Sixty-seven patients underwent retrieval of biopsies between 6 and 117 months after replacement of the ACL from 2001 up to 2007. The biopsies were carried out during second-look arthroscopies, which were not associated with the initial ACL reconstruction. Inclusion criteria were the following: 1. Previous ACL reconstruction by one senior orthopaedic surgeon (HS) using quadruple hamstring autograft with Bone Mulch Screw fixation in femur and WasherLoc fixation on tibia (Arthrotec, Warsaw, IN, USA); 2. No signs of abnormal laxity on clinical examination nor at examination under anaesthesia at the time of second-look arthroscopy. A knee with normal laxity is defined as a knee without giving way sensation, KT 1000 < 3 mm differences on 133 N side-to-side testing (MEDmetric Co., San Diego, CA, USA) and a negative pivot shift test; 3. Informed consent to participate in the study. Exclusion criteria were the following: 1. Unwillingness to participate in the study; 2. Previous ACL reconstruction by another orthopaedic surgeon or different method of fixation; 3. Abnormal laxity on clinical examination or found in examination under anaesthesia at the time of second-look arthroscopy. Abnormal laxity is defined as a knee with giving way sensation, KT-1000 >3 mm differences on 133 N side-to-side testing and/or positive pivot shift test; 4. Cyclops lesion, extension deficit or other reasons related to possible ACL graft problems; 5. Inadequate tissue sample for histological analysis. The study was approved by the medical ethics board of the Máxima Medical Center Eindhoven-Veldhoven, the Netherlands. Written informed consent was documented in all patients.
The ACL reconstructions were performed by the same senior orthopaedic surgeon (HS). The surgical technique for ACL reconstruction was identical in all patients: a trans-tibial technique with Bone Mulch Screw fixation on the femur and WasherLoc fixation on the tibia (surgical technique by S.M. Howell, MD. Fixation materials by Arthrotec, Warsaw, IN, USA). The graft was a double-strand semitendinosus and double-strand gracilis tendon. Tension on the hamstring autograft at the time of fixation was 90–100 N, with the knee in full extension.
A standardized accelerated rehabilitation programme started on the first day postsurgery. Patients were allowed full weight bearing as tolerated. Crutches were used for the first 2 weeks. In addition to active flexion and extension exercises, the knee was flexed to 90 degrees by means of continuous passive motion machine (OrthoRehab, Oakville, Ontario, Canada). Closed-chain exercises were prescribed on the first day postsurgery. Full range of motion was allowed. After discharge, physiotherapy was continued 2–3 times a week according to a standardized, brace-free, protocol. Unrestricted closed- and open-chain knee-extension exercises were allowed. Resumption of running in a straight line was allowed at 8–10 weeks. Unrestricted return to heavy work activities was allowed after 3 months and competitive contact sports after 4–6 months.
Histological examination
A single tissue sample was collected from one of the four strands of the quadruple hamstring autograft. The synovial layer was cleared from the middle section of the graft bundle. A Shutt mini-tip straight forceps (2.75 mm diameter, Linvatec, Fl, USA) were used through the anteromedial portal to take a superficial mid-substance biopsy of the hamstring tendon graft bundle. Size of the biopsies was 2–3 mm.
In order to create a timeline of ACL autograft remodelling belonging to different individuals, samples were allocated to one of the following groups depending on the time point of their retrieval after ACL reconstruction: group 1 = 6–12 months, group 2 = 13–24 months and group 3 = greater than 24 months. Two control groups were made: native hamstring tendon (HT) and native ACL. The HT control group biopsies were taken in patients (non-related to groups 1–3) at the end of ACL reconstructive surgery. A biopsy was taken from the excess of the hamstring autograft exiting the tibia tunnel after tensioning and fixation of the graft. This excess tendon was normally discarded. The ACL control biopsies were taken from patients (non-related to groups 1–3) who underwent ACL reconstruction after an acute femoral ACL tear (not later than 8 weeks after injury). Therefore, it was possible to obtain tissue samples from non-injured mid-substance areas. All patients gave written consent that tissue samples were allowed to be obtained and to be processed histologically. After the biopsies were taken, the remaining tissue was removed to continue with the ACL reconstruction procedure. Directly after retrieval, samples were fixed in formalin for 2 to 3 days, automatically dehydrated for 3 days and embedded in paraffin. Serial cuts (4 μm) were prepared and mounted on slides with 3% silane (Sigma Chemical, St. Louis, MO, USA).
For descriptive and quantitative cell analysis, haematoxylin–eosin (HE) and Masson-Goldner-Trichrom (MG) staining were used following standard histological protocols.
Vascular density was evaluated by immunostaining sections with a polyclonal rabbit anti-human von Willebrandt factor (factor VIII) antibody (cat.-no. M0851, Dako, Glostrup, Denmark). This antibody binds on the endothelial surface of blood vessels.
For the detection of myofibroblasts, tissue sections were immunostained with a mouse anti-human ASMA monoclonal antibody (cat. no. M0851, Dako, Glostrup, Denmark), which binds to ά-smooth muscle actin (ά –sma) especially present in myofibroblast.
All tissue samples were hydrated and pre-treated with 0,1% pronase for 10 min at 37°C for factor-VIII analysis. Myofibroblast detection did not require pre-treatment. For both analyses, 10% normal horse serum (Vector Laboratories Inc., Burlington, CA, USA) was used for 20 min at room temperature to block non-specific binding sites. The antibody was diluted 1:200 for factor-VIII and 1:100 for ά-sma and added to the tissue samples overnight in a humidity chamber at 4°C. After rinsing the samples with tris-buffered saline, they were incubated with the biotinylated horse anti-mouse immunoglobulin G secondary antibody for 30 min. This was followed by incubation with an avidin–biotin complex (ABC Kit, Vectors Laboratories Inc., Burlington, CA, USA) linked with alkaline phosphatase as a reporter enzyme for 50 min. Staining was achieved with Neurofuchsin as a chromogen. Tissues were counterstained with Mayers Haematoxylin, dehydrated and mounted in a xylol-soluble mount (Vitroclud, R Langenbrinck, Emme Both Endingen, Germany).
For all assessments (cellular density, vessel density and myofibroblast density), sections were automatically digitized and saved using a digital video analysis system (KS 400 3.0, Carl Zeiss AG, Göttingen, Germany). Ten regions of interest (ROI) of different sizes, depending on the sample size, were placed on the sample at random. Cells, vessels and myofibroblasts were counted per mm² at ×100 power. Values are reported with 1 decimal. Myofibroblasts were morphologically differentiated from perizytes. These cells vary by their cell shape, the proximity to vessels and show a different distribution between matrix fibres.
For descriptive analysis, cell distribution pattern (uniform/not uniform), morphology (oblong/spindled/rounded, ovoid) and the evidence of inflammatory reactions were analysed at ×50, ×100, ×200 and ×400 power. Collagen fibril alignment was also assessed.