Histomorphological Alterations of Human Anterior Cruciate Ligament Grafts During Mid-Term and Long-Term Remodeling

Abstract

Objective: The aim of the present paper is to analyze mid-term and long-term alterations of human anterior cruciate ligament (ACL) grafts during the remodeling process with special regards to cellularity, α-smooth muscle protein (αSMP) expression, and crimp length in comparison to the native ACL.

Methods: A total of 34 patients were included (23 male and 11 female). Biopsies of 13 semitendinosus tendon and 14 patellar tendon autografts were obtained during surgical revision secondary to an ACL reconstruction. According to the interval between the index procedure and sample collection, the patients were divided into four groups: 4-12 months, 13-60 months, 61-108 months, and >108 months. Seven samples of native ruptured ACL tissue obtained during surgical intervention served as control. All biopsies were taken from the intraligamentous part of the ACL or the graft. Histomorphological and immunohistochemical analyses were conducted after samples were stained using hematoxylin-eosin, Giemsa, and αSMP enzyme-labeled antibodies. The total cell density, the numbers of fibroblasts and fibrocytes, the fibroblast/fibrocyte ratio, the number of αSMP+ cell nuclei, and the percentage of αSMP+ cells per fibroblast as well as the crimp lengths were determined using light microscopy.

Results: In the early phase of remodeling, the grafts featured extensively high total cell counts (1021.2 ± 327.8, P = 0.001), with high numbers of fibroblasts (841.4 ± 245.2, P = 0.002), fibrocytes (174.5 ± 113.0, P = 0.04), and αSMP+ cells (78.3 ± 95.0, P = 0.02) compared to controls (390.1 ± 141.7, 304.5 ± 160.8, 65.6 ± 31.4 and 2.3 ± 2.6, respectively). Thereafter, the numbers of all cell entities decreased. After more than 108 months, the percentage of αSMP+ cells per fibroblast reached physiological values (ratio 1.3 ± 1.0, P = 0.41; control 0.8 ± 0.8), while the total cell count (834.3 ± 183.7, P = 0.001) as well as the numbers of fibroblasts (663.5 ± 192.6, P = 0.006) and fibrocytes (134.1 ± 73.0, P = 0.049) remained significantly high. The fibroblast/fibrocyte ratio showed no significant alterations over the course of time compared to the controls. The collagen crimp lengths were elongated by tendency in the early phase (28.8 ± 12.9 mm, P = 0.15; control 20.7 ± 2.2 mm) and significantly shortened over time, with the lowest values in the long term (14.8 ± 2.0 mm, P = 0.001). The comparison of biopsies from semitendinosus tendon and patellar tendon autografts revealed no significant differences for any of the histomorphological parameters investigated.

Conclusion: This study reveals distinctive mid-term and long-term immunomorphological alterations during human ACL graft remodeling. These data clearly indicate that the remodeling is a process that continues for 9 years or more. Furthermore, it seems to be a process of adaptation rather than full restoration. Even in the long run, several biological properties of the native ACL are not completely reestablished.

Keywords: ACL; Anterior cruciate ligament; Ligamentization; Reconstruction; Remodeling.