Chondrocytes respond to mechanical stimuli through regulating gene expression, proliferation, and metabolic functions. However, little is known about the key genes, signalling pathways, and proteins. Chondrocytes have been considered a post-mitotic tissue with nearly no cellular turnover. They are surrounded by an extracellular matrix comprised of glycosaminoglycan (GAG) and collagen and are subjected to daily dynamic compression. During the in vitro culture, growth factors such as bone morphogenetic protein (BMP) and the TGF-β superfamily are indispensable for the chondrogenic differentiation of MSCs [16]. However, compared to native cartilage, cartilage induced by TGF-β alone showed inferior mechanical properties [17]. Dynamic compression was proved to stabilise the chondrogenic phenotype by inhibiting hypertrophy in the presence of TGF-β3 [18]. To sum up, dynamic compression is essential for inducing non-hypertrophic chondrogenesis of MSCs.
Furthermore, in Huang’s [15] original study, the results revealed that the timing of applying dynamic compression was important. The loading initiated soon after MSCbeing encapsulated into agarose, led to reduced mechanical properties. In contrast, loading initiated after chondrogenic induction and ECM elaboration in the presence of TGF-β3, enhanced the mechanical properties of MSC-seeded constructs. This may be attributed to different mechanotransduction pathways between differentiated and undifferentiated MSCs. Following a shift from the 2% agarose to a denser, cartilage-like construct, the stresses induction was higher. The microarray analysis of the original study showed that several genes from the MMP/TIMP family were significantly modulated. However, the original microarray analysis merely took the fold change of genes into consideration when evaluated the gene importance. This may lead to an inadequate revelation of actual hub genes, as the fold change of genes is not always reliable and proportional to the actual influence on cells. Considering the availability of original data, and the fact that dynamic loading with TGF-β3 is the proven condition that promoted a stable chondrogenic phenotype, this study was built up on one of Huang’s series experiments for further bioinformatics analysis. It explored how compressive stimuli influence the gene expression after chondrogenic induction using TGF-β3, to shed important insight on the mechanism behind. Although the study was initially intended to collect a series of datasets at different time points, the uploaded datasets involving mechanical loading were only available at the time point of day 42. As consequence, a possible loss of some gene information at the initial time point might become inevitable, nevertheless, the long-term gene modulation data at the ending time point was indispensable for analysis. New understanding resulting from the data excavation may contribute towards developing a better strategy to enhance chondrogenic efficiency, quality, and stability.
The high-throughput microarray technology combined with bioinformatics analysis has been widely used in providing new insight into gene expression changes and molecular mechanisms. In the present study, the GEO database was utilised to obtain microarray raw data. A total of 236 DEGs were identified between TGF-β3-induced and TGF-β3/dynamic-compression-induced MSCs, including 178 up-regulated genes and 58 down-regulated genes. After that, the DEGs were analysed by GO functional enrichment analysis and classified into three groups, which were subsequently further clustered, based on functions and signalling pathways.
The results of GO functional enrichment analysis showed that the DEGs were mainly enriched in the GO terms of inflammatory response, in utero embryonic development and negative regulation of angiogenesis. This conforms to previous studies showing that the inflammatory response was involved in chondrogenic regulation. Inflammatory factors have been recognised as an important driving force leading to cartilage breakdown, and their down-regulation is vital for constructing initial collagen networks. A previous animal study revealed that the three-day cyclic compression of 0.5 MPa at 0.5 Hz on bovine chondrocytes counteracted the cartilage degradation induced by IL-1 [19]. Therefore, dynamic loading is not only a stimulator for chondrogenesis, but also an anti-inflammatory factor against pro-inflammatory cytokines. In this study, there were two other GO terms – GO:0001701 (in utero embryonic development) and GO:0016525 (negative regulation of angiogenesis) – that were significantly abnormal between the TGF-β3-induced and TGF-β3/dynamic-compression-induced MSCs. This demonstrates that dynamic compression may affect the anatomical structure development of chondrogenesis. During the early embryogenesis and cartilage maturation, various mechanical stimuli in the microenvironment promote chondrogenesis and limb formation and are responsible for adult chondrocyte phenotype maintenance [20]. Generally, biomechanics has been widely regarded as a promoter of angiogenesis and osteogenesis [21, 22]. On the other hand, cartilage is an avascular system [3], however, the understanding regarding how cartilage maintains avascularity under a mechanical load is limited in the literature, and the underlying biomechanics have not yet been fully established. This study suggests that appropriate mechanical stimuli are vital for inducing less angiogenesis.
Moreover, KEGG pathways enrichment analysis was performed. Because the KEGG database integrates data on genomes, chemical molecules and biochemical systems, including pathways, drug, disease, gene sequences, and genomes, some irrelevant disease clusters might be unexpectedly enriched. These disease-related clusters were screened and removed from the results and discussion. The KEGG pathway enrichment of DEGs and module analysis showed that the PI3K-Akt signalling pathway, toll-like receptor signalling pathway and TNF signalling pathway were highly enriched. Studies have demonstrated that the activation of the PI3K-Akt pathway promotes the terminal differentiation of chondrocytes and inhibits the hypertrophic differentiation of chondrocytes [23, 24]. The toll-like receptors mainly use MyD88-dependent signalling to activate NF-κB to transcript pro-inflammatory cytokines. Moreover, the activation of the toll-like receptor-2 induces the chondrogenic differentiation of MSCs [25, 26]. On the other hand, the mechanical load may promote chondrogenesis by inhibiting the TNF signalling pathway to reduce cartilage degradation. Further investigation is desired to support these findings. In brief, the findings of identified GO terms and the KEGG pathways may provide a theoretical basis on how dynamic compression regulates chondrogenesis.
The PPI network was constructed to predict the connections of proteins encoded by DEGs. The top 10 hub genes were screened according to connection degree as follows: IL6, UBE2C, TOP2A, MCM4, PLK2, SMC2, BMP2, LMO7, TRIM36, and MAPK8. Nine of them functioned in two of the top three most significant modules, suggesting that these genes play a more important role in chondrogenesis and are enhanced by dynamic compression. The Modules 1 and 3 were extracted from the PPI network. UBE2C, TOP2A, MCM4, PLK2, SMC2 LMO7, and TRIM36 were contained in Module 1, which were mainly enriched in GO terms related to the cellular metabolic process. These genes have closed relationships with the cell cycle and proliferation, and some of them were found overexpressed in various tumours. Moreover, UBE2C [27], TOP2A [28] and MCM4 [29] were identified as DEGs in OA. However, to the best of our knowledge, there is as yet no study on how these genes function in MSCs differential regulation were enhanced by mechanical load. This needs further investigation.
It was reported that the downregulation of PLK2 inhibited the degree of inflammation of knee joint synovial tissue and inhibited the cartilage collagen destruction in rats [30]. In recent years, studies have revealed that the SMC family might regulate bone development via mitogenic signals and the Wnt pathway, which is a central pathway in the bone and cartilage differentiation [31]. However, little is known on the specific function of SMC2 in response to mechanical stimuli, which requires further study. LMO7 and TRIM36 are both cell cycle-related genes. The overexpression of TRIM36 decelerates the cell cycle and attenuates cell growth [32], however, their functions in chondrogenesis have not been identified. The IL6 and MAPK8 showed vital roles in Module 3, which GO terms were mainly enriched in response to stimuli and the immune system. The pro-inflammatory cytokine IL6 constitutes an important factor involved in inflammation, immunoregulation, haematopoiesis and tumorigenesis. Its function in chondrogenesis remains controversial. Some studies reported that IL6 inhibited the chondrogenic differentiation [33, 34], while others demonstrated that activating the IL6/STAT3 signalling pathway promoted homeostasis maintenance and cartilage regeneration [35]. It is speculated that mechanical stimulus within the appropriate range of intensity, duration, and frequency may function as a potent anti-inflammatory signal and impose a positive influence on chondrogenesis, while overloading and unloading may lead to cartilage degradation. MAPK8 belongs to the c-Jun N-terminal kinase (JNK), a family which is one of the three main categories of MAPK families. JNK activation represents a protective response to external stimuli. Mechanical stress may activate the JNK pathway by phosphorylating ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1), resulting in chondrogenic differentiation [36] and apoptosis regulation [37]. Collectively, the comprehensive findings from this study show that UBE2C, IL6, and MAPK8 may play more important roles in dynamic compression enhanced chondrogenesis, unlike the original study which suggested the MMP/TIMP family might be the key genes (15).