Experimental animals
Thirty two (32) female, Swiss Alpine sheep with a mean age of 2.5 years (range 2–3 years) and 70 kg of body weight (range 48–91 kg) served as experimental animals. Prior to surgery the animals were adapted to the new stable environment for 14 days, checked for their overall health (physical exam and blood check), dewormed (Cestocur®; Bayer AG, Leverkusen, Germany; Endex®; Novartis AG; Bale; Switzerland), vaccinated against Clostridium difficile and tetanus (Pulpyvax®-T s.c.; Essex Veterianaria AG; Bern, Switzerland) and their claws were inspected. The sheep were randomly assigned to 6 groups with 4 animals each and 1 group with 8 animals according to the distribution of the osteochondral grafts [see Additional file 1
]. In each animal, independent of group distribution, 2 grafts of the same origin and treatment were implanted into the weight bearing area of the distal medial and lateral femoral condyle alternating between right and left limbs. Thus, a total of 64 osteochondral grafts were implanted. All animal experiments were conducted according to the national laws of animal welfare and protection with the permission of the local Ethical Committee and official veterinary authorities (# 08/2002).
Osteochondral transplants
Mushroom-structured, photooxidized osteochondral grafts were used as transplants originating from different species. Equine (EN) and bovine (BO, BN) grafts were harvested from shoulder joints of healthy animals (age: 10–18 months for heifers, between 2 – 5 years of age for horses) in the local slaughterhouse within 12 hours after death of the animals, while ovine grafts (OO,ON) were taken at the university facilities immediately after sacrifice of experimental sheep (age between 2–3 years) killed for other reasons than infectious or joint disease. While bovine, ovine and equine grafts were collected at the slaughterhouse, the human grafts were received from a registered bone bank and treated with the photooxidation process using the exact same protocol. Graft harvesting and preparation was performed as described before [3–5]. Briefly, cylindrical osteochondral plugs were harvested from the weight bearing area of the proximal humerus using a diamond coated drill bit (Draenert, Munich, Germany). Before harvesting, the cartilage surface was assessed macroscopically for absence of signs of cartilage degradation, such as fibrillation, discoloration and loss of shiny appearance. The cartilage plugs were then further processed through an automated process resulting in mushroom-shaped grafts with a 6 mm overall diameter of the mushroom head and a 3 mm width of the mushroom stem. The overall length of the grafts was 7 mm divided in 3 mm length of the mushroom head including the calcified cartilage zone and a minimal amount of subchondral bone, and a 4 mm long stem. Thereafter, the osteochondral mushroom-structured grafts were photooxidized, where a cleansing procedure was followed by a prolonged bathing process in 0.01% methylene blue solution in buffered saline while exposure to a halogen light source took place under stable room temperature conditions (patent # EP0768332A1, US5,817,153) [6]. Harvesting and preparation of the grafts were performed under sterile conditions. Grafts were lyophilized and kept under sterile conditions before surgical implantation. The original process was applied for bovine, ovine and human grafts (BO,OO,HO). For the other groups (BN, EN, HN and ON), the cleansing process was intensified, such that a more rigorous washing procedure with alcohol was used compared to the other 3 original groups (BO, OO, HO). Equine grafts were prepared only with the new cleansing procedure.
Animal anesthesia and surgery
The sheep were routinely prepared for surgery and operated as described before [4, 5]. Twentyfour (24) hours prior to surgery the animals were fasted, the surgery field clipped and cleansed. Anesthesia was induced with ketamine hydrochloride (2 mg/kg bw. i.v., Narketan, Chassot AG, Switzerland) in combination with valium (0.01 mg/kg bw., Diazepam, Roche, Switzerland) after sedating the sheep with medetomidine (5 μg/kg bw., im., Domitor, Orion-Farmos, Turku, Finland). Anesthesia was maintained with isoflurane (Forene, Abbott, AG, Baar, Switzerland) in 100% oxygen using endotracheal intubation.
A parapatellar approach to the lateral and medial condyles, with the animals in lateral recumbency and the surgery limb in supine and maximally bent position was chosen. With a specially designed drill bit [5] (Centerpulse Biologics, Winterthur, Switzerland) the bed for the mushroom-shaped grafts was prepared in the weight bearing area of both condyles (Fig. 1). The grafts were applied according to the pressfit technique[14, 15], where the cartilage surface was left flush with the adjacent cartilage and the stem and the mushroom head were stable within the host bed (Fig. 2). The wound closure was routine.
After surgery the sheep were kept in small stalls for 2 weeks, thereafter released to a larger pen for another 4 weeks and subsequently allowed to roam freely on the pasture throughout the rest of the study. At 6 months after surgery all animals were sacrificed and the stifle joints immediately harvested.
Evaluation of samples
The stifle joints were carefully opened, the patellar ligaments severed and reflected dorsally and the joint capsule was evaluated macroscopically for signs of inflammation, such as reddening of the tissue, hypertrophy of the villous part of the synovial membrane, tissue adhesions, appearance of the fat pad and color of the synovial fluid. The cartilage surface was studied for position, surface and staining of the original graft, graft-host cartilage interface as well as the surface of the host cartilage. Obvious signs of matrix degradation, such as fibrillation, cleft formation, cobble stone appearance and discoloration of the hyaline cartilage surface were recorded and compared among groups. All samples were documented using a digital camera (Minolta® Dimage7).
Sample preparation
The stifle joints were then further prepared for sampling of probes such, that synovial membrane samples were harvested within the villous part immediately cranial to the lateral and medial aspects of the proximal tibia plateau. Then the femoral condyles were freed from the soft tissues and 1 × 1 cm blocks containing the original grafts were cut using a bone saw (K 410, Kolbe GmbH, Elchingen, Germany).
Radiology
The bone blocks were radiographed using a faxitron (Cabinet x-ray-faxitron series, model 43855A, Hewlett Packard®, USA) with Fuji Photo Films (Fuji Photo Film Co.®, Ltd, Tokyo, Japan) from a cranio-caudal and latero-medial view before further processing.
Histology
The bone and synovial membrane samples were fixed in 4% paraformaldehyde at 4°C and further processed for histology. The synovial membrane samples were prepared for routine histology using paraffin blocks, where sections were stained with hematoxilin eosine (HE). Bone samples were embedded in acrylic resin based on methyl methacrylate as described before [4, 5, 16, 17]. Briefly, the bone samples were fixed for ca. 5–7 days, washed in phosphate buffered saline (PBS), before they were dehydrated in a series of alcohol, defatted in xylene, infiltrated with methyl methacrylate (methacrylacid-methylester; dibuthylphtalate and perkadox in a proportion 89,5: 10: 0,5) and then embedded in the same solution using special Teflon molds (custom made in-house by Centerpulse) that were placed in a water bath at 30°C. Ground (30–40 μm) and thin (5 μm) sections were cut (Leica® SP 1600 and Leica® RM 2155; Leica Instruments GmbH, Nussloch, Germany). Ground sections were surface stained with toluidine blue, while thin sections were deplastified with Methoxyethyl-acetate (Merck, Schweiz AG) and stained with toluidine blue or von Kossa/McNeal. Toluidine blue allows assessing metachromatic staining of cartilage matrix due to proteoglycan content, while the staining method according to von Kossa /McNeal distinguishes between calcified bone matrix and non-mineralized osteoid. Furthermore, cytoplasms of cells are stained turquoise blue.
Evaluation of histology sections
Synovial membranes were evaluated for signs of immunological reactions and inflammations through 3 independent reviewers. A semi-quantitative score system was developed for morphology and proliferation of synoviocytes, appearance of neutrophilic and eosinophilic granulocytes, lymphocytes, plasma cells and macrophages. The neoformation of capillaries and small vessels, amount of fibrin exsudate and metaplasia of fibrous tissue was also assessed. The score system for each variable is depicted in [see Additional file 2
]. If the assessment of the reviewers differed, the mean value was used for further statistical analysis.
Cartilage repair was evaluated qualitatively, semi-quantitatively and by means of histomorphometry. In the qualitative evaluation by means of a light microscope (Leica, DMR, Glattbrugg, Switzerland) focus was placed on cell types involved in mechanisms of cartilage repair, viability of cell grafts, quality of interface between host and grafts, as well as subchondral bone remodeling and cyst development. For semi-quantitative evaluation a score system was developed that was based on a previously published system by our research group [4, 5]. Scores were divided in regenerative and degenerative aspects ranging between 0–3, where low scores represented good and high scores bad results [see Additional file 3
]. Two reviewers evaluated the sections independently, before scores were compared. If differences occurred, mean values were used for final statistical analysis. For quantitative evaluation by means of histomorphometry ground sections were used that were captured through a macroscope (Leica, M420, Glattbrugg, Switzerland) as digital images in TIF-format (DC 200, Leica. Glattbrugg, Switzerland) and at a 5.8 times magnification. As reported in an earlier study with those type of grafts, the digitalized pictures were colored before measuring for better detection of the fractions cartilage matrix, bone, fibrous tissue, resp. bone marrow [5]. Using a special software program (Leica Qwin®, Leica Quips®) the areas of the different fractions were measured in μm and the percentage of each was calculated.
Statistical analysis
Results were analyzed using a factorial analysis of variance (ANOVA, StatView®, Version 4.5, Abacus concepts, California, USA) to assess overall differences between the various groups. Specific differences between individual groups were calculated using post hoc Scheffé tests. Significance was set at p-values < 0.05.