Differentiation Of Bone Marrow Stem Cells Research Articles

PCAT-1' s role in wound healing impairment: Mitochondrial dysfunction and bone marrow stem cell differentiation inhibition via PKM2/β-catenin pathway and its impact on implant osseo-integration.

This study focused on unravelling the role of PCAT-1 in wound-healing process, particularly its impact on regenerative and osteogenic abilities of mesenchymal stem cells (MSCs). We delved into how PCAT-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) and interacts with pivotal molecular pathways, especially β-catenin and PKM2, using human bone marrow-derived MSCs. MSCs were cultured under specific conditions and PCAT-1 expression was modified through transfection. We thoroughly assessed several critical parameters: MSC proliferation, mitochondrial functionality, ATP production and expression of wound healing and osteogenic differentiation markers. Further, we evaluated alkaline phosphatase (ALP) activity and mineral deposition, essential for bone healing. Our findings revealed that overexpressing PCAT-1 significantly reduced MSC proliferation, hampered mitochondrial performance and lowered ATP levels, suggesting the clear inhibitory effect of PCAT-1 on these vital wound-healing processes. Additionally, PCAT-1 overexpression notably decreased ALP activity and calcium accumulation in MSCs, crucial for effective bone regeneration. This overexpression also led to the reduction in osteogenic marker expression, indicating suppression of osteogenic differentiation, essential in wound-healing scenarios. Moreover, our study uncovered a direct interaction between PCAT-1 and the PKM2/β-catenin pathway, where PCAT-1 overexpression intensified PKM2 activity while inhibiting β-catenin, thereby adversely affecting osteogenesis. This research thus highlights PCAT-1's significant role in impairing wound healing, offering insights into the molecular mechanisms that may guide future therapeutic strategies for enhancing wound repair and bone regeneration.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

Morroniside Inhibits Inflammatory Bone Loss through the TRAF6-Mediated NF-κB/MAPK Signalling Pathway.

Osteoporosis is a chronic inflammatory disease that severely affects quality of life. Cornus officinalis is a Chinese herbal medicine with various bioactive ingredients, among which morroniside is its signature ingredient. Although anti-bone resorption drugs are the main treatment for bone loss, promoting bone anabolism is more suitable for increasing bone mass. Therefore, identifying changes in bone formation induced by morroniside may be conducive to developing effective intervention methods. In this study, morroniside was found to promote the osteogenic differentiation of bone marrow stem cells (BMSCs) and inhibit inflammation-induced bone loss in an in vivo mouse model of inflammatory bone loss. Morroniside enhanced bone density and bone microstructure, and inhibited the expression of IL6, IL1β, and ALP in serum (p < 0.05). Furthermore, in in vitro experiments, BMSCs exposed to 0-256 μM morroniside did not show cytotoxicity. Morroniside inhibited the expression of IL6 and IL1β and promoted the expression of the osteogenic transcription factors Runx2 and OCN. Furthermore, morroniside promoted osteocalcin and Runx2 expression and inhibited TRAF6-mediated NF-κB and MAPK signaling, as well as osteoblast growth and NF-κB nuclear transposition. Thus, morroniside promoted osteogenic differentiation of BMSCs, slowed the occurrence of the inflammatory response, and inhibited bone loss in mice with inflammatory bone loss.

Identification of cell-type-specific genes in multimodal single-cell data using deep neural network algorithm

The emergence of single-cell RNA sequencing (scRNA-seq) technology makes it possible to measure DNA, RNA, and protein in a single cell. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) is a powerful multimodal single-cell research innovation, allowing researchers to capture RNA and surface protein expression on the same cells. Currently, identification of cell-type-specific genes in CITE-seq data is still challenging. In this study, we obtained a set of CITE-seq datasets from Kaggle database, which included the sequencing dataset of seven cell types during bone marrow stem cell differentiation. We used Student's t-test to analyze these transcription RNAs and pick out 133 significantly differentially expressed genes (DEGs) among all cell types. Functional enrichment revealed that these DEGs were strongly associated with blood-related diseases, providing important insights into the cellular heterogeneity within bone marrow stem cells. The relation between RNA and protein levels was performed by deep neural network (DNN) model and achieved a high prediction score of 0.867. Based on their coefficients in the DNN model, three genes (LGALS1, CENPV, TRIM24) were identified as cell-type-specific genes in erythrocyte progenitor. Our works provide a novel perspective regarding the differentiation of stem cells in the bone marrow and provide valuable insights for further research in this field.

Research progress on the regulation of bone marrow stem cells by noncoding RNAs in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity in young women, but its pathogenesis remains unclear. The primary pathogenic factors contributing to its development include genetics, abnormal bone metabolism, and endocrine factors. Bone marrow stem cells (BMSCs) play a crucial role in the pathogenesis of AIS by regulating its occurrence and progression. Noncoding RNAs (ncRNAs) are also involved in the pathogenesis of AIS, and their role in regulating BMSCs in patients with AIS requires further evaluation. In this review, we discuss the relevant literature regarding the osteogenic, chondrogenic, and lipogenic differentiation of BMSCs. The corresponding mechanisms of ncRNA-mediated BMSC regulation in patients with AIS, recent advancements in AIS and ncRNA research, and the importance of ncRNA translation profiling and multiomics are highlighted.

Impaired autophagy activity-induced abnormal differentiation of bone marrow stem cells is related to adolescent idiopathic scoliosis osteopenia.

Osteopenia has been well documented in adolescent idiopathic scoliosis (AIS). Bone marrow stem cells (BMSCs) are a crucial regulator of bone homeostasis. Our previous study revealed a decreased osteogenic ability of BMSCs in AIS-related osteopenia, but the underlying mechanism of this phenomenon remains unclear. A total of 22 AIS patients and 18 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Bone marrow blood was collected for BMSC isolation and culture. Osteogenic and adipogenic induction were performed to observe the differences in the differentiation of BMSCs between the AIS-related osteopenia group and the control group. Furthermore, a total RNA was extracted from isolated BMSCs to perform RNA sequencing and subsequent analysis. A lower osteogenic capacity and increased adipogenic capacity of BMSCs in AIS-related osteopenia were revealed. Differences in mRNA expression levels between the AIS-related osteopenia group and the control group were identified, including differences in the expression of LRRC17 , DCLK1 , PCDH7 , TSPAN5 , NHSL2 , and CPT1B . Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed several biological processes involved in the regulation of autophagy and mitophagy. The Western blotting results of autophagy markers in BMSCs suggested impaired autophagic activity in BMSCs in the AIS-related osteopenia group. Our study revealed that BMSCs from AIS-related osteopenia patients have lower autophagic activity, which may be related to the lower osteogenic capacity and higher adipogenic capacity of BMSCs and consequently lead to the lower bone mass in AIS patients.

Sericin/Nano-Hydroxyapatite Hydrogels Based on Graphene Oxide for Effective Bone Regeneration via Immunomodulation and Osteoinduction.

Immune responses and osteogenesis differentiation induced by implants are crucial for bone tissue regeneration. Consideration of only one of those properties is not sufficient. To investigate the synergistic actions, we designed alginate/graphene oxide/sericin/nanohydroxyapatite (Alg/GO/Ser/nHAP) nanocomposite hydrogels with both osteoimmunomodulatory and osteoinductive activities. This study aimed to explore the effect of hydrogel with osteoimmunomodulatory properties on promoting osteogenesis of bone marrow stem cells (BMSCs). Alg/GO/Ser/nHAP nanocomposite hydrogel was fabricated and was characterized by SEM, FTIR, XRD, stress-strain, rheology, swelling and degradation. After the impact of sericin on M2 macrophage polarization was identified, the BMSCs viability and adhesion were evaluated by CCK8 assay, live/dead staining, cytoskeleton staining. The cell osteogenic differentiation was observed by ALP/ARS staining, immunofluorescence staining, RT-PCR, and Western blotting, respectively. Rat cranial defect model was used to assess osteoimmunomodulatory effects of scaffolds in vivo by micro‑computed tomographic, histological, and immunohistochemical analyses after 8 weeks of healing. In vitro experiments revealed that the hydrogel presented desirable mechanical strength, stability, porosity, and biocompatibility. Significantly, sericin and nHAP appeared to exert synergistic effects on bone regeneration. Sericin was observed to inhibit the immune response by inducing macrophage M2-type polarization to create a positive osteoimmune microenvironment, contributing to improving osseointegration at the bone-implant interface to promote osteogenesis. However, the osteogenic differentiation in rat BMSCs was further enhanced by combining nHAP and sericin in the nanocomposite hydrogel. Eventually, the hydrogel was implanted into the rat cranial defect model, assisting in the reduction of local inflammation and efficient bone regeneration. The nanocomposite hydrogel stimulated bone formation by the synergistic effects of immunomodulation of macrophage polarization by sericin and direct osteogenic induction by nHAP, demonstrating that such a scaffold that modulates the osteoimmune microenvironment to promote osteogenesis is a promising approach for the development of bone tissue engineering implants in the future.

Electromagnetic Field Effect on Bone Marrow Stem Cells Differentiation and Nucleoli AgNOR

Purpose: Numerous studies have described the effect of Electromagnetic Fields (EMFs) in the promotion of Bone Marrow Stem Cell (BMSC) differentiation. We aimed to investigate the influence of frequency (10 and 100 Hz) and different pulse shapes (sine, rectangular, and triangular) of EMF on rats' BMSCs.&#x0D; Materials and Methods: The BMSCs in 6 groups were exposed to EMF for 1 h/ 7 days. The BMSCs viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cresyl violet labeled the Nissl bodies, and the silver nitrate staining was done to evaluate the BMSCs nucleoli AgNORs.&#x0D; Results: The MTT test verified that EMF and pulse shape did not affect cell viability. In Nissl bodies staining most of the large neurons were related to the rectangular 10 Hz EMF group. The majority of the differentiated BMSCs were astrocytes, microglia, and oligodendrocyte in the triangular 100 Hz EMF group. Although the silver nitrate staining confirmed the effect of 10 Hz EMF, pulse shape alteration did not affect AgNOR parameters. In conclusion, we presented a low-magnetic flux density EMF (400 µT) to assess the responses of BMSCs nuclei.&#x0D; Conclusion: The findings showed that BMSCs differentiation was frequency-dependent. Further investigations are recommended for recognizing the function of EMF on BMSCs.

Experimental Autoimmune Encephalomyelitis of Mice: IgGs from the Sera of Mice Hydrolyze miRNAs

It was shown that the spontaneous development of experimental encephalomyelitis (EAE) in C57BL/6 mice occurs due to changes in the profile of bone marrow stem cells differentiation. This leads to the appearance of lymphocytes producing antibodies-abzymes that hydrolyze DNA, myelin basic protein (MBP), and histones. The activity of abzymes in the hydrolysis of these auto-antigens slowly but constantly increases during the spontaneous development of EAE. Treatment of mice with myelin oligodendrocyte glycoprotein (MOG) leads to a sharp increase in the activity of these abzymes with their maximum at 20 days (acute phase) after immunization. In this work, we analyzed changes in the activity of IgG-abzymes hydrolyzing (pA)23, (pC)23, (pU)23, and six miRNAs (miR-9-5p, miR-219a-5p, miR-326, miR-155-5p, miR-21-3p, and miR-146a-3p) before and after mice immunization with MOG. Unlike abzymes hydrolyzing DNA, MBP, and histones, the spontaneous development of EAE leads not to an increase but to a permanent decrease of IgGs activity of hydrolysis of RNA-substrates. Treatment of mice with MOG resulted in a sharp but transient increase in the activity of antibodies by day 7 (onset of the disease), followed by a sharp decrease in activity 20-40 days after immunization. A significant difference in the production of abzymes against DNA, MBP, and histones before and after mice immunization with MOG with those against RNAs may be since the expression of many miRNAs decreased with age. This can lead to a decrease in the production of antibodies and abzymes that hydrolyze miRNAs with age mice.

Dental pulp stem cells induce anti-inflammatory phenotypic transformation of macrophages to enhance osteogenic potential via IL-6/GP130/STAT3 signaling.

Periodontitis is a major oral condition and current treatment outcomes can be unsatisfactory. Macrophages are essential to the regeneration process, so we investigated the influence of human dental pulp stem cells (hDPSCs) on macrophage differentiation and the microenvironment and the underlying mechanism. hDPSCs were isolated from healthy third molars extracted from patients undergoing maxillofacial surgery. The surface antigens CD73, CD45, CD90 and CD11b of the hDPSCs were detected using flow cytometry. hDPSCs were induced for osteogenic and adipogenic differentiation, and the outcome was assessed by alizarin red staining or Oil Red O staining. The IL-6 level released by hDPSCs was measured by enzyme linked immunosorbent assay (ELISA). Tohoku Hospital Pediatrics-1 (THP-1) cells were cultured and induced into macrophages by phorbol-12-myristate-13-acetate. After coculture of THP-1-derived macrophages with hDPSCs, interleukin 6 (IL-6), Argininase-1 (Arg-1), Mannose receptor C-1 (Mrc-1), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) levels in the medium were measured using ELISA and quantificational RT-PCR (qRT-PCR). The numbers of CD80+ and CD163+ macrophages were counted by immunofluorescence, and GP130/STAT3 signaling protein expression was detected. After coculturing the culture medium of hDPSCs with human bone marrow stem cells (BMSCs), scratch assays and transwell assays were performed to evaluate cell migration and invasion. Alkaline phosphatase (ALP) staining, alizarin red staining, and western blots were performed to assess osteoblast differentiation. The hDPSCs were positive for surface antigens CD73 and CD90 and negative for CD45 and CD11b expression. The level of IL-6 secreted by hDPSCs significantly increased the number of CD80+ cells as well as the levels of Arg-1 and Mrc-1. It also promoted M2 macrophage polarization and activated GP130/STAT3 signaling. However, the medium cocultured with THP-1-derived macrophages by hDPSCs facilitated the migration, invasion, and osteogenic abilities of human bone marrow-derived stem cells (hBMSCs). hDPSCs can regulate the periodontal microenvironment through IL-6 by inducing phenotypic transformation of M2 macrophages and stimulating osteogenic differentiation of BMSCs.

Activating Wnt/β-Catenin Signaling in Osteocytes Promotes Osteogenic Differentiation of BMSCs through BMP-7.

Bone formation is critically needed in orthopedic clinical practice. We found that, bone morphogenetic protein-7 (BMP-7) gene expression was significantly increased in fractured mice, which activates canonical Wnt signaling exclusively in osteocytes. Wnt and BMP signaling appear to exhibit synergistic or antagonistic effects in different kinds of cells. However, the communication between Wnt/β-catenin signaling and BMP signaling in osteocytes is almost unknown. Our study verified in vitro that BMP-7 expression was significantly increased when Wnt signaling was activated in osteocytes. Next, BMP-7 in osteocytes was overexpressed using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was enhanced, when cocultured with osteocytes. On the contrary, BMP-7 in osteocytes was silenced using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was weakened. In addition, the osteogenesis of BMSCs was no longer promoted by Wnt-activated osteocytes when BMP-7 was silenced. Therefore, the results showed that BMP-7 mediated the anabolic actions of Wnt/β-catenin signaling in osteocytes. Our study provides new evidence for the clinical application of BMP-7-overexpressed osteocytes.

DLP-based bioprinting of void-forming hydrogels for enhanced stem-cell-mediated bone regeneration.

The integration of 3D bioprinting and stem cells is of great promise in facilitating the reconstruction of cranial defects. However, the effectiveness of the scaffolds has been hampered by the limited cell behavior and functions. Herein, a therapeutic cell-laden hydrogel for bone regeneration is therefore developed through the design of a void-forming hydrogel. This hydrogel is prepared by digital light processing (DLP)-based bioprinting of the bone marrow stem cells (BMSCs) mixed with gelatin methacrylate (GelMA)/dextran emulsion. The 3D-bioprinted hydrogel can not only promote the proliferation, migration, and spreading of the encapsulated BMSCs, but also stimulate the YAP signal pathway, thus leading to the enhanced osteogenic differentiation of BMSCs. In addition, the in vivo therapeutic assessments reveal that the void-forming hydrogel shows great potential for BMSCs delivery and can significantly promote bone regeneration. These findings suggest that the unique 3D-bioprinted void-forming hydrogels are promising candidates for applications in bone regeneration.

Micro/nano-topography promotes osteogenic differentiation of bone marrow stem cells by regulating periostin expression

Micro/nano-topography (MNT) is an important factor affecting cell response. Earlier studies using titania (TiO2) nanotube as a model of MNT found that they mediated the differentiation of BMSCs into osteoblasts, but the mechanisms are not fully understood. Surprisingly, Periostin (Postn), a secreted protein involved in extracellular matrix (ECM) construction and promoting osteogenic differentiation of bone marrow stem cells (BMSCs), was previously observed to significantly up-regulated on TiO2 nanotube. We proposed that Postn may act as a MNT signal transduction role. In this study, we investigated the effect of MNT on Postn, and the influence of Postn on osteogenic differentiation-related genes through focal adhesion and downstream signals. It was found that, titanium (Ti) plates carrying TiO2 nanotubes with diameters of ∼100 nm (TNT-100) significantly up-regulated the expression of Postn compared with flat Ti. Furthermore, Postn activated the downstream focal adhesion kinase (FAK) signal pathway and β-catenin into the nucleus by interacting with integrin αV. Surprisingly, TNT-100 up-regulated the transcription level of Wnt3a, which was independent of the up-regulation of Postn. This new Postn signaling pathway may provide more insights into the signal transduction mechanism of MNT and development of biomaterials with improved osteogenic properties.

Local Administration of Metformin Improves Bone Microarchitecture and Biomechanical Properties During Ruptured Canine Achilles Tendon–Calcaneus Interface Healing

Background: Tendon-bone interface (TBI) healing is a clinical dilemma that is closely relevant to new bone formation and remodeling at the repair site. Previous studies showed that metformin is an osteogenic inducer for stem cells in vitro and capable of stimulating bone regeneration in vivo. Hypothesis: Metformin would be effective for promoting TBI healing by enhancing new bone formation and remodeling. Study Design: Controlled laboratory study. Methods: Canine bone marrow stem cells (BMSCs) were cultured with various concentrations of metformin (0, 10, 50, 100, 200 μM). The effect of metformin on the osteogenic differentiation of canine BMSCs was evaluated via alizarin red staining and osteogenic gene expression. Eighteen mature beagles were included in a bilateral Achilles tendon–calcaneus (ATC) interface injury model. The right interface was reattached via surgical repair only, while the left was surgically reattached after implanting a fibrin glue containing metformin. At postoperative week 4 or 8, the healing quality of the wounded ATC interfaces was evaluated. Results: In vitro experiments determined that metformin was an osteogenic inducer for canine BMSCs. In vivo experiments showed that the metformin-treated ATC interfaces were repaired with significantly greater failure load and stiffness than was the no-metformin control site (P < .05 for all). Micro–computed tomography analysis showed that the metformin-treated specimens presented significantly higher bone volume/total volume and trabecular thickness than did the no-metformin control specimens (P < .05 for all), as confirmed via hematoxylin and eosin staining. Immunohistochemical staining showed that significantly more osteocalcin-positive cells were located at the newly formed bones treated with metformin than at the no-metformin control site at week 4 (P < .05). Masson trichrome staining showed that significantly more oriented collagen fibers anchored into the newly formed bone of the metformin-treated site than the no-metformin control site (P < .05). Conclusion: Metformin induced the osteogenesis of canine BMSCs in vitro, and local administration of metformin provided an improvement of bone microarchitecture at the calcaneus as well as an increase in the tensile properties of the repaired ATC interfaces in canines. Clinical Relevance: Findings of the study indicate that local administration of metformin may be an effective strategy for TBI healing in clinic.

LncRNA metastasis-associated lung adenocarcinoma transcript-1 promotes osteogenic differentiation of bone marrow stem cells and inhibits osteoclastic differentiation ofMø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/β-catenin axis.

Osteoporosis is defined as a skeletal disorder characterized by impairment in bone strength. The potential application of lncRNAs as therapeutic targets for osteoporosis has been unveiled. This study investigated the regulatory mechanism of lncRNA MALAT1 in the differentiation of bone marrow stem cells (BMSCs) and macrophages (Mø) in osteoporosis. MALAT1 expression in peripheral blood of elderly osteoporosis patients and healthy volunteers was detected. BMSCs and mononuclear Mø were isolated and cultured. Osteogenic differentiation of BMSCs and osteoclastic differentiation of Mø were induced. BMSCs and Mø were transfected with si-MALAT1, miR-124-3p mimics, miR-124-3p inhibitor, or pcDNA IGF2BP1, followed by detection of cell differentiation. The target microRNAs (miRs) and downstream genes and signaling pathways of MALAT1 were examined. The ovariectomy-induced mouse model of osteoporosis was established, and the mice were injected with pcDNA-MALAT1. MALAT1 was downregulated in osteoporosis patients, increased in BMSCs after osteogenic differentiation, and diminished in Mø after osteoclastic differentiation. Downregulation of MALAT1 repressed osteogenic differentiation of BMSCs and facilitated osteoclastic differentiation of Mø. MALAT1 upregulated IGF2BP1 expression by competitively binding to miR-124-3p. miR-124-3p silencing reversed the effect of si-MALAT1 on BMSCs and Mø differentiation, and IGF2BP1 upregulation averted the effect of overexpressed-miR-124-3p by activating the Wnt/β-catenin pathway. Upregulation of MALAT1 activated the Wnt/β-catenin pathway and attenuated bone injury in mice. In conclusion, lncRNA MALAT1 promoted the osteogenic differentiation of BMSCs and inhibited osteoclastic differentiation of Mø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/β-catenin axis.

Inhibition of miRNA-100 facilitates bone regeneration defects of mesenchymal stem cells in osteoporotic mice through the protein kinase B pathway

ABSTRACT Osteoporotic patients suffer from bone microstructure damage and are prone to fracture and bone defect. Due to the damage of bone healing ability, the bone repair of osteoporotic patients is usually slow. Here we aimed to explore the function and potential molecular mechanism of miR-100 in osteogenic differentiation ability of bone marrow stem cells (BMSCs). Ovariectomy was performed on mice to induce osteoporosis. BMSCs were extracted from normal and ovariectomized (OVX) mice to examine the effect of microRNA (miR)-100 on BMSC osteogenic differentiation. Hematoxylin and eosin (H&E) staining and safranin O-fast green staining assays were performed on femur tissues to reveal pathological changes. The osteogenic differentiation of BMSCs were determined by Alkaline Phosphatase and Alizarin red staining assays. The results showed that miR-100 expression was significantly upregulated in bone tissues and BMSCs from osteoporotic mice. MiR-100 knockdown partially improved osteogenic function of OVX mice-derived BMSCs. Next, mechanistic target of rapamycin kinase (MTOR) was identified as the target downstream miR-100. MiR-100 deficiency can activate the protein kinase B (AKT)/mTOR pathway. MiR-100 controlled the osteogenic function of BMSCs by the AKT/mTOR pathway. Collectively, our findings demonstrate that inhibition of miR-100 facilitates bone regeneration defects of BMSCs in osteoporotic mice through AKT pathway, indicating that miR-100 might be an effective target for the treatment of osteoporotic mandibular injury and bone defect diseases.

Enhanced bone formation in rat critical-size tibia defect by a novel quercetin-containing alpha-calcium sulphate hemihydrate/nano-hydroxyapatite composite

We developed an innovative method to include quercetin into alpha-calcium sulphate hemihydrate/nano-hydroxyapatite (α-CSH/n-HA), to prepare a novel quercetin-containing α-CSH/n-HA composite (Q-α-CSH/n-HA). The physicochemical properties, and ability of Q-α-CSH/n-HA to promote cell proliferation, migration, and osteogenic differentiation of bone marrow stem cells (BMSCs) in vitro were examined. Further, the potential of Q-α-CSH/n-HA to promote bone defect repair was studied using a Sprague-Dawley rat model of critical tibial defects. Imaging was conducted by radiography and micro-CT, and bone defect repairs were observed by histopathological staining. Addition of quercetin clearly increased the porosity of the degraded composite, which elevated the cell proliferation rate, migration ability, osteogenesis differentiation, and mineralisation of BMSCs. Further, quercetin-containing composite increased the expression levels of OSX, RUNX2, OCN, ALP, BMP-2, OPN, BSP, SMAD2, and TGF-β in BMSCs, while it downregulated TNF-α. X-ray and micro-CT imaging showed that the quercetin-containing composite significantly enhanced bone defect repair and new bone in formation. Haematoxylin and eosin, Goldner, and Safranin O staining also showed that quercetin significantly increased new bone generation and promoted composite degradation and absorption. Moreover, immunofluorescence assay revealed that quercetin significantly increased the number of RUNX2/OSX/OCN-positive cells. Overall, our data demonstrate that Q-α-CSH/n-HA has excellent biocompatibility, bone conductivity, and osteo-induction performance in vitro and mediates enhanced overall repair effects and bone reconstruction in vivo, indicating that it is a promising artificial bone graft to promote bone regeneration.

Research on the Mechanism of Asiaticoside Inhibiting Osteoclast Differentiation and Promoting the Recovery of Steroid-Induced Osteonecrosis of the Femoral Head (SIONFH)

Our study aims to assess whether asiaticoside promotes the recovery of SINOFH by inhibiting bone marrow stem cells (BMSCs) differentiation into osteoclasts (OC). BMMs were induced to form OC system by dexamethasone in vitro and ELISA detected the expression of OC-related genes formation by asiaticoside. BMSCs were cultured followed by analysis of BMSCs morphology under microscope, gene expression by qRT-PCR. TRACP and c-Src level by western blot, RANKL, OPG and TRACP5b level by ELISA. Asiaticoside inhibited the expression of OC formation in SIONFH. The expression of OC-related genes increased with the induction days. With the increasing of induction days, asiaticoside level in culture fluid was decreased. While after asiaticoside interference, OCrelated genes and proteins levels were significantly down-regulated. Aasiaticoside can significantly increase the RANKL signaling protein expression. In conclusion, asiaticoside promotes the recovery of SINOFH by inhibiting BMSCs differentiation into OC.

Investigation into the effects of leukemia inhibitory factor on the bone repair capacity of BMSCs-loaded BCP scaffolds in the mouse calvarial bone defect model.

Leukemia inhibitory factor (LIF) is known to play a major role in bone physiology. In the present study, we examined the in vitro effects of LIF on osteoblast differentiation of bone marrow stem cells (BMSCs) and explored in vivo effects of LIF on the bone repair capacity of BMSCs-loaded biphasic calcium phosphate (BCP) scaffolds in mouse calvarial bone defect model. The mRNA and protein expression levels in the BMSCs were determined by quantitative real-time PCR and western blot, respectively; the in vitro osteoblast differentiation of the BMSCs was evaluated by using Alizarin Red S staining. The bone volume and bone density in the repaired calvarial bone defect were determined by Micro-CT. Bone regeneration was also histologically evaluated by hematoxylin and eosin staining and Masson's trichrome staining. Hypoxia treatment induced the up-regulation of Lif mRNA and LIF protein in the BMSCs. Lif overexpression up-regulated the mRNA expression levels of osteopontin and Runt-related transcription factor 2, and increased intensity of Alizarin Red S staining in the BMSCs; while Lif silence exerted the opposite effects. The in vivo studies showed that implantation of Lif-overexpressing BMSCs-loaded BCP scaffolds significantly increased the bone volume and bone density at 4 and 8weeks after transplantation, and promoted the regeneration of bone tissues in the mouse calvarial bone defect at 8weeks after transplantation when compared to the BMSCs-loaded BCP scaffolds group; while Lif-silencing BMSCs-loaded BCP scaffolds had the opposite effects. The present study for the first time demonstrated that LIF promoted the in vitro osteoblast differentiation of hypoxia-treated BMSCs; and further studies revealed that LIF exerted enhanced effects on the bone repair capacity of BMSCs-load BCP scaffolds in mouse calvarial bone defect model. However, future studies are warranted to determine the detailed mechanisms of LIF in the large-scale bone defect repair.

Exosome derived from bone marrow mesenchymal stem cells pre-treated with curcumin alleviates osteoporosis by promoting osteogenic differentiation via regulating the METTL3/microRNA-320/RUNX2 signaling pathway

IntroductionCurcumin (CUR) was reported to stimulate the expression of methyltransferase‐like 3 (METTL3), a potential new target for the replacement therapy of osteoporosis. Besides, marrow stem cells (MSC)-derived exosomes (EXO) were proven to improve osteoporosis by promoting the proliferation of osteoblasts. In this study, we aimed to study the effect of CUR and BMSC-derived exosomes in the treatment of osteoporosis.Material and methodsMicroscopy was used to characterize exosomes derived from bone marrow stem cells (BMSCs). MicroCT was carried out to analyze the parameters of bone formation. Western blot was carried out to analyze the expression of surface markers on BMSC-derived exosomes and METTL3/Runt-related transcription factor 2 (RUNX2) proteins under different conditions. Real-time PCR was used to assess the gene expression under different circumstances.ResultsThe exosomes derived from CUR-treated BMSCs showed an enhanced therapeutic effect on osteoporosis mice compared with exosomes derived from untreated BMSCs. Mechanistically, the effect of BMSC-derived exosomes on restoring the de-regulated expression of METTL3, miR-320 and RUNX2 was significantly enhanced by CUR treatment upon BMSCs. Besides, CUR treatment upon BMSCs showed an obvious effect on enhancing the stimulatory role of BMSC-derived exosomes in the expression of METTL3, miR-320, RUNX2, BGLAP and LAP in BMSCs. Furthermore, luciferase assay demonstrated that miR-320 was capable of suppressing the expression of RUNX2 through binding to the 3’ UTR of RUNX2.ConclusionsOur study demonstrated that BMSC-derived exosomes could modulate the METTL3/miRNA-320/RUNX2 axis to attenuate osteoporosis by promoting osteogenic differentiation of BMSCs. Moreover, the CUR treatment upon BMSCs promoted the therapeutic effect of BMSC-derived exosomes.