Tissue-Engineered Bone Regeneration for Medium-to-Large Osteonecrosis of the Femoral Head in the Weight-Bearing Portion: An Observational Study.

Background: Stem cell therapy for the treatment of osteonecrosis of the femoral head (ONFH) showed promising outcomes. However, ONFH with a large lesion in the weight-bearing portion is a poor prognostic factor and still challenging issue to be solved. We aimed to evaluate the effect of tissue-engineered bone regeneration for this challenging condition to preserve the femoral head. Methods: A total of 7 patients (9 hips) with ONFH who received osteoblasts expanded ex vivo from bone marrow-derived mesenchymal stem cells (BMdMSCs) and calcium metaphosphate (CMP) as scaffolds from March 2002 to March 2004 were retrospectively reviewed. The median age was 27.0 years (interquartile range [IQR], 23.0-34.0 years), and the median follow-up period was 20.0 years (IQR, 11.0-20.0 years). After culture and expansion of stem cells, we performed core decompression with BMdMSC implantation at a median number of 10.1 ×107 (IQR, 9.9-10.9 ×107). To evaluate radiographic outcomes, the Association Research Circulation Osseous (ARCO) classifications, the Japanese Investigation Committee (JIC) classification, and modified Kerboul combined necrotic angle (mKCNA) were evaluated preoperatively and during follow-up. Clinical outcomes were evaluated by a visual analog scale (VAS) and Harris Hip Score (HHS). Results: The preoperative stage of ONFH was ARCO 2 in 5 hips and ARCO 3a in 4 hips. The ARCO staging was maintained in 3 hips of ARCO 2 and 4 hips of ARCO 3a. Two hips of ARCO 2 with radiographic progression underwent total hip arthroplasty. According to mKCNA, 2 hips showed medium lesions, and 7 hips showed large lesions. The size of necrotic lesion was decreased in 4 hips (2 were ARCO 2 and 2 were ARCO 3a). There were no significant changes in JIC classification in all hips (type C1: 3 hips and type C2: 6 hips) (p = 0.655). Clinically, there were no significant changes in the VAS and HHS between preoperative and last follow-up (p = 0.072 and p = 0.635, respectively). Conclusions: Tissue engineering technique using osteoblasts expanded ex vivo from BMdMSC and CMP showed promising outcomes for the treatment of pre-collapsed and early-collapsed stage ONFH with medium-to-large size, mainly located in weight-bearing areas.

Ank-mediated pyrophosphate regulates shear stress-induced small extracellular vesicle production in 3D-cultured osteocytes.

Osteocytes are located in the lacunae of fluid-filled bone and communicate with neighboring or distant cells by secreting small extracellular vesicles (sEVs) and growth factors as well as via dendrite-dendrite direct connections. However, the mechanism regulating sEV production in osteocytes is yet to be elucidated. In this study, we investigated sEV production and its underlying mechanism in osteocytes cultured on a three dimensional (3D) scaffold. We employed a perfusion system to apply shear stress stimulation to MLO-Y4 cells cultured on a 3D biphasic calcium phosphate (BCP) scaffold and analyzed sEV production and gene expression using RNA sequencing. We found that the expression of genes associated with sEV biogenesis and the secretory pathway were enhanced by fluid shear stress in MLO-Y4 cells cultured on a 3D BCP scaffold. In particular, fluid shear stress induced the expression of Ank, a pyrophosphate transporter, in 3D-cultured MLO-Y4 cells. The role of Ank in sEV production was further examined. Probenecid, an Ank inhibitor, significantly suppressed shear stress-induced sEV production, whereas Ank cDNA overexpression stimulated it. The inhibition of shear stress-induced sEV production by probenecid was recovered by the exogenous addition of pyrophosphate to MLO-Y4 cells. These findings suggest that shear stress-mediated sEV production in 3D-cultured osteocytes is regulated by extracellular pyrophosphate transported by Ank.

S100P binds to RAGE and activates ERK/NF-κB signaling to promote osteoclast differentiation and activity.

S100 calcium-binding protein P (S100P) is a secretory protein that is expressed in various healthy tissues and tumors. Megakaryocyte-secreted S100P promotes osteoclast differentiation and function; however, its receptor and cellular signaling in osteoclasts remain unclear. Receptor for advanced glycation end products (RAGE), which is the receptor for S100P on cancer cells, was expressed in osteoclast precursors, and S100P-RAGE binding was confirmed through co-immunoprecipitation. Additionally, the phosphorylation of ERK and NF-κB was increased in S100P-stimulated osteoclast precursors but was inhibited by addition of the RAGE antagonistic peptide (RAP). S100P-induced osteoclast differentiation and excessive bone resorption activity were also reduced by the addition of RAP. This study demonstrates that S100P, upon binding with RAGE, activates the ERK and NF-κB signaling pathways in osteoclasts, leading to increased cell differentiation and bone resorption activity.

Glycinamide Facilitates Nanocomplex Formation and Functions Synergistically with Bone Morphogenetic Protein 2 to Promote Osteoblast Differentiation In Vitro and Bone Regeneration in a Mouse Calvarial Defect Model.

BACKGROUND: This study aimed to identify glycine analogs conducive to the formation of cell-absorbable nanocomplexes, enhancing collagen synthesis and subsequent osteogenesis in combination with BMP2 for improved bone regeneration. METHODS: Glycine and its derivatives were assessed for their effects on osteogenic differentiation in MC3T3-E1 cells and human bone marrow mesenchymal stem cells (BMSCs) under osteogenic conditions or with BMP2. Osteogenic differentiation was assessed through alkaline phosphatase staining and real-time quantitative polymerase chain reaction (RT-qPCR). Nanocomplex formation was examined via scanning electron microscopy, circular dichroism, and ultraviolet-visible spectroscopy. In vivo osteogenic effects were validated using a mouse calvarial defect model, and bone regeneration was evaluated through micro-computed tomography and histomorphometric analysis. RESULTS: Glycine, glycine methyl ester, and glycinamide significantly enhanced collagen synthesis and ALP activity in conjunction with an osteogenic medium (OSM). GA emerged as the most effective inducer of osteoblast differentiation marker genes. Combining GA with BMP2 synergistically stimulated ALP activity and the expression of osteoblast markers in both cell lines. GA readily formed nanocomplexes, facilitating cellular uptake through strong electrostatic interactions. In an in vivo calvarial defect mouse model, the GA and BMP2 combination demonstrated enhanced bone volume, bone volume/tissue volume ratio, trabecular numbers, and mature bone formation compared to other combinations. CONCLUSION: GA and BMP2 synergistically promoted in vitro osteoblast differentiation and in vivo bone regeneration through nanocomplex formation. This combination holds therapeutic promise for individuals with bone defects, showcasing its potential for clinical intervention.

The beneficial effects of lupeol on particulate matter-mediated pulmonary inflammation.

Particulate matter (PM) poses significant health risks, especially fine particles (PM2.5) that can cause severe lung injuries. Lupeol, a phytosterol from medicinal plants, has potential anti-cancer properties. This study investigated lupeol's protective effects against PM2.5-induced lung damage. Mice received lupeol following intratracheal PM2.5 exposure. Results showed lupeol reduced lung damage, lowered wet/dry (W/D) weight ratio, and suppressed increased permeability caused by PM2.5. Additionally, lupeol decreased plasma inflammatory cytokines, total protein concentration in bronchoalveolar lavage fluid (BALF), and PM2.5-induced lymphocyte proliferation. Lupeol also reduced expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and autophagy-related proteins microtubule-associated protein 1 A/1 B-light chain 3 (LC3) II and Beclin 1, while increasing phosphorylated mammalian target of rapamycin (mTOR) phosphorylation. These findings suggest lupeol's potential as a therapeutic agent for PM2.5-induced lung damage via modulation of the TLR4-MyD88 and mTOR-autophagy pathways.

Unique cartilage matrix-associated protein inhibits osteoclast differentiation by alleviating RANKL-induced reactive oxygen species.

Unique cartilage matrix-associated protein (UCMA) is a γ-carboxyglutamic acid-rich secretory protein primarily expressed in adult cartilage. UCMA promotes osteoblast differentiation and reduces high glucose-induced reactive oxygen species (ROS) production in osteoblasts; however, its role in osteoclasts remains unclear. Since Ucma is not expressed in osteoclasts, treatment with recombinant UCMA protein (rUCMA) was employed to investigate the effect of UCMA on osteoclasts. The rUCMA-treated osteoclasts exhibited significantly reduced osteoclast differentiation, resorption activity, and osteoclast-specific gene expression. Moreover, rUCMA treatment reduced RANKL-induced ROS production and increased the expression of antioxidant genes in osteoclasts. This study demonstrates that UCMA effectively inhibits RANKL-stimulated osteoclast differentiation and oxidative stress.

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Britanin inhibits titanium wear particle­induced osteolysis and osteoclastogenesis.

Wear particle­induced osteolysis is a serious complication that occurs in individuals with titanium (Ti)­based implants following long­term usage due to loosening of the implants. The control of excessive osteoclast differentiation and inflammation is essential for protecting against wear particle­induced osteolysis. The present study evaluated the effect of britanin, a pseudoguaianolide sesquiterpene isolated from Inula japonica, on osteoclastogenesis in vitro and Ti particle­induced osteolysis in vivo. The effect of britanin was examined in the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) using TRAP staining, RT­PCR, western blotting and immunocytochemistry. The protective effect of britanin was examined in a mouse calvarial osteolysis model and evaluated using micro­CT and histomorphometry. Britanin inhibited osteoclast differentiation and F­actin ring formation in the presence of macrophage colony­stimulating factor and receptor activator of nuclear factor kB ligand in BMMs. The expression of osteoclast­specific marker genes, including tartrate­resistant acid phosphatase, cathepsin K, dendritic cell­specific transmembrane protein, matrix metallopeptidase 9 and nuclear factor of activated T­cells cytoplasmic 1, in the BMMs was significantly reduced by britanin. In addition, britanin reduced the expression of B lymphocyte­induced maturation protein­1, which is a transcriptional repressor of negative osteoclastogenesis regulators, including interferon regulatory factor­8 and B­cell lymphoma 6. Conversely, britanin increased the expression levels of anti­oxidative stress genes, namely nuclear factor erythroid­2­related factor 2, NAD(P)H quinone oxidoreductase 1 and heme oxygenase 1 in the BMMs. Furthermore, the administration of britanin significantly reduced osteolysis in a Ti particle­induced calvarial osteolysis mouse model. Based on these findings, it is suggested that britanin may be a potential therapeutic agent for wear particle­induced osteolysis and osteoclast­associated disease.

Therapeutic Effects of Cornuside on Particulate Matter-Induced Lung Injury.

Particulate matter (PM) is a mixture comprising both organic and inorganic particles, both of which are hazardous to health. The inhalation of airborne PM with a diameter of ≤2.5 µm (PM2.5) can cause considerable lung damage. Cornuside (CN), a natural bisiridoid glucoside derived from the fruit of Cornus officinalis Sieb, exerts protective properties against tissue damage via controlling the immunological response and reducing inflammation. However, information regarding the therapeutic potential of CN in patients with PM2.5-induced lung injury is limited. Thus, herein, we examined the protective properties of CN against PM2.5-induced lung damage. Mice were categorized into eight groups (n = 10): a mock control group, a CN control group (0.8 mg/kg mouse body weight), four PM2.5+CN groups (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight), and a PM2.5+CN group (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight). The mice were administered with CN 30 min following intratracheal tail vein injection of PM2.5. In mice exposed to PM2.5, different parameters including changes in lung tissue wet/dry (W/D) lung weight ratio, total protein/total cell ratio, lymphocyte counts, inflammatory cytokine levels in the bronchoalveolar lavage fluid (BALF), vascular permeability, and histology were examined. Our findings revealed that CN reduced lung damage, the W/D weight ratio, and hyperpermeability caused by PM2.5. Moreover, CN reduced the plasma levels of inflammatory cytokines produced because of PM2.5 exposure, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and nitric oxide, as well as the total protein concentration in the BALF, and successfully attenuated PM2.5-associated lymphocytosis. In addition, CN substantially reduced the expression levels of Toll-like receptors 4 (TLR4), MyD88, and autophagy-related proteins LC3 II and Beclin 1, and increased protein phosphorylation of the mammalian target of rapamycin (mTOR). Thus, the anti-inflammatory property of CN renders it a potential therapeutic agent for treating PM2.5-induced lung injury by controlling the TLR4-MyD88 and mTOR-autophagy pathways.

Gulp1 deficiency augments bone mass in male mice by affecting osteoclasts due to elevated 17ß-estradiol levels.

The engulfment adaptor phosphotyrosine-binding domain containing 1 (GULP1) is an adaptor protein involved in the engulfment of apoptotic cells via phagocytosis. Gulp1 was first found to promote the phagocytosis of apoptotic cells by macrophages, and its role in various tissues, including neurons and ovaries, has been well studied. However, the expression and function of GULP1 in bone tissue are poorly understood. Consequently, to determine whether GULP1 plays a role in the regulation of bone remodeling in vitro and in vivo, we generated Gulp1 knockout (KO) mice. Gulp1 was expressed in bone tissue, mainly in osteoblasts, while its expression is very low in osteoclasts. Microcomputed tomography and histomorphometry analysis in 8-week-old male Gulp1 KO mice revealed a high bone mass in comparison with male wild-type (WT) mice. This was a result of decreased osteoclast differentiation and function in vivo and in vitro as confirmed by a reduced actin ring and microtubule formation in osteoclasts. Gas chromatography-mass spectrometry analysis further showed that both 17ß-estradiol (E2) and 2-hydroxyestradiol levels, and the E2/testosterone metabolic ratio, reflecting aromatase activity, were also higher in the bone marrow of male Gulp1 KO mice than in male WT mice. Consistent with mass spectrometry analysis, aromatase enzymatic activity was significantly higher in the bone marrow of male Gulp1 KO mice. Altogether, our results suggest that GULP1 deficiency decreases the differentiation and function of osteoclasts themselves and increases sex steroid hormone-mediated inhibition of osteoclast differentiation and function, rather than affecting osteoblasts, resulting in a high bone mass in male mice. To the best of our knowledge, this is the first study to explore the direct and indirect roles of GULP1 in bone remodeling, providing new insights into its regulation.

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption.

Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases.

rhBMP-2-Conjugated Three-Dimensional-Printed Poly(L-lactide) Scaffold is an Effective Bone Substitute.

BACKGROUND: Bone growth factors, particularly bone morphogenic protein-2 (BMP-2), are required for effective treatment of significant bone loss. Despite the extensive development of bone substitutes, much remains to be desired for wider application in clinical settings. The currently available bone substitutes cannot sustain prolonged BMP-2 release and are inconvenient to use. In this study, we developed a ready-to-use bone substitute by sequential conjugation of BMP to a three-dimensional (3D) poly(L-lactide) (PLLA) scaffold using novel molecular adhesive materials that reduced the operation time and sustained prolonged BMP release. METHODS: A 3D PLLA scaffold was printed and BMP-2 was conjugated with alginate-catechol and collagen. PLLA scaffolds were conjugated with different concentrations of BMP-2 and evaluated for bone regeneration in vitro and in vivo using a mouse calvarial model. The BMP-2 release kinetics were analyzed using ELISA. Histological analysis and micro-CT image analysis were performed to evaluate new bone formation. RESULTS: The 3D structure of the PLLA scaffold had a pore size of 400 µm and grid thickness of 187-230 µm. BMP-2 was released in an initial burst, followed by a sustained release for 14 days. Released BMP-2 maintained osteoinductivity in vitro and in vivo. Micro-computed tomography and histological findings demonstrate that the PLLA scaffold conjugated with 2 µg/ml of BMP-2 induced optimal bone regeneration. CONCLUSION: The 3D-printed PLLA scaffold conjugated with BMP-2 enhanced bone regeneration, demonstrating its potential as a novel bone substitute.

Fermented Oyster (Crassostrea gigas) Extract Cures and Prevents Prednisolone-Induced Bone Resorption by Activating Osteoblast Differentiation.

Osteoporosis is a bone resorptive disease characterized by the loss of bone density, causing an increase in bone fragility. In our previous study, we demonstrated that gamma aminobutyric acid-enriched fermented oyster (Crassostrea gigas) extract (FO) stimulated osteogenesis in MC3T3-E1 preosteoblast cells and vertebral formation in zebrafish. However, the efficacy of FO in prednisolone (PDS)-induced bone resorption remains unclear. In this study, we evaluated the osteogenic potential of FO in MC3T3-E1 preosteoblast cells and zebrafish larvae under both PDS-pretreated and PDS-post-treated conditions. We found that FO recovered osteogenic activity by upregulating osteoblast markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2, and osterix, in both PDS-pretreated and post-treated MC3T3-E1 osteoblast cells and zebrafish larvae. In both conditions, PDS-induced decrease in calcification and ALP activity was recovered in the presence of FO. Furthermore, vertebral resorption in zebrafish larvae induced by pretreatment and post-treatment with PDS was restored by treatment with FO, along with the recovery of osteogenic markers and downregulation of osteoclastogenic markers. Finally, whether FO disturbs the endocrine system was confirmed according to the Organization for Economic Cooperation and Development guideline 455. We found that FO did not stimulate estrogen response element-luciferase activity or proliferation in MCF7 cells. Additionally, in ovariectomized mice, no change in uterine weight was observed during FO feeding. These results indicate that FO effectively prevents and treats PDS-induced osteoporosis without endocrine disturbances.

Osteogenic Differentiation of Human Mesenchymal Stem Cells Modulated by Surface Manganese Chemistry in SLA Titanium Implants.

The manganese (Mn) ion has recently been probed as a potential candidate element for the surface chemistry modification of titanium (Ti) implants in order to develop a more osteogenic surface with the expectation of taking advantage of its strong binding affinity to the integrins on bone-forming cells. However, the exact mechanism of how Mn enhances osteogenesis when introduced into the surface of Ti implants is not clearly understood. This study investigated the corrosion resistance and potential osteogenic capacity of a Mn-incorporated Ti surface as determined by electrochemical measurement and examining the behaviors of human mesenchymal stem cells (MSCs) in a clinically available sandblasted/acid-etched (SLA) oral implant surface intended for future biomedical applications. The surface that resulted from wet chemical treatment exhibited the formation of a Mn-containing nanostructured TiO2 anatase thin film in the SLA implant and improved corrosion resistance. The Mn-incorporated SLA surface displayed sustained Mn ion release and enhanced osteogenesis-related MSC function, which enhanced early cellular events such as spreading, focal adhesion, and mRNA expression of critical adhesion-related genes and promoted full human MSC differentiation into mature osteoblasts. Our findings indicate that surface Mn modification by wet chemical treatment is an effective approach to produce a Ti implant surface with increased osteogenic capacity through the promotion of the osteogenic differentiation of MSCs. The improved corrosion resistance of the resultant surface is yet another important benefit of being able to provide favorable osseointegration interface stability with an increased barrier effect.

The Neuropeptide Spexin Promotes the Osteoblast Differentiation of MC3T3-E1 Cells via the MEK/ERK Pathway and Bone Regeneration in a Mouse Calvarial Defect Model.

BACKGROUND: The neural regulation of bone regeneration has emerged recently. Spexin (SPX) is a novel neuropeptide and regulates multiple biological functions. However, the effects of SPX on osteogenic differentiation need to be further investigated. Therefore, the aim of this study is to investigate the effects of SPX on osteogenic differentiation, possible underlying mechanisms, and bone regeneration. METHODS: In this study, MC3T3-E1 cells were treated with various concentrations of SPX. Cell proliferation, osteogenic differentiation marker expressions, alkaline phosphatase (ALP) activity, and mineralization were evaluated using the CCK-8 assay, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), ALP staining, and alizarin red S staining, respectively. To determine the underlying molecular mechanism of SPX, the phosphorylation levels of signaling molecules were examined via western blot analysis. Moreover, in vivo bone regeneration by SPX (0.5 and 1 µg/µl) was evaluated in a calvarial defect model. New bone formation was analyzed using micro-computed tomography (micro-CT) and histology. RESULTS: The results indicated that cell proliferation was not affected by SPX. However, SPX significantly increased ALP activity, mineralization, and the expression of genes for osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), Alp, collagen alpha-1(I) chain (Col1a1), osteocalcin (Oc), and bone sialoprotein (Bsp). In contrast, SPX downregulated the expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). Moreover, SPX upregulated phosphorylated mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2). In vivo studies, micro-CT and histologic analysis revealed that SPX markedly increased a new bone formation. CONCLUSION: Overall, these results demonstrated that SPX stimulated osteogenic differentiation in vitro and increased in vivo bone regeneration via the MEK/ERK pathway.

Recombinant Unique Cartilage Matrix-associated Protein Potentiates Osteogenic Differentiation and Mineralization of MC3T3-E1 Cells.

OBJECTIVE: The relative balance of osteoblasts in bone formation and osteoclasts in bone resorption is crucial for maintaining bone health. With age, this balance between osteoblasts and osteoclasts is broken, resulting in bone loss. Anabolic drugs are continuously being developed to counteract this low bone mass. Recombinant proteins are used as biotherapeutics due to being relatively easy to produce on a large scale and are cost-effective through various expression systems. This study aimed to develop a recombinant protein that would positively impact osteoblast differentiation and mineralized nodule formation using unique cartilage matrix-associated protein (UCMA). METHODS: A recombinant glutathione-S-transferase (GST)-UCMA fusion protein was generated in an E.coli system, and purified by affinity chromatography. MC3T3-E1 osteoblast cells and Osterix (Osx)-knockdown stable cells were cultured for 14 days to investigate osteoblast differentiation and nodule formation in the presence of the recombinant GST-UCMA protein. The differentiated cells were assessed by alizarin red S staining and quantitative PCR of the osteoblast differentiation marker osteocalcin. In addition, cell viability in the presence of the recombinant GST-UCMA protein was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell adhesion assay. RESULTS: The isolation of both purified recombinant GST-only and GST-UCMA proteins were confirmed at 26 kDa and 34 kDa, respectively, by Coomassie staining and western blot analysis. Neither dose-dependent nor time-dependent presence of recombinant GST-UCMA affected MC3T3-E1 cell viability. However, MC3T3-E1 cell adhesion to the recombinant GST-UCMA protein increased dose-dependently. Osteoblast differentiation and nodule formation were promoted in both MC3T3-E1 osteoblast cells and Osxknockdown stable cells when cultured in the presence of recombinant GST-UCMA protein. CONCLUSION: A recombinant GST-UCMA protein induces osteogenic differentiation and mineralization, suggesting its potential use as an anabolic drug to increase low bone mass in osteoporotic patients.

Suppressive activity of RGX-365 on HMGB1-mediated septic responses

Abstract RGX-365 is the main fraction of black ginseng conmprising protopanaxatriol (PPT)-type rare ginsenosides (ginsenosides Rg4, Rg6, Rh4, Rh1, and Rg2). No studies on the antiseptic activity of RGX-365 have been reported. High mobility group box 1 (HMGB1) is recognized as a late mediator of sepsis, and the inhibition of HMGB1 release and recovery of vascular barrier integrity have emerged as attractive therapeutic strategies for the management of sepsis. In this study, we examined the effects of RGX-365 on HMGB1-mediated septic responses and survival rate in a mouse sepsis model. RGX-365 was administered to the mice after HMGB1 challenge. The antiseptic activity of RGX-365 was assessed based on the production of HMGB1, measurement of permeability, and septic mouse mortality using a cecal ligation and puncture (CLP)-induced sepsis mouse model and HMGB1-activated human umbilical vein endothelial cells (HUVECs). We found that RGX-365 significantly reduced HMGB1 release from LPS- activated HUVECs and CLP-induced release of HMGB1 in mice. RGX-365 also restored HMGB1-mediated vascular disruption and inhibited hyperpermeability in the mice. In addition, treatment with RGX-365 reduced sepsis-related mortality in vivo. Our results suggest that RGX- 365 reduces HMGB1 release and septic mortality in vivo, indicating that it is useful in the treatment of sepsis.

Enzymatic transformation products of phloretin as potent antiadipogenic compounds.

Enzymatic structure modification of the representative chalcone phloretin (1) with polyphenol oxidase from Agaricus bisporus origin produced 2 new biphenyl-type phloreoxin (2) and phloreoxinone (3), and a previously undescribed (2R)-5,7,3',5'-tetrahydroxyflavanone (4). The structure of these new oxidized products 2-4 elucidated by interpreting the spectroscopic data (NMR and FABMS) containing the absolute stereochemistry is established by the analysis of the circular dichroism spectrum. Compared to the original phloretin, the new products (2) and (3) showed highly improved antiadipogenic potencies both toward pancreatic lipase and accumulation of 3T3-L1 cells. Also, phloreoxin (2) effectively inhibited the expression of C/EBPß, PPARγ, and aP2 at the mRNA level in the 3T3 adipocytes. Thus, phloreoxin (2), containing a biphenyl moiety catalyzed by A. bisporus polyphenol oxidase, have the potential to influence the antiadipogenic capacity.

Combined effects of soy isoflavone and lecithin on bone loss in ovariectomized mice.

BACKGROUND/OBJECTIVES: Isoflavones (ISFs) are effective in preventing bone loss, but not effective enough to prevent osteoporosis. Mixtures of soy ISF and lecithin (LCT) were prepared and characterized in an attempt to improve the bone loss. MATERIALS/METHODS: The daidzein (DZ) and genistein (GN) solubility in soy ISF were measured using liquid chromatography-mass spectrometry. The change in the crystalline characteristics of soy ISF in LCT was evaluated using X-ray diffraction analysis. Pharmacokinetic studies were conducted to evaluate and compare ISF bioavailability. Animal studies with ovariectomized (OVX) mice were carried out to estimate the effects on bone loss. The Student's t-test was used to evaluate statistical significance. RESULTS: The solubility of DZ and GN in LCT was 125.6 and 9.7 mg/L, respectively, which were approximately 25 and 7 times higher, respectively, than those in water. The bioavailability determined by the area under the curve of DZ for the oral administration (400 mg/kg) of soy ISF alone and the soy ISF-LCT mixture was 13.19 and 16.09 µg·h/mL, respectively. The bone mineral density of OVX mice given soy ISF-LCT mixtures at ISF doses of 60 and 100 mg/kg daily was 0.189 ± 0.020 and 0.194 ± 0.010 g/mm3, respectively, whereas that of mice given 100 mg/kg soy ISF was 0.172 ± 0.028 g/mm3. The number of osteoclasts per bone perimeter was reduced by the simultaneous administration of soy ISF and LCT. CONCLUSIONS: The effect of preventing bone loss and osteoclast formation by ingesting soy ISF and LCT at the same time was superior to soy ISF alone as the bioavailability of ISF may have been improved by the emulsification and solvation of LCT. These results suggest the possibility of using the combination of soy ISF and LCT to prevent osteoporosis.

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function.

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.