Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy.

Anterior cruciate ligament (ACL) injuries lead to great morbidity in sports and daily life activities and may cause instability of the knee joint, leading to meniscus injuries and the subsequent development of degenerative joint disease1. Reconstruction surgery is chosen for ACL injuries in most cases because of the limited capacity of these injuries for self-healing. The pivotal healing process after ACL reconstruction largely depends on the integration of the graft with the host bone2,3,4. However, slow and incomplete healing of the tendon-to-bone interface may result in inferior functional rehabilitation and even worse osteoarthritic changes5,6,7. Thus, it is important to explore better strategies to accelerate and improve tendon-to-bone healing.

The gene transfer of therapeutic factors has been intensively developed to accelerate the early healing of the tendon-to-bone interface, which overcomes the limitations of the direct use of growth factors, including repeated applications and the short biological half-life of these proteins, and ensures a sustained delivery of growth factors at the injury site8. Basic fibroblast growth factor (bFGF) has been widely acknowledged for substantial roles in numerous cellular functions, including cell proliferation, angiogenesis, and tissue remodeling9,10,11,12. However, the effect of bFGF gene therapy for ACL repair remains unknown. To date, numerous studies have reported positive effects of bone morphogenetic protein 2 (BMP2) on healing after ACL reconstruction13,14. Kohno et al. reported that endogenous bFGF and BMP2 were both found at the tendon-to-bone interface after ACL reconstruction in a rabbit model15: in their findings, bFGF was expressed at the first 3 weeks of graft incorporation, but was absent at the 12 weeks, which contributed to fibrous integration between the tendon and bone via vascularization during the early postoperative phases. BMP2 was expressed throughout the 12-week study period, which was responsible for remodeling process of the bone, leading to osseous integration between the tendon and bone. Low levels of growth factors might be partly responsible for the slow or weak healing responses in the injured tendons. These discoveries indicated that a combination of these beneficial genes might have additive roles to achieve optimal efficacy in the molecular network system of the tendon-bone interface. Despite the promising role of bFGF and BMP2, there are very few studies that have assessed the effects of single growth factor bFGF on healing, or the functional implications of the combination bFGF-BMP2 for ACL reconstruction. Furthermore, our previous study demonstrated the superiority of using multiple growth factors via gene transfer to treat experimental osteoarthritis16. Thus, it was reasonable for us to hypothesize that sustained delivery of bFGF and BMP2 by gene transfer at the tendon-to-bone insertion site could achieve the best biological and biochemical effects on healing after ACL reconstruction.

The purpose of this study was to investigate the efficacy of promoting tendon-to-bone healing using bone marrow-derived mesenchymal stem cells (BMSCs) that were genetically modified with bFGF and BMP2. We hypothesized that transplanted cells genetically modified with either bFGF alone or the combination of BMP2 and bFGF would significantly enhance the healing process and that the combined growth factors would achieve the best effects.

To determine the optimal infection efficiency of BMSCs with the proper dosage of adenovirus, BMSCs were infected with adenovirus encoding enhanced green fluorescent protein (EGFP). After 72 h, a high efficiency of BMSCs with adenovirus at MOI 40 was observed via fluorescence microscopy (Fig. 1A). Furthermore, the percentage of EGFP-positive cells was 94.6% ± 3.24%, as demonstrated by flow cytometry analysis (Fig. 1B).