In Vitro and In Vivo Evaluation of Heparin Mediated Growth Factor Release from Tissue-Engineered Constructs for Anterior Cruciate Ligament Reconstruction Natalie L. Leong, 1 Armin Arshi, 1 Nima Kabir, 1 Azadeh Nazemi, 2 Frank A. Petrigliano, 1 Ben M. Wu, 2 David R. McAllister 1 1 Department of Orthopaedic Surgery, University of California, Los Angeles, California, 2 Department of Biomedical Engineering, University of California, Los Angeles, California Received 7 July 2014; accepted 29 September 2014 Published online 31 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22757 ABSTRACT: Anterior cruciate ligament (ACL) rupture is a common injury often necessitating surgical treatment with graft reconstruction. Due to limitations associated with current graft options, there is interest in a tissue-engineered substitute for use in ACL regeneration. While they represent an important step in translation to clinical practice, relatively few in vivo studies have been performed to evaluate tissue-engineered ACL grafts. In the present study, we immobilized heparin onto electrospun polycaprolactone scaffolds as a means of incorporating basic fibroblast growth factor (bFGF) onto the scaffold. In vitro, we demonstrated that human foreskin fibroblasts (HFFs) cultured on bFGF-coated scaffolds had significantly greater cell proliferation. In vivo, we implanted electrospun polycaprolactone grafts with and without bFGF into athymic rat knees. We analyzed the regenerated ACL using histological methods up to 16 weeks post- implantation. Hematoxylin and eosin staining demonstrated infiltration of the grafts with cells, and picrosirius red staining demonstrated aligned collagen fibers. At 16 weeks postop, mechanical testing of the grafts demonstrated that the grafts had approximately 30% the maximum load to failure of the native ACL. However, there were no significant differences observed between the graft groups with or without heparin-immobilized bFGF. While this study demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture, it also demonstrates that in vitro results do not always predict what will occur in vivo. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:229–236, 2015. Keywords: ACL; drug delivery; scaffold; heparin; bFGF Anterior cruciate ligament (ACL) tears are a common surgical problem. Though high success rates can be achieved with autograft or allograft reconstruction, there are also serious complications associated with these options. 1 Autograft harvest can cause donor site morbidity, and supply is limited, particularly in cases of re-rupture or multi-ligamentous injuries. However, allografts are limited in supply and increase the risk of disease transmission and adverse inflammatory response. 2 Due to limitations with existing options and to recent advances in bioengineering and regenerative medicine, there has been great interest in a tissue- engineered ACL graft. Current strategies employ degr- adable biological and synthetic materials to allow for host tissue ingrowth while avoiding limitations associ- ated with permanent synthetic material implantation. 3 Polycaprolactone (PCL) is a biodegradable polymer that is currently used for a number of medical and tissue engineering applications. 2,4–11 Its popularity in tissue engineering can be attributed to the favorable biocompatibility, in vivo half-life, mechanical strength, and elasticity of this polymer. Our laboratory has developed a synthetic scaffold by electrospinning PCL and showed that this scaffold was biocompatible, integrated with native tissue, and had increasing mechanical strength over time when implanted into rat knees. 12 To further improve upon this scaffold, we considered the integration of growth factors. Specifically, we were interested in bFGF, also known as FGF2, because it has previously been shown to increase proliferation, and synthesis of extracellular matrix components inclu- ding collagen in fibroblasts. 13–15 Because a ligament takes weeks to months to regenerate, and growth factor may not be efficacious during the initial inflammatory stage of healing, 3 there exists a need for controlled, prolonged growth factor release in vivo. Heparin is a naturally occurring highly sulfated glycosaminoglycan. Because of its unique structure, and consequent ability to bind, release, and protect multiple growth factors, heparin has been investigated as a drug delivery mechanism. 16–20 Our group has developed a method of immobilizing heparin onto a PCL scaffold for drug delivery and showed that immo- bilized heparin resulted in sustained release of VEGF, which resulted in a dose-dependent angiogenic effect when implanted subcutaneously in mice. 16,17 Heparin binds bFGF in vitro. 21 Specific binding of heparin and bFGF has also been demonstrated in vivo, 22 and this interaction protects bFGF from proteol- ysis. 23–25 Additionally, it is known that a mechanism by which heparin promotes the mitogenic activity of bFGF is by inducing dimerization of one of its receptors, FGFR2. 26,27 Consequently, heparin-based strategies have been used to deliver bFGF for a wide variety of clinical applications. Heparin conjugated fibrin has been used to deliver bFGF in murine models, demonstrating successful enhancement of neurovascularization and Disclosure statement: No competing financial interests exist for any of the authors. Grant sponsors: OREF Clinician Scientist Training, H. H. Lee Surgical Research, The Veterans Administration, and Musculo- skeletal Transplant Foundation. Correspondence to: Natalie L. Leong (T: 310-825-6557; F: 310- 825-1311; E-mail: nleong@mednet.ucla.edu) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2015 229