Comparison of micro-CT post-processing methods for evaluating the trabecular bone volume fraction in a rat ACL-transection model.

Trabecular bone volume fraction assessments are likely sensitive to the analysis method and selection of the region of interest. Currently, there are several methods for selecting the region of interest to analyze trabecular bone in animal models of post-traumatic osteoarthritis. The objective of this study was to compare three published methods for determining the trabecular bone volume fraction of the medial tibial epiphyses in ACL transected and contralateral ACL intact knees. Micro-computed tomography images of both knees were obtained five weeks post-operatively and evaluated using three methods: (1) the Whole Compartment Method that captured the entire medial compartment, (2) the centrally located Single Core Method, and (3) the Triplet Core Method that averaged focal locations in the anterior, central, and posterior regions. The Whole Compartment Method detected significant bone loss in the ACL transected knee compared to the ACL intact knee (p<0.001), with a loss of 15.2±3.9%. The Single Core and the Triplet Core Methods detected losses of 7.5±10.5% (p=0.061) and 14.1±13.7%(p=0.01), respectively. Details regarding segmentation methods are important for facilitating comparisons between studies, and for selecting methods to document trabecular bone changes and treatment outcomes. Based on these findings, the Whole Compartment Method is recommended, as it was least variable and more sensitive for detecting differences in the bone volume fraction in the medial compartment.

Loss of extracellular matrix from articular cartilage is mediated by the synovium and ligament after anterior cruciate ligament injury.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) occurs after anterior cruciate ligament (ACL) injury. PTOA may be initiated by early expression of proteolytic enzymes capable of causing degradation of the articular cartilage at time of injury. This study investigated the production of three of these key proteases in multiple joint tissues after ACL injury and subsequent markers of cartilage turnover. METHODS: ACL transection was performed in adolescent minipigs. Collagenase (MMP-1 and MMP-13) and aggrecanase (ADAMTS-4) gene expression changes were quantified in the articular cartilage, synovium, injured ligament, and the provisional scaffold at days 1, 5, 9, and 14 post-injury. Markers of collagen degradation (C2C), synthesis (CPII) and aggrecan synthesis (CS 846) were quantified in the serum and synovial fluid. Histologic assessment of the cartilage integrity (OARSI scoring) was also performed. RESULTS: MMP-1 gene expression was upregulated in the articular cartilage, synovium and ligament after ACL injury. MMP-13 expression was suppressed in the articular cartilage, but upregulated 100-fold in the synovium and ligament. ADAMTS-4 was upregulated in the synovium and ligament but not in the articular cartilage. The concentration of collagen degradation fragments (C2C) in the synovial joint fluid nearly doubled in the first five days after injury. CONCLUSION: We conclude that upregulation of genes coding for proteins capable of degrading cartilage ECM is seen within the first few days after ACL injury, and this response is seen not only in chondrocytes, but also in cells in the synovium, ligament and provisional scaffold.

Mesenchymal stem cells from the retropatellar fat pad and peripheral blood stimulate ACL fibroblast migration, proliferation, and collagen gene expression.

Mesenchymal stem cells (MSCs) have been of recent interest as adjuncts for ligament repair. However, the effect of these cells on the resident ligament fibroblasts has not yet been defined. In this study, we hypothesized that co-culture of MSCs and ligament fibroblasts would result in increases in the proliferative rate of the ligament fibroblasts and their expression of collagen-related genes, as well as differentiation of the MSCs down a fibroblastic pathway. In addition, we hypothesized that these effects would be dependent on the source of the MSCs. Porcine MSCs were isolated from both the retro-patellar fat pad (ADSCs) and the peripheral blood (PBMCs) and co-cultured with porcine anterior cruciate ligament (ACL) fibroblasts. Fibroblast migration, proliferation, and collagen gene expression were evaluated at time points up to 14 days. ADSCs had a greater effect on stimulating ACL-fibroblast proliferation and procollagen production, while PBMCs were more effective in stimulating ligament fibroblast migration. In addition, co-culture with the ACL fibroblasts led to significant increases in collagen gene expression for ADSCs, suggesting a differentiation of these cells down a fibroblastic pathway during the co-culture period. This was not seen for the PBMCs. Thus, the effects of MSCs on in situ ACL fibroblasts were found to be source dependent, and the choice of MSC source should take into account the different performance characteristic of each type of MSC.

Histologic Predictors of Maximum Failure Loads Differ between the Healing ACL and ACL Grafts after 6 and 12 Months In Vivo.

BACKGROUND: Bio-enhanced ACL repair, where the suture repair is supplemented with a biological scaffold, is a promising novel technique to stimulate healing after ACL rupture. However, the histological properties of a successfully healing ACL and how they relate to the mechanical properties have not been fully described. HYPOTHESIS/PURPOSE: The purpose of the study was to determine which histological features best correlated with the mechanical properties of the healing ACL repairs and ACL grafts in a porcine model at six and twelve months after injury. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-eight Yucatan mini-pigs underwent ACL transection followed by: 1) conventional ACL reconstruction with bone-patellar tendon-bone (BPTB) allograft, 2) bio-enhanced ACL reconstruction with BPTB allograft using a bioactive scaffold, or 3) bio-enhanced ACL repair using the same bioactive scaffold. After 6 and 12 months of healing, structural properties of the ACL or graft (yield & failure load, linear stiffness) were measured. Following mechanical testing, ACL specimens were histologically analyzed for cell and vascular density and qualitatively assessed using the advanced Ligament Maturity Index. RESULTS: We found that after six months of healing, the cellular organization sub-score was most predictive of yield load (r2=0.98), maximum load (r2=0.89) and linear stiffness (r2=0.95) of the healing ACL, while at 12 months, the collagen sub-score (r2=0.68) became the best predictor of maximum load. For ACL grafts, the reverse was true, with the collagen sub-score predictive of yield and maximum loads at six months (r2=0.55), and graft cellularity predictive of maximum load of the graft at 12 months (r2=0.50). CONCLUSIONS: These findings suggest there may be key biologic differences in development and maintenance of ACL tissue after repair or reconstruction with early ligament function dependent on cellular population of the repair but early graft function dependent on the maintenance of organized collagen.