Interspecies Single-Cell RNA-Seq Analysis Reveals the Novel Trajectory of Osteoclast Differentiation and Therapeutic Targets.

Bone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone-resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor-kappa-Β ligand (RANKL) stimulation. We performed single-cell RNA-sequencing analyses of human and murine osteoclast-lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Clinical Outcomes After First Metatarsophalangeal Joint Arthrodesis by Flat Cut Joint Preparation With Individual Adjustment for Sagittal Alignment.

Sagittal misalignment is a major cause of patient dissatisfaction and re-operation after first metatarsophalangeal (MTP) joint arthrodesis. The stereotypical application of the fixed angle would be undesirable, especially in cases of flat or cavus foot. We retrospectively reviewed 31 cases (27 patients) in which first MTP joint arthrodesis was performed using the flat cut joint preparation technique with reference to the plantar clearance beneath the pulp of the toe while simulating weightbearing by pushing a board against the sole. The most common underlying cause of surgery was rheumatoid arthritis (22 cases [71%]). Clinical outcomes were evaluated by the Japanese of Surgery of the Foot (JSSF) hallux scale and the self-administered foot evaluation questionnaire (SAFE-Q). Twenty-three cases were also examined by pedobarography to evaluate postoperative walking plantar pressure. At the most recent follow-up of a mean 19.6 months, the toe-to-floor distance of the hallux in static standing posture was a mean of 2.5 mm (range, 0-10 mm). All but 1 foot (97%) achieved bone union. There were no complications or revisions due to misalignment of the fused MTP joint. JSSF hallux scales improved significantly from 47 preoperatively to 82 postoperatively. All subscale scores except general health and well-being in the SAFE-Q improved significantly at final follow-up versus preoperative period. Plantar pressure under the hallux was correlated with the toe-to-floor distance but not radiographic parameter. In conclusion, first MTP joint arthrodesis achieved good clinical outcomes when using toe-to-floor distance and Kirschner wire template for flat cut joint preparation.

Excision Arthroplasty With Interpositional Achilles Tendon Autograft: A Novel Approach to Talonavicular Joint Osteoarthritis Associated With Ankle Arthrodesis.

Talonavicular joint arthritis is a great concern after ankle fusion. Although arthrodesis is the gold standard treatment for this complication, it could initiate a vicious cycle of further adjacent joint arthritis. An alternative that may delay or eliminate the need for arthrodesis is excision arthroplasty; however, there are only a few reports on its application on a talonavicular joint. We report 3 cases of excision arthroplasty with interpositional Achilles tendon autograft for the treatment of end-stage talonavicular osteoarthritis in low-demand elderly patients. In 1 patient, excision arthroplasty was performed after tibiotalocalcaneal arthrodesis, and in 2 patients, it was performed after tibiotalar arthrodesis, in which the subtalar joints were also damaged and fused simultaneously on performance of the interpositional arthroplasty of the talonavicular joint. In all cases, pain relief and functional activities of daily living improvement were achieved with this procedure. At a minimum follow-up of 1 year, no patient reported adjacent joint symptoms or flatfoot progression. These cases show that interpositional arthroplasty with Achilles tendon autograft is an effective treatment for end-stage talonavicular arthritis in patients with fused ankle and subtalar joints. This procedure was helpful in relieving pain and improving activities of daily living function in low-demand elderly patients with the preservation of movement of the talonavicular joint. Autograft was considered to be superior to other grafts with respect to availability, graft rejection, or allergy development. Fused subtalar joint resolved the concerning issues, such as flatfoot progression and muscular weakness of ankle plantar flexion, associated with this procedure.

Cooperation of PU.1 With IRF8 and NFATc1 Defines Chromatin Landscapes During RANKL-Induced Osteoclastogenesis.

Receptor activator of nuclear factor κB ligand (RANKL) induces osteoclast (OC) differentiation from bone marrow-derived macrophages (BMMs). The transcription factors nuclear factor of activated T cells 1 (NFATc1) and interferon regulatory factor (IRF) 8 play positive and negative roles, respectively, in this process. However, genomewide mapping of the active cis-regulatory elements regulating OC differentiation has not been performed, and little is known about the global landscape of OC-specific gene regulation. We used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements followed by sequencing to show that PU.1 transcription factor binding motifs were overrepresented at active cis-regulatory regions in both murine BMMs and OCs, while IRF and NFAT binding motifs were selectively enriched at these regions in BMMs and OCs, respectively. We also found that RANKL induced the downregulation of Irf8 and upregulation of Nfatc1 expression, which was associated with dramatic alterations in histone modification. BMM-specific PU.1 binding sites were observed to overlap with IRF8 binding sites in BMMs, and this also occurred for OC-specific PU.1 binding sites and NFATc1 binding sites in OCs. The expression of genes with IRF8 peaks within BMM-specific PU.1 binding sites was significantly higher in BMMs than in OCs, while that of genes with NFATc1 peaks within OC-specific PU.1 binding sites was significantly higher in OCs than in BMMs. Our results suggest that PU.1 switches its transcription partner from IRF8 to NFATc1 and alters the binding regions during RANKL-induced osteoclastogenesis, which is associated with changes in epigenetic profiles and the control of cell type-specific gene expression. © 2019 American Society for Bone and Mineral Research.

The utility of 25-question Geriatric Locomotive Function Scale for evaluating functional ability and disease activity in Japanese rheumatoid arthritis patients: A cross-sectional study using NinJa database.

OBJECTIVES: To investigate the distribution of 25-question Geriatric Locomotive Function Scale (GLFS-25) scores in Japanese rheumatoid arthritis (RA) patients and evaluate relationships with clinical variables. METHODS: Among 15,115 patients registered in the NinJa database for fiscal year 2015, 1710 with complete GLFS-25 and disease activity score-28 (DAS28) data were analyzed. Correlations between GLFS-25 score and clinical variables were assessed by Spearman coefficients. Mean GLFS-25 scores were compared among DAS28 groups (<2.6, 2.6-3.1, 3.2-5.0, ≥5.1) using the Kruskal-Wallis test. To evaluate the performance of the GLFS-25 and Health Assessment Questionnaire Disability Index (HAQ-DI) for predicting DAS28 ≥ 3.2 (moderate/high disease activity), receiver operator characteristic (ROC) curves were constructed. RESULTS: GLFS-25 score was significantly correlated with age, disease duration, DAS28, and HAQ-DI. GLFS-25 score increased in parallel with DAS28. The proportion of patients with locomotive syndrome stage 2 also increased with DAS28. Area under the curve values for HAQ-DI and GLFS-25 score were 0.739 and 0.768, respectively. At a GLFS-25 positive cutoff score ≥16, sensitivity was 0.716 and specificity was 0.661 for predicting DAS28 ≥ 3.2. CONCLUSION: This study documents the GLFS-25 score distribution in Japanese RA patients and demonstrates that GLFS-25 is a useful measure for evaluating functional ability in RA.

The effect of switching from teriparatide to anti-RANKL antibody on cancellous and cortical bone in ovariectomized mice.

We examined the effect of teriparatide, and switching from teriparatide to anti-RANKL (receptor activator of nuclear factor κB ligand) monoclonal antibody, in ovariectomized mice. Twelve-week-old female C57BL/6 mice were ovariectomized or sham operated. Four weeks after surgery, ovariectomized mice were subjected to one of the following four treatments: phosphate-buffered saline (PBS) for 8weeks; teriparatide for 4weeks followed by PBS for 4weeks (PTH4W group); teriparatide for 8weeks (PTH8W group); or teriparatide for 4weeks followed by anti-RANKL antibody (single subcutaneous injection of 5mg/kg) (SWITCH group). Twelve weeks after the operation, bone mineral density was increased in PTH8W and SWITCH groups to broadly comparable levels, but these were significantly decreased in the PTH4W group after discontinuation of teriparatide. Histomorphometric analysis demonstrated that cancellous bone formation and resorption were profoundly suppressed in the SWITCH group. Bone formation was also suppressed on the endocortical surface of cortical bone but was maintained on the periosteal surface. Anti-RANKL antibody suppressed osteoclast activity immediately after treatment, while bone formation was only gradually decreased. These results suggest that anti-RANKL antibody may be a therapeutic option after discontinuation of teriparatide therapy.

Trends in Treatment, Outcomes, and Incidence of Orthopedic Surgery in Patients with Rheumatoid Arthritis: An Observational Cohort Study Using the Japanese National Database of Rheumatic Diseases.

OBJECTIVE: In this study, we investigated the changes in clinical outcome, treatment, and incidence of orthopedic surgery in patients with rheumatoid arthritis (RA) from 2004 to 2014. METHODS: Data were studied from the Japanese nationwide cohort database, NinJa (National Database of Rheumatic Diseases by iR-net in Japan), from 2004 to 2014. The time trends in the incidence of orthopedic procedures were analyzed using linear regression analysis. The cross-sectional annual data were compared between 2004 and 2014 to analyze the changes in clinical outcome and treatment. RESULTS: The incidence of orthopedic surgeries in patients with RA consistently decreased from 72.2 procedures per 1000 patients in 2004 to 51.5 procedures per 1000 patients in 2014 (regression coefficient = -0.0028, 95% CI -0.0038 to -0.0019, p < 0.001). The greatest reduction was found in total knee arthroplasty and total hip arthroplasty. Disease activity and functional disability improved significantly over this decade. The proportions of patients receiving methotrexate and biologic disease-modifying antirheumatic drugs significantly increased from 39.6% and 1.7% in 2004 to 63.8% and 27.4% in 2014, respectively. CONCLUSION: The overall incidence of orthopedic surgeries in patients with RA significantly decreased, accompanied by improved clinical outcomes because of the expanded use of effective drugs; however, the declining trend differed between procedures or locations. The results from the present study suggest that there might be a change in supply and demand for orthopedic surgeries.

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

Interdigital Neuroma in the Second Intermetatarsal Space Associated with Metatarsophalangeal Joint Instability.

The entrapment theory is the most commonly accepted theory concerning the development of interdigital neuroma; it incriminates the deep transverse metatarsal ligament as the major causative factor of the condition. This report presents a patient with interdigital neuroma in the second intermetatarsal space, which was strongly suspected to be caused by the metatarsophalangeal joint instability due to plantar plate injury. Surgical intervention revealed that the neuroma was located more distally and dorsally than the deep transverse metatarsal ligament and was pinched between the adjacent metatarsal heads, suggesting the involvement of the metatarsophalangeal joint instability and chronic trauma as etiologies in this case.

Time trends and risk factors for perioperative complications in total ankle arthroplasty: retrospective analysis using a national database in Japan.

BACKGROUND: Total ankle arthroplasty (TAA) has become increasingly popular worldwide as an alternative to ankle arthrodesis for surgical treatment of end-stage ankle arthritis. The aim of this epidemiological study, using a national inpatient database in Japan, was to describe the volume, utilization, patient characteristics, and temporal trends regarding these procedures in Japan, and to identify the risk factors associated with perioperative adverse events in TAA. METHODS: This was a population-based, retrospective cohort study. We retrospectively identified 2775 patients in the Diagnosis Procedure Combination database who underwent ankle arthrodesis or TAA for ankle arthritis at 559 hospitals in Japan from 2007 to 2013. Information on sex, age, main diagnosis, use of blood transfusion, duration of anesthesia, length of hospital stay, in-hospital mortality, hospitalization costs, additional procedures after primary surgery, and use of negative pressure wound therapy was extracted. Multivariable logistic regression analysis was performed to analyze the effect of various factors on the incidence of perioperative adverse events in TAA, including additional procedure during hospitalization, negative pressure wound therapy, blood transfusion, and in-hospital death. RESULTS: We identified 465 patients who underwent TAA and 2310 patients who underwent ankle arthrodesis. There was no apparent increase in the proportion of TAAs performed during the survey period. Patients undergoing TAA tended to be older, female, and have rheumatoid arthritis compared with those undergoing ankle arthrodesis. Patients undergoing TAA had shorter length of stay, higher hospitalization costs, and more blood transfusions compared with those undergoing ankle arthrodesis. Lower hospital volume and shorter anesthesia time were associated with higher rates of adverse events after TAA. CONCLUSIONS: Despite an increase in the popularity of TAA internationally, the number of TAAs performed remains low in Japan. Lower hospital volume and anesthesia time were associated with higher rates of perioperative adverse events after TAA. LEVEL OF EVIDENCE: IV, Cross-sectional study.

Identification of Nedd9 as a TGF-ß-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis.

TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis.

Individual and combining effects of anti-RANKL monoclonal antibody and teriparatide in ovariectomized mice.

We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand) monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX) or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab) alone (5 mg/kg single injection at 4 weeks after OVX, Ab group), teriparatide alone (80 µg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group), or mAb plus teriparatide (Ab + PTH group). Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD) was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

Genomewide comprehensive analysis reveals critical cooperation between Smad and c-Fos in RANKL-induced osteoclastogenesis.

We have previously reported that transforming growth factor ß (TGF-ß) plays an essential role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-ß, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF-ß signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF-ß regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos.

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell-cell fusion.

Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(-) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell-cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell-cell fusion.

A Japanese patient with Li-Fraumeni syndrome who had nine primary malignancies associated with a germline mutation of the p53 tumor-suppressor gene.

We describe a patient who had nine primary malignant tumors and a germline mutation in the p53 tumor-suppressor gene, characteristically found in the Li-Fraumeni syndrome (LFS). A 15-year-old girl with no family history of cancer was referred to our hospital because of pain and swelling of the right knee. Osteosarcoma was diagnosed. The patient received chemotherapy followed by surgery and had a remission. After the age of 28 years, nine primary malignant tumors developed successively, including right breast cancer, colon cancer, malignant fibrous histiocytoma (MFH) of the abdominal wall, right lung double cancers, bilateral breast cancers, and MFH of the left thigh. This is the second highest number of types of primary malignant tumors to be reported in LFS. All tumors were treated by a multidisciplinary approach, including surgery. Genetic analysis revealed a germline missense mutation in the p53 gene (c.659 A > G), resulting in Y220C, which has been reported in three families with LFS. The patient died of lung metastasis from MFH at the age of 37 years. Despite the multiple tumors, repeated induction of remissions resulted in long survival. Our findings suggest that a multidisciplinary approach to treatment, including surgery, is beneficial in patients with LFS.

Malignant change secondary to fibrous dysplasia.

BACKGROUND: Malignant change in fibrous dysplasia (FD) is very rare. This study was carried out to establish some characteristic clinical information about this disorder. METHODS: Four cases with a malignant change in FD out of 128 cases with FD were surgically treated and followed up for a median period of 61.3 months. The mean age of the patients was 39.8 years. Clinical features, radiological findings, and the outcome were analyzed for each of the four cases. RESULTS AND CONCLUSION: The sites of the lesions were tibia (2 cases), femur (1 case), and rib (1 case). The forms of FD were monostotic in one case and polyostotic in three cases. Radiologically, plain films and computed tomography (CT) showed osteolytic lesions with poorly delineated margins within and/or near areas having a ground-glass appearance. In the osteolytic lesions, simple cystic changes associated with old FD could be excluded by enhanced magnetic resonance imaging (MRI). Histopathologically, two cases were osteosarcoma, one case was malignant fibrous histiocytoma (MFH), and one case was fibrosarcoma. The management of this disease should be decided according to the type of primary high-grade bone sarcoma. One patient, with MFH, was dead of lung metastasis 13 months after surgery. The others are alive without disease.

Oncologic outcome of parosteal osteosarcoma.

BACKGROUND: This retrospective review evaluated the clinical features and surgical outcomes of parosteal osteosarcoma (POS). METHODS: Nine patients were surgically treated and followed up. Their mean age was 30.8 years. Clinical information and oncologic outcomes of each case were analyzed. RESULTS: Sites of involvement were all in the femur, and all tumors arose from the metaphyseal area of the distal femur. Biopsies for definite diagnoses were performed in just two of the nine cases. Wide resection was applied for all tumors. Surgical evaluations were a 1-cm-wide procedure in six cases and a 2-cm-wide procedure (or more) in three cases. All patients were found to be continuously disease free during the follow-up period of 115.1 months. CONCLUSION: POS showed characteristic findings on radiographic images. Therefore, wide resection without biopsy could be performed in 77.8% (7/9) of the cases. This procedure may contribute to attaining better limb function, because of preventing contamination of healthy surrounding tissue and minimizing the extent of resection. The safety margin was evaluated as a 1-cm-wide procedure. For the choice of reconstruction, indication of autobone grafting (3/9) or total knee replacement (TKR) (6/9) depended on tumor size, location, and shape. With no adjuvant treatments, all cases have shown good clinical courses during the entire follow-up period of about 10 years.