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RISS 인기검색어
- 검색키워드
- 저자 : ( Sayan Deb Dutta ) (검색결과 8 건)
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( Sayan Deb Dutta ),( Tusan Park ),( Keya Ganguly ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Most bone tissue-engineering models fail to demonstrate the complex cellular functions of living bone; therefore, most translational studies on bone tissue are performed in live models. To reduce the need for live models, we developed a stimulated micro-chip model for monitoring protein secretion during osteogenesis using human mesenchymal stem cells (hBMSCs). We established a bone micro-chip system for monitoring the in vitro differentiation and sensing the secreted proteins of hMSCs under a sinusoidal electromagnetic field (SEMF), which ameliorates bone healing in a biomimetic natural bone matrix. A 3V-1Hz SEMF biophysically stimulated osteogenesis by activating ERK-1/2 and promote phosphorylation of p38 MAPK kinases. Exposure to a 3V-1Hz SEMF upregulated the expression of osteogenesis-related genes, and enhanced the expression of key osteoregulatory proteins. hBMSCs undergoing osteoblastic differentiation are responsive to a well-defined SEMF stimulation at 3 V and 1 Hz, with an amplitude of 0.1 mT, for 20 minutes once a day. SEMF-treated plates contained the highest number of viable cells (>11%) compared to the other groups. Constant treatment with higher voltage, amplitude, or for a longer duration is generally counterproductive, suggesting that these parameters are optimized for this cell type. Our findings suggest that low voltage-frequency SEMF-guided osteoblast differentiation is not only triggered by the exposure time or dosage, but is also affected by various physicochemical factors. The activation of ERK1/2 and phosphorylation of p38 factors regulates the osteogenic differentiation during SEMF stimulation. Moreover, the bioinformatics analysis revealed the activation of various genes that are differentially expression only during SEMF stimulation but not in unstimulated condition. Notably, the gene expression of ALP (1.3 fold) and OCN (5.2 fold) was significantly higher (*p<0.05, **p<0.01) in cells receiving 3 V-1 Hz (0.1 mT, 20 min/day) stimulation compared to in the control. Significant increases in the expression of osteogenic gene markers were also observed for Runx2, OSX, BSP, OPN, and COL1, compared to in the unstimulated cells (*p<0.05). Six secretome proteins were upregulated in stimulated hBMSCs compared to in unstimulated hBMSCs. Overall, 23 proteins were either upregulated or downregulated by SEMF stimulation. These proteins are predicted to be secreted or included in secretory vesicles according to annotations in the Uniport database. Out of the upregulated proteins in SEMF-stimulated hBMSCs, 36% or 50% are involved in the 'immune response' or 'extracellular matrix function,' respectively. The unprecedented efficacy of our low-voltage-frequency SEMF exposure protocol for achieving hBMSC osteogenesis has broad clinical and practical implications, and could form the basis of SEMF-based therapeutic strategies for stem cell-based bone tissue regeneration. Our on-chip stimulation technology is easy to use, versatile, and non-disruptive, and should have diverse applications in regenerative medicine and cell-based therapies.
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( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
3D bioprinted hydrogel has gained enormous attention, especially in tissue engineering, owing to its attractive structure and excellent biocompatibility. In this study, we demonstrated that 3D bioprinted cell-laden ‘thermoresponsive’ poloxamer-407 (P407) gels have the potential to stimulate osteogenic differentiation of apical papilla stem cells (SCAPs) under the influence of low voltage- frequency 1V-1Hz (0.14 mT), 5V-1Hz (0.62 mT), and 10V-1Hz (1.21 mT) electromagnetic fields (EMFs). Their exposure time was 5 min/day, 10 min/day, 20 min/day, and 30 min/day for each hydrogel group, respectively. The developed hydrogel exhibited higher mechanical strength as well as good printability, showing high-quality micro-architecture. Moreover, the as-printed hydrogels (5 mm × 5 mm) were loaded with plasminogen activator inhibitor-1 (PAI-1) for testing the combined effect of PAI-1 and EMFs on SCAP differentiation. Interestingly, the 3D hydrogels showed improved viability and differentiation of SCAPs under EMFs' influence as examined by live/dead assay and alizarin Red-S staining, respectively. Our results demonstrated that DSPP and DMP-1 markers' expression significantly increased (3.8-fold) in 5V (0.62 mT) EMFs treatment. A similar fashion was also observed in ALP and Col-1(2.80-fold), comparable to the control groups. Therefore, the higher expression of theses gene markers (DSPP, DMP-1 ALP, and Col-1) indicated their better osteogenic efficiency of P407-encapsulated SCAPs in the presence of EMFs. The results confirmed that P407 hydrogels are non-toxic for encapsulation of SCAPs, yielding high cell viability and accelerate the cell migration potential. The 3D hydrogels with PAI-1 exhibited high mRNA expression levels for osteogenic/odontogenic gene markers (ALP, Col-1, DSPP, and DMP-1) vis-à-vis control after 14 days of in vitro culture. Our findings suggest that 3D bioprinted P407 hydrogels are biocompatible for SCAP encapsulation, and the applied low voltage-frequency EMFs could effectively improve dental tissue regeneration, particularly for oral applications.
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( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Mesenchymal stem cells (hMSCs) are considered as one of the promising approaches for treating several diseases in cell-based therapeutics. The current strategies for hMSCs expansion include the in vitro static culture system yielding a low number of cells. Therefore, a scalable, dynamic bioreactor-based culture system is needed to produce an adequate number of cells for clinical applications. In this study, a new strategy for hMSCs expansion is employed with a modified wave-motion bioreactor system coupled with continuous electromagnetic field (EMF) stimulation. For this purpose, hMSCs were tested in various EMF exposure (1V, 5V, and 10V-1Hz) to find the maximum viability for cell expansion. Interestingly, the hMSCs tend to form aggregate-like structures during the wave motion culture. We show that a 10V-1Hz EMF exposure (magnetic field: 5.82 G) during wave motion culture (35 rpm) induces the viability of hMSCs up to 15% than static culture as evaluated by WST-8 assay. Moreover, the long-term cell culture of hMSCs in wave bioreactor significantly enhanced the expression of osteogenic transcription factors, such as Runx2 (6.8-8.0 fold), ALP (8.0 fold), BSP (5-6.0 fold), and OCN (8-10.0 fold) compared to the static culture (28% higher as of control). We also found that the average glucose consumption was significantly higher compared to static culture as predicted by Six biosensor B.LV5. In the initial phase of cell culture, the average detectable level of glucose was 8.35±0.34 mM/μL and lactate was 0.63±0.05 mM/μL. However, after 7 days of dynamic culture, the available glucose in the media was estimated about 2.57±0.32 mM/μL and lactate was 7.67±0.26 mM/μL which was significantly higher than the static control group. The RNA-sequencing results further indicate the upregulation of major osteogenic/mechanically stimulated signaling pathways during wave motion culture of hMSCs. These results suggested that the use of 10V-1Hz (5.82 G) EMF is non-toxic for hMSCs, and the modification of wave bioreactor with externally controlled EMF has good potential for hMSCs proliferation and expansion.
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( Sayan Deb Dutta ),( Keya Ganguly ),( Dinesh Patel ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Most bone tissue-engineering models fail to demonstrate the complex cellular functions of living bone; therefore, most translational studies on bone tissue are performed in live models. To reduce the need for live models, we developed a stimulated micro-chip model for monitoring protein secretion during osteogenesis using human mesenchymal stem cells (hBMSCs). We established a bone micro-chip system for monitoring the in vitro differentiation and sensing the secreted proteins of hMSCs under a sinusoidal electromagnetic field (SEMF), which ameliorates bone healing in a biomimetic natural bone matrix. A 3V-1Hz SEMF biophysically stimulated osteogenesis by activating ERK-1/2 and promote phosphorylation of p38 MAPK kinases. Exposure to a 3V-1Hz SEMF upregulated the expression of osteogenesis-related genes, and enhanced the expression of key osteoregulatory proteins. We identified 23 proteins that were differentially expressed in stimulated hBMSC secretomes, or were absent in the control groups. Our on-chip stimulation technology is easy to use, versatile, and non-disruptive, and should have diverse applications in regenerative medicine and cell-based therapies.
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( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Electrical stimulation has been shown to ameliorate bone healing for a long time. This study developed a hybrid and 3D printable conductive methacrylated gelatin-polypyrrole (GelMA-PPy)-based photocurable and self-healing hydrogel inks for continuous microcurrent stimulation. For this, a custom-made electrical stimulation device (DC stimulation) was used to evaluate the osteogenic differentiation of human bone mesenchymal stem cells (hMSCs). The pyrrole was chemically grafted onto the surface of GelMA via a one-step conjugation reaction with ammonium persulfate (APS) and cross-linked with iron (III) chloride. The fabricated hydrogel was characterized by proton nuclear magnetic resonance (1H-NMR), scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) to analyze the grafting and chemical interaction. The as-prepared hydrogel exhibited higher mechanical and swelling properties compared to pure GelMA scaffolds. The 3D printed GelMA-PPy hydrogel showed improved cell viability (~12.6%) when exposed to 500 mV/day current, compared to pure GelMA after 7 and 14 days of cell culture. Interestingly,16.0~20.0-fold higher expression of osteogenic genes and protein markers (Runx2, ALP, OCN, and OPN) were observed in the GelMA-PPy treated groups than control suggest that pyrrole incorporation into GelMA matrix significantly improved the conductivity and osteogenic differentiation of hMSCs. Therefore, this study shows that the hMSCs react differentially to low-voltage DC electrical stimulation in the presence of GelMA-PPy scaffolds, which could be used as an ideal material for electrical stimulation for tissue engineering.
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( Keya Ganguly ),( Sayan Deb Dutta ),( Dinesh Patel ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
The rapid infiltration and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) on 3D printed osteogenic scaffolds represents a promising strategy to promote bone remodeling. However, the effect of constant wear and tear resulting from physical activities on mechanical integrity and functionality of tissue engineering constructs is crucial to determine before scaffold implantation. Therefore, we have focused on constructing an indigenous pulsatile bioreactor system creating artificial pressure loading microenvironment on GelMA/cellulose nanocrystals 3D-printed hydrogel to analyze the osteogenic differentiation of hBMSCs under a constant pulsatile pressure stimulation. The osteogenic differentiation potential of the hBMSCs seeded onto the fabricated scaffold was monitored after 7 and 14 days of cell culture under a pulsatile regiment of 10 ± 0.8 kPa pressure stimulation. Concurrently, hBMSCs maintained under static condition was taken as the control set. Our results indicated a 14.8% increase in the hBMSCs viability analyzed through the WST-8 assay and Live-Dead assay. Besides, increased adherence efficiency was evident by the morphometric analysis of the actin cytoskeletal arrangements and the expression of focal adhesion protein (Paxillin) determined by the immunocytochemistry analysis. Moreover, the qRT-PCR results after 7 days of cell culture indicated a higher expression of the early osteogenic marker genes, including ALP (7-fold), Runx2 (10.0-fold), OSX (5.8-fold), and mechanosensitive marker genes YAP/TAZ (4.2-fold), confirming the massive onset of early osteogenic differentiation in the hBMSCs. Similarly, the expression of the late osteogenic marker genes, including COL1 (8.0-fold), OPN (15.0-fold), and BSP (12.0-fold), was noted to be higher in cells cultured under pressure stimulation after 14 days of cell culture. Besides, the transcriptome analysis revealed the regulation of mechanosensitive genes during pressure stimulation than static group. The pulsatile pressure mechanical stimulation can be used to predetermine the mechanical potential of bone constructs and rapid osteogenic differentiation of hBMSCs.
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( Keya Ganguly ),( Sayan Deb Dutta ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Bone tissue engineering has emerged as an excellent alternative to the traditional treatment strategies of bone injuries. However, current in-vitro systems do not fully mimic the biological environment of the bone defects. Hence, the design of bioreactor system is worth investing to allow monitoring of the de-novo bone formation in vitro. Herein, we have evaluated the osteogenic differentiation of human bone marrow derived mesenchymal stem cells seeded on to 3D printed nanohydroxyapatite-GelMA (nHAp-GelMA) hydrogel under the different flow rates (0.1 mL/ min, 0.25 mL/ min, and 0.5 mL/ min) in a custom designed perfusion type bioreactor. The mechanical property of the as-fabricated hydrogel increased as the concentration of hydroxyapatite increased from 0.5 to 2%, respectively. Moreover, the incorporation of nHAp into GelMA matrix also improved the swelling efficiency and mineralization potential in vitro. Our results indicated a higher osteogenic differentiation in the hBMSCs under the fluid shear stress as compared to the static cultures. A 10~15% increase in the cell viability was observed under the varying fluid shear stress as determined by the WST-8 assay and the Live-Dead cell viability assay. Moreover, an enhancement in the overall osteogenic differentiation markers in the hBMSCs under the fluid shear stress (as of control) was evident by the qRT-PCR analysis of the osteogenic marker genes including ALP (7.0~9.5-fold), Runx2 (16.5-fold), OSX (6.5-fold), Col1 (18.0-fold), OPN (10.8-fold), and BSP (12.5-fold). The upregulation of the respective proteins was also supported by the transcriptome analysis. Also, applied shear stress induced the secretion of several growth factors and cytokines. Taken together, our study indicated fluid-shear stress by the custom-designed perfusion chamber induces hBMSC differentiation and in vitro bone regeneration.
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( Ki-taek Lim ),( Dinesh Patel ),( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2
Human mesenchymal stem cells (hMSCs) have the potential to differentiate into different types of mesodermal tissues. In vitro proliferation and differentiation of hMSCs are necessary for bone regeneration in tissue engineering. The present study aimed to design and develop a fluid flow mechanically-assisted cartridge device to enhance the osteogenic differentiation of hMSCs. We used the fluorescence-activated cell-sorting method to analyze the multipotent properties of hMSCs and found that the cultured cells retained their stemness potential. We also evaluated the cell viabilities of the cultured cells via water-soluble tetrazolium salt 1 (WST-1) assay under different rates of flow (0.035, 0.21, and 0.35 mL/min) and static conditions and found that the cell growth rate was approximately 12% higher in the 0.035 mL/min flow condition than the other conditions. Moreover, the cultured cells were healthy and adhered properly to the culture substrate. Enhanced mineralization and alkaline phosphatase activity were also observed under different perfusion conditions compared to the static conditions, indicating that the applied conditions play important roles in the proliferation and differentiation of hMSCs. Furthermore, we determined the expression levels of osteogenesis-related genes, including the runt-related protein 2 (Runx2, 1.3-fold), collagen type I (Col1, 1.4-fold), osteopontin (OPN, 1.5-fold), and osteocalcin (OCN, 1.5-fold), under various perfusion vis-à-vis static conditions and found that they were significantly affected by the applied conditions. Furthermore, the fluorescence intensities of OCN and OPN osteogenic gene markers were found to be enhanced with 38% in the 0.035 mL/min flow condition compared to the control, indicating that it was a suitable condition for osteogenic differentiation. Taken together, the findings of this study reveal that the developed cartridge device promotes the proliferation and differentiation of hMSCs and can potentially be used in the field of tissue engineering.
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