Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep

Margarete K Akens; Brigitte von Rechenberg; Pedro Bittmann; Daniel Nadler; Kati Zlinszky; Jörg A Auer

doi:10.1186/1471-2474-2-9

Research article
Open Access
Open Peer Review
Published: 30 November 2001

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep

Margarete K Akens¹,
Brigitte von Rechenberg¹,
Pedro Bittmann²,
Daniel Nadler²,
Kati Zlinszky¹ &
Jörg A Auer¹

BMC Musculoskeletal Disordersvolume 2, Article number: 9 (2001) | Download Citation

Abstract

Background

Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success.

Methods

Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery.

Results

The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts.

Conclusions

The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients.

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Background

Destruction of articular cartilage due to acute trauma or degenerative joint disease (DJD) results in most instances in repair with inferior cartilage tissue [1–3]. The repair mechanisms depend on several parameters: 1. the size of the original lesion; 2. the overall status of the articular cartilage; 3. the condition of the synovial membrane; and 4. the successful removal of the causative factors, such as mechanical overload, loose bone fragments between the opposing articular surfaces, or instability of the joint due to ligament damage [4, 5].

Hyaline cartilage lesions smaller than 3 mm of size in diameter repair with normal hyaline cartilage, whereas lesions greater than 3 mm are initially replaced by fibrous cartilage that eventually degenerates into fibrous tissue [4, 6]. This inferior repair tissue is not capable of withstanding the mechanical loads exerted on the tissue during motion and the result is eburnation of the subchondral bone [7]. Clinical symptoms of joint disease in human and animal patients include joint distension, decreased range of motion, and pain during weight bearing associated with lameness. In severe cases the pain may be excruciating. Joint replacement with artificial joint prostheses is an option for several joints in human patients and in some instances also in small animals. In young human patients, however, a better option than metal implants is required, because the clinical problem of aseptic loosening and wearing out of joint prostheses have not yet been solved.

Therefore, grafting procedures to replace damaged cartilage with biological tissue is considered an option to delay the implantation of artificial joints [8–12].

Different grafts, osteochondral – [13–15], periosteal – [16, 17], and chondral and chondrocytes in various carrier systems [18, 19], have been used, but the results are questionable [20]. Although initially they showed some promise, problems with matrix degradation of the grafts, graft dislocation and replacement with inferior tissue prevented their successful clinical application [20]. Nevertheless, to date, osteochondral -, fresh – and cryopreserved grafts are used in man and animals, either as a single transplant [21–24] or as multiple transplants [25]. Fresh autografts proved to be superior to fresh or cryopreserved allografts [12, 23, 26, 27]. Cryopreservation reduces the viability of chondrocytes within the grafts, which results in inferior remodelling activity of the subchondral bone compared to autografts [13, 23, 26, 27].

Apart from graft survival, another crucial parameter of transplantation is the transition between graft and host tissue. The gap between transplant and host tissue was held responsible for the initiation of cartilage degeneration, possibly as a result of micromotion between transplant and host tissue [8, 20, 28]. Additionally, penetration of synovial fluid into the host-graft interface results in resorption within the subchondral bone area and cyst formation [29]. A good surgical technique that results in a tight fit between the host and graft tissue is, therefore, essential [11, 20, 30].

The search for the ideal graft for articular cartilage replacement is continuing. The reduced ability of articular cartilage to regenerate and integrate an osteochondral transplant, and the subsequent degradation of the transplanted matrix are problems that remain to be solved [20].

The use of an osteochondral xenograft, pre-treated with photo-oxidation was investigated in an experimental sheep model. Photo-oxidation involves the immersion of the grafts in methylene blue solution and exposure to light. This process affects the sulphur-containing and aromatic amino acids of the collagen fibrils. An increase of oxygen uptake and CO₂ evolution are due to the cyclic nucleus of these amino acids and leads to a rupture of the ring system. In studies with cardiac valve replacement, photo-oxidation was shown to reduce antigenicity of porcine pericardial tissue [31, 32]. The photo-oxidation procedure has not previously been applied to bone and articular cartilage. Previous in-vitro studies with the photo-oxidized cartilage showed that this process leaves a transplant with a mechanically stronger matrix devoid of living chondrocytes [33]. In addition, it was demonstrated that the incubation of photo-oxidized cartilage in combination with native, untreated cartilage resulted in decreased release of local mediators, such as nitric oxide (NO) and prostaglandin E₂ (PGE₂), and reduced activity of neutral metalloproteinases that are known to be involved in cartilage degeneration [34, 35].

Using the press-fit technique [11], photo-oxidized osteochondral transplants were implanted into the femoral condyles of sheep to evaluate the feasibility of in-vivo transplantation. Short and long term survival of the grafts and the adjacent host matrix were followed macro- and microscopically at six, twelve and eighteen months.

Methods

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.

Results

Clinical evaluation

The sheep showed a slight lameness on the operated limb for eight to ten days after surgery. Furthermore, the joint swelling evident in six sheep regressed within two weeks after surgery. All except one of the sheep walked without lameness when turned out to pasture. In this sheep, the collateral stifle ligament was accidentally severed during the surgical approach. The ligament was reconstructed using resorbable suture material (Vicryl, Ethicon, Norderstedt, Germany) and this sheep was kept inside until sacrifice. One sheep from the eighteen-month; xenogeneic osteochondral graft group acquired a clostridium enterotoxaemia infection fourteen months after surgery and died. No other complications occurred.

Macroscopic evaluation of cartilage surface

The joints of all groups appeared normal except in two sheep (six month untreated xenografts) where joint effusion and slight synovitis was found. One effusion was fresh and of unknown origin, whereas the second was caused by joint instability because of the severed collateral stifle ligament (see above). The synovial membrane of the other sheep showed no signs of chronic or acute inflammation macroscopically.

6 – months group

At six months all photo-oxidized grafts (n = 10) were still blue in colour, although less intensive (Figure 2). The blue colour results from the process of photo-oxidation, because the methylene blue solution cannot be removed completely. Four of ten transplants collapsed slightly into the defect, whereas six stayed in place. The interface between graft and host cartilage was still visible and a slight discoloration and loss of glossy appearance was noted in the adjacent host cartilage in four cases. The autogeneic osteochondral transplants (n = 2) stayed in place and the graft-host interface was still visible. One graft showed a rough surface. Three of the xenogeneic osteochondral transplants (n = 4) had collapsed 1 to 2 mm into the defect.

12 – months group

In nine of the photo-oxidized osteochondral grafts (n = 10) the blue colour had disappeared (Figure 3). The surface of cartilage on the grafts appeared white, slightly uneven and, in seven cases shiny. Only the graft that still showed a bluish colour was surrounded by 1 to 2 mm of dull host cartilage and the host-graft interface was still visible. Seven photo-oxidized osteochondral transplants showed evidence of a union between the graft cartilage and the adjacent host cartilage. The autogeneic osteochondral transplants (n = 2) stayed in place, showed a slightly uneven surface and the graft-host interface was still visible. Half of the xenogeneic transplants (n = 4) stayed in place and were surrounded by 1 to 2 mm of dull host cartilage. The other half of the transplants had sunk and had a very rough surface (Figure 4).

18 – months group

All photo-oxidized osteochondral transplants (n = 10) appeared white. Six transplants showed evidence for a union between the graft cartilage and host cartilage. The surface of the cartilage of the transplants was slightly uneven and only one transplant was surrounded by dull host cartilage. The autogeneic osteochondral transplants (n = 2) stayed in place, showed a slightly uneven surface and cartilage flakes at the interfaces. Interfaces between graft and host cartilage were still visible in three xenogeneic osteochondral grafts (n = 4) and there graft surface appeared uneven.

Microradiography

At six months in photo-oxidized (Figure 5), autogeneic and xenogeneic transplants showed radiological changes from slight inhomogenous radiolucency to severe cyst like lesions. No signs of cyst like lesions were seen at twelve months in six of 10 photo-oxidized osteochondral transplants. In the autogeneic osteochondral transplant a slight uneven subchondral contour and slight radiolucency were seen, whereas two xenogeneic osteochondral transplants showed moderate cyst like lesions. The radiological results of the osteochondral transplants at eighteen months were comparable in most cases with the twelve months results, except that in one case of the photo-oxidized transplants and in one case of the xenogeneic transplants a severe cyst like lesion at the graft-host interface was found. A microradiograph of a photo-oxidized transplant at eighteen months is shown in figure 6.