Tissue-Engineered ACL
From the Laboratory to the Knee Joint

Francine Goulet
Lucie Germain
Rjean Cloutier
Jean Lamontagne
Hubert Robitaille
Ludovic Bouchard
Franois A. Auger
Laboratoire d'Organognse Exprimentale (LOEX)
Qubec, QC

Tissue-engineering of Anterior Cruciate Ligament (ACL) substitutes involves some technical challenges. Considering native ACL biomechanical properties, including strength, the production of a tissue-engineered ACL in vitro seems impossible to achieve. In addition, strong links are required at the matrix-bone interface of ACL substitutes to allow proper ligament functional stability in the joint. Other issues may be raised post-implantation. Collagen, the main component of any soft tissue matrix, must be synthesized and remodeled in situ, to ensure a continuous regeneration of the broken matrix fibers following physical activities. The alignment and ultrastructural organization of the collagen fiber network that supports the tissue must be reproduced in vitro, similar to native ACL in vivo. These issues greatly influence the strategy used to develop tissue-engineered ACL substitutes. Various approaches can be proposed to reach this goal, creating different acellular and cellularized ACL substitutes.

Our tissue-engineering team aims at reconstructing tissues that could serve both experimental and clinical interests. Our main concept is to produce bACLs in vitro, to permanently implant them in vivo, such that their properties will be improved by local and systemic physiological stimuli. We have produced a bioengineered ACL (bACL), using biocompatible materials to facilitate integration in the knee joint. Up to now, we have succeeded in constructing a bACL that shares several histological and mechanical features of the natural ACL. A type I collagen gel seeded or not with living cells and anchored with two bone plugs is the basic definition of the bACL that we initially developed (Figure 1). The main differences between our bACL over other tissue-engineered ACL substitutes include: 1) reconstruction is entirely achieved in vitro, 2) the collagen scaffold, 3) the addition of autologous cells within its scaffold, allowing early matrix secretion and remodeling, 4) the absence of chemical crosslinking agents, and 5) the use of porous bone plugs to anchor the bACL in culture, facilitating its implantation in the tibia and femur of the knee joint.

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Figure 1
Macroscopic view of a bACL in culture.

The strength of such bACL in vitro is too low for implantation in a knee joint. However, it is possible to reinforce the bACL collagen scaffold in vitro, to reach acceptable strength ranges for implantation. Static tension, applied in a direction parallel to the longitudinal axis of the bACL in culture, induces alignment of the collagen fibers between its two bone anchors, before or after cell seeding in its matrix. Cyclic stretching of cell-seeded bACLs, cultured in ascorbic acid supplemented medium, leads to matrix strengthening by stimulating collagen synthesis by the cells. Dehydration of the scaffold, notably by lyophilization, induces the aligned matrix fibers to cluster. Freeze-drying also reinforces the links between the collagen matrix of the bACL and its bone plugs, since dehydration favours attachment of the collagen fibers to the bone, after initial polymerization into the porous structure of the bone plugs. After rehydration of lyophilized bACL in culture medium, its strength is increased by at least ten times in vitro. The addition of a surgical thread between the two bone plugs of the bACL, resorbable within a month post-implantation in a knee joint, increases 15 times its ultimate resistance to rupture. Such level of strength allows the surgical implantation of the bACL in a knee joint.

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Figure 2
Histological section, stained with the method of Massons trichrome, of a vascularized bACL, grafted for six months in a goat knee joint. The red spots are hematies distributed in small blood vessels. The collagen fibers are stained in blue (40X).

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Figure 3
Histological section, stained with the method of Massons trichrome, of a bACL grafted for six months in a goat knee joint. The red spots correpond to hematies, distributed in small blood vessels (40X).

Most animal experimentations with the new ACL substitutes are performed on goats and rabbits. When the bACL is grafted in the caprine model, several changes occur within its structure in situ. Only one month post-grafting, the goats were walking and hopping normally. The integration of the bACL was so successful that it was difficult to differentiate the graft from the contralateral native ACL macroscopically. Histological data showed bACL integration in the goat knee joints, with vascularization (Figure 2), and neoformation of structured fibrocartilage, populated with several chondrocytes (Figure 3). Electron microscopy analysis revealed the formation of multiple and large collagen fibers, organized as in the native ACL. Interestingly, the size of these fibers was equivalent to the natural ACL fibers1. Consequently, a major gain in strength had occurred after implantation. The one-year-old grafts reached up to 40% of the strength of a natural ACL. We postulate that early vascularization and the fibrocartilage regeneration at the bACL bone insertions, possibly in response to biomechanical stimuli, contributed to this strengthening. Two goats were grafted with autologous central portion of the patellar tendon, as is frequently performed in humans. The strength of these grafts reached up to 30% of the strength of a natural ACL. Thus, these data suggest that tissue-engineered ACLs could eventually be evaluated in human knee joints and possibly become a new surgical option for torn ACL replacement.

References

1. Goulet F., Rancourt D., Cloutier R., McKee M., Tremblay J., Bouchard M., Stevens L.-M., Labrosse J., Dupuis D., Lamontagne J. Implantation of Bioengineered Anterior Cruciate Ligament Substitutes: Histological, Ultrastructural and Biomechanical Analyses. Applied Bionics Biomech. 1(2): 115-121, 2004.

2. Germain L., Berthod F., Moulin V., Goulet F., Auger F.A. Principles of living organs reconstruction by tissue engineering. In Tissue Engineering and Novel Delivery Systems. (Yaszemski M.J., Trantolo D.J., Lewandrowski K.U., Hasirci V, Altobelli D.E., Wise D.L., Eds.), Marcel Dekker, Inc., New-York. Chap. 10 : pp. 197-228, 2004

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