A cellular stimulation device utilizing an AT-cut quartz coverslip mounted on an ultrasonic live imaging chamber is developed to investigate the effect of piezoelectric stimulation. Two types of chambers deliver ultrasound at intensities ranging from 1 to 20 mW/cm2 to mesenchymal stem cells (MSCs) seeded on the quartz coverslip. The quartz coverslip imposes additionally localized electric charges as it vibrates with the stimulation. The device was applied to explore whether piezoelectric stimulation can facilitate chondrogenesis of MSCs. The results suggest piezoelectric stimulation drove clustering of MSCs and consequently facilitated chondrogenesis of MSCs without the use of differentiation media.

Chondrogenesis of mesenchymal stem cells (MSCs) is seen as a crucial step in the regenerative tissue engineering approach for cartilage repair (Somoza et al., 2014; Lolli et al., 2019; Kim et al., 2020) and for callus formation in bone fractures (Einhorn and Gerstenfeld, 2015; Xue et al., 2019). Different stimulations have been investigated to specify proper conditions for MSC differentiation into chondrocytes, including growth factors, cell-cell interaction, and biomaterials (Yu et al., 2012). Experimental evidence has shown that SOX9 plays a critical role in chondrogenesis, especially to secure chondrocyte lineage commitment (Lefebvre and Dvir-Ginzberg, 2017). The Wnt signaling pathway is known to play a role in chondrogenic differentiation of MSCs (Naito et al., 2015; Xie et al., 2018; Diederichs et al., 2019). As non-canonical Wnt signaling is also relevant to planar cell polarity (Wallingford and Mitchell, 2011), one possible way to facilitate chondrogenesis from MSCs is to artificially induce cell polarity in MSCs with external stimuli. This can be done, for example, by an electrical charge distribution in a piezoelectric substrate mediated by ultrasound across the MSC culture.

Piezoelectricity is widely available in our body and plays a role in bone physiology due to a high concentration of collagen (Ahn and Grodzinsky, 2009). Piezoelectricity of type I (Minary-Jolandan and Yu, 2009) and type II (Denning et al., 2014) collagen has been measured and reported as shear piezoelectric. Collagen distribution in bone is the basis for a biologically inspired engineering approach to create an artificial implant for bone regeneration (Yu et al., 2017). To extend the concept in vitro, ultrasonically mediated piezoelectric substrates were used to promote differentiation of neuron-like PC12 cells (Hoop et al., 2017) and human osteosarcoma SaOS-2 cells (Genchi et al., 2018). Ultrasonically excited piezoelectric nanoparticles were also suggested to inhibit proliferation of breast cancer cells (Marino et al., 2018). These experiments used piezoelectric films as cell substrates, requiring high intensity (>1 W/cm2) ultrasound for stimulation. At this intensity level, other physical effects accompanying the wave propagation, such as heat generation, cavitation, radiation force, or acoustic streaming (Padilla et al., 2014), are also affecting cells during the stimulation. Ultrasound alone has been reported to facilitate the effect of progranulin (Uddin et al., 2015) and transforming growth factor beta (Ebisawa et al., 2004) on chondrogenesis of MSCs. In addition to ultrasound, low frequency dynamic loading to piezoelectric substrates was also reported to promote osteogenetic differentiation from MSCs (Ribeiro et al., 2015) and proliferation of pre-osteoblastic cells (Ribeiro et al., 2012). In addition to planar substrates, three dimensional piezoelectric scaffolds were also used to promote MSC chondrogenic differentiation (Damaraju et al., 2017).

While a direct link between piezoelectric stimulation and cell differentiation is lacking, the study by Hoop et al. (2017) suggested piezoelectric stimulation affected cell polarity at a voltage level of 400 mV, but not at 20 mV. The voltage was induced in the piezoelectric PVDF film by immersing the cell culture in an ultrasound bath (80 W, 132 kHz) for 10 min. In order to deliver a broader range of piezoelectric stimulation for chondrogenesis of MSCs, we extended the previously designed live imaging chamber (LIC), with intensity around 1 mW/cm2 (Chu et al., 2019), to a higher intensity of 10–20 mW/cm2. We refer to this new chamber as LIC-Pro. The design of LIC-Pro is motivated by bringing the ultrasound stimulation energy level close to those used in therapeutic applications (30 mW/cm2). While stimulation by LIC is sufficient to engage cell adhesion related mechanisms (Chu et al., 2019), it is difficult to relate the observations to clinical applications due to the large energy difference. In order to make biological observations clinically relevant, we developed LIC-Pro with output of the same order as the clinical applications. Wee replaced the glass coverslip of both LIC and LIC-Pro with an AT-cut quartz coverslip to create piezoelectric stimulation. An AT-cut quartz plate is a thickness shear mode resonator (He et al., 2013) and has been used to monitor cell/substrate adhesion (Hong et al., 2006). Upon ultrasound excitation, the quartz coverslip exhibits a dynamic charge distribution according to the excitation mode (He et al., 2013). In this work, we report the effect of piezoelectric stimulations on chondrogenesis of MSCs using these two chambers.

In our previous LIC chamber design, four disk-type 1 MHz transducers (15 mm in diameter, 2.1 mm in thickness, Eleceram Tech Co. Ltd, Taoyuan, Taiwan) were placed on top of the base plate to generate Lamb waves propagating along the glass coverslip [Fig. 1(a), left panel]. When the coverslip is loaded with media, ultrasound energy is leaked into the media for stimulation. This design offers the unique advantage of uniform acoustic field across the chamber space. To keep this advantage while increasing ultrasound intensity, we redesigned the top cover and placed four transducers directly on top of it [Fig. 1(a), right panel]. This allows a direct transmission of ultrasound energy from the top plate to the coverslip. The difference in acoustic paths between the two chambers is illustrated in Fig. 1(b). We validated and characterized the design using a hydrophone (HGL-1000 by Onda, Sunnyvale, US) immersed in the chamber to measure ultrasound radiated from the coverslip. Input of the transducer was controlled by a portable control unit based on the STEVAL-IME011V1 ultrasound pulser evaluation board (STMicroelectronics, Geneva, Switzerland).

Fig. 1.

(a) Schematic diagrams of LIC and LIC-Pro chambers. AT-cut quartz coverslips were used to replace glass coverslips. (b) Illustration of acoustic paths inside LIC and LIC-Pro chambers. (c) Hydrophone output (mVpp) measured across the chamber space of LIC-Pro. (d) Comparison of measured ultrasound intensity with 1 MHz continuous waves between LIC and LIC-Pro chambers. (e) Measured temperature changes during ultrasound exposure in LIC-Pro: under the transducer, inside the chamber, and ambient. Calculated displacement mode (f), shear stress mode (g), and electric field mode (h) for 1 MHz forced oscillation of AT-cut quartz coverslip in LIC-Pro.

Fig. 1.

(a) Schematic diagrams of LIC and LIC-Pro chambers. AT-cut quartz coverslips were used to replace glass coverslips. (b) Illustration of acoustic paths inside LIC and LIC-Pro chambers. (c) Hydrophone output (mVpp) measured across the chamber space of LIC-Pro. (d) Comparison of measured ultrasound intensity with 1 MHz continuous waves between LIC and LIC-Pro chambers. (e) Measured temperature changes during ultrasound exposure in LIC-Pro: under the transducer, inside the chamber, and ambient. Calculated displacement mode (f), shear stress mode (g), and electric field mode (h) for 1 MHz forced oscillation of AT-cut quartz coverslip in LIC-Pro.

Close modal

The measured amplitude of a short burst (to avoid interference with chamber boundaries) across the chamber space [Fig. 1(c)] shows a reasonably uniform acoustic field, suggesting that direct excitation from the top plate does not disturb the uniformity. We also compared the intensity of continuous waves measured in two chambers [Fig. 1(d)]. The results show higher variation due to reverberation from the chamber boundaries and a tenfold increase in energy level by LIC-Pro, while the intensity remains lower than those used in therapeutic applications (>30 mW/cm2). This increase is mainly due to a direct acoustic path in LIC-Pro in comparison to multiple reflections in the acoustic path of LIC. As the transducers in LIC-Pro were placed closer to the incubation space, we also measured the temperature profile during ultrasound exposure. The temperature of LIC-Pro increases 1.5 °C after 5 min of ultrasound stimulation [Fig. 1(e)]. This is higher compared to the 0.4 °C increase for the same stimulation period in LIC (Chu et al., 2019). For this reason, we limit the exposure time for LIC-Pro to be within one minute so that the temperature increase is less than 0.5 °C.

Circular AT-cut quartz coverslips of 30 mm in diameter and 0.17 mm in thickness were obtained from Mustec Corp (Hsinchu, Taiwan). The diameter is chosen to fit LIC and LIC-Pro chambers and the thickness is selected to meet the microscopic imaging needs. We calculated forced excitation patterns of the AT-cut quartz plate at 1 MHz with comsol multiphysics (Comsol, Stockholm, Sweden). The maximal displacements [Fig. 1(f)] is 0.1 μm, maximal shear stresses [Fig. 1(g)] 4 MPa, and maximal electrical field [Fig. 1(h)] 400 kV/m on the quartz coverslip in LIC-Pro. As expected, shear stress and electric field share a similar distribution pattern.

To explore the effect of piezoelectric stimulation, we used bone marrow derived mesenchymal stem cells (Lonza, Basel, Switzerland). The MSCs were seeded on a glass or quartz coverslip 24 h before experiments in MSC growth media as suggested by the cell provider. Two cell cultures were exposed to piezoelectric stimulation for one minute in the LIC-Pro chamber and then kept in an incubator (Touch 190 from LEEC, Nottingham UK) for observations. One cell culture was added with chondrogenic differentiation media suggested by the cell provider after the piezoelectric stimulation, while the other one was kept in the MSC growth media. Cell morphology and distribution did not change immediately after the piezoelectric stimulation [day 1 in Fig. 2(a)]. However, day 2 after piezoelectric stimulation cell cultures with differentiation media started to show a clear clustering pattern [Fig. 2(a)] while the control group remains evenly distributed.

Fig. 2.

(a) Images of mesenchymal stem cells (MSCs) after 1 MHz piezoelectric stimulation with LIC-Pro for one minute. Clustering of MSCs occurred since day three. (b) Images of mica power patterns created by 1 MHz piezoelectric stimulation. (c) Comparison between patterns of mica powders (left), cell clustering by piezoelectric stimulation without differentiation media (upper right panel), and cell nodules by piezoelectric stimulation with differentiation media (lower right panel). (d) Alcian blue tests confirmed chondrogenesis of MSCs is possible by 1 MHz piezoelectric stimulation in the LIC-Pro chamber with (lower panel) and without differentiation media (upper panel). (e) Images of β-catenin in control and piezoelectric stimulation (cells fixed right after 5-min piezoelectric stimulation in LIC) groups. The β-catenin signal increases in both cytoplasm and nucleus in response to piezoelectric stimulation. Upper panels are β-catenin and lower panels are merged nucleus and β-catenin images. Scale bar refers to 50 μm. (f) Comparison of nuclear β-catenin signal level (immunofluorescent grey level) in control and piezoelectric stimulation.

Fig. 2.

(a) Images of mesenchymal stem cells (MSCs) after 1 MHz piezoelectric stimulation with LIC-Pro for one minute. Clustering of MSCs occurred since day three. (b) Images of mica power patterns created by 1 MHz piezoelectric stimulation. (c) Comparison between patterns of mica powders (left), cell clustering by piezoelectric stimulation without differentiation media (upper right panel), and cell nodules by piezoelectric stimulation with differentiation media (lower right panel). (d) Alcian blue tests confirmed chondrogenesis of MSCs is possible by 1 MHz piezoelectric stimulation in the LIC-Pro chamber with (lower panel) and without differentiation media (upper panel). (e) Images of β-catenin in control and piezoelectric stimulation (cells fixed right after 5-min piezoelectric stimulation in LIC) groups. The β-catenin signal increases in both cytoplasm and nucleus in response to piezoelectric stimulation. Upper panels are β-catenin and lower panels are merged nucleus and β-catenin images. Scale bar refers to 50 μm. (f) Comparison of nuclear β-catenin signal level (immunofluorescent grey level) in control and piezoelectric stimulation.

Close modal

We visualized the forced oscillation mode of the quartz coverslip using mica powders (First Chemical Works, Taipei, Taiwan) and compared the mica powder pattern with the MSC clustering pattern. The quartz oscillation mode under piezoelectric stimulation shows a weaver pattern [Fig. 2(b)] similar to the simulated shear stress/electric field mode [Fig. 1(f)–1(g)]. When compared with the MSC clustering patterns, the mica powder pattern matches nicely with both cell cultures with and without the differentiation media [Fig. 2(c)]. The match between the forced oscillation mode and the MSC clustering patterns suggests the electric charge distribution is well represented by the mica powder pattern. It is possible that these spatial charge peaks create a center to align cell polarity of its neighboring cells and to induce cell clustering subsequently. Furthermore, the clustered cells with and without differentiation media were verified to have undergone chondrogenesis [Fig. 2(d)] using Alcian blue staining (Merck, Darmstadt Germany). The results showed that piezoelectric stimulation alone can induce chondrogenesis of MSCs without differentiation media.

Our hypothesis is that cell clustering observed in the chondrogenesis process [Fig. 2(a)] might be caused by noncanonical planar cell polarity Wnt signaling. On the other hand, Wnt/ β-catenin signaling also plays various roles in different stages of cartilage development (Candela et al., 2014); we thus compared β-catenin signals between control and piezoelectric stimulation. Activation of the canonical Wnt signaling pathway is known to safeguard β-catenin in cytoplasm and eventually translocate β-catenin to the nucleus (Rao and Kuhl, 2010). Our results [Figs. 2(e) and 2(f)] demonstrate clearly that piezoelectric stimulation elevates β-catenin, indicating activation of the canonical Wnt signaling pathway in MSCs. We argue that piezoelectric stimulation might interact with Wnt receptors in MSCs and activate canonical and noncanonical signaling pathways throughout the chondrogenesis process.

Because both ultrasound and piezoelectric stimulation have been reported to facilitate chemically induced chondrogenesis of MSCs, we performed a comparative experiment to systematically analyze the effectiveness of chondrogenic induction by differentiation media (DM), ultrasound (US), and piezoelectric (PE) stimulation. Experiments of six cell culture groups with three factors, i.e., the DM, US, and PE, were designed. The MSCs were seeded on glass or quartz coverslips and received ultrasound or piezoelectric stimulation of 5 min on day one and day two. The differentiation media were added in day 3 [see Fig. 3(a) for experimental protocol]. The cell culture with differentiation media and piezoelectric stimulation started clustering on the fifth day and nodule forming on the seventh day [Fig. 3(b)] while other groups did not show visible changes in this stage. This suggests the combination of differentiation media with piezoelectric stimulation speeds up chondrogensis of MSCs. Alcian blue staining was performed on day 11 to examine the differentiation results. Among the six groups tested, two groups (control without differentiation media and ultrasound without differentiation media) failed to undergo chondrogenic differentiation [Fig. 3(c)]. The other four groups underwent chondrogenic differentiation to a certain degree.

Fig. 3.

(a) Experimental protocols for a comparative study of MSC chondrogenesis in different combinations of DM, US, and PE stimulation. (b) Images of MSCs on days 5 and 7. The group with piezoelectric stimulation and differentiation media shows cell clustering (indicated by red arrows) on day 5 and nodule forming (indicated by red arrow) on day 7. (c) Images of Alcian blue tests on day 11. Two groups (control and ultrasound without differentiation media) failed to produce chondrocytes. (d) Area ratio (blue cell area divided by total cell area) for four groups (control and ultrasound with differentiation media, piezoelectric stimulation with and without differentiation media) showed the degree of successful chondrogenesis. (e) SOX9 protein levels measured on day 3 as an early indication of differentiation.

Fig. 3.

(a) Experimental protocols for a comparative study of MSC chondrogenesis in different combinations of DM, US, and PE stimulation. (b) Images of MSCs on days 5 and 7. The group with piezoelectric stimulation and differentiation media shows cell clustering (indicated by red arrows) on day 5 and nodule forming (indicated by red arrow) on day 7. (c) Images of Alcian blue tests on day 11. Two groups (control and ultrasound without differentiation media) failed to produce chondrocytes. (d) Area ratio (blue cell area divided by total cell area) for four groups (control and ultrasound with differentiation media, piezoelectric stimulation with and without differentiation media) showed the degree of successful chondrogenesis. (e) SOX9 protein levels measured on day 3 as an early indication of differentiation.

Close modal

We analyzed the percentage of cell areas of chondrogenesis upon treatment of ultrasound and/or piezoelectric stimulation. The percentage of differentiated cell area against the total cell area was normalized with respect to the control group with differentiation media [Fig. 3(d)]. It is clear that piezoelectric stimulation without differentiation media creates a sixfold result compared to the control group, and 13 folds with differentiation media. To validate piezoelectric stimulation facilitates chondrogenesis of MSCs independently, we analyze the SOX9 protein level with western blotting among different groups on the third day as an early sign of differentiation. All groups gave a twofold or higher SOX9 level than the control group [Fig. 3(e)]. The results confirmed piezoelectric stimulation produces an elevated SOX9 level similar to the group with differentiation media. However, we also observed SOX9 elevation in ultrasound stimulation group without differentiation media, suggesting that an increase in SOX9 does not necessarily result in chondrogenesis.

In this study, we demonstrated the integration of an AT-cut quartz coverslip with two types of imaging chambers for piezoelectric stimulation. Our cellular experiment showed that both energy levels of piezoelectric stimulation can induce chondrogenesis of MSCs even without differentiation media. This points to the possibility for a cost effective in vitro chondrogenesis approach based on MSCs. Furthermore, the results also suggest that piezoelectricity might play a role in creating a physiological niche for chondrogenesis. While piezoelectricity is abundantly available in bones, its relevance in physiology was unclear (Ahn and Grodzinsky, 2009). Our results provide the first direct evidence that piezoelectricity might play a role in chondrogenesis.

Clustering of MSCs after piezoelectric stimulation [Figs. 2(b) and 3(a)] indicated possible formation of cell polarity as migration itself already demonstrates front-rear polarity (Li and Dudley, 2009; Gao et al., 2011; Randall et al., 2012; Kuss et al., 2014; Vassilev et al., 2017). Clustering of MSCs cells and concentric arrangement of the clustered cells may be mimicking the in vivo environment of chondrogenic cells aligned to planar cell polarity (Randall et al., 2012). Since mesenchymal cell aggregation may be required for chondrogenesis (Randall et al., 2012), the sequential events after piezoelectric stimulation likely were initiated by establishment of cell polarity axis, followed by cell clustering and up-regulation of chondrogenic marker such as SOX9. Planar cell polarity represents one of the noncanonical Wnt signaling pathways (Rao and Kuhl, 2010). Together with the canonical Wnt signaling results induced by piezoelectric stimulation [Figs. 2(e) and 2(f)], we argue piezoelectric stimulation might interact with Wnt receptors in MSCs and consequently activate canonical and noncanonical Wnt signaling pathways throughout the whole chondrogenesis process. The canonical Wnt signaling causes translocation of β-catenin from cytoplasm to nucleus while noncanonical planar cell polarity Wnt signaling causes cell clustering. Our results suggest that the cell polarity in this experimental setting is associated with the dynamic charge distribution on the quartz plate due to piezoelectric stimulation. Such a dynamic charge distribution might also occur in physiology during ultrasound treatments for bone fracture healing. Further study is needed to clarify the role of piezoelectricity in chondrogenesis of tissue regeneration or ultrasound bone fracture treatment.

We thank Dr. Dun-Yen Kang at National Taiwan University for the access to the comsol package. This study was supported by Ministry of Science and Technology, Taiwan (Grant Nos. MOST 107-2221-E-002-068-MY3, MOST107-2321-B-002-040, MOST 107-2321-B-010-012, 108-2321-B-002-061-MY2), National Health Research Institute, Taiwan (Grant No. NHRI-EX109-10924EI), and National Taiwan University (Grant No. NTU-CC-107L891105).

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