Tendon to bone healing after an ACL reconstruction in mouse: A gene expression analysis Camila Carballo, MSc, Nathan Coleman, MD, Amir Lebaschi MD, Jianchun Zong MD, Guang-Ting Cong, BS, Xiang-Hua Deng, MD, Scott A. Rodeo, MD.

Hospital for Special Surgery, New York, New York Introduction: Achieving successful anterior cruciate ligament (ACL) reconstruction remains a significant challenge in orthopedic surgery. Reconstruction of the ACL requires the use of bone tunnels to securely attach tendon grafts to bone. However, the unique microstructure of the normal ligament-bone insertion site is not recreated following such repairs [1]. The ultimate function of an ACL graft is dependent on healing at the tendon-bone interface in the bone tunnels. The purpose of this study is to identify the molecular mechanisms of tendon-to-bone healing after ACL reconstruction by gene expression assays using a mouse ACL reconstruction model. Methods: ACL reconstruction was performed in the right knee of 15 male 12 week-old wild-type C57BL/6 mice. The ipsilateral flexor digitorum longus tendon was harvested and used for autograft ACL reconstruction. Bone tunnels were drilled in the tibia and femur using a G23 needle (0.64mm diameter) and the graft was passed through the tunnels and secured using a surgical clip and a 3-0 wire bone bridge at the femoral and tibial metaphyseal bone-exit sites, respectively. Postoperatively, all mice were allowed to have free-range cage activity and 50 cycles of CPM was applied manually on a daily basis .The mice were euthanized on postoperative days 7, 14, and 28 (n=3 per time point). After sacrifice, the tendon grafts were removed and tissue from the tendon-bone interface from within the bone tunnels was collected using a G22 needle for RNA extraction. cDNA was prepared from 50ng total RNA. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed using mouse-specific primers for 7 genes of interest: Aggrecan (ACAN), Collagen1α1 (COL1), MMP3, MMP13, MMP14, Scleraxis (Scx) and Sox9. The data was normalized to a housekeeping gene (GAPDH) and analyzed using the delta CT method. Temporal changes in gene expression were calculated using intact flexor digitorum longus tendon as a day 0 control, and statistical differences were analyzed by ANOVA and Tukey's post hoc test. Significance was set to p<0.05. 2- and 4-week specimens were also prepared for routine histologic analysis with Safranin-O (n=3 per time point). Results: Matrix remodeling genes MMP3 and MMP13 were transiently and significantly upregulated at postoperative days 7 and 14, respectively, and returned to baseline thereafter (Figure 1). MMP14 expression increased at postoperative day 14 (p = 0.1) and remained high at day 28. There was a trend for increased expression of extracellular matrix genes aggrecan (ACAN) and collagen type 1a1 (COL1) over time (p=n.s.), consistent with postoperative healing at the bone tunnel interface. There was no change in expression of the differentiation factors scleraxis (Scx) or SOX-9 compared to the time zero control tendon. A representative Safranin-O histology image is shown (Fig. 2), demonstrating the fiber orientation of the tendon autograft in the femoral bone tunnel. Healing at the tendon-bone interface between the 2- and 4-week time points was demonstrated by an increase in the number of positive staining chodroprogenitor cells. Discussion: Secure tendon to bone healing is required for a successful ACL reconstruction. Factors involved in chondrogenesis and tenogenesis are thought to be implicated in healing of the tendon-bone interface [3,4]. We have shown that expression of matrix metalloproteinase MMP3 occurs early in the healing process (Figure 1). This result is consistent with the literature finding that matrix metalloproteinases are increased during the early remodeling phase of healing [2]. However, we also found that expression of MMP13 and 14 occurs later in the postoperative period. This may be explained by the worsening tendinosis of the ACL graft observed at the later time points, possibly occurring due to increased overuse injury incurred to the graft secondary to the lack of postoperative immobilization of the knee. Further studies employing a period of post-surgical immobilization may resolve this delay in tissue turnover and healing. Interestingly, we did not see statistically significant changes in expression of scleraxis or SOX-9 compared to the control by qRT-PCR, though our histological data demonstrates existence of chondrocyte-like cells at the tendon-bone interface up to 4 weeks postoperatively. Increased collagen 1 and aggrecan expression over the course of the study suggests that anabolic processes at the tendon-bone interface continues through at least the first 4 weeks of the postoperative period in this model. Further study is needed to more conclusively define the gene expression profiles. Significance: This is the first study to quantitatively evaluate the gene expression profile of the healing tendon-bone interface using a mouse ACL reconstruction model. Development of this model will allow further studies using transgenic strains to further define the molecular mechanisms of ACL graft healing. References: [1] Thomopoulos S. et al., 2003. J of Biomech Eng. 125: 106-13. [2] Manning CN. et al., 2014. J Orthop Res. 32(5): 645-652. [3] Asou Y. et al., 2002. J Orthop Res. 20: 827-33. [4] Thomopoulos S. et al., 2010. J Musculoskelet Neuronal Interact. 10(1): 35-45.

 Figure   1:  qRT-­‐PCR  analysis  of  MMP3,  MMP13,  MMP14,  SCX,  SOX9,  COL1,  and  ACAN  gene  expression  at   the  bone  tunnel   interface   following  ACL  reconstruction,  using native flexor digitorum longus tendon as day 0 control. * P<0.05.

Figure 2: Safranin-O staining showing the hypercellularity of the interface substance at 2wks (A-A’) and the progressive graft healing by 4wks, with chondrocyte like cells proliferating from the interface towards the inner tendon (B-B’). Scale bar: 100µm  

ORS 2016 Annual Meeting Poster No. 0954