Time-dependent gene expression and immunohistochemical analysis of the injured anterior cruciate ligament

Abstract

Objectives: This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation.

Methods: Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-β1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3).

Results: Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-β1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III.

Conclusions: Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs' expressions.

Keywords: ACL; Anterior cruciate ligament; Gene expression; Immunohistochemical analysis; Matrix metalloproteinases (MMPs); Stat3; Synovial fibroblast-like cell.

Fig. 1

Box plots showing the relative mRNA expression of collagen type 1 (COL 1), COL 3, biglycan, decorin, α-smooth muscle actin (α-SMA), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinases-1 (TIMP-1). In each box plot the y-axis represents the normalised ratio, the box the median and interquartile range, the whiskers the 10th and 90th percentiles and ° the outliers. (* p < 0.05, ** p < 0.01).

Figs. 2a - 2c

Figure 2a – haemotoxylin and eosin staining (H & E) and cell distributions positive for CD68, CD55, Stat3 and P-Stat3 of the ruptured end of the ligament in each phase. Figures 2b and 2c – box plots showing quantification using ImageJ software of b) the CD55 positive area and c) the P-Stat3 positive areas for the ruptured and mid areas of the ligament for each phase. The box represents the median and interquartile range, the whiskers the 10th and 90th percentiles and ° the outliers (*, significant difference (p < 0.01) compared with phases I and II; **, significant difference compared with phases I, II and IV).

Figs. 2a - 2c

Figure 2a – haemotoxylin and eosin staining (H & E) and cell distributions positive for CD68, CD55, Stat3 and P-Stat3 of the ruptured end of the ligament in each phase. Figures 2b and 2c – box plots showing quantification using ImageJ software of b) the CD55 positive area and c) the P-Stat3 positive areas for the ruptured and mid areas of the ligament for each phase. The box represents the median and interquartile range, the whiskers the 10th and 90th percentiles and ° the outliers (*, significant difference (p < 0.01) compared with phases I and II; **, significant difference compared with phases I, II and IV).

Figs. 2a - 2c

Figure 2a – haemotoxylin and eosin staining (H & E) and cell distributions positive for CD68, CD55, Stat3 and P-Stat3 of the ruptured end of the ligament in each phase. Figures 2b and 2c – box plots showing quantification using ImageJ software of b) the CD55 positive area and c) the P-Stat3 positive areas for the ruptured and mid areas of the ligament for each phase. The box represents the median and interquartile range, the whiskers the 10th and 90th percentiles and ° the outliers (*, significant difference (p < 0.01) compared with phases I and II; **, significant difference compared with phases I, II and IV).

Figs. 3a - 3b

Figure 3a – western blotting of Stat3 and P-Stat3 in each phase. Figure 3b – bar chart showing the mean P-Stat3 quantification using ImageJ software. Error bars represent the standard deviation.

Figs. 3a - 3b

Figure 3a – western blotting of Stat3 and P-Stat3 in each phase. Figure 3b – bar chart showing the mean P-Stat3 quantification using ImageJ software. Error bars represent the standard deviation.