Enhancement of Tendon Graft Osteointegration Using Mesenchymal Stem Cells in a Rabbit Model of Anterior Cruciate Ligament Reconstruction Jit-Kheng Lim, F.R.C.S., James Hui, F.R.C.S., Li Li, M.D., Ashvin Thambyah, M.Sc., James Goh, Ph.D., and Eng-Hin Lee, F.R.C.S.C. Purpose: To study the effect of coating tendon grafts with mesenchymal stem cells (MSCs) on the rate and quality of graft osteointegration in anterior cruciate ligament (ACL) reconstruction. Type of Study: Animal model. Methods: Bilateral ACL reconstructions using hamstring tendon autografts were performed on 48 adult rabbits. Grafts were coated with MSCs in a fibrin glue carrier in one limb, and fibrin glue only in the other. Assessment was done at 2, 4, and 8 weeks. Histologic analysis was carried out using standard and immunohistochemical stains. Biomechanical testing of force and stiffness during loading to ultimate failure was performed. Results: Control reconstructions showed mature scar tissue with some Sharpey’s-like fibers spanning the tendon-bone interface at 8 weeks. The MSC-enhanced reconstructions had large areas of cartilage cells at the tendon-bone junction at 2 weeks. By 8 weeks, a mature zone of cartilage was seen gradually blending from bone into the tendon grafts. This zone stained strongly for type II collagen and showed histologic characteristics similar to normal rabbit ACL insertions. Biomechanically, there was no statistical difference between limbs at 2 and 4 weeks. At 8 weeks, the MSC-enhanced grafts had significantly higher failure load and stiffness. Conclusions: Coating of tendon grafts with MSCs results in healing by an intervening zone of cartilage resembling the chondral enthesis of normal ACL insertions rather than collagen fibers and scar tissue. MSC-enhanced ACL reconstructions perform significantly better than controls on biomechanical testing. Clinical Relevance: Enhancement of tendon graft osteointegration with MSCs is a novel method offering the potential for more physiologic and biomechanically stronger ligament reconstructions. Key Words: Graft healing—Stem cell—Mesenchymal—ACL reconstruc- tion—Bone tunnel—Tendon graft. T endon grafts anchored within bony tunnels are used in a wide variety of orthopaedic procedures. Tendon pullout from bony tunnels occurs up to 12 weeks after hamstring graft anterior cruciate ligament (ACL) reconstruction in animal models, 1-4 suggesting that mechanical strength of reconstructions before that time are dependent to a large extent on the method of fixation. For this reason, a large emphasis of recent research has been on the relative benefits and biome- chanical profiles of various fixation methods for ham- string tendon autografts. 5-7 There is increasing interest in the use of biological agents to enhance tendon-bone osteointegration within the bony tunnel. Recent studies have shown that ap- plication of bone morphogenic protein-2 1 and a mix- ture of bone growth proteins 8 to the surface of tendon autografts resulted in enhanced tendon-bone healing and improved biomechanical properties. Mesenchy- mal stem cells (MSCs) are pluripotential cells that are From the Department of Orthopaedic Surgery, National Univer- sity Hospital, Singapore, Republic of Singapore. Supported in part by Grant No. EF2001/33/ROC-009 from the National University Hospital, Singapore. Address correspondence and reprint requests to Jit-Kheng Lim, F.R.C.S., Department of Orthopaedic Surgery, National University Hospital, Lower Kent Ridge Road, Singapore 119074, Republic of Singapore. E-mail: nitrogenbubble@mac.com © 2004 by the Arthroscopy Association of North America 0749-8063/04/2009-3814$30.00/0 doi:10.1016/j.arthro.2004.06.035 899 Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 20, No 9 (November), 2004: pp 899-910