Osteogenic Phenotype Research Articles

Influence of Cell Spreading Area on the Osteogenic Commitment and Phenotype Maintenance of Mesenchymal Stem Cells

Osteogenic differentiation and commitment of mesenchymal stem cells (MSCs) is a complex process that is induced and regulated by various biological factors and biophysical cues. Although cell spreading area, as a biophysical cue, has been demonstrated to play a critical role in the regulation of osteogenic differentiation of MSCs, it is unclear how it affects the maintenance of the committed phenotype after osteogenic differentiation of MSCs. In this study, poly (vinyl alcohol) was micropatterned on a tissue culture polystyrene surface, and the micropatterns were used to culture MSCs to control their cell spreading area. The influence of cell spreading area on osteogenic differentiation and maintenance of the differentiated phenotype of MSCs was investigated. MSCs with a larger spreading area showed a higher degree of osteogenic differentiation, slower loss of differentiated phenotype and slower re-expression of stem cell markers compared with MSCs with a smaller spreading area. A large cell spreading area was beneficial for osteogenic differentiation of MSCs and maintenance of their differentiated phenotype.

Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells

Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in co-cultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling.

Lipopolysaccharide and interferon-γ team up to activate HIF-1α via STAT1 in normoxia and exhibit sex differences in human aortic valve interstitial cells

In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis.

Differential inflammatory landscape stimulus during titanium surfaces obtained osteogenic phenotype.

Molecular mechanism governing inflammatory scenario in response to titanium (Ti)-nanotexturing surfaces needs to be better addressed. Thus, we subjected pre-osteoblast to different Ti-texturing surfaces, as follows: machined (Mac), double acid-etching (DAE), and nanoscaled hydroxyapatite-blasted titanium surface (nHA), considering the cells chronically responding either directly (when the cells were cultured onto the surfaces) or indirectly (when the cells were challenged with the conditioned medium by the surfaces), up to 10 days. Our results showed that there is a dynamic requirement of inflammatory-related genes activation in response to nHA by up expressing IL1ß, IL6, IL10, and IL33 (direct condition) and IL6, IL10, IL18 (indirect condition). Importantly, our data show that there is inflammasome involvement, once NLRP3, ASC1, and CASP1 genes were also required. As we found a strong signal of IL10, an anti-inflammatory cytokine, we further investigated Sonic Hedgehog (Shh) signaling cascade. Surprisingly, Shh ligand and Smoothened (Smo) genes were up-modulated in response to nHA, while Patched (Ptc) was down-modulated. Finally, an interactome was built using bioinformatics reinforcing Shh signaling cascade on modulating IL10 transcripts by Src mediating this process and this prevalence of anti-inflammatory picture might explain the low profile of RANKL transcripts in response to nHA, compromising the osteoclastogenesis surrounding the implants. Taking our results into account, our data show that the inflammatory landscape promoted by nHA is strictly modulated by Shh signaling promoted anti-inflammatory pathways. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1597-1604, 2019.

Growth factor sustained delivery from poly-lactic-co-glycolic acid microcarriers and its mass transfer modeling by finite element in a dynamic and static three-dimensional environment bioengineered with stem cells.

Poly-lactic-co-glycolic acid (PLGA) microcarriers (0.8 ± 0.2 μm) have been fabricated with a load of 20 μg/gPLGA by an emulsion-based-proprietary technology to sustained deliver human bone morphogenetic protein 2 (hBMP2), a growth factor largely used for osteogenic induction. hBMP2 release profile, measured in vitro, showed a moderate "burst" release of 20% of the load in first 3 days, followed by a sustained release of 3% of the load along the following 21 days. PLGA microbeads loaded with fluorescent marker (8 mg/gPLGA ) and hydroxyapatite (30 mg/gPLGA ) were also fabricated and successfully dispersed within three-dimensional (3D) alginate scaffold (Ca-alginate 2% wt/wt) in a range between 50 and 200 mg/cm3 ; the presence of microcarriers within the scaffold induced a variation of its stiffness between 0.03 and 0.06 MPa; whereas the scaffold surface area was monitored always in the range of 190-200 m2 /g. Uniform microcarriers dispersion was obtained up to 200 mg/cm3 ; higher loading values in the 3D scaffold produced large aggregates. The release data and the surface area were, then, used to simulate by finite element modeling the hBMP2 mass transfer within the 3D hydrogel bioengineered with stem cells, in dynamic and static cultivations. The simulation was developed with COMSOL Multiphysics® giving a good representation of hBMP2 mass balances along microbeads (bulk eroded) and on cell surface (cell binding). hBMP2 degradation rate was also taken into account in the simulations. hBMP2 concentration of 20 ng/cm3 was set as a target because it has been described as the minimum effective value for stem cells stimulation versus the osteogenic phenotype. The sensitivity analysis suggested the best microbeads/cells ratio in the 3D microenvironment, along 21 days of cultivations in both static and dynamic cultivation (perfusion) conditions. The simulated formulation was so assembled experimentally using human mesenchymal stem cells and an improved scaffold stiffness up to 0.09 MPa (n = 3; p ≤ 0.01) was monitored after 21 days of cultivation; moreover a uniform extracellular matrix deposition within the 3D system was detected by Von Kossa staining, especially in dynamic conditions. The results indicated that the described tool can be useful for the design of 3D bioengineered microarchitecture by quantitative understanding.

Coronary Smooth Muscle Cytodifferentiation is Associated with an Increase in Pro‐Inflammatory, Pro‐Calcifying Gene Expression in an Organ Culture Model of Coronary Artery Disease

IntroductionDuring coronary artery disease (CAD) progression, coronary smooth muscle (CSM) cells dedifferentiate into synthetic or osteogenic phenotypes that induce CSM proliferation and vascular calcification, respectively. This phenotypic switching is in parallel to the upregulation of intracellular [Ca2+]i handling observed in early CAD and downregulation observed in late CAD. These [Ca2+]i handling alterations may play a role in promoting coronary artery calcification (CAC), which is associated with increased risk of cardiovascular health complications and mortality. Therefore, the aims of this study are two‐fold: 1) utilize an in vitro organ culture model of CAD to elucidate the extent of CSM phenotypic switching and 2) identify what genes are upregulated in each major CSM phenotype. Collectively, the data show possible signaling mechanisms and cellular processes that contribute to CAD pathophysiology, specifically CAC.MethodsCoronary rings from lean Ossabaw miniature swine were either used fresh or cultured in osteogenic media containing 10% FBS, 180 mg/dL glucose, 1% penicillin/streptomycin, 3.8 mM inorganic phosphate, and 7.5 U/mL alkaline phosphatase in DMEM for 3 days. To identify cells, cell population heterogeneity, and gene expression, CSM were enzymatically dispersed and used for single‐cell RNA‐sequencing.ResultsThe presence of differentiated, contractile CSM phenotypic markers including actin (ACTA2), myosin light chain (MYL9), myosin heavy chain (MYH11), and calponin (CNN1) were drastically reduced in the transcriptome of cells in cultured arterial rings. CSM from cultured rings also exhibited higher levels of transforming growth factor beta 1 (TGFB1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin (SMARCA4), both markers of the synthetic, proliferative phenotype. Furthermore, genes related to inflammation, chemotaxis, oxidative stress, extracellular matrix degradation, and vascular calcification were upregulated in CSM from cultured arterial rings when compared to CSM from fresh, noncultured rings.ConclusionAfter 3 days cultured in osteogenic media, CSM in whole arterial rings undergo dedifferentiation, as evidenced by decreased contractile markers and increased proliferative markers. This is associated with the upregulation of several genes associated with CAD pathophysiology, including genes involved in inflammation, calcification, and oxidative stress. This study will further help to parse out the complex cellular mechanisms involved in the progression of CAD and CAC.Support or Funding InformationThis work was supported by the CorVus Biomedical Research Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A pilot study of human mesenchymal stem cells from visceral and sub-cutaneous fat tissue and their differentiation to osteogenic phenotype.

To evaluate the different behavior of two different human adult adipocytes derived stem cells (hASCs) during proliferation and osteogenic differentiation. Human adult adipocytes stem cells (hAT-SCs) from visceral (hAV-SCs) and subcutaneous (hAS-SCs) sites were obtained after surgery procedures of seven patients. All samples were fully investigated and the different proliferation rates were evaluated. All MSCs clusters were cultured with an osteogenic and adipogenic differentiation medium. Homogeneous pools of Mesenchymal Stem Cells (MSCs) were confirmed by Flow-Cytometry Analysis (FACS) and Spectrophotometric Assay. The differentiated cells were eventually assessed for the expression of Alkaline Phosphatase (ALP), Alizarin Red (AR) and Oil Red-O (OR-O) detection, and analyzed by the Spectrophotometric Assay. After osteogenic differentiation, the cell clusters were incubated and analyzed with Real Time-Polymerase Chain Reaction (qRT-PCR) and fluorescence microscopy. The FACS analysis performed on hAT-SCs confirmed the homogenous presence of MSCs in all samples. The ALP, AR stain confirmed the osteogenic differentiation capacity of MSCs towards osteoblast-like-cells. The colorimetric cell metabolic activity (MTS) assay showed an increase in the proliferation rate with different values in both sets hAS-SCs vs. hAV-SCs. These in vitro findings of both hAS-SCs and hAV-SCs suggested an important role of these stem cells for future clinical use in bone regeneration. Indeed, the final outcomes suggested a better performance of cells coming from subcutaneous adipose tissue vs. those from visceral fat tissue.

Modulatory effects of silibinin in cell behavior during osteogenic phenotype.

Natural molecules, such as flavonoid, are very welcome strategies to modulate bone turnover. This prompted us to comprehend better the effect of silibinin on osteoblast metabolism, mainly considering intracellular pathways able to drive cell adhesion to differentiation. By exploring in vitro approaches, our data show a modulatory effect of the silibinin (200 μg/mL) on the osteoblast intracellular signaling, contributing with decisive pathways governing cell adhesion, differentiation, and further mineralization, recapitulating important stages of osteogenesis. Within the first 24 hours of adhesion (acute stage), osteoblasts respond to silibinin by rearranging their cytoskeleton and start mechanisms responsible to extracellular matrix (ECM) remodeling, which reach intense profile at 28 days of treatment (chronic stage) by favoring matrix metalloproteinases (MMPs-2, and -9) activities, concomitant to mineralizing phenotype. Importantly, silibinin seems to reprogram genes related to inflammatory landscape and significantly upmodulating osteoprotegerin (>25 fold-changes), signaling molecule involved with osteoclastogenesis. Altogether, our results show for the first time that silibinin drives in vitro osteoblast differentiation by requiring specific intracellular signaling. In conjunction, this molecular landscape contributes to understand the effect of silibinin on osteoblasts performance and open novel therapeutic possibilities to silibinin in bone disorders, such as osteoporosis.

Tissue-Specific Influence of Lamin A Mutations on Notch Signaling and Osteogenic Phenotype of Primary Human Mesenchymal Cells.

Lamin A is involved in many cellular functions due to its ability to bind chromatin and transcription factors and affect their properties. Mutations of LMNA gene encoding lamin A affect the differentiation capacity of stem cells, but the mechanisms of this influence remain largely unclear. We and others have reported recently an interaction of lamin A with Notch pathway, which is among the main developmental regulators of cellular identity. The aim of this study was to explore the influence of LMNA mutations on the proosteogenic response of human cells of mesenchymal origin and to further explore the interaction of LMNA with Notch pathway. Mutations R527C and R471C in LMNA are associated with mandibuloacral dysplasia type A, a highly penetrant disease with a variety of abnormalities involving bone development. We used lentiviral constructs bearing mutations R527C and R471C and explored its influence on proosteogenic phenotype expression and Notch pathway activity in four types of human cells: umbilical vein endothelial cells (HUVEC), cardiac mesenchymal cells (HCMC), aortic smooth muscle cells (HASMC), and aortic valve interstitial cells (HAVIC). The proosteogenic response of the cells was induced by the addition of either LPS or specific effectors of osteogenic differentiation to the culture medium; phenotype was estimated by the expression of osteogenic markers by qPCR; activation of Notch was assessed by expression of Notch-related and Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. Overall, we observed different reactivity of all four cell lineages to the stimulation with either LPS or osteogenic factors. R527C had a stronger influence on the proosteogenic phenotype. We observed the inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling. Our results suggest that the effect of a LMNA mutation is strongly dependent not only on a specific mutation itself, but also might be influenced by the intrinsic molecular context of a cell lineage.

Poly(ADP-ribose) polymerase 1 accelerates vascular calcification by upregulating Runx2

Vascular calcification is highly prevalent in end-stage renal diseases and is predictive of cardiovascular events and mortality. Poly(ADP-ribose) polymerase 1 (PARP1) inhibition or deletion is vasoprotective in several disease models. Here we show that PARP activity is increased in radial artery samples from patients with chronic renal failure, in arteries from uraemic rats, and in calcified vascular smooth muscle cells (VSMCs) in vitro. PARP1 deficiency blocks, whereas PARP1 overexpression exacerbates, the transdifferentiation of VSMCs from a contractile to an osteogenic phenotype, the expression of mineralization-regulating proteins, and calcium deposition. PARP1 promotes Runx2 expression, and Runx2 deficiency offsets the pro-calcifying effects of PARP1. Activated PARP1 suppresses miRNA-204 expression via the IL-6/STAT3 pathway and thus relieves the repression of its target, Runx2, resulting in increased Runx2 protein. Together, these results suggest that PARP1 counteracts vascular calcification and that therapeutic agents that influence PARP1 activity may be of benefit to treat vascular calcification.

Death-associated protein kinase 3 deficiency alleviates vascular calcification via AMPK-mediated inhibition of endoplasmic reticulum stress

Vascular calcification (VC) is a critical feature of chronic kidney disease (CKD), diabetes, hypertension, and atherosclerosis. Death-associated protein kinase 3 (DAPK3) is involved in vascular remodeling in hypertension. However, it remains to be clarified whether DAPK3 controls vascular smooth muscle cell (VSMC) phenotypic transition into an osteogenic cell phenotype, which is an important process for VC. In vivo VC was induced in rats by vitamin D3 and nicotine. VSMCs were incubated with calcifying media containing β-glycerophosphate and Ca2+ to induce VC in vitro. Herein, we demonstrated increased expression of DAPK3 in the aortas of VC rats and VSMCs cultured in calcifying media. Knockdown of DAPK3 significantly inhibited calcifying media-induced VSMC mineralization and retarded the phenotypic transformation of VSMCs into osteogenic cells. Silencing of DAPK3 suppressed endoplasmic reticulum stress (ERS) related protein expressions, but upregulated the phosphorylation level of AMP-activated protein kinase (AMPK) in calcified VSMCs. Moreover, pretreatment with AMPK inhibitor Compound C abolished DAPK3 shRNA-mediated inhibition of ERS in VSMCs. In vivo, DAPK inhibitor significantly prevented calcium deposition in the aortas of VC rats. The present results revealed that DAPK3 modulated VSMC calcification through AMPK-mediated ERS signaling.

Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification.

To investigate the effects of Bushen Huoxue Decoction (BSHXD) and its underlying molecular mechanisms on inhibiting osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vascular calcification via regulating the mRNA expression of osteoprotegerin (OPG) and the receptor activator of the nuclear factor-kappa B ligand (RANKL). VSMCs from the aortas of rats were cultured in vitro. Osteogenic differentiation of VSMCs was induced by high levels of an inorganic phosphate medium (2.4 mM). BSHXD-containing serum was prepared using the serum-pharmacological method. VSMCs were plated using 6-well plates at an approximate density of 4.0×104 cells/mL and cultured for 10 days. This was followed by the application of different concentrations of BSHXD-containing serum. The percentage of concentrations of BSHXD-containing serum in high, middle and low dosage group was 20%, 10% and 5%, respectively. Calcium nodules were evaluated by alizarin red S staining, and alkaline phosphatase (ALP) activity and calcium deposition were both examined as per the instruction of the test kits on the 3rd, 6th, and 10th days. Protein expression level of ALP and α-smooth muscle actin (α-SMA) were detected by Western blot on the 3rd, 6th, and 10th days. The mRNA expression of the OPG and RANKL were also detected by real-time PCR on the 3rd, 6th, and 10th days. Compared with the control group, BSHXD significantly attenuated the calcium nodules that were examined by alizarin red-S staining. Protein expression levels of α-SMA were up-regulated and ALP were down-regulated on the BSHXD group (P<0.05). BSHXD also attenuated the ALP activity and calcium deposition of the VSMCs (P<0.05). These changes were associated with the effect of BSHXD on up-regulating the expression of OPG mRNA and down-regulating the expression of RANKL mRNA in the process of osteogenic differentiation of VSMCs. BSHXD has a beneficial effect on inhibiting osteogenic differentiation of VSMCs induced by high levels of phosphate. The underlying mechanism appears to be related to the modulation of expressions of OPG mRNA and RANKL mRNA in the VSMCs, thereby preventing the phenotypic changes of VSMCs to an osteogenic phenotype.

Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation

Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype.

Regulation of cell deformation induced by RhoA/ROCK signaling pathway in osteogenic differentiation of human mesenchymal stem cells

Objective: To investigate whether the cell deformation induced by RhoA/ROCK signaling pathway plays a regulatory role in the osteogenic differentiation in human mesenchymal stem cells (hMSCs). Methods: The primary hMSCs were cultured until the P3 generation was added to the osteogenic induction solution for 14 days, and the expression levels of RhoA and ROCK-1 proteins were detected by immunofluorescence. Another P3 generation hMSCs cells were divided into induction group, blank plus drug group and medicated group, and the induction group was induced by simple osteogenic induction; the blank drug-added group was added to the osteogenic induction solution and the solvent dimethyl sulfoxide (DMSO) for induction culture; the medicinal group was added with osteogenic induction solution and Y-27632 (RhoA/ROCK signaling pathway inhibitor) dissolved in DMSO for induction culture. The proliferation of the cells was detected by CCK-8 methods. The changes of cytoskeleton in each group were observed by fluorescence microscope. The expression changes of osteogenic genes alkaline phosphatase (ALP), osteocalcin (OCN) and runt-relatedtranscriptionfactor-2 (RUNX-2) in each group were detected by real time polymerase chain reaction (RT-PCR) and ALP staining. The t test was used for data comparison between the two groups. Results: There were significant differences in the expression of RhoA and ROCK-1 protein before and after osteogenic induction of hMSCs (22.7±2.1 vs 14.7±0.6 and 24.3±1.5 vs 20.3±0.6, t=-6.414, -4.243, both P<0.05). Mesenchymal stem cell proliferation on day 1, 5, 9 and day 14 in the medicated group were comparable with those in the induction group (F=0.427, 1.000, 0.298, 1.314, all P>0.05). The cytoskeleton of the medicated group showed obvious spindle-shaped changes, and the cell spreading area became smaller. From the three groups of ALP staining on the 14th day, it can be seen that the color of the medicated group was significantly lighter than that of the induction group. The results of RT-PCR showed that the expression of osteogenic phenotypic genes increased with the induction time in the induction group and the blank drug-treated group, the expression levels of osteogenic phenotype genes ALP, OCN and RUNX-2 in the drug-treated group were gradually decreased with the induction time. The expression of ALP in the drug-treated group was significantly lower than those in the other two groups at 14th day (F=25.891, P=0.001); the expression of OCN in the drug-treated group was significantly lower than those in the other two groups at 11th and 14th days (F=5.773, 25.382, both P<0.05); the expression of RUNX-2 in the drug group was significantly lower than those in the other two groups at 11th and 14th days (F=34.972,10.808, both P<0.05). Conclusion: The RhoA/ROCK signaling pathway may play a role in promoting osteogenic differentiation of hMSCs through mediating cytoskeletal deformation.

Scalable MSC-derived bone tissue modules: In vitro assessment of differentiation, matrix deposition, and compressive load bearing

Enhancements to the mechanical properties of modular designs for bone tissue engineering could increase their clinical applications. In this study, bone marrow mesenchymal stem cells (MSCs) and hydroxyapatite (HAP) microgranules were encapsulated in polyelectrolyte complex membranes composed of chondroitin 4-sulfate (C4S), carboxymethyl cellulose (CMC) and chitosan. Microcapsules were formed with and without HAP microgranules, and cultured in either osteoinduction medium (Osteo) or expansion medium (Exp) to produce four microcapsule conditions: Osteo, Osteo+HAP, Exp, and Exp+HAP. Microcapsules facilitated alkaline phosphatase secretion and deposition of bone specific proteins (osteocalcin and osteopontin) by encapsulated MSCs over 28 days of osteogenic culture. SEM and micro-CT analysis showed cell-deposited mineral covering the surfaces of the HAP microgranules and interior of the microcapsule membrane. The mineralized microcapsules could be combined and fused into cylindrical constructs (4 × 5 mm, W × H), and uniaxial compression tests confirmed that microcapsule mineralization greatly enhanced the yield stresses of Osteo and Osteo+HAP fused constructs (10.4 ± 4.4 MPa and 6.4 ± 2.8 MPa), compared to only HAP microgranules (Exp+HAP, 0.5 ± 0.3 MPa). The C4S/CMC/Chitosan microcapsules provide a platform allowing pre-mineralization of microcapsules in vitro for later assembly of larger load-bearing constructs, or for use as an injectable bone regeneration strategy. Statement of SignificanceClinical translation of bone tissue engineering is limited by the difficulty of generating space filling implants that both resist compressive loading, and simultaneously deliver cells throughout the bone defect. Here, we present the design of a microcapsule system containing both stem cells capable of rebuilding bone tissue, and a mechanically tough bone-like mineral, that imparts compression resistance to the microcapsules. The microcapsules support stem cell differentiation to an osteogenic phenotype, that can mineralize the microcapsule membrane and interior. The mineralized microcapsules can be assembled into larger bone constructs, and have mechanical properties on par with trabecular bone.

Multiscale Stem Cell Technologies for Osteonecrosis of the Femoral Head.

The last couple of decades have seen brilliant progress in stem cell therapies, including native, genetically modified, and engineered stem cells, for osteonecrosis of the femoral head (ONFH). In vitro studies evaluate the effect of endogenous or exogenous factor or gene regulation on osteogenic phenotype maintenance and/or differentiation towards osteogenic lineage. The preclinical and clinical outcomes accelerate the clinical translation. Bone marrow mesenchymal stem cells and adipose-derived stem cells have demonstrated better effects in the treatment of femoral head necrosis. Various materials have been used widely in the ONFH treatment in both preclinical and clinical trials. In a word, in vivo and multiscale efforts are expected to overcome obstacles in the approaches for treating ONFH and provide clinical relevance and commercial strategies in the future. Therefore, we will discuss the above aspects in this paper and present our opinions.

Fabrication and characterization of the 3D-printed polycaprolactone/fish bone extract scaffolds for bone tissue regeneration.

Fish bone extract (FBE) containing a trioligopeptide (FBP-KSA, Lys-Ser-Ala) isolated from Johnius belengerii could induce osteogenic activities on MC3T3-E1 pre-osteoblasts in our previous study. Regarding the osteogenic effect of FBE, in the present study, we fabricated the three-dimensional (3D) interconnected polycaprolactone (PCL)/FBE scaffolds for bone tissue regeneration. After fabrication of PCL scaffolds using 3D printing, FBE was coated on the surface of PCL scaffolds by self-assembly process. In the physical characteristic and mechanical property tests, the results demonstrated that the fabricated scaffolds have the strut diameter (between 340 and 345 μm), pore size (between 470 and 480 μm), porosity (between 50% and 55%), and tensile properties (Young's modulus: 9.18-9.42 MPa; max tensile strengths 82.3-87.4 MPa) were similar to those of PCL scaffold. In the cell proliferation and osteogenic assay, the results showed that PCL/FBE scaffolds could significantly induce cell proliferation, calcium deposition, and the expression of osteogenic phenotype markers such as alkaline phosphatase, osteopontin, osteocalcin, and bone morphogenetic protein-2 in the osteoblasts. These results suggest that FBE-coated PCL scaffolds are promising materials for use in biomedical application to promote bone tissue regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1937-1944, 2019.

A Perfusion Culture System for Assessing Bone Marrow Stromal Cell Differentiation on PLGA Scaffolds for Bone Repair.

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration.

In vitro enhancement and functional characterization of neurite outgrowth by undifferentiated adipose-derived stem cells.

Adipose‑derived stem cells(ASCs) can easily be obtained and expanded invitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion(DRG) neurons were co‑cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co‑culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co‑culture and compared with single‑culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co‑culture with ASCs, resulting in increased neurite length after 24h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell‑to‑cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue‑engineered constructs may be promising for peripheral nerve repair.

Osteogenesis-Related Behavior of MC3T3-E1 Cells on Substrates with Tunable Stiffness.

Osteogenic differentiation of cells has considerable clinical significance in bone defect treatment, and cell behavior is linked to extracellular matrix stiffness. This study aimed to determine how matrix stiffness affects cell morphology and subsequently regulates the osteogenic phenotype of osteogenesis precursor cells. Four PDMS substrates were prepared with stiffness corresponding to the elastic modulus ranging from 0.6 MPa to 2.7 MPa by altering the Sylgard 527 and Sylgard 184 concentrations. MC3T3-E1 cells were cultured on the matrices. Cell morphology, vinculin expression, and key osteogenic markers, Col I, OCN, OPN, and calcium nodule, were examined. The activity and expression level of Yes-associated protein (YAP) were evaluated. Results showed that cell spreading exhibited no correlation with the stiffness of matrix designed in this paper, but substratum stiffness did modulate MC3T3-E1 osteogenic differentiation. Col I, OPN, and OCN proteins were significantly increased in cells cultured on soft matrices compared with stiff matrices. Additionally, cells cultured on the 1:3 ratio matrices had more nodules than those on other matrices. Accordingly, cells on substrates with low stiffness showed enhanced expression of the osteogenic markers. Meanwhile, YAP expression was downregulated on soft substrates although the subcellular location was not affected. Our results provide evidence that matrix stiffness (elastic modulus ranging from 0.6 MPa to 2.7 MPa) affects the osteogenic differentiation of MC3T3-E1, but it is not that “the stiffer, the better” as showed in some of the previous studies. The optimal substrate stiffness may exist to promote osteoblast differentiation. Cell differentiation triggered by the changes in substrate stiffness may be independent of the YAP signal. This study has important implications for biomaterial design and stem cell-based tissue engineering.