The authors have declared that no competing interests exist.
Conceived and designed the experiments: SP WS BM SY AK DK. Performed the experiments: SP WS. Analyzed the data: SP WS BM SY AK DK. Contributed reagents/materials/analysis tools: SP WS. Wrote the paper: SP WS BM SY AK DK.
Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.
The stromal component of bone marrow is known to contain stem cell populations capable of differentiating into adipocytes, chondrocytes, myoblasts and osteoblasts. Despite their therapeutic potential in tissue engineering
Stem cells actively sense, adapt and respond to their surrounding microenvironment and interactively responding to external signals. Stem cell differentiation
In previous macroscale studies, dynamic fluid flow increased mineralized matrix deposition in 3D perfusion culture of marrow stromal cells
Recently, microscale engineering has been increasingly used to mimic the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals
In the present study, a microscale stem cell chip was developed to culture stem cells loaded into separated micro chambers and to assess their comparative responses by dynamic compressive stimulation using a microchip. The osteogenic outcomes of hASCs were compared with hMSCs under the same mechanical stimulation which was assessed using this microscale stem cell chip system. The stem cell microchip was designed to culture the two different kinds of stem cells (hMSCs and hASCs) loaded into separated cell culture chambers, but to apply uniform dynamic compressive stimulation simultaneously. After exposure to mechanical stimulation, the ability of the hASCs towards osteogenic differentiation was assessed by histochemical and immunofluorescent staining, osteogenic related cluster of differentiation (CD) markers and gene expression, all in comparison to the hMSCs. The stem cell microchip developed in this research offers advantages, including those that are generic (i.e. minimizing size, cost, and usage of materials) for microscale systems, as well as new features such as the concentric design of holes and cell chambers for uniform mechanical stimulation, embedded microvalve systems to improve convenience and minimize manual intervention in closing fluidic channels, and compartmentally paired cell culture chambers for collecting statistically relevant data from two different cell types in single experiments.
The new stem cell microchip bioreactor was designed and fabricated (
(A) The microchip is comprised of a cover, an air chamber, looped microvalves, and twelve cell culture chambers. These paired cell chambers share the inlet/outlet channel. The cells (hMSCs and hASCs) are loaded into half of the chip, individually. Scale bar = 1 cm. (B) Schematic diagram of top view (I) and simplified cross-sectional view (II) of the device. The device was designed to culture two different stem cells simultaneously and to apply mechanical stimulation using cyclic pneumatic force. (C) The experimental setup for mechanical stimulation, including a controlled nitrogen gas pressurized air chamber. The frequency of pneumatic pressure is controlled with a switching solenoid valve derived by a control circuit. During mechanical stimulation, microvalves are closed with higher pressure (P2>P1) to prevent undesired shear stress in the cell chambers.
hASCs and hMSCs cultured in the microchip with osteogenic medium for 7 days were stained with ALP and Alrizarin red. The stimulated group of BMSCs resulted in significantly enhanced ALP activity and calcium deposits. (Scale bars: ALP staining 100 (m, Alrizarin red staining 200 (m).
Immunocytochemistry confocal images were taken to examine expression of BSP, OP (representative proteoglycans in osteogenesis) and Col I (representative collagen type in osteogenesis) after 7 days
(A) The expression of osteogenic markers after 1 and 7 days. Bone sialoprotein (BSP), Osteopotin (OP), and Collagen type I (Col I) were stained with GFP and strongly expressed in the stimulated BMSCs. Blue = DAPI Nucleic Acid Stain. (Scale bars: 100 (m) Green fluruorecent expression intensity (B) and area (C) of ECMs in hMSCs and hASCS. Data presented in the line graph represent mean value with SD (n = 12). *p<0.05. Star (*) indicates comparison of statistical difference of stimulation to control and statistical difference between stimulated hMSCs and hASCs.
External stress caused by mechanical stimulation is known to change CD markers of cells. To examine the changes of cell surface receptors of the two stem cell types, CD31 (PE-CAM) and CD11b of β2 integrin were evaluated
(A) Fluorescent expression intensity and area of CD11b in hMSCs and hASCS. (B) Green fluorescent expression intensity and area of CD31 in hMSCs and hASCS. Star (*) indicates comparison of statistical difference of stimulation to control in the same cell type. *
Transcript levels of osteogenic markers such as BSP, OP, Runx-2 and β1 integrin were analyzed by real-time PCR (Brilliant II, Stratagene, USA)
(A) Bone sialoprotein (BSP), (B) Osteopontin (OP), (C) Runt-related transcription factor 2 (Runx2), (D) β1 integrin (*
The importance of mechanical stimulation in the regulation of stem cell differentiation has been identified, thus increasing the need for efficient systems to perform mechanical stimulation on cells in a rapid and controllable manner. The physiological mechanisms by which bone and progenitor cells sense mechanical forces
Previously, we developed microscale stimulation systems actuated by electromagnetic and pneumatic forces for studying osteogenesis of rabbit and human MSCs under mechanical stimulation
hMSCs and hASCs have shown promise as a source of expandable and pluipotent cells for tissue engineering and regenerative medicine
The embedded structure adopted in this microchip also has an advantage in minimizing external contamination. In addition, an on-chip control microvalve system is integrated to minimize unexpected shear stress inside the cell chamber during stimulation by closing the inlet and outlet channels of the cell chamber with relatively high pressure. In the previous experiment using the pneumatic chip, we manually clamped silicone tubes connected to each inlet/outlet ports with locking forceps. This was time consuming and labor-intensive, as well as tricky, because it can expose the cultures to contaminants. The on-chip control microvalve can close multiple inlet and outlet channels simultaneously by applying pressure into the valve control line. The embedded microvalve system is more efficient, convenient, time-saving and safe than the manual method.
The molecular mechanisms responsible for the adaptation of connective tissue to mechanical loading is clinically relevant, especially for bone, but also for other mechanically sensitive tissues
The integrins are composed of noncovalently linked α and β subunits. We analyzed the expression of adherence molecules using CD31 (PE-CAM), CD11b of β2 integrin after exposure to mechanical stimulation. CD11b/CD18 (Mac-1, αmβ2, mo1, CR3) receptors can recognize a wide range of structurally unrelated ligands and transfer the information from the outside to the inside of the cell
CD31 is a cell-adhesion molecule involved in the amplification of integrin-mediated cell adhesion, maintenance of the adherent junction integrity, organization of the intermediate filament cytoskeleton, regulation of transcriptional activities, and control of apoptotic events
Furthermore, we confirmed elevated β1 integrin gene expression by mechanical stimulation (
ALP is a cell surface glycoprotein that is involved with mineralization
Osteogenic genes (BSP, OP and Runx 2) were examined with real-time PCR after 7 days. Cbfa1/Runx2 and Protein C-ets-1 (Ets-1) are transcription factors, which play important roles in regulating the expression of a wide variety of genes responsible for the osteoblast phenotype
The feasibility of using this pneumatically actuated microscale chip was demonstrated as a convenient and effective tool for comparative stem cell studies responsive to mechanical stimulation. The chip reduces the quantity of stem cells required for screening, reduces process costs and time, and increases throughput for various stimulation conditions. In addition, the device has many advantages compared to the previous systems, such as concentric-located holes on each cell chamber for the uniform stimulation, embedded microvalve system for improving convenience and minimizing contamination, and compartmentalized cell chambers for the culture of different types of cells for collecting reliable and statistical data in two different cell types. Mechanical stimuli affect many different physical and biochemical phenomena at the cellular level, including proliferation and biosynthetic activity. With the knowledge gained through this type of bioreactor system and study, new options to understand mechanotransduction and cellular responses to mechanical stimulation can be developed and used to investigate optimal conditions for osteogenesis for bone tissue engineering and regenerative medicine needs.
hMSCs exposed to mechanical stimulation showed distinct ALP and Alrizarin outcomes, while hASCs did not show positive staining under the same experimental conditions. Dynamic compressive mechanical stimulation (1 Hz, 1 psi) increased osteogenic ECM formation (BSP, OP, Col I) and integrin (CD11b and CD31) levels in both stem cell types (hMSCs and hASCs). Upon application of mechanical stimulation to the two types of stem cells, integrin (β1) and osteogenic gene transcripts were upregulated. The results demonstrated that hMSCs were more sensitive to mechanical stimulation compared to hASCs. The microchip presented here, which has embedded concentric-located holes on each cell chamber and a microvalve system, was demonstrated in terms of utility for comparative stem cell studies in response to mechanical stimulation. Further studies are needed to identify the primary osteogenic signals associated with cyclic compressive mechanical stimulation and to determine the mechanism by which these influence commitment to and progression through the osteogenic lineage. By selectively applying specific mechanical stimuli
The stem cell microchip was designed to be able to apply uniform dynamic compressive stimulation to hMSCs and hASCs generated by a pulsatile pneumatic pressure. A photograph and schematics (top and cross-sectional views) of the system are shown in
The device had two main components: one consisting of three poly(methylmethacrylate) (PMMA) substrates and the other with two poly(dimethylsiloxane) (PDMS) layers and one glass substrate. The dimensions of the device were 30 mm×30 mm×10 mm. Both PDMS and PMMA are biocompatible and transparent, so that the cell cultures can be observed with a microscope. The PMMA components, including the cover with air inlet, plate for the air chamber and hole-plate were prepared with a computer controlled laser-cutting machine (VersaLASER, USA). The cover has one air inlet (green circle) at the center, two access holes for the valve control line (blue line) and twelve inlets and outlets (black circles) to access the cell culture chambers (
hMSCs were isolated and expanded using our previously published protocols
hASCs were obtained from a 30 year-old female donor abdomen lipoaspirate (Pennington Biomedical Research Center, Baton Rouge, USA). The hASCs were expanded from collagenase-digested stromal vascular fraction cells in stromal medium consisting of DMEM/F12 Ham's medium, 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin. Cell number and viability were determined using trypan blue exclusion. The resuspended cells were plated at a density of 1.5×105 cells/cm2 and placed in a 5% CO2 incubator at 37°C. The culture medium was changed every other day. Passage two cells were dissociated with 0.25% trypsin–EDTA at 80% confluency before being used for experiments.
To investigate the osteogenic differentiation potential ability of hASCs compared to hMSCs under the dynamic mechanical stimulation, hASCs and hMSCs were separately mixed with medium (density: 2.5×106 cells/mL), and manually loaded into each cell culture chamber through microchannels (width: 300 µm, height: 200 µm) with a 1 mL plastic syringe (BD Medical, USA). The loaded stem cell chips were placed in a humidified incubator (5% CO2, 37°C) overnight. To apply the hydraulic compressive pressure to the stem cells (hMSCs and hASCs), the chips were connected to the pneumatic control setup describe in
To analyze the osteogenic differentiation of stem cells in the microchips, ALP was assessed by histochemical analysis using staining kit (Sigma). Alkaline assay mixture was prepared with the standard recipe (2.4 mg fast violet B salt (Sigma) and 0.4 mL naphthol AS-MX phosphate alkaline solution (Sigma) in 9.6 mL of distilled water). Cells in all culture chambers were incubated in a dark room for 45 min with the alkaline-mixture by injecting solution (over 250 µL in each device) into chambers. For alizarin red staining, cells were fixed in 4% formaldehyde after washing twice with PBS. The cells were stained with 40 mM alizarin red S (pH 4.2, Sigma) for 10 min. All stained cells were observed with a Leica DMIL light microscope (Watzlar, Germany) and Leica Application Suite (v3.1.0) software after washing twice with PBS.
To stain for cell response, cell culture medium was gently removed and cell culture chambers were gently washed twice with PBS (pH 7.4). Subsequently, the samples were fixed with 4% paraformaldehyde solution for 10 min at room temperature. The 4% paraformaldehyde was removed with three PBS washes. The cells were then permeabilized with PBS (pH 7.4) containing 0.2% Triton X-100 for 10 min, and blocked with PBS (pH 7.4) containing 1% bovine serum albumin (BSA) for 30 min. After dilution, the solution was placed onto each sample for 30 min with two subsequent PBS rinses. Primary antibodies for type I collagen (rabbit, Abcam, Inc., Cambridge, MA), bone sialoprotein (BSP) (rabbit, Abcam, Inc., Cambridge, MA), and osteopontin (OP) (rabbit, Abcam, Inc., Cambridge, MA) were diluted from their respective stock solutions to 5–10 µg/mL concentrations in PBS. Then 250 µL of antibody solution was placed into each chamber on devices and incubated at 4°C for 3 hours. The samples were then washed 3 times with PBS and stained using fluorescein isothiocyanate (FITC) (anti-rabbit, Abcam, Inc., Cambridge, MA) as secondary antibody, in which a 10 µg/mL dilution was prepared. A 250 µL aliquot of secondary antibody solution was added into each chamber for 1 hour with two subsequent PBS rinses.
The changes in surface markers on the stimulated stem cells were examined by immunofluorescence staining on cells in a monolayer using FITC-conjugated anti-human monoclonal antibodies, CD11b (Thy-1, Abcam) and CD31 (PECAM1, Abcam). Cells were fixed for 5 min in 4% paraformaldehyde and washed twice with PBS. FITC-monoclonal anti-CD11b and CD31 were applied for assessment of the expression of proteins. After diluting, washing twice with PBS for 10 min each time, actin filaments were stained using Texas Red-X phalloidin stain (Invitrogen, Inc., Grand Island, NY), which was diluted using 10 µL of methanol stock reagent and 400 µL of PBS for each sample.
Confocal microscopy was carried out to examine cytoskeleton and extracellular matrix (ECM) structures. The middle z-section images of cells were taken using a Leica TCS SP2 AOBS confocal microscope (Leica, Mannheim, Germany) equipped with 488 nm argon and 543 nm He/Ne lasers. Phalloidin staining was excited at 543 nm and emission collected between 580 and 650 nm. FITC secondary antibody excitations were at 488 nm, and emission collected between 500 and 550 nm. Image J software (Ver. 1.44P, NIH) was used to quantify the mean fluorescent intensity and the area (%) occupied by positive staining, following immunohistology of osteogenic ECMs and CD markers.
Total RNAs from each specimen were extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and Micro RNeasy Micro kit (Qiagen, Hilden, Germany). hMSCs and hASCs culture chambers were filled with Trizol. All detached cells were collected into 1.5 mL tubes after 30 min. Chloroform (100 µL) was added to the solution and incubated for 5 min at room temperature. Tubes were again centrifuged at 12,000 g for 15 min and the upper aqueous phase was transferred to a new tube. All samples were homogenized by vortexing for 1 min after adding 20 ng carrier RNA (5 µL of a 4 ng/µL solution). Continuously, one volume of 70% ethanol (v/v) was added and applied to an RNeasy minElute spin column.
The RNA samples were reverse transcribed into cDNA using oligo (dT)-selection according to the manufacturer's protocol (High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA). Runx 2, BSP and OP levels were quantified using the Mx3000 Quantitative Real Time PCR system (Stratagene, La Jolla, CA) for osteogenesis and β1-integrin for a cell surface marker. All data analysis employed the Mx3500 software (Stratagene) based on fluorescence intensity values after normalization with an internal reference dye and baseline correction. Differences of gene expression were generated by a using comparative Ct method (Ct [delta][delta] Ct comparison). Ct values for samples were normalized to the endogenous housekeeping gene. PCR reaction conditions were 2 min at 50°C, 10 min at 95°C, and then 50 cycles at 95°C for 15 s, and 1 min at 60°C. The data were normalized to the expression of the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) within the linear range of amplification and differences
Statistical difference in biochemical and mechanical quantitative analysis were determined using the Mann-Whitney U test (Independent t-test, SPSS). Statistical significance was assigned as *
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