Abstract
BackgroundA detailed evaluation focusing on the fibrocartilage layers in the anterior cruciate ligament (ACL) insertion is necessary to consider regeneration of the insertion. This study examined the development of the fibrocartilage layers in the ACL tibial insertion in rabbits by quantitative morphometric evaluations based on histological and immunohistochemical analyses.MethodsMale Japanese white rabbits were used because of their history of use for histomorphometric analyses of the ACL insertion and to eliminate the influence of female hormones on the ACL. Six animals were euthanized at each age (1 day and 1, 2, 4, 6, 8, 12, and 24 weeks); in total, 48 animals were used. Proliferation rate, apoptosis rate, Sox9-positive rate, and chondrocyte number were evaluated. Safranin O-stained glycosaminoglycan (GAG) areas, tidemark length, ACL insertion width, and ACL length were also evaluated. All parameters were compared with those at age 24 weeks of age.ResultsHigh levels of chondrocyte proliferation and Sox9 expression continued until 4 and 8 weeks of age, respectively, and then gradually decreased. Chondrocyte apoptosis increased up to 8 weeks. The chondrocyte number, ACL insertion width, ACL length, safranin O-stained GAG areas, and tidemark length gradually increased up to 12 weeks.ConclusionChondrocytes that displayed chondrocyte proliferation and Sox9 expression increased until 12 weeks of age, in accordance with development of the ACL length and its insertion width. The GAG production and tidemark length also increased until 12 weeks of age. The development of fibrocartilage layers in the ACL insertion was complete at 12 weeks of age.
Highlights
A detailed evaluation focusing on the fibrocartilage layers in the anterior cruciate ligament (ACL) insertion is necessary to consider regeneration of the insertion
We considered that a more detailed quantitative evaluation using these parameters when focusing on the fibrocartilage layers in the ACL insertion is crucial to understanding the formation process and the anatomical structural differences of the fibrocartilage layers during growth and healing of the ACL insertion
The chondrocyte apoptosis rate was determined by the numbers of Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL)-positive chondrocytes (Fig. 4)
Summary
A detailed evaluation focusing on the fibrocartilage layers in the anterior cruciate ligament (ACL) insertion is necessary to consider regeneration of the insertion. We showed that mechanical unloading and knee immobilization increased chondrocyte apoptosis, decreased chondrocyte proliferation, and decreased the GAGs in the fibrocartilage layers in the patellar tendon insertion and ACL insertion in rabbits [8, 9]. Over load via an ACL partial tear and gradual elongation using external fixation decreased chondrocyte apoptosis, increased chondrocyte proliferation, and increased the GAGs in the fibrocartilage layers in the patellar tendon insertion and ACL insertion in rabbits [10, 11]. We consider that chondrocyte apoptosis, chondrocyte proliferation, and GAGs can effectively reflect differences in the mechanical environment at the insertion site. Sex-determining region Y box 9 (Sox9) directly regulates the type-II collagen gene and is a master regulator of chondrogenesis by promoting proliferation and differentiation of mesenchymal stem cells into chondrocytes [12,13,14]
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