Promotion Of Osteogenic Differentiation Research Articles

METTL7A-mediated m6A modification of corin reverses bisphosphonates-impaired osteogenic differentiation of orofacial BMSCs

Bisphosphonate-related osteonecrosis of jaw (BRONJ) is characterized by impaired osteogenic differentiation of orofacial bone marrow stromal cells (BMSCs). Corin has recently been demonstrated to act as a key regulator in bone development and orthopedic disorders. However, the role of corin in BRONJ-related BMSCs dysfunction remains unclarified. A m6A epitranscriptomic microarray study from our group shows that the CORIN gene is significantly upregulated and m6A hypermethylated during orofacial BMSCs osteogenic differentiation. Corin knockdown inhibits BMSCs osteogenic differentiation, whereas corin overexpression or soluble corin (sCorin) exerts a promotion effect. Furthermore, corin expression is negatively regulated by bisphosphonates (BPs). Corin overexpression or sCorin reverses BPs-impaired BMSCs differentiation ability. Mechanistically, we find altered expression of phos-ERK in corin knockdown/overexpression BMSCs and BMSCs under sCorin stimulation. PD98059 (a selective ERK inhibitor) blocks the corin-mediated promotion effect. With regard to the high methylation level of corin during osteogenic differentiation, we apply a non-selective m6A methylase inhibitor, Cycloleucine, which also blocks the corin-mediated promotion effect. Furthermore, we demonstrate that METTL7A modulates corin m6A modification and reverses BPs-impaired BMSCs function, indicating that METTL7A regulates corin expression and thus contributes to orofacial BMSCs differentiation ability. To conclude, our study reveals that corin reverses BPs-induced BMSCs dysfunction, and METTL7A-mediated corin m6A modification underlies corin promotion of osteogenic differentiation via the ERK pathway. We hope this brings new insights into future clinical treatments for BRONJ.

A moldable hydrogel based on sericin and Zn2+/F- dual-doped hydroxyapatite promotes skull defect repair through the synergistic effects of immunoregulation, enhanced angiogenesis and osteogenesis

Skull defect repair typically involves multiple stages, including immunomodulation, angiogenesis, osteogenic differentiation, and biomineralization, etc. Most existing therapeutic biomaterials fail to show functional effects across all these stages, leading to unsatisfactory repair effects. To address this challenge, an organic–inorganic hybrid HT(SA)HAp moldable hydrogel with the aforementioned functional properties was developed. The moldable hydrogel consisted of phenylboronic acid-grafted hyaluronic acid, tannic acid, alendronate sodium-grafted sericin (Ser-AL), and hydroxyapatite co-doped with 5 % Zn and 10 % F (Zn5%/F10%-HAp). These materials were cross-linked due to phenylborate ester bonds, metal-phenol interactions, and the AL-mediated chelation of divalent metal cations. Sericin-mediated immunomodulation serves as a precursor to micro-environmental regulation, reducing inflammation at the site of injury and creating an environment conducive to angiogenesis and osteogenic differentiation. The degradation products of the hydrogel’s organic skeleton promoted the proliferation of mesenchymal stem cells and vascular endothelial cells. The enhanced paracrine effect of mesenchymal stem cells and the release of Zn2+ from modified Zn5%/F10%-HAp promoted angiogenesis, while the degradation products of these materials ensured continued promotion of osteogenic differentiation and biomineralization. This organic–inorganic hybrid hydrogel had the synergistic effects of immunoregulation, enhanced angiogenesis, osteogenic differentiation, and biomineralization, which could significantly accelerate the repair of skull defects, and demonstrate completely repaired skull defects at 8 weeks. Thus, this multifunctional moldable hydrogel provided an effective and stable treatment strategy for the rapid repair of skull defects.

Hai-Honghua medicinal liquor is a reliable remedy for fracture by promotion of osteogenic differentiation via activation of PI3K/Akt pathway

Ethnopharmacological relevanceHai-Honghua medicinal liquor (HHML), an external Chinese herbal formula preparation, is often applied to treat freshly closed tibia/fibular fractures, ankle fractures, and other bone-related disorders, but the related molecular mechanism is unclear. Aim of the studyTo evaluate the therapeutic effect of HHML in patients with tibial/fibular and ankle fractures, and to explore its related possible mechanism. Methods and materialsA total of 182 patients with tibia/fibular fractures and 183 patients with ankle fractures were enrolled in this study. A randomized, controlled, unblinded clinical trial was designed to evaluate the therapeutic effect of HHML on tibial/fibular and ankle fractures. The chemical compositions of HHML were analyzed by the HPLC-Q-Extractive MS/MS. Furthermore, a rat tibial fracture model was established to evaluate the therapeutic effects of HHML in promoting fracture healing, and the mouse embryonic osteoblasts cell line of MC3T3-E1 was further carried out to explore the mechanisms of HHML on osteoblast differentiation. ResultsIn the clinical evaluation, HHML treatment significantly shortened the time for pain and swelling in patients with tibial/fibular fractures (P < 0.01) and ankle fractures (P < 0.01), and the incidence of complications was significantly reduced as well. Subsequently, 116 constituents were identified from HHML via HPLC-Q-TOF-MS/MS analysis. In vivo, no obvious changes in weight were observed in HHML-treated rats. Moreover, the levels of bone formation markers (including osteocalcin (OCN), N-terminal propeptide of type I procollagen (PINP), alkaline phosphatase (ALP), calcium (Ca) and substance P) in rat serum were significantly increased in HHML-treated rats compared with model rats (P < 0.05). Micro-CT analysis showed bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th) of the HHML-treated rats were significantly increased (P < 0.05, vs. Model) while trabecular separation (Tb.Sp) and structure model index (SMI) values were significantly reduced (P < 0.05, vs. Model). Histological analysis showed that HHML treatment promoted the healing of fractures and cartilage repair, and increased the osteoblasts and collagen fibers. Furthermore, our results also revealed HHML could promote MC3T3-E1 cells proliferation and osteoblast differentiation via regulation of the runt-related transcription factor 2 (RUNX2), bone alkaline phosphatase (BALP), and OCN by activating phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, which confirmed by adding PI3K chemical inhibitor of LY294002. ConclusionHHML treatment is a reliable remedy for fractures in tibial and ankle by promotion of osteogenic differentiation via activation of PI3K/Akt pathway.

MAGP2 promotes osteogenic differentiation during fracture healing through its crosstalk with the β-catenin pathway.

Osteogenic differentiation is important for fracture healing. Microfibrial-associated glycoprotein 2 (MAGP2) is found to function as a proangiogenic regulator in bone formation; however, its role in osteogenic differentiation during bone repair is not clear. Here, a mouse model of critical-sized femur fracture was constructed, and the adenovirus expressing MAGP2 was delivered into the fracture site. Mice with MAGP2 overexpression exhibited increased bone mineral density and bone volume fraction (BV/TV) at Day 14 postfracture. Within 7 days postfracture, overexpression of MAGP2 increased collagen I and II expression at the fracture callus, with increasing chondrogenesis. MAGP2 inhibited collagen II level but elevated collagen I by 14 days following fracture, accompanied by increased endochondral bone formation. In mouse osteoblast precursor MC3T3-E1 cells, MAGP2 treatment elevated the expression of osteoblastic factors (osterix, BGLAP and collagen I) and enhanced ALP activity and mineralization through activating β-catenin signaling after osteogenic induction. Besides, MAGP2 could interact with lipoprotein receptor-related protein 5 (LRP5) and upregulated its expression. Promotion of osteogenic differentiation and β-catenin activation mediated by MAGP2 was partially reversed by LRP5 knockdown. Interestingly, β-catenin/transcription factor 4 (TCF4) increased MAGP2 expression probably by binding to MAGP2 promoter. These findings suggest that MAGP2 may interact with β-catenin/TCF4 to enhance β-catenin/TCF4's function and activate LRP5-activated β-catenin signaling pathway, thus promoting osteogenic differentiation for fracture repair. mRNA sequencing identified the potential targets of MAGP2, providing novel insights into MAGP2 function and the directions for future research.

Loss of PA28γ exacerbates imbalanced differentiation of bone marrow stromal cells during bone formation and bone healing in mice.

Proteasome activator subunit 3 (PA28γ) is a member of the proteasome activator family, which mainly regulates the degradation and stability of proteins. Studies have shown that it plays crucial roles in lipid formation, stemness maintenance, and blood vessel formation. However, few studies have clarified the association between PA28γ and bone diseases. Herein, we identified PA28γ as a previously unknown regulator of bone homeostasis that coordinates bone formation and lipid accumulation. PA28γ-knockout mice presented with the characteristics of low bone mass and accumulation of lipids. Suppressed expression of PA28γ restrained the osteogenic differentiation and enhanced the adipogenic differentiation of bone marrow stromal cells (BMSCs). Overexpression of PA28γ promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. Mechanistically, PA28γ interacted with Wnt5α, and the two interactors appeared to be positively correlated. PA28γ mainly activated the downstream Wnt/β-catenin signaling pathway, which affects BMSCs differentiation homeostasis. Deletion of Wnt5α significantly delayed the promotion of osteogenic differentiation and partially alleviated the inhibitory effect of adipogenic differentiation of BMSCs in the PA28γ-overexpressing group. Furthermore, we demonstrated that PA28γ-knockout mice had an inhibited rate of bone healing in a drill-hole femoral bone defect model in vivo. Therefore, our results confirm the effects of PA28γ on bone formation and bone defect repair, indicating that PA28γ mainly interacts with Wnt5α to activate the Wnt/β-catenin signaling pathway regulating BMSCs differentiation homeostasis. Our results reveal the function of PA28γ in bone diseases and provide a new theoretical basis for expanding the treatment of bone diseases.

3D PRINTED POLYETHER-ETHER-KETONE (PEEK) MECHANICAL-ADAPTIVE IMPLANTS FOR IMMUNOMODULATORY OSSEOINTEGRATION

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering.

Preparation of cerium-containing nanoporous titanium coatings for osseointegration and their promotion of osteogenic differentiation and anti-inflammatory properties

Conventional implants face many problems, such as failure to form good osseointegration and peri-implantitis in the early stage of implantation. It has been shown that hydroxyapatite (HA) has good osteoconductive ability. Also considering the potential of cerium oxide in osteogenesis and anti-inflammation, we prepared cerium-containing nanoporous HA coatings on the surface of pure titanium using the micro-arc oxidation (MAO) technique. The surface morphology of the coating was observed by scanning electron microscopy (SEM). The mechanical strength of the coating was examined by the ultrasonic oscillator. The elemental composition of the coating was examined by energy spectrum analysis (EDS). The ion release capacity of the cerium-containing coatings was examined by inductively coupled plasma emission spectrometry (ICP-OES). The hydrophilic properties of the coatings were examined by the hydrophilic angle test. In vitro, experiments were performed using rat bone marrow mesenchymal stem cells (BMSCs) and mouse mononuclear macrophages (Raw264.7). The biosafety of the cerium-containing coating was verified by live-dead staining and CCK-8 proliferation assay. The osteogenic and anti-inflammatory ability of the cerium-containing coating was examined by immunofluorescence staining and quantitative polymerase chain reaction (q-PCR). In addition, ion experiments were used to further demonstrate the osteogenic and anti-inflammatory potential of cerium ions. Subsequent animal experiments further demonstrated the osteoinductive potential of the cerium-containing HA coating. In conclusion, our cerium (HA)-containing coatings prepared by micro-arc oxidation on titanium surfaces show excellent osteogenic and anti-inflammatory properties, which are expected to improve the functionality of clinical implants and also lay the foundation for the clinical application of cerium ions.

A bone-targeting near-infrared luminescence nanocarrier facilitates alpha-ketoglutarate efficacy enhancement for osteoporosis therapy

Osteoporosis (OP), which largely increases the risk of fractures, is the most common chronic degenerative orthopedic disease in the elderly due to the imbalance of bone homeostasis. Alpha-ketoglutaric acid (AKG), an endogenous metabolic intermediate involved in osteogenesis, plays critical roles in osteogenic differentiation and mineralization and the inhibition of osteoclastogenic differentiation. However, the low bioavailability and poor bone-targeting efficiency of AKG seriously limit its efficacy in OP treatment. In this work, a bone-targeting, near-infrared emissive lanthanide luminescence nanocarrier loaded with AKG (β-NaYF4:7%Yb, 60%Nd@NaLuF4@mSiO2-EDTA-AKG, abbreviated as LMEK) is developed for the enhancement of AKG efficacy in OP therapy. By utilizing the NIR-II luminescence (>1000 nm) of LMEK, whole-body bone imaging with high spatial resolution is achieved to confirm the bone enrichment of AKG noninvasively in vivo. The results reveal that LMEK exhibits a remarkable OP therapeutic effect in improving the osseointegration of the surrounding bone in the ovariectomized OP mice models, which is validated by the enhanced inhibition of osteoclast through hypoxia-inducible factor-1α suppression and promotion of osteogenic differentiation in osteoblast. Notably, the dose of AKG in LMEK can be reduced to only 0.2 % of the dose when pure AKG is used in therapy, which dramatically improves the bioavailability of AKG and mitigates the metabolism burden. This work provides a strategy to conquer the low utilization of AKG in OP therapy, which not only overcomes the challenges in AKG efficacy for OP treatment but also offers insights into the development and application of other potential drugs for skeletal diseases. Statement of significanceAlpha-ketoglutarate (AKG) is an intermediate within the Krebs cycle, participating in diverse metabolic and cellular processes, showing potential for osteoporosis (OP) therapy. However, AKG's limited bioavailability and inefficient bone-targeting hinder its effectiveness in treating OP. Herein, a near-infrared emissive nanocarrier is developed that precisely targets bones and delivers AKG, bolstering its effectiveness in OP therapy. Thanks to this efficient bone-targeting delivery, the AKG dosage is reduced to 0.2 % of the conventional treatment level. This marks the first utilization of a bone-targeting nanocarrier to amplify AKG's bioavailability and OP therapy efficacy. Furthermore, the mechanism of AKG-loaded nanocarrier regulating the biological behavior of osteoclasts and osteoblasts mediated is tentatively explored.

Identification and validation of ferroptosis-related biomarkers in steroid-induced osteonecrosis of the femoral head

ObjectivesTreatment of steroid-induced osteonecrosis of the femoral head (SIONFH) is challenging. Due to the limited understanding of its molecular mechanisms, investigating the potential mechanisms of ferroptosis will shed light on SIONFH and provide directions for treating this disease. MethodsThe GSE123568 dataset was utilized to apply various bioinformatics methodologies to identify ferroptosis-related hub genes (FRHGs). Subsequently, the importance of these genes and the reliability of the results were confirmed using protein data-independent acquisition (DIA) and cell experiments. Finally, we assessed the correlation between FRHG expression and immune cell infiltration. ResultsThirty-one hub genes were identified and validated by constructing a protein–protein interaction network and subsequent screening using experimentally determined interactions. These 31 hub genes were enriched in immunity, the AMPK signaling pathway, and the Toll-like receptor signaling pathway. Next, we identified a diagnostic marker comprising two ferroptosis-related genes, NCF2 and SLC2A1. The differential expression of these two genes in healthy and necrotic regions was confirmed by protein DIA analysis. Cell experiments verified the link between FRHGs and ferroptosis and preliminarily explored the potential mechanism of the antioxidant vitexin in promoting osteogenic differentiation in cells. The diagnostic efficiency of these two markers was confirmed by receiver operating characteristic curve (ROC) curves, yielding an area under the curve of 1.0. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated enrichment of FRHGs in the superoxide anion and HIF-1 signaling pathways. A significant correlation was observed between FRHGs and various immune cell populations. ConclusionNCF2 and SLC2A1 are promising ferroptosis-related diagnostic biomarkers of SIONFH. Concurrently, we embarked on a preliminary investigation to elucidate the potential mechanism underlying the promotion of osteogenic differentiation by the antioxidant vitexin. Moreover, these biomarkers are associated with distinct immune cell populations.

Nano-/micro-scaled hydroxyapatite ceramic construction and the regulation of immune-associated osteogenic differentiation.

Hydroxyapatite (HA) bioceramic is a promising substitute for bone defects, and the surface properties are major factors that influence bioactivity and osteoinductivity. In this study, two kinds of HA bioceramics with nanoscale (n-HA) and microscale (m-HA) surface topography were designed to mimic the natural bone, thus enhancing the stimulation of osteogenic differentiation and revealing the potential mechanism. Compared to m-HA, n-HA owned a larger surface roughness, a stronger wettability, and reduced hardness and indentation modulus. Based on these properties, n-HA could maintain the conformation of vitronectin better than m-HA, which may contribute to higher cellular activities and a stronger promotion of osteogenic differentiation of mesenchymal stem cells (MSCs). Further RNA sequencing analysis compared the molecular expression between n-HA and m-HA. Six hundred twenty-seven differentially expressed genes were identified in MSCs, and 17 upregulated genes and 610 downregulated genes were included when n-HA compared to m-HA. The GO cluster analysis and enriched Kyoto encyclopedia of genes and genome signaling pathways revealed a close correlation with the immune process in both upregulated (chemokine signaling pathway and cytokine-cytokine receptor interaction) and downregulated pathways (osteoclasts differentiation). It suggested that the nanoscale surface topography of HA enhanced the osteoinductivity of MSCs and could not be separated from its regulation of immune function and the retention of adsorbed protein conformation.

Osteogenic induction of asiatic acid derivatives in human periodontal ligament stem cells

Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration.

Osteocytes autophagy mediated by mTORC2 activation controls osteoblasts differentiation and osteoclasts activities under mechanical loading

Autophagy is an important mechanosensitive response for cellular homeostasis and survival in osteocytes. However, the mechanism and its effect on bone metabolism have not yet clarified. The objective of this study was to evaluate how compressive cyclic force (CCF) induced autophagic response in osteocytes and to determine the effect of mechanically induced-autophagy on bone cells including osteocytes, osteoblasts, and osteoclasts. Autophagic puncta observed in MLO-Y4 cells increased after exposure to CCF. The upregulated levels of the LC3-II isoform and the degradation of p62 further confirmed the increased autophagic flux. Additionally, ATP synthesis and release, osteocalcin (OCN) expression, and cell survival increased in osteocytes as well. The Murine osteoblasts MC3T3-E1 cells and RAW 264.7 macrophage cells were cultured in conditioned medium collected from MLO-Y4 cells subjected to CCF. The concentration of FGF23 increased and the concentrations of SOST and M-CSF and RANKL/OPG ratio decreased significantly in the conditioned medium. Moreover, the promotion of osteogenic differentiation in MC3T3-E1 cells and inhibition of osteoclastogenesis and function in RAW 264.7 cells were significantly attenuated when osteocytes autophagy was inhibited by siAtg7. Our findings suggested that CCF induced protective autophagy in osteocytes and subsequently enhanced osteocytes survival and osteoblasts differentiation and downregulated osteoclasts activities. Further study revealed that CCF induced autophagic response in osteocytes through mechanistic target of rapamycin complex 2 (mTORC2) activation. In conclusion, CCF-induced osteocytes autophagy upon mTORC2 activation promoted osteocytes survival and osteogenic response and decreased osteoclastic function. Thus, osteocytes autophagy will provide a promising target for better understanding of bone physiology and treatment of bone diseases.

Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide.

This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism. Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6. We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor). Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Peiminine regulates bone-fat balance by canonical Wnt/β-catenin pathway in an ovariectomized rat model.

Peiminine is a major biologically active component of Fritillaria thunbergii Miq that exhibits good anticancer, antiinflammatory, and anti-osteoclast effects. However, its effects on osteoporosis (OP) remain unknown. This study aimed to explore whether Peiminine was able to regulate osteogenesis and adipogenesis in ovariectomized (OVX) rat. The effects on the differentiation of bone marrow stem cells (BMSCs), function of Wnt/β-catenin pathway, ALP activity, calcium nodule deposition, as well as adipocyte formation in vitro by Peiminine at different concentrations, were detected. The curative effects of Peiminine on the ovariectomy-induced osteoporosis model by micro-CT and bone histomorphology assays were analyzed. The promotion of osteogenic differentiation and inhibition of adipogenic differentiation by Peiminine (5-40 μg/mL) was detected and the optimum concentration was 20 μg/mL. Mechanistically, Peiminine regulated the fate of BMSCs in vitro, and activated Wnt/β-catenin signaling pathway by restraining phosphorylation of β-catenin and promoting the nuclear translocation of β-catenin. Moreover, Peiminine prevented ovariectomy-induced osteoporosis by alleviating trabecular bone loss and inhibiting adipose formation. Our data suggested that Peiminine could attenuate ovariectomy-induced osteoporosis by alleviating trabecular bone loss and inhibiting adipose formation. These encouraging discoveries could lay the foundation for Peiminine to be a promising preventive treatment strategy for skeletal diseases, such as osteoporosis.

A Novel Zwitterionic Hydrogel Incorporated with Graphene Oxide for Bone Tissue Engineering: Synthesis, Characterization, and Promotion of Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

Zwitterionic materials are widely applied in the biomedical field due to their excellent antimicrobial, non-cytotoxicity, and antifouling properties but have never been applied in bone tissue engineering. In this study, we synthesized a novel zwitterionic hydrogel incorporated with graphene oxide (GO) using maleic anhydride (MA) as a cross-linking agent by grafted L-cysteine (L-Cys) as the zwitterionic material on maleilated chitosan via click chemistry. The composition and each reaction procedure of the novel zwitterionic hydrogel were characterized via X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FT-IR), while the morphology was imaged by scanning electron microscope (SEM). In vitro cell studies, CCK-8 and live/dead assay, alkaline phosphatase activity, W-B, and qRT-CR tests showed zwitterionic hydrogel incorporated with GO remarkably enhanced the osteogenic differentiation of bone mesenchymal stem cells (BMSCs); it is dose-dependent, and 2 mg/mL GO is the optimum concentration. In vivo tests also indicated the same results. Hence, these results suggested the novel zwitterionic hydrogel exhibited porous characteristics similar to natural bone tissue. In conclusion, the zwitterionic scaffold has highly biocompatible and mechanical properties. When GO was incorporated in this zwitterionic scaffold, the zwitterionic scaffold slows down the release rate and reduces the cytotoxicity of GO. Zwitterions and GO synergistically promote the proliferation and osteogenic differentiation of rBMSCs in vivo and in vitro. The optimal concentration is 2 mg/mL GO.

LncRNA MALAT1 mediates osteogenic differentiation in osteoporosis by regulating the miR-485-5p/WNT7B axis.

Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis. This study aimed to investigate the effects of MALAT1 on osteogenic differentiation and cell apoptosis in osteoporosis. MALAT1 level, detected by RT-qPCR, was downregulated in hindlimb unloading (HU) mice and simulated microgravity (MG)-treated MC3T3-E1 cells. Moreover, osteogenic differentiation-related factor (Bmp4, Col1a1, and Spp1) levels were measured by RT-qPCR and Western blot. ALP activity was detected, and ALP staining was performed. Cell apoptosis was assessed by flow cytometry. The results revealed that MALAT1 upregulated the expression of Bmp4, Col1a1, and Spp1, and enhanced ALP activity. Knockdown of MALAT1 suppressed their expression and ALP activity, suggesting that MALAT1 promoted osteogenic differentiation. Additionally, MALAT1 inhibited apoptosis, increased Bax and caspase-3 levels, and decreased Bcl-2 level. However, knockdown of MALAT1 had opposite results. In MG cells, MALAT1 facilitated osteogenic differentiation and suppressed apoptosis. Furthermore, miR-485-5p was identified as a target of MALAT1, and WNT7B was verified as a target of miR-485-5p. Overexpression of miR-485-5p rescued the promotion of osteogenic differentiation and the inhibition of apoptosis induced by MALAT1. Knockdown of WNT7B abolished the facilitation of osteogenic differentiation and the suppression of apoptosis induced by downregulation of miR-485-5p. In conclusion, MALAT1 promoted osteogenic differentiation and inhibited cell apoptosis through the miR-485-5p/WNT7B axis, which suggested that MALAT1 is a potential target to alleviate osteoporosis.

Bimetallic Metal–Organic Framework for Mitigating Aseptic Osteolysis

The disability rate of joint diseases can be reduced by the use of artificial joints, but joint loosening at a late state limits the lifespan and surgical efficacy of the joints. Wear particles can be recognized by macrophages and induce cells to produce reactive oxygen species (ROS) and inflammatory factors, causing persistent inflammation and decreased osteogenic activity, which ultimately leads to loosening of joint prostheses. Here, the platinum (Pt) nanozymes with excellent ROS scavenging and anti-inflammatory capabilities were encapsulated in zinc imidazolium zeolite framework-8 (ZIF-8), and then the osteogenic active element lanthanum (La) was introduced through ion exchange to finally construct a bimetallic metal-organic framework (Pt@ZIF-8@La). In vitro and in vivo experiments demonstrated that this multifunctional nanoplatform possessed the functions of efficient scavenging of ROS, immune regulation, and promotion of osteogenic differentiation. Meanwhile, the mechanism is explored that Pt@ZIF-8@La can also promote osteogenic mineralization by upregulating the ratio of the osteoprotegerin (OPG)/receptor activator of the NF-κB ligand (RANKL), which can achieve a synergistic therapeutic effect of immunomodulation and osteogenesis, thereby realizing the purpose of relieving aseptic osteolysis.

MicroRNA-181a-5p Curbs Osteogenic Differentiation and Bone Formation Partially Through Impairing Runx1-Dependent Inhibition of AIF-1 Transcription.

Evidence has revealed the involvement of microRNAs (miRNAs) in modulating osteogenic differentiation, implying the promise of miRNA-based therapies for treating osteoporosis. This study investigated whether miR-181a-5p influences osteogenic differentiation and bone formation and aimed to establish the mechanisms in depth. Clinical serum samples were obtained from osteoporosis patients, and MC3T3-E1 cells were treated with osteogenic induction medium (OIM) to induce osteogenic differentiation. miR-181a-5p-, Runt-related transcription factor 1 (Runx1)-, and/or allograft inflammatory factor-1 (AIF-1)-associated oligonucleotides or vectors were transfected into MC3T3-E1 cells to explore their function in relation to the number of calcified nodules, alkaline phosphatase (ALP) staining and activity, expression levels of osteogenesis-related proteins, and apoptosis. Luciferase activity, RNA immunoprecipitation, and chromatin immunoprecipitation assays were employed to validate the binding relationship between miR-181a-5p and Runx1, and the transcriptional regulatory relationship between Runx1 and AIF-1. Ovariectomy (OVX)-induced mice were injected with a miR-181a-5p antagonist for in vivo verification. miR-181a-5p was highly expressed in the serum of osteoporosis patients. OIM treatment decreased miR-181a-5p and AIF-1 expression, but promoted Runx1 expression in MC3T-E1 cells. Meanwhile, upregulated miR-181a-5p suppressed OIM-induced increases in calcified nodules, ALP content, and osteogenesis-related protein expression. Mechanically, miR-181a-5p targeted Runx1, which acted as a transcription factor to negatively modulate AIF-1 expression. Downregulated Runx1 suppressed the miR-181a-5p inhibitor-mediated promotion of osteogenic differentiation, and downregulated AIF-1 reversed the miR-181a-5p mimic-induced inhibition of osteogenic differentiation. Tail vein injection of a miR-181a-5p antagonist induced bone formation in OVX-induced osteoporotic mice. In conclusion, miR-181a-5p affects osteogenic differentiation and bone formation partially via the modulation of the Runx1/AIF-1 axis.

Photothermal extracellular matrix based nanocomposite films and their effect on the osteogenic differentiation of BMSCs.

Mild thermal stimulation in vivo could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In this study, nano-functionalized photothermal extracellular matrix (ECM) nanocomposite films were obtained through adding graphene during cell culture, so that graphene could directly integrate with the ECM secreted by cells. Owing to the similarity of the ECM to the in vivo microenvironment and the apparent photothermal effect of graphene nanoflakes, heat could be generated and transferred at the material-cell interface in a biomimetic way. It was demonstrated that such nanocomposite films achieved an interface temperature rise with light illumination. This could be easily sensed by BMSCs through the ECM. According to alkaline phosphatase, osteogenic related gene expression, mineral deposition, and upregulated expression of heat shock protein (HSP70) and p-ERK, composite films with proper illumination significantly promoted the differentiation of BMSCs into osteoblasts. This work endeavors to study the thermal regulation of BMSC differentiation and provide a new perspective on biocompatible osteo-implant materials which can be remotely controlled.

Experimental study of resveratrol-solid lipid nanoparticles in promotion of osteogenic differentiation of bone marrow mesenchymal stem cells

To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. Res-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 μmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. Live/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 μmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 μmol/L and 1 μmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 μmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). Encapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases.