Effects of Weight Bearing on Marrow Adipose Tissue and Trabecular Bone after Anterior Cruciate Ligament Reconstruction in the Rat Proximal Tibial Epiphysis

Abstract

The effects of mechanical unloading after anterior cruciate ligament (ACL) reconstruction on bone and marrow adipose tissue (MAT) are unclear. We investigated weight bearing effects on bone and MAT after ACL reconstruction. Rats underwent unilateral knee ACL transection and reconstruction, followed by hindlimb unloading (non-weight bearing), no intervention (low-weight bearing, the hindlimb standing time ratio (STR; operated/contralateral) during treadmill locomotion ranging from 0.55 to 0.91), or sustained morphine administration (moderate-weight bearing, STR ranging from 0.80 to 0.95). Untreated rats were used as controls. At 7 or 14 days after surgery, changes in trabecular bone and MAT in the proximal tibial were assessed histologically. Histological assessments at 7 or 14 days after surgery showed that ACL reconstruction without post-operative intervention did not significantly change trabecular bone and MAT areas. Hindlimb unloading after ACL reconstruction induced MAT accumulation with adipocyte hyperplasia and hypertrophy within 14 days, but did not significantly affect trabecular bone area. Increased weight bearing through morphine administration did not affect trabecular bone and MAT parameters. Our results suggest that early weight bearing after ACL reconstruction is important in reducing MAT accumulation, and that reduction in weight bearing alone is not sufficient to induce bone loss early after ACL reconstruction.

Keywords: ACL reconstruction; apoptosis; marrow adipose tissue; trabecular bone; weight bearing.

Fig. 1.

Experimental protocol.

Fig. 2.

Histological features in the epiphysis of the proximal tibia. Representative images of Safranin-O Fast Green-stained sections are presented for the control (A, E, I, M), ACLR (B, F, J, N), ACLR + HU (C, G, K, O), and ACLR + M (D, H, L, P) groups. Images in panels (A–H) and (I–P) represent 7 and 14 days post-surgery, respectively. Images in panels (E–H) and (M–P) show high magnification views of the boxes in images (A–D) and (I–L), respectively. Arrowheads indicate empty lacunae. Circular cavities with absence of cytoplasmic staining are adipocytes. Bars = 200 μm and 100 μm in low magnification images (A–D and I–L) and high magnification images (E–H and M–P), respectively.

Fig. 3.

Trabecular bone parameters. (A) Trabecular bone area, (B) empty lacuna ratio, (C) caspase-3 positive cell ratio, and (D) osteoclast surface length. Values are presented as mean ± standard deviation. §: significant main effect of intervention. Different letters indicate statistically significant differences between groups, whereby groups not sharing the same letter are significantly different from one another.

Fig. 4.

Expression of caspase-3. Representative images of caspase-3-immunostained sections are presented for the control (A, E), ACLR (B, F), ACLR + HU (C, G), and ACLR + M (D, H) groups. Images in panels (A–D) and (E–H) represent 7 and 14 days post-surgery, respectively. Arrowheads indicate caspase-3-positive cells. Bars = 100 μm.

Fig. 5.

TRAP staining. Representative images of TRAP-stained sections are presented for the control (A, E), ACLR (B, F), ACLR + HU (C, G), and ACLR + M (D, H) groups. Images in panels (A–D) and (E–H) represent 7 and 14 days post-surgery, respectively. Osteoclasts are stained in purple. Bars = 200 μm.

Fig. 6.

Bone marrow adiposity. (A) Adipocyte number, (B) adipocyte size, and (C) marrow adipose tissue area. Values are presented as mean ± standard deviation. †: significant interactions between time and intervention. ‡: significant main effect of time. §: significant main effect of intervention. Different letters indicate statistically significant differences between groups, whereby groups not sharing the same letter are significantly different from one another. *: significant difference compared to seven days post-surgery.

Fig. 7.

Correlations between empty lacuna ratio and caspase-3 positive cell ratio (A, B), empty lacuna ratio and osteoclast surface length (C, D), trabecular bone area and empty lacuna ratio (E, F), and trabecular bone area and marrow adipose tissue area (G, H). (A), (C), (E), and (G) represent 7 days post-surgery, and (B), (D), (F), and (H) represent 14 days post-surgery.