Joint and Epiphyseal Progenitor Cells Revitalize Tendon Graft and Form Mineralized Insertion Sites in Murine ACL Reconstruction Model

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1 Joint and Epiphyseal Progenitor Cells Revitalize Tendon Graft and Form Mineralized Insertion Sites in Murine ACL Reconstruction Model Yusuke Hagiwara 1,2, Nathaniel A. Dyment 3, Douglas J. Adams 3, Shinro Takai 2, David W. Rowe 3. 1 Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan, 2 Nippon Medical School Hospital, Tokyo, Japan, 3 University of Connecticut Health Center, Farmington, CT, USA. Disclosures: Y. Hagiwara: None. N.A. Dyment: None. D.J. Adams: None. S. Takai: None. D.W. Rowe: None. Introduction: Poor osseointegration of tendon grafts to the bone tunnel is a major concern when using soft tissue grafts for anterior cruciate ligament (ACL) reconstructions [1]. While ACL reconstruction (ACLR) preclinical models have been used to improve our understanding of graft to bone healing, these models have been limited to larger animal models that lack the genetic tools provided by the mouse. We have utilized a combination of lineage tracing and GFP reporter mice, fluorescent stains for alkaline phosphatase (AP), and a number of cryohistological imaging steps all applied to the same nondecalcified section of the knee joint to characterize the lineage and expression of cells in the repair. We also developed a surgical bone tunnel model for free autologous tendon reconstruction of the ACL in the mouse. The objectives of this study were to trace the origin and phenotype of cells that revitalize the tendon graft following ACL reconstruction and to examine whether these cells are capable of creating mineralized attachments within the bone tunnels. Methods: All protocols were IACUC approved. Experimental Design: 1) Cre mice driven by Growth and Differentiation Factor 5 (GDF5Cre) were crossed with Ai9-tdTomato Cre reporter mice (GDF5-9) in order to label mesenchymal cells in the joint and epiphysis. ACLR surgery was performed and mice were assessed at 1 and 4 weeks post-surgery. 2) ACLR surgery was also performed in triple transgenic Dkk3- GFP (ligament and enthesis cells), Col3.6-CFP (strong in osteoblasts and weaker in fibrochondrocytes), αsma-rfp (muscular blood vessels) or double transgenic Dkk3-GFP, αsmaa-rfp mice. These mice were injected with demeclocycline (DMC) at 1, calcein (CAL) at 2, and alizarin complexone (AC) at 4 weeks of age and were assessed on the following day. ACL Reconstruction: All surgical procedures were performed under a microscope. One knee joint of each mouse was subjected to surgical transection of the ACL followed by reconstruction (ACLR). The contralateral knee joint was either left intact or subjected to a sham procedure consisting of a mid-vastus approach, joint opening, and closure. Three 3-4 cm long tail tendon fascicles were harvested from 2 or 3 small incisions in the proximal tail. The three bundles were maintained in PBS, sutured at both ends with 7-0 nylon, and threaded through a makeshift endobutton crafted from 1 mm of 28G needle. Surgical transection of the ACL was achieved via midvastus incision, subsequently cutting of the ACL with a 27G needle [2]. After confirmation of significant anterior drawer and intact PCL, 28G needles were used to drill tunnels originating at the ACL femoral and tibial footprints through the femur and tibia. The sutured ends of the tail tendon graft were passed through a needle and pulled back through the two tunnels, securing the button on the upper lateral femoral epicondyle. External tibial fixation was achieved by passing one free end through neighboring muscle membrane fascia, tensioning the graft with the knee in extension, and knotting 3 times. This fixation was augmented with a figure 8 suture pattern through these knots and surrounding muscle to

2 complete the external tibial anchor. Repetitive Imaging Cryohistology: Frozen mineralized sagittal or coronal sections of the knee using cryotape were made to include the full length of the tendon graft through bone tunnel. Each section went through up to 4 rounds of imaging. These rounds included imaging 1) fluorescent GFP reporters and/or mineralization labels, 2) collagen SHG signal via two photon imaging, 3) alkaline phosphatase (AP) fluorescent staining, and 4) toluidine blue (TB) staining. Layered composite images of all 4 rounds were made in Photoshop. Results: Both at 1 and 4 weeks, stabilized knee joints and surviving grafted tail tendons were confirmed macroscopically. Expansion of cells in bone marrow and joint. At 1 week post-surgery, GDF5-9+ cells in the bone marrow of the epiphysis expanded greatly as well as cells within the joint. The tail tendons, which were originally GDF5-9 negative, still contained GDF-9 negative cells within the body of the tendon within the bone tunnel, but cells were isolated to the surface of the graft within the joint space. There were no GDF5-9+ cells within the tendon graft at 1 week. GDF5-9+ cells revitalize the tendon graft. At 4 weeks, a significant number of GDF5-9+ cells had infiltrated and were embedded within collagen fibers within the tendon graft, especially near the bone tunnels. It appears the majority of original graft cells had died and were replaced by external GDF5-9+ cells except in the central region of the graft that was still acellular (Fig. 1A1 ). GDF5-9+ cells create mineralized attachments in bone tunnels. The infiltrating cells anchor collagen fibers to adjacent bone within the tunnel, mineralize the fibrocartilage (arrow in Fig. 1B3, yellow DMC label given one day before sacrifice), and have high AP activity (Fig. 1B4, magenta). Two photon second harmonic generation (SHG) shows continuous collagen fibers of grafted tendon anchored down to bone in the tunnel (Fig. 2B2 blue, and B3 white). The attachment between grafted tendon and the bone tunnel shows TB+ chondrocytes (Fig. 2B1), and Dkk3+ cells within the collagen fibers appear to have migrated from the joint surface and gradually transition into fibrochondrocytes (Fig. 2B2 green). Cells at the base of the fibrocartilage also are mineralizing (Fig. 2B2, red AC label given day before sacrifice), indicating that a mineralized fibrocartilage zone of the attachment was initiating. Discussion: There are three characteristic stages of ACL graft healing seen clinically and in preclinical models: 1) early graft healing leading to central graft necrosis without revitalization, 2) proliferative phase where the graft revitalizes with intense remodeling and revascularization, and 3) ligamentous phase where the graft remodels to increase mechanical properties towards that of the native ACL [3]. In the present model, cells within the graft are replaced by GDF5-9+ cells from the epiphyseal marrow or joint space, which suggests similar populations may revitalize ACL grafts clinically. GDF5-9+ progenitors within the joint are capable of anchoring down collagen fibers to underlying bone, differentiating into fibrocartilage (Dkk3+ and TB+), and mineralizing the surrounding matrix (AP+ with AC and DMC labeling). These characteristics are seen in the normal ACL enthesis during growth and development and should be an initial requirement for a functional ACL reconstruction. Significance: Understanding the origin and phenotype of cells that revitalize ACL grafts has tremendous impact in guiding future surgical and/or therapeutic strategies. Cells from outside the graft must first infiltrate the graft tissue and then anchor collagen fibers down to form new attachments with bone in the tunnels. Next, the cells at the attachment site must differentiate to form a zonal insertion with unmineralized and mineralized fibrocartilage to create a functional enthesis.

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5 ORS 2015 Annual Meeting Poster No: 0821