Osteogenesis Of Adipose-derived Mesenchymal Stem Cells Research Articles

TRAF6 promotes osteogenesis in ADSCs through Raf-Erk-Merk-Hif1-a pathway

ABSTRACT Critical-size defects (CSDs) are challenging oral clinical issues that need to be solved. Adipose-derived mesenchymal stem cells (ADSCs) and gene therapy offer a new target to solve these issues. Consequently, ADSCs attract more and more attention because of advantages such as easy obtainability and no ethical concerns. TNF receptor-associated factor 6 (TRAF6) is a significant binding protein both of tumour necrosis factor superfamily and of the toll/interleukin-1 receptor superfamily. Evidence is accumulating that TRAF6 inhibited osteoclast formation and promoted the proliferation of multiple myeloma cell lines and bone resorption. Here, we reported that overexpression of TRAF6 enhanced the proliferation, migration and osteogenesis of ADSCs through Raf-Erk-Merk-Hif1a pathway. Cell sheet of ADSCs combined with TRAF6 accelerated the healing of CSDs. In a word, TRAF6 enhanced osteogenesis, migration and proliferation through Raf-Erk-Merk-Hif1a pathway.

Adipose-derived stem cells with miR-150-5p inhibition laden in hydroxyapatite/tricalcium phosphate ceramic powders promote osteogenesis via regulating Notch3 and activating FAK/ERK and RhoA

Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stromal cells and play huge role in forming and repairing bone tissues. Emerging evidence shows that MicroRNAs (miRNAs) are involved in ADSCs differentiation. Here, we explored the role of miR-150-5p and its related mechanisms in ADSCs osteogenesis. Real-time PCR was used to determine miR-150-5p expression during ADSCs osteogenesis. miR-150-5p inhibitors, miR-150-5p ADV or short hairpin RNA (shRNA) of Notch3 were transfected to ADSCs for analyzing the effects on osteogenesis. The mixture of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders and transfected ADSCs was implanted into BALB/C nude mice. Micro-CT and histological methods were performed to evaluate the new bone formation. Compared with negative control (NC) and miR-150-5p overexpression, inhibition of miR-150-5p increased ADSCs osteogenesis by regulating Notch3. MiR-150-5p overexpression decreased the expression of pFAK, pERK1/2, and RhoA, while these were up-regulated when miR-150-5p was inhibited, or notch3 was silenced. Furthermore, miR-150-5p inhibition partially reversed the suppression effect of notch3 knockdown on osteogenesis in vitro and in vivo. This study demonstrated the critical function of miR-150-5p during osteogenesis. The combination of ADSCs with miR-150-5p inhibition and HA/TCP might be a promising strategy for bone damage repair. Statement of significanceOsteoporosis is a common chronic metabolic bone disease in humans. Bone tissue engineering based on mesenchymal stem cells, biomaterials, and growth factors, provides a promising way to treat osteoporosis and bone defects. ADSCs commonly differentiate into adipose cells, they can also differentiate into osteogenic cell lineages. Nucleic acids and protein have usually been considered as regulators of ADSCs osteogenic differentiation. In the current study, we demonstrated the combination of ADSCs with miR-150-5p inhibition and hydroxyapatite/tricalcium phosphate ceramic powders enhanced bone regeneration. Furthermore, miR-150-5p/Notch3 axis regulating osteogenesis via the FAK/ERK1/2 and RhoA pathway was assessed. The current study showed the application of ADSCs in bone regeneration might be a promising strategy for osteoporosis and bone damage repairing.

CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin \u03b15 expression

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.

Hydrostatic pressure induces osteogenic differentiation of adipose-derived mesenchymal stem cells through increasing lncRNA-PAGBC.

Induced osteogenesis of adipose-derived mesenchymal stem cells (AMSCs) has been used to facilitate bone regeneration. Specifically, hydrostatic pressure (HP) has been implicated as a key regulator of AMSC differentiation, whereas the mechanisms that underlie the effects of HP on osteogenesis of AMSCs are not fully understood. Long noncoding RNAs (lncRNAs) are emerging regulators for osteogenic differentiation from AMSCs. In the current study, we found that lncRNA-PAGBC was a specific lncRNA that significantly upregulated during osteogenic differentiation of AMSCs based on published database. HP increased lncRNA-PAGBC, which is a competitive endogenous RNA (ceRNA) that binds to the osteogenesis-inhibitory microRNA, miR-133b, to regulate osteogenic differentiation of AMSCs. Moreover, a key osteogenesis-trigger gene, runt-related transcription factor 2 (RUNX2), was identified as a target gene for miR-133b. Suppression of RUNX2 by miR-133b caused impaired osteogenic differentiation of AMSCs. Furthermore, lncRNA-PAGBC overexpression upregulated, whereas lncRNA-PAGBC silencing decreased the expression of RUNX2 through miR-133b. Together, these data suggest that HP induces osteogenic differentiation of AMSCs through increasing lncRNA-PAGBC.

Knockdown of MiR-140-5 promotes osteogenesis of adipose-derived mesenchymal stem cells by targeting TLR4 and BMP2 and promoting fracture healing in the atrophic nonunion rat model.

This study aims to assess the effect and mechanism of genetically modified adipose-derived mesenchymal stem cells (ASCs) with recombinant lentiviruses mediated knockdown of miR-140-5p in ASCs' osteogenesis in vitro and atrophic nonunion rat model. This study included 36 male adult Sprague-Dawley (SD) rats weighing 400 g to 450 g from the experimental animal facility of our university. Approval was obtained from the University Animal Care Committee before the study. Rats' ASCs were prepared and genetically modified with lentivirus (Lv)-empty (NC) or Lv-miR-140-5p-TuD (inhibitors). After that, the expressions of RUNX2 and osteocalcin (OCN) were detected in the ASCs. To confirm the mechanisms of miR-140-5p in ASCs, we predicted the target genes by bioinformatics analysis and then the target genes were verified by luciferase reporting assay. The artificial atrophic nonunion was created in the rat's femoral bone. Animals were randomly divided into three groups according to the material implanted into bone defects space: AT scaffolds (AT group, n=12), AT scaffold with Lv-NC modified (AT+ASCs+Lv-NC group, n=12), AT scaffold with the Lv-miR-140-5p-TuD modified ASCs (AT+ASCs+Lv-miR-140-5p-TuD group, n=12). After four weeks, the rats were euthanized for the following radiographic examination, histologic study and biomechanical testing. MiR-140-5p was down-regulated during osteogenic differentiation of ASCs, and inhibition of MiR-140-5p promoted osteogenesis of ASCs in vitro. Inhibition of MiR-140-5p promoted osteogenesis of ASCs and enhanced fracture in the atrophic nonunion rat model: AT+ASCs+Lv-NC group, AT+ASCs+Lv-miR-140-5p-TuD group resulted in a better bone formation and higher BMD and BMC than AT group, while excellent bone formation and the highest BMD and BMC were observed in AT+ASCs+Lv-miR-140-5p-TuD group. Both AT+ASCs+Lv-NC group and AT+ASCs+Lv-miR-140-5p-TuD group presented more mature characteristics in the micro-architecture than AT group, whereas AT+ASCs+Lv-miR-140-5p-TuD group presented the highest BV/TV, Tb.Th and Tb.N as well as the lowest Tb.Sp. The peak load of the operated femur increased by 94.43% AT+ASCs+Lv-miR-140-5p-TuD group, 50.68% in AT+ASCs+Lv-NC group compared to the control AT group, respectively. The result of luciferase reporting assay showed that miR-140-5p could directly target TLR4 and BMP2. This study demonstrates that lentiviruses-mediated knockdown of miR-140-5p can significantly promote osteogenesis of ACSs by directly regulating its' target genes, TLR4 and BMP2, and that combined adipose scaffold with genetically modified ASCs can significantly enhance fracture-healing and bone formation in the atrophic nonunion rat model.

Three-Dimensional Graphene-RGD Peptide Nanoisland Composites That Enhance the Osteogenesis of Human Adipose-Derived Mesenchymal Stem Cells.

Graphene derivatives have immense potential in stem cell research. Here, we report a three-dimensional graphene/arginine-glycine-aspartic acid (RGD) peptide nanoisland composite effective in guiding the osteogenesis of human adipose-derived mesenchymal stem cells (ADSCs). Amine-modified silica nanoparticles (SiNPs) were uniformly coated onto an indium tin oxide electrode (ITO), followed by graphene oxide (GO) encapsulation and electrochemical deposition of gold nanoparticles. A RGD–MAP–C peptide, with a triple-branched repeating RGD sequence and a terminal cysteine, was self-assembled onto the gold nanoparticles, generating the final three-dimensional graphene–RGD peptide nanoisland composite. We generated substrates with various gold nanoparticle–RGD peptide cluster densities, and found that the platform with the maximal number of clusters was most suitable for ADSC adhesion and spreading. Remarkably, the same platform was also highly efficient at guiding ADSC osteogenesis compared with other substrates, based on gene expression (alkaline phosphatase (ALP), runt-related transcription factor 2), enzyme activity (ALP), and calcium deposition. ADSCs induced to differentiate into osteoblasts showed higher calcium accumulations after 14–21 days than when grown on typical GO-SiNP complexes, suggesting that the platform can accelerate ADSC osteoblastic differentiation. The results demonstrate that a three-dimensional graphene–RGD peptide nanoisland composite can efficiently derive osteoblasts from mesenchymal stem cells.

Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture.

ObjectivesAdipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs.MethodsHuman ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS).ResultsHPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages.ConclusionHPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs.Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414–422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1.

Effects of miR-146a on the osteogenesis of adipose-derived mesenchymal stem cells and bone regeneration

Increasing evidence has indicated that bone morphogenetic protein 2 (BMP2) coordinates with microRNAs (miRNAs) to form intracellular networks regulating mesenchymal stem cells (MSCs) osteogenesis. This study aimed to identify specific miRNAs in rat adipose-derived mesenchymal stem cells (ADSCs) during BMP2-induced osteogenesis, we selected the most significantly down-regulated miRNA, miR-146a, to systematically investigate its role in regulating osteogenesis and bone regeneration. Overexpressing miR-146a notably repressed ADSC osteogenesis, whereas knocking down miR-146a greatly promoted this process. Drosophila mothers against decapentaplegic protein 4 (SMAD4), an important co-activator in the BMP signaling pathway, was miR-146a’s direct target and miR-146a exerted its repressive effect on SMAD4 through interacting with 3′-untranslated region (3′-UTR) of SMAD4 mRNA. Furthermore, knocking down SMAD4 attenuated the ability of miR-146a inhibitor to promote ADSC osteogenesis. Next, transduced ADSCs were incorporated with poly(sebacoyl diglyceride) (PSeD) porous scaffolds for repairing critical-sized cranial defect, the treatment of miR-146a inhibitor greatly enhanced ADSC-mediated bone regeneration with higher expression levels of SMAD4, Runt-related transcription factor 2 (Runx2) and Osterix in newly formed bone. In summary, our study showed that miR-146a negatively regulates the osteogenesis and bone regeneration from ADSCs both in vitro and in vivo.

Enhanced Osteogenesis of ADSCs by the Synergistic Effect of Aligned Fibers Containing Collagen I.

The topographical features and material composition of scaffolds have a powerful influence on cell behaviors such as proliferation and differentiation. Here, scaffolds consisting of aligned fibers with incorporated bioactive collagen I were tested for their ability to enhance osteogenesis in vitro. Rat adipose-derived mesenchymal stem cells (ADSCs) were seeded on the scaffolds and their morphology, proliferation, and osteogenic differentiation were examined. Aligned scaffolds with collagen I showed the best osteogenic properties. Also, adhesion-related genes showed the higher expression on aligned scaffolds with collagen I. Our findings indicate that fiber alignment combined with incorporation of collagen I increases the capacity of electrospun scaffolds to induce enhanced and directed osteogenesis. Such scaffolds may, therefore, have potential for improving guided oral bone regeneration.