Osteogenic Differentiation Of Mesenchymal Stem Cells Research Articles

IGF-1 Induces Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Promoting SOX4 via the MAPK/ERK Pathway.

Tissue engineering envisions functional substitute creation for damaged tissues. Insulin-like growth factor-1 (IGF-1) plays roles in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation (OD), and we investigated its specific mechanism. BMSCs were cultured and OD was induced. Surface antigens (CD105, CD90, CD44, CD45, CD34) were identified by flow cytometry. Adipogenic, chondrogenic, and osteogenic differentiation abilities of BMSCs were observed. BMSCs were cultured in osteogenic medium containing 80 ng/mL IGF-1 for 3 weeks. Alkaline phosphatase activity, calcification level, osteogenic factor (runt related protein 2 [RUNX2], osteocalcin [OCN], osterix [OSX]), total (t-) ERK1/2 and phosphorylated- (p-) ERK1/2 levels, and SRY-related high-mobility-group box 4 (SOX4) levels were assessed by alkaline phosphatase staining and Alizarin Red staining, Western blot, and reverse transcription-quantitative polymerase chain reaction. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibitor (PD98059) was used to inhibit the MAPK/ERK pathway in IGF-1-treated BMSCs. Small interfering-SOX4 was transfected into BMSCs to down-regulate SOX4. IGF-1 increased alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels in BMSCs, indicating that IGF-1 induced rat BMSC OD. SOX4, and p-ERK1/2 and t-ERK1/2 levels were elevated in IGF-1-induced BMSCs, which were annulled by PD98059. PD98059 partly averted IGF-1-induced rat BMSC OD. SOX4 levels, alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels were reduced after SOX4 down-regulation, showing that downregulation of SOX4 averted the effect of IGF-1 on inducing rat BMSC OD. IGF-1 induced rat BMSC OD by stimulating SOX4 via the MAPK/ERK pathway.

Icariin accelerates bone regeneration by inducing osteogenesis-angiogenesis coupling in rats with type 1 diabetes mellitus

BACKGROUND Icariin (ICA), a natural flavonoid compound monomer, has multiple pharmacological activities. However, its effect on bone defect in the context of type 1 diabetes mellitus (T1DM) has not yet been examined. AIM To explore the role and potential mechanism of ICA on bone defect in the context of T1DM. METHODS The effects of ICA on osteogenesis and angiogenesis were evaluated by alkaline phosphatase staining, alizarin red S staining, quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence. Angiogenesis-related assays were conducted to investigate the relationship between osteogenesis and angiogenesis. A bone defect model was established in T1DM rats. The model rats were then treated with ICA or placebo and micron-scale computed tomography, histomorphometry, histology, and sequential fluorescent labeling were used to evaluate the effect of ICA on bone formation in the defect area. RESULTS ICA promoted bone marrow mesenchymal stem cell (BMSC) proliferation and osteogenic differentiation. The ICA treated-BMSCs showed higher expression levels of osteogenesis-related markers (alkaline phosphatase and osteocalcin) and angiogenesis-related markers (vascular endothelial growth factor A and platelet endothelial cell adhesion molecule 1) compared to the untreated group. ICA was also found to induce osteogenesis-angiogenesis coupling of BMSCs. In the bone defect model T1DM rats, ICA facilitated bone formation and CD31hiEMCNhi type H-positive capillary formation. Lastly, ICA effectively accelerated the rate of bone formation in the defect area. CONCLUSION ICA was able to accelerate bone regeneration in a T1DM rat model by inducing osteogenesis-angiogenesis coupling of BMSCs.

Effect of polydimethylsiloxane surface morphology on osteogenic differentiation of mesenchymal stem cells through SIRT1 signalling pathway.

Constructing surface topography with a certain roughness is a widely used, non-toxic, cost-effective and effective method for improving the microenvironment of cells, promoting the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs), and promoting the osseointegration of grafts and further improving their biocompatibility under clinical environmental conditions. SIRT1 plays an important regulatory role in the osteogenic differentiation of bone marrow-derived MSCs (BM-MSCs). However, it remains unknown whether SIRT1 plays an important regulatory role in the osteogenic differentiation of BM-MSCs with regard to surface morphology. Polydimethylsiloxane (PDMS) with different surface morphologies were prepared using different grits of sandpaper. The value for BMSCs added on different surfaces was detected by cell proliferation assays. RT-qPCR and Western blotting were performed to detect SIRT1 activation and osteogenic differentiation of MSCs. Osteogenesis of MSCs was detected by alkaline phosphatase (ALP) and alizarin red S staining. SIRT1 inhibition experiments were performed to investigate the role of SIRT1 in the osteogenic differentiation of MSCs induced by surface morphology. We found that BM-MSCs have better value and osteogenic differentiation ability on a surface with roughness of PDMS-1000M. SIRT1 showed higher gene and protein expression on a PDMS-1000M surface with a roughness of 13.741 ± 1.388 µm. The promotion of the osteogenic differentiation of MSCs on the PDMS-1000M surface was significantly decreased after inhibiting SIRT1 expression. Our study demonstrated that a surface morphology with certain roughness can activate the SIRT1 pathway of MSCs and promote the osteogenic differentiation of BMSCs via the SIRT1 pathway.

Mijiao formula regulates NAT10-mediated Runx2 mRNA ac4C modification to promote bone marrow mesenchymal stem cell osteogenic differentiation and improve osteoporosis in ovariectomized rats

Ethnopharmacological relevanceThe Mijiao (MJ) formula, a traditional herbal remedy, incorporates antlers as its primary constituent. It can effectively treat osteoporosis (OP), anti-aging, enhance immune activity, and change depression-like behavior. In this study, we investigated that MJ formula is a comprehensive treatment strategy, and may provide a potential approach for the clinical treatment of postmenopausal osteoporosis. Aim of the studyThe purpose of this study was to determine whether MJ formula promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and improved osteoporosis in ovariectomized rats by regulating the NAT10-mediated Runx2 mRNA ac4C modification. Materials and MethodsFemale Sprague-Dawley (SD) rats were used to investigate the potential therapeutic effect of MJ formula on OP by creating an ovariectomized (OVX) rat model. The expression of osteogenic differentiation related proteins in BMSCs was detected in vivo, indicating their role in promoting bone formation. In addition, the potential mechanism of its bone protective effect was explored via in vitro experiments. ResultsOur study showed that MJ formula significantly mitigated bone mass loss in the OVX rat model, highlighting its potential as an OP therapeutic agent. We found that the possible mechanism of action was the ability of this formulation to stabilize Runx2 mRNA through NAT10-mediated ac4C acetylation, which promoted osteogenic differentiation of BMSCs and contributed to the enhancement of bone formation. ConclusionsMJ formula can treat estrogen deficiency OP by stabilizing Runx2 mRNA, promoting osteogenic differentiation and protecting bone mass. Conceivably, MJ formulation could be a safe and promising strategy for the treatment of osteoporosis.

Tantalum Particles Promote M2 Macrophage Polarization and Regulate Local Bone Metabolism via Macrophage-Derived Exosomes Influencing the Fates of BMSCs.

In this study, the regulatory role and mechanisms of tantalum (Ta) particles in the bone tissue microenvironment are explored. Ta particle deposition occurs in both clinical samples and animal tissues following porous Ta implantation. Unlike titanium (Ti) particles promoting M1 macrophage (Mϕ) polarization, Ta particles regulating calcium signaling pathways and promoting M2 Mϕ polarization. Ta-induced M2 Mϕ enhances bone marrow-derived mesenchymal stem cells (BMSCs) proliferation, migration, and osteogenic differentiation through exosomes (Exo) by upregulating miR-378a-3p/miR-221-5p and downregulating miR-155-5p/miR-212-5p. Ta particles suppress the pro-inflammatory and bone resorption effects of Ti particles in vivo and in vitro. In a rat femoral condyle bone defect model, artificial bone loaded with Ta particles promotes endogenous Mϕ polarization toward M2 differentiation at the defect site, accelerating bone repair. In conclusion, Ta particles modulate Mϕ polarization toward M2 and influence BMSCs osteogenic capacity through Exo secreted by M2 Mϕ, providing insights for potential bone repair applications.

Plasma exosomal miR-30a-5p inhibits osteogenic differentiation of bone marrow mesenchymal stem cells from a chronic unpredictable mild stress-induced depression rat model

With rising society stress, depression-induced osteoporosis is increasing. However, the mechanism involved is unclear. In this study, we explored the effect of plasma exosomal miRNAs on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation in a chronic unpredictable mild stress (CUMS)-induced depression rat model. After 12 weeks of CUMS-induced depression, the pathological changes in the bone tissue and markers of osteogenic differentiation were tested by micro-computed tomography, hematoxylin-eosin staining, and quantitative real-time reverse transcription PCR (qRT-PCR). Plasma exosomes from rats were isolated and co-incubated with BMSCs for 14 d to detect the effect on osteogenic markers. Next-generation sequencing identified the miRNAs in the plasma exosomes, and the differential miRNAs were analyzed and verified by qRT-PCR. BMSCs were infected with lentivirus to upregulate miRNA-30a-5p and incubated in a medium that induced osteogenic differentiation for 14 d. The effect of miR-30a-5p on osteogenic differentiation was determined by qPCR and alizarin red staining. CUMS-induced depression rat model was established successfully, and exhibited reduced bone mass and damaged bone microstructure compared to that of the controls. The observed pathological changes suggested the occurrence of osteoporosis in the CUMS group, and the mRNA expression of osteogenic markers was also significantly reduced. Incubation of BMSCs with plasma exosomes from the CUMS group for 14 d resulted in a significant decrease in the expression of osteogenic markers. Twenty-five differentially expressed miRNAs in plasma exosomes were identified and upregulation of miR-30a-5p was observed to significantly inhibit the expression of osteogenic markers in BMSCs. Our findings contributed to a comprehensive understanding of the mechanism of osteoporosis caused by depression, and demonstrated the potential of miR-30a-5p as a novel biomarker or therapeutic target for the treatment of osteoporosis.

SGMS1 facilitates osteogenic differentiation of MSCs and strengthens osteogenesis-angiogenesis coupling by modulating Cer/PP2A/Akt pathway

Mesenchymal stem cell (MSC)-mediated coupling of osteogenesis and angiogenesis is a critical phenomenon in bone formation. Herein, we investigated the role and mechanism of SGMS1 in the osteogenic differentiation of MSCs and, in combination with osteogenesis and angiogenesis, to discover new therapeutic targets for skeletal dysplasia and bone defects. SGMS1 addition accelerated MSC osteogenic differentiation, whereas SGMS1 silencing suppressed this process. Moreover, SGMS1 overexpression inhibited ceramide (Cer) and promoted sphingomyelin (SM) levels. SM treatment neutralized the suppressive effect of shSGMS1 on osteogenesis. SGMS1 restrained PP2A activity by regulating Cer/SM metabolism and thus enhanced the levels of phosphorylated Akt, Runx2, and vascular endothelial growth factor (VEGF). Furthermore, SGMS1 transcription was regulated by Runx2. SGMS1 increased MSC-mediated angiogenesis by promoting VEGF expression. SGMS1 addition promoted rat bone regeneration invivo. In conclusion, SGMS1 induces osteogenic differentiation of MSCs and osteogenic-angiogenic coupling through the regulation of the Cer/PP2A/Akt signaling pathway.

The Effect of Clopidogrel and Ticagrelor on Human Adipose Mesenchymal Stem Cell Osteogenic Differentiation Potential: In Vitro Comparative Study.

Ticagrelor (TICA) and clopidogrel (CLP) are extensively used antiplatelet drugs that act by antagonizing the P2Y12 receptors that are found on platelets in addition to bone cells. Aim. The purpose of this study was to investigate the effect of clopidogrel and ticagrelor on stem cells osteogenic differentiation in vitro. Methods. Human adipose-derived mesenchymal stem cells (hAd-MSCs) were divided into (1) control group, (2) osteogenic group (osteo group), (3) clopidogrel group (CLP group), and (4) ticagrelor group (TICA group). The osteogenic differentiation potential was determined by mineralization nodule formation using Alizarin Red S staining, measuring ALP enzyme activity by alkaline phosphatase assay. Quantitative determination for osteogenic markers included osteocalcin (OC); runt-related transcription factor 2 (RUNX2) performed using western blot; osteoprotegerin (OPG) using enzyme-linked immunosorbent assay (ELISA) and inflammatory markers; and tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) measured using real-time polymerase chain reaction quantitative (RT-PCR) and ELISA. Results. In comparison to all study groups, the TICA group showed significant increase in the mineralized extracellular matrix, ALP enzyme activity, and bone markers expression as RUNX2 (P < 0.0001), OC, and OPG (P < 0.05). The expression of IL-6 and TNF-α was determined by RT-qPCR and ELISA techniques. TICA and CLP significantly decreased both markers compared to the control group. The TICA group showed statistically significant lower levels of both markers (P < 0.0001) than the CLP and control groups via the ELISA technique. Conclusion. TICA may possess a positive effect on hAd-MSCs osteogenic differentiation compared to CLP.

Metformin rejuvenates Nap1l2-impaired immunomodulation of bone marrow mesenchymal stem cells via metabolic reprogramming.

Ageing and cell senescence of mesenchymal stem cells (MSCs) limited their immunomodulation properties and therapeutic application. We previously reported that nucleosome assembly protein 1-like 2 (Nap1l2) contributes to MSCs senescence and osteogenic differentiation. Here, we sought to evaluate whether Nap1l2 impairs the immunomodulatory properties of MSCs and find a way to rescue the deficient properties. We demonstrated that metformin could rescue the impaired migration properties and T cell regulation properties of OE-Nap1l2 BMSCs. Moreover, metformin could improve the impaired therapeutic efficacy of OE-Nap1l2 BMSCs in the treatment of colitis and experimental autoimmune encephalomyelitis in mice. Mechanistically, metformin was capable of upregulating the activation of AMPK, synthesis of l-arginine and expression of inducible nitric oxide synthase in OE-Nap1l2 BMSCs, leading to an increasing level of nitric oxide. This study indicated that Nap1l2 negatively regulated the immunomodulatory properties of BMSCs and that the impaired functions could be rescued by metformin pretreatment via metabolic reprogramming. This strategy might serve as a practical therapeutic option to rescue impaired MSCs functions for further application.

Nicotinamide mononucleotide based hyaluronic acid methacryloyl hybrid hydrogel regulating stem cells fate for bone regeneration via SIRT1/RUNX2 signaling

Efficient bone reconstruction, especially of the critical size after bone damage, remains a challenge in the clinic. Bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation is considered as a promising strategy for bone repair. Nicotinamide adenine dinucleotide (NAD+) regulating BMSC fate and cellular function enhance osteogenesis, but is hardly delivered and lack of targeting. Herein, a novel and biocompatible scaffold was fabricated to locally deliver a precursor of NAD+, nicotinamide mononucleotide (NMN) to the bone defect site, and its bone repair capability and healing mechanism were clarified. NMN-based hyaluronic acid methacryloyl hybrid hydrogel scaffold (denoted as NMN/HAMA) was prepared via photopolymerization. In vitro RT-qPCR analysis, western blotting, Elisa and alizarin red S staining assays demonstrated that the NMN/HAMA hybrid hydrogel regulated BMSCs cellular function in favour of osteogenic differentiation and mineralization by upregulating the mRNA and proteins expression of the osteogenic genes type I pro-collagen (Col-1), bone morphogenic protein 4 (BMP4), and runt-related transcription factor 2 (RUNX2) via the SIRT1 pathway. Implantation of such hybrid hydrogels significantly enhanced bone regeneration in rodent critical calvarial defect models. Furthermore, restoration of the bone defect with NMN administration was inhibited in Prx1 Cre+; SIRT1flox/flox mice, confirming that the NMN/HAMA hybrid hydrogel scaffold promoted bone regeneration via the SIRT1-RUNX2 pathway. These results imply that NMN-based scaffold may be a promising and economic strategy for the treatment of bone defects.

The effect of nano-natural biomaterial on stimulation of RADMSCs toward osteogenesis: an in vitro study

In recent years, natural biomaterials have been the focal point of most biomedical investigations. To prepare the Ostrich Eggshell/Hydroxyapatite (OE/HA) powder scaffolds, OE powder was immersed in 1% Triton X-100 for 24 h and then autoclaved twice. Medical grade HA was combined with pulverized OE at different ratios to yield three different OE/HA powder scaffolds. The resultant scaffolds underwent characterization experiments using different techniques, including FTIR, XRD, radiography, and SEM analyses. Rabbit adipose-derived mesenchymal stem cells (RADMSCs) were isolated from adipose tissues and characterized by flow cytometry. A direct contact test was performed for the cytotoxicity test using L929 cells. Cell adhesion was validated using SEM imaging, viability was assessed using DAPI and Annexin V/PI staining, and osteogenic differentiation was investigated using the ALP assay. The cytotoxicity assay using L929 cells verified that the OE/HA powder scaffold is safe and further functional experiments could be carried out. Behavioral assays, radiography, surface markers, surface topography analyses, and viability tests yielded promising results confirming cellular acceptance of the scaffold. In the present study, we provided evidence that the prepared OE/HA scaffold is not only cytocompatible but also can enhance mesenchymal stem cell adhesion, growth, and osteogenic differentiation in vitro.

YAP mechanotransduction under cyclic mechanical stretch loading for mesenchymal stem cell osteogenesis is regulated by ROCK.

While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAP-mediated MSC osteogenic transcription.

Effects of Oral Cavity Stem Cell Sources and Serum-Free Cell Culture on Hydrogel Encapsulation of Mesenchymal Stem Cells for Bone Regeneration: An In Vitro Investigation.

To develop a stem cell delivery model and improve the safety of stem cell transplantation for bone regeneration, this study aimed to determine the effects of stem cell sources, serum-free cell culture, and hydrogel cell encapsulation on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs) from the oral cavity. The study groups were categorized according to stem cell sources into buccal fat pad adipose (hBFP-ADSCs) (Groups 1, 4, and 7), periodontal ligament (hPDLSCs) (Groups 2, 5, and 8), and dental pulp-derived stem cells (hDPSCs) (Groups 3, 6, and 9). MSCs from each source were isolated and expanded in three types of sera: fetal bovine serum (FBS) (Groups 1-3), human serum (HS) (Groups 4-6), and synthetic serum (SS) (StemPro™ MSC SFM) (Groups 7-9) for monolayer (m) and hydrogel cell encapsulation cultures (e). Following this, the morphology, expression of MSC cell surface antigens, growth, and osteogenic differentiation potential of the MSCs, and the expression of adhesion molecules were analyzed and compared. SS decreased variations in the morphology and expression levels of cell surface antigens of MSCs from three cell sources (Groups 7m-9m). The levels of osteoblastic differentiation of the hPDLSCs and hBFP-ADSCs were increased in SS (Groups 8m and 7m) and the cell encapsulation model (Groups 1e, 4e, 7e-9e), but the promoting effects of SS were decreased in a cell encapsulation model (Groups 7e-9e). The expression levels of the alpha v beta 3 (ITG-αVβ3) and beta 1 (ITG-β1) integrins in the encapsulated cells in FBS (Group 1e) were higher than those in the SS (Group 7e). Human PDLSCs and BFP-ADSCs were the optimum stem cell source for stem cell encapsulation by using nanohydroxyapatite-calcium carbonate microcapsule-chitosan/collagen hydrogel in serum-free conditions.

Regulatory mechanisms of circular RNAs during human mesenchymal stem cell osteogenic differentiation.

Human osteogenic differentiation is a complex and well-orchestrated process which involves a plethora of molecular players and cellular processes. A growing number of studies have underlined that circular RNAs (circRNAs) play an important regulatory role during human osteogenic differentiation. CircRNAs are single-stranded, covalently closed non-coding RNA molecules that are acquiring increased attention as epigenetic regulators of gene expression. Given their intrinsic high conformational stability, abundance, and specificity, circRNAs can undertake various biological activities in order to regulate multiple cellular processes, including osteogenic differentiation. The most recent evidence indicates that circRNAs control human osteogenesis by preventing the inhibitory activity of miRNAs on their downstream target genes, using a competitive endogenous RNA mechanism. The aim of this review is to draw attention to the currently known regulatory mechanisms of circRNAs during human osteogenic differentiation. Specifically, we provide an understanding of recent advances in research conducted on various human mesenchymal stem cell types that underlined the importance of circRNAs in regulating osteogenesis. A comprehensive understanding of the underlying regulatory mechanisms of circRNA in osteogenesis will improve knowledge on the molecular processes of bone growth, resulting in the potential development of novel preclinical and clinical studies and the discovery of novel diagnostic and therapeutic tools for bone disorders.

Tescalcin modulates bone marrow-derived mesenchymal stem cells osteogenic differentiation via the Wnt/β-catenin signaling pathway.

Bone mesenchymal stem cells (BMSCs) are recognized for their intrinsic capacity for self-renewal and differentiation into osteoblasts, adipocytes, and chondrocytes, making them pivotal entities within the field of bone research. Tescalcin (TESC) is known to play a role in specific cellular processes related to proliferation and differentiation. However, the precise involvement of TESC in the regulation of BMSCs remains unclear. The present study was designed to verify the functional implications of TESC in BMSCs. An adenovirus vector was engineered to downregulate TESC expression, and the Wnt/β-catenin signaling pathway was activated using BML-284. The assessment of mRNA was conducted by quantitative real-time polymerase chain reaction (qRT-PCR). The assessment of protein expression was conducted by Western blotting and immunofluorescence techniques (IF), respectively. Alkaline phosphatase (ALP) staining and activity assays were performed to verify ALP changes, while Alizarin Red S (ARS) staining and quantitative analysis were employed to assess mineralization capacity. Initially, we observed an upregulation of TESC expression during osteogenic differentiation. Subsequently, TESC knockdown was demonstrated to decrease the osteogenic-related genes expression and diminish BMSCs mineralization. Concomitantly, we identified the inhibition of Wnt/β-catenin signaling following the TESC knockdown. Furthermore, the administration of BML-284 effectively activated the Wnt/β-catenin pathway, successfully rescuing the compromised TESC-mediated osteogenic differentiation. Our findings indicate that TESC knockdown exerts an inhibitory effect on the osteogenic differentiation of BMSCs through the modulation of the Wnt/β-catenin signaling pathway. This study unveils a novel target with potential applications for enhancing the regenerative potential of BMSCs in the realm of regenerative medicine.

Sustained Release of Dexamethasone from 3D-Printed Scaffolds Modulates Macrophage Activation and Enhances Osteogenic Differentiation.

Enhancing osteogenesis via modulating immune cells is emerging as a new approach to address the current challenges in repairing bone defects and fractures. However, much remains unknown about the crosstalk between immune cells and osteolineage cells during bone formation. Moreover, biomaterial scaffold-based approaches to effectively modulate this crosstalk to favor bone healing are also lacking. This study is the first to investigate the interactions between macrophages and mesenchymal stem cells (MSCs) in co-cultures with the sustained release of an anti-inflammatory and pro-osteogenesis drug (dexamethasone) from three-dimensional (3D)-printed scaffolds. We successfully achieved the sustained release of dexamethasone from polycaprolactone (PCL) by adding the excipient-sucrose acetate isobutyrate (SAIB). Dexamethasone was released over 35 days in the 17-163 nM range. The osteogenic differentiation of MSCs was enhanced by M1 macrophages at early time points. The late-stage mineralization was dominated by dexamethasone, with little contribution from the macrophages. Besides confirming BMP-2 whose secretion was promoted by both dexamethasone and M1 macrophages as a soluble mediator for enhanced osteogenesis, IL-6 was found to be a possible new soluble factor that mediated osteogenesis in macrophage-MSC co-cultures. The phenotype switching from M1 to M2 was drastically enhanced by the scaffold-released dexamethasone but only marginally by the co-cultured MSCs. Our results offer new insight into macrophage-MSC crosstalk and demonstrate the potential of using drug-release scaffolds to both modulate inflammation and enhance bone regeneration.

Mesenchymal Stromal Cell-Derived MMP14 Facilitates Leukemia Progression and Chemotherapy Resistance

Abstract i ntroduction:Acute Myeloid Leukemia (AML) stands as a menacing hematological malignancy, characterized by the aberrant proliferation and development of myeloid cells, entailing formidable therapeutic complexities. Matrix metalloproteinases (MMP) represent pivotal enzymes that dismantle the extracellular matrix by disintegrating tissue barriers. Among these, MMP14 assumes significance as it governs the invasion and metastasis of cancer cells, exhibiting heightened expression in solid tumors, including prostate cancer, colon cancer, pancreatic cancer, breast cancer, and skin cancer, thereby endorsing the invasiveness and metastatic potential of angiogenic inflammatory cancer cells. Nonetheless, the effect of abnormal expression of MMP14 derived from mesenchymal stem cells (MSCs) in AML patients upon the bone marrow microenvironment and AML pathogenesis, along with its underlying mechanism, remains unexplored. Methods: AML-MSCs were extracted from bone marrow samples of AML patients through whole bone marrow adherent culture. Lentiviral vectors carrying cDNA of the MMP14 gene and empty lentiviral vectors were meticulously constructed and subsequently introduced into AML-MSCs. Brdu and CFU-F assayed the proliferative capacity of knockdown and control MSCs, and osteogenic induced differentiation was performed in vitro on both groups of MSCs.AML cell lines Kasumi-1 and Molm-13 were subjected to co-culture with AML-MSCs to evaluate the migratory and invasive capabilities of AML cell lines , while the proliferative activity of AML cell lines was assessed via BrdU. Following co-cultivation, AML cell lines were exposed to 1μM cytarabine for 48 hours, and cell viability was gauged using flow cytometry. Moreover, CD34+ cells were isolated from leukemia patients' bone marrow using magnetic beads and co-cultivated with the aforementioned MSC groups to explore the impact of MMP14 derived from MSCs on the resistance of leukemia stem cells to cytarabine. Results: Knockdown of MMP14 reduces MSC proliferation and colony-forming ability while also impeding MSC osteogenic differentiation. AML cell lines subjected to co-culture with the MSC knockdown group exhibited a remarkable reduction in both invasion and migration abilities. The BrdU assay demonstrated a significant decrease in the proliferation rate of AML cell lines within the MMP14 knockdown group. Moreover, following 48 hours of cytarabine administration, flow cytometric analysis revealed a lower proportion of viable cells in the knockdown group when compared to the control group. The same trend was observed in primary cells, as evidenced by a decreased percentage of proliferating CD34+ cells within the knockdown group, along with a reduced proportion of viable cells following cytarabine treatment. Conclusion: The MMP14 derived from MSCs fosters AML progression and confers chemoresistance. Our study provides valuable insights into the significance of MMP14 in AML drug resistance and underscores its potential as a promising preclinical target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

3D printed PLGA/MgO/PDA composite scaffold by low-temperature deposition manufacturing for bone tissue engineering applications

IntroductionBones are easily damaged. Biomimetic scaffolds are involved in tissue engineering. This study explored polydopamine (PDA)-coated poly lactic-co-glycolic acid (PLGA)-magnesium oxide (MgO) scaffold properties and its effects on bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation. MethodsPLGA/MgO scaffolds were prepared by low-temperature 3D printing technology and PDA coatings were prepared by immersion method. Scaffold structure was observed by scanning electron microscopy with an energy dispersive spectrometer (SEM-EDS), fourier transform infrared spectrometer (FTIR). Scaffold hydrophilicity, compressive/elastic modulus, and degradation rates were analyzed by water contact angle measurement, mechanical tests, and simulated-body fluid immersion. Rat BMSCs were cultured in scaffold extract. Cell activity on days 1, 3, and 7 was detected by MTT. Cells were induced by osteogenic differentiation, followed by evaluation of alkaline phosphatase (ALP) activity on days 3, 7, and 14 of induction and Osteocalcin, Osteocalcin, and Collagen I expressions. ResultsThe prepared PLGA/MgO scaffolds had dense microparticles. With the increase of MgO contents, the hydrophilicity was enhanced, scaffold degradation rate was accelerated, magnesium ion release rate and scaffold extract pH value were increased, and cytotoxicity was less when magnesium mass ratio was less than 10%. Compared with other scaffolds, compressive and elastic modulus of PLGA/MgO (10%) scaffolds were increased; BMSCs incubated with PLGA/MgO (10%) scaffold extract had higher ALP activity and Osteocalcin, Osteopontin, and Collagen I expressions. PDA coating was prepared in PLGA/MgO (10%) scaffolds and the mechanical properties were not affected. PLGA/MgO (10%)/PDA scaffolds had better hydrophilicity and biocompatibility and promoted BMSC osteogenic differentiation. ConclusionLow-temperature 3D printing PLGA/MgO (10%)/PDA scaffolds had good hydrophilicity and biocompatibility, and were conducive to BMSC osteogenic differentiation.

A novel degradable PCL/PLLA strapping band for internal fixation of fracture

Early fracture fixation is the critical factor in fracture healing. Common internal fracture implants are made of metallic materials, which often affects the imaging quality of CT and MRI. Most patients will choose secondary surgery to remove the internal fixation implants, which causes secondary damage to them. The development of new degradable internal fracture implants has attracted more and more attention from orthopedic surgeons and researchers. Based on these problems, we improved the various properties of medical grade polycaprolactone (PCL) by adding poly(L-lactide) (PLLA). We produced PCL/PLLA strapping bands with different mass ratios by injection molding. We compared the mechanical properties, degradation properties, cell biocompatibility, bone marrow mesenchymal stem cells (BMSCs) adhesion, proliferation, osteogenic differentiation and fracture fixation effect of these strapping bands. The results showed that the tensile strength and yield force of the strapping bands increased with the increase of the content of PLLA. The addition of PLLA could significantly improve the mechanical strength in the early stage and accelerate the degradation rate of the strapping band. PCL/PLLA (80/20) strapping band had no significant cytotoxicity toward rBMSCs and could promote osteogenic differentiation of rBMSCs. The strapping band could ensure femoral fracture healing of beagles in 3 months and didn’t cause damage to the surrounding tissues and main organs. This study will provide some new insights into the biodegradable products of PCL/PLLA blends for internal fixation of fracture.Graphical We produced novel degradable PCL/PLLA strapping bands with different mass ratios by injection molding. We tested the biological safety of the prepared internal fixation strapping bands for fracture, such as cell experiment in vitro and animal experiment, and studied the degradation behavior in vitro. The strapping bands could ensure femoral fracture healing of beagles. This study will provide some new insights into the biodegradable products of PCL/PLLA blends for internal fixation of fracture. A Immunofluorescence staining of rBMSCs (live cells: green; dead cells: red). B Young’s modulus change curve during strapping bands degradation. C The implantation process of strapping bands. D Micro-CT images of the beagle’s fracture recovery after the operation.

HuR-mediated nucleocytoplasmic translocation of HOTAIR relieves its inhibition of osteogenic differentiation and promotes bone formation

Bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoblast function play critical roles in bone formation, which is a highly regulated process. Long noncoding RNAs (lncRNAs) perform diverse functions in a variety of biological processes, including BMSC osteogenic differentiation. Although several studies have reported that HOX transcript antisense RNA (HOTAIR) is involved in BMSC osteogenic differentiation, its effect on bone formation in vivo remains unclear. Here, by constructing transgenic mice with BMSC (Prx1-HOTAIR)- and osteoblast (Bglap-HOTAIR)-specific overexpression of HOTAIR, we found that Prx1-HOTAIR and Bglap-HOTAIR transgenic mice show different bone phenotypes in vivo. Specifically, Prx1-HOTAIR mice showed delayed bone formation, while Bglap-HOTAIR mice showed increased bone formation. HOTAIR inhibits BMSC osteogenic differentiation but promotes osteoblast function in vitro. Furthermore, we identified that HOTAIR is mainly located in the nucleus of BMSCs and in the cytoplasm of osteoblasts. HOTAIR displays a nucleocytoplasmic translocation pattern during BMSC osteogenic differentiation. We first identified that the RNA-binding protein human antigen R (HuR) is responsible for HOTAIR nucleocytoplasmic translocation. HOTAIR is essential for osteoblast function, and cytoplasmic HOTAIR binds to miR-214 and acts as a ceRNA to increase Atf4 protein levels and osteoblast function. Bglap-HOTAIR mice, but not Prx1-HOTAIR mice, showed alleviation of bone loss induced by unloading. This study reveals the importance of temporal and spatial regulation of HOTAIR in BMSC osteogenic differentiation and bone formation, which provides new insights into precise regulation as a target for bone loss.