Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor.
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ABSTRACT:
SUBMITTER: Yamada S
PROVIDER: S-EPMC8243246 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:INTRODUCTION:Articular cartilage (AC) is a viscoelastic tissue with a limited regenerative capability because of the lack of vasculature. Mechanical stimulation contributes to the homeostasis of functional AC since it promotes the delivery of nutrients, cytokines and growth factors between the distant chondrocytes. We hypothesized that biomechanical stimulation might enhance mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from neighboring niches as the bone marrow. AIM:This study aimed to introduce a bioreactor for inducing mobilization of MSCs from one compartment to another above by mechanical stimulation in vitro. METHODS:A novel mechanical system for evaluating mobilization of cells in a 3D context in vitro is presented. The system consists of a compression bioreactor able to induce loading on hydrogel-based scaffolds, custom-made software for settings management and data recording, and image based biological evaluation. Intermittent load was applied under a periodic regime with frequency of 0.3 Hz and unload phases of 10 seconds each 180 cycles over 24 hours. The mechanical stimulation acted on an alginate scaffold and a cell reservoir containing MSCs below it. The dynamic compression exerted amplitude of 200 ?m as 10% strain regarding the original height of the scaffold. RESULTS:The bioreactor was able to stimulate the scaffolds and the cells for 24.4 (±1.7) hours, exerting compression with vertical displacements of 185.8 (±17.8) ?m and a force-amplitude of 1.87 (±1.37; min 0.31, max 4.42) N. Our results suggest that continuous mechanical stimulation hampered the viability of the cells located at the cell reservoir when comparing to intermittent mechanical stimulation (34.4 ± 2.0% vs. 66.8 ± 5.9%, respectively). Functionalizing alginate scaffolds with laminin-521 (LN521) seemed to enhance the mobilization of cells from 48 (±21) to 194 (±39) cells/mm3 after applying intermittent mechanical loading. CONCLUSION:The bioreactor presented here was able to provide mechanical stimulation that seemed to induce the mobilization of MSCs into LN521-alginate scaffolds under an intermittent loading regime.
| S-EPMC6953860 | biostudies-literature
Project description:Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation.
| S-EPMC4917651 | biostudies-literature
Project description:Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal chondral defects in situ by opening small holes in the subchondral bone. However, the original function of AC is not obtained yet. We hypothesize that mechanical stimulation can mobilize mesenchymal stromal cells (MSCs) from BM reservoirs upon MF of the subchondral bone. Thus, the aim of this study was to compare the counts of mobilized human BM-MSCs (hBM-MSCs) in alginate-laminin (alginate-Ln) or collagen-I (col-I) scaffolds upon intermittent mechanical loading. The mechanical set up within an established bioreactor consisted of 10% strain, 0.3 Hz, breaks of 10 s every 180 cycles for 24 h. Contrary to previous findings using porcine MSCs, no significant cell count was found for hBM-MSCs into alginate-Ln scaffolds upon mechanical stimulation (8 ± 5 viable cells/mm3 for loaded and 4 ± 2 viable cells/mm3 for unloaded alginate-Ln scaffolds). However, intermittent mechanical stimulation induced the mobilization of hBM-MSCs into col-I scaffolds 10-fold compared to the unloaded col-I controls (245 ± 42 viable cells/mm3 vs. 22 ± 6 viable cells/mm3, respectively; p-value < 0.0001). Cells that mobilized into the scaffolds by mechanical loading did not show morphological changes. This study confirmed that hBM-MSCs can be mobilized in vitro from a reservoir toward col-I but not alginate-Ln scaffolds upon intermittent mechanical loading, against gravity.
| S-EPMC7672606 | biostudies-literature
Project description:The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild-type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild-type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild-type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence.
| S-EPMC4376591 | biostudies-literature
Project description:IntroductionAcute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis.MethodsWe studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system.ResultsWe found that AML cell lines and primary blasts, but not normal hematopoietic CD34+ cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation.DiscussionTogether, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.
| S-EPMC10725948 | biostudies-literature
Project description:Alendronate inhibits osteoclastic activity. However, some studies suggest alendronate also has effects on osteoblast activity. We hypothesized alendronate would enhance osteoblastic differentiation without causing cytotoxicity of the osteoblasts. We evaluated the effect of alendronate on the osteogenic differentiation of mouse mesenchymal stem cells. D1 cells (multipotent mouse mesenchymal stem cells) were cultured in osteogenic differentiation medium for 7 days and then treated with alendronate for 2 days before being subjected to various tests using MTT assays, Alizarin Red, enzyme-linked immunosorbent assay, energy-dispersive xray spectrophotometry, reverse transcriptase-polymerase chain reaction, confocal microscopy, and flow cytometric analysis. D1 cells differentiated into osteoblasts in the presence of osteogenic differentiation medium as confirmed by positive Alizarin Red S staining, increased alkaline phosphatase activity and osteocalcin mRNA expression, a calcium peak by energy-dispersive xray spectrophotometry, and by positive immunofluorescence staining against CD44. Osteogenic differentiation was enhanced after treatment with alendronate as confirmed by Alizarin Red S staining, elevated alkaline phosphatase activity and osteocalcin mRNA expression, a greater calcium peak by energy-dispersive xray spectrophotometry, and by immunofluorescence staining against CD44 by flow cytometric analysis. These data suggest alendronate enhances osteogenic differentiation when treated with mouse mesenchymal stem cells in osteogenic differentiation medium.
| S-EPMC2772902 | biostudies-other
Project description:One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.
| S-EPMC4617101 | biostudies-literature
Project description:Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 989-1001, 2017.
| S-EPMC6349397 | biostudies-literature
Project description:Nutritional microenvironment determines the specification of progenitor cells, and lipid availability was found to modulate osteogenesis in skeletal progenitors. Here, we investigated the implications of lipid scarcity in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and the role of low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor transducing canonical Wnt/beta-catenin signals, in BMSC lipid uptake during osteogenesis. The osteogenic differentiation of murine BMSCs was suppressed by lipid scarcity and partially rescued by additional fatty acid treatment with oleate. The enhancement of osteogenesis by oleate was found to be dosage-dependent, along with the enhanced activation of beta-catenin and Wnt target genes. Conditional knockout (CKO) of Lrp5 gene in murine mesenchymal lineage using Lrp5 fl/fl ;Prrx1-cre mice led to decreased bone quality and altered fat distribution in vivo. After Lrp5 ablation using adenoviral Cre-recombinase, the accumulation of lipid droplets in BMSC cytoplasm was significantly reduced, and the osteogenesis of BMSCs was suppressed. Moreover, the impaired osteogenesis due to either lipid scarcity or Lrp5 ablation could be rescued by recombinant Wnt3a protein, indicating that the osteogenesis induced by Wnt/beta-catenin signaling was independent of LRP5-mediated lipid uptake. In conclusion, lipid scarcity suppresses BMSC osteogenic differentiation. LRP5 plays a role in the uptake of lipids in BMSCs and therefore mediates osteogenic specification.
| S-EPMC8593169 | biostudies-literature
Project description:Incorporation of hydroxyapatite (HA) within a degradable polymeric scaffold may provide a favorable synthetic microenvironment that more closely mimics natural bone tissue physiology. Both incorporation of HA nanoparticles and alterations of the paracrine cell-cell signaling distance may affect the intercellular signaling mechanism and facilitate enhanced osteogenic signal expression among the implanted cell population. In this study we investigate the effect of the incorporation of HA nanoparticles into poly(propylene fumarate) (PPF) scaffolds on the surface properties of composite scaffolds and early osteogenic growth factor gene expression in relation to initial cell seeding density. The results of surface characterization indicated that HA addition improved the surface properties of PPF/HA composite scaffolds by increasing the roughness, hydrophilicity, protein adsorption, and initial cell attachment. Rat bone marrow stromal cells (BMSCs), which were CD34-, CD45-, CD29+, and CD90+, were cultured on three-dimensional (3-D) macroporous PPF/HA scaffolds at two different initial cell seeding densities (0.33 and 1.00 million cells per scaffold) for 8 days. The results demonstrated that endogenous osteogenic signal expression profiles, including bone morphogenetic protein-2, fibroblast growth factor-2, and transforming growth factor-?1, as well as the transcriptional factor Runx2, were affected by both HA amount and initial cell seeding density. Up-regulated expression of osteogenic growth factor genes was related to subsequent osteoblastic differentiation of rat BMSCs on 3-D scaffolds, as characterized by alkaline phosphatase activity, osteocalcin mRNA expression, and calcium deposition. Thus, the PPF/HA composite scaffold construction parameters, including amount of HA incorporated and initial cell seeding density, may be utilized to induce the osteoblastic differentiation of transplanted rat BMSCs.
| S-EPMC3031657 | biostudies-literature