Calcium Content Assay Research Articles

MO565M2 MACROPHAGES REGULATE MOUSE AORTIC SMOOTH MUSCLE CELL CALCIFICATION BY TRANSMITTING SENESCENCE INFORMATION

Abstract Background and Aims Vascular calcification is highly prevalent in chronic kidney disease (CKD)with high morbidity and mortality. Complex pathological mechanisms are involved in the development of vascular calcification, including aging. Previous work have indicated that M2-macrophage promote mouse aortic smooth muscle cells (MAoSMCs) calcification. The present study aimed to understand the contribution of M2-macrophage to MAoSMCs calcification by focusing on the mechanisms underlying the transmitting senescence information Method The expression of IFITM3 and P16, marker of aging, in aging macrophages were confimed by RT-qPCR, western blotting and immunofluorescence staining. Then, the MAoSMCs were co-cultured with the supernatant of M2 macrophages. The expression of IFITM3 and P16 in MAoSMCs also were detected. Alizarin red staining and calcium content assay were used to analyse MAoSMCs calcification. We modulated the expression of IFITM3 using siRNAs to study M2-macrophages function in regulating MAoSMCs calcification.We detected the expression of IFITM3 and P16 in co-cultivate MAoSMCs and validate intracellular calcium contents by calcium test kit. Results In the present study, we observed a significant increase in the expression of IFITM3 and P16 expression in M2 macrophages.Compared with M0 and M1 macrophages, the expression of IFITM3 and P16 at lowlevel. Alizarin red staining and calcium content assay revealed that M2 macrophage-mediated IFITM3 and P16 overexpression led to an apparent VSMC calcification in the presence of M2 macrophages medium.By contrast, inhibition of IFITM3 by siRNAs significantly blocked calcification of VSMCs in vitro. Moreover, we found that aging macrophages that overexpressed of IFITM3 and P16 increased in rats with CKD. Furthermore, pharmacological inhibition of aging signal was shown to block IFITM3-induced VSMC calcification. These findings demonstrate for the first time that M2 macrophage-mediated IFITM3 contributes to vascular calcification through a mechanism involving transmitting Senescence signalling. Conclusion Collectively, our present study demonstrated that the functional importance of M2 macrophage-IFITM3 dependent vascular calcification and provided a novel mechanistic insight to the macrophage senescence in CKD.

MO569MACROPHAGE PROMOTE VASCULAR CALCIFICATION VIA THE MIGRASOMES/ INTEGRIN Α5Β1 PATHWAY IN CKD

Abstract Background and Aims Patients with chronic kidney disease (CKD) have a predisposition to develop vascular calcification due to dysregulated homeostatic mechanisms. Macrophages can promote vascular calcification by releasing diverse extracellular vehicles including the newly found migrasomes (M-mig). Our previous research had found that M-mig provide nucleating foci for calcific mineral formation and initiating bone mineralization process. However, the specific mechanism by which M-mig influence the formation of vascular calcification remains incompletely understood. Method To study calcifying M-mig, we exposed M-mig to high Ca/P (Ca/P=3 mmol/L calcium/2 mmol/L phosphate) and/or with LPS for 1, 3, 5,7 days. The expression of M-mig surface integrin α5β1 was determined by fluorescence staining. To block the M-mig-integrin α5β1 mediated calcification, we modulated the expression of integrin α5 using siRNAs to produce M-migintegrin α5- or using 20 nM ATN-161 (small peptide antagonist of integrin α5β1) or integrin α5 antibody under high Ca/P stimulation. The stray mice artery co-cultivate with M-mig integrin α5- under high level Ca/P. Then the calcifying M-mig were assessed by TEM, Fluo-3 staining and calcium content assay. Results We discovered that Ca/P-stimulated macrophages released M-mig capable of mineralization. Amorphous calcium phosphate mineral deposit the surface or internal of M-mig. The M-mig exhibited increased Ca/P mineral content, implying aggregate larger calcifying M-mig that develop over time. Significantly, following a 7 days incubation with high level Ca/P, fiber tube and vesicle structure of M-mig showed rupture or fragmentation and the expression of M-mig surface integrin α5β1 increased. Pre-treatment with integrin α5β1 antagonist or block by integrin α5 antibody significantly reduced the calcifying M-mig formation. Further investigation showed that M-mig induced stray mice artery microcalcification while M-migintegrin α5- exhibited a reduce microcalcification. Conclusion Our finding revealed an association between microcalcification and integrin α5β1 signalling in the fiber tube and vesicle structure of M-mig and provide a new insight into vascular calcification in CKD.

RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/\u03b2-Catenin Signaling Pathway

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

A novel hydrogel scaffold contained bioactive glass nanowhisker (BGnW) for osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro

Employing hydrogels as an alternative strategy for repairing bone defects has received great attention in bone tissue engineering. In this study, hydrogel scaffold based on collagen, gelatin, and glutaraldehyde was combined with bioactive glass nanowhiskers (BGnW) to differentiate human mesenchymal stem cells (hMSCs) into the osteogenic lineage and inducing biomineralization. Pure Gel-Glu-Col and bioactive glass nanowhiskers were used as control throughout the paper. Chemical, physical and morphological characteristics of the nanocomposite scaffold were assessed meticulously using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), porosity measurement, water uptake ability, tensile test, and scanning electron microscopy (SEM). To determine the cytotoxicity and cell viability of the hydrogel, MTT assay and Acridine orange (AO) staining were performed. hMSCs seeded on Gel-Glu-Col/BGnW were then incubated with osteogenic differentiation media for 14 days. Biomineralization assays (alkaline phosphatase (ALP) activity, calcium content assay, von Kossa, and Alizarin red staining) were carried out, and osteogenic genes and protein markers were examined using real time-PCR and immunocytochemistry. Results showed that the components of the hydrogel were properly integrated. The mechanical property of hydrogel was enhanced following the addition of BGnW. Cell viability assays confirmed the biocompatibility of the scaffold and increasing the proliferation after incorporating BGnW into pure Ge1-Glu-Col. Our nanocomposite maintained an enhanced ability of biomineralization as compared to its pure counterparts. Molecular investigations revealed an elevated level of osteogenic markers as compared to Ge1-Glu-Col and BGnW. All in all, Gel-Glu-Col/BGnW seems to be a potential candidate for the regeneration of bone tissue.

Carnosine attenuates vascular smooth muscle cells calcification through mTOR signaling pathway.

ObjectiveVascular calcification is prevalent in the aging population, as we know that arterial calcification is associated with aging. Recent studies have demonstrated that carnosine, a naturally occurring dipeptide, performs the treatment of aging‐related diseases, such as atherosclerosis and type 2 diabetes. Here, we investigated the role of carnosine in a calcification model of vascular smooth muscle cells (VSMCs).MethodsIn this research, we used an in vitro model of VSMC calcification to investigate the role of carnosine in the progression of rat VSMC calcification.ResultsCarnosine treatment attenuated calcium deposition in a dose‐dependent manner, detected by Alizarin Red S staining and calcium content assay. Carnosine also reduced the protein level of Runx2, bone morphogenetic protein 2 (BMP‐2), and cellular reactive oxygen species (ROS) production. Further, carnosine inhibited the activation of the mammalian target of rapamycin (mTOR) pathway.ConclusionCarnosine attenuated the VSMC calcification via inhibition of osteoblastic transdifferentiation and the mTOR signaling pathway.

Adipose-Derived Stem Cells Conditioned Media Promote In Vitro Osteogenic Differentiation of Hypothyroid Mesenchymal Stem Cells

Background: Thyroid hormones have many effects on the physiological functions of cells, including growth, differentiation, and metabolism. Objectives: Recently, studies have shown that the adipose-derived mesenchymal stem cells conditioned medium (ADMSCs-CM) has many osteogenic factors, such as IGF-1, IL-6, and FGFs. Methods: In the current study, mesenchymal stem cells (MSCs) were isolated from two sources; the adipose tissue of the testicular fat pad and the bone marrow of rat, and then characterized by flow cytometry. ADMSCs-CM was collected from the ADMSC in the healthy adult male rats. Hypothyroidism was induced by the administration of the Methimazole during 60 days and confirmed by the analysis of the serum level of T4 and TSH hormones. Cell proliferation and osteogenic differentiation potential of bone marrow stem cells (BMSCs) derived from hypothyroid rats were investigated in the presence and absence of the CM by MTT assay, alkaline phosphatase (ALP) activity, calcium content assay, and bone-related gene expression. Healthy BMSCs were assigned to the control group. Results: Although Cell proliferation was decreased in the hypothyroid BMSCs, there was no significant difference between the control and the hypothyroid-CM groups. Similarly, osteogenic potential was significantly reduced in the hypothyroid group compared to the control and hypothyroid-CM groups according to the ALP, calcium content assays, and gene expression results. There was no significant difference between the hypothyroid-CM group and control. Conclusions: Our results indicated that hypothyroidism can decrease cell proliferation and osteogenic differentiation of BMSCs. Although ADMSCs-CM improved these parameters, it may be a promising candidate for the bone regeneration of the hypothyroidism cases.

Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells

AimsIn this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. Main methodsBy controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. Key findingsThe highest loading efficiency percentage (%LE) was %32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and %LE were 142 ± 6.1 nm, 198 ± 16.56 nm, %16.76 ± 3.91 and %32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. SignificanceWe concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds.

25-Hydroxycholesterol promotes vascular calcification via activation of endoplasmic reticulum stress

Vascular calcification is a highly regulated process similar to osteogenesis involving phenotypic change of vascular smooth muscle cells (VSMCs). 25-Hydroxycholesterol (25-HC), one of oxysterols synthesized by the enzyme cholesterol 25-hydroxylase, has been shown to promote bovine calcifying vascular cells (CVC) calcification. However, whether and how 25-HC regulates vascular calcification are not completely understood. In this study, in vitro and ex vivo models of vascular calcification were used to determine whether 25-HC regulates vascular calcification. Alizarin red staining and calcium content assay showed that 25-HC treatment promoted calcification of rat and human VSMCs in a dose-dependent manner. Similarly, ex vivo study further confirmed that 25-HC accelerated calcification of rat aortic rings. In addition, western blot analysis showed that 25-HC significantly up-regulated the expression of endoplasmic reticulum stress (ERS) signaling molecules including ATF4 and CHOP in VSMCs and flow cytometry analysis revealed that 25-HC increased apoptosis of VSMCs. Moreover, knockdown of CHOP by siRNA blocked 25-HC-induced mineral deposition in VSMCs. Collectively, this study for the first time demonstrates that 25-HC promotes vascular calcification via ATF4/CHOP signaling using in vitro and ex vivo models, suggesting that ERS is involved in the regulation of 25-HC-induced vascular calcification.

Inhibition of vascular calcification by microRNA-155-5p is accompanied by the inactivation of TGF-β1/Smad2/3 signaling pathway

Vascular calcification (VC) is a vital factor for cardiovascular morbidity and mortality. Accumulating data suggest that microRNA (miR) is implicated in the VC. The main purpose of this study is to study the influence of miR-155-5p overexpression on VC development in vitro and in vivo. Immunofluorescence staining, real-time PCR, alizarin red staining, alkaline phosphatase (ALP) activity assay, western blot, luciferase assay, hematoxylin-eosin (HE), Masson’s trichrome staining, and calcium content assay were used in this research. The results showed that miR-155-5p was decreased in the rat vascular smooth muscle cells (rVSMCs) undergoing calcification in vitro. MiR-155-5p overexpression reversed the increase of calcification and ALP activity in calcified cells. Further, overexpression of miR-155-5p inhibited the transforming growth factor-β1 (TGF-β1)/Smad2/3 signaling pathway, as evidenced by decreased protein expression of TGF-β1, pSmad-2 and pSmad-3 in rVSMCs. MiR-155-5p was showed to target Smad2 directly. Moreover, miR-155-5p upregulation reduced vascular thickening, fibrosis and calcium content of aorta abdominalis in CaCl2-mediated VC model. Collectively, our results suggest that miR-155-5p overexpression may inhibit VC development through suppressing TGF-β1/Smad2/3 signaling pathway in vivo and in vitro, indicating that miR-155-5p may act as a potential therapeutic target for VC-related disease.

Trimethylamine-N-Oxide Promotes Vascular Calcification Through Activation of NLRP3 (Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3) Inflammasome and NF-κB (Nuclear Factor κB) Signals.

Vascular calcification is highly prevalent in patients with chronic kidney disease. Increased plasma trimethylamine N-oxide (TMAO), a gut microbiota-dependent product, concentrations are found in patients undergoing hemodialysis. However, a clear mechanistic link between TMAO and vascular calcification is not yet established. In this study, we investigate whether TMAO participates in the progression of vascular calcification using in vitro, ex vivo, and in vivo models. Approach and Results: Alizarin red staining revealed that TMAO promoted calcium/phosphate-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner, and this was confirmed by calcium content assay. Similarly, TMAO upregulated the expression of bone-related molecules including Runx2 (Runt-related transcription factor 2) and BMP2 (bone morphogenetic protein-2), suggesting that TMAO promoted osteogenic differentiation of vascular smooth muscle cells. In addition, ex vivo study also showed the positive regulatory effect of TMAO on vascular calcification. Furthermore, we found that TMAO accelerated vascular calcification in rats with chronic kidney disease, as indicated by Mico-computed tomography analysis, alizarin red staining and calcium content assay. By contrast, reducing TMAO levels by antibiotics attenuated vascular calcification in chronic kidney disease rats. Interestingly, TMAO activated NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome and NF-κB (nuclear factor κB) signals during vascular calcification. Inhibition of NLRP3 inflammasome and NF-κB signals attenuated TMAO-induced vascular smooth muscle cell calcification. This study for the first time demonstrates that TMAO promotes vascular calcification through activation of NLRP3 inflammasome and NF-κB signals, suggesting the potential link between gut microbial metabolism and vascular calcification. Reducing the levels of TMAO could become a potential treatment strategy for vascular calcification in chronic kidney disease.

Polyvinyl alcohol modified polyvinylidene fluoride-graphene oxide scaffold promotes osteogenic differentiation potential of human induced pluripotent stem cells.

Tissue engineering is fast becoming a key approach in bone medicine studies. Designing the ideally desirable combination of stem cells and scaffolds are at the hurt of efforts for producing implantable bone substitutes. Clinical application of stem cells could be associated with serious limitations, and engineering scaffolds that are able to imitate the important features of extracellular matrix is a major area of challenges within the field. In this study, electrospun scaffolds of polyvinylidene fluoride (PVDF), PVDF-graphene oxide (GO), PVDF-polyvinyl alcohol (PVA) and PVDF-PVA-GO were fabricated to study the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs) while cultured on fabricated scaffolds. Scanning electron microscopy study, viability assay, relative gene expression analysis, immunocytochemistry, alkaline phosphates activity, and calcium content assays confirmed that the osteogenesis rate of hiPSCs cultured on PVDF-PVA-Go is significantly higher than other scaffolds. Here, we showed that the biocompatible, nontoxic, flexible, piezoelectric, highly porous and interconnected three-dimensional structure of electrospun PVDF-PVA-Go scaffold in combination with hiPSCs (as the stem cells with significant advantageous in comparison to other types) makes them a highly promising scaffold-stem cell system for bone remodeling medicine. There was no evidence for the superiority of PVDF-GO or PVDF-PVA scaffold for osteogenesis, compared to each other; however both of them showed better potentials as to PVDF scaffold.

Bone morphogenic protein-2 immobilization by cold atmospheric plasma to enhance the osteoinductivity of carboxymethyl chitosan-based nanofibers

Electrospun polycaprolactone/carboxymethyl chitosan (PCL/CMC) nanofibers treated by helium cold atmospheric plasma (CAP) and grafted with bone morphogenic protein-2 (BMP-2) were used scaffolds for the osteodifferentiation of stem cells to. For in vitro study, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on these scaffolds, and their behaviors were assessed via optical microscopy, MTT assay, and SEM. The osteogenic differentiation of the hMSCs was evaluated by calcium content and alkaline phosphatase assays, Alizarin red and immunofluorescence (ICC) staining, and RT-PCR. The results showed that scaffolds not only can support the proliferation of hMSCs but also can promote their differentiation to osteoblasts without using any external osteogenic differential agent. The RT-PCR and ICC data revealed that the CAP treatment and BMP-2-functionalization have synergic enhancement on the ossification of hMSCs. These fabricated scaffolds can be used as promising candidates for bone tissue engineering applications.

Micro-RNA-incorporated electrospun nanofibers improve osteogenic differentiation of human-induced pluripotent stem cells.

Smart scaffolds have a great role in the damaged tissue reconstruction. The aim of this study was developing a scaffold that in addition to its fiber's topography has also content of micro-RNAs (miRNAs), which play a regulatory role during osteogenesis. In this study, we inserted two important miRNAs, including miR-22 and miR-126 in the electrospun polycaprolactone (PCL) nanofibers and after scaffold characterization, osteoinductivity of the fabricated nanofibers was investigated by evaluating of the osteogenic differentiation potential of induced pluripotent stem cells (iPSCs) when grown on miRNAs-incorporated PCL nanofibers (PCL-miR) and empty PCL. MiRNAs incorporation had no effect on the fibers size and morphology, cell attachment, and protein adsorption, although viability and proliferation rate of the human iPSCs were increased after a week in PCL-miR compared to the empty PCL. The results obtained from alkaline phosphatase activity, calcium content, bone-related genes, and proteins expression assays demonstrated that the highest osteogenic markers were observed in iPSCs grown on the PCL-miR compared to the cells cultured on PCL and culture plate. According to the results, miR-incorporated PCL nanofibers could be considered as a promising potential tissue-engineered construct for the treatment of patients with bone lesions and defects.

Comparative impact of platelet rich plasma and transforming growth factor-β on chondrogenic differentiation of human adipose derived stem cells

Introduction: Transforming growth factor-beta (TGF-β) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-β and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-β. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-β groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-β with lesser inflammatory and hypertrophic side effects.

CDC42 promotes vascular calcification in chronic kidney disease.

Vascular calcification is prevalent in patients with chronic kidney disease (CKD) and a major risk factor of cardiovascular disease. Vascular calcification is now recognised as a biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Cell division cycle 42 (CDC42), a Rac1 family member GTPase, is essential for cartilage development during endochondral bone formation. However, whether CDC42 affects osteogenic differentiation of VSMCs and vascular calcification remains unknown. In the present study, we observed a significant increase in the expression of CDC42 both in rat VSMCs and in calcified arteries during vascular calcification. Alizarin red staining and calcium content assay revealed that adenovirus-mediated CDC42 overexpression led to an apparent VSMC calcification in the presence of calcifying medium, accompanied with up-regulation of bone-related molecules including RUNX2 and BMP2. By contrast, inhibition of CDC42 by ML141 significantly blocked calcification of VSMCs in vitro and aortic rings ex vivo. Moreover, ML141 markedly attenuated vascular calcification in rats with CKD. Furthermore, pharmacological inhibition of AKT signal was shown to block CDC42-induced VSMC calcification. These findings demonstrate for the first time that CDC42 contributes to vascular calcification through a mechanism involving AKT signalling; this uncovered a new function of CDC42 in regulating vascular calcification. This may provide a potential therapeutic target for the treatment of vascular calcification in the context of CKD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Platelet-rich plasma incorporated electrospun PVA-chitosan-HA nanofibers accelerates osteogenic differentiation and bone reconstruction

Biologically active materials and polymeric materials used in tissue engineering have been one of the most attractive research areas in the past decades, especially the use of easily accessible materials from the patients that reduces or eliminates any patient's immune response. In this study, electrospun nanofibrous scaffolds were fabricated by using polyvinyl-alcohol (PVA), chitosan and hydroxyapatite (HA) polymers and platelet-rich plasma (PRP) as a bioactive substance isolated from human blood. Fabricated scaffold's structure and cytotoxicity were evaluated using scanning electron microscope and MTT assays. Scaffolds osteoinductivity was investigated by osteogenic differentiation of the mesenchymal stem cells (MSCs) at the in vitro level and then its osteoconductivity was examined by implanting at the critical-sized rat calvarial defect. The in vitro results showed that scaffolds have a good structure and good biocompatibility. Alkaline phosphatase activity, calcium content and gene expression assays were also demonstrated that their highest amount was detected in MSCs-seeded PVA-chitosan-HA(PRP) scaffold. For this reason, this scaffold alone and along with the MSCs was implanted to the animal defects. The in vivo results demonstrated that in the animals implanted with PVA-chitosan-HA(PRP), the defect was repaired to a good extent, but in those animals that received MSCs-seeded PVA-chitosan-HA(PRP), the defects was almost filled. It can be concluded that, PVA-chitosan-HA(PRP) alone or when stem cells cultured on them, has a great potential to use as an effective bone implant.

Osteogenic differentiation of hMSCs on semi-interpenetrating polymer networks of polyurethane/poly(2‑hydroxyethyl methacrylate)/cellulose nanowhisker scaffolds

Poly (2‑hydroxyethyl methacrylate) (PHEMA) was crosslinked in the presence of biocompatible and biodegradable poly(caprolactone) (PCL) based polyurethanes (PUs) and cellulose nanowhiskers (CNWs). The CNWs were obtained from wastepaper. In order to crosslink PHEMA (10 wt%), a novel acrylic-urethane cross-linker was produced by a condensation reaction of PHEMA and hexamethylene diisocyanate (HDI). The PU-PHEMA-CNWs scaffolds were prepared by solvent casting/particulate leaching method in different weight percentages of CNWs (i.e., 0, 0.1, 0.5, and 1 wt%). The structural, mechanical, and in vitro biological properties of bio-nanocomposites were evaluated via FTIR, SEM, tensile, and MTT assay. The tensile strength of PU-PHEMA-0, PU-PHEMA-0.1, PU-PHEMA-0.5, and PU-PHEMA-1 were 76.2, 95.8, 98.1, and 89.8 kPa, respectively. Incorporation of CNWs also resulted in improved cell proliferation on PU-PHEMA-CNWs scaffolds. The bone marrow derived human mesenchymal stem cells (hMSCs) were seeded on the prepared porous scaffolds and incubated in osteogenic medium. Based on the results including calcium content assay, alkaline phosphatase assay, and mineralization staining, PU-PHEMA-CNW scaffolds were introduced as a suitable election for imitating the behavior of cellular niche. Bone mineralization and osteogenesis differentiation of hMSCs on PU-PHEMA-CNW scaffolds were significantly more than control after 14 days.

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers.

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.

A new nanocomposite scaffold based on polyurethane and clay nanoplates for osteogenic differentiation of human mesenchymal stem cells in vitro

Bone tissue engineering as an alternative strategy, provides a great opportunity for regeneration of large bone tissue lesions. The use of biodegradable porous scaffolds along with stem cells, cytokines and growth factors improves cell survival, adhesion, proliferation and differentiation. In the present study, clay nanoplates (CNPs) were surface-modified (MCNPs) using phosphoric acid and calcium hydroxide, then porous polyurethane (PU) scaffolds and PU-MCNPs nanocomposite scaffolds were synthesized using solvent evaporation-dissolution technique. Physicochemical and morphological properties of scaffolds and MCNPs were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Moreover, thermal behavior of scaffolds was assessed by differential scanning calorimetry (DSC). Degradability, water uptake and mechanical behaviors of scaffolds were evaluated and hydrophilicity properties of them were obtained by contact angle technique. MTT assay and Acridine Orange/Ethidium Bromide (AO/EB) staining were used to assess the biocompatibility of MCNPs and PU scaffolds regarding cell attachment and proliferation support. Osteogenic differentiation of cultured human adipose derived mesenchymal stem cells (hADSCs) on MCNPs, PU and PU-MCNPs scaffolds was evaluated using common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content assay, Alizarin Red staining, immunocytochemical analysis (ICC) and quantitative real-time PCR (qPCR). According to the results, the surface modification of CNPs and their presence into the PU scaffolds significantly enhanced proliferation and osteogenic differentiation of hADSCs. These results were obtained by higher ALP enzyme activity, biomineralization and expression of osteogenic related proteins and genes in differentiated hADSCs on PU-MCNPs scaffolds. In conclusion, our results revealed that these biocompatible nanocomposites porous scaffolds with proper cell adhesion and proliferation as well as effective osteogenic differentiation and which are able to provide a new and useful matrix for bone tissue engineering purposes.

Puerarin inhibits vascular calcification of uremic rats

The risk of cardiovascular events in patients with chronic kidney disease is tremendously increased due to vascular calcification. Local vascular inflammation significantly promotes vascular calcification. A previous study has shown that puerarin inhibits calcification of mouse vascular smooth muscle cells (VSMCs) in vitro, but whether puerarin can inhibit vascular calcification in vivo and the underlying mechanisms remain unclear. In this study, we found that rat VSMCs treated with calcifying medium showed more mineral deposition, and this effect was inhibited by puerarin in a dose-dependent manner, as detected by alizarin red staining and calcium content assay. Puerarin also prevented the trans-differentiation of VSMCs into osteoblast-like cells, indicated by down-regulation of bone-related genes including Runx2 and BMP2. In vivo study of uremic rats induced by renal nephrectomy further confirmed the inhibitory effect of puerarin on vascular calcification in uremic rats. Puerarin treatment significantly prevented mineral deposition in rat aortas and down-regulated the expression of Runx2 and BMP2. Furthermore, we detected the levels of pro-inflammatory cytokines including IL-1β, IL-6, IL-18 and TNFα in vitro and in vivo. The levels of IL-1β were remarkably increased in both calcified VSMCs and aortas of uremic rats, while puerarin treatment markedly decreased the expression of IL-1β. In addition, we found that puerarin reduced IL-1β possibly through targeting NLRP3/Caspase1/IL-1β and NF-κB signaling pathways and inhibiting the generation of reactive oxygen species. Taken together, we demonstrated that puerarin has the capability of inhibiting vascular calcification in uremic rats by inhibiting inflammation.