New Neuromuscular Training for Peripheral Nerve Disorders Using an Ankle Joint Hybrid Assistive Limb: A Case Series.

Peripheral nerve disorder of the lower extremities causes drop foot and disturbs the daily living activities of patients. The ankle joint hybrid assistive limb (HAL) provides voluntary ankle joint training using surface bioelectrical signals from the muscles of the lower extremities. We investigated the neurological effects of ankle joint HAL training in three patients. Sensory nerve action potentials (SNAPs) and compound muscle action potentials (CMAPs) were analyzed for the peroneal and tibial nerves prior to the first ankle joint HAL training session. Integrated surface electromyography EMG signals were recorded before and after the HAL training sessions to evaluate the effects of training for neuromuscular disorders. The patients were hospitalized to receive rehabilitation with HAL training for 2 weeks. The HAL training was performed daily with two 60 min sessions. All cases demonstrated severe neuromuscular impairment according to the result of the CMAP. All integrated EMG measurements of antagonistic muscle activities decreased after the ankle joint HAL training. The manual muscle testing (MMT) scores of each muscle were slightly increased after the HAL intervention for Case 2(tibialis anterior, from 2 to 2+; gastrocnemius muscles, from 2- to 2; extensor digitorum longus, and extensor hallucis longus, from 1 to 3). The MMT scores were also slightly increased except for gastrocnemius muscle for Case 3 (tibialis anterior, extensor digitorum longus, and extensor hallucis longus, from 2- to 2). These two patients demonstrated voluntary muscle contractions and nerve signals in the CMAP before the HAL training. Even though the amplitude of CMAPs was low, the HAL training may provide voluntary ankle joint movements by reducing the antagonistic muscle contraction via computer processing. The HAL training may enhance muscle movement and coordination through motor learning feedback.

The anterior center edge angle has limited ability to predict three-dimensional coverage of the femoral head in patients with developmental dysplasia of the hip undergoing curved periacetabular osteotomy.

INTRODUCTION: Femoral head coverage in patients with hip dysplasia (DDH) is typically quantified using 2D measurements of the lateral center edge angle (LCEA) and anterior center edge angle (ACEA). However, as the morphology of DDH is complex and varies between patients, 2D measurements may not predict the true 3D femoral head coverage. Herein, 2D and 3D coverage were quantified before and after curved periacetabular osteotomy (CPO) and their relationships were assessed. MATERIALS AND METHODS: Forty-three hips that underwent CPO for DDH were analyzed. For 2D evaluation, LCEA was quantified from X-rays and CT images. The ACEA was measured from CT images (CT-ACEA) and digitally reconstructed radiographs generated from CT images (DRR-ACEA). Three-dimensional coverage was quantified from CT reconstructions of the hip and evaluated in the anterior, superior, posterior, and inferior regions of the femoral head. Two-dimensional measurements were correlated to 3D coverage to assess their relationships. RESULTS: The median preoperative 3D percent coverage was 17.7, 36.1, 56.1, and 14.6% for the anterior, superior, posterior, and inferior region, respectively. After CPO, all LCEAs and ACEAs increased significantly (all p < 0.001). For the 3D coverage, anterior and superior coverage significantly increased while the posterior and inferior coverage decreased (all p < 0.001). Moderate to strong correlations were detected between the two LCEAs and the 3D superior coverage in both the preoperative and postoperative period. For the correlation between 3D anterior coverage, no significant correlation was found between the CT-ACEA while a moderate correlation was found between the DRR-ACEA (rs = 0.41, p = 0.023). CONCLUSIONS: Our results indicate that the LCEA can be used to predict 3D coverage in the superior region of the femoral head. However, as the CT-ACEA or DRR-ACEA had no or only moderate correlation between the 3D anterior coverage, these measurements are not recommended for evaluating/estimating the 3D anterior coverage in patients with DDH.

Implications of the uncertainty of postoperative functional parameters for the preoperative planning of total hip arthroplasty.

The functional parameters pelvic tilt (PT) and hip joint force (HJF) are required to calculate patient-specific target zones based on the range of motion (ROM) and implant loading for preoperative planning of total hip arthroplasty (THA). Both functional parameters may change after THA. The preoperative prediction of the postoperative PT and HJF is associated with a specific amount of uncertainty. The prediction uncertainty has to be considered in the preoperative planning process to avoid a suboptimal implantation. So far, very little attention has been paid to the necessary reduction of patient-specific target zones by the prediction uncertainties of postoperative functional parameters. Prediction models for the postoperative PT in standing position and for the HJF during one-leg stance as a surrogate for the peak force phase during level walking were used to quantify the reduction of the ROM- and load-based target zones of 196 Japanese THA patients. The prediction uncertainty was about 14° for the postoperative standing PT and ranged from 17% body weight to 37% body weight for the components of the HJF. On average, the prosthetic ROM-based target zone had to be significantly reduced by 43% and the load-based target zone by 39%. This led to a median reduction of the combined prosthetic ROM- and load-based target zone of 96%. The study sharpens the awareness for the substantial reduction of ROM- and load-based target zones by prediction uncertainties of the postoperative PT and HJF and highlights the importance of further research to improve prediction models for both functional parameters.

Prediction of femoral head coverage from articulated statistical shape models of patients with developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is commonly described as reduced femoral head coverage due to anterolateral acetabular deficiency. Although reduced coverage is the defining trait of DDH, more subtle and localized anatomic features of the joint are also thought to contribute to symptom development and degeneration. These features are challenging to identify using conventional approaches. Herein, we assessed the morphology of the full femur and hemi-pelvis using an articulated statistical shape model (SSM). The model determined the morphological and pose-based variations associated with DDH in a population of Japanese females and established which of these variations predict coverage. Computed tomography (CT) images of 83 hips from 47 patients were segmented for input into a correspondence-based SSM. The dominant modes of variation in the model initially represented scale and pose. After removal of these factors through individual bone alignment, femoral version and neck-shaft angle, pelvic curvature, and acetabular version dominated the observed variation. Femoral head oblateness and prominence of the acetabular rim and various muscle attachment sites of the femur and hemi-pelvis were found to predict 3D CT-based coverage measurements (R2 = 0.5-0.7 for the full bones, R2 = 0.9 for the joint). Statement of Clinical Significance: Currently, clinical measurements of DDH only consider the morphology of the acetabulum. However, the results of this study demonstrated that variability in femoral head shape and several muscle attachment sites were predictive of femoral head coverage. These morphological differences may provide insight into improved clinical diagnosis and surgical planning based on functional adaptations of patients with DDH.

The Patient-Specific Combined Target Zone for Morpho-Functional Planning of Total Hip Arthroplasty.

Background Relevant criteria for total hip arthroplasty (THA) planning have been introduced in the literature which include the hip range of motion, bony coverage, anterior cup overhang, leg length discrepancy, edge loading risk, and wear. The optimal implant design and alignment depends on the patient's anatomy and patient-specific functional parameters such as the pelvic tilt. The approaches proposed in literature often consider one or more criteria for THA planning. but to the best of our knowledge none of them follow an integrated approach including all criteria for the definition of a patient-specific combined target zone (PSCTZ). Questions/purposes (1) How can we calculate suitable THA implant and implantation parameters for a specific patient considering all relevant criteria? (2) Are the resulting target zones in the range of conventional safe zones? (3) Do patients who fulfil these combined criteria have a better outcome score? Methods A method is presented that calculates individual target zones based on the morphology, range of motion and load acting on the hip joint and merges them into the PSCTZ. In a retrospective analysis of 198 THA patients, it was calculated whether the patients were inside or outside the Lewinnek safe zone, Dorr combined anteversion range and PSCTZ. The postoperative Harris Hip Scores (HHS) between insiders and outsiders were compared. Results 11 patients were inside the PSCTZ. Patients inside and outside the PSCTZ showed no significant difference in the HHS. However, a significant higher HHS was observed for the insiders of two of the three sub-target zones incorporated in the PSCTZ. By combining the sub-target zones in the PSCTZ, all PSCTZ insiders except one had an HHS higher than 90. Conclusions The results might suggest that, for a prosthesis implanted in the PSCTZ a low outcome score of the patient is less likely than using the conventional safe zones by Lewinnek and Dorr. For future studies, a larger cohort of patients inside the PSCTZ is needed which can only be achieved if the cases are planned prospectively with the method introduced in this paper. Clinical Relevance The method presented in this paper could help the surgeon combining multiple different criteria during THA planning and find the suitable implant design and alignment for a specific patient.

Preoperative factors improving the prediction of the postoperative sagittal orientation of the pelvis in standing position after total hip arthroplasty.

The aims of this study were to investigate if the sagittal orientation of the pelvis (SOP) in the standing position changes after total hip arthroplasty (THA) and evaluate what preoperative factors may improve the prediction of the postoperative standing SOP in the context of a patient-specific functional cup orientation. 196 primary THA patients from Japan were retrospectively selected for this study. Computed tomography imaging of the pelvis, EOS imaging of the lower body and lateral radiographs of the lumbar spine in the standing position were taken preoperatively. Common biometrics and preoperative Harris Hip Score were recorded. The EOS imaging in the standing position was repeated three months following THA. A 3D/2.5D registration process was used to determine the standing SOP. Thirty-three preoperative biometric, morphological and functional parameters were measured. Important preoperative parameters were identified that significantly improve the prediction of the postoperative standing SOP by using multiple linear LASSO regression. On average, the SOP changed significantly (p < 0.001) between the preoperative and postoperative standing position three months after THA by 3° ± 4° in the posterior direction. The age, standing lumbar lordosis angle (LLA) and preoperative supine and standing SOP significantly (p < 0.001) improve the prediction of the postoperative standing SOP. The linear regression model for the prediction of the postoperative standing SOP is significantly (p < 0.001) improved by adding the parameters preoperative standing SOP and LLA, in addition to the preoperative supine SOP, reducing the root mean square error derived from a leave-one-out cross-validation by more than 1°. The mean standing SOP in Japanese patients changes already three months after THA in comparison to the preoperative value. The preoperative factors age, LLA, supine and standing SOP can significantly improve the prediction of the postoperative standing SOP and should be considered within the preoperative planning process of a patient-specific functional cup orientation.

Can measurements from an anteroposterior radiograph predict pelvic sagittal inclination?

Pelvic sagittal inclination (PSI) is often evaluated in patients with hip pathology using lateral radiographs. However, it would be useful if PSI could be predicted from an anteroposterior radiograph since this film is ubiquitous in the evaluation of hip pathology. Herein, computer-modeling was applied to predict PSI from radiographic measurements assessed in the anteroposterior plane. Three-dimensional surface models of the pelvis, femur, and sacrum were reconstructed from computed tomography images of 50 women with hip dysplasia. This study cohort was selected as changes in PSI alter femoral head coverage, which is relevant to the diagnosis and treatment of hip dysplasia, a known cause of hip osteoarthritis. Five radiographic parameters commonly used to independently estimate PSI were evaluated after bone surfaces were projected to an anteroposterior plane, including the symphysis to sacrococcygeal joint distance (S-S distance), the pelvic foramen aspect ratio (PF ratio), the distance between the symphysis and a line connecting the femoral head centers (S-H distance), the sacro-femoral-pubic angle (SFP angle), and the pelvic vertical ratio (PVR). Regression models determined the ability of these parameters to predict PSI from -20° to 20° at 1° increment. All five parameters showed a strong correlation with the PSI (all r > 0.9). From the regression models, PSI was estimated with a median (maximum) absolute error of 3.6° (18.4°), 3.8° (17.7°), 5.2° (17.9°), 5.8° (28.8°), and 3.2° (23.5°) for the S-S distance, PF ratio, S-H distance, SFP angle, and PVR, respectively. The regression model for S-S distance had a mean slope of 2.18 that ranged from 1.98 to 2.41 when the sacrococcygeal joint was located superior to the symphysis. Results indicated that substantial errors occur when estimating the actual value of PSI from an anteroposterior radiograph. However, the change in PSI could be estimated from the S-S distance, which may aid surgeons to successfully increase head coverage through periacetabular osteotomy and to locate the acetabular cup in a functional position for total hip arthroplasty.

Implant Orientation Measurement After THA Using the EOS X-Ray Image Acquisition System.

We investigated the accuracy of measuring implant orientation after THA in standing position using EOS system (EOS Imaging Inc., Paris, France). Ninety patients who underwent THA were subjected to this study by comparing angles measured by EOS system and those measured from CT scans using 3D image analyzing software, ZedHip (LEXI, Tokyo, Japan). The radiographic cup inclination and anatomical cup anteversion were measured with respect to the anterior pelvic plane (APP) coordinate. The femoral stem antetorsion was analyzed by measuring the angles between the stem neck axis and the post-condylar axis in the femoral functional axis coordinate.The differences (mean ± SD) (range of 95%CI) between angles measured by EOS system and those from CT scans in the cup inclination, cup anteversion, and stem antetorsion were - 2.3° ± 2.7° (-2.8°âˆ¼ - 1.7°), -0.1° ± 5.0° (-1.2°âˆ¼0.9°), and - 1.3° ± 6.5° (-2.7°âˆ¼0.1°), respectively. Cup inclination measured on 14 hips, cup anteversion measured on 28 hips, and stem antetorsion measured on 27 hips were classified as outliers whose differences were over 5°. Difficulties in defining the reference points for APP correlated with the incidences of the outliers in cup orientation measurements.We could not set new reference points on the 3D bone surface models reconstructed by EOS system, so we have to use reference points defined on 2D images. In addition, the APP coordinate in EOS system was not the same as the standard definition. EOS system may not be used to measure the implant positions after THA until these problems will be improved.

Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats.

Idiopathic osteonecrosis of the femoral head (ION) is a painful disease of the hip, the pathogenic mechanism of which is still unclear. The most common extraneous factor is steroid treatment. Steroids have inhibiting effects on bone formation and resorption. When bone regenerative treatments are indicated for ION patients who are exposed to steroids, we cannot ignore the effects of corticosteroid itself on bone healing. The aim of this study was to investigate the effects of glucocorticoid on bone regeneration after osteonecrosis of the femoral head in a rat model. Osteonecrosis was induced surgically on the left femoral heads of aged female rats (about 6 months old) on day 0. Methylprednisolone sodium succinate (MPSS) or normal saline was administrated at a dose of 100 mg/kg/day from day 7 to 11. Femoral heads were analyzed histologically. There were no pathological findings in the right femoral heads of the MPSS-treated and saline-treated rats, as control for the contralateral injury. The newly formed bone volume and the osteoclast number were significantly smaller in the MPSS-treated group. The normal bone marrow was regenerated in the saline-treated group, whereas most of the bone marrow space still contained fibroblast-like spindle-shaped cells in the MPSS-treated group on day 42. Alkaline phosphatase-positive cells were only seen in the areas around the regenerated bone marrow cavities. Thus, MPSS inhibits bone formation by suppressing osteoblast proliferation and resorption by suppressing the recruitment of osteoclast precursors. These findings may be useful when designing treatments for ION patients exposed to steroids.

Intermittent administration of human parathyroid hormone (1-34) increases new bone formation on the interface of hydroxyapatitecoated titanium rods implanted into ovariectomized rat femora.

BACKGROUND: As hydroxyapatite (HA) has good osteoconductive properties, HA is used as coating material for the implants in cementless arthroplasty. However, its effect is not sufficient for osteoporotic bone. Parathyroid hormone (PTH) is known to have anabolic effects on bone formation. Intermittent administration of PTH increases both cancellous and cortical bone mass. The aim of this study was to confirm the effect of the fixation strength of HA-coated implants in the osteoporotic condition with a mechanical test and a bone histomorphometric method. METHODS: Female Sprague-Dawley rats were used for this study. Four weeks after ovariectomy (OVX) or sham surgery, HA-coated titanium rods were inserted into the distal femoral canal (Sham+HA group and OVX+HA group). PTH was administered immediately after the implantation of the HAcoated rods (OVX+HA+P group). We measured the shear strength at the bone-implant interface by a push-out test and the newly formed bone volume on the implant (BV.Im) by bone histomorphometry at 2 and 4 weeks after implantation. RESULTS: The bone-implant shear strength in the OVX+HA group was significantly lower than that in the Sham+HA group at 2 weeks after implantation of the rods. In the OVX+HA+P group, the strength was significantly higher than that in the other groups. Similarly, at 4 weeks, statistically significant differences were confirmed in the bone-implant shear strength among the Sham+HA group, the OVX+HA group, and the OVX+HA+P group. BV.Im in the OVX+HA group was significantly lower than that in the Sham+HA group at 2 weeks after implantation. BV.Im was significantly higher in the OVX+HA+P group than that in the OVX+HA group. However, there was no difference in BV.Im between the Sham+HA group and the OVX+HA+P group. At 4 weeks after implantation, BV.Im was significantly lower in the OVX+HA group than that in the other groups, but no difference was found between the Sham+HA group and the OVX+HA+P group. CONCLUSIONS: Intermittent administration of PTH has an effect to increase new bone formation on the surface of HA-coated implants in the osteoporotic condition. This finding suggests that PTH administration is useful to improve the initial fixation of HA-coated implants even in osteoporotic patients.

The effect of cup placement in cementless total hip arthroplasty on the wear rate of polyethylene.

There are many reports on cup placement, inclination, anteversion, and position. This article strictly defines the area of cup placement based on data from 185 healthy hip centers and Ranawat's triangle. Linear wear rate (LWR) was measured in 55 total hip arthroplasty (THA) cases and categorized as low or high. The relationships between these categories and cup size, inclination, anteversion, position, age, follow-up period, bone graft, cup osteolysis, and stem osteolysis were investigated.

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat.

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.

Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation.

The aim of the study was to examine the chronological histology of osteoinduction of highly purified beta-tricalcium phosphate (beta-TCP) implanted in dog dorsal muscles. Specimens were harvested on days 14, 28, 42, 56, 112 and 168 after implantation, and were analyzed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, in situ hybridization, and silver impregnation. After day 28, abundant TRAP- and cathepsin K-positive multinucleated cells adhered to beta-TCP, suggesting that these cells are osteoclasts that can resorb beta-TCP. On day 56, new bone was formed and alpha1 chain of type I procollagen mRNA-positive osteoblasts lined the newly formed bone. Silver impregnation showed abundant collagen fibrils within the beta-TCP micropores. These results suggest that micropores function as a storage space for extracellular matrix components, including collagen. Newly formed bone never degenerated in the late stage, suggesting that beta-TCP has good biocompatibility and this material retains the conditions appropriate for osteointegration and bioresorption. In conclusion, beta-TCP has osteoinductivity after implantation in dog dorsal muscles without use of bone marrow cells or osteoinductive cytokines. The appearance of a large number of active osteoclasts precedes new bone formation.

Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones.

Prominent osteoconductive activity and the biodegradable nature of commercially available beta-tricalcium phosphate (beta-TCP, OSferion) have been documented in animal experiments. We analyzed four cases of involving grafted OSferion in human bone with respect to histological features by routine hematoxylin and eosin staining, silver impregnation, immunohistochemistry and in situ hybridization. OSferion affords early bioresorption by osteoclasts, vascular invasion of macropores and osteoblastic cell attachment on the surface on the ceramic surface 14 days after grafting. Prominent bone formation and direct bone connection between preexisting bone and OSferion were evident 28 days after grafting. Nearly the entire TCP surface was covered by lamellar bone; additionally, active osteoblastic lining and attachment of the osteoclast-like giant cells were not observed 72 weeks after grafting. Silver impregnation revealed the presence of collagen fibrils within probable micropores of OSferion.

Bone formation and resorption of highly purified beta-tricalcium phosphate in the rat femoral condyle.

The aim of this study was to examine the chronological histology associated with highly purified beta-tricalcium phosphate (beta-TCP) implanted in the rat femoral condyle. Specimens were harvested on days 4, 7, 14, 28 and 56 after implantation, and were analyzed by tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry of the ED1 protein as a marker of the phagocyte system, and in situ hybridization with digoxigenin-labeled alpha1 chain of type I procollagen (COL1A1), osteopontin and osteocalcin. beta-TCP was resorbed in a chronological manner. Although new bone was not observed on day 4, fibroblast-like cells around beta-TCP were positive for COL1A1 and osteopontin mRNA. New bone formation presented after day 7. In the double-staining for OPN and ED1 on day 7, most cells around beta-TCP were positive for either osteopontin mRNA or ED1 protein. However, there were some doubly positive multinucleated cells, suggesting that they belonged to the mononuclear phagocyte system. After day 28, the implanted region was replaced with bone marrow. Multinucleated TRAP-positive and ED1-positive cells which adhered to beta-TCP at all stages seemed to be osteoclasts and they continuously resorbed beta-TCP. beta-TCP has a good biocompatibility since both bioresorption and bone formation started at an early stage after implantation.

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis.

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.

High-level expression of the coxsackievirus and adenovirus receptor messenger RNA in osteosarcoma, Ewing's sarcoma, and benign neurogenic tumors among musculoskeletal tumors.

PURPOSE: The sensitivity of human tumor tissues to infection with recombinant adenoviruses correlates with the expression of the coxsackievirus and adenovirus receptor (CAR). CAR has been shown to function as the primary receptor for adenoviruses and to play a critical role in adenovirus entry into host cells. It is important for clinical gene therapy to determine the expression level of CAR in tumor tissues. EXPERIMENTAL DESIGN: We analyzed the expression of CAR mRNA in 154 musculoskeletal tumor tissues from 154 patients and 10 normal mesenchymal tissues from 3 patients using reverse transcription-PCR and real-time quantitative PCR. An adenovirus infection assay was performed in two cell lines that were established from CAR-positive osteosarcoma tissue and CAR-negative malignant fibrous histiocytoma tissue. RESULTS: Ninety-nine of 154 tumors were detected as CAR positive by reverse transcription-PCR. We found that the expression levels of CAR mRNA varied markedly between different tumors as determined by real-time quantitative PCR. CAR mRNA was expressed at high levels in osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma; at intermediate levels in exostosis, giant cell tumor, liposarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, and hemangioma; and at low levels in alveolar soft part sarcoma and desmoid. Whereas the osteosarcoma cell line that expressed a high level of CAR mRNA, like its parent tumor, had a high efficiency of adenovirus infection, the malignant fibrous histiocytoma cell line with almost undetectable expression of CAR mRNA, like its parent tumor, had a low efficiency of infection. CONCLUSIONS: Our data showed the great variations in CAR mRNA expression among human musculoskeletal tumors and mesenchymal tissues and implicated the potential usefulness of adenoviral vectors in gene therapy for osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma. Efficient transduction with adenovirus for gene therapy could be realized in appropriate, sensitive tumor types.

Activating Gs alpha mutation at the Arg201 codon in liposclerosing myxofibrous tumor.

Liposclerosing myxofibrous tumor (LSMFT) is a benign fibro-osseous lesion that is characterized by mixture of histologic elements including lipoma, fibroxanthoma, myxoma, ischemic ossification, and fibrous dysplasia (FD)-like features. These tissue components are seen in the original reports of FD; however, the relationship between LSMFT and FD is not clear. Point mutation of the alpha subunit of G protein (Gs alpha), which increases cyclic adenosine monophosphate formation, has been recognized as the cause of McCune-Albright syndrome as well as polyostotic and monostotic FD of bone. Gs alpha mutation at the Arg201 codon in 2 patients of LSMFT was demonstrated in the present study. Although direct sequencing analysis using the fresh-frozen materials could not detect the mutation, the polymerase chain reaction fragmentation length polymorphism (PCR-RFLP) disclosed the missense point mutation Gs alpha at the Arg201 codon in 2 cases involving LSMFT. This result strongly suggests that a subset of LSMFT is a variant form of FD.

Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis.

OBJECTIVE: Chondromodulin-I (ChM-I), a cartilage derived anti-angiogenic factor, has been shown to regulate the vascular invasion during endochondral bone formation. We evaluated the expression and localization of ChM-I in articular cartilage during the progression of osteoarthritis (OA) in the rat, and correlated ChM-I expression with the increase in vascular invasion into OA articular cartilage. METHODS: Expression of ChM-I, type II collagen, basic fibroblast growth factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases MMP-9 and MMP-13 were examined in articular cartilage of intact growing and adult rats and in the surgically induced OA model using in situ hybridization, Western blot analysis, and immunohistochemistry. Co-immunostaining for ChM-I and CD-31 was performed to localize ChM-I and neovascularization in articular cartilage at advanced stage of OA. RESULTS: Abundant expression of ChM-I protein was detected in avascular regions of the developing and adult healthy articular cartilage. In early OA, ChM-I expression decreased in the superficial zone of articular cartilage, while levels of proteoglycan and type II collagen were comparable to control. In advanced OA, ChM-I expression was reduced in all zones of articular cartilage, and the number of VEGF-expressing cells was increased. Immunohistochemical studies showed that vascular invasion occurred in proximity to chondrocytes with high expression of pro-angiogenic markers, and decreased expression of ChM-I. CONCLUSION: High expression of ChM-I was detected in articular cartilage of growing and normal adult joints, implicating its role in the maintenance of avascularity of intact articular cartilage. Expression of ChM-I decreased, while expression of VEGF and other pro-angiogenic factors increased, in OA cartilage. These findings suggest the loss of ChM-I from articular cartilage might be responsible in part for promoting blood vessel invasion into the cartilage during progression of OA.

Chondromodulin-I expression in rat articular cartilage.

The localization and expression of chondromodulin-I (ChM-I), an angiogenesis inhibitor, in the rat articular cartilage during maturation from 2 to 10 weeks of age were examined by immunohistochemistry, Western blot analysis and ribonuclease protection assay, and the results were compared with those in the epiphyseal cartilage. ChM-I was found to be diffusely immunostained in the inter-territorial space of the cartilage matrix from the intermediate to the deep layers at the immature stage. As the articular cartilage matured, the immunoreactivity was localized around the hypertrophic chondrocytes in the deep layer and the immunoreactivity became weak after maturation. In contrast, the ChM-I immunoreactivity was intense in the epiphyseal cartilage at all ages examined. ChM-I was detected by Western blotting as a broad band or occasionally as a cluster of multiple bands (approximately 25 kDa) in both the articular and the epiphyseal cartilage. The intensity of the bands decreased gradually with age in the articular cartilage, but was unchanged in the epiphyseal cartilage at all ages. Ribonuclease protection assay revealed that ChM-I mRNA also decreased gradually with age in the articular cartilage in parallel with the maturation of the articular cartilage, while no decrease in ChM-I mRNA was found in the epiphyseal cartilage. The expression of ChM-I mRNA in the articular cartilage was less than that in the epiphyseal cartilage at all ages. The decrease in amount of ChM-I in the mature articular cartilage suggests that ChM-I plays a more important role in the maintenance of avascularity in the immature articular cartilage than in the mature one. The avascular condition may be preserved by angiogenic inhibitors or mechanisms other than ChM-I in the mature articular cartilage.