A Case of Fibula Regeneration after Below-the-knee Amputation in an Adult.

We report the case of an adult with fibula regeneration after below-the-knee amputation. Fibula regeneration conventionally occurs at the donor site of children after autogenous fibula transplantation when the periosteum is preserved. However, the patient was an adult, and the regenerated fibula was 7-cm long and grew directly from the stump. A 47-year-old man was referred to the plastic surgery department owing to stump pain. He had an open comminuted fracture of the right fibula and tibia due to a traffic accident when he was 44 years old and underwent below-the-knee amputation and negative pressure wound therapy for skin defects. The patient recovered and was able to walk using a prosthetic limb. Upon radiography, the fibula was found to have regenerated 7 cm directly from the stump. Pathological examination revealed that the regenerated fibula contained normal bone tissue and neurovascular bundles in the cortex. The periosteum, mechanical stimuli with limb proteases, and negative pressure wound therapy were suspected to have accelerated bone regeneration. He had no inhibitory factors for bone regeneration, including diabetes mellitus, peripheral arterial disease, or active smoking status. After the resection of the regenerated fibula, the patient was ambulatory without further bone regeneration or pain. This case report suggests that bone regeneration may occur even in adults. The surgeon should not leave any part of the periosteum behind in patients undergoing amputation. In adult amputees complaining of stump pain, the possibility of bone regeneration may be considered.

Cancer may accelerate locomotive syndrome and deteriorate quality of life: a single-centre cross-sectional study of locomotive syndrome in cancer patients.

BACKGROUND: Thanks to recent advancement in cancer treatment, an increasing number of cancer patients are expected to live longer with cancer. The ambulatory ability is essential for cancer patients to spend their own independent lives, but locomotive syndrome (LS), a condition of reduced mobility due to impairment of locomotive organs, in cancer patients has been seldom examined. METHODS: This was a single-institutional cross-sectional study. Cancer patients receiving cancer therapy between April 2020 and March 2021 were asked to participate. LS was classified as stage 0-3, and compared with their performance status (PS). Physical component summary (PCS) and mental component summary (MCS) were calculated from the results of Short Form-8. Logistic regression analysis was performed to identify risk factors for LS stage 3. RESULTS: One hundred and seventy-six cancer patients were included. The rate of LS was 96.0%. That of LS stage 3 was 40.9% and as high as 29.7% even if limited to those with PS 0. The mean PCS and MCS were both inferior to the national averages. PCS decreased as the LS stage advanced. Old age and underweight were revealed as independent risk factors for LS stage 3. CONCLUSIONS: The ratio of LS in cancer patients was extremely high, and the LS stage correlated with physical QOL. Even those with PS 0 can have severe LS; thus, LS can be a sensitive detector of physical disability of cancer patients than PS. The improvement of LS can be a key to the preservation of their ADL and QOL.

Involvement of orthopaedic surgeons for cancer patients in orthopaedic training facilities certified by the Japanese Orthopaedic Association - A nationwide survey.

BACKGROUND: In order to improve cancer care in Japan further, it is now required for orthopaedic surgeons to get actively involved in managing locomotive organs such as bones, muscles and nerves in cancer patients. In 2018, the Japanese Orthopaedic Association (JOA) conducted a questionnaire survey to investigate the current status of cancer treatment at the orthopaedic training facilities certified by the JOA. We analyzed the results of that questionnaire survey, focusing on the data from the core hospitals for cancer care (designated cancer hospitals), to clarify the involvement of orthopaedic surgeons in cancer treatment. MATERIALS AND METHODS: A nationwide survey was conducted in the orthopaedic training facilities certified by the JOA using an online questionnaire from March 15th to 31st, 2018. To clarify the involvement of orthopaedic surgeons in cancer treatment, we analyzed the results of that questionnaire survey, focusing on the data from the designated cancer hospitals in Japan. RESULTS: From the questionnaire survey, it became clear that 24% of the orthopaedic training facilities certified by the JOA are designated cancer hospitals. There were significant differences concerning cancer treatment and the prospect of orthopaedic surgeons' involvement in the treatment for bone metastases between institutions classified according to number of both certified orthopaedic surgeons by the JOA and specialists for bone and soft tissue tumors. In addition, in 45% of the designated cancer hospitals, orthopaedic surgeons treated bone metastases that occur in cancer patients, but in the rest of the institutions, orthopaedic surgeons did not yet adequately respond. CONCLUSION: In order to further improve the locomotive function and quality of life (QOL) in cancer patients, it was seemed to be necessary that all medical professionals engaged in cancer treatment, including orthopaedic surgeons, recognize the importance of locomotive management for cancer patients. In addition, the results of this study suggested that the presence of more than six certified orthopaedic surgeons by the JOA, including one or more specialists for bone and soft tissue tumors, may be able to create an environment conducive to the involvement of orthopaedic surgeons in cancer treatment at the facility.

Accuracy and reliability of a smartphone application for measuring the knee joint angle.

[Purpose] Recently, a photo-based smartphone application for angle measurement-"Grid line imaging application Professional"-was developed to evaluate joint disease treatments. The aim of this study was to determine the accuracy and reliability of the application. [Participants and Methods] We measured the knee joint of a mannequin using an application and a universal goniometer. Twelve examiners measured eight knee joints of mannequins at different arbitrary angles using the application and a universal goniometer. Correlations between the application and universal goniometer measurements were examined using scatter plots and correlation coefficients. Systematic errors of the application were visually confirmed using the Bland-Altman method. Intra-class correlation coefficients were used to evaluate the inter-examiner reliability of the application. [Results] The application and universal goniometer measurements showed a good correlation (r=0.99) and no systematic error. The intra-class correlation coefficient for inter-examiner reliability was 0.999. Furthermore, to evaluate intra-examiner reliability, six examiners measured six different knee joints twice using the application on a 2-day interval. The intra-class correlation coefficient for intra-examiner reliability was 0.982. [Conclusion] The accuracy of the application was equivalent to that of a universal goniometer, and both the inter- and intra-examiner reliabilities of the application were almost perfect.

Three-dimensional motion analysis of lumbopelvic rhythm during lateral trunk bending.

[Purpose] To examine the variations in the lumbopelvic rhythm and lumbar-hip ratio in the frontal plane. [Subjects and Methods] Markers were placed on the T10 and T12 spinous processes, bilateral paravertebral muscles at the T11 level, the pelvis, and the femur. Lumbar spine and hip angles were measured during lateral trunk bending using three-dimensional motion analysis. Data from the trunk lateral bending movement were categorized into descending (start of hip movement to when the hip angle reached its maximum value) and ascending (from the maximum hip angle to the end of movement) phases. The lumbar-hip ratio was calculated as the ratio of the lumbar spine angle to the hip angle. [Results] The lumbar-hip ratio decreased from 5.9 to 3.6 in the descending phase, indicating lumbar spinal movement was less than hip movement. In the ascending phase, the lumbar-hip ratio was reversed. The lumbopelvic rhythm was better expressed by a cubic or quadratic function rather than a linear function. These functions indicate that when the hip inclines by 1° that the lumbar spine bends laterally by 2.4°. [Conclusion] The lumbopelvic rhythm and lumbar-hip ratio indicate lumbar lateral bending instead of a limitation of hip inclination.

Mortality-Reducing Effect of Rehabilitation for COPD: Observational Propensity-Matched Cohort Study Using a Nationwide Database.

BACKGROUND: In the course of therapy of patients with COPD, non-pharmacologic treatment, such as rehabilitation, plays an important role. Although some studies have provided concrete evidence of the effectiveness of rehabilitation in improving functional outcomes in subjects with COPD, evidence of its mortality-reducing effect has been insufficient. In the present study, we examined whether rehabilitation had positive effects on in-hospital mortality of subjects with COPD. METHODS: We used the Japanese Diagnosis Procedure Combination nationwide administrative claims database. This was a retrospective cohort study, and there were 18,037 eligible subjects with COPD from 1,055 hospitals. The main outcome was in-hospital mortality rates. A one-to-one propensity score matching method was used to compare hospital mortality rates after admission between rehabilitation and non-rehabilitation groups. RESULTS: A total of 3,356 pairs of subjects were selected from the rehabilitation and non-rehabilitation groups (n = 6,712). Subjects in the rehabilitation program showed a reduction in the odds of mortality (odds ratio = 0.80, 95% CI 0.65-1.00, P = .045). In the subgroup analyses, the rehabilitation group had a lower in-hospital mortality in the pre-obese subgroup (body mass index 25.0-29.9) than the non-rehabilitation group (P = .02). Although not significant, the rehabilitation group showed a relatively low in-hospital mortality in the Hugh-Jones dyspnea scale class 5 subgroup (P = .066). CONCLUSIONS: This large nationwide cohort study showed that rehabilitation indeed contributed to a reduction of in-hospital mortality. These findings underscore the importance of adopting rehabilitation as part of the treatment of COPD.

Three-Dimensional Motion Analysis of Lumbopelvic Rhythm During Trunk Extension.

Hip-spine coordination, known as the lumbopelvic rhythm, can be expressed as the lumbar-hip ratio. The lumbopelvic rhythm and lumbar-hip ratio can be used to assess lower limb function. We clarified the lumbopelvic rhythm and lumbar-hip ratio during trunk extension. We established a novel set of marker positions for three-dimensional motion analysis to assess the lumbar spinal angle. The original markers were placed on both paravertebral muscle groups at the 11th thoracic spinous process level, the 10th and 12th thoracic spinous processes, and the pelvis. We measured angle data during trunk extension using three-dimensional motion analysis, and the data for eight healthy male subjects were categorized into backward and forward phases. The lumbar-hip ratio increased significantly from 1.2 to 1.9 (mean, 1.6) in the backward phase, indicating considerable movement of the lumbar spine compared with hip movement in the latter phase. In the forward phase, the ratio decreased significantly from 1.9 to 0.5 (mean, 1.5). After completion of 80% of the forward phase, the lumbar-hip ratio decreased to <1.0. The lumbopelvic rhythm for trunk extension was better expressed by a cubic or quadratic function than a linear function. According to a linear function, when the hip extends by 1°, lumbar spine extends by 1.9°. Therefore, lumbar spinal movement was greater than hip movement in the sagittal plane. The implication of the curved line would indicate lumbar extension instead of the limitation of hip extension.

Neck range of motion measurements using a new three-dimensional motion analysis system: validity and repeatability.

PURPOSE: Neck movement is important for many activities of daily living (ADL). Neck disorders, such as cervical spondylosis and whiplash can limit neck movement and ADL. The cervical range of motion (CROM) device has been recently used to measure neck range of motion (ROM); however, this measurement includes trunk motion, and therefore does not represent a pure neck ROM measurement. The authors aimed to develop a new method to establish pure neck ROM measurements during flexion, extension, lateral bending, and rotation using a three-dimensional motion analysis system, VICON. METHODS: Twelve healthy participants were recruited and neck ROMs during flexion, extension, lateral bending, and rotation were measured using VICON and the CROM device. Test-retest repeatability was assessed using interclass correlation coefficients (ICCs), standard error of measurement (SEM), and minimal detectable change (MDC). Validity between two measurements was evaluated using a determination coefficient and Pearson's correlation coefficient. RESULTS: ICCs of neck ROM measured using VICON and the CROM device were all at substantial or almost perfect levels [VICON: ICC(1,2) = 0.786-0.962, the CROM device: ICC(1,2) = 0.736-0.950]. Both SEMs and MDCs were low in all measurement directions (VICON: SEM = 1.3°-4.5°, MDC = 3.6°-12.5°; the CROM device: SEM = 2.2°-3.9°, MDC = 6.1°-10.7°). Determination coefficients (R(2)s) and Pearson's correlation coefficients (rs) between the two measurement methods were high (R(2) = 0.607-0.745, r = 0.779-0.863). CONCLUSIONS: VICON is a useful system to measure neck ROMs and evaluate the efficacy of interventions, such as surgery or physiotherapeutic exercise.

Education and related support from medical specialists for Japanese patients with major skeletal dysplasias.

BACKGROUND: Skeletal dysplasias manifest various clinical symptoms. Age at onset, severity, and progression of symptoms differ even among individuals with the same diagnosis. Though necessary support in education is presumed to differ among patients with different disorders, few articles report on education in patients with skeletal dysplasias. OBJECTIVE: To clarify what types of schools children with major skeletal dysplasias attend, what kind of support they needed at schools, and how the advice on such support was conveyed from medical specialists to schools. METHODS: Questionnaire study on patients with achondroplasia or hypochondroplasia (A/HCH), and osteogenesis imperfecta (OI). RESULTS: In A/HCH childhood locomotion ability was high and most patients had received general education, irrespective of their generation. Children with OI showed a lower level of locomotion ability; only about half of them had received general education. In selecting schools, the patients received advice from pediatricians, physiatrists, and orthopedic surgeons. The degree of necessity and content of support at the schools differed between A/HCH and OI. Remodeling of the lavatory, washbasin, and chair and support during swimming lessons were common in A/HCH patients. Support in school for OI patients was more frequent and included propelling wheelchairs, assisting in the use of the bathroom, and remodeling the lavatory. Most children were restricted from participating in physical education classes. CONCLUSIONS: Locomotion ability and the necessary support at school differed between A/HCH and OI. Support and advice from medical specialists who recognize disability of patients with skeletal dysplasias may improve patients' participation and education in schools.

Novel 3-dimensional motion analysis method for measuring the lumbar spine range of motion: repeatability and reliability compared with an electrogoniometer.

STUDY DESIGN: Repeatability and reliability for measuring methods for real-time lumbar range of motion. OBJECTIVE: We established a novel set of marker positions for 3-dimensional motion analysis (VICON system) to determine lumbar spine range of motion (LROM) and lumbar motion precisely; we compared the repeatability and reliability of VICON system with those of an electrogoniometer. SUMMARY OF BACKGROUND DATA: The assessment of the LROM using x-ray is still one of the most precise methods, despite the radiation exposure. To avoid this, alternative methods, such as the VICON system and electrogoniometer, have been widely used. No study has reported the repeatability and reliability of LROM measurements using a VICON system and electrogoniometer. METHODS: The VICON system and electrogoniometer measured LROM and lumbar motion in 7 healthy males during 7 days. Differences between both systems were analyzed using Bland-Altman plots. Repeatability and reliability of the LROM measurements was assessed using coefficients of multiple correlations and intraclass correlation coefficients, respectively. Standard error of measurement was calculated to quantify the systematic error in LROM measurements. RESULTS: The mean maximum LROM values using the VICON system/electrogoniometer were 42°/52° for flexion, 17°/24° for extension, 16°/16° for lateral bending, and 8°/2° for axial rotation, respectively. Between VICON system and the electrogoniometer, Bland-Altman plots revealed no discrepancies in LROM values except for flexion.Coefficients of multiple correlations for LROM showed excellent repeatability. LROM measurements with VICON system showed excellent reliability for flexion and extension and fair-to-good reliability for other motions. LROM measurements with the electrogoniometer showed excellent reliability for flexion and fair-to-good reliability for other motions. Except for axial rotation, maximum intraclass correlation coefficients using VICON system were more reliable than the electrogoniometer for measuring lumbar motion. CONCLUSION: VICON system with our novel marker set allows practical and reliable longitudinal assessment of dynamic LROM. LEVEL OF EVIDENCE: N/A.

Two-dimensional video gait analyses in patients with congenital insensitivity to pain.

PURPOSE: To clarify the features of gait in patients with congenital insensitivity to pain (CIP) by analyzing the video-recorded gait. METHODS: Eleven patients with the diagnosis of hereditary sensory and autonomic neuropathy (HSAN) type 4 or 5 and 15 healthy participants were enrolled in this study. Gait was analyzed using two-dimensional (2D) digital video and video analysis software. Walking speed, cadence, step length, stance phase duration, and heel contact to foot flat period were compared between patients and healthy participants. RESULTS: The results showed that walking speed and heel contact angular velocity were significantly higher (p < 0.05), and step length was significantly longer (p < 0.05) in CIP patients, especially in the younger age group. CONCLUSION: Young patients with CIP walked faster, with a longer step length and higher heel contact angular velocity than young control participants. These results may explain the frequency of lower extremity injuries in CIP patients.

ß-catenin regulates parathyroid hormone/parathyroid hormone-related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C-terminal region of the receptor.

OBJECTIVE: To investigate the underlying mechanisms of action and functional relevance of ß-catenin in chondrocytes, by examining the role of ß-catenin as a novel protein that interacts with the intracellular C-terminal portion of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor type 1 (PTHR-1). METHODS: The ß-catenin-PTHR-1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C-terminus, using a mammalian two-hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain- and loss-of-function of ß-catenin, transfection experiments were performed to induce overexpression of the constitutively active form of ß-catenin (ca-ß-catenin) and to block ß-catenin activity with small interfering RNA, in cells cotransfected with either wild-type PTHR1 or mutant forms (lacking binding to ß-catenin). Activation of the G protein α subunits G(αs) and G(αq) in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca(2+) concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay. RESULTS: In differentiated chondrocytes, ß-catenin physically interacted and colocalized with the cell membrane-specific region of PTHR-1 (584-589). Binding of ß-catenin to PTHR-1 caused suppression of the G(αs)/cAMP pathway and enhancement of the G(αq)/Ca(2+) pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca-ß-catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of ß-catenin (via the Cre recombinase) in chondrocytes from ß-catenin-floxed mice. Mutagenesis analyses to block the binding of ß-catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers. CONCLUSION: ß-catenin binds to the PTHR-1 C-tail and switches the downstream signaling pathway from G(αs)/cAMP to G(αq)/Ca(2+), which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.

A genome-wide sib-pair linkage analysis of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common musculoskeletal disease among people after middle age. The OPLL presents with serious neurological abnormalities due to compression of the spinal cord and nerve roots. The OPLL is caused by genetic and environment factors; however, its etiology and pathogenesis still remain to be elucidated. To determine the susceptibility loci for OPLL, we performed a genome-wide linkage study using 214 affected sib-pairs of Japanese. In stratification analyses for definite cervical OPLL, we found loci with suggestive linkage on 1p21, 2p22-2p24, 7q22, 16q24 and 20p12. Fine mapping using additional markers detected the highest non-parametric linkage score (3.43, P = 0.00027) at D20S894 on chromosome 20p12 in a subgroup that had no complication of diabetes mellitus. Our result would shed a new light on genetic aspects of OPLL.

G alpha(q) signal in osteoblasts is inhibitory to the osteoanabolic action of parathyroid hormone.

This study examined the role of the Gα(q) signal constituted by Gα(q) and Gα(11) (encoded by Gnα(q) and Gnα(11), respectively), a major intracellular pathway of parathyroid hormone (PTH), in the PTH osteoanabolic action by the gain- and loss-of-function analyses. Transgenic mice with osteoblast-specific overexpression of the constitutively active Gnα(q) gene under the control of 2.3-kb type I collagen α1 chain (Col1a1) promoter exhibited osteopenia with decreased bone formation parameters and did not respond to the daily PTH treatment. We then established osteoblast-specific Gnα(q) and Gnα(11) double-knock-out (cDKO) mice by crossing the 2.3-kb Col1a1 promoter-Cre recombinase transgenic mice and those with Gnα(q) gene flanked with loxP and global ablation of Gnα(11) (Col1a1-Cre(+/-);Gna(q)(fl/fl);Gna(11)(-/-)) and found that the cDKO and single knock-out littermates of Gnα(q) or Gnα(11) exhibited normal bone volume and turnover under physiological conditions. With a daily injection of PTH, however, the cDKO mice, but not the single knock-out mice, showed higher bone volume and turnover than the wild-type littermates. Cultures of primary osteoblasts derived from cDKO and wild-type littermates confirmed enhancement of the PTH osteoanabolic action by the Gα(q) signal deficiency in a cell-autonomous mechanism, in association with the membrane translocation of protein kinase Cδ. This enhancement was reproduced by overexpression of regulator of G protein signaling-2, a Gα(q) signal inhibitor, in osteoblastic MC3T3-E1 cells. Hence, the Gα(q) signal plays an inhibitory role in the PTH osteoanabolic action, suggesting that its suppression may lead to a novel treatment in combination with PTH against osteoporosis.

[Cytokines in bone diseases. Genetic defects of PTH/PTHrP receptor in chondrodysplasia].

Parathyroid hormone-related protein (PTHrP) signaling plays important roles in regulating the differentiation of chondrocytes in endochondral bone development. PTHrP signaling functions as an inhibitory effect on chondrocyte hypertrophy which is a terminal stage of differentiation at a growth plate. Mutations of the PTH÷PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP and its receptor genes in mice have demonstrated the critical roles of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes.

Antiapoptotic molecule Bcl-2 is essential for the anabolic activity of parathyroid hormone in bone.

The antiapoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Since Bcl-2(-/-) mice die before six weeks of age on account of renal failure and cannot be compared to adult wild-type mice, we generated Bcl-2(-/-)Bim(+/-) mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2(+/-)Bim(+/-) littermates. Bcl-2(-/-)Bim(+/-) mice grew normally, but exhibited decreased bone mass compared to Bcl-2(+/-)Bim(+/-) mice, mainly due to impaired osteoblast function. Interestingly, the anabolic effect of parathyroid hormone (PTH) was not observed in Bcl-2(-/-)Bim(+/-) mice. This data demonstrates that Bcl-2 is indispensable for the anabolic activity of PTH in bone.

Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditions.

OBJECTIVE: To examine the role of the phosphoinositide-dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification. METHODS: Skeletal phenotypes of homozygous Akt1-deficient (Akt1(-/-)) mice and their wild-type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1. RESULTS: Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1(-/-) chondrocytes, indicating a dominant role of Akt1. Akt1(-/-) mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild-type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1(-/-) joints. Calcification was significantly suppressed in cultures of Akt1(-/-) chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1. CONCLUSION: Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells.

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co-culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co-culture and in the bone marrow cell layer of the separated co-culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co-culture, accompanied by IGF-I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin-receptor substrate-1 (IRS-1-/-) or when osteoblasts were from PTH1R-/- mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell-to-cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF-I which activates IRS-1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation.

Screening of chondrogenic factors with a real-time fluorescence-monitoring cell line ATDC5-C2ER: identification of sorting nexin 19 as a novel factor.

OBJECTIVE: To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors. METHODS: The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor beta (TGFbeta) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene. RESULTS: We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGFbeta, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19. CONCLUSION: We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.

Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type X collagen gene.

OBJECTIVE: Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells. METHODS: Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation. RESULTS: RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor beta in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between -89 and -60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells. CONCLUSION: Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.