The authors have declared that no competing interests exist.
Conceived and designed the experiments: GS RR JP GJM MS HB JB MM EH AB HH. Performed the experiments: RR JB EH MM HH JS GS AB. Analyzed the data: GS JB RR EH MM MS GJM HH AB JP HB JS. Contributed reagents/materials/analysis tools: JB RR HH MM EH JS GS AB. Wrote the paper: MM GS MS RR GJM.
Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young’s modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices.
Most cells transform mechanical stimuli into intracellular signals in a process termed mechanotransduction
In all previous works the mechanical properties of PDMS-based cell substrates cannot be changed after fabrication. Magnetoactive elastomers (MAE)
Therefore, our goals were (1) to generate inherently stable non-viscous PDMS-based cell culture substrata with a Young’s modulus in a biologically relevant range (<100 kPa), which could enhance standard cell culture techniques and (2) to establish compliant MAE cell culture substrata to enable a magnetically tunable elastic modulus and for dynamic mechanotransduction in a cell culture environment.
Elastomeric silicone is generally prepared through platinum-catalyzed addition of vinyl-terminated PDMS to a cross-linker, resulting in a comb-like hydride-functionalized PDMS (
The PDMS is cross-linked via Pt-catalyzed hydrosilylation.
By hydrolysis of the high molecular cross-linking agent, the number of cross-linking sites is reduced and an excess of silanol groups generated. In this way the reaction sites (OH groups) at the surface of the PDMS are created. The surface is consequently enabled for the bonding of aminopropyl-triethoxysilane (APTES) without plasma treatment. This is advantageous, because the plasma treatment may alter mechanical properties of PDMS surface
All indentation data are average values over the entire surface and the indentation depth is 200 µm. (A) Unfilled PDMS matrix; (B) MAE filled with 30 vol. % of CIP. Values represent the average of five separate experiments. Error bars show the standard deviations from the mean.
To avoid iron particle sedimentation during the vulcanization process, a fast reacting room-temperature-vulcanizing Pt-catalyst (Karstedt catalyst) was employed at elevated temperatures. The distribution of iron particles in the elastomer was isotropic. With combined methods it was possible to prepare dimensionally stable substrata with low Young’s moduli (<20 kPa,
We developed devices for the application of magnetic fields to MAE in a standard cell culture environment. The devices are compatible with 24 well cell culture plates or 35 mm Petri dishes, allow placement under an upright microscope and are robust enough to easily withstand 37°C in the humid atmosphere of a cell culture incubator.
(A) Schematic of the static device for generating different
The oscillatory shear test is commonly used to characterize the dynamic mechanical properties of MAE. It allows determination of the complex shear modulus
(A) Shear modulus |
The static device for magnetic field application to MAE in a 24-well cell culture plate and the corresponding values of
(A) Schematic diagram of the device in combination with the well24 cell carrier in a cut illustration. (B) Photograph of the assembled prototype. (C) Dimensions of the device and the positions of the magnetic circuits (top view). (D)
(A) Areas with different levels of
In the absence of an applied magnetic field, good agreement (discrepancy of about 10% was within the uncertainty of measurements) between indentation test and uniaxial compression test results were found for penetration depths from 180 µm.
Varying the magnetic fields generated by a magnetic device such as that shown in
(A) Distribution of engineering strain εxx = Δ
To explore whether these novel, soft PDMS-based substrata of different Young’s moduli would result in elasticity-dependent biological effects, we studied smooth muscle actin (α-SMA) expression in human fibroblasts. Earlier observations had established that α-SMA expression decreases in mesenchymal cells with decreasing substratum stiffness
Cells were plated on substrata of different elasticity, allowed to relax for 4–7 days before being harvested for western blot or prepared for immunofluorescence microscopy. Since rigid glass or plastic dishes constitute the current standard substratum for cell culture, glass coverslips were used for control measurements.
Here, we found that the α-SMA expression was lower when cells were cultivated on the newly devised very soft U1/U2 & M1/M2 (<20 kPa) as compared to more rigid PDMS U3 and MAE M3 (120 kPa) substrata and was highest on hard glass. We made similar observations with MAE of comparable pliability (
(A) Western Blot and (B) immunofluorescence analysis show decreased expression of the myofibroblast marker α-smooth muscle actin with decreasing
To study possible effects of magnetic field-induced changes in MAE properties on adherent cells, we used a device as characterized in
(A) Mean fibroblast cell area 60 min after plating increases with substratum E-modulus. (B) Transcription of the muscle satellite cell marker PAX-7 in human mesenchymal stem cells decreases with increasing substratum E-modulus. Triplicate mean ±SEM. Asterisks indicate significance of difference from controls **p<0.01, *p<0.05.
Substratum elasticity has also been shown to influence mesenchymal stem cell and myotube differentiation
Magnetic fields of time-dependent strength can induce displacements in the MAE surface. In the pilot systems used, the movements were anisometric (
(A) Composite image of (B, red and C, green) depicting displacement extremes. (D) Cell movement in 7 h: 56 min. Composite image of first (E, red in D) and last frame (F, green in D) of a time lapse series with 15 frames/h.
Ultra-soft MAE may be used to generate cell-coated valves or pump systems actuated by magnetic field generators. Furthermore, actuated cell culture substrata may allow to improve the cultivation of specific cell types e.g. to generate muscle or tendon constructs in vitro. To this end, modifications of surface topography or composite constructs with hydrogel surfaces offer additional possibilities.
Our data indicate that dimensionally stable, PDMS-based cell culture substrata with
Vinyl-terminated polydimethylsiloxane can be purchased either from Hanse Chemie or Gelest and Hydride functional silane from Hanse Chemie. The Karstedt catalyst was purchased from Gelest. The plasticizer, low molecule weight silicone oil AK10, was obtained from Wacker Chemie and the carbonyl iron powder purchased from BASF (type SQ, mean diameter of 4,5 µm). All chemicals were used without further purification. The MAE substrates were prepared by the cross-linking of a liquid silicone rubber dispersion containing 30% of CIP by volume.
After thorough compounding using a speed mixer (Hausschild DAC 150.1 FVZ) for 3 minutes at 2500 RPM and removal of air in vacuum (20 min), curing was completed after only one hour at 100°C. Due to the high reactivity of the catalyst system a platinum concentration of 10 ppm was sufficient to accomplish the vulcanization process completely. In order to obtain precisely shaped MAE sheets, high-quality PTFE-coated tools (45×45×2) mm, were used for molding. Finally, the test specimen for tissue cultivation, (12×2) mm, compression test (20×6) mm and rheological characterization, (20×2) mm, was simply die cut.
Three different base elastomers of PDMS/MAE samples were prepared during this work. (cf.
The silicone matrix of U3 and of the MAE sample M3 differ only by the type of cross-linker applied. The ratio of vinyl-polymer to chain extender and the amount of plasticizer were maintained constant. The MAE samples M3 utilized a high-molecular weight cross-linker whereas a comparatively low-molecular-weight cross-linker was applied in the unfilled sample U3. This has been done in order to accommodate for the stiffening of MAE samples due to the presence of CIP.
Three different methods were used to measure the mechanical properties of PDMS-based elastomers. Two of them, namely oscillatory shear test (OST)
The rheological characterization was carried out on a MCR 501 rheometer from Anton Paar, Austria. The shear modulus
To locally examine the mechanical properties of the MAE surface, conventional micro-hardness measurement by the so-called penetration method was used. In this case a Vickers pyramid shaped diamond head penetrates to a depth of 200 µm into the sample surface which barely damages the sample. The automated surface characterization has been performed using a FISCHERSCOPE® HM2000 device with the corresponding software package WIN-HCU® 4.4 supplied by Helmut Fischer GmbH, Sindelfingen, Germany. The measurement parameters using an indenter type H2N 17201110 were as follows: test load 15–20 mN, application duration 20 s. The Poisson’s ratio for MAE samples has been estimated from the literature to
Moreover the indentation device was complemented by calibrated magnetic circuits (cf.
The static magnetic device in
The deformations of the MAE surface (
In the final step image sequences centered at several points equally distributed over the entire MAE surface were digitally recorded using the fluorescence microscope (Zeiss AxioScope.A1, camera AVT Stingray F-125B, frame rate 18 frames/s). The visibility field of the microscope was about 1.7×1.3 mm. The images were processed according to the conventional PIV procedure using JPIV
Human dermal fibroblasts were obtained from Provitro (Berlin, Germany) and human mesenchymal stem cells from Lonza (Basel, Switzerland). Cells were cultured in Dulbeccós Modified Eagle’s Medium (DMEM, PAA Laboratories GmbH; Pasching, Austria) supplemented with 10% heat inactivated fetal calf serum (FCS, Biochrom, Berlin, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin (both from PAA) as suggested by the supplier and used in passages 3–10. Experiments were performed at least three times with similar results. To provide ECM coating for cell attachment, PDMS and MAE surfaces were treated as previously described
Cells were serum-starved for 16 h, plated on the substrata and allowed to adjust for 5 days. Cells were rinsed with cold (4°C) PBS and total cell protein extracts were prepared using a RIPA lysis buffer (20 mM TRIS, 150 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, 1% Deoxycholate, 0.1% SDS) containing phosphatase and protease inhibitors (Phosphatase Inhibitor Cocktail III, Calbiochem/Merck, Bad Soden, Germany; Complete Protease Inhibitor, Roche, Mannheim, Germany). Protein extracts were boiled in Laemmli sample buffer, subjected to SDS polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Amersham, Braunschweig, Germany) using a BioRad gel blotting apparatus. Membranes were blocked in 3% BSA in TBST (10 mM TrisHCl, 150 mM NaCl, 0.1% Tween 20) for 1 hour. Membranes were incubated with primary antibody to α-SMA (Sigma, Schnelldorf) overnight at 4°C and with a peroxidase-conjugated secondary antibody (Jackson Immuno Research, Newmarket, UK) for 60 min at room temperature. After each incubation step, membranes were washed in TBST for 30 min. Peroxidase was visualized by Enhanced Chemoluminescence and exposure to Hyperfilm ECL films (both Amersham, Braunschweig, Germany) for appropriate times.
Cells were rinsed with PBS, gently scraped off the substrate and collected by centrifugation. The cell pellet was then processed using the RNeasy kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. Two µg of extracted RNA were reverse transcribed (Superscript II, Qiagen) using Oligo-dT primers (Promega). A commercially available kit (SYBR Premix Ex Taq II, Takara Bio Inc., Otsu, Japan) was used for SYBR-green-monitored real-time PCR amplification performed in triplicates on a Step One plus cycler (Applied Biosystems, Foster City, U.S.A.). Primers were: β-Macroglobuline (left:
Human dermal fibroblasts were trypsinized, maintained in suspension in a cell culture incubator for 1 hour to allow for equal retraction of all cells and subsequently plated on collagen-coated substrata. 40 min after plating the cells were fixed in 2% paraformaldehyde (Merck, Mannheim, Germany), permeabilized with 0.1% Triton X100 and F-actin was stained with Phalloidin-TRITC (Sigma). After washing in PBS the stained samples were mounted in Vectashield (Vector, Burlingame, U.S.A.) and viewed with a fluorescence microscope (Axio, Zeiss, Oberkochen, Germany). To assess cell spreading, slide labels were blinded and cell area was measured in all cells of three random fields capturing at least 30 cells using NIH-image software. The groups were analyzed in a two-tailed students t-test.
Dermal human fibroblasts were fluorescently labeled using CMTMR celltracker dye following the manufacturer’s instructions (Invitrogen), plated on fibronectin-coated MAE and allowed to spread overnight. Next, the cells were transferred to L - 15 medium (PAA, Pasching, Austria) containing 10% FCS and mounted on the custom-built magnetic field stimulator (