Expression Of Osteogenic Markers Research Articles

VCAM-1 and GFPT-2: Predictive markers of osteoblast differentiation in human dental pulp stem cells.

Dental pulp stem cells (DPSCs) have high proliferative and multilineage differentiation potential in mesenchymal stem cells. However, several studies have indicated that there are individual differences in the potential for osteogenic differentiation of DPSCs, and the factors determining these differences are unknown. To identify the genes responsible for the individual differences in the osteogenic differentiation ability of DPSCs. We divided DPSCs into high and low osteogenic differentiation ability groups (HG or LG) with ALP and von Kossa stain, and compared the gene expression patterns using RNA-seq. In addition, genes that may affect osteogenic differentiation were knocked down using small interfering RNA (siRNA) and their effects were investigated. The RNA-seq patterns revealed that VCAM1 and GFPT2 were significantly expressed at higher levels in the HG than in the LG. The results of siRNA analysis showed that VCAM1 and GFPT2 knockdown significantly reduced the expression of osteogenic markers. Furthermore, we analyzed the involvement of these two genes in cell signaling in DPSC differentiation. The results indicated that the VCAM1-mediated Ras-MEK-Erk and PI3K/Akt pathways are involved in the osteogenic differentiation of DPSCs, and that GFPT2-mediated HBP signaling influences the osteogenic differentiation of DPSCs. These findings indicate that DPSCs that highly express VCAM1 and GFPT2 have a high capacity for osteogenic differentiation. Evaluation of VCAM1 and GFPT2 expression in undifferentiated DPSCs may predict the outcome of bone regenerative therapy using DPSCs. Moreover, the expression levels of VCAM1 and GFPT2 in DPSCs may be useful in setting criteria for selecting donors for allogeneic cell transplantation for bone regeneration.

Sulfasalazine, a potent cystine-glutamate transporter inhibitor, enhances osteogenic differentiation of canine adipose-derived stem cells.

Cystine-glutamate transporter (xCT) is a plasma membrane transporter that imports cystine and indirectly contributes to the oxidative stress resistance associated with increased intracellular glutathione levels. Canine adipose-derived stem cells (CADSCs) include an xCT-positive subpopulation and show relatively low expression of osteogenic markers during in vitro osteogenic differentiation. Sulfasalazine (SSZ), a drug used to treat rheumatoid arthritis, suppresses xCT expression in cancer cells. In this study, we found that the SSZ treatment at 100 µM significantly suppressed xCT mRNA expression in CADSCs but did not significantly affect cell proliferation under the same conditions. Additionally, this treatment decreased the intracellular glutathione concentration. During in vitro osteogenic differentiation, the SSZ treatment at 50 µM and 100 µM significantly increased alizarin red staining and its quantification, as well as the concentration-dependent osteogenic differentiation markers (BMP1 and SPP) mRNA expression. Our results suggested that SSZ enhances the osteogenic differentiation potential of CADSCs and can potentially exhibit a superior therapeutic profile in canine bone regenerative medicine.

LncRNA AC108925 promotes osteoblast differentiation of tendon-derived stem cells by targeting miR-146a-3p.

It has been reported that tendon-derived stem cells(TDSCs) conduce to the ostosis in tendon diseases, and the molecular mechanism needs to be discussed. To investigate the function and mechanism of LncRNA in tendinopathy. Tendon of tendinopathy patients and health controls were obtained, and sequencing analysis have been performed to detect the significantly expressed genes and non-coding RNAs. Moreover, to further discuss LncRNA AC108925 in tendinopathy, tendinopathy animal models have been established, and the expression of LncRNA AC108925 expression was examined by RT-qPCR methods. Furthermore, hTDSCs have been treated by osteogenic medium, and the modulating function of LncRNA AC108925 on the osteoblast differentiation of hTDSCs have been examined. Sequencing analysis showed that AC108925 a dramatically elevated LncRNA, and results of animal and cells studies confirmed the finding. Knockdown AC108925 inhibited the osteogenic differentiation of osteogenic medium treated TDSCs by decreasing the expression of osteogenic markers. Furthermore, miR-146a-3p is a target of AC108925 in TDSCs, and miR-146a-3p is a negative modulator of osteogenic differentiation of hTDSCs by inhibiting the effects of AC108925 shRNA on osteogenic differentiation of hTDSCs. AC108925 can regulate the osteogenic differentiation of hTDSCs via regulating the miR-146a-3p. Targeting the AC108925/miR-146a-3p axis might be a latent way to treat tendinopathy.

Loss of N-acetylglucosaminyl transferase V is involved in the impaired osteogenic differentiation of bone marrow mesenchymal stem cells.

The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of β-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the β-catenin, BMP type 2 (BMP2) and TGF-β signals and involved in the process of OP.

Maternal hyperthyroidism increases the synthesis activity and the osteogenic markers expression of calvarial osteoblasts from offspring in a murine model

To evaluate the characteristics and synthesis activity of osteoblasts extracted from the calvaria of offspring of rats exposed to maternal hyperthyroidism. Twelve adult Wistar rats were divided into two groups, one control and one treated with daily administration of L-thyroxine by an orogastric tube (50 µg/animal/day) during pregnancy. Three days after delivery and confirmation of the mothers’ hyperthyroidism, the offspring were euthanized for the extraction of osteoblasts from the calvaria. At 7, 14, and 21 days, proliferation activity was assessed using MTT assay, while alkaline phosphatase (ALP) activity was assessed by the BCIP/NBT method. At 21 days, the total area of the mineralized matrix stained by von Kossa was evaluated by morphometry. The expression of gene transcripts for Runx2, Bmp2, Fgfr1, collagen type 1 (Col1), osteocalcin (Oc), and osteopontin (Op) were evaluated by real-time RT-PCR. Means were compared using the Student’s t-test. FA activity was significantly higher at 14 and 21 days in cultures of osteoblasts extracted from offspring exposed to maternal hyperthyroidism, while MTT conversion was significantly lower at 21 days in this group. Osteoblast cultures of neonates exposed to maternal hyperthyroidism also showed a larger total area of mineralized matrix and greater expression of gene transcripts for Oc and Op. Maternal hyperthyroidism increases the activity of matrix synthesis, alkaline phosphatase activity, and expression of gene transcripts for osteocalcin and osteopontin in the osteoblasts, extracted from the calvaria of the offspring, which may be one of the mechanisms of premature fusion of cranial sutures.

Effect of strontium substitution on functional activity of phosphate-based glass.

Phosphate-based glass (PBG) is a bioactive agent, composed of a glass network with phosphate as the primary component and can be substituted with various therapeutic ions for functional enhancement. Strontium (Sr) has been shown to stimulate osteogenic activity and inhibit pro-inflammatory responses. Despite this potential, there are limited studies that focus on the proportion of Sr substituted and its impact on the functional activity of resulting Sr-substituted PBG (PSr). In this study, focusing on the cellular biological response we synthesized and investigated the functional activity of PSr by characterizing its properties and comparing the effect of Sr substitution on cellular bioactivity. Moreover, we benchmarked the optimal composition against 45S5 bioactive glass (BG). Our results showed that PSr groups exhibited a glass structure and phosphate network like that of PBG. The release of Sr and P was most stable for PSr6, which showed favorable cell viability. Furthermore, PSr6 elicited excellent early osteogenic marker expression and inhibition of pro-inflammatory cytokine expression, which was significant compared to BG. In addition, compared to BG, PSr6 had markedly higher expression of osteopontin in immunocytochemistry, higher ALP expression in osteogenic media, and denser alizarin red staining in vitro. We also observed a comparable in vivo regenerative response in a 4-week rabbit calvaria defect model. Therefore, based on the results of this study, PSr6 could be identified as the functionally optimized composition with the potential to be applied as a valuable bioactive component of existing biomaterials used for bone regeneration.

PI3K-Akt signaling regulates BMP2-induced osteogenic differentiation of mesenchymal stem cells (MSCs): A transcriptomic landscape analysis

Bone morphogenetic protein 2 (BMP2) effectively induced mesenchymal stem cells (MSCs) osteogenic differentiation hold great potential for bone tissue engineering. However, a global mechanistic view of BMP2-induced osteogenic differentiation of MSCs remains to be fully elucidated. Here, human umbilical cord-derived MSCs (UC-MSCs) were induced with BMP2, three days and five days later, total RNA were extracted and subjected to RNA-sequencing (RNA-Seq) followed with bioinformatic analysis. Osteogenic differentiation abilities were evaluated with Alkaline phosphatase (ALP) staining and osteogenic differentiation marker expression at both mRNA and protein levels. We identified that adenoviral vectors effectively transduced in UC-MSCs and expressed BMP2 in high efficiency. Both on day 3 and day 5, differentially expressed genes (DEGs) were highly enriched in PI3K-Akt signaling pathway. As for the common DEGs among total BMP2 group vs control group, BMP2 (day 3) versus control (day 3) and BMP2 (day 5) versus control (day 5), there were 105 DGEs and highly enriched in PI3K-Akt signaling pathway. Finally, we found that PI3K-Akt signaling inhibitor dramatically inhibited BMP2-iduced osteogenic differentiation of UC-MSCs. We firstly identified that PI3K-Akt signaling pathway plays a pivotal role in BMP2-induced osteogenic differentiation of MSCs, which may apply a new perspective for BMP2 based bone tissue engineering.

SOX2 suppresses osteoblast differentiation of MC3T3-E1 cells through activating the transcription of LGR4.

Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers' expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.

Hybrid 3D Printed and Electrospun Multi-Scale Hierarchical Polycaprolactone Scaffolds to Induce Bone Differentiation.

Complex scaffolds composed of micro- and nano-structures are a key target in tissue engineering and the combination of sequential 3D printing and electrospinning enables the fabrication of these multi-scale structures. In this work, dual 3D printed and electrospun polycaprolactone (PCL) scaffolds with multiple mesh layers were successfully prepared. The scaffold macro- and micro-porosity were assessed by optical and scanning electron microscopy, showing that electrospun fibers formed aligned meshes within the pores of the scaffold. Consequently, the hydrophilicity of the scaffold increased with time, enhancing cell adhesion and growth. Additionally, compression tests in back and forth cycles demonstrated a good shape recovery behavior of the scaffolds. Biological results indicated that hybrid PCL scaffolds are biocompatible and enable a correct cell culture over time. Moreover, MC3T3-E1 preosteoblast culture on the scaffolds promoted the mineralization, increased the alkaline phosphatase (ALP) activity and upregulated the expression of early and late osteogenic markers, namely ALP and osteopontin (OPN), respectively. These results demonstrate that the sequential combination of 3D printing and electrospinning provides a facile method of incorporating fibers within a 3D printed scaffold, becoming a promising approach towards multi-scale hierarchical scaffolds capable of guiding the osteogenic differentiation.

Sex-dependent expression of neutrophil gelatinase-associated lipocalin in aortic stenosis

BackgroundAccumulating evidence suggest the existence of sex-related differences in the pathogenesis of aortic stenosis (AS) with inflammation, oxidative stress, fibrosis and calcification being over-represented in men. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in a myriad of tissues and cell types, and it is associated with acute and chronic pathological processes comprising inflammation, fibrosis or calcification. Sex-dependent signatures have been evidenced for NGAL which expression has been associated predominantly in males to metabolic and cardiovascular disorders. We aimed to analyse sex-related differences of NGAL in AS and its role in the inflammatory and fibrocalcific progression of AS.Methods and results220 (60.45% men) patients with severe AS elective for surgical aortic valve (AV) replacement were recruited. Immunohistochemistry revealed higher expression of NGAL in calcific areas of AVs and that was validated by qPCR in in 65 (60% men) donors. Valve interstitial cells (VICs) were a source of NGAL in these samples. Proteome profiler analyses evidenced higher expression of NGAL in men compared to women, and that was further validated by ELISA. NGAL expression in the AV was correlated with inflammation, oxidative stress, and osteogenic markers, as well as calcium score. The expression of NGAL, both intracellular and secreted (sNGAL), was significantly deregulated only in calcifying male-derived VICs. Depletion of intracellular NGAL in calcifying male-derived VICs was associated with pro-inflammatory profiles, dysbalanced matrix remodelling and pro-osteogenic profiles. Conversely, exogenous NGAL mediated inflammatory and dysbalanced matrix remodelling in calcifying VICs, and all that was prevented by the pharmacological blockade of NGAL.ConclusionsOwing to the over-expression of NGAL, the AV from men may be endowed with higher expression of inflammatory, oxidative stress, matrix remodelling and osteogenic markers supporting the progression of calcific AS phenotypes. The expression of NGAL in the VIC emerges as a potential therapeutic checkpoint, with its effects being potentially reverted by the pharmacological blockade of extracellular NGAL.

Fluid Shear Stress Regulates Osteogenic Differentiation via AnnexinA6-Mediated Autophagy in MC3T3-E1 Cells.

Fluid shear stress (FSS) facilitates bone remodeling by regulating osteogenic differentiation, and extracellular matrix maturation and mineralization. However, the underlying molecular mechanisms of how mechanical stimuli from FSS are converted into osteogenesis remain largely unexplored. Here, we exposed MC3T3-E1 cells to FSS with different intensities (1 h FSS with 0, 5, 10, and 20 dyn/cm2 intensities) and treatment durations (10 dyn/cm2 FSS with 0, 0.5, 1, 2 and 4 h treatment). The results demonstrate that the 1 h of 10 dyn/cm2 FSS treatment greatly upregulated the expression of osteogenic markers (Runx2, ALP, Col I), accompanied by AnxA6 activation. The genetic ablation of AnxA6 suppressed the autophagic process, demonstrating lowered autophagy markers (Beclin1, ATG5, ATG7, LC3) and decreased autophagosome formation, and strongly reduced osteogenic differentiation induced by FSS. Furthermore, the addition of autophagic activator rapamycin to AnxA6 knockdown cells stimulated autophagy process, and coincided with more expressions of osteogenic proteins ALP and Col I under both static and FSS conditions. In conclusion, the findings in this study reveal a hitherto unidentified relationship between FSS-induced osteogenic differentiation and autophagy, and point to AnxA6 as a key mediator of autophagy in response to FSS, which may provide a new target for the treatment of osteoporosis and other diseases.

Hypoxia Inhibits Osteogenesis and Promotes Adipogenesis of Fibroblast-like Synoviocytes via Upregulation of Leptin in Patients with Rheumatoid Arthritis.

Hypoxia is associated with the pathogenesis of rheumatoid arthritis (RA). RA fibroblast-like synoviocytes (FLSs) are able to differentiate into osteoblasts and adipocytes. In this study, we aimed to investigate the role of hypoxia in the osteogenesis or adipogenesis of RA-FLSs. Bioinformatics analysis was performed to profile gene expression in the datasets of GSE21959, GSE32006, and GSE55875, and flow cytometry was performed for FLS characterization, while Alizarin Redand Oil Red O staining for osteogenic or adipogenic differentiation of FLSs, respectively. RNA interference leptin knockdown was used to determine the role of leptin in the osteogenesis and adipogenesis of RA-FLSs, and the expression of osteogenic and adipogenic markers was quantified by RT-qPCR and Western blotting. FLSs exhibited a mesenchymal stem cell (MSC)-like phenotype and we observed a limited self-renewal capacity in RA-FLSs compared to that in MSCs, but it was still greater than osteoarthritis (OA)-FLSs. Hypoxia did not change the RA-FLS MSC-like phenotype but inhibited the osteogenic differentiation and promoted the adipogenic differentiation of RA-FLSs. From the bioinformatics analysis ofGSE21959, GSE32006, and GSE55875 datasets, we found leptin, the only perturbed hypoxia-mediated upregulated gene across the three profiled datasets. Leptin knockdown in RA-FLSs reversed the hypoxia-mediated reduction of osteogenesis and hypoxia-mediated enhancement of adipogenesis by elevated expression of osteogenic markers and reduced expression of adipogenic markers, respectively. Therefore, hypoxia-leptin regulation of the osteogenic and adipogenic differentiation of RA-FLSs advances our understanding of RA pathogenesis, meanwhile also provides opportunities for future therapeutic intervention of RA.

Biomimetic Hydroxyapatite Composite Coatings with a Variable Morphology Mediated by Silk Fibroin and Its Derived Peptides Enhance the Bioactivity on Titanium.

Various modifications performed on titanium alloy surfaces are shown to improve osteointegration and promote the long-term success of implants. In this work, a bioactive nanostructured hydroxyapatite (HA) composite coating with a variable morphology mediated by silk fibroin (SF) and its derived peptides (Cs) was prepared. Numerous experimental techniques were used to characterize the constructed coatings in terms of morphology, roughness, hydrophilicity, protein adsorption, in vitro biomineralization, and adhesion strength. The mixed protein layer with different contents of SF and Cs exhibited different secondary structures at different temperatures, effectively mediating the electrodeposited HA layer with different characteristics and finally forming proteins/HA composite coatings with versatile morphologies. The addition of Cs significantly improved the hydrophilicity and protein adsorption capacity of the composite coatings, while the electrodeposition of the HA layer effectively enhanced the adhesion between the composite coatings and Ti surface. In the in vitro mineralization experiments, all the composite coatings exhibited excellent apatite formation ability. Moreover, the composite coatings showed excellent cell growth and proliferation activity. Osteogenic induction experiments revealed that the coating could significantly increase the expression of specific osteogenic markers, including ALP, Col-I, Runx-2, and OCN. Overall, the proposed modification of the Ti implant surface by protein/HA coatings had good potential for clinical applications in enhancing bone induction and osteogenic activity of implants.

Use of CGF in Oral and Implant Surgery: From Laboratory Evidence to Clinical Evaluation

Edentulism is the condition of having lost natural teeth, and has serious social, psychological, and emotional consequences. The need for implant services in edentulous patients has dramatically increased during the last decades. In this study, the effects of concentrated growth factor (CGF), an autologous blood-derived biomaterial, in improving the process of osseointegration of dental implants have been evaluated. Here, permeation of dental implants with CGF has been obtained by using a Round up device. These CGF-coated dental implants retained a complex internal structure capable of releasing growth factors (VEGF, TGF-β1, and BMP-2) and matrix metalloproteinases (MMP-2 and MMP-9) over time. The CGF-permeated implants induced the osteogenic differentiation of human bone marrow stem cells (hBMSC) as confirmed by matrix mineralization and the expression of osteogenic differentiation markers. Moreover, CGF provided dental implants with a biocompatible and biologically active surface that significantly improved adhesion of endothelial cells on CGF-coated implants compared to control implants (without CGF). Finally, data obtained from surgical interventions with CGF-permeated dental implants presented better results in terms of optimal osseointegration and reduced post-surgical complications. These data, taken together, highlight new and interesting perspectives in the use of CGF in the dental implantology field to improve osseointegration and promote the healing process.

Magnetic Systems for Regenerative Medicine

Magnetic Systems for Regenerative Medicine

Abnormally elevated EZH2-mediated H3K27me3 enhances osteogenesis in aortic valve interstitial cells by inhibiting SOCS3 expression

Background and aimsThe osteogenic transition of aortic valve interstitial cells (AVICs) plays a critical role for the progression of calcific aortic valve disease (CAVD). Enhancer of zeste homolog 2 (EZH2) is an important methyltransferase for histone H3 Lys27 (H3K27) that has been found to be involved in osteogenesis. Here, we investigated the effect and mechanism of EZH2 in CAVD progression. MethodsHigh throughout mRNA sequencing, qRT-PCR and immunoblot were performed to screen differentially expressed genes in non-CAVD and CAVD aortic valves. To investigate the role of EZH2 and SOCS3 in osteogenesis, AVICs were treated with siRNA, adenovirus and specific inhibitors, then osteogenic markers and mineralized deposits were examined. In vivo, the morphology and function of aortic valves were investigated by HE stain and echocardiography in ApoE−/− mice fed a long-term western diet (WD). ResultsWe discovered that EZH2 was upregulated and SOCS3 was downregulated in calcified aortic valves. In AVICs, inhibition or silencing of EZH2 attenuated the osteogenic responses. On the other hand, demethylases inhibitor (GSK-J4) enhanced osteogenic transition of AVICs. Moreover, SOCS3 knockdown enhanced the expression of osteogenic markers, while SOCS3 overexpression suppressed osteogenesis and calcification. The chromatin immunoprecipitation and restored experiments indicated that EZH2 directly targeted SOCS3 to promote osteogenic responses of AVICs. In vivo, treatment with EZH2 inhibitor through intraperitoneal injection attenuated aortic valve thickening, calcification and dysfunction induced by WD. ConclusionsCollectively, we found that EZH2-mediated H3K27me3 enhanced osteogenesis and microcalcification of AVICs via inhibiting SOCS3 expression, which provides potential targets for future therapeutic interventions of CAVD.

Marine plankton exoskeleton-derived honeycombed hydroxyapatite bone granule for bone tissue engineering

The recovery of biocompatible implantable biomaterials from biowaste is a significant method for protecting both human health and the environment concerns. This study aimed to evaluate the possibility of using hydroxyapatite (HAp) granules derived from planktonic exoskeleton bone in the ocean (OceanBone-HAp) as a bone graft substitute. The morphology and physiochemical properties of the OceanBone-HAp were evaluated using SEM, XRD and FT-IR. In addition, the absence of heavy metals and abundance of trace elements in OceanBone-HAp was confirmed using ICP-OES analysis. Moreover, in vitro results showed that OceanBone-HAp improved cell proliferation, adhesion, differentiation, and infiltration, compared to stoichiometric synthetic HAp granules (pure HAp). Additionally, OceanBone-HAp improved the expression of osteogenic related markers, by upregulation of ALP, collagen type-1αI(Col1αI), osteocalcin (OCN), and bone sialoprotein (BSP), which was confirmed using quantitative real-time polymerase chain reaction. Furthermore, in vivo bone regeneration was also evaluated using a rabbit calvarial defect model, and the results showed that the area of mature bone formation was significantly higher for OceanBone-HAp than pure HAp. These results demonstrated that OceanBone-HAp granules have good potential for use in bone healing as a bone graft material.

Nano porous polycarbonate membranes stimulating cell adhesion and promoting osteogenic differentiation and differential mRNA expression

Tissue engineering is thought to be the ideal therapy for bone defect reconstructive treatment. In this study, we present a method of utilizing micro/nano porous polycarbonate membranes (PCMs) as the extracellular matrix to cultivate the human periodontal ligament cells (hPDLCs) and investigate the osteogenic differentiation of those cells. We also compared the osteogenic enhancing abilities of different pore size PCMs. The pore diameters of the candidate membranes are 200 nm, 800 nm, 1200 nm, and 10 μm respectively, and their physical properties are identified. After seeding and cultivating on the PCMs, hPDLCs can be stimulated to undergo osteogenic differentiation, in which the 200 nm PCM is proved to have the most optimal osteo-induction ability. The results of in vivo experiments provide strong evidence suggesting that the hPDLCs stimulated by 200 nm PCM greatly accelerates the healing of bone reconstruction in mice skull defects, as well as promote the process of ectopic osteogenesis. RNA-sequencing was conducted to determine the differential mRNA expression profile during the osteogenesis process of hPDLCs on PCMs. GO and KEGG enrichment analysis were conducted to study the regulatory mechanisms, in which osteogenic marker expression such as Hippo, TGF-β, and PI3K-Akt signaling pathways were significantly up-regulated. The up-regulation indicates the promising potential of nano porous PCMs for promoting osteogenesis for bone regeneration applications. Ultimately, signaling pathways that promote osteogenesis warrants further exploration.

Thermal treatment of magnesium particles in polylactic acid polymer films elicits the expression of osteogenic differentiation markers and lipidome profile remodeling in human adipose stem cells

The efficacy of polylactic acid (PLA)/Magnesium (Mg)-based materials for driving stem cells toward bone tissue engineering applications requires specific Mg surface properties to modulate the interface of stem cells with the film. Here, we have developed novel PLA/Mg-based composites and explored their osteogenic differentiation potential on human adipose stem cells (hASCs). Mg-particles/polymer interface was improved by two treatments: heating in oxidative atmosphere (TT) and surface modification with a compatibilizer (PEI). Different contents of Mg particles were dispersed in PLA and composite surface and bulk properties, protein adsorption, stem cell-PLA/Mg interactions, osteogenic markers expressions, and lipids composition profile were evaluated. Mg particles were uniformly distributed on the surface and in the bulk PLA polymer. Improved and modulated particle-polymer adhesion was observed in Mg particle-treated composites.After 21 days in canonical growth culture conditions, hASCs on PLA/MgTT displayed the highest expression of the general osteogenic markers, RUNX2, SSP1, and BGLAP genes, Alkaline Phosphatase, type I Collagen, Osteopontin, and Calcium deposits. Moreover, by LC/MS QTOF mass-spectrophotometry lipidomic analysis, we found in PLA/MgTT-cells, for the first time, a remodeling of the lipid classes composition associated with the osteogenic differentiation. We ascribed these results to MgTT characteristics, which improve Mg availability and composite osteoinductive performance.

Abstract 15254: Acta2 Missense Variant Increases Atherosclerotic Plaque Burdern by Heat Shock Factor 1-Driven Augmented Phenotypic Modulation of Smooth Muscle Cells

ACTA2 encodes the contractile protein smooth muscle α-actin expressed in smooth muscle cells (SMCs). A heterozygous pathogenic variant in ACTA2 , p.R149C, predisposes to premature coronary artery disease (CAD) in the absence of risk factors. Recent studies have determined that SMCs contribute to atherosclerotic plaque formation after undergoing phenotypic modulation, characterized by de-differentiation and increased expression of macrophage, fibroblast, stem, and osteogenic markers. This SMC modulation is driven in part by exogenous cholesterol entering the endoplasmic reticulum (ER) and activating ER stress, specifically PERK signaling. To determine how a mutation in SM α-actin predisposes to atherosclerosis, Acta2 R149C/+ mice were crossed into Apoe -/- mice and fed a high fat diet for 3 months. Despite no differences in serum lipid levels, the Acta2 R149C/+ Apoe -/- mice have a 3-fold increased plaque burden compared to Apoe -/- mice. SMCs explanted from Acta2 R149C/+ mice have phenotypic modulation at baseline while WT SMCs do not. We determined that misfolding of the R149C SMα-actin activates heat shock factor 1 (HSF1), which increases endogenous cholesterol biosynthesis through increasing HMG-CoA reductase expression. The increase in endogenous cholesterol levels induces ER stress, activates PERK, and augments both modulation of SMCs and atherosclerotic plaque formation. ScRNA sequencing confirms increased HSF1 and PERK signaling and modulation in SMCs in the mutant mice. Importantly, pravastatin treatment dramatically lowers plaque burden to similar low levels of plaque burden in both the Acta2 R149C/+ Apoe -/- and Apoe -/- mice (Fig. 1a) . These studies identify a novel mechanism by which mutant SM α-actin increases endogenous cholesterol biosynthesis and drives atherosclerotic plaque formation, a mechanism that also informs as to why statins are effective in individuals at risk for CAD who do not have elevated plasma cholesterol levels (Fig. 1b) .