Abstract
Modern intra-articular anterior cruciate ligament (ACL) reconstructive techniques produce clinically stable ligament reconstruction in the majority of cases. In ACL reconstruction, however, the strength of the grafted tendon is reduced in the early phase after surgery, and then it gradually increases. In this chapter, the authors will review recent studies that are intended to enhance intraarticular and intraosseous graft healing after ACL reconstruction using growth factors, gene therapy, and cell-based therapy. Previous animal studies have suggested that application of growth factors, in particular transforming growth factor (TGF) -β, is a possible strategy to prevent graft deterioration in ACL reconstruction and that several types of Bone morphogenetic proteins (BMPs) and TGF-β enhances tendon–bone healing. As a concentration of autologous growth factors, Platelet rich plasma (PRP) has gained increased use in musculoskeletal applications. However, clinical results of PRP application to ACL reconstruction have been mixed and disappointing. Gene therapy and cell-based approaches may represent new alternatives in delivering these specific growth factors to the grafted tendon and the interface between the graft and the bone after ACL reconstruction. These advancements in ACL graft biology may bring new strategies and additional therapeutic options to accelerate the remodeling of the graft after ACL reconstruction.
Keywords
ACL reconstruction, cell-based therapy, gene therapy, graft healing, graft remodeling, growth factors
Modern intra-articular anterior cruciate ligament (ACL) reconstructive techniques produce clinically stable ligament reconstruction in the majority of cases. In ACL reconstruction, however, the strength of the grafted tendon is reduced in the early phase after surgery, and then it gradually increases. A problem is that this graft remodeling occurs very slowly. The slow graft maturation may result in graft failure or elongation during the postoperative rehabilitation period due to unknown causes. In addition, a firm attachment of a tendon graft to the bone is a significant factor for success in ACL reconstruction. In procedures using a hamstring tendon graft, however, the anchoring strength of the soft tissue in a bone tunnel is the weakest in the femur–graft–tibia complex in the early phase after surgery. To improve these problems after ACL reconstruction in the near future, we should try to develop a new strategy to accelerate the intra-articular and intraosseous remodeling of the tendon graft. This may enable more aggressive rehabilitation and an earlier return to rigorous sports for patients with ACL reconstruction. In this chapter, the authors will review recent studies that are intended to enhance intra-articular and intraosseous graft healing after ACL reconstruction using growth factors, gene therapy, and cell-based therapy.
Keywords
ACL reconstruction, cell-based therapy, gene therapy, graft healing, graft remodeling, growth factors
Modern intra-articular anterior cruciate ligament (ACL) reconstructive techniques produce clinically stable ligament reconstruction in the majority of cases. In ACL reconstruction, however, the strength of the grafted tendon is reduced in the early phase after surgery, and then it gradually increases. A problem is that this graft remodeling occurs very slowly. The slow graft maturation may result in graft failure or elongation during the postoperative rehabilitation period due to unknown causes. In addition, a firm attachment of a tendon graft to the bone is a significant factor for success in ACL reconstruction. In procedures using a hamstring tendon graft, however, the anchoring strength of the soft tissue in a bone tunnel is the weakest in the femur–graft–tibia complex in the early phase after surgery. To improve these problems after ACL reconstruction in the near future, we should try to develop a new strategy to accelerate the intra-articular and intraosseous remodeling of the tendon graft. This may enable more aggressive rehabilitation and an earlier return to rigorous sports for patients with ACL reconstruction. In this chapter, the authors will review recent studies that are intended to enhance intra-articular and intraosseous graft healing after ACL reconstruction using growth factors, gene therapy, and cell-based therapy.
Basic Knowledge To Enhance the Graft Remodeling in Anterior Cruciate Ligament Reconstruction
In ACL reconstruction, intrinsic fibroblasts of the tendon graft are necrotized immediately after transplantation, and then numerous extrinsic fibroblasts infiltrate the graft with revascularization. Delay et al. reported a clinical case in which the core portion of the patellar tendon graft still remained necrotic even at the 18-month period after ligament reconstruction. On the other hand, biomechanically, the mechanical properties of the graft deteriorate in the early phase after transplantation and then are very gradually restored over a long period.
Concerning the graft deterioration mechanism, the fibroblast necrosis itself does not deteriorate the mechanical properties of the tendon matrix, but extrinsic fibroblasts proliferating after the necrosis reduce the strength properties. In the extrinsic fibroblasts, type III collagen is overexpressed, even under physiological stress in areas where extrinsic fibroblasts infiltrate. In the matrix of the autograft after transplantation, ultrastructurally, fibrils having a diameter less than 90 nm predominantly increase in the graft matrix, and these fibrils with small diameters still remain predominant at the 4-year period after surgery. Such ultrastructural changes due to type III collagen production are considered to be one of the causes of mechanical deterioration of autografts.
What molecular mechanisms control the autograft remodeling? In a rabbit ACL reconstruction model, vascular endothelial growth factor (VEGF) is overexpressed in the extrinsic fibroblasts at 2 weeks after graft implantation, followed by vascular formation at 3 weeks. On the other hand, basic fibroblast growth factor, transforming growth factor (TGF)-β, and platelet-derived growth factor (PDGF) are overexpressed in the autogenous patellar tendon graft used to reconstruct the ACL in the canine model, reaching their greatest expression 3 weeks after implantation. This fact suggests that a complex growth factor network controls the fibroblasts, resulting in remodeling of the graft matrix, and implies that control of the fibroblasts using growth factors is a potential strategy to accelerate the graft remodeling after ACL reconstruction.
Enhancement of Graft Healing with Growth Factors
Intra-articular Healing
Platelet-Derived Growth Factor-BB
It has been known that PDGF-BB enhances proliferation and migration of ligament fibroblasts. Woo et al. and Hildebrand et al. described that 20-μg PDGF-BB is the most effective agent to enhance the extra-articular medial collateral ligament healing in the rabbit. Regarding intra-articular ACL reconstruction, Weiler et al. showed that the PDGF-BB application significantly increased the load to failure and vascular density of the graft at 6 weeks after ACL reconstruction, although they found no significant effects at 24 weeks. Nagumo et al. investigated the effect of PDGF-BB using fibrin sealant as a carrier on the in situ frozen-thawed rabbit ACL, an idealized intra-articular autograft model. They reported that an application of 4-μg PDGF-BB did not significantly affect the mechanical properties of the frozen-thawed ACL at 12 weeks. Therefore the effect of PDGF-BB in ACL reconstruction is controversial.
Vascular Endothelial Growth Factor
VEGF is a potent mediator of angiogenesis, which involves activation, migration, and proliferation of endothelial cells, in various pathological conditions. There is a possibility that an application of VEGF to the necrotized tendon graft enhances angiogenesis in the graft and accelerates remodeling of the graft. Ju et al. showed that the application of VEGF significantly enhanced vascular endothelial cell infiltration and revascularization in the ACL at 3, 6, and 12 weeks after the in situ freeze thaw treatment, respectively ( Fig. 141.1 ). Concerning the effects of the VEGF application on the mechanical properties of the hamstring graft in a sheep ACL reconstruction model, Yoshikawa et al. showed that that the linear stiffness of the VEGF-treated femur–graft–tibia complex was significantly lower than that of the saline-treated graft at 12 weeks. Therefore we should take into account this adverse effect of exogenous VEGF application on the mechanical characteristics of the grafted tendon, while VEGF has a potential to enhance revascularization of the autograft after ACL reconstruction surgery.
Transforming Growth Factor-β and Endothelial Growth Factor
A number of in vitro studies have shown that TGF-β enhances collagen and noncollagenous protein synthesis in fibroblasts. Endothelial growth factor (EGF) also stimulates fibroblast proliferation in vitro. A combined application of these two growth factors enhances these effects. Sakai et al. and Yasuda et al. found that a combined application of TGF-β and EGF significantly inhibited the natural deterioration that occurred in the in situ frozen-thawed ACL and in the BTB graft after ACL reconstruction, respectively. Azuma et al. reported that the effect was significantly greater when TGF-β and EGF were applied at 3 weeks than when they were applied at 0 and 6 weeks. Nagumo et al. distinguished between the effect of 4-ng TGF-β and the effect of 100-ng EGF on the autograft model. According to them, the effect of 100-ng EGF was not significant, but the effect of 4-ng TGF-β was significant.
Platelet Rich Plasma
Platelet rich plasma (PRP) is a concentrated extract of platelets from autologous blood. PRP contains growth factors in concentration 3 to 5 times the normal plasma level. Growth factors are secreted from PRP within 1 hour following intra-articular administration. The success of PRP in dental and oral surgery has led to its application in orthopaedic surgery. In order to allow early return to sports activity after ACL reconstruction, some investigators conducted clinical studies about application of PRP to ACL reconstruction for acceleration of healing process and integration of the graft. However, these clinical studies of application of PRP to ACL reconstruction showed mixed results. Radice et al. found that application of PRP to ACL reconstruction shortens the time to homogeneous appearance of the intra-articular portion of the graft on magnetic resonance imaging from 369 to 179 days. On the other hand, Nin et al. reported that the use of PDGF on the graft in patients treated with bone–patellar tendon–bone allografts has no discernable clinical or biomechanical effect at 2 years’ follow-up. In addition, Sánchez et al. also showed that there were no significant differences in arthroscopic findings between plasma rich in growth factors-treated and control ACL grafts at 6–24 months after ACL reconstruction.
Some investigators conducted clinical studies to clarify whether application of PRP to ACL reconstruction can enhance the bone-graft healing process and reduce tunnel widening after ACL reconstruction. Darabos et al. reported that a series of postoperative intra-articular injections of PRP significantly decreased IL-1β synovial fluid concentration and tunnel widening after ACL reconstruction using a bone-tendon-bone or hamstring tendon graft, while they found no significant correlation between IL-1β synovial fluid concentration and tunnel widening. Vogrin et al. showed that the platelet gel (PG) used for ACL reconstruction using double hamstring tendon graft enhances early revascularization in the interface zone between the graft and the bone tibial tunnel. The same investigators also found that enhanced cortical bone formation encircling the tibial tunnel at 2.5 and 6 months after ACL graft reconstruction results from locally applied PG. On the other hand, other studies showed no significant effects of PRP application on tendon-bone healing or tunnel widening after ACL reconstruction.
Intraosseous Healing
Bone Morphogenetic Proteins
The process of tendon-bone healing involves bone ingrowth into the interface tissue between the tendon and bone. Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and are factors that have a strong osteoinductive and osteogenic capacity. They induce endochondral bone formation at extraskeletal sites. The BMPs have been successfully used to regenerate bone defects, to stimulate bone ingrowth into soft tissues and metal implants, and to regenerate articular cartilage defects in large animals. Concerning intraosseous healing of the tendon, Rodeo et al. showed that the BMP-2 treatment resulted in earlier and more abundant bone ingrowth into the tendon–bone interface and that it biomechanically increased the anchoring strength. Anderson et al. described that a bone-derived extract (Bone Protein, Sulzer Biologics, Wheat Ridge, Colorado) was effective in augmenting healing of a tendon graft within a bone tunnel in a rabbit ACL reconstruction model. Mihelic et al. reported that BMP-7 (osteogenic protein-1) induced the new bone formation at the bone-tendon interface, creating a dense trabecular network in a sheep ACL reconstruction model. Their mechanical testing showed greater strength in the knees treated with BMP-7 than in control specimens. Thus BMPs have potential growth factors to enhance intraosseous graft healing.
Transforming Growth Factor-β
It has been shown that TGF-β can enhance bone ingrowth into biomaterial implants. Yamazaki et al. found that administration of exogenous TGF-β1 significantly increased the bonding strength of the flexor tendon graft to the tunnel wall at 3 weeks in a canine ACL replacement model. This result was accompanied by the histological findings that the administration appeared to enhance not only synthesis or maturation of the perpendicular collagen fibers connecting the graft to the bone. Thus TGF-β1 also has the potential to enhance intraosseous graft healing.
Enhancement of Graft Healing with Gene Therapy
Gene therapy approaches may represent a new alternative in delivering these specific growth factors to the grafted tendon after ACL reconstruction. Martinek et al. found that the BMP-2 gene transfer histologically improved the integration of semitendinosus tendon grafts at the tendon-bone interface with higher pullout strength after reconstruction of the ACL in rabbits. Concerning gene therapy for the intra-articular portion of the graft healing, Martinek et al. reported that in the intra-articular portion of the AdLacZ-infected grafts, infected cells were observed only in the surface portion, and the number of the cells decreased between 2 and 8 weeks after ACL reconstruction with the autologous semitendinosus tendon graft in the rabbit. Gerich et al. also reported that after injection of the adenovirus, high-level expression of lacZ was observed only in the portion adjacent to the injection site. It may be difficult to successfully perform gene therapy by itself to enhance the graft healing of the intra-articular portion in ACL reconstruction. In 1999 Menetrey et al. reported a myoblast-mediated gene transfer method for a persistent expression of selected growth factors to enhance ACL healing following injury.
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Acute Anterior Cruciate Ligament Rupture: A Biological Approach through Primary Anterior Cruciate Ligament Repair, Augmentation with Bone Marrow Stimulation, and Growth Factor Injection
Allografts Have Higher Failure Rates Than Autografts in Anterior Cruciate Ligament Reconstruction in Young, Active Patients
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