Instruments for reproducible setting of defects in cartilage and harvesting of osteochondral plugs for standardisation of preclinical tests for articular cartilage regeneration

Reproducibility and standardisation of procedures in experimental surgery has more limitations the smaller the animals are, especially when articular cartilage is examined. But experiments with large animals are expensive in comparison to smaller ones like rodents or rabbits. A harmonisation of surgical procedures could deliver comparability between various experiments published in literature, leading to fewer animals needed in the end. Mainly regulatory requirements can be met if tools for standardisation are provided for surgeons, experimenters and lab personnel. But the quality of the particular scientific study will also benefit from tools that enable secure and quick work with animals and samples, respectively.

Information on tools used for creating defects in cartilage layer is given in publications of studies in the field [14, 15], and some authors describe the surgical technique in particular for validation [19, 20]. Frequently, the curettage is the recommended tool for creating the defects in combination with knives or circular cutting devices to determine the edges of the defect [19, 20]. The curette seems to be appropriate for removing tissue of the inner part of the defect. But its use is limited on the edges as its diameter has to be smaller than the diameter of an intended circular defect leading to irregular edges of the defect when looked at from above [20]. Seen from the side, the required perpendicularity between the sides and the bottom of the defect is not always possible. This is also the case if the curette has an offset (15°) [20] and bits of tissue may be found at the bottom depending on the technique used [19, 20]. If tissue remains in the “corners”, thus creating an inclined plane, it is a possibility that the implant slips out through the shear forces arising from the joint while moving. Bits of tissue are also found in the inner part of the defects after the use of a curette if e.g. the applied force was not strong enough [20]. However, the correct geometry of the defect is crucial for the stability of the construct. It is either glued, sutured or covered with a periosteal flap [26, 15], because the construct has to be protected through stable flanks for shouldering [18], and sutures need to find anchorage. Thus, primary stability of the implant can be supported by an appropriate quality of the defect avoiding a dislocation of the implanted construct. Punches or trephines are also used to create the defects as described [14, 15] to deliver well-shaped defects with regard to the defect edges. But controlling the depth is not always possible in situ during surgery due to the parallax of view in deeper levels. The removal of the core of the defect cylinder can be difficult if the bottom of the defect core stays in contact with the substrate. Chondral defects are prepared by custom-made instruments like an angulated raspatorium [14]. However, circular punches or trephines provide no centring components like drilling tools and depth control in high resolution. Both specifications are implemented in the presented instrument: a precise and reproducible centring and adjustment of the depth in 0.05-mm steps.

The developed and presented instruments fulfil the requirements of perpendicularity of the defect seen from the side, creation of straight and smooth surfaces of the sides and complete resection of the cartilage layer (Fig. 7). The instruments were used to create defects in the facets of the trochlea, offering a quite flat plane in the stifle joint (Fig. 8). Therefore, the convexity plays a lower role looking at the shape of the defect. Looking at the curvatures of e.g. condyles of a stifle joint, the shape changes due to different radii of the convexity of the condyles. We considered this situation in the construction of a concave front mill in another version (not presented here), but the problem remains unsolved, as one has to respect at least two different radii of the condyles in the sagittal plane and in the horizontal plane, respectively. However, the incongruence depends on the extension of a circular defect in terms of the relation between the diameter of the defect and the radius of the surface. In this case, the inner part of the defect can be taken out with the curette after the crown mill is used. As the width of the groove is only 1 mm, the form of the surface has only little influence on the shape.

The crucial issue in creating the defect is the depth. From the biological point of view, the anatomical edges are important like subchondral lamella or the tidemark. Both are hard to detect in the situs of operation, and surrogate criteria are used like the sound (“metal on stone” [20]) or training the surgeon to handle the curette properly [20]. However, experienced surgeons are in demand to create the defects. But the results show that the intended depths are not always reached depending on the applied technique [19, 20].

The presented instruments work independently of the force applied [20] as they are adjusted via positioning before use. Thus, reproducibility is given for experts and less experienced users as the data show in the validation experiment (Fig. 6). Hence, the disadvantage of this procedure may be seen in the fact that the depth of the defect has to be determined before creating it. But information on the height of the articular cartilage can be achieved by imaging or species-specific data [36, 37]. In addition, one can proceed stepwise respecting the expected depth as the cutting behaviour will change depending on the tissue while using the crown mill; also, isolated bleeding indicates a partial opening of the bone marrow. Thus, the advantage is that the crown mill is determining the final depth and can also be used as pilot instrument. The front mill will finish the defect while respecting the previously identified depth.

However, we have not compared our results to the results achieved through conventional techniques, neither before nor after healing processes. But we assume that the new technique we developed is less prone to influences such as the expertise of the surgeon executing the operation. We also believe that the new technique is likely to be a lot faster. Another disadvantage of the new technique could be that only defects of one predetermined diameter can be created during any one operation. Changing the diameter is thus not possible while operating. As the instruments were designed for the use in preclinical studies, where the size of the defect is determined before the operation, this is not a disadvantage. As our previous tests have shown, so far the diameter cannot be smaller than 4 mm. However, it is possible to create a crown mill and corresponding front mill of a larger diameter than the 6 mm we used.

Histopathological examination is indicated as the predominant outcome measurement tool in OA [26, 38, 39, 25, 16] and for regenerative therapeutic procedures, analysing the cartilage layer or regenerative tissue, the subchondral plate and the combination of both with other aspects [25]. Thus, the suitable harvesting of osteochondral samples is crucial for the preparation of histological sections. Proper sampling protocols need to respect comparable anatomical locations, or one has to focus on the most severe lesions [40, 25, 41, 42]. There are up to 12 regions requested for examination in a medial tibial condyle if a complex analysis is required, including several issues like biochemistry, biomechanics, gene expression, etc. [25]. The presented tool for the harvesting of osteochondral samples allows the isolation of several specimens even if they lie close together (Fig. 5). While sawing or grinding creates debris in the process, punching separates the tissue without leaving any debris. More importantly, it seems that the fact that the edges of the samples are smooth and defined, frayed and destroyed texture and cells can be avoided. Thermal alterations of the tissue can also be excluded as punching is executed under the application of PBS which, in addition, avoids drying out of the sample.

The presented punching procedure allows the exact harvesting of regenerated tissue or tissue originating from other locations, as the used apparatus allows the individual adjustment of the punch as desired. In combination with a 6-mm defect in diameter, a 5-mm sample in diameter can be extracted exactly as shown (Fig. 5). Further preparations of the articular layer on top are possible, as contamination is excluded by the used of “no touch” technique. Thus, a good starting point is also given for gene expression analysis or cell culture. The latter is possible as sterile preparation can be guaranteed.

In conclusion, we can provide new instruments and procedures for standardisation of creating defects in vitro and in vivo and for exact harvesting of samples in a quick, secure and sterile manner.