Electron beam sterilization does not have a detrimental effect on the ability of extracellular matrix scaffolds to support in vivo ligament healing

Abstract

Extracellular matrix (ECM) scaffolds have been used to enhance anterior cruciate ligament (ACL) repair in large animal models. To translate this technology to clinical care, identifying a method which effectively sterilizes the material without significantly impairing in vivo function is desirable. Sixteen Yorkshire pigs underwent ACL transection and were randomly assigned to bridge-enhanced ACL repair-primary suture repair of the ACL with addition of autologous blood soaked ECM scaffold--with either (i) an aseptically processed ECM scaffold, or (ii) an electron beam irradiated ECM scaffold. Primary outcome measures included sterility of the scaffold and biomechanical properties of the scaffold itself and the repaired ligament at 8 weeks after surgery. Scaffolds treated with 15 kGy electron beam irradiation had no bacterial or fungal growth noted, while aseptically processed scaffolds had bacterial growth in all tested samples. The mean biomechanical properties of the scaffold and healing ligament were lower in the electron beam group; however, differences were not statistically significant. Electron beam irradiation was able to effectively sterilize the scaffolds. In addition, this technique had only a minimal impact on the in vivo function of the scaffolds when used for ligament healing in the porcine model.

Keywords: anterior cruciate ligament; bridge-enhanced ACL repair; collagen scaffold; electron beam irradiation.

Figure 1. Surgical technique

A – Knee capsule is opened through a lateral arthrotomy, part of the retropatellar fat pad is resected and the ACL is transected in the mid section. B - Bone tunnels for the suture stent are drilled through the tibial and femoral ACL attachment and an endobutton loaded with three suture loops is passed through the femoral drill hole. C – A separate suture loop is stitched to the tibial ACL stump and the ECM scaffold is threaded on two of the suture loops loaded on the femoral endobutton. D – The same two suture loops are passed through the tibial drill hole and knotted over another endobutton. Care is taken to keep the suture stent under tension with the knee in extension. The scaffold is soaked with autologous whole blood. One end of the suture loop sutured to the tibial ACL stump is tied to one end of the suture loop from the femoral endobutton that has not been passed through the scaffold or the tibial drill hole. The suture connection is tensioned by pulling the lose end of the femoral suture end. E – The free suture ends from tibial stump and femoral endobutton are tied together and the knee is closed in layers. (Published with permission from Vavken et al. 2012). ACL – Anterior cruciate ligament ECM –extracellular matrix

Figure 2. Representative histological sections from ACL, synovium, and popliteal lymph node 8 weeks after ACL injury and repair utilizing aseptically processed (ASEPTIC) and electron beam irradiated (EBEAM) ECM scaffolds

A – H&E stained ACL sections at 20× magnification with smaller pictures showing sections recorded using polarized light to visualize collagen crimp (black bar = 800 μm). Moderate to marked fibrosis with large bands of fibrosis in both groups (black arrows). Additionally, under polarized light, mature collagen appears well organized (white arrows); B - H&E stained ACL sections at 400× magnification to visualize cells (Black bar = 40μm). Little areas of inflammation may represent foci in which suture material was recently cleared or in which suture material was present just out of the plane of section in the aseptically processed group (black arrows). Areas of inflammation surrounding small pieces of suture characteristic of a foreign body reaction in the EBEAM group (black arrows). C - H&E stained synovium sections at 40× magnification for a general view (Black bar = 400 μm). Intimal hyperplasia was minimal and most of the synovium was covered by a normal, thin layer of synovial cells (black arrows) and blood vessels (green arrows) consistent with neovascularization were visible in some sections in both groups; D - H&E stained popliteal lymph node sections at 40× magnification for a general view (Black bar = 400 μm). Prominent sinus histiocytosis consistent with mild lymphadenopathy as a result of draining inflammation from the implant site and is not considered an adverse reaction in both groups. ACL – Anterior cruciate ligament ECM – Extra cellular matrix H&E – Hematoxylin and Eosin

Figure 3. Histological Scoring of ACL, Synovium, and Popliteal Lymph Node

Scatterplots (empty circles, n=8 per group) with mean (diamond) and 95% CI (range plots with caps) of ACL inflammation, neovascularization, fibrosis, and fatty infiltration, as well as synovial inflammation, neovascularization, fibrosis, and hypertrophy, and also popliteal lymph node sinus histiocytosis and follicular hyperplasia ACL – Anterior cruciate ligament PLN – Popliteal lymph node Pts – Points