In the present study we found an improvement in histological and biomechanical integration of articular cartilage after treatment with a combination of hyaluronidase and collagenase, a protocol that was previously shown to increase chondrocyte densities in wound edges in vitro
]. Our setup of a 3-mm disc placed in an annulus provides a reasonable representation of the in vivo
situation, in which cartilage is transplanted into a defect with wound edges perpendicular to the surface. Because an in vitro
culture system might not provide the optimal environment for tissue growth and repair [24
], we decided to perform our experiments in the well established nude mouse model [25
], creating an environment in which there is an ample supply of nutrients.
In this setup, cellularity in nontreated wound edges reached the levels of unwounded cartilage, and cellularity of unwounded cartilage was increased to a level similar to that before implantation, which is in contrast to results from our previous in vitro
]. We believe that this is due to the nutrient-rich in vivo
environment. However, in this model we confirmed [22
] that the enzymatic treatment protocol enhanced the number of cells near the wound edges as compared with nontreatment, and resulted in better histological integration, as assessed by the percentage of matrix connection in the interfacial area. Furthermore, the repair tissue exhibited collagen fibres crossing the wound edges, and the matrix in both experimental groups exhibited cartilage specific collagen type II, limited (pro-)collagen type I and no collagen type III. This improved integration following enzymatic treatment was further supported by push-out tests, which are similar to tests described by others [24
Although enzymatic treatment significantly increased mechanical strength to 1.32 MPa, the interfacial strength was still almost sevenfold less than the 8.8 MPa intrinsic failure strength values observed for intact cartilage. It should be appreciated that the fairly simple normalization to interface area is a rather crude method because the interface stress is not uniformly distributed. Therefore, tests using different sizes or shapes of specimens cannot readily be compared. Because the average thickness of our samples was 1.14 ± 0.28 mm for the treated group and 1.14 ± 0.21 mm for controls, and no correlation could be found between sample thickness and failure strength, we may compare strength values within the present study.
Our findings indicate a relation between interfacial strength and cellular activity at the interface. This confirms the results reported by DiMicco and coworkers [28
], who used fetal, calf and adult bovine cartilage; after 14 days of culture those investigators found the highest failure stress in calf cartilage at 77 kPa in a single lap shear test. However, Reindel and coworkers [29
] found an interface strength of 34 kPa after 3 weeks of culture, and showed that integrative strength was highly dependent on the use of fetal bovine serum in culture, which can influence cellular activity. Dependence of integration on active cell processes is also demonstrated by lack of adhesive strength when combining two lyophilized explant blocks [30
]. In an 8-week bioreactor culture of tissue engineered cartilage core constructs with surrounding native cartilage, Obradovic and coworkers [24
] found better mechanical integration of very young (5 days) constructs (254 kPa) as compared with more mature constructs (5 weeks; approximating 150 kPa). Peretti and coworkers [26
] also used lyophilized explants, which were seeded with chondrocytes and then held together using fibrin glue and placed subcutaneously in nude mice for up to 6 weeks. Tensile testing showed a clear increase of failure strength to 77 kPa, which is 10 times higher than unseeded control explants, with failure always occurring at the interface between new tissue and devitalized matrix. Because cellular activity is clearly an important factor in integration, we should appreciate that in most studies young bovine cartilage is used, which is more cellular than human cartilage. In a previous study, however, we did see similar effects of enzymatic treatment on cell density in human adult articular cartilage [22
]. It can be anticipated that, because of the lower cell numbers, the overall repair process might be slower than in the present study but can still be stimulated using enzyme treatment.
Our findings suggest that enzymatic treatment may be a promising technique with which to improve cartilage integration, in addition to currently developing clinical and experimental articular cartilage repair techniques. The cell counts along the wound edge in the control group were comparable to those of native tissue. However, a close look at the histological pictures (Fig. ) shows that a thin acellular band is still visible. In the enzyme-treated group cell counts were even higher than those in native cartilage, and histology did not reveal a large acellular band, as seen in the controls (Fig. ), thus fulfilling one of the prerequisites for integration, namely the presence of active chondrocytes close to the lesion site. The high cellularity at the wound edge observed in the present study probably resulted in the increased collagen fibre deposition across the wound gap of adjacent cartilage surfaces, as shown in the picro-Sirius Red slides (Fig. ). Normally, cross-gap deposition of collagen between native and repair tissue is insufficient in the reparative process that occurs after full-thickness defects [2
]. The observed cross-gap deposition of collagen in the present study coincides with increased interfacial strength, as shown previously in integration experiments using fetal, calf and adult bovine cartilage explants [28
]. Those studies showed a correlation between increased adhesive strength and an increased hydroxyproline incorporation in the interface area. Furthermore, inhibition of collagen cross-link formation by β-aminopropionitrile resulted in almost complete loss of integrative repair.
The explanation for the success of the enzymatic treatment technique may be found by examining wound healing in vascularized tissues. In nonvascularized articular cartilage, proteolytic enzyme activity is either lacking or insufficient to degrade and remove the observed acellular band in the wounded areas, as occurs with debris and necrotic tissue in vascularized tissues. Application of enzymes may remove this layer, uncovering an activated area of chondrocytes that are capable of integration. Another possible underlying mechanism of this enzymatic treatment may be the partial degradation of extracellular matrix surrounding the wound edge chondrocytes, which frees chondrocytes from the tight extracellular matrix in which they were entrapped. Because chondrocytes have been shown to have the ability to migrate [33
], this may enable them to move to the wound edge in need of repair. A third possible mechanism of the enzymatic degradation of wound edge extracellular matrix may be the stimulation of local chondrocyte proliferation, which can be seen by looking at the cell clusters in the histological images (e.g. Fig. ). Although we did observe cell division, the exact mechanism by which the enzymatic treatment exerts its effects is still unclear. A more detailed mechanistic study is needed to further elucidate this.
In the present study we demonstrated the potential of hyaluronidase and collagenase treatment in a screening 'in vivo' environment. Animal experiments with actual articular cartilage defects are needed to determine the value of our findings. Further studies must be undertaken to optimize the enzymatic treatment protocol (e.g. shorter treatment duration) and learn more about the mechanisms involved, such as cell migration to the wound area and matrix deposition, and to improve mechanical interface strength further to the level of intact cartilage, which is still almost an order of magnitude higher. Therefore, longer term studies are required to judge the success of different integration enhancing techniques against the mechanical strength of intact cartilage, and to develop protocols that may become clinically applicable, which in our view could be a valuable addition to existing repair strategies.