Induced collagen cross-links enhance cartilage integration

Abstract

Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2-2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.

Figure 1. Schematic of the experiment examining integration of tissue engineered cartilage to native cartilage.

For Group B, LOX was applied during construct formation, t = 15–28 d. For Group C, LOX was applied after forming the construct-to-native assemblies, t = 29–35 d. For Group D, LOX was applied both before and after the formation of the construct-to-native assemblies, t = 15–35 d.

Figure 2. Gross morphology and histology of constructs/explant assemblies.

Straight from culture, most controls resembled LOX-treated samples, though gaps were seen in one-third of the controls (upper left panel). None of the LOX-treated samples displayed gaps that were grossly visible; a representative sample (Group D) is shown (upper right). Gaps in the controls were also seen after histological processing using picrosirius red (lower left) versus LOX-treated samples (lower right, Group D).

Figure 3. Tensile mechanical data of construct/explant interface.

Significantly higher apparent stiffness (top) was seen when LOX was applied during t = 15–35 d (Group D) than controls (Group A). Significantly higher apparent strength was obtained across the integration interface when engineered cartilage was treated with LOX before being press-fitted into the native cartilage (bottom). Bars with different letters are significantly different (p<0.05).

Figure 4. Gross morphology and histology of explant/explant assemblies.

Neither control nor LOX-treated native-to-native assemblies displayed grossly visible gaps when removed from culture (top row). However, gaps can be seen after histological processing using picrosirius red in the control group, unlike the LOX-treated group (bottom).

Figure 5. Tensile mechanical data of explant/explant interface.

No differences in apparent stiffness (top) or apparent strength (bottom) were seen when LOX was applied to native-to-native integration.