to investigate the pathomechanism of the signal changes based on the stresses in pedicles, as predicted using finite-element analyses. Furthermore, to evaluate the usefulness of the signal change to predict the bony healing following conservative treatment. Summary of Background Data. Since early-stage spondylolysis can achieve osseous healing conservatively, it is important to diagnose this disorder as early as possible. Presently, there is no well-established, noninvasive, and reliable diagnostic tool for the early diagnosis. Methods. Thirty-seven pediatric patients with spondylolysis were included. Sixty-eight defects were examined and their stages as revealed on CT scans were recorded. High signal changes (HSC) of the pedicles on axial T2-weighted MRI were compared with the CT-based stages of the defect. Among them, 16 patients, including 15 boys and 1 girl, were treated conservatively for at least a 3-month period. Bony healing of the fracture site was evaluated on CT, and the results were compared between two groups with or without HSC at the initial consultation. Using a three-dimensional nonlinear finite-element model of the L3–L5 segment, stress distributions in the pars and pedicle regions were analyzed in response to 400 N compression and 10.6 Nm moment. Results. Based on CTs, 68 pars defects were classified as follows: 8 very early, 24 late-early, 16 progressive, and 20 terminal stages. All defects in very early and late-early stages (100%) showed HSC on T2-weighted MRI at the ipsilateral pedicle. Among 16 progressive stages, eight (50%) showed HSC, while no defects of the terminal stage (0%) were found to have HSC. In total, 29 pars defects were treated conservatively out of 16 patients. In 19 of the HSC positive defects, 15 (79%) showed bony healing after the conservative treatment, whereas none of the 10 HSC negative defects (0%) showed any healing. The results were statistically significant at P < 0.05 (&khgr;2). Stress results from the finite-element model indicated that pars interarticularis showed the highest value in all loading modes, and the pedicle showed the second highest. Conclusions. The correlation between the high stresses in the pedicle and the corresponding HSC suggest that signal changes in MRI could be used as an indicator for early diagnosis of spondylolysis. The HSC of the pedicle provided useful information to diagnose early stage spondylolysis. Furthermore, the HSC may be a good indicator as to whether a bony union will result from conservative treatment.

Athletes With Unilateral Spondylolysis Are at Risk of Stress Fracture at the Contralateral Pedicle and Pars Interarticularis A Clinical and Biomechanical Study

Background Unilateral spondylolysis is common in youths; its clinical and biomechanical features, especially effects on the contralateral side, are not fully understood. Hypothesis Unilateral spondylolysis predisposes the contralateral side to stress fracture, especially in athletes actively engaged in sporting activities involving torsion of the trunk. Study Design Case series and descriptive laboratory study. Methods Thirteen athletes younger than age 20 with unilateral spondylolysis were included. The contralateral pedicle and pars of spondylolytic vertebrae were examined using computed tomography and magnetic resonance imaging. Using a finite element model of the intact ligamentous L3-S1 segment, stress distributions were analyzed in response to 400-N axial compression and 10.6-N.m moment in flexion, extension, lateral bending, and axial rotation. Unilateral spondylolysis was created in the model at L5. The stress results from the unilateral defect model were compared to the intact model predictions and correlated to the contralateral defects seen in patients. Results Among 13 patients, there were 6 early-, 2 progressive-, and 5 terminal-stage defects. Three (23.1%) showed contralateral stress fracture. Among them, 2 belonged to the progressive-stage and 1 to the terminal-stage spondylolysis group. The remaining 4 patients in the terminal defect group showed stress reactions, such as sclerosis at the contralateral pedicle. In the finite element analysis model with an L5 left spondylolysis, the stresses at the contralateral and pars interarticularis were found to increase in all loading modes, with increases as high as 12.6-fold compared to the intact spine. Conclusions Unilateral spondylolysis could lead to stress fracture or sclerosis at the contralateral side due to an increase in stresses in the region. Clinical Relevance Surgeons should be aware of possibility of contralateral stress fractures in cases in which patients, especially athletes engaged in active sports, show unilateral spondylolysis and persistent low back pain complaints.

Union of defects in the pars interarticularis of the lumbar spine in children and adolescents THE RADIOLOGICAL OUTCOME AFTER CONSERVATIVE TREATMENT

Lumbar spondylolysis can heal with conservative treatment, but few attempts have been made to identify factors which may affect union of the defects in the pars. We have evaluated, retrospectively, the effects of prognostic variables on bony union of pars defects in 134 young patients less than 18 years of age with 239 defects of the pars who had been treated conservatively. All patients were evaluated by CT scans when first seen and more than six months later at follow-up. The results showed that the spinal level and the stage of the defects were the predominant factors. The site of the defects in the pars, the presence or development of spondylolisthesis, the condition of the contralateral pars, the degree of lumbar lordosis and the degree of lumbar inclination all significantly affected union.