Three-Dimensional Engineered Bone–Ligament–Bone Constructs for Anterior Cruciate Ligament Replacement

Abstract

The anterior cruciate ligament (ACL), a major stabilizer of the knee, is commonly injured. Because of its intrinsic poor healing ability, a torn ACL is usually reconstructed by a graft. We developed a multi-phasic, or boneligament-bone, tissue-engineered construct for ACL grafts using bone marrow stromal cells and sheep as a model system. After 6 months in vivo, the constructs increased in cross section and exhibited a well-organized microstructure, native bone integration, a functional enthesis, vascularization, innervation, increased collagen content, and structural alignment. The constructs increased in stiffness to 52% of the tangent modulus and 95% of the geometric stiffness of native ACL. The viscoelastic response of the explants was virtually indistinguishable from that of adult ACL. These results suggest that our constructs after implantation can obtain physiologically relevant structural and functional characteristics comparable to those of adult ACL. They present a viable option for ACL replacement.

Fresh Versus Frozen Engineered Bone–Ligament–Bone Grafts for Sheep Anterior Cruciate Ligament Repair

“…We have shown that after 6 months in vivo, these engineered scaffoldless BLB constructs undergo significant remodeling to develop similar mechanical and morphological properties to those of native ACLs and are capable of inducing vascular and neural development. 5 These results show that our BLB constructs offer a promising alternative technology for ACL reconstruction.…”

“…5,11 Animals were acclimated to the Unit for Laboratory Animal Medicine (ULAM) husbandry facilities at the University of Michigan for at least 1 week before any procedure. Sheep were given access to food and water ad libitum.…”

“…5 All solutions and media were prepared and stored at 4°C and were warmed to 37°C in a heated bead bath before use. Briefly, the growth medium (GM) consisted of 78% Dulbecco's modified Eagle medium (DMEM; Gibco, Grand Island, NY) with 20% fetal bovine serum (Gibco, Grand Island, NY), 2% antibiotic anti-mycotic (ABAM; Gibco), 6 ng/mL basic fibroblast growth factor (Peprotech, Rocky Hill, NJ), 0.13 mg/mL ascorbic acid-2-phosphotase (Sigma-Aldrich, St. Louis, MO), and 0.05 mg/mL L-proline (Sigma-Aldrich); the differentiation medium (DM) consisted of 91% DMEM, 7% horse serum albumin (Gibco), 2% ABAM, 0.13 mg/mL asc-2-phos, 0.05 mg/mL L-proline, and 2 ng/mL transforming growth factor beta (Peprotech).…”

“…For the culture of bone cells, 10 -8 M dexamethasone (Sigma-Aldrich) was added to the GM and DM. 13 Construct dishes were prepared as described previously 5 to house and constrain the formed 3D constructs. Briefly, 100-mm-diameter cell culture plates were filled with 12 mL Sylgard (type 184 silicon elastomer; Dow Chemical Corp., Midland, MI) and allowed to cure for 3 weeks at room temperature.…”

“…5 Briefly, after induction of general anesthesia and preparation of the surgical site, the native ACL of the left limb was removed with remnants of the stump on both the femur and tibia used to aid in anatomical positioning of the BLB. Drill guides were used to position Steinmann pins for precise placement at the center of the tibial and femoral footprints.…”