The effect of light compressive and tensile mechanical forces on SOST and POSTN expressions in human periodontal ligament cells: an in vitro study.

Periodontal ligament (PDL) cells play an important role in mechanosensing and secretion of signaling molecules during bone remodeling. However, the regulatory mechanism is unknown. The aim of the present study is to investigate the expression pattern of periostin and sclerostin in response to orthodontic forces in periodontal ligament cells in vitro. PDL cells were isolated from extracted teeth and treated with compressive forces of 25 gr/cm2 or equiaxial tension forces at frequency 1 Hz for 0, 24, 48, and 72 h. qRT-PCR was applied to evaluate the gene expressions. The secretion of sclerostin and periostin was assessed using ELISA. DAPI staining was used to evaluate apoptosis. The expression of sclerostin elevated significantly at protein and gene levels under compression forces after 24 h, while the application of tensile forces induced the expression of periostin and its upstream regulator RUNX2 (p < 0.05). Gene expression up-regulation was significant for POSTN and RUNX2 after 48 and 72 h tensile forces. Also, the gene expression of sclerostin reduced in a time-dependent manner after application of tensile force. The compression forces enhanced apoptosis to 7.5 ± 3.5% and induced gene expression of apoptotic markers of CASP9, and BCL2 within 72 h of exposure. Periostin and sclerostin play an important role in orthodontic loads and their expressions are affected oppositely by compressive and tensile forces that might be suggested as a biomarker for assessment of bone remodeling during orthodontic treatment.

The Role of Low-Frequency Electromagnetic Fields on Mesenchymal Stem Cells Differentiation: A Systematic Review.

BACKGROUND: Low-frequency electromagnetic fields (EMFs) influence biological processes. This present study was aimed at the scientific literature on the use of EMFs in the mesenchymal stem cell differentiation process. MATERIALS AND METHODS: The electronic search was carried out in PubMed and Web of Science, a database with a combination of the sinusoidal and pulsed low- and extremely low-frequency electromagnetic fields stimulation and mesenchymal stem cells differentiation, considering the period of publication until December 2021. The literature search identified 118 references in PubMed and Web of Science of which 46 articles were selected, respectively, according to the eligibility requirements. CONCLUSION: The analysis of research indicated that EMFs are an easy-to-apply and practical way in cell therapy and tissue engineering when regulation of stem cells is required. Studies have shown that EMFs have positive effects on stem cell differentiation, accelerating its process regardless of the parameters and type of stem cells. However, the exact amplitude, frequency, duration of the electrical field, and application method remain elusive and need more study in future work.

Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates.

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

Evaluation of alginate modification effect on cell-matrix interaction, mechanotransduction and chondrogenesis of encapsulated MSCs.

Mesenchymal stem cells (MSCs) are promising cell candidates for cartilage regeneration. Furthermore, it is important to control the cell-matrix interactions that have a direct influence on cell functions. Providing an appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of MSCs. In this study, hydrogels consisted of different proportions of alginates that were modified using gelatin, collagen type I and arginine-glycine-aspartic acid (RGD) and were evaluated regarding their effects on mesenchymal stem cells. The effect of applying hydrostatic pressure on MSCs encapsulated in collagen-modified alginate with and without chondrogenic medium was evaluated 7, 14 and 21 days after culture, which is a comprehensive evaluation of chondrogenesis in 3D hydrogels with mechanical and chemical stimulants. Alcian blue, safranin O and dimethyl methylene blue (DMMB) staining showed the chondrogenic phenotype of cells seeded in the collagen- and RGD-modified alginate hydrogels with the highest intensity after 21 days of culture. The results of real-time PCR for cartilage-specific extracellular matrix genes indicated the chondrogenic differentiation of MSCs in all hydrogels. Also, the synergic effects of chemical and mechanical stimuli are indicated. The highest expression levels of the studied genes were observed in the cells embedded in collagen-modified alginate by loading after 14 days of exposure to the chondrogenic medium. The effect of using IHP on encapsulated MSCs in modified alginate with collagen type I is equal or even higher than using TGF-beta on encapsulated cells. The results of immunohistochemical assessments also confirmed the real-time PCR data.

The chromene derivative 4-Clpgc inhibits cell proliferation and induces apoptosis in the K562 cell line.

Chronic myeloid leukemia (CML) is a malignant blood disease with a particular chromosomal aberration that is known as a common form of leukemia. The chromene family exhibits strong anti-cancer effects. Therefore, the effects of six members of the dihydropyrano [2,3-g] chromene family on cell toxicity and apoptosis induction in K562 cancer cells were investigated and compared with those of normal peripheral blood mononuclear cells (PBMCs). The K562 cells were cultured in the presence of the aforementioned chromene derivatives at concentrations of 40 to 200 µM for 24 to 72 h. The effects of these compounds on the growth and viability of the K562 cell line and PBMCs were studied through MTT assay. Furthermore, apoptosis induction was investigated using flow cytometry. Real-time PCR was used for relative quantification of BCL2, Bax, TP53 and BCR- ABL genes after 48 h of exposing K562 cells and PBMCs to 4-Clpgc. Based on the results, these chromene derivatives inhibited the growth of K562 cells. According to the obtained data, 4-Clpgc was the strongest compound with IC50 values of 102 ± 1.6 µM and 143 ± 9.41 µM in K562 cells and PBMCs, while pgc was the weakest one with IC50 levels of 278 ± 2.7 µM and 366 ± 47 µM in K562 cells and PBMCs (after 72 h), respectively. The results demonstrated that the apoptotic cell percentage in the control group increased from 6.09% to 84.10% and 17.2% to 20.06% in K562 cells and PBMCs after 48 h of treatment, respectively. Moreover, 4-Clpgc treatment increased the expression of Bax and TP53 genes by 42.74 and 35.88 folds in K562 cells and 9.60 and 7.75 folds in PBMCs, respectively. On the other hand, the expression of BCL2 was reduced by 1.47 and 1.38 folds in K562 cells and PBMCs, respectively. These compounds were associated with less toxic effects on normal cells, compared to the cancer cells. In conclusion, these derivatives can be considered as appropriate candidates for leukemia treatment.

Combination of Mechanical and Chemical Methods Improves Gene Delivery in Cell-based HIV Vaccines.

OBJECTIVE: Novel vaccination approaches are required to control human immunodeficiency virus (HIV) infections. The membrane proximal external region (MPER) of Env gp41 subunit and the V3/glycans of Env gp120 subunit were known as potential antigenic targets for anti-HIV-1 vaccines. In this study, we prepared the modified dendritic cells (DCs) and mesenchymal stem cells (MSCs) with HIV-1 MPER-V3 gene using mechanical and chemical approaches. METHODS: At first, MPER-V3 fusion DNA delivery was optimized in dendritic cells (DCs) and mesenchymal stem cells (MSCs) using three mechanical (i.e., uniaxial cyclic stretch, equiaxial cyclic stretch and shear stress bioreactors), and two chemical (i.e., TurboFect or Lipofectamine) methods. Next, the modified DCs and MSCs with MPER-V3 antigen were compared to induce immune responses in vivo. RESULTS: Our data showed that the combination of equiaxial cyclic stretch loading and lipofectamine twice with 48 h intervals increased the efficiency of transfection about 60.21 ± 1.05 % and 65.06 ± 0.09 % for MSCs and DCs, respectively. Moreover, DCs and MSCs transfected with MPER-V3 DNA in heterologous DC or MSC prime/ peptide boost immunizations induced high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses as well as an increased level of Granzyme B. Indeed, the modified MSCs and DCs with MPER-V3 DNA could significantly enhance the MPER/V3-specific T-cell responses compared to MPER/V3 peptide immunization. CONCLUSIONS: These findings showed that the modified MSC-based immunization could elicit effective immune responses against HIV antigen similar to the modified DC-based immunization.

Efficacy of mechanical vibration in regulating mesenchymal stem cells gene expression.

This study aimed at investigating the expression of osteoblast and chondrocyte-related genes in mesenchymal stem cells (MSCs), derived from rabbit adipose tissue, under mechanical vibration. The cells were placed securely on a vibrator's platform and subjected to 300 Hz of sinusoidal vibration, with a maximum amplitude of 10 µm, for 45 min per day, and for 14 consequent days, in the absence of biochemical reagents. The negative control group was placed in the conventional culture medium with no mechanical loading. The expression of osteoblast and chondrocyte-related genes was investigated using real-time polymerase chain reaction (real-time PCR). In addition, F-actin fiber structure and alignment with the help of actin filament fluorescence staining were evaluated, and the level of metabolic activity of MSCs was determined by the methyl thiazolyl tetrazolium assay. The real-time PCR study showed a significant increase of bone gene expression in differentiated cells, compared with MSCs (P < 0.05). On the other hand, the level of chondrocyte gene expression was not remarkable. Applying mechanical vibration enhanced F-actin fiber structure and made them aligned in a specific direction. It was also found that during the differentiation process, the metabolic activity of the cells increased (P < 0.05). The results of this work are in agreement with the well-accepted fact that the MSCs, in the absence of growth factors, are sensitive to low-amplitude, high-frequency vibration. Outcomes of this work can be applied in cell therapy and tissue engineering, when regulation of stem cells is required.

An investigation into osteogenic differentiation effects of silk fibroin-nettle (Urtica dioica L.) nanofibers.

The purpose of this study was to investigate physical, mechanical, and osteogenic properties of silk fibroin (SF) nanofibers containing Urtica dioica L. (nettle) extract at different concentrations. In this respect, the successful incorporation of nettle in SF nanofibers was analyzed and then confirmed through Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The mean fiber diameter, water uptake, breaking strain, cellular attachment, and proliferation of the given nanofibers also increased as the nettle content was added, while this trend was opposite in terms of tensile strength and modulus. The in vitro release studies correspondingly demonstrated that the nettle release had been controlled according to Fickian diffusion and it was faster in the samples including more nettle. Furthermore, both ARS staining and real-time RT-PCR results suggested that nettle had enhanced the expression of both early and late markers of osteoblast differentiation in a dose-dependent manner.

Modified DCs and MSCs with HPV E7 antigen and small Hsps: Which one is the most potent strategy for eradication of tumors?

Immunotherapy with DCs as antigen-presenting vehicles have already improved patients' outcome against a variety of tumors. Moreover, MSCs were recently used to develop anti-cancer therapeutic or anti-microbial prophylactic vaccines. The current study evaluated immune responses and anti-tumor effects generated by DCs and MSCs derived from mouse bone marrow which were modified with small heat shock proteins 27 and 20 (sHsp27 and sHsp20) and also E7 oncoprotein in tumor mouse model. Two vaccination strategies were utilized including homologous DC or MSC prime/ DC or MSC boost, and heterologous MSC or DC prime/ protein boost vaccinations. Our data revealed that DCs pulsed with E7+Hsp27 and/or E7+Hsp20 in homologous and heterologous prime/ boost vaccinations could stimulate high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses. Moreover, these regimens induced an increased level of Granzyme B, and displayed complete protection more than 60 days after treatment. On the other hand, MSCs transfected with E7+Hsp27 DNA in homologous and heterologous prime/ boost vaccinations could significantly enhance the E7-specific T-cell responses and suppress tumor growth in mice. However, MSCs transfected with E7+Hsp20 DNA did not induce a complete protection against TC-1 tumor compared to DCs pulsed with E7+Hsp20 protein complexes. These results indicated that DC- and MSC-based vaccinations with specific modalities will be a useful approach for immunotherapy and protection against HPV-associated cancers.

Enhanced gene delivery in tumor cells using chemical carriers and mechanical loadings.

Intracellular delivery of DNA is considered a challenge in biological research and treatment of diseases. The previously reported transfection rate by commercially available transfection reagents in cancer cell lines, such as the mouse lung tumor cell line (TC-1), is very low. The purpose of this study is to introduce and optimize an efficient gene transfection method by mechanical approaches. The combinatory transfection effect of mechanical treatments and conventional chemical carriers is also investigated on a formerly reported hard-to-transfect cell line (TC-1). To study the effect of mechanical loadings on transfection rate, TC-1 tumor cells are subjected to uniaxial cyclic stretch, equiaxial cyclic stretch, and shear stress. The TurboFect transfection reagent is exerted for chemical transfection purposes. The pEGFP-N1 vector encoding the green fluorescent protein (GFP) expression is utilized to determine gene delivery into the cells. The results show a significant DNA delivery rate (by ~30%) in mechanically transfected cells compared to the samples that were transfected with chemical carriers. Moreover, the simultaneous treatment of TC-1 tumor cells with chemical carriers and mechanical loadings significantly increases the gene transfection rate up to ~ 63% after 24 h post-transfection. Our results suggest that the simultaneous use of mechanical loading and chemical reagent can be a promising approach in delivering cargoes into cells with low transfection potentials and lead to efficient cancer treatments.

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems.

OBJECTIVE: In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. RESULTS: The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p < 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~ 83.94 and ~ 77.01% for HEK-293T), and cancer cells (~ 71.69 and ~ 67.19% for TC-1, and ~ 73.86 and ~ 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~ 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~ 63.66%) compared to TurboFect (~ 32.95%); however, the efficiency level of hPP10 was only ~ 17.51 and ~ 16.36% in TC-1 and A549 cells. CONCLUSIONS: Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

Mechanical and Chemical Predifferentiation of Mesenchymal Stem Cells Into Cardiomyocytes and Their Effectiveness on Acute Myocardial Infarction.

Myocardial infarction is one of the leading causes of death all over the world. Mesenchymal stem cells (MSCs) transplantation has shown a promising potential to recovery of ischemic heart disease due to their capability in differentiating into cardiac cells. However, various investigations have been performed to optimize the efficacy of cardiac cell therapy in recent years. Here, we sought to interrogate the effect of autologous transplantation of undifferentiated and predifferentiated adipose and bone marrow-derived MSCs in a rabbit model of myocardial infarction and also to investigate whether cardiac function could be improved by mechanically induced MSCs via equiaxial cyclic strain. The two sources of MSCs were induced toward cardiomyocyte phenotype using mechanical loading and chemical factors and thereafter injected into the infarcted myocardium of 35 rabbits. Echocardiography and histopathology studies were used to evaluate cardiac function after 2 months. The results demonstrated significant scar size reduction and greater recovery of left ventricle ejection fraction after transplantation of predifferentiated cells, though the differences were not significant when comparing mechanically with chemically predifferentiated MSCs. Thus, although there was no significant improvement in infarcted myocardium between chemically and mechanically predifferentiated MSCs, mechanically induced cells are more preferred due to lack of any chemical intervention and cost reasonableness in their preparation method. Outcomes of this study may be useful for developing future therapeutic strategies, however long-term assessments are still required to further examine their effectiveness.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-ß1.

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-ß1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

The stability evaluation of mesenchymal stem cells differentiation toward endothelial cells by chemical and mechanical stimulation.

Adipose-derived mesenchymal stem cells (ADSCs) are adult multipotent cells able to differentiate into several cell lineages. Vascular endothelial growth factor (VEGF) and the shear stress associated with blood flow are considered as the most important chemical and mechanical cues that play major roles in endothelial differentiation. However, the stability of endothelial-specific gene expression has not been completely addressed yet. ADSCs in passage 3 were cultured inside the tubular silicon tubes and then exposed to VEGF or shear stress produced in a perfusion bioreactor. To investigate the differentiation, the expression levels of Flk-1, von Willebrand factor (vWF), and vascular endothelial-cadherin (VE-cadherin) were studied using Real-Time PCR. For studying the endothelial differentiation stability, mRNA levels of the genes were evaluated in certain time intervals after completion of the tests so as to determine whether the expression level of each gene in different time points was stable and remained constant or not. Application of VEGF and shear stress caused an elevation in endothelial cells' specific genes. Although there are some changes following the days after application of mechanical and chemical stimuli, the gene expression results depicted significantly higher gene expression between sequential chemically and mechanically incited groups. In conclusion, stress alone can be a differentiating factor, by itself. Our results verified the efficient stable differentiation ability of the chemical and mechanical factors.

Computational simulation of static/cyclic cell stimulations to investigate mechanical modulation of an individual mesenchymal stem cell using confocal microscopy.

It has been found that cells react to mechanical stimuli, while the type and magnitude of these cells are different in various physiological and pathological conditions. These stimuli may affect cell behaviors via mechanotransduction mechanisms. The aim of this study is to evaluate mechanical responses of a mesenchymal stem cell (MSC) to a pressure loading using finite elements method (FEM) to clarify procedures of MSC mechanotransduction. The model is constructed based on an experimental set up in which statics and cyclic compressive loads are implemented on a model constructed from a confocal microscopy 3D image of a stem cell. Both of the applied compressive loads are considered in the physiological loading regimes. Moreover, a viscohyperelastic material model was assumed for the cell through which the finite elements simulation anticipates cell behavior based on strain and stress distributions in its components. As a result, high strain and stress values were captured from the viscohyperelastic model because of fluidic behavior of cytosol when compared with the obtained results through the hyperelastic models. It can be concluded that the generated strain produced by cyclic pressure is almost 8% higher than that caused by the static load and the von Mises stress distribution is significantly increased to about 150kPa through the cyclic loading. In total, the results does not only trace the efficacy of an individual 3D model of MSC using biomechanical experiments of cell modulation, but these results provide knowledge in interpretations from cell geometry. The current study was performed to determine a realistic aspect of cell behavior.

Effect of extremely low frequency electromagnetic field on MAP2 and Nestin gene expression of hair follicle dermal papilla cells.

INTRODUCTION: In recent years, the extremely low frequency electromagnetic field (ELF-EMF) has attracted a great deal of scientific interest. The ELF-EMF signal is able to control ion transport across ion channels and therefore induce cell differentiation. AIM: The purpose of this study was to investigate the effect of ELF-EMF (50 Hz, 1 mT) on MAP2 and Nestin gene expression of dermal papilla mesenchymal cells (DPCs). METHODS: In order to examine the effect of chemical and electromagnetic factors on gene expression, 4 experimental groups, namely chemical (cell exposure to chemical signals), EMF (exposing cells to ELF-EMF), chemical-EMF (subjecting cells to chemical signals and ELF-EMF) and control (with no treatment) groups, were prepared, treated for 5 days, and studied. To assess the effect of extended test time on the expression of neural differentiation markers (Nestin and MAP2), an EMF group was prepared and treated for a period of 14 consecutive days. The beneficial role of EMF in inducing neural differentiation was shown by real-time PCR analysis. RESULTS: The higher expression of MAP2 after 14 days compared to that after 5 days and decrease of cell proliferation on days 5 to 20 were indicative of the positive effect of extending treatment time on neural differentiation by evaluation of gene expression in EMF group.

Extremely Low Frequency Electromagnetic Field in Mesenchymal Stem Cells Gene Regulation: Chondrogenic Markers Evaluation.

There is little evidence demonstrating the effects of electromagnetic fields (EMFs) generated within the biological entity and the effect of extrinsic fields on cellular programing. Taking the path of the more studied stimuli into attention, mechanical forces, it could be understood that nonchemical factors play a consequential role in transcriptional regulatory networks. Cartilaginous tissue consists of collagen protein that is considered as a piezoelectric substrate and is influenced by electric fields making chondrogenic specific genes an exciting candidate for bioelectromagnetic studies. As electromagnetic properties highly depend on the frequencies applied, this study delves into the ability of two EMFs with the frequency of 25 Hz and 50 Hz in inducing SOX9 and COL2 gene expressions in a three-dimensional (3D) mesenchymal stem cell (MSC)-alginate construct. Cell-alginate beads were divided into six groups and treated for a time period of 21 days. To determine the results, qualitative and quantitative data were both reviewed. On observation of real-time polymerase chain reaction (PCR) data, it was apparent that TGF-ß1 treatment had a greater COL2 and SOX9 gene expression impact on MSCs compared to pulsed electromagnetic field (PEMF) treatments alone. COL2 was shown to have a greater transcriptional tendency to PEMF, whereas under defined electromagnetic parameters applied in this study, no significant difference was detected in SOX9 gene expressions compared to the control group. PEMF co-treatments enhanced the deposition of extracellular matrix molecules, as the matrix-rich beads were positively stained by Alcian blue. This genre of study is the venue for the control and healing of connective tissue defects.

Fluid-Structure Interactions Analysis of Shear-Induced Modulation of a Mesenchymal Stem Cell: An Image-Based Study.

Although effects of biochemical modulation of stem cells have been widely investigated, only recent advances have been made in the identification of mechanical conditioning on cell signaling pathways. Experimental investigations quantifying the micromechanical environment of mesenchymal stem cells (MSCs) are challenging while computational approaches can predict their behavior due to in vitro stimulations. This study introduces a 3D cell-specific finite element model simulating large deformations of MSCs. Here emphasizing cell mechanical modulation which represents the most challenging multiphysics phenomena in sub-cellular level, we focused on an approach attempting to elicit unique responses of a cell under fluid flow. Fluorescent staining of MSCs was performed in order to visualize the MSC morphology and develop a geometrically accurate model of it based on a confocal 3D image. We developed a 3D model of a cell fixed in a microchannel under fluid flow and then solved the numerical model by fluid-structure interactions method. By imposing flow characteristics representative of vigorous in vitro conditions, the model predicts that the employed external flow induces significant localized effective stress in the nucleo-cytoplasmic interface and average cell deformation of about 40%. Moreover, it can be concluded that a lower strain level is made in the cell by the oscillatory flow as compared with steady flow, while same ranges of effective stress are recorded inside the cell in both conditions. The deeper understanding provided by this study is beneficial for better design of single cell in vitro studies.

The synergistic effects of shear stress and cyclic hydrostatic pressure modulate chondrogenic induction of human mesenchymal stem cells.

PURPOSE: In this study, we examined chondrogenic regulation of 2 types of mesenchymal stem cells seeded on the bioengineered substrate in monolayer cultures under mechanically defined conditions to mimic the in vivo microenvironment of chondrocytes within articular cartilage tissues. METHODS: Human adipose-derived mesenchymal stem cells (ASCs) and bone marrow mesenchymal stem cells (BSCs) were exposed to 0.2 Pa shear stress, 3 MPa cyclic hydrostatic pressure, and combined loading with different sequences on chemically designed medical-grade silicone rubber, while no soluble growth factors were added to the culture medium. The expression levels of chondrogenic-specific genes of SOX9, aggrecan, and type II collagen (Col II) were measured. Results were compared to those of cells treated by biological growth factor. RESULTS: Gene expression patterns were dependent on the loading regime. Moreover, the source of mesenchymal stem cells (adipose or bone marrow) was influential in gene expression. Overall, enhanced expression of chondrogenic markers was found through application of mechanical stimuli. The response was generally found to be significantly promoted when the 2 loading regimes were superimposed. CONCLUSIONS: Differentiation of ASCs was shown by a modest increase in gene expression profiles. In general, BSCs expressed higher levels of chondrogenic gene expression than ASCs after 3 weeks. A greater effect on Col II and SOX9 mRNA expression was observed when combined loadings were applied. Results may be applied in determining the proper loading sequence for obtaining functional target cells in cartilage engineering applications.

Evaluation of Mechanical and Chemical Stimulations on Osteocalcin and Runx2 Expression in Mesenchymal Stem Cells.

The osseous tissue repair and regeneration have great importance in orthopedic and maxillofacial surgery. Tissue engineering makes it possible to cure different tissue abnormalities using autologous grafts. It is now obvious that mechanical loading has essential role in directing cells to differentiation. In this study, the influence of cyclic uniaxial loading and its combination with chemical factors on expression of osteogenic markers was investigated. Rat bone marrow-derived stem cells were isolated and cultured. In one group cells were maintained in chemical induction medium. In another group cells were subjected to cyclic uniaxial strain with 3% amplitude and 0.3 Hz frequency for 24 hours and in the last group cells were affected by induction medium and physical stimulation. TaqMan Real time PCR and immunocytochemistry were done to evaluate gene expression variations. Moreover, a small incision was made to access the bone of the cranium and induced cells were seeded on collagen based scaffolds and finally the cell seeded scaffolds were implanted. Results indicated that mechanical loading alone caused a phenomenal increase in Runx2 and osteocalcin expression. Remarkable increment in gene expression was gained when induction medium were added to mechanical stimulation. The order of chemical and mechanical stimulation caused different effects and results were much better when the cells were affected by mechanical strain at first. Histological analysis showed mechanical stimulation could promote bone ingrowth in vivo. These evidences demonstrated that combination of chemical factors with mechanical strain was much more effective for directing osteogenesis since these elements have synergistic effects.