In this study, the effects of compression of the cartilage interface, surface degradation, and biochemical cross-linking on articular cartilage bonding were investigated. Specific emphasis was put on the resulting mechanical stability of the bonded interface due to molecular bridging of opposing surface structures. This immediate repair technique might provide one further option for the therapeutic treatment of articular cartilage wounds.
Cross-linking
EDC/NHS can non-specifically catalyse covalent binding of the amino or carboxyl groups of collagen; furthermore, carboxylic groups of GAGs may also be involved. In our study, EDC/NHS was the best cross-linking reagent with regard to bonding of articular cartilage blocks in all investigations, that is, when comparing the cross-linkers alone and in combination with degrading pre-treatment. The combinations of EDC/NHS with pepsin or guanidine pre-treatment led to the highest adhesive strengths detected in this study. To date, cell-based articular cartilage repair
in vitro has been correlated with cell metabolism [
2], collagen deposition [
1] and collagen cross-linking [
3,
31]. However, the impressive results yielded with EDC/NHS, the only cross-linker in this study able to additionally catalyse binding of GAGs, may support the idea that molecules other than those involved in the collagen network can also contribute to the integrative process
in vitro.
With regard to cytoxicity, even after exposure for two hours, EDC/NHS elicited no significant effects, whereas glutaraldehyde and genipin compromised cell vitality considerably. In previous investigations, EDC/NHS has also been observed to be advantageous in this respect, compared to glutaraldehyde [
32]. Furthermore, the presented adhesive strength data resulted from a total incubation with cross-linking reagents for two hours. In additional experiments in which guanidine or pepsin pre-treated samples were exposed to EDC/NHS for only 30 or 10 minutes, an adhesive strength between 58 and 56 kPa was observed, that is, there was no significant difference to the prolonged treatment of two hours. An exposure for only 10 minutes would further reduce the risk of any cytotoxic effects.
Glutaraldehyde and genipin cross-link amino groups of proteins; glutaraldehyde molecules can cross-link to each other and the chain building properties may have beneficial effects in comparison to genipin, which can only cross-link in pairs [
33]. In this study, glutaraldehyde alone yielded higher adhesive strengths than genipin alone and also had slight advantages in combination with pre-treatments. With regard to cytotoxicity, a better tolerance for genipin in comparison to glutaraldehyde has been shown in other investigations utilising 3T3 mouse fibroblasts [
34], human osteoblasts [
35] and a subcutaneous chamber in mice [
36]. In our study, both agents exhibited similarly strong cytotoxic effects.
Transglutaminase, a naturally occurring enzyme in articular cartilage, catalyses a specific collagen cross-linking reaction between lysine and glutamine residues. This enzyme has been previously introduced, combined with compressive load, to enhance integrative bonding of articular cartilage wounds [
26]. In our investigation, bonding was detectable only in combination with guanidine pre-treatment; however, compared to the other cross-linking options investigated in this study, transglutaminase resulted in rather weak bonding. The protocol employed in this study was initially described by Chen and colleagues [
27] for cross-linking collagen matrices and may not be well transferable to articular cartilage. The reduced reaction time in this study (2 h) compared to that reported previously (12 h) may also have contributed to the reduced effect. Nevertheless, transglutaminase may still play an important role in
in vitro and
in vivo integrative repair. Transglutaminase has been previously shown to be biocompatible [
37], which was also found in this study, with no significant differences to the control. This enzyme, with its specific catalysing mechanism, may be especially beneficial in an ongoing integrative repair process in which newly synthesised collagen fibrils are present, in contrast to a static experimental setting such as used in this study.
For many years, soft tissue adhesives like fibrin [
38] have been used for cartilage repair or as an additive in autologous chondrocyte transplantation [
39,
40]. They have been found to be supportive in chondrocyte transplantation or to seal the periosteum flap to the cartilage
in vitro, but
in vivo fibrin glue did not provide enough mechanical strength to hold the periosteum flap in place [
41]. As a further alternative, several synthetic materials have been employed as glues for soft tissues, for example, aminopropyltrimethoxysilane-methylenebisacrylamide siloxane or n-butylcyanoacrylate [
42]. In general, the bonding mechanisms of these polymeric substances differ from those of the chemical reagents used in the present work. The polymers penetrate the soft tissue to a certain extent and adhesion is achieved through an interpenetrating network that is irremovable and may impair tissue development at the integration site. In contrast, the chemical cross-linker induces formation of covalent bonds on the surface of the soft tissue. In our opinion, EDC/NHS may be beneficial compared to polymer glue and other chemical cross-linkers (glutaraldehyde and genipin) due to its pure catalysing function. EDC/NHS will not be incorporated into the cartilage and can be easily removed and the scar tissue can be remodelled by cell and extracellular matrix turnover.
Degradation
Degradation or swelling of articular cartilage surfaces have been reported to be beneficial in cell-based integrative repair
in vitro [
6,
9‐
12]. In our study, bonding between cartilage blocks did not occur by merely treating the blocks with trypsin, pepsin, or guanidine (even under compressive conditions). However, pre-treatment with the endopeptidases pepsin or trypsin before cross-linking led to distinct improvements in bonding compared to the use of cross-linkers alone. This was particularly the case for the combination of pepsin with EDC/NHS, for which high values for adhesive strength were achieved. With regard to cytoxicity, pepsin led to no significant effects, whereas trypsin treatment compromised cell vitality considerably.
Pre-treatment with guanidine led to the highest adhesive strengths in combination with all cross-linkers compared to the endopeptidase pre-treatments. Unfortunately, guanidine elicited the strongest cytotoxic effects of all reagents in the study. It is noteworthy that the achieved mechanical bonding is comparable to previous studies employing a similar model. Reindel and colleagues [
4] first reported a mechanical adhesive strength of 34 kPa in integrative experiments. Subsequently, studies including degradation with trypsin followed by cultivation reported enhanced adhesive strengths up to 100 kPa [
6]. In the present study, adhesive strengths up to 65 kPa were achieved by guanidine or pepsin pre-treatment and EDC/NHS cross-linking.
Previously, it was assumed that degrading surface treatment led to cell proliferation or stimulation of cell metabolism [
12]. The observation from our investigations that cross-linking reagents lead to significantly stronger bonding of cartilage blocks after degradation or swelling pre-treatment implies another hypothesis. The accessibility of functional groups is enhanced by both treatments and, therefore, may have led to a better bonding in our study and better integrative repair in previously described studies. It remains to be clarified on which components of the extracellular matrix these functional groups are located. The endopeptidase trypsin was clearly the most effective at releasing GAGs from the cartilage blocks, whereas pepsin released only a minor fraction of the GAGs. The treatment of cartilage blocks with guanidine prior to biochemical cross-linking leads, in theory, primarily to reduction of non-covalent bonding between molecules of the extracellular matrix. The tertiary structure of matrix molecules, especially GAGs, becomes more open after hydrogen bonds are broken. Additionally, elevated water uptake may occur due to more accessible functional groups or a loosened collagen network with increased pore diameter (swelling). Nevertheless, in our study, guanidine also led to the release of a significant fraction of GAGs from the cartilage blocks. As trypsin led to the lowest adhesive strength values of all surface-degrading agents in this study, the bonding observed can not be directly correlated with the amounts of GAGs released. On the contrary, the large amount of GAGs released by trypsin may have compromised the cartilage structure. For total collagen, no significant release was detected for all the pre-treatments. However, only small changes in the structure of the cartilage surface, which are triggered by the pre-treatment agents, but which are not detectable by the assays employed in this study, may be necessary to elicit distinctly improved responses to the cross-linkers.
Clarification of the mechanisms involved appears to be a worthwhile subject for further investigation. Future studies should also address the fact that immature and mature cartilage differ in extracellular matrix content, structure and mechanical properties [
43‐
45]. In aging, cartilage undergoes structural changes that affect the susceptibility to degradation [
46‐
48]. It is also known that integrative bonding is influenced by the developmental stage of articular cartilage [
4]. Therefore, in future studies, the introduced treatment may have to be adjusted to adult cartilage. Furthermore, it has to be noted that for clinical applications special care should be taken to limit any treatment with degrading and cross-linking agents to the area close to the cartilage wound surface. In addition, cell culture experiments after bonding should assure the long-term viability of the treated cartilage.