Promotes Osteoblast Differentiation Research Articles

Formulate Adaptive Biphasic Scaffold via Sequential Protein-Instructed Peptide Co-Assembly.

To ensure compositional consistency while mitigating potential immunogenicity for stem cell therapy, synthetic scaffolds have emerged as compelling alternatives to native extracellular matrix (ECM). Substantial progress has been made in emulating specific natural traits featuring consistent chemical compositions and physical structures. However, recapitulating the dynamic responsiveness of the native ECM involving chemical transitions and physical remodeling during differentiation, remains a challenging endeavor. Here, the creation of adaptive scaffolds is demonstrated through sequential protein-instructed molecular assembly, utilizing stage-specific proteins, and incorporating in situ assembly technique. The procedure is commenced by introducing a dual-targeting peptide at the onset of stem cell differentiation. In response to highly expressed integrins and heparan sulfate proteoglycans (HSPGs) on human mesenchymal stem cell (hMSC), the peptides assembled in situ, creating customized extracellular scaffolds that adhered to hMSCs promoting osteoblast differentiation. As the expression of alkaline phosphatase (ALP) and collagen (COL-1) increased in osteoblasts, an additional peptide is introduced that interacts with ALP, initiating peptide assembly and facilitating calcium phosphate (CaP) deposition. The growth and entanglement of peptide assemblies with collagen fibers efficiently incorporated CaP into the network resulting in an adaptive biphasic scaffold that enhanced healing of bone injuries.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

Zhuang-Gu-Fang promotes osteoblast differentiation via myoblasts and myoblast-derived exosomal miRNAs:miR-5100, miR-126a-3p, miR-450b-5p, and miR-669a-5p

Zhuang-Gu-Fang promotes osteoblast differentiation via myoblasts and myoblast-derived exosomal miRNAs:miR-5100, miR-126a-3p, miR-450b-5p, and miR-669a-5p

Yunnan Baiyao Might Mitigate Periodontitis Bone Destruction by Inhibiting Autophagy and Promoting Osteoblast Differentiation in vivo, ex vivo and in vitro.

Periodontitis is an inflammatory disease that eventually destroys tooth-supporting tissue. Yunnan Baiyao (YNBY), a traditional Chinese medicine compound with haemostatic and anti-inflammatory properties has shown therapeutic potential in several diseases. Our previous study revealed that YNBY suppressed osteoclast differentiation in periodontitis. The purpose of this study is to investigate the influences of YNBY on osteoblasts and explore its potential mechanisms. A rat periodontitis model was established by ligation of maxillary second molars. After the end of modelling, histopathological observation by hematoxylin-eosin (HE) staining and Masson trichrome staining, detection of bone resorption by Micro-CT scanning, detection of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining, expression of osteocalcin (OCN) and microtubule-associated protein 1 light chain 3 (LC3) by immunohistochemistry. Lipopolysaccharides was used to irritate MC3T3-E1 osteoblastic cells and ex vivo calvarial organ as an in vitro model of inflammation. CCK-8 assay was performed to examine the toxicity of YNBY to MC3T3-E1 osteoblastic cells. Osteogenesis was assessed with alizarin red staining, immunofluorescence staining, Western blot and immunohistochemical staining. Transmission electron microscopy, fluorescent double staining, Western blot and immunohistochemical staining were employed to detect autophagy. Histological and micro-CT analyses revealed that YNBY gavage reduced bone loss caused by experimental periodontitis and upregulated osteogenic proteins in vivo. YNBY attenuated the production of autophagy-related proteins in periodontitis rats. Additionally, YNBY promoted osteogenesis by inhibiting inflammation-induced autophagy in vitro. Furthermore, YNBY suppressed LPS-mediated bone resorption and promoted the production of osteoblast-related proteins in inflamed calvarial tissues ex vivo. This study demonstrated, through in vivo, in vitro and ex vivo experiments, that YNBY promoted osteoblast differentiation by suppressing autophagy, which markedly alleviated bone destruction caused by periodontitis.

Osteoblast Biospecific Extraction Conjugated with HPLC Analysis for Screening Bone Regeneration Active Components from Moutan Cortex.

The function of promoting bone regeneration of Moutan Cortex (MC), a traditional Chinese medicine, has been widely known but, the effective components of MC in promoting osteoblast-mediated bone regeneration were still unclear. The method of osteoblast membrane bio-specific extraction conjugated with HPLC analysis was established to screen bone regeneration active components from MC. The fingerprints, washing eluate and desorption eluate of MC extract were analyzed by the established HPLC-DAD method. The established MC3T3-E1 cells membrane chromatography method was used for the bio-specific extraction of MC. The isolated compounds were identified by MS spectrometry. The effects and possible mechanisms of the isolated compounds were evaluated by molecular docking, ALP activity, cell viability by MTT Assay and proteins expression by Western Blot Analysis. The active compound responsible for bone regeneration from MC was isolated using the established method of osteoblast membrane bio-specific extraction conjugated with HPLC analysis, and it was identified as 1,2,3,4,6-penta-O-β-galloyl-D-glucose (PGG) by MS spectrometry. It was further demonstrated through molecular docking that PGG could fit well into the functional ALP, BMP2, and Samd1 binding pocket. The proliferation of osteoblasts was promoted, the level of ALP was increased, and the protein expression of BMP2 and Smad1 was increased as shown by further pharmacological verification. It was concluded that PGG, the bone regeneration active compound from MC, could stimulate the proliferation of osteoblasts to promote osteoblast differentiation, and its mechanism might be related to the BMP/Smad1 pathway.

Vanillin Promotes Osteoblast Differentiation, Mineral Apposition, and Antioxidant Effects in Pre-Osteoblasts.

Antioxidant vanillin (4-hydroxy-3-methoxybenzaldehyde) is used as a flavoring in foods, beverages, and pharmaceuticals. Vanillin possesses various biological effects, such as antioxidant, anti-inflammatory, antibacterial, and anticancer properties. This study aimed to investigate the biological activities of vanillin purified from Adenophora triphylla var. japonica Hara on bone-forming processes. Vanillin treatment induced mineralization as a marker for mature osteoblasts, after stimulating alkaline phosphatase (ALP) staining and activity. The bone-forming processes of vanillin are mainly mediated by the upregulation of the bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, and runt-related transcription factor 2 (RUNX2) pathway during the differentiation of osteogenic cells. Moreover, vanillin promoted osteoblast-mediated bone-forming phenotypes by inducing migration and F-actin polymerization. Furthermore, we validated that vanillin-mediated bone-forming processes were attenuated by noggin and DKK1. Finally, we demonstrated that vanillin-mediated antioxidant effects prevent the death of osteoblasts during bone-forming processes. Overall, vanillin has bone-forming properties through the BMP2-mediated biological mechanism, indicating it as a bone-protective compound for bone health and bone diseases such as periodontitis and osteoporosis.

The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts

Schizophyllan (SPG), a β-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1β/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor–κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Functionalized chitosan hydrogel promotes osseointegration at the interface of3D printed titanium alloy scaffolds

Autogenous bone transplantation is a prevalent clinical method for addressing bone defects. However, the limited availability of donor bone and the morbidity associated with bone harvesting have propelled the search for suitable bone substitutes. Bio-inspired scaffolds, particularly those fabricated using electron beam melting (EBM) deposition technology, have emerged as a significant advancement in this field. These 3D-printed titanium alloy scaffolds are celebrated for their outstanding biocompatibility and favorable elastic modulus. Thermosensitive chitosan hydrogel, which transitions from liquid to solid at body temperature, serves as a popular carrier in bone tissue engineering. Icariin (ICA), known for its efficacy in promoting osteoblast differentiation from bone marrow mesenchymal stem cells (BMSCs), plays a crucial role in this context. We developed a system combining a 3D-printed titanium alloy with a thermosensitive chitosan hydrogel, capable of local bone regeneration and integration through ICA delivery. Our in vitro findings reveal that this system can gradually release ICA, demonstrating excellent biocompatibility while fostering BMSC proliferation and osteogenic differentiation. Immunohistochemistry and Micro-CT analyses further confirm the effectiveness of the system in accelerating in vivo bone regeneration and enhancing osseointegration. This composite system lays a significant theoretical foundation for advancing local bone regeneration and integration.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

Osteoblastgenic and Osteogenic Effects of KY-273 with CDK8/19 Inhibitory Activity in Bone Marrow Mesenchymal Stem Cells and Female Rats.

Osteoporosis is caused by imbalance between osteogenesis and bone resorption, thus, osteogenic drugs and resorption inhibitors are used for treatment of osteoporosis. The present study examined the effects of (R)-4-(1-hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-273), a diphenyl ether derivative, on CDK8/19 activity, osteoblast differentiation and femoral bone using micro-computed tomography in female rats. KY-273 potently inhibited CDK8/19 activity, promoted osteoblast differentiation with an increase in alkaline phosphatase (ALP) activity, and gene expression of type I collagen, ALP and BMP-4 in mesenchymal stem cells (ST2 cells). In female rat femur, ovariectomy decreased metaphyseal trabecular bone volume (Tb.BV), mineral content (Tb.BMC), yet had no effect on metaphyseal and diaphyseal cortical bone volume (Ct.BV), mineral content (Ct.BMC) and strength parameters (BSPs). In ovaries-intact and ovariectomized rats, oral administration of KY-273 (10 mg/kg/d) for 6 weeks increased metaphyseal and diaphyseal Ct.BV, Ct.BMC, and BSPs without affecting medullary volume (Med.V), but did not affect Tb.BV and Tb.BMC. In ovariectomized rats, alendronate (3 mg/kg/d) caused marked restoration of Tb.BV, Tb.BMC and structural parameters after ovariectomy, and increased metaphyseal but not diaphyseal Ct.BV, Ct.BMC, and BSPs. In ovaries-intact and ovariectomized rats, by the last week, KY-273 increased bone formation rate/bone surface at the periosteal but not the endocortical side. These findings indicate that KY-273 causes osteogenesis in cortical bone at the periosteal side without reducing Med.V. In conclusion, KY-273 has cortical-bone-selective osteogenic effects by osteoblastogenesis via CDK8/19 inhibition in ovaries-intact and ovariectomized rats, and is an orally active drug candidate for bone diseases such as osteoporosis in monotherapy and combination therapy.

BMP2-ERK-ATF4 Axis-Based 6-methoxybenzofuran Compound I-9 Acts as Candidate Drug for Bone Formation and Anti-Osteoporosis.

As the global population ages, the number of patients with osteoporosis is rapidly rising. The existing first-line clinical drugs are bone resorption inhibitors that have difficulty restoring the bone mass of elderly patients to the safe range. The range and period of use of existing peptides and monoclonal antibodies are limited, and small-molecule bone formation-promoting drugs are urgently required. We established an I-9 synthesis route with high yield, simple operation, and low cost that was suitable for future large-scale production. I-9 administration promoted bone formation and increased bone mass in mice with low bone mass in an aged C57 mouse model. Our findings revealed a hitherto undescribed pathway involving the BMP2-ERK-ATF4 axis that promotes osteoblast differentiation; I-9 has favorable biosafety in mice. This study systematically investigated the efficacy, safety, and mechanism of I-9 for treating osteoporosis and positions this drug for preclinical research in the future. Thus, this study has promoted the development of small-molecule bone-promoting drugs.

Knockdown of the UL-16 binding protein 1 promotes osteoblast differentiation of human mesenchymal stem cells by activating the SMAD2/3 pathway

Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-β signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-β signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles’ heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis.

In vivo study on osteogenic efficiency of nHA/ gel porous scaffold with nacre water-soluble matrix

Background/purposeNano-hydroxyapatite (nHA)/ gel porous scaffolds loaded with WSM carriers are promising bone replacement materials that can improve osseointegration ability. This investigation aimed to evaluate the osteoinductive activity by implanting the composition of nano-hydroxyapatite (nHA)/ Gel porous scaffolds as a carrier of WSM via an animal model. Materials and methods: WSM was extracted and nHA was added to the matrix to construct porous composite scaffolds. The dose-effect curve of WSM concentration and alkaline phosphatase (ALP) activity was made by culturing rat osteoblasts and examining the absorbance. Three different materials were implanted into critical size defects (CSD) in the skulls of rats, which were further divided into four groups: WSM nHA /Gel group, n-WSM nHA /Gel group, HA powder group, and control group. Results: WSM (150 μg/mL-250μg/mL) effectively improved the activity of ALP in rat osteoblasts. All rats in each group had normal healing. WSM-loaded nHA /Gel group showed better performance on newly-formed bone tissue of rat skull and back at 4th week and 8th week, respectively. At the 4th week, the network of woven bone formed in the WSM-loaded nHA/Gel scaffold material. At 8th week, the reticular trabecular bone in the WSM-loaded scaffold material became dense lamellar bone, and the defect was mature lamellar bone. In the subcutaneous implantation experiment, WSM-loaded nHA/Gel scaffold material showed a better performance of heterotopic ossification than the pure nHA/Gel scaffold material. Conclusion: WSM promotes osteoblast differentiation and bone mineralization. The results confirm that the nHA/ Gel Porous Scaffold with Nacre Water-Soluble Matrix has a significant bone promoting effect and can be used as a choice for tissue engineering to repair bone defects.

Sympathetic innervation induces exosomal miR-125 transfer from osteoarthritic chondrocytes, disrupting subchondral bone homeostasis and aggravating cartilage damage in aging mice

IntroductionOsteoarthritis (OA) is a progressive disease that poses a significant threat to human health, particularly in aging individuals: Although sympathetic activation has been implicated in bone metabolism, its role in the development of OA related to aging remains poorly understood. Therefore, this study aimed to investigate how sympathetic regulation impacts aging-related OA through experiments conducted both in vivo and in vitro. MethodsTo analyze the effect of sympathetic regulation on aging-related OA, we conducted experiments using various mouse models. These models included a natural aging model, a medial meniscus instability model, and a load-induced model, which were used to examine the involvement of sympathetic nerves. In order to evaluate the expression levels of β1-adrenergic receptor (Adrβ1) and sirtuin-6 (Sirt6) in chondrocytes of naturally aging OA mouse models, we performed assessments. Additionally, we investigated the influence of β1-adrenergic receptor knockout or treatment with a β1-adrenergic receptor blocker on the progression of OA in aging mice and detected exosome release and detected downstream signaling expression by inhibiting exosome release. Furthermore, we explored the impact of sympathetic depletion through tyrosine hydroxylase (TH) on OA progression in aging mice. Moreover, we studied the effects of norepinephrine(NE)-induced activation of the β1-adrenergic receptor signaling pathway on the release of exosomes and miR-125 from chondrocytes, subsequently affecting osteoblast differentiation in subchondral bone. ResultsOur findings demonstrated a significant increase in sympathetic activity, such as NE levels, in various mouse models of OA including natural aging, medial meniscus instability, and load-induced models. Notably, we observed alterations in the expression levels of β1-adrenergic receptor and Sirt6 in chondrocytes in OA mouse models associated with natural aging, leading to an improvement in the progression of OA. Critically, we found that the knockout of β1-adrenergic receptor or treatment with a β1-adrenergic receptor blocker attenuated OA progression in aging mice and the degraded cartilage explants produced more exosome than the nondegraded ones, Moreover, sympathetic depletion through TH was shown to ameliorate OA progression in aging mice. Additionally, we discovered that NE-induced activation of the β1-adrenergic receptor signaling pathway facilitated the release of exosomes and miR-125 from chondrocytes, promoting osteoblast differentiation in subchondral bone. ConclusionIn conclusion, our study highlights the role of sympathetic innervation in facilitating the transfer of exosomal miR-125 from osteoarthritic chondrocytes, ultimately disrupting subchondral bone homeostasis and exacerbating cartilage damage in aging mice. These findings provide valuable insights into the potential contribution of sympathetic regulation to the pathogenesis of aging-related OA.

DDIT3 deficiency accelerates bone remodeling during bone healing by enhancing osteoblast and osteoclast differentiation through ULK1-mediated autophagy

The coordination of osteoblasts and osteoclasts is essential for bone remodeling. DNA damage inducible script 3 (DDIT3) is an important regulator of bone and participates in cell differentiation, proliferation, autophagy, and apoptosis. However, its role in bone remodeling remains unexplored. Here, we found that Ddit3 knockout (Ddit3-KO) enhanced both bone formation and resorption. The increased new bone formation and woven bone resorption, i.e., enhanced bone remodeling capacity, was found to accelerate bone defect healing in Ddit3-KO mice. In vitro experiments showed that DDIT3 inhibited both osteoblast differentiation and Raw264.7 cell differentiation by regulating autophagy. Cell coculture assay showed that Ddit3-KO decreased the ratio of receptor activator of nuclear factor-κβ ligand (RANKL) to osteoprotegerin (OPG) in osteoblasts, and Ddit3-KO osteoblasts inhibited osteoclast differentiation. Meanwhile, DDIT3 knockdown (DDIT3-sh) increased receptor activator of nuclear factor-κβ (RANK) expression in Raw264.7 cells, and DDIT3-sh Raw264.7 cells promoted osteoblast differentiation, whereas, DDIT3 overexpression had the opposite effect. Mechanistically, DDIT3 promoted autophagy partly by increasing ULK1 phosphorylation at serine555 (pULK1-S555) and decreasing ULK1 phosphorylation at serine757 (pULK1-S757) in osteoblasts, thereby inhibiting osteoblast differentiation. DDIT3 inhibited autophagy partly by decreasing pULK1-S555 in Raw264.7 cells, thereby suppressing osteoclastic differentiation. Taken together, our data indicate that DDIT3 is one of the elements regulating bone remodeling and bone healing, which may become a potential target in bone defect treatment.

Calcium absorption by Alaska pollock surimi protein hydrolysate promotes osteoblast differentiation.

The calcium-binding capacity and osteoblast proliferation and differentiation were studied in Alaska pollock surimi hydrolysate (APSH) using a system that mimics the gastrointestinal digestive system. Evaluation of the calcium absorption-promoting ability of APSH revealed that the best calcium-binding ability was achieved after hydrolysis with a combination of pepsin, α-chymotrypsin, and trypsin, and separation into <3kDa (APSH-I), 3-5kDa (APSH-II), 5-10kDa (APSH-III), and <10kDa (APSH-IV) fractions. Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis confirmed that the hydrolysate and calcium ions formed a complex. Comparison of the calcium absorption capacity using Caco-2 cells showed that calcium absorption was promoted by these hydrolysates. Measurement of the osteoblast activation revealed higher alkaline phosphatase activity, collagen synthesis, and mineralization effect for the low-molecular-weight hydrolysate (LMH) than for the other hydrolysates. In addition, LMH promoted the expression of osteocalcin, osteopontin, and bone morphogenetic protein-2 and -4, which are hormones related to bone formation. Expression of the Runx2 transcription factor, which regulates the expression of these hormones, also increased. These results suggest that Alaska pollock surimi protein hydrolysates prepared using a system that mimics gastrointestinal hydrolysis may result in better osteoblast proliferation and bone health than those prepared using other proteases.

MicroRNA-877-5p promotes osteoblast differentiation by targeting EIF4G2 expression

Stimulating bone formation potentially suggests therapeutics for orthopedic diseases including osteoporosis and osteoarthritis. Osteoblasts are key to bone remodeling because they act as the only bone-forming cells. miR-877-5p has a chondrocyte-improving function in osteoarthritis, but its effect on osteoblast differentiation is unknown. Here, miR-877-5p-mediated osteoblast differentiation was studied. Real-time reverse transcriptase-polymerase chain reaction was performed to measure miR-877-5p expression during the osteogenic differentiation of MC3T3-E1 cells. Osteoblast markers, including alkaline phosphatase (ALP), collagen type I a1 chain, and osteopontin, were measured and detected by alizarin red staining and ALP staining. Potential targets of miR-877-5p were predicted from three different algorithms: starBase (http://starbase.sysu.edu.cn/), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), and miRanda (http://www.microrna.org/microrna/home.do). It was further verified by dual luciferase reporter gene assay. The experimental results found that miR-877-5p was upregulated during the osteogenic differentiation of MC3T3-E1 cells. Overexpression of miR-877-5p promoted osteogenic differentiation, which was characterized by increased cell mineralization, ALP activity, and osteogenesis-related gene expression. Knockdown of miR-877-5p produced the opposite result. Dual luciferase reporter gene assay showed that miR-877-5p directly targeted eukaryotic translation initiation factor 4γ2 (EIF4G2). Overexpression of EIF4G2 inhibited osteogenic differentiation and reversed the promoting effect of overexpression of miR-135-5p on osteogenic differentiation. These results indicate that miR-877-5p might have a therapeutic application related to its promotion of bone formation through targeting EIF4G2.

RING finger E3 ligase, RNF138 inhibits osteoblast differentiation by negatively regulating Runx2 protein turnover.

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin-proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme-like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18-58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome-dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin-mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis.

Enhanced Osteogenic Differentiation Based on Combining Pulp Stem Cells with Ultralong Hydroxyapatite Nanowires and Cellulose Fibers

Previous studies have confirmed the excellent biocompatibility, osteogenic properties, and angiogenic ability of hydroxyapatite (HAP), as well as the good osteoblast differentiation ability of dental pulp stem cells. We hypothesized that combining dental pulp stem cells with ultralong hydroxyapatite nanowires and cellulose fibers could more effectively promote osteoblast differentiation, making it a potential biomaterial for enhancing bone wound healing. Therefore, based on the optimal ratio of ultralong hydroxyapatite nanowires and cellulose fibers (HAPNW/CF) determined in previous studies, we added human dental pulp stem cells (hDPSCs) to investigate whether this combination can accelerate cell osteogenic differentiation. hDPSCs were introduced into HAPNW/CF scaffolds, and in vitro experiments revealed that: (1) HAPNW/CF scaffolds exhibited no cytotoxicity toward hDPSCs; (2) HAPNW/CF scaffolds enhanced alkaline phosphatase staining activity, an early marker of osteogenic differentiation, and significantly upregulated the expression level of osteogenic-related proteins; (3) co-culturing with hDPSCs in HAPNW/CF scaffolds significantly increased the expression of angiogenesis-related factors compared to hDPSCs alone when tested using human umbilical vein endothelial cells (hUVECs). Our study demonstrates that combining hDPSCs with HAPNW/CF can enhance osteogenic differentiation more effectively, potentially through increased secretion of angiogenesis-related factors promoting osteoblast differentiation.

Planar catechin increases bone mass by regulating differentiation of osteoclasts in mice

ObjectivesWhile catechins have been reported to exhibit potential to benefit osteoporosis patients, the effects of planar catechin (PCat), synthesized during the development of drugs for Alzheimer's disease, have not been clearly elucidated. Here, we examined the effects of PCat on mouse bone metabolism both in vivo and in vitro. MethodsSix week old female mice were orally administered PCat (30 mg/kg) every other day for four weeks, and their femurs were analyzed using micro-computed tomography imaging. Osteoclasts and osteoblasts were collected from mice and cultured with PCat. Subsequently, osteoclast formation and differentiation and osteoblast differentiation were observed. ResultsMice orally administered PCat displayed significantly increased femur bone mass compared to the control group. Quantitative polymerase chain reaction findings indicated that PCat addition to osteoclast progenitor cultures suppressed osteoclast formation and decreased osteoclast marker expression without affecting the proliferative potential of the osteoclast progenitor cells. Addition of PCat to osteoblast cultures increased osteoblast marker expression. ConclusionsPCat inhibits osteoclast differentiation and promotes osteoblast differentiation, resulting in increased bone mass in mice. These results suggest that PCat administration is a promising treatment option for conditions associated with bone loss, including osteoporosis.