Preparation of photo-oxidized grafts
Bovine shoulder joints from eighteen-month old cattle containing macroscopically normal articular cartilage were collected twelve hours after slaughter. Cartilage was considered normal if it was shining bluish – white with no discoloration, wear lines, or other signs of degradation present. The osteochondral transplants were harvested under aseptic conditions. All soft tissues were removed and remnants of synovial fluid flushed away with sterile physiological saline (0.9 % w/v). Using a diamond-coated hollow drill bit (Draenert, Munich, Germany) with built-in irrigation, standardised osteochondral transplants (5.5 mm in diameter; 6 mm in length) were harvested from the articular surface of the humeral head and immediately transferred into sterile vials filled with physiologic saline. After extensive rinsing, the transplants were placed in a 0.01 percent methylene blue solution in phosphate buffered saline (PBS) (Life Technologies AG, Basel, Switzerland) for 12 h to allow dye penetration. The transplants were then transferred into a customised device and photo-oxidized under aseptic conditions by exposure to light under controlled conditions (light intensity, temperature and exposition time (patent pending)). After completion of the photo-oxidation process the transplants were transferred to a second closed and sterile circuit where the excess methylene blue dye was washed off with PBS. The photo-oxidized osteochondral transplants were stored in sterile ethanol (50 % v/v) at 4°C. The maximal storage time before transplantation was two weeks.
Control xenogeneic, untreated osteochondral grafts were harvested immediately prior to transplantation from fresh bovine shoulder joints under aseptic conditions, washed and kept in saline for only a few minutes before use. Care was taken to assure short storage time in saline solution to avoid osmotic swelling of the cartilage. Even harvesting grafts from a different joint, congruency was not a major problem because of the size of the graft (5.5 mm in diameter). Problems with joint congruency were mainly related to surgical procedure when the graft was not placed perpendicular to the joint surface. Attention was paid to the cartilage thickness of the graft, but differences between graft and host cartilage could not always be avoided.
The autogeneic ovine graft was harvested from the weight bearing area of the femoral condyle, washed in PBS and transplanted either from the medial to the lateral condyle or vice versa in the same joint. A photo-oxidized graft was used to fill the defect resulting at the autograft-harvesting site.
Experimental animals
Twenty-four adult female Swiss crossbred sheep (dentes incisivi had completely changed) were randomly allocated to six-, twelve-, and eighteen-month survival groups (eight animals per group). Grafts that were placed in the lateral and medial femoral condyles of one stifle joint in the weight bearing area. The total number of grafts per group was sixteen, ten were photo-oxidized, two autogeneic and four untreated xenogeneic. In a preliminary study, the results at two months after implantation with auto- and xenografts were poor, and therefore only two condyles were transplanted with autogeneic and four condyles with untreated xenogeneic grafts in each group (six, twelve and eighteen months). Since experimental animals were used and the main focus of this time course study was placed on the photo-oxidized grafts, the use of fewer control sheep was justified for ethical reasons. Furthermore, it was not expected that the results with the control transplants would improve with longer experimental times.
An animal utilisation study committee has approved the study and the sheep were kept in compliance with the law of animal welfare (Permission number: 145/97) Two weeks prior to surgery the sheep were examined clinically, dewormed and accustomed to their new environment. Prior to induction of anaesthesia, food was withheld for thirty-six hours and the hind limbs of the sheep were clipped.
Anaesthesia and surgical procedure
The animals were sedated using xylazine intramuscularly (0.02 mg/kg; Rompun®, Bayer AG, Germany) and anaesthesia was induced with ketamine intravenously (4 mg/kg; Narketan®, Chassot AG, Switzerland). Anaesthesia was maintained with isoflurane (Forene®, Abbott AG, Baar, Switzerland) in oxygen. The sheep were positioned in laterodorsal recumbency with the hyperflexed hindlimb fixed to the table in upright position. A long diagonal skin incision in the shape of a stretched S was made over the knee joint. The incision was started proximally medial to the patella and ended laterally, 2 cm distal to the tibia plateau. Subsequently, an arthrotomy was performed into the medial and lateral joint compartment, parallel to the straight patellar ligament. A partial resection of the intra-articular fat pad facilitated the exposure of the weight-bearing surface of both condyles where the host bed for the transplant was created with a specially designed drill bit conforming to the prepared grafts. The drill bit had an outer diameter of 5.4 mm, 0.1 mm less than the outer diameter of the photo-oxidized – and xenografts. In addition there was a stop mechanism, so that the depth (6 mm) drilled was exactly the same as that of the graft, attached, allowing an exact fit of the graft (pressfit technique). Small variation in placing the graft within the weightbearing area could not be avoided. The autograft was harvested with a drill bit, which had the outer diameter of the photo-oxidized graft. The diameter of the autograft was smaller because of the tissue loss due to drill hollow bit kerf (Draenert, Munich, Germany). Therefore the hole for the autograft was 4.4 mm). The size of the graft was chosen to achieve congruency of the joint surface. The convexity of the ovine femoral is greater compared to the human femoral condyle.
Arthrotomy closure was routine using 2–0 polyglycolic acid suture material (Vicryl, Ethicon, Norderstedt, Germany) for adaptation of the joint capsule, the fascia and subcutaneous tissue. Each layer was closed in a simple continuous pattern. Surgical staples (Signet 35 W®, Auto Suture, Connecticut, USA) were used to close the skin. Additionally, a gauze sponge was stapled over the incision. Postoperative antibiotics (penicillin G (30.000 IU/kg, Hoechst AG, Germany) and gentamicin (7 mg/kg, Streuli & Co. AG, Switzerland) were administered prophylactically twice a day and nonsteroidal analgesic (flunixin meglumine, 1.1 mg/kg, Essex Pharma GmbH, Germany) once a day for three days.
After surgery the animals were confined in groups in a small stable for the first 5 days and allowed normal weight bearing thereafter in a larger indoor pen for six weeks. For the rest of the experimental period they were allowed to roam free on a pasture. After six-, twelve-, and eighteen-months respectively a group of eight sheep each were sacrificed. The stifle joints were immediately harvested for further evaluation.
Macroscopic evaluation
The joints were dissected carefully and the appearance of the articular cartilage assessed immediately. Emphasis was placed on 1) graft position 2) graft surface 3) graft-host interface and 4) adjacent host cartilage. Additionally osteophyte development, synovial membrane pathology and degree of joint effusion were recorded. The position of the graft was considered normal if no obvious dislocation was present and if the graft surface was at the same level as the adjacent host cartilage. The surface of the graft was considered normal if no clefts, no dull, blood tinged, discoloration with a cobblestone-like appearance of the articular cartilage was present. In case of the autogeneic or xenogeneic grafts a shiny, whitish colour was judged normal. In the pre-treated, photo-oxidized grafts the intensity of the bluish colour was recorded. The host-graft interface was evaluated closely. Special emphasis was placed on whether clefts were present, or whether a cartilage-like bond had formed between the graft and the host cartilage. The host cartilage was checked for signs of early degeneration, such as change of colour, fibrillation, cleft formation or flaking cartilage. Again, the host cartilage was considered normal if the surface was glossy and bluish-white, and no signs of matrix degradation were noticed. Each joint was recorded photographically.
Microradiographs
Blocks 1.5 × 1.5 cm containing the graft with the adjacent cartilage and bone were cut with of a bone saw (Biro, Modell 3334, Matti AG, Lenzburg, Switzerland). Microradiographs (Faxitron Series – Cabinet X-ray System, Hewlett Packard, Oregon, USA) of the blocks were taken to assess the trabecular bone structure.
Histology
The bone samples were then fixed in 4 percent buffered formaldehyde for at least one week at 4°C, followed by washing and dehydration with graded ethanols and clearing with xylene and embedding in methylmethacrylate (HistoDur®, Leica, Switzerland) as described elsewhere [36]. Sections 200 μm thick were cut from the polymerised blocks, using a sawing microtome (Sawing microtome Leica SP 1600) and mounted on acrylglas slides (Perspex GS Acrylglas PMMA/Opal 1013, Wachendorf AG, Basel, Switzerland). The sections were ground with a grinding machine (Planopol-V, Struers, Rødovre, Denmark), to an appropriate thickness of 30–40 μm. The sections were subsequently polished with polishing cloths (DP-MOL, DP-NAP with adhesive back), containing polycrystalline diamonds (Diamantpaste P® (3 μM and 1 μM grain size) and lubricants (DP-Lubricant Blue and Red®). The sections were subsequently surface stained with toluidine blue for histological evaluation. One of the ten photo-oxidized transplants from the eighteen-month group was used for a viability test and could not be evaluated histologically.
Evaluation of sections
Qualitative, blind evaluation of the histological sections was carried out using the scoring system listed in table 1. The assessment was based on the appearance of 1) the cartilage, 2) the mineralised cartilage, 3) the subchondral bone and 4) the general appearance of the graft and the surrounding tissue. Each category was subdivided in further parameters (Table 1). The appearance of the graft included its overall position and integrity relative to the surrounding tissues, whereas its bonding to the host tissue was judged by the presence or absence of a cleft between the graft and the adjacent cartilage matrix. Matrix staining of articular cartilage, cell morphology and density, and the integrity of the tidemark were evaluated for the cartilaginous regions. For the general appearance (four above) new bone formation, remodelling of the old subchondral bone and the interface between graft and host bone matrix were assessed.
Scores were given ranging from 0 to 2, with 0 indicating the lowest and 2 the best results (Table 1). Two independent scientists in blinded fashion evaluated the sections and the scores were averaged. Each parameter was evaluated in graft and host except the border of the graft. The average mean of all scores for each group and time point is shown in figure 1.
Statistical analysis of scores was performed using the Kruskal – Wallis – test followed by Dunns post test only for photo-oxidized grafts at the different time points: six, twelve and eighteen months; because of the low numbers of xenografts and autografts in the long term survival groups. A p value < 0.05 was considered to be significant.