Effects of mechanical stress on chondrocyte phenotype and chondrocyte extracellular matrix expression

Mechanical factors play a key role in regulating the development of cartilage degradation in osteoarthritis. This study aimed to identify the influence of mechanical stress in cartilage and chondrocytes. To explore the effects of mechanical stress on cartilage morphology, we observed cartilages in different regions by histological and microscopic examination. Nanoindentation was performed to assess cartilage biomechanics. To investigate the effects of mechanical stress on chondrocytes, cyclic tensile strain (CTS, 0.5 Hz, 10%) was applied to monolayer cultures of human articular chondrocytes by using Flexcell-5000. We quantified the mechanical properties of chondrocytes by atomic force microscopy. Chondrocytes were stained with Toluidine blue and Alcian blue after exposure to CTS. The expression of extracellular matrix (ECM) molecules was detected by qPCR and immunofluorescence analyses in chondrocytes after CTS. Our results demonstrated distinct morphologies and mechanical properties in different cartilage regions. In conclusion, mechanical stress can affect the chondrocyte phenotype, thereby altering the expression of chondrocyte ECM.


Results
Histologic examination of cartilage in different regions. In order to observe the morphologies of cartilage in intact and damage region, we examined the cartilage by histologically. The sections were stained with safranin-O, hematoxylin and eosin, as well as Toluidine blue. The cartilage samples with smooth surface, intact hyaline cartilage architecture, and no loss of proteoglycan staining were defined as intact regions of the cartilage with OA. By contrast, cartilage samples with fibrillation, clefts, several cell clusters, and extensive loss of proteoglycan staining were defined as damaged regions of the cartilage with OA ( Fig. 1a-c). The histologic score of the damaged region (mean ± SD, 8 ± 1) was significantly higher than that of the intact region (3 ± 1) (Fig. 1d).

Observation of cartilage in different regions with TEM and SEM.
In order to observe the microstructure of the cartilage in the intact and damaged regions, we examined the cartilage by TEM and SEM. In the intact regions, the size of the chondrocyte was relatively normal, and the cell outline was intact and clear; the cellular projections were visible, and the collagen texture was slightly indistinct. In the damaged regions, the amount of finely granular matter increased; similarly, the projections on the chondrocyte surface increased. Meanwhile, the chondrocyte nuclei were deformed, with uneven chromatin distribution and visible fat droplets. The matrix fibers became more cluttered, and the cell outline was unclear or even missing (Fig. 2a). SEM images showed that the articular cartilage in the intact region indicated a relatively normal organization with an undulated course on the surface; this cartilage also showed a superficial cobblestone-like appearance. SEM images showed changes in the structural organization of the damaged region; it exhibited a rough surface with irregular grooves, and the matrix fibers were arranged more loosely; furthermore, the connections were blocked (Fig. 2b).

Assessment of cartilage biomechanics with nanoindentation.
In order to detect biomechanics of the intact and damage cartilage, we performed nano-indentation to assess cartilage biomechanics. Micro-scanning images showed that the articular surface in the damaged region was scraggier and rougher than the intact region, which is consistent with SEM and TEM analyses (Fig. 3a). By using load-displacement curves, we quantified the biomechanical properties of the two regions (Fig. 3b). The cartilage in the intact region was harder than that in the damaged region (Fig. 3c). Moreover, the reduced modulus value of the intact cartilage was significantly higher than that of the damaged cartilage (Fig. 3d). Therefore, nanoindentation testing indicated that the different regions of cartilage exhibited distinct biomechanical properties.
Chondrocytes were stimulated by CTS and observed via TEM and AFM. In order to mimic the cartilage stimulated with stress in vivo, we established a model of chondrocytes exposed to CTS in vitro and observed chondrocytes by SEM. To investigate the biomechanics of chondrocytes, cells were exposed to pre-optimized magnitudes of CTS by using Flexcell-5000 (Fig. 4a). Chondrocytes were exposed to CTS with a magnitude of 10% (0.5 Hz) for 24 h throughout the study. According to TEM analysis, unstimulated chondrocytes showed a euchromatic nucleus, a cytoplasm with an abundant rough endoplasmic reticulum, and several cytoplasmic processes. However, chondrocytes exposed to mechanical stress showed a reduced size of the nucleus with partially condensed chromatin; the cytoplasm contained the swollen rough endoplasmic reticulum and numerous vesicles (Fig. 4b).  In order to determine the biomechanical property of chondrocytes exposed to CTS, we performed atomic force microscopy (AFM) and detected the elastic modulus. Topography images showed that the surface of chondrocytes with CTS was rough, compared with the unstimulated chondrocytes with smooth surface ( Fig. 5a and b). The results indicated that the elastic modulus decreased in chondrocytes stimulated with CTS compared with the unstimulated chondrocytes (Fig. 5c).

Effects of biomechanical stress on chondrocyte ECM expression.
We presume that biomechanical stress could induce the matrix degradation and decrease the matrix synthesis. As mentioned above, chondrocytes were exposed to pre-optimized magnitudes of CTS; the expression levels of COL2A1 and aggrecan were significantly downregulated, whereas the expression levels of MMP1 and MMP13 were significantly upregulated, and ADAMTS5 expression was essentially unaffected by CTS (Fig. 6a). Furthermore, chondrocytes were stained with Toluidine blue and Alcian blue. The results showed that the chondrocytes stimulated with CTS included fewer ECM components than unstimulated cells (Fig. 6b). The ECM-related proteins COL II and MMP13 were also evaluated by immunofluorescence staining. The results showed that the expression of COL II decreased, whereas the expression of MMP13 increased in chondrocytes after CTS (Fig. 7).  Discussion OA is a multifactorial disease characterized by progressive inflammation, pain, and cartilage destruction in load-bearing surfaces of knee joints 16 . Among various pathogenic factors, biomechanical factor is critical for cartilage development, homeostasis, and functionality 17 . Aberrant mechanical stimulation results in a physiological imbalance between mechanical stress on the joint and the joint's ability to withstand such stress. Chondrocytes are directly exposed to compression force during cartilage loading; thus, collagen and other matrix components of the ECM interaction linked to the chondrocytes will likely stretch the cells during cartilage compression 18 . During normal movements in vivo, articular chondrocytes are exposed to compression loads of 15%, leading to 5% tensile strain in chondrocytes 19,20 . Previous studies have confirmed that chondrocytes perceive mechanical stress and respond to it in a magnitude-dependent manner. The mechanical stressors of low magnitude (2% to 8% CTS) were not perceived as inflammatory signals, whereas CTS of high magnitudes was associated with proinflammatory gene expression, which markedly upregulated the matrix degradation and decreased the matrix synthesis [20][21][22][23] . Our study applied CTS with 10% elongation for 24 h in mechanical stress.
A previous study found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15 nm to 2000 nm). The elastic modulus of adult chondrocytes was significantly greater than that of neonatal cells at indentations greater than 500 nm. Furthermore, the intrinsic viscosity was lower in geriatric chondrocytes than in neonatal ones 24 . In addition, the stiffness of chondrocytes obtained from older human individuals (> 55 years old) was greater than that of chondrocytes obtained from younger human individuals    25 . The changes in the elastic and viscoelastic mechanical properties of the cells may influence the synthetic activity of the chondrocytes, which are known to respond to their mechanical environment [26][27][28] . Our study is the first to investigate the mechanics of chondrocytes stimulated by CTS; the elastic moduli decreased in chondrocytes stimulated with CTS compared with the unstimulated chondrocytes.
Many biological processes were involved in mechanical stress, which included but were not limited to the recruitment of adapter proteins and the activation of protein kinases, such as extracellular-signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Consequently, these kinases activate certain transcription factors, thereby resulting in changes in gene expression and metabolism 29 . Mechanical stress has also been reported in chondrocytes, but the present study focused on the effects of biomechanical stress on the cell phenotype and ECM expression in human chondrocytes. The mechanisms by which mechanical stress regulates these changes are not fully understood but are likely interconnected because the chondrocyte mechanical properties, phenotype, and ECM had all been linked to the cytoskeleton structure 30,31 . Many studies have proposed a relationship between the cytoskeleton and biological phenotype from healthy differentiated cells to cancer cells [32][33][34] . These findings showed that both the elastic and viscoelastic properties of chondrocytes can be significantly altered by treatment with agents that disrupted or enhanced the structure of F-actin microfilaments 31,35 . Thus, we presumed that mechanical stress can induce ECM expression by regulating the cytoskeleton arrangement and expression. Collectively, our results demonstrated that different cartilage regions indicated distinct morphologies and mechanical properties. Furthermore, mechanical stress can affect the chondrocyte phenotype and alter the expression of chondrocyte ECM.

Materials and Methods
Patients and specimens. OA cartilage was isolated from the knee joints of 30 patients undergoing total knee arthroplasty. The tissues were processed for histological examination. The informed consent was obtained from all subjects. This study was approved by the Human Ethics Committee of Peking University Third Hospital (China). The methods were performed in accordance with the approved guidelines.
Histological examination. Cartilage specimens were dehydrated in graded alcohols and xylene, embedded in paraffin, and cut serially into 5 mm sagittal sections. The sections were stained with Toluidine blue, safranin-O, as well as hematoxylin and eosin as routine protocol. Changes were graded based on the modified Mankin scale 36 . A score of < 4 points was considered intact cartilage, and a score of > 6 represented damaged cartilage 37 . TEM/SEM. Cartilage specimens were subjected to fixation, gradient dehydration, and embedment. The specimens were sliced into ultra-thin sections, mounted on copper grids, and then stained with uranyl acetate and lead citrate 38 . The cells were grown on pronectin-coated Bioflex six-well culture plates and they were collected by scraping from the plates before fixing in cold Karnovsky fixative for TEM analysis. Afterward, the specimens were observed by TEM, with a Philips EM 208 TEM (Philips Scientifics, Eindhoven, Netherlands). For SEM, the specimens were dehydrated, mounted on aluminum stubs, coated with gold, and then examined using high-resolution SEM S-2500 (Hitachi Ltd., Tokyo, Japan). Nanoindentation/AFM. Biomechanical analysis of cartilage tissues was performed using nanoindentation.
Samples were isolated from different cartilage regions. All indentations were performed using the TriboIndenter (Hysitron Inc., Minneapolis, MN, USA) with a 100-mm radius curvature conospherical diamond probe tip. A trapezoidal load function was applied to each indent site with loading (10 s), holding (2 s), and unloading (10 s). The microscopic geomorphology of the indentation zones was captured using a micro-scanning apparatus 39 . The mechanical properties of the chondrocytes were measured using a Bioscope Resolve AFM (Bruker AXS Corporation, Santa Barbara, CA), following previously established techniques 40 . The light microscope used was DMI6000 (Leica, Wetzlar, Germany). For elastic modulus measurements, chondrocytes were maintained in the medium at room temperature. All presented data were acquired on Bioscope Catalyst TM (Veeco Instruments, Santa Barbara CA, USA).
Culture of primary chondrocytes and exposure to mechanical stress. Donor chondrocytes were isolated as previously described 41 . The chondrocytes were at passage 2 and fibroblasts were used as control. We detect specific markers in both cells by immunofluorescence analysis. The results showed that the expression of Sox9 is high in our chondrocytes, while low in fibroblasts (Supplementary Figure 1A). Meanwhile, expression of S100A4 is low in our chondrocytes, while high in fibroblasts (Supplementary Figure 1B). Chondrocytes (5 × 10 5 / well) were grown on pronectin-coated Bioflex six-well culture plates (Flexcell International, Hillsborough, NC) to 80% confluence. Cyclic tensile strain (CTS) experiments were performed using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA). CTS was enforced at 5%, 10%, and 15% elongation (0.5 Hz) for 24 h 42,43 .
Statistics. Statistically significant differences from multiple groups were calculated using analysis of variance.
Results from the Mankin Score were evaluated using non-parametric tests (chi-square test). Results from the same group were evaluated using t-tests. The results are reported as mean ± SEM. P values less than 0.05 were considered statistically significant. All experiments were performed and analyzed in triplicate. Data analysis was conducted using SPSS software.