The RT-PCR results showed that ADSCs could be differentiated into chondroid cells expressing chondrocyte-specific markers such as COL II, Aggrecan, and SOX9. When differentiated to the 12th day, the expression of COL II, Aggrecan, and SOX9 was close to that in normal chondrocytes, but subsequently fell. Therefore, through our PCR results, we inferred that ADSCs might be differentiated to mature chondroid cells at 12th day after induction, but after that their differentiated state is not maintained. Additionally, expression of the dedifferentiated marker gene COL I increased, behaving in an opposite manner to the differentiation markers. From this, we see that the extension of differentiation time does not improve the differentiation rate and indeed leads to dedifferentiation. Because no clear morphological markers of dedifferentiation are apparent under an inverted microscope, we employed other methods to observe the sequential morphological variation over the course of differentiation at nanometer scale. Because the cell membrane is not only a barrier between the intracellular environment and extracellular world but also a regulator of many important biological processes such as signal transduction, material transportation, and energy exchange, we looked for variation in the cell membrane structure accompanying with the change of cellular function; in this case, the level of differentiation.
AFM is a powerful tool for nanobiological studies, so we first used AFM to compare the ultrastructure of chondroid cells and NC and attempt to explain the relationship between cell dysfunction and its ultrastructure.
We obtained visual data of appearance and size, as well as dynamic changes of R a and R q on the nanometer scale using this method. In our experiment, we observed that ADSCs were irregular, long spindle shape with a round and extruded nucleus, but 12DD and NC were triangular or polygonal with flat and compact nuclei and endochylema. Both R a and R q in 12DD were close to those NC. Though there was no obvious morphological change with 21DD, we still obtained the change of R a data. The R a value of 21DD was reduced distinctly and membrane protein arrangement changed from regular porous arrangement to more of line and clusters. Taking the PCR data, we conclude that dedifferentiation after the 12th day is responsible for the ultrastructure changes. We hope the visual and quantitative data will be helpful in analyzing the differentiation process of ADSCs to mature chondroid cells and revealing a mechanism of cell destabilization in the late stage.
Obtaining of cell biomechanical data was another strength of AFM. Recent studies found that mechanical properties of a cell may be used as phenotypic biomarkers. Therefore, we inferred that the functional change of cells caused by late stage dedifferentiation may also be observed through the cellular mechanics. To test this, we measured adhesion force and Young’s modulus across the whole differentiation process to further support the changes in function and cell surface ultrastructure.
Adhesion force mostly represents the number and distribution of cell surface adhesion molecules. Our force-distance curve shows that during chondrogenic differentiation, adhesion force gradually increases to the maximum at the 12th day, but this value is slightly lower than that of NC, and then the value decreases as differentiation continues. Adhesion force corresponds to the change of R a. Our data demonstrate a trend of adhesion force that is in accordance with R a in the process of chondrogenic differentiation. Quantity and distribution of cell surface proteins directly affects R a data. Surface particle numbers increased, causing the cell membrane to be uneven and rough thereby increasing R a. The higher adhesion force and R a value of 12th day are due to the increase of biomacromolecule particles on the mature chondroid cells, which interact more with the AFM needle. Likewise, as differentiation continued, there were fewer cell surface adhesion proteins, and the adhesion force and R a decreased. Thus, the dedifferentiation of chondroid cells was relative to the decrease of cell surface proteins.
Expression of adequate adhesion proteins is important for cells to attach in cartilage lacuna, which is necessary for stable synthesis and secretion of extracellular matrix (ECM) proteins. It is crucial for chondrocytes to remain differentiated to function properly. We chose integrin β1 as a representative adhesion protein for this experiment because it is widely expressed and is the main adhesion molecule in chondrocytes[26, 27]. Then, we detected the distribution of integrin β1 through LCSM. We found integrin β1 on the cell membrane and the dynamic tracing of integrin β1 revealed a maximum fluorescence intensity of integrin β1 on the 12th day. In parallel, we used flow cytometry to test the quantity of integrin β1, and this supported the maximum at day 12, although the quantity did not reach that of NC. The qualitative and quantitative changes of integrin β1 in these groups correspond to R a and adhesion force results, so we conclude that dedifferentiation of chondroid cells may be directly related to loss or involution of integrin β1.
Acting as a bridge between ECM and the cytoskeleton, integrin not only transmits signals between the cell and the ECM but also regulates cytoskeletal arrangement and therefore cell rigidity[28, 29]. We then wanted to test if the change of integrin β1 is accompanied with the change of cell rigidity, and we did so using AFM to measure cell Young’s modulus of each differentiation stage. We found that Young’s modulus increased gradually throughout the differentiation process. It came to the maximum at 21DD and was higher than NC in 15DD, 18DD, and 21DD. Young’s modulus of 12DD was similar to that of NC, having no statistically significant difference. Our data imply that 12DD had the most ideal stiffness and elasticity for chondrocytes.
The stiffness of cells is related to their physiological roles, and cartilage cells in particular require stiffness to bear and transmit a stress load. Reduction in elasticity would prevent the cartilage from buffering the vibrations from stress loads. We observed that the stiffness of chondroid cells increased continuously in the late stage differentiation, reducing cell deformability and perhaps causing cell degeneration. This is an important consideration in tissue engineering of cartilage as opposed to normal cartilage, because the continual increase in stiffness could negate the therapeutic effect of regenerative cartilage tissue. We speculate the improper rigidity of 21DD chondroid cells might be an objective manifestation and the intrinsic factor of degeneration.