KIF22 regulates mitosis and proliferation of chondrocyte cells.

Point mutations in KIF22 have been linked to spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2). Skeletal features of SEMDJL2 include short stature and joint laxity. Mechanisms underlying these limb abnormalities are unknown. Here in this manuscript, we have investigated the function of KIF22 in chondrocytes. Quantitative PCR and immunostaining revealed that Kif22 was highly expressed in proliferating-zone growth-plate chondrocytes. Kif22 knockdown resulted in defective mitotic spindle formation and reduced cell proliferation. Forced expression of SEMDJL-associated mutant Kif22 constructs likewise induced abnormal mitotic spindle morphology and reduced proliferation. Mice expressing a KIF22 truncation mutant had shorter growth plates and shorter tibial bones compared to wild-type mice. These results suggest that KIF22 regulates mitotic spindle formation in proliferating chondrocytes thereby linking the stunted longitudinal bone growth observed in SEMDJL2 to failures of chondrocyte division.

Dietary restriction plus exercise change gene expression of Cxcl12 abundant reticular cells in female mice.

INTRODUCTION: Low energy availability due to excessive exercise lowers bone mass and impairs various physiological functions, including immunity and hematopoiesis. We focused on Cxcl12 abundant reticular (CAR) cells, which are bone marrow mesenchymal stem cells and are essential for the maintenance of hematopoietic and immune cells in bone marrow. We examine the functional changes in CAR cells resulting from dietary restriction combined with exercise. MATERIALS AND METHODS: Five-week-old wild-type female mice were divided into an ad libitum group (CON), a 60% dietary restriction group (DR), an ad libitum with exercise group (CON + ex), and a 60% dietary restriction with exercise group (DR + ex). Blood parameters, bone structure parameters, and bone marrow fat volume were evaluated after 5 weeks. In addition, bone marrow CAR cells were isolated by cell sorting and analyzed for gene expression by RT-qPCR. RESULTS: Bone mineral density (BMD) was significantly decreased in DR and DR + ex compared to CON and CON + ex. Especially, cortical bone mass and thickness were significantly decreased in DR and DR + ex groups, whereas trabecular bone mass was significantly increased. Bone marrow fat volume was significantly increased in DR and DR + ex groups compared to CON and CON + ex. The number of leukocytes in the blood was significantly decreased in the DR + ex group compared to the other three groups. RT-qPCR showed a significant decrease in gene expression of both Foxc1 and Runx2 in CAR cells of the DR + ex group compared to CON. CONCLUSION: Dietary restriction combined with exercise promotes CAR cell differentiation into bone marrow adipocyte and suppresses osteoblast differentiation.

Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1ß treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cell­specific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.

Cell-specific functions of androgen receptor in skeletal muscles.

Androgens play a vital role not only in promoting the development of male sexual characteristics but also in exerting diverse physiological effects, including the regulation of skeletal muscle growth and function. Given that the effects of androgens are mediated through androgen receptor (AR) binding, an understanding of AR functionality is crucial for comprehending the mechanisms of androgen action on skeletal muscles. Drawing from insights gained using conditional knockout mouse models facilitated by Cre/loxP technology, we review the cell-specific functions of AR in skeletal muscles. We focus on three specific cell populations expressing AR within skeletal muscles: skeletal muscle cells, responsible for muscle contraction; satellite cells, which are essential stem cells contributing to the growth and regeneration of skeletal muscles; and mesenchymal progenitors, situated in interstitial areas and playing a crucial role in muscle homeostasis. Furthermore, the indirect effects of androgens on skeletal muscle through extra-muscle tissue are essential, especially for the regulation of skeletal muscle mass. The regulation of genes by AR varies across different cell types and contexts, including homeostasis, regeneration and hypertrophy of skeletal muscles. The varied mechanisms orchestrated by AR collectively influence the physiology of skeletal muscles.

Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation.

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Ingestion of a collagen peptide containing high concentrations of prolyl-hydroxyproline and hydroxyprolyl-glycine reduces advanced glycation end products levels in the skin and subcutaneous blood vessel walls: a randomized, double-blind, placebo-controlled study.

In this randomized, double-blind, placebo-controlled study, we investigated the effects of collagen peptides (CP) containing high concentrations of prolyl-hydroxyproline and hydroxyprolyl-glycine on advanced glycation end products (AGEs) levels in the skin and subcutaneous blood vessel walls. A total of 31 individuals aged 47-87 years were randomly assigned to receive either 5 g/day of fish-derived CP or a placebo for 12 weeks. Body and blood compositions and AGEs levels were measured at the beginning and end of the study. No adverse events were observed, and both groups' blood and body compositions did not change significantly. However, the CP group had significantly lower AGEs levels and a slightly lower insulin resistance index (homeostasis model assessment ratio [HOMA-R]) than the placebo group. In addition, the percentage changes in AGEs and HOMA-R levels were positively and strongly correlated in both groups. These findings suggest that fish-derived CP may be effective in reducing AGEs levels and improving insulin resistance.

Crosstalk between synovial macrophages and fibroblasts in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with chronic inflammation of joints. Abnormally activated cells such as synovial macrophages and synovial fibroblasts induce RA pathogenesis and ultimately joint destruction. Since macrophages can change their own characteristics depending on the microenvironmental condition, it has been suggested that activation and remission of RA are regulated by crosstalk between synovial macrophages and other cells. Moreover, recent findings of heterogeneity of synovial macrophages and fibroblasts support the idea that complex interactions regulate RA from its onset to remission. Importantly, an understanding of the intercellular crosstalk in RA is far from complete. Here, we summarize the molecular mechanisms underlying the pathological development of RA with particular reference to the crosstalk between synovial macrophages and fibroblasts.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

LIM1 contributes to the malignant potential of endometrial cancer.

Introduction: The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor. Methods: Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advanced-stage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay. Results: Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signaling-related genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation. Conclusion and discussion: Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC.

Periostin increased by mechanical stress upregulates interleukin-6 expression in the ligamentum flavum.

Ligamentum flavum (LF) hypertrophy is a major cause of lumbar spinal canal stenosis. Although mechanical stress is thought to be a major factor involved in LF hypertrophy, the exact mechanism by which it causes hypertrophy has not yet been fully elucidated. Here, changes in gene expression due to long-term mechanical stress were analyzed using RNA-seq in a rabbit LF hypertrophy model. In combination with previously reported analysis results, periostin was identified as a molecule whose expression fluctuates due to mechanical stress. The expression and function of periostin were further investigated using human LF tissues and primary LF cell cultures. Periostin was abundantly expressed in human hypertrophied LF tissues, and periostin gene expression was significantly correlated with LF thickness. In vitro, mechanical stress increased gene expressions of periostin, transforming growth factor-ß1, α-smooth muscle actin, collagen type 1 alpha 1, and interleukin-6 (IL-6) in LF cells. Periostin blockade suppressed the mechanical stress-induced gene expression of IL-6 while periostin treatment increased IL-6 gene expression. Our results suggest that periostin is upregulated by mechanical stress and promotes inflammation by upregulating IL-6 expression, which leads to LF degeneration and hypertrophy. Periostin may be a pivotal molecule for LF hypertrophy and a promising therapeutic target for lumbar spinal stenosis.

Effect of hormonal therapy on the otoconial changes caused by estrogen deficiency.

Benign paroxysmal positional vertigo (BPPV) is associated with menopause and/or osteopenia. Morphological changes in the otoconial layer have been reported after ovariectomy (OVX). Moreover, hormone replacement therapy decreases BPPV risk. However, knowledge concerning the effect of hormonal therapy on the otoconial changes caused by estrogen deficiency is limited. We aimed to examine the effect of hormonal therapy on otoconial changes caused by estrogen deficiency. We hypothesized that hormonal therapy could reduce otoconial changes caused by OVX. Eight-week-old C57BL/6 mice were divided into four groups: sham operation with implantation of vehicle (sham + v), OVX with implantation of vehicle (OVX + v), OVX with implantation of estradiol (E2) (OVX + E2), and OVX with implantation of raloxifene (RAL) (OVX + RAL) groups. Otoconial layer volume was measured by micro-CT at 4 weeks after OVX or the sham operation. The otic bullae were removed; immunohistochemistry was performed for estrogen receptor alpha and 4-hydroxynonenal. Otoconial layer volume was significantly higher in the OVX + v than in the sham + v group. E2 and RAL significantly reduced these changes in the endometrial layer. The staining of estrogen receptor alpha and 4-hydroxynonenal were stronger in the OVX + v than in the sham + v group but equal in the sham + v, OVX + E2, and OVX + RAL groups. These results indicate that E2 and RAL are effective against morphological changes of the otoconial layer caused by estrogen deficiency via oxidative stress reduction.

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells.

BACKGROUND: Lubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation. METHODS: To generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon, surrounding the Achilles tendons and knee joints of severe combined immunodeficient mice and assessed lubricin expression. RESULT: Wnt3a, activin A, TGF-ß1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints. CONCLUSIONS: The present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.

Reduced dynamic loads due to hip dislocation induce acetabular cartilage degeneration by IL-6 and MMP3 via the STAT3/periostin/NF-κB axis.

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVß3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn-/- DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.

Novel animal model of soft tissue tumor due to aberrant hedgehog signaling activation in pericyte lineage.

Pericytes are pluripotent cells that enclose the endothelium of small blood vessels in the whole body. These cells are thought to play a limited role in vascular development and blood pressure regulation; however, current evidence from numerous studies suggests several significant biologic aspects of pericytes in animals. One viewpoint is that pericytes are also known as potential cellular origin of multiple soft tissue tumors. Experimental evidence of the cellular origin of pericytic tumors is still insufficient, however, and their molecular pathogenesis is poorly understood. Here, we used a conditional constitutively active Smoothened allele (Rosa-SmoM2) and Cre recombinase mice to activate hedgehog (Hh) signaling, exclusively in the monocyte/macrophage and osteoclast lineage (LysMcre) or in RANK expressing cells (RANKcre) that are recognized as osteoclast precursor cells. Mice conditionally expressing SmoM2 with LysMcre displayed no significant skeletal phenotype; surprisingly, however, RANKcre; Rosa-SmoM2 mice frequently developed progressive soft tissue tumors in regions of the leg. Genetic lineage tracing analysis uncovered a new domain of RANKcre-expressing cells in the skeletal muscle interstitial cells that display markers consistent with vascular pericytes. Neoplasms arising from these cells showed increased expression of Matrix metalloproteinases (MMPs) that are molecular indicators of malignancy. Moreover, the tumors displayed strong bone invasive potency associated with osteoclastic bone resorption. Thus, these findings provide a novel insight into tumor pathology: Hh signal activated-pericytes can be a potential cellular origin of multiple soft tissue tumors.

Macrophages are requisite for angiogenesis of type H vessels during bone regeneration in mice.

Macrophages are progenitors of osteoclasts as well as regulators of bone metabolism. Macrophages mediate not only bone formation by osteoblasts under physiological conditions, but also bone regeneration after fracture. The mechanisms of macrophages regulation of bone formation and regeneration remain unclear, however. Here, we demonstrate that the liposome-encapsulated Clodronate (Clod-lip) injected mouse model with cortical bone defect induced by drill-hole injury and targeted depletion of phagocytic macrophages exhibits impaired angiogenesis of type H vessels that couple angiogenesis and osteogenesis. Moreover, we identify Tgfbi (encoding TGFBI), Plau (encoding uPA) and Tgfb1 (encoding TGF-ß1), through RNA-seq analysis, as genes of macrophage-secreted factors mediating angiogenesis and wound healing. The relevant mRNA was highly expressed in bone marrow-derived macrophages among bone cells, as determined through qRT-PCR. Finally, we disclose that treatment with uPA inhibitor or TGF-ß receptor I, receptor II inhibitor impairs bone regeneration after injury, confirming the importance of uPA and TGF-ß1 during bone regeneration. Our findings reveal a novel mechanism of bone regeneration mediated by macrophages.

Estrogen receptor α in mature osteoblasts regulates the late stage of bone regeneration.

Estrogen deficiency impairs fracture healing and homeostasis of bone tissue. OVX-induced estrogen deficiency in mice attenuates fracture healing and changes the expression ratio of estrogen receptor (ER) α and ERß in callus during the process of fracture healing. Therefore, ERs may be involved in the regulation of fracture healing. However, the roles of ERs in fracture healing are largely unknown. The purpose of this study was to clarify the significance of ERs during fracture healing using osteoblast-specific ER knockout mice in a mono-cortical drill hole bone regeneration model. The mature osteoblast-specific ER knockout mice were generated using osteocalcin (OCN)-Cre mice, and ERα and ERß flox mice (OCN-Cre; ERαf/f, ERαΔOb/ΔOb and OCN-Cre; ERßf/f, ERßΔOb/ΔOb). Drill hole surgery was conducted on the tibiae of 8-week-old female mice. The mice were sacrificed 10 or 14 days after surgery and the bones were analyzed by DXA, µCT and bone histomorphometry. DXA analysis revealed that intact femoral BMD was significantly decreased in ERαΔOb/ΔOb mice compared with ERαf/f mice, but there was no difference in bone mass between ERßΔOb/ΔOb and ERßf/f mice. Micro CT analyses showed that the callus volume at the restricted drill hole site in tibiae was significantly less in ERαΔOb/ΔOb compared to ERαf/f mice only at day 14 but not at day 10. In addition to femoral BMD, there was no significant difference in callus volume between ERßΔOb/ΔOb and ERßf/f mice. Bone histomorphometric analyses showed that Ob.S/BS and N.Ob/B.Pm were significantly less in ERαΔOb/ΔOb mice compared with ERαf/f mice only at day 10. In addition, Oc.S/BS and N.Oc/B.Pm were significantly less in ERαΔOb/ΔOb mice compared with ERαf/f mice only at day 14. These results suggest that ERα but not ERß in osteocalcin-positive osteoblasts may contribute to the late stage of bone regeneration.

Topical co-administration of zoledronate with recombinant human bone morphogenetic protein-2 can induce and maintain bone formation in the bone marrow environment.

BACKGROUND: Bone morphogenetic proteins (BMPs) induce osteogenesis in various environments. However, when BMPs are used alone in the bone marrow environment, the maintenance of new bone formation is difficult owing to vigorous bone resorption. This is because BMPs stimulate the differentiation of not only osteoblast precursor cells but also osteoclast precursor cells. The present study aimed to induce and maintain new bone formation using the topical co-administration of recombinant human BMP-2 (rh-BMP-2) and zoledronate (ZOL) on beta-tricalcium phosphate (ß-TCP) composite. METHODS: ß-TCP columns were impregnated with both rh-BMP-2 (30 µg) and ZOL (5 µg), rh-BMP-2 alone, or ZOL alone, and implanted into the left femur canal of New Zealand white rabbits (n = 56). The implanted ß-TCP columns were harvested and evaluated at 3 and 6 weeks after implantation. These harvested ß-TCP columns were evaluated radiologically using plane radiograph, and histologically using haematoxylin/eosin (H&E) and Masson's trichrome (MT) staining. In addition, micro-computed tomography (CT) was performed for qualitative analysis of bone formation in each group (n = 7). RESULTS: Tissue sections stained with H&E and MT dyes revealed that new bone formation inside the ß-TCP composite was significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05). Micro-CT data also demonstrated that the bone volume and the bone mineral density inside the ß-TCP columns were significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05). CONCLUSIONS: The topical co-administration of both rh-BMP-2 and ZOL on ß-TCP composite promoted and maintained newly formed bone structure in the bone marrow environment.

Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions.

BACKGROUND: Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear. METHODS: Immunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM. RESULTS: SM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM. CONCLUSIONS: These findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.

Estrogen Receptor ß Controls Muscle Growth and Regeneration in Young Female Mice.

Estrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor ß (ERß) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERß-knockout (mKO) and satellite cell-specific ERß-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERß in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERß pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice.

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.