2015 ISAKOS Biennial Congress ePoster #1376

In Vivo Evaluation of Tissue-Engineered Constructs for Anterior Cruciate Ligament Reconstruction

Natalie Luanne Leong, MD, Los Angeles, CA UNITED STATES
Nima Kabir, MD, Los Angeles, CA UNITED STATES
Armin L Arshi, MD, Los Angeles, CA UNITED STATES
Azadeh Nazemi, BS, Los Angeles, CA UNITED STATES
Ben M. Wu, DDS, PhD, Los Angeles, CA UNITED STATES
Frank Petrigliano, Los Angeles, CA UNITED STATES
David R. McAllister, MD, Los Angeles, CA UNITED STATES
University of California, Los Angeles, Los Angeles, California, USA

FDA Status Not Applicable

Summary: In this study, a tissue-engineered biodegradable polymer graft with and without the growth factor bFGF and human foreskin fibroblasts is evaluated in a rat model of ACL reconstruction; it represents one of the few in vivo studies evaluating a tissue-engineered ACL, with many animals and long time points, and demonstrates aligned collagen production and promising mechanical properties.

Abstract:

There is interest in a tissue-engineered substitute for use in ACL regeneration. However, there have been relatively few in vivo studies to date. Previously, our laboratory developed a synthetic scaffold by electrospinning PCL to produce a biodegradable aligned fiber scaffold for ACL reconstruction. We showed that this scaffold, when implanted into rat knees, was biocompatible, integrated with native tissue, and had increasing mechanical strength over time. In this study, we report on the implantation and evaluation of tissue-engineered scaffolds augmented with basic fibroblast growth factor (bFGF) and human foreskin fibroblasts (HFF) in an athymic rat model. Polycaprolactone was dissolved in organic solvent and electrospun around a rotating lathe mandrel. Electrospun mats were laser cut into strips, plasma etched, sterilized with EtOH, and coated with collagen. For scaffolds in bFGF containing groups, bFGF was added to the collagen solution, for a total quantity of 100 ng bFGF loaded per scaffold. After drying, four scaffold layers were stacked and affixed to one another using 5-0 vicryl sutures. Human foreskin fibroblasts were seeded at a density of 100,000 cells/cm2. An adaptation of a published rat model of ACL reconstruction was employed for implanting engineered grafts in 44 nine week old male athymic rats. The graft was implanted in the left hind limb, with n=11 for each of four graft groups: collagen coated scaffolds, collagen + bFGF coated scaffolds, collagen coated scaffolds seeded with cells, and collagen coated scaffolds with growth factor and cells. Of the 11 rats in each graft group, n=3 for histology at weeks 8 and 16, and n=5 for biomechanical testing at week 16. At the time of sacrifice, the contralateral native ACLs were used as controls for 11 rats (n=3 for histology/immunohistochemistry at weeks 8 and 16 and n=5 for mechanical testing at week 16). Additionally, the right knees of 5 rats sacrificed at week 16 were used for post-mortem ACL reconstruction using the graft with immediate mechanical testing thereafter as an additional control. All rats underwent surgery without any complications. Hematoxylin and eosin staining demonstrated infiltration of the grafts with cells, with production of aligned collagen. There was minimal inflammatory response as seen with CD68 IHC. At 16 weeks postop, mechanical testing of the grafts in most groups demonstrated significantly higher stiffness and peak load as compared to immediately post-implantation. Acellular grafts loaded with bFGF, achieved 58.8% of the stiffness and 40.7% of the peak load of native ACL. There were no significant differences among the different experimental groups in terms of mechanical properties at 16 weeks post-implantation. This study evaluated an electrospun PCL graft with and without bFGF and/or HFFs in a rodent model of ACL reconstruction. By 16 weeks after implantation, neo-ligaments had begun to form, with infiltration of the scaffolds by native cells and elaboration of aligned collagen. Mechanical strength improved over time, but was not affected by the addition of bFGF or seeding the scaffolds with cells prior to implantation. This study demonstrates the potential of a regenerative medicine approach to ligament tissue engineering.