Temporal changes in collagen cross-links in spontaneous articular cartilage repair

Abstract

OBJECTIVE: Little is known about how the biochemical properties of collagen change during tissue regeneration following cartilage damage. In the current study, temporal changes in cartilage repair tissue biochemistry were assessed in a rabbit osteochondral defect. DESIGN: Bilateral full thickness 3mm osteochondral trochlear groove defects were created in 54 adult male skeletally mature New Zealand white rabbits and tissue repair monitored over 16 weeks. Collagen content, cross-links, lysyl hydroxylation, gene expression, histological grading, and FTIR analyses were performed at 2, 4, 6, 8, 12, and 16 weeks. RESULTS: Defect fill occurred at ~4 weeks post-injury, however, histological grading showed that the repair tissue never became normal, primarily due to the presence of fibrocartilage. Gene expression levels of Col1a1 and Col1a2 were higher in the defect compared to adjacent regions. Collagen content in the repair tissue reached the level of normal cartilage at 6 weeks, but it took 12 weeks for the extent of lysine hydroxylation to return to normal. Divalent immature cross-links markedly increased in the early stages of repair. Though the levels gradually diminished thereafter, they never returned to the normal levels. The mature cross-link, pyridinoline, gradually increased with time and nearly reached normal levels by week 16. Infrared imaging data of protein content paralleled the biochemical data. However, collagen maturity, a parameter previously shown to reflect collagen cross-link ratios in bone, did not correlate with the biochemical determination of cross-links in the repair tissue.. CONCLUSION: Collagen biochemical data could provide markers for clinical monitoring in a healing defect.

Figure 1.

Alcian blue-stained histological sections of repair and adjacent cartilage at (A) 2 weeks, (B) 4 weeks, (C) 12 weeks, and (D) 16 weeks of postrepair. (E) Average O’Driscoll scores (mean ± SEM) (maximum score = 24) from 2 blinded independent observers. *Significantly different from 2-week scores, P < 0.05. Minimal tissue fill was evident at 2 weeks but was nearly complete at 4 weeks. However, as indicated by the O’Driscoll scores, even at 16 weeks postrepair, normal, integrated hyaline cartilage was not found in most tissues.

Figure 2.

Gene expression levels of (A) Type II collagen, (B) Type I collagen, (C) Aggrecan, and (D) Sox9 were measured in RNA isolated from repair tissue in the defect (region A), peri-lesion tissue surrounding the defect (region B), or control tissue from an unaffected part of the joint (region C) at 2, 4, 6, 8, 12, and 16 weeks after surgery to create the experimental cartilage defect in the rabbit trochlea. Relative gene expression levels were measured in triplicate, normalized to the expression level of the housekeeping gene GAPDH, and expressed as mean ± SEM. Asterisk indicates a significant difference between groups and/or time points with P < 0.05.

Figure 3.

(A) Collagen content per protein evaluated as hydroxyproline/1,000 amino acids. (B) Extent of lysine hydroxylation of collagen evaluated as hydroxylysine/collagen at repair time points (2-16 weeks). Region A, within the healing defect; Region B, which included cartilage around the healing defect (peri-lesion or adjacent cartilage); and Region C, control tissue harvested from cartilage on either of the femoral condyles, which was normal in appearance at all time points. Tissues from 5 samples per time point were pooled to obtain enough tissue for analysis, and therefore statistical analysis could not be performed. Collagen content and lysine hydroxylation of collagen were initially lower in repair tissue compared with control and increased to normal levels over time.

Figure 4.

Collagen cross-links expressed as moles of cross-links per mole of collagen for all repair time points (2-16 weeks). DHLNL = dihydroxylysinonorleucine (immature cross-link); HLNL = hydroxylysinonorleucine (immature cross-link); Pyr = pyridinoline (mature cross-link). Region A, within the healing defect; Region B, which included cartilage around the healing defect (perilesion or adjacent cartilage); and Region C, control tissue harvested from cartilage on either of the femoral condyles, which was normal in appearance at all time points. Tissues from 5 samples per time point were pooled to obtain enough tissue for analysis, and therefore statistical analysis could not be performed. Immature cross-links were higher in repair tissue versus control and tended to diminish over time. Pyr was lower in repair compared with controls and appeared to normalize over time.

Figure 5.

FT-IRIS-determined protein content (A) and collagen maturity (B) in repair tissue and in tissue away from the defect. Mean ± SEM were plotted, with some SEM values too low for error bars to be visualized. Significant differences in repair tissue over time as determined by ANOVA at P < 0.05 are shown with ▼. *Significant difference by paired t test for repair and normal tissue at the same time point. By week 8, both total protein and collagen maturity had normalized to levels similar to those in the control tissue. However, the collagen maturity parameters indicated a higher maturity level in the repair tissue compared with controls initially, which is in contrast to the biochemically-determined cross-link data.