Abstract：Abstract Endochondral ossification (EO), the main form of bone formation, has been well studied, and collagen type X (Col X) is considered as an essential component for EO, and its expression has been detected in the hypertrophied chondrocytes in developing cartilage tissues. In this regard, deer antlers arguably fit this purpose the best. In this paper, an in vitro model was established using antler stem cell to investigate the role of Col X in EO through RNAi approach. High score shRNA sequences were designed and four (S1~S4) were selected after removing off the target ones based on general design rules. Then, oligo DNA comprising both sense and antisense strands of each shRNA was ligated into pLVTHM (lentiviral carrier plasmid using green fluorescent protein (GFP) as a maker) using T4 DNA ligase. Positive clones named as pLVTHM S1~S4 were selected by using PCR screening and then sequenced. The carrier plasmid containing shRNA (pLVTHM S1~S4), packaging plasmid (pCMV-dr8.91) and enveloping plasmid (pMD2.G) were co-transfected into a 293T cell line. The lentiviral particles produced from the co-transfected 293T cells were harvested and concentrated. Antlerogenic periosteum (AP), within which antler stem cells reside, was sampled from a 1-year-old male sika deer and enzymatically digested to release cells for in vitro culture. The resultant cells were subsequently infected by the lentiviral particles (S1~S4), or a negative control construct (PBS), and then seeded in a high-density (10 /mL) to establish micromass culture. Knock-down efficiency of Col X was examined using both qRT-PCR and Western blot analysis. The results showed that cell nodules were formed around 70 h after micromass seeding; These AP cell nodules strongly expressed GFP, implicating that shRNA that targets Col X gene had been expressed in these nodules, whereas negative control was completely dark under UV light. The qRT-PCR results showed that Col X gene was significantly silenced in micromass-cultured AP cells compared to the negative control. Among these shRNAs, S3 was the one that had most dramatic effects on down-regulating Col X gene expression reaching 94.6%. Western blot analysis further confirmed the results of qRT-PCR. Histological examination revealed that, in the absence of Col X, the matrix of the cellular nodules of pLVTHM-S3 infected group had little alcian blue staining with empty lacunas, whereas nodules of the negative control were darkly stained, which indicated that Col X plays a key role in the process of EO through maintaining the matrix of chondrocytes. Overall, this study successfully established an in vitro model using antler stem cells for the investigation of the role of Col X in EO through RNAi approach. Down regulation of Col X could promote the process of chondrocyte death in EO to occur, and result in empty lacunas. Further study shall use antler models to determine how bone replacement is affected after Col X silencing in vivo .
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