Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology

There is a burgeoning interest in cartilage repair worldwide, with particular focus on tissue engineering and cell-based therapies. While much effort goes into developing novel culture conditions and support mechanisms or scaffolds, autologous chondrocyte implantation (ACI) [1] remains the most commonly used cell-based therapy for the treatment of cartilage defects in young humans [2‐4], although no randomised trials have been completed as yet [5]. Objective measures of the properties of the grafted regions are necessary for long-term follow-up of this procedure and to evaluate how closely the treated region resembles normal articular cartilage. Useful outcome measures that assess the overall function, structure, and composition of chondral tissue [6] include mechanical properties or its appearance in arthroscopy, histology, and magnetic resonance imaging (MRI), in addition to clinical assessment of the patient. However, there has been little standardisation of such outcome measures [7]. We have therefore developed histological and MRI scoring schemes and used them to assess the quality of repair tissue at varying time points up to 34 months after the grafting procedure. In addition, immunohistochemistry has been used to assess whether the tissue in the grafted site resembled normal articular cartilage, not only in its matrix organisation but also in its chemical composition.

Cartilage function reflects its biochemical composition [8]. A small biopsy specimen such as is used for histochemical assessment can provide only limited information, as it is from a discrete location. MRI, in contrast, can provide information on the whole area. In addition, it is noninvasive and successive scans can be carried out, so allowing longitudinal monitoring at different time points. MR images have been shown to correlate with biochemical composition in other tissues, in cartilage in vivo, and even in engineered cartilage generated in a bioreactor [9‐11]. Thus in this study we have used both forms of assessment of articular cartilage and correlated them where they are available at the same time points post-treatment. We have previously reported on the immunohistochemical appearance of such biopsy specimens, but only on two individuals and at 12 months after implantation [12]. Here we report on a much more extensive sample group, obtained up to 3 years after treatment, and compare histological assessments with those obtained by MRI.

Although ACI has been carried out as a treatment for cartilage defects for 14 years [26], there remains much discussion about the efficacy of the procedure, despite 74–90% of patients having good to excellent results clinically in a 2–10-year follow-up study of more than 200 patients [27]. Objective outcome measures are required to assess any form of treatment and to date there is a substantial lack of information on the biochemical nature of cartilage repair tissue [28]. We have used MRI and histology as a means of assessing the quality of repair tissue in patients treated with ACI, sometimes in conjunction with mosaicplasty. In an attempt to render the observations more objective and, to some extent, quantitative, we have designed scoring systems specifically for patients who have had cartilage repair. Immunohistochemistry has been used to facilitate some assessment of the biochemical components within the repair tissue.

Many histological scoring systems have been published, but these have primarily been designed for animal studies of cartilage repair in rabbits [29‐35] or dogs [36]. The scores assess parameters such as cell and tissue morphology, degree of chondrocyte clustering, surface regularity, structural integrity, thickness, metachromasia, bonding to adjacent cartilage, filling of the defect, and degree of cellularity. Some of these parameters can be assessed only on whole joints, which are commonly available in the animal models but not appropriate for humans. Here, where histological examination is carried out on biopsy specimens of the repair tissue, these specimens must be as small as possible and usually obtained only at one time point (thereby having certain inherent limitations, e.g. only representing a small area at one location within the treated area). Scoring systems for human tissue have been published, but these have, in the main, been devised for studies on osteoarthritis [37, 38]. Hence many of the parameters assessed, such as growth of pannus, may be inappropriate for cartilage repair. Thus, in this study we have devised a histology score specifically for small, discrete biopsy specimens obtained from human patients undergoing treatment to induce repair of cartilage. We have identified characteristics that, in our opinion, are important to monitor and assess the quality of repair tissue. These include features such as the presence of blood vessels or mineralisation, in addition to the more obvious parameters such as integration with the underlying bone and tissue morphology. Other features should perhaps be considered for inclusion in the assessment procedure, such as the predominant type of collagen present or whether a higher degree of matrix organisation is present; i.e. whether hyaline cartilage has developed the zonal organisation typical of adult articular cartilage. While the latter is easily identifiable and could be included in the scoring scheme, the former is not necessarily routinely available in all support laboratories.

Nonetheless, it was felt to be of some benefit to compare the purpose-devised scoring system to one previously devised and described in the literature. Therefore, a scoring system used by many groups researching cartilage repair was chosen: the modified O'Driscoll (MOD) score. This utilises parameters identified by O'Driscoll et al. [29] in their study of periosteal grafts to treat cartilage defects in rabbits. The correlation between the modified O'Driscoll score (but restricted to the parameters that could be assessed on small core biopsy specimens) and the OsScore was reasonable (r = 0.91, P = 0.0001, n = 26) and they could be deemed to achieve their purpose, in that control samples of 'normal' hyaline tissue scored 94 ± 3% of maximum for OsScore and 90 ± 9% for the MOD score. However, all three observers found the OsScore much easier, quicker, and more reproducible to use.

Other workers have reported that hyaline cartilage is often formed in people treated by ACI [26, 27]. In the present study, three of the five samples showing hyaline cartilage morphology were from individuals treated with ACI and mosaicplasty combined. If the biopsy specimen was taken through a transplanted mosaic plug (which makes up approximately 80% or more of the treated area), one would expect it to be hyaline cartilage. The other two specimens that were hyaline cartilage were both obtained much longer after the ACI treatment (30 and 34 months) than 16 of the 17 other cores. In addition, the average time interval between graft and biopsy was greatest for biopsies of hyaline morphology (19.8 months) and least for those of fibrocartilage morphology (12.0 months). This suggests that the cartilage that forms initially is often more fibrocartilaginous but may transform with time to remodel to form hyaline cartilage, possibly in response to loading. The appearance of zonal organisation (sample 22) typically found in normal adult articular cartilage suggests that this technique can indeed lead to regeneration of articular cartilage and may not require the use of a scaffold as is necessary in animal models [39].

The most ubiquitous type of collagen in normal adult articular cartilage is type II [40], both in calcified and uncalcified tissue [41]. The fact that this was commonly found in all but two samples of repair tissue in the present study is encouraging, even though production of type II collagen is not exclusive to hyaline cartilage and is also produced by some fibrocartilages such as the intervertebral disc [42]. The other collagen types examined in the present study (types I, III, VI, and X) have all been described in normal articular cartilage [40, 43]. Collagen types III and VI are typically pericellular, particularly in the deep zone [43, 44] as was found in hyaline cartilage in the biopsy specimens in the present study. Type I collagen has also been reported in articular cartilage: in the normal tissue it is usually restricted to the upper surface layer and the bone, similar to that found in the control samples (see Fig. 8). Similarly, type X collagen has been found in normal articular cartilage, predominantly in the deep zone and sometimes in the surface layer [45]. All of these collagen types – I, III, VI, and X – have been reported to occur at increased levels in diseased cartilage such as osteoarthritis [44, 46, 47]. The presence of type X collagen is considered by some people to be undesirable as it is found in the growth plate, for example, in the hypertrophic zone, which goes on to calcify. However, it is also found in extracellular matrices in cartilage [45] and intervertebral disc [48], which do not often proceed to mineralisation.

Chondroitin sulfate and keratan sulfate glycosaminoglycans are typically found in both articular cartilage [49] and fibrocartilage [50], their distribution and intensity varying with age and stage of development. The presence of 7-D-4 in 'control' hyaline cartilage seen here is likely to reflect the youth of the control subjects, as other studies have shown this and other abnormally sulfated chondroitin-6-sulfate epitopes to be expressed in developing and growing articular cartilage [51]. Lin et al. [52] found the expression of 7-D-4 to be greatest of all the proteoglycan epitopes in repair tissue in animal models of cartilage repair. They found it was able to differentiate repair hyaline tissue from both normal and fibrous repair tissue. Certainly in the present study there was no staining with the antibody 7-D-4 in totally fibrocartilaginous samples (either the ACI biopsy specimens or the meniscus).

MRI is considered by some to be the optimal modality for assessing articular cartilage [11, 53], being able to evaluate the volume of repair tissue filling the cartilage defect, the restoration of the surface contour, the integration of the repair tissue to the subchondral plate, and the status of the subchondral bone [11]. MRI can reliably detect overgrowth or hypertrophy or graft delamination. It can also detect oedema-like signal in the marrow underlying the autologous chondrocyte repair. The significance of these marrow changes has yet to be clarified, but persistent or increasing oedema-like signal may indicate that the repair tissue is failing.

The use of MRI is limited to some extent, however, by the lack of standardisation and consensus on which sequences should be used [11]. 3D fat-suppressed echo MRI sequences provide a high contrast-to-noise ratio between cartilage and subchondral bone [54, 55], thus allowing the interface to be clearly assessed. MRI has been shown previously to correlate with cartilage histology [55]. 3D requires a gradient echo sequence and thus there is an increase in the potential for susceptibility artefacts in the follow-up studies; consequently, there is a compromise between the greater degree of resolution obtained in such 3D sequences and the increase in obvious postoperative artefacts. This is of particular relevance in this group of patients, because so many of them have had previous surgical procedures.

The grading scheme used for the MR changes in this study is at best only semiquantitative and may oversimplify and lose information that could be obtained by more sophisticated analysis. Fifty percent of patients had had between one and five procedures on their knee before undergoing ACI grafting. This will obviously influence the MRIs of that joint, often rendering their interpretation more difficult – for example, in defining the edge of the graft to assess the degree of overgrowth or incorporation. The fact that the MRI scores were lower for the patients treated with ACI and mosaicplasty combined almost certainly reflects more interference within the joint for these patients than occurred in patients treated using ACI only. Patients with mosaicplasty as part of their treatment would generally only score 75% of maximum, as they would usually score zero on the subchondral bone parameter.

Several animal studies on ACI have shown that while relatively good cartilage forms initially, it often breaks down and degenerates with time. For example, in dogs [36], the remodelling phase at 3–6 months is followed by a degradative phase, during which the repair tissue and surrounding cartilage appear to become progressively damaged. Results from our studies suggest the opposite may be true in humans treated with ACI, in whom the repair tissue appears to 'mature' with increasing time and tend more towards hyaline cartilage than fibrocartilage [56]. This is similar to the impression obtained from clinical results in long-term follow-up of patients, up to 10 years after ACI [26]. Why there should be this apparent difference in progression between animals and humans is unclear. One common finding in animal studies, however, is delamination of repair tissue from the surrounding 'native' or original cartilage with time [57]. One can imagine that if this occurs, it can only deteriorate further with movement and may be the cause of the subsequent failure of the graft tissue. Observations on patients treated with ACI in this study, and others within our centre, indicate that there is good integration between native and repair tissue. Certainly histological examination demonstrates that the cartilage integrates fully with the underlying bone. Lateral integration is not assessed routinely by the histological samples, because they are taken from the centre of the graft region. However, in the single case where a sample was taken obliquely in a patient treated with ACI and mosaicplasty combined, this showed complete integration across all regions of the sample (see Fig. 1). Lateral integration appears to be good generally, at least in the surface layers, when ascertained by its appearance and resistance to probing at arthroscopy (JB Richardson, unpublished observation).

Why integration might be more successful in humans than other species is unclear. Several factors may contribute, such as the way certain aspects of the procedure are performed – for example, where and how the periosteum is obtained or fixed in place. Alternatively, the type or amount of loading and mobilisation post-treatment may prove to be influential. For example, limited mobilisation, which may be easier to control in patients than in animal models, may be important immediately postoperation in allowing protection of the surgical site in the early weeks. In addition, cells can be mechanically induced to transfer from fibroblastic to chondrocytic cells, at least in tendon [58], and synthesis of proteins and proteoglycans by cartilage cells is inhibited by static compression but not by intermittent loading [59]. Other, more basic, differences between animal species and mankind may be important, such as variations in cartilage thickness, cellularity, or mechanical properties [6].

In summary, we have used histology and MR imaging in an attempt to assess objectively the quality and hence success of ACI in eliciting repair of articular cartilage. Despite more than 6000 ACI procedures being carried out worldwide, the understanding of the biology of cartilage repair remains poor. Further long-term study of patients treated with ACI, together with the use of objective outcome measures, should improve this understanding, and is vital in allowing comparison of the long-term success of this technique with others such as debridement and subchondral drilling for the treatment of cartilage defects. It is only after true objective and scientific study [7] or after the completion of randomised trials [5] that informed judgements on the effectiveness of ACI can be made. In addition, establishing objective, standarised outcome measures will be important to compare and assess future generations of treatment regimes incorporating scaffolds and support matrices, or other, more advanced, tissue-engineered therapies.