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Purpose: The purpose of this study was to clarify femoral and tibial tunnel angles in anatomic double-bundle anterior cruciate ligament (ACL) reconstruction. Methods: A prospective study was performed in 50 consecutive patients who underwent anatomic double-bundle ACL reconstruction via hamstring tendon grafts for chronic ACL deficiency in the unilateral knee. For this procedure, 2 hamstring tendon grafts were secured with EndoButtons (Smith & Nephew Endoscopy, Andover, MA) on the femur and with 2 staples on the tibia. Each patient underwent radiologic examination 1 year after surgery. Results: The tibial tunnel angles of the posterolateral bundle averaged 40.7° in the anteroposterior view and 35.4° in the lateral view. The tibial tunnel angles of the anteromedial bundle averaged 15.6° in the anteroposterior view and 41.4° in the lateral view. The femoral tunnel angles of the posterolateral bundle averaged 44.0° in the anteroposterior view and 52.0° in the lateral view. The femoral tunnel angles of the anteromedial bundle averaged 18.0° in the anteroposterior view and 49.8° in the lateral view. Conclusions: The given data will be useful for surgeons to evaluate the tunnel positions and standardization postoperatively in anatomic double-bundle reconstruction. Level of Evidence: Level II, development of diagnostic criteria on the basis of consecutive patients with universally applied reference gold standard.

A tissue-cultured tendon matrix infiltrated with cultured fibroblasts can be regarded as an ideal tissue-engineered tendon model. To clarify the role of TGF-beta in a tissue-cultured tendon matrix during ex vivo cellular infiltration, the present ex vivo study was conducted to test the following hypothesis that antibody neutralization of TGF-beta enhances weakening of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. In skeletally mature female rabbits, fibroblasts were isolated from the right patellar tendons using an explant culture technique, and the left patellar tendons underwent multiple freeze/thaw treatment with liquid nitrogen to obtain an acellular tendon matrix. Each acellular tendon was placed in a collagen gel containing cultured fibroblasts and then incubated with or without anti-TGF-beta1 antibody for 6 weeks. We found that antibody neutralization of TGF-beta enhanced the decrease in the tensile strength and tensile modulus of the collagen fascicles of the patellar tendon matrix in response to ex vivo fibroblast infiltration. The present study indicates a possibility that TGF-beta may have a role in suppressing the material deterioration of the fascicles in the tendon during ex vivo cellular infiltration.

expansion with proliferation medium. Osteogenic assay Cells (n1.0x105) were cultured in 6-well plates in osteogenic medium suppremented with BMP4 (100ng/ ml) and stained for alkaline phosphatase (ALP) at day 7. As well, cells were also cultured as pellets for 21 days in osteogenic medium and evaluated with a micro-CT and stained with von Kossa for assessment of mineralization. Chondrogenic assay Cells (n2.5x105) were placed in a 15ml conical tube, centrifuged at 600g, and cultured in chondrogenic medium (Lonza) supplementted with TGF-3 (10ng/ml). Pellets were assessed macroscopically at day 14, and stained with Alcian blue/nuclear fast red. Adipogenic assay Cells (n1.0x105) were cultured in 6-well plates for 14 days in adipogenic medium and stained for Oil Red O. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Differentiation potential of these cells were confirmed by RT-PCR using osteogenic (osteocalcin and type 1 collagen), chondrogenic (Sox9 and type 2 collegen), and adipogenic (lipoprotein lipase (LPL) and Peroxisome proliferator activated receptor (PPAR))-related markers. Results: Immunohistochemical staining Tissues showed more positive staining for CD34 and CD146 in the ruptured site than in the mid-substance. Flow cytometry analysis The percentage of CD34CD45 and CD146 CD45 cells were significantly higher in the ruptured site compared to the mid-substance (CD34CD45 cells: reptured site, 44.3%, mid-substance, 8.3%; CD146CD45 cells: reptured site, 6.5%, mid-substance, 0.5%) (p0.01). Proliferation potential CD34 and CD146 cells from the ruptured site showed high expansion potential, however, those from the mid-substance did not expand well. Based on these results, we used those from the ruptured site for multilineage differentiation assay. Osteogenesis Cultured CD34 and