Osteogenesis Of Adipose-derived Stem Cells Research Articles

Preosteoclast plays a pathogenic role in syndesmophyte formation of ankylosing spondylitis through the secreted PDGFB — GRB2/ERK/RUNX2 pathway

ObjectivesAnkylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the sacroiliac joint and spine. However, the real mechanisms of immune cells acting on syndesmophyte formation in AS are not well identified. We aimed to find the key AS-associated cytokine and assess its pathogenic role in AS.MethodsA protein array with 1000 cytokines was performed in five AS patients with the first diagnosis and five age- and gender-matched healthy controls to discover the differentially expressed cytokines. The candidate differentially expressed cytokines were further quantified by multiplex protein quantitation (3 AS-associated cytokines and 3 PDGF-pathway cytokines) and ELISA (PDGFB) in independent samples (a total of 140 AS patients vs 140 healthy controls). The effects of PDGFB, the candidate cytokine, were examined by using adipose-derived stem cells (ADSCs) and human fetal osteoblast cell line (hFOB1.19) as in vitro mesenchymal cell and preosteoblast models, respectively. Furthermore, whole-transcriptome sequencing and enrichment of phosphorylated peptides were performed by using cell models to explore the underlying mechanisms of PDGFB. The xCELLigence system was applied to examine the proliferation, chemotaxis, and migration abilities of PDGFB-stimulated or PDGFB-unstimulated cells.ResultsThe PDGF pathway was observed to have abnormal expression in the protein array, and PDGFB expression was further found to be up-regulated in 140 Chinese AS patients. Importantly, PDGFB expression was significantly correlated with BASFI (Pearson coefficient/p value = 0.62/6.70E − 8) and with the variance of the mSASSS score (mSASSS 2 years − baseline, Pearson coefficient/p value = 0.76/8.75E − 10). In AS patients, preosteoclasts secreted more PDGFB than the healthy controls (p value = 1.16E − 2), which could promote ADSCs osteogenesis and enhance collagen synthesis (COLI and COLIII) of osteoblasts (hFOB 1.19). In addition, PDGFB promoted the proliferation, chemotaxis, and migration of ADSCs. Mechanismly, in ADSCs, PDGFB stimulated ERK phosphorylation by upregulating GRB2 expression and then increased the expression of RUNX2 to promote osteoblastogenesis of ADSCs.ConclusionPDGFB stimulates the GRB2/ERK/RUNX2 pathway in ADSCs, promotes osteoblastogenesis of ADSCs, and enhances the extracellular matrix of osteoblasts, which may contribute to pathological bone formation in AS.

Wnt10b regulates osteogenesis of adipose-derived stem cells through Wnt/β-catenin signalling pathway in osteoporosis.

Our previous finding revealed that the Wnt10b RNA expression of osteoporotic adipose-derived stem cells (OP-ASCs) with impaired osteogenic capacity was significantly reduced than that of ASCs. There are no ideas that the relationship between the OP-ASCs' impaired osteogenic potential and Wnt10b expression. This study aimed to indicate the potential molecular mechanisms and functional role of Wnt10b in OP-ASCs, as well as to investigate a potential application to reverse the OP-ASCs' impaired osteogenic differentiation potential. The OP-ASCs and ASCs were harvested from the inguinal fat of osteoporosis (OP) mice with bilateral ovariectomy (OVX) and normal mice. qPCR and WB were used to detect the different levels of the expression of the Wnt10b RNA in both OP-ASCs and ASCs. Lentiviral-mediated regulation of Wnt10b expression was employed for OP-ASCs, and the detection of the expression levels of key molecules in the Wnt signalling pathway and key osteogenic factors was performed through qPCR and WB in vitro experiments. The capacity of OP-ASCs to osteogenesis was determined using alizarin red staining. Lastly, the repair effect of the BCP scaffolds incorporating modified OP-ASCs on the critical-sized calvarial defects (CSCDs) in OP mice was scanned and detected by micro-computed tomography, haematoxylin and eosin staining, Masson's trichrome staining and immunohistochemistry. First, we discovered that both the RNA and protein expression levels of Wnt10b were significantly lower in OP-ASCs than that in ASCs. In vitro experiments, upregulation of Wnt10b could activate the Wnt signalling pathway, and increase expression of β-catenin, Lef1, Runx2 and osteopontin (Opn), thereby enhancing the osteogenic ability of OP-ASCs. In addition, the OP-ASCs with Wnt10b-overexpressing could promote the repair of CSCD in osteoporotic mice with increasing new bone volume, bone mineral density, and increased expression of Opn in new bone in vivo. Taken together, overexpression of Wnt10b could partially facilitate the differentiation of OP-ASCs towards osteogenesis and accelerated the healing of bone defects by activating the Wnt/β-catenin signalling pathway in vitro and in vivo experiments. This study confirmed the important role of Wnt10b in regulating the osteogenic differentiation capability of OP-ASCs and indicated Wnt10b could be a potential therapeutic target for reversing the impaired osteogenic capabilities of OP-ASCs to therapy bone defects of OP patients.

Lactoferrin Mediates Enhanced Osteogenesis of Adipose-Derived Stem Cells: Innovative Molecular and Cellular Therapy for Bone Repair.

A prospective source of stem cells for bone tissue engineering is adipose-derived stem cells (ADSCs), and BMP-2 has been proven to be highly effective in promoting the osteogenic differentiation of stem cells. Rarely has research been conducted on the impact of lactoferrin (LF) on ADSCs' osteogenic differentiation. As such, in this study, we examined the effects of LF and BMP-2 to assess the ability of LF to stimulate ADSCs' osteogenic differentiation. The osteogenic medium was supplemented with the LF at the following concentrations to culture ADSCs: 0, 10, 20, 50, 100, and 500 μg/mL. The Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation of ADSCs. Calcium deposition, alkaline phosphatase (ALP) staining, real-time polymerase chain reaction (RT-PCR), and an ALP activity assay were used to establish osteogenic differentiation. RNA sequencing analysis was carried out to investigate the mechanism of LF boosting the osteogenic development of ADSCs. In the concentration range of 0-100 μg/mL, LF concentration-dependently increased the proliferative vitality and osteogenic differentiation of ADSCs. At a dose of 500 μg/mL, LF sped up and enhanced differentiation, but inhibited ADSCs from proliferating. LF (100 and 500 μg/mL) produced more substantial osteoinductive effects than BMP-2. The PI3 kinase/AKT (PI3K/AKT) and IGF-R1 signaling pathways were significantly activated in LF-treated ADSCs. The in vitro study results showed that LF could effectively promote osteogenic differentiation of ADSCs by activating the PI3K/AKT and IGF-R1 pathways. In our in vitro investigation, an LF concentration of 100 μg/mL was optimal for osteoinduction and proliferation. Our study suggests that LF is an attractive alternative to BMP-2 in bone tissue engineering. As a bioactive molecule capable of inducing adipose stem cells to form osteoblasts, LF is expected to be clinically used in combination with biomaterials as an innovative molecular and cellular therapy to promote bone repair.

Irisin Promotes Osteogenesis by Modulating Oxidative Stress and Mitophagy through SIRT3 Signaling under Diabetic Conditions.

Advanced glycation end products (AGEs) accumulate in the bone tissue of patients with diabetes mellitus, resulting in oxidative stress, poor bone healing, or regeneration. Irisin, a novel exercise-induced myokine, is involved in the regulation of bone metabolism. However, the effects of irisin on adipose-derived stem cell (ASC) osteogenic differentiation and bone healing under diabetic conditions remain poorly understood. ASCs were obtained from inguinal fat of Sprague-Dawley rats and treated with different concentrations of AGEs and irisin. Cell proliferation, apoptosis, and osteogenic differentiation abilities of ASCs were detected. To explore the regulatory role of sirtuin 3 (SIRT3), ASCs were transfected with lentivirus-mediated SIRT3 overexpression or knockdown vectors. Next, we investigated mitochondrial functions, mitophagy, and mitochondrial biogenesis in different groups. Moreover, SOD2 acetylation and potential signaling pathways were assessed. Additionally, a diabetic rat model was used to evaluate the effect of irisin on bone healing in calvarial critical-sized defects (CSDs) in vivo. Our results showed that irisin incubation mitigated the inhibitory effects of AGEs on ASCs by increasing cell viability and promoting osteogenesis. Moreover, irisin modulated mitochondrial membrane potential, intracellular ROS levels, mitochondrial O2·− status, ATP generation, complex I and IV activities, mitophagy, and mitochondrial biogenesis via a SIRT3-mediated pathway under AGEs exposure. Furthermore, in calvarial CSDs of diabetic rats, transplantation of gels encapsulating irisin-pretreated ASCs along with irisin largely enhanced bone healing. These findings suggest that irisin attenuates AGE-induced ASC dysfunction through SIRT3-mediated maintenance of oxidative stress homeostasis and regulation of mitophagy and mitochondrial biogenesis. Thus, our studies shed new light on the role of irisin in promoting the ASC osteogenesis and targeting SIRT3 as a novel therapeutic intervention strategy for bone regeneration under diabetic conditions.

Enhancement of Osteoblast Function through Extracellular Vesicles Derived from Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cell that is investigated in bone tissue engineering (BTE). Osteoblasts are the main cells responsible for bone formation in vivo and directing ADSCs to form osteoblasts through osteogenesis is a research topic in BTE. In addition to the osteogenesis of ADSCs into osteoblasts, the crosstalk of ADSCs with osteoblasts through the secretion of extracellular vesicles (EVs) may also contribute to bone formation in ADSC-based BTE. We investigated the effect of ADSC-secreted EVs (ADSC-EVs) on osteoblast function. ADSC-EVs (size ≤ 1000 nm) were isolated from the culture supernatant of ADSCs through ultracentrifugation. The ADSC-EVs were observed to be spherical under a transmission electron microscope. The ADSC-EVs were positive for CD9, CD81, and Alix, but β-actin was not detected. ADSC-EV treatment did not change survival but did increase osteoblast proliferation and activity. The 48 most abundant known microRNAs (miRNAs) identified within the ADSC-EVs were selected and then subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The GO analysis revealed that these miRNAs are highly relevant to skeletal system morphogenesis and bone development. The KEGG analysis indicated that these miRNAs may regulate osteoblast function through autophagy or the mitogen-activated protein kinase or Ras-related protein 1 signaling pathway. These results suggest that ADSC-EVs enhance osteoblast function and can contribute to bone regeneration in ADSC-based BTE.

Knockdown of PDX1 enhances the osteogenic differentiation of ADSCs partly via activation of the PI3K/Akt signaling pathway

BackgroundOsteoporosis (OP) is a systemic bone disease manifested as low bone mass, destruction of bone microstructure, increased bone fragility and fracture risk. The purpose of this study was to explore the role and mechanism of PDX1 for osteogenic differentiation of adipose derived stem cells (ADSCs).MethodsGSE37329 dataset was retrieved from NCBI Gene Expression Omnibus (GEO) database and performed bioinformatic analyses. ADSCs were incubated with normal medium, osteogenic induction medium (OIM) and OIM+si-PDX1. Then, alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS) were performed to assess the role of PDX1 for osteogenesis of ADSCs. PI3K inhibitor, LY294002 was then added to further explore the mechanism of PDX1 for osteogenic differentiation of ADSCs. Western blot assay was used to assess the osteogenic-related markers. Graphpad software was used to perform statistically analysis.ResultsA total of 285 DEGs were obtained from analysis of the dataset GSE37329, of which 145 were upregulated and 140 were downregulated genes. These differentially expressed genes mainly enriched in cell differentiation and PI3K/Akt signaling pathway. Moreover, PDX1 was decreased in osteogenic induced ADSCs. Knockdown of PDX1 significantly increased osteogenic differentiation capacity and p-PI3K and p-Akt protein levels. Administration with LY294002 could partially reversed the promotion effects of si-PDX1.ConclusionIn conclusion, knockdown of PDX1 promotes osteogenic differentiation of ADSCs through the PI3K/Akt signaling pathway.

LncRNA-AK137033 inhibits the osteogenic potential of adipose-derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation.

ObjectivesBone tissue engineering based on adipose‐derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON‐ASCs), osteogenic potential of DOP‐ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP‐ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.Materials and methodsAn animal model of DOP was established in mice. CON‐ASCs and DOP‐ASCs were isolated from CON and DOP mice, respectively. AK137033 small interfering RNA (SiRNA) and an AK137033 overexpression plasmid were used to regulate the expression of AK137033 in CON‐ASCs and DOP‐ASCs in vitro. Lentiviruses that carried shRNA‐AK137033 or AK137033 cDNA were used to knockdown or overexpress AK137033, respectively, in CON‐ASCs and DOP‐ASCs in vivo. Hematoxylin and eosin (H&E), Masson's, alizarin red, and alkaline phosphatase (ALP) staining, micro‐computed tomography (Micro‐CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite‐specific PCR (BSP) were used to analyze the functional changes of ASCs.ResultsThe DOP mouse model was established successfully. Compared with CON‐ASCs, AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP‐ASCs. In vitro experiments showed that AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON‐ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of AK137033 in DOP‐ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.ConclusionsLncRNA‐AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients.

3D-printed bioresorbable poly(lactic-co-glycolic acid) and quantum-dot nanocomposites: Scaffolds for enhanced bone mineralization and inbuilt co-monitoring.

Multifunctional 3D-printed nanocomposites based on poly(lactic-co-glycolic acid), that is, PLGA (RESOMER® LG857S) were developed for simultaneous monitoring of cells and scaffold as a function of time and spectral responses. These were achieved by impregnating carbon quantum dots (CQDs) on PLGA using melt-blending, plasticating extrusion, and 3D-printing. The nanocomposites enabled enhanced bio-affinity and cellular interactions for bone tissue engineering (TE). PLGA (control) and PLGA-CQD scaffolds were used for growing human adipose-derived-stem-cells (ADSCs) and tested for cell biocompatibility, cellular adhesion, growth, and osteogenesis. CQDs were found to enhance the hydrophilicity of nanocomposites and promote cellular nesting. MTS assays confirmed that CQDs on PLGA act as cell anchoring sites, thereby enhancing seeding efficiency and cell proliferation. Alkaline phosphate tests showed increased osteogenesis and Alizarin assays confirmed enhanced bone mineralization on PLGA-CQD. The qPCR tests based on selected mRNA expressions showed that the incorporation of CQDs significantly enhanced osteogenesis of ADSCs during all three phases of cell differentiation. The intrinsic luminescence of the composites allowed label-free monitoring of cell proliferation and bone mineralization on the scaffolds. Thus, the CQDs facilitated significant enhancements in composite processability with customized fabrication of 3D printed scaffolds, bone tissue osteoconductivity, and monitoring of cell-scaffold activities, offering multifunctional benefits for bone TE.

Association of Fpr1 gene expression with osteogenesis and adipogenesis of adipose derived stem cells

Formyl peptide receptors (Fprs) play fundamental roles in multiple cell functions including promotion of osteogenesis and bone fracture healing. In this study, the role of Fpr1 gene in osteogenic and adipogenic differentiation of adipose derived stem cells (ADSCs) was investigated. Primary ADSCs (mADSCs) from either Fpr1 knockout (KO) or wild type (WT) mice and human ADSCs (hADSCs) were treated by osteogenic (OM) or adipogenic (AM) medium, with basal medium as control. Osteogenesis and adipogenesis were measured by histological and biochemical methods. In both hADSCs and mADSCs, Fpr1 gene expression, osteogenic gene expression, as well as mineralization were increased after osteogenic induction. The osteogenic capacity of KO ADSCs was remarkably reduced compared to WT ADSCs, with decreased levels of expression of osteogenic markers, alkaline phosphatase activity, and mineralization. In contrast, the adipogenesis of KO ADSCs was remarkably enhanced compared with WT ADSCs, forming more lipid droplets, and increasing expression of adipogenic markers PPARγ and aP2. Expression of the nuclear transcription factor Forkhead box protein O1 (FoxO1) was decreased in KO ADSCs, while OM and AM caused increase and decrease in FoxO1 expression, respectively. The current study revealed a correlation of Fpr1 gene expression with osteogenesis and adipogenesis of mADSCs, underlying a mechanism involving FoxO1. Our present research suggests that targeting Fpr1 might be a novel strategy to enhance osteogenesis of ADSCs.

Overexpression of lncRNA-NEF regulates the miR-155/PTEN axis to inhibit adipogenesis and promote osteogenesis.

Osteoporosis is characterized by osteopenia and bone tissue microstructure degradation. Adipose-derived stem cells (ADSCs) are multipotent adult stem cells that have the ability to yield mesenchymal stem cells and have the potential to undergo osteogenesis and bone regeneration. Therefore, ADSCs have the potential to treat osteoporosis, but the molecular mechanism of these cells in the process of osteogenesis and osteoclasts is still not clear. In the present study, we collected serum samples from 10 clinical osteoporosis patients to detect long noncoding RNA-neighboring enhancer of FOXA2 (lncRNA-NEF) and miR-155 expression levels. Half of these patients were senile and half were postmenopausal women, and nine of them have used steroids for a long time, in which ADSCs were cultured and induced to adipogenic and osteogenic differentiations. Quantitative real-time polymerase chain reaction was used to detect the expression of genes in ADSCs. Overexpression of lncRNA-NEF in ADSCs were undertaken to verify its regulatory function on cell osteogenic and adipogenic differentiations. A luciferase activity experiment was performed to determine the relationship between miR-155 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN). The level of lncRNA-NEF was downregulated, and miR-155 was upregulated, in serum samples from patients with clinical osteoporosis. LncRNA-NEF showed different expression levels in the induction of osteogenic or adipogenic differentiation, which increased during osteogenic induction and decreased during adipogenic induction. Overexpression of lncRNA-NEF or downregulation of miR-155 in ADSCs promoted osteogenic differentiation and inhibited adipogenesis progression. PTEN was the direct target of miR-155 and was involved in the regulation of osteogenic differentiation. Overexpression of lncRNA-NEF regulated the miR-155/PTEN axis to inhibit adipogenesis and promote osteogenesis in ADSCs.

Long noncoding RNA AC092155 facilitates osteogenic differentiation of adipose-derived stem cells through the miR-143-3p/STMN1 axis.

Numerous studies have demonstrated that long noncoding RNAs (lncRNAs) induce osteogenesis in adipose-derived stem cells (ADSCs). This study aimed to explore the role of lncRNAs AC092155 in promoting osteogenic differentiation of ADSCs. MicroRNA (miRNA) and lncRNA sequencing were performed in ADSCs that underwent normal or osteogenic induction. Differentially expressed miRNAs and lncRNAs were identified using R software. The relative expression levels of lncRNA AC092155, miR-143-3p, and STMN1 during the process of osteogenic induction were determined by real-time polymerase chain reaction (RT-PCR). ADSCs were then transfected with agomiR-143-3p and pcDNA3.1-sh-lncRNA AC092155. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were used to confirm the regulatory function of the lncRNA AC092155/miR-143-3p/STMN1 axis in osteogenic differentiation of ADSCs. lncRNA AC092155 was significantly upregulated in ADSCs following induction in the osteogenic medium. lncRNA AC092155 and STMN1 mimics increase the markers of osteogenic differentiation in the early and late phases, which was reflected in increased ALP activity as well as the higher deposition of calcium nodules. An miR-143-3p mimic showed the opposite effect. Luciferase reporter gene analysis demonstrated that lncRNA AC092155 directly targets miR-143-3p. Moreover, the lncRNA AC092155/miR-143-3p/STMN1 regulatory axis was found to activate the Wnt/β-catenin signaling pathway. lncRNA AC092155 contributes to the osteogenic differentiation of ADSCs. The lncRNA AC092155/miR-143-3p/STMN1 axis may be a new therapeutic target for bone-related diseases.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment.

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) was reported to promote both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis is initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM augments HAM-induced chondrogenesis but not osteogenesis of ADSCs. ADSCs were treated with SIM in a HAM (SIM plus HAM) by HA-coated wells or HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs was evaluated. SIM plus HAM increased chondrogenesis more than HAM or SIM alone, including cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and cartilaginous tissue formation (collagen type II and sulfated glycosaminoglycan). In contrast, SIM-induced osteogenesis in ADSCs was reduced in SIM plus HAM, including mRNA expression of osteogenic genes, osteocalcin and alkaline phosphatase (ALP), ALP activity and mineralization. SIM plus HAM also showed the most effective increases in the mRNA expression of BMP-2 and transcription factors of SOX-9 and RUNX-2 in ADSCs, while these effects were reversed by CD44 blockade. HAM suppressed the levels of JNK, p-JNK, P38 and p-P38 in ADSCs, and SIM plus HAM also decreased SIM-induced phosphorylated JNK and p38 levels. In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. The results from this study indicate that SIM may be an enhancer of HAM-initiated MSC-based chondrogenesis and avoid osteogenesis.

MiR-1249-5p regulates the osteogenic differentiation of ADSCs by targeting PDX1

BackgroundOsteoporosis (OP) is an age-related systemic bone disease. MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation. The purpose of this study was to explore the role and mechanism of miR-1249-5p for promoting osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsGSE74209 dataset was retrieved from NCBI Gene Expression Omnibus (GEO) database and performed bioinformatic analyses. OP tissue and healthy control tissues were obtained and used for RT-PCR analyses. ADSCs were incubated with miR-1249-5p mimic, inhibitor and corresponding negative control (NC), alkaline phosphatase (ALP) staining, and Alizarin Red Staining (ARS) were then performed to assess the role of miR-1249-5p for osteogenesis of ADSCs. Targetscan online website and dual-luciferase reporter assay were performed to verify that the 3′-UTR of PDX1 mRNA is a direct target of miR-1249-5p. RT-PCR and western blot were also performed to identify the mechanism of miR-1249-5p for osteogenesis of ADSCs.ResultsA total of 170 differentially expressed miRNAs were selected, among which, 75 miRNAs were downregulated and 95 miRNAs were upregulated. Moreover, miR-1249-5p was decreased in OP patients, while showed a gradual increase with the extension of induction time. miR-1249-5p mimic significantly increased osteogenic differentiation capacity and p-PI3K and p-Akt protein levels. Luciferase activity in ADSCs co-transfected of miR-1249-5p mimic with PDX1-WT reporter plasmids was remarkably decreased, but there was no obvious change in miR-1249-5p mimic with PDX1-MUT reporter plasmids co-transfection group. Overexpression PDX1 could partially reverse the promotion effects of miR-1249-5p on osteogenesis of ADSCs.ConclusionIn conclusion, miR-1249-5p promotes osteogenic differentiation of ADSCs by targeting PDX1 through the PI3K/Akt signaling pathway.

Temperature-activated PRP–cryogel for long-term osteogenesis of adipose-derived stem cells to promote bone repair

A cryogel was used to encapsulate PRP for the temperature activation of PRP and long-term release of growth factors, which could further promote the osteogenesis of costless ADSCs. The smart integration resulted in efficient bone repair.

Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/β-catenin axis.

ObjectivesIt is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose‐derived stem cells (ADSCs) will be probably promoted by exosomes.Materials and MethodsBy means of lentiviral transfection, miR‐130a‐3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis‐related genes, to verify the effect of miR‐130a‐3p on the osteogenic differentiation of ADSCs. CCK‐8 was used to detect the effect of miR‐130a‐3p on the proliferation of ADSCs. The target binding between miR‐130a‐3p and SIRT7 was verified by dual‐luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR‐130a‐3p was further verified by detecting osteogenic‐related genes and proteins and alkaline phosphatase activity.Results(a) Overexpression of miR‐130a‐3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR‐130a‐3p. (b) Our study further found that overexpression of miR‐130a‐3p leads to down‐regulation of SIRT7 expression with up‐regulation of Wnt signalling pathway‐associated protein. (c) Overexpression of miR‐130a‐3p inhibited proliferation of ADSCs, while knockdown promoted it.ConclusionsThe obtained findings indicate that exosomal miR‐130a‐3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/β‐catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.

Osteogenesis of adipose-derived stem cells from patients with glucose metabolism disorders

BackgroundAdipose derived stem cells (ADSCs) are clinically widely used somatic stem cells obtained from white adipose tissue. They are characterized by ability to differentiate e.g. into osteoblasts and might successfully regenerate bone tissue in fracture repair. However, the main problem of somatic stem cells is a documented influence of various diseases, drugs or age which can inhibit cells activity. Therefore, in the present study, we investigated the influence of insulin resistance (IR) and type 2 diabetes (T2D) on the proliferation and differentiation potential of ADSCs.MethodsThe fat from subcutaneous abdominal adipose tissue was acquired by lipoaspiration from 23 voluntary participants, divided into three groups: with diabetes type 2, with insulin resistance and control healthy donors. The proliferative potential was analyzed by cell cytotoxicity assays and by mRNA expression of genes connected with proliferation. Flow cytometry was done for identifying proteins characteristic for mesenchymal stem cells and an analysis of osteogenic differentiation potential based on the assessment of osteogenic markers by real time RT-qPCR, and the evaluation of calcium deposition were also performed.ResultsThe results showed that diabetes type 2 lowered the activity of ADSCs in proliferation assays and changed their phenotypical characteristics. Interestingly, we observed differences in the proliferation potential of ADSCs in patients with insulin resistance, which is often the first phase of diabetes, compared to the control. It might suggest that insulin resistance, early-stage T2D, alters the activity of cells. Moreover, expression of osteogenesis markers was higher in cells from T2D patients than in cells from patients with IR and control.ConclusionWe conclude that type 2 diabetes changes the activity of stem cells, and insulin resistance influences on the proliferation of ADSCs.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) is a lipid-lowering drug that also affects both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis can be initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM can augment the HAM-induced chondrogenesis but not osteogenesis of ADSCs and that this synergism can be applied for articular cartilage regeneration. ADSCs were treated with SIM in a HAM in HA-coated wells or an HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs and formation of cartilage were evaluated. Treatment with SIM in a HAM (SIM plus HAM) induced the expression of BMP-2 and transcription factors for chondrogenesis (SOX-9) and osteogenesis (RUNX-2) in ADSCs, and these effects were reversed by CD44 blockade. Furthermore, ADSCs exposed to SIM plus HAM exhibited cell aggregation, chondrogenic gene (collagen type II and aggrecan) expression and sulfated glycosaminoglycan formation. By contrast, SIM-induced osteogenesis in ADSCs was reduced in a HAM, as determined by measuring osteogenic gene (osteocalcin and alkaline phosphatase) expression and calcium deposition. These inhibitory effects were not altered by changes in the HA molecular weight (80~2000 kDa). In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. These results indicate that a HAM may be used as a chondroinductive agent to enhance SIM-induced MSC chondrogenesis but prevent osteogenesis.

CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.

Extracellular vesicles derived from human dental pulp stem cells promote osteogenesis of adipose-derived stem cells via the MAPK pathway

Recent studies have shown that co-culture systems play an important role in bone tissue engineering. In this study, human dental pulp stem cells (hDPSCs) were co-cultured with human adipose-derived stem cells (hADSCs), and osteoblastic phenotypes were found to be enhanced in co-cultures compared with monocultures of hDPSCs or hADSCs. Furthermore, GW4869, an inhibitor of extracellular vesicle (EV) formation, suppressed the mineralization of co-cultured cells. Studies indicate that the therapeutic potential of DPSCs is realized through paracrine action, in which EVs play an important role. To study their role, we successfully obtained and identified hDPSC-derived extracellular vesicles (hDPSC-EVs), and further investigated their effects on hADSCs and the underlying mechanism. hADSCs were stimulated with hDPSC-EVs, which were found to promote the migration and mineralization of hADSCs. Moreover, hDPSC-EVs promoted osteogenic differentiation by enhancing the phosphorylation of ERK 1/2 and JNK in hADSCs. To investigate the specific proteins in EVs that might play a role in hADSC osteogenic differentiation, we performed proteomic analysis of hDPSC-EVs. We determined the top 30 enriched pathways, which notably included the insulin signaling pathway. The number of genes enriched in the insulin signaling pathway was the largest, in addition to the “protein processing in endoplasmic reticulum” term. The MAPK cascade is a typical downstream pathway mediating insulin signaling. To further study the effects of hDPSC-EVs on maxillofacial bone regeneration, we used hDPSC-EVs as a cell-free biomaterial in a model of mandibular defects in rats. To assess the therapeutic potential of EVs, we analyzed their proteome. Animal experiments demonstrated that hDPSC-EVs promoted the regeneration of bone defects. Overall, these results highlight the potential of hDPSC-EVs to induce lineage specific differentiation of hADSCs. The results also indicated the importance of considering hDPSC-EVs as biomimetic materials for clinical translation of treatments for oral maxillofacial defects.

Promoting Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Altering the Expression of Exosomal miRNA.

Human adipose-derived stem cells (ADSCs) can release exosomes; however, their specific functions remain elusive. In this study, we verified that exosomes derived from osteogenically differentiated ADSCs can promote osteogenic differentiation of ADSCs. Furthermore, in order to investigate the importance of exosomal microRNAs (miRNAs) in osteogenic differentiation of ADSCs, we used microarray assays to analyze the expression profiles of exosomal miRNAs derived from undifferentiated as well as osteogenically differentiated ADSCs; 201 miRNAs were upregulated and 33 miRNAs were downregulated between the two types of exosomes. Additionally, bioinformatic analyses, which included gene ontology analyses, pathway analysis, and miRNA-mRNA-network investigations, were performed. The results of these analyses revealed that the differentially expressed exosomal miRNAs participate in multiple biological processes, such as gene expression, synthesis of biomolecules, cell development, differentiation, and signal transduction, among others. Moreover, we found that these differentially expressed exosomal miRNAs connect osteogenic differentiation to processes such as axon guidance, MAPK signaling, and Wnt signaling. To the best of our knowledge, this is the first study to identify and characterize exosomal miRNAs derived from osteogenically differentiated ADSCs. This study confirms that alterations in the expression of exosomal miRNAs can promote osteogenic differentiation of ADSCs, which also provides the foundation for further research on the regulatory functions of exosomal miRNAs in the context of ADSC osteogenesis.