The Relative Gene Expression Profile of human Anterior versus Posterior Cruciate Ligament

Krismer, Anna; Cabra, Romina Silvia; Kohl, Sandro; Ahmad, Sufian; Gantenbein, Benjamin (2 March 2016). The Relative Gene Expression Profile of human Anterior versus Posterior Cruciate Ligament (Unpublished). In: ORS Annual Meeting. Orlando. 02.03.2016.

ABSTRACT INTRODUCTION: The knee and in particular ruptures of the anterior cruciate ligament (ACL) still represents a major challenge in orthopaedics. Ex vivo organ culture models of human ACLs are a valuable tool to test degenerative and regenerative scenarios.1,2 These models require an internal control tissue for judging relative gene expression of intact ACL organ culture. In this context, the gene expression profile of donor-matched ACL versus posterior cross ligament (PCL) has never been surveyed. Here, we investigated selected RNA transcripts of teno-specific gene markers to evaluate whether the expression between ACL and PCL is relatively similar and could be used as an internal control and reference for future in vitro mechano- biological models of whole ACL in organ culture.
METHODS: 9 donor-matched ACLs and PCLs were obtained from full-knee prosthesis surgery with written consent from the patients (ethically approved). Tissue was immediately transferred to RNA later, snap-frozen in liquid nitrogen and stored at -80°C prior extraction. RNA was then extracted from pulverized tissue in liquid nitrogen by piston and mortar. The grinded powder was then soaked in TRI, RNA was extracted following a combined TRI- reagent phase separation protocol and further purified using silicon column as previously described.3 Total mammalian RNA was then translated to cDNA using the iScript kit (Bio-Rad) and amplified by the iQ5 PCR cycler using SYBR green master mix (Bio-Rad). The expression of 12 human genes was measured, i.e. tenocytes important anabolic maintenance genes: biglycan (BGN), collagen type 1 and 3, (col1 & 3), scleraxis (SCX), tenascin (TNC), tenomodulin (TNMD), two genes were screened for potential dedifferentiation of fibroblasts, i.e. aggrecan (ACAN) and col2. Four genes of the catabolic side of the extracellular matrix turnover were screened, i.e. ADAMTS4 and 5, MMP3 and MMP13, respectively. Human 18S was used as an internal control. Data were tested for normality and due to deviation of it analyzed using non-parametric Wilcoxon-Signed ranked exact test.
RESULTS SECTION: The overview of the donor characteristics is given in Table 1. From the totally 12 screened genes two anabolic genes, col1 and TNC, showed levels of mRNA expression deviating significantly from the baseline of ACL and PCL comparison (gene expression = 1.0, Figure 1). On the side of the catabolic genes, there were no deviations obtained from the baseline, however, the exact P-value of MMP13 was borderline (P = 0.054).
DISCUSSION: For future mechano-biological experiments, caution must be taken to use the PCL as an internal control. There seem to be donor-matched differences in col1 and TNC, which indicate ligament-specific gene expression differences. In the future, gene expression profiles should be even further distinguished in different sub-zones of the ACL and PCL, which might also cause the heterogeneity presented here.4
SIGNIFICANCE: This research uses intact primary biological samples of the ACL, which has to be considered highly relevant for clinical research. Future research will focus on in vitro degenerative rupture models and testing regenerative approaches.
REFERENCES: 1. Altman GH, Lu HH, Horan RL, et al. 2002. Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering. J Biomech Eng 124:742-749. 2. Hohlrieder M, Teuschl AH, Cicha K, et al. 2013. Bioreactor and scaffold design for the mechanical stimulation of anterior cruciate ligament grafts. Biomed Mater Eng 23:225-237. 3. Gantenbein B, Gadhari N, Chan SCW, et al. 2015. Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair. World J Stem Cells 7:537-550. 4. Murray MM, Fleming BC. 2013. Biology of anterior cruciate ligament injury and repair: Kappa delta ann doner vaughn award paper 2013. J Orthop Res 31:1501-1506.
ACKNOWLEDGEMENTS: We thank Eva Roth for assistance with the biochemical assays. This project was partially funded by the Swiss Orthopedic Society grant No. S99083814080618560 to SS Ahmad.

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