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.^7,8 However, it is possible that the cell infiltration into a core portion of the graft occasionally occurs very slowly. Delay et al⁵ 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.^2–4

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.⁹ In the extrinsic fibroblasts, type III collagen is overexpressed even under physiological stress in areas where extrinsic fibroblasts infiltrate.¹⁰ 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.¹¹ 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.¹² This fact shows that revascularization in the graft is induced by VEGF produced in the fibroblasts. 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.¹³ This fact suggests that a complex growth factor network controls the fibroblasts, resulting in remodeling of the graft matrix,^14,15 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

Intraarticular Healing

PDGF-BB

It has been known that PDGF-BB enhances proliferation and migration of ligament fibroblasts in vitro.^16,17 In addition, an in vivo rabbit study showed that the strong expression of PDGF correlates with the observed increased cellularity around the wound site in the medial collateral ligament (MCL), suggesting potent mitogenic and chemotactic properties of PDGF in vivo.¹⁸ Concerning the in vivo effect of application of PDGF-BB on ligamentous tissues, Woo et al¹⁹ and Hildebrand et al²⁰ described that 20-μg PDGF-BB alone is the most effective agent to enhance the extraarticular MCL healing in the rabbit. Regarding intraarticular ACL reconstruction, Weiler et al²¹ applied PDGF-BB of approximately 60 μg using coated sutures as a vehicle on the flexor tendon autograft in a sheep ACL reconstruction model. They 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. However, Nagumo et al²² investigated the effect of PDGF-BB using fibrin sealant as a carrier on the in situ frozen-thawed rabbit ACL, an idealized intraarticular autograft model.^23–25 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. Hildebrand et al²⁰ suggested that the dose is critical to evaluate the effect of growth factors on ligament healing. Application of a high dose of PDGF-BB appears to be effective for MCL healing. However, the effect of PDGF-BB in ACL reconstruction is controversial.

VEGF

VEGF is a potent mediator of angiogenesis, which involves activation, migration, and proliferation of endothelial cells, in various pathological conditions.²⁶ A rabbit study showed that extrinsic cells newly proliferating in the necrotized tendon graft express VEGF at 3 weeks after surgery, when the revascularization does not occur.¹² This finding suggests the high possibility that an application of VEGF to the necrotized tendon graft enhances angiogenesis in the graft and accelerates remodeling of the graft. Ju et al²⁷ histologically and mechanically examined the effect of an application of 30-μg VEGF to the in situ frozen-thawed rabbit ACL. The application of VEGF significantly enhanced vascular endothelial cell infiltration and revascularization in the ACL at 3 and 12 weeks, respectively (Fig. 81-1). On the other hand, the application provided no significant effect on the mechanical properties of the ACL at 12 weeks, although the mechanical properties of the in situ frozen-thawed rabbit ACL were significantly weaker than those of the normal ACL at 12 weeks. Thus VEGF has a potential to be used as a treatment to enhance only revascularization of the autograft after ACL reconstruction surgery.

Fig. 81-1 Immunohistochemistry for CD31 to identify vascular endothelial cells in the anterior cruciate ligament (ACL) after the in situ freeze-thaw treatment without vascular endothelial growth factor (VEGF) application (A, 3 weeks; B, 6 weeks; C, 12 weeks) and with VEGF application (D, 3 weeks; E, 6 weeks; F, 12 weeks). A, In the ACL after in situ freeze-thaw treatment, few vascular endothelial cells were found at 3 weeks. B, At 6 weeks, several vascular endothelial cells formed vessels in the superficial portion of the ACL. C, The number of the vessels with endothelial cells decreased in the ACLs from 6 weeks to 12 weeks. D, Several vessels formed by endothelial cells were observed in the superficial portion of the ACL with VEGF application at 3 weeks after the in situ freeze-thaw treatment. E and F, The vessels with endothelial cells in the ACL with VEGF application were more abundant than those in the ACL without VEGF application until 12 weeks.

From Ju YJ, Tohyama H, Kondo E, et al. Effects of local administration of vascular endothelial growth factor on properties of the in situ frozen-thawed anterior cruciate ligament in rabbits. Am J Sports Med 2006;34:84–91.

TGF-β and EGF

A number of in vitro studies have shown that TGF-β enhances collagen and noncollagenous protein synthesis in fibroblasts.^28–30 EGF also stimulates fibroblast proliferation in vitro.³¹ A combined application of these two growth factors enhances these effects.³² Sakai et al²⁵ examined in vivo effects of an application of TGF-β and EGF on the in situ frozen-thawed ACL, using fibrin sealant as a vehicle. They found that a combined application of 4-ng TGF-β and 100-ng EGF significantly inhibited the natural deterioration that occurred in this autograft model with significant reduction of the water content and significant changes of the ultrastructural profile. Azuma et al³³ investigated the effect of the timing of this combined application on the same rabbit model. They reported that the effect was significantly greater when 4-ng TGF-β and 100-ng EGF were applied at 3 weeks than when they were applied at 0 and 6 weeks. This study suggested that the timing is critical in application of these growth factors. Recently, Nagumo et al²² 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. Concerning an intraarticular application of TGF-β and EGF on the bone–patellar tendon–bone (BPTB) autograft in a canine ACL reconstruction model, Yasuda et al³⁴ evaluated the effect on the structural properties of the graft. They stated that a combined application of 4-ng TGF-β and 100-ng EGF significantly inhibited the natural reduction of the structural properties of the autograft at 12 weeks 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.³⁵ Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and are factors that have a strong osteoinductive and osteogenic capacity.³⁶ They induce endochondral bone formation at extraskeletal sites. They are mostly expressed at sites of epithelial–mesenchymal interaction and serve as signaling molecules during mammalian embryogenesis and morphogenesis, suggesting their capability for differentiation of mesenchymal cells into chondrocytes and osteoblasts. 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.³⁶ Concerning intraosseous healing of the tendon, Rodeo et al³⁷ showed that tendon healing in a bone tunnel was enhanced by BMP-2 in the canine. Their histological analysis 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 al³⁸ described that a bone-derived extract (Bone Protein, Sulzer Biologics, Wheat Ridge, CO) was effective in augmenting healing of a tendon graft within a bone tunnel in a rabbit ACL reconstruction model. Recently, Mihelic et al³⁹ 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.

TGF-β

TGF-β is a multifunctional growth factor that induces new matrix synthesis in numerous types of cells. It has been shown that TGF-β can enhance bone ingrowth into biomaterial implants.^40,41 Recently, Yamazaki et al⁴² 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, but also new bone formation from the tunnel wall (Fig. 81-2). Thus, TGF-β1 also has the potential to enhance intraosseous graft healing.

Fig. 81-2 The new bone formation at the anterior wall of the bone tunnel at 3 weeks after anterior cruciate ligament (ACL) reconstruction using flexor tendon graft. The rectangle (500 × 1000 μm) was drawn in the adjacent trabecular area to the tunnel wall. The newly formed bone was generated more richly in the interface with transforming growth factor (TGF)-β application (A) than in that without TGF-β application (B). (B, Bone; G, granulation tissue in tendon–bone gap) (hematoxylin and eosin, original magnification ×20).

From Yamazaki S, Yasuda K, Tomita F, et al. The effect of transforming growth factor-beta1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy 2005;21:1034–1041.

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.⁴³ Martinek et al⁴⁴ examined the capacity of BMP-2 gene transfer to improve the integration of semitendinosus tendon grafts at the tendon–bone interface after reconstruction of the ACL in rabbits. They found that in the intraosseous portion of the grafts, the number of infected cells did not decrease until 8 weeks after surgery. Moreover, a number of transduced cells were found in the deeper layers of the tendon in the bone tunnel. In the AdBMP-2–infected ACL graft, the tendon–bone interface in the osseous tunnel was similar to that of a normal ACL insertion. The pullout load and the stiffness were significantly greater in the AdBMP-2–transduced graft than the control graft.

Concerning gene therapy for the intraarticular portion of the graft healing after ACL reconstruction, however, Gerich et al⁴⁵ evaluated methods for gene delivery to patellar tendons in the rabbits using the lacZ marker gene. They found that after injection of the adenovirus, high-level expression of lacZ was observed only in the portion adjacent to the injection site. Martinek et al⁴⁴ reported that in the intraarticular 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. Therefore it seems difficult to directly transfer genes for specific proteins to the core portion of the intraarticular graft and to maintain the number of the cells for a long period. Thus it may be difficult to successfully perform gene therapy by itself to enhance the graft healing of the intraarticular portion in ACL reconstruction. In 1999, Menetrey et al⁴⁶ reported a myoblast-mediated gene transfer method for a persistent expression of selected growth factors to enhance ACL healing following injury.

Enhancement of Graft Healing with Cell-Based Therapy

TGF-β1 and EGF significantly inhibited the deterioration of the mechanical properties of the BPTB autograft in ACL reconstruction.³⁴ Nagumo et al²² suggested that only TGF-β1 is a key in this effect on the intraarticular graft. However, Mi et al⁴⁷ reported that gene transfer of TGF-β induced arthritic changes of the articular cartilage in the knee joint. Thus intraarticular administration of TGF-β is considered to be unsuitable for clinical application with an ACL reconstruction procedure. Therefore Okuizumi et al⁴⁸ conducted the rabbit study to clarify the effect of cell therapy with autologous synovial tissue–derived fibroblasts activated by TGF-β1 on the necrotized ACL (Fig. 81-3). They wrapped the fibrin glue with autologous synovial tissue–derived fibroblasts after TGF-β stimulation around the necrotized ACL following the freeze-thaw treatment. Histological observation found that implantation of fibroblasts after TGF-β stimulation accelerated cellular infiltration into the ACL following fibroblast necrosis (Fig. 81-4). Biomechanically, the transplantation of synovial tissue–derived autologous fibroblasts activated by TGF-β inhibited deterioration in the tangent modulus of the ACL after the freeze-thaw treatment. Therefore this cell-based therapy using fibroblasts activated by TGF-β may be a potential solution against this problem in order to inhibit the deterioration of the mechanical properties of the graft after ACL reconstruction.

Fig. 81-3 The experimental protocol of the cell-based therapy with autologous synovial tissue–derived fibroblasts with transforming growth factor (TGF)-β application. ACL, Anterior cruciate ligament.

From Okuizumi T, Tohyama H, Kondo E, et al. The effect of cell-based therapy with autologous synovial fibroblasts activated by exogenous TGF-beta1 on the in situ frozen-thawed anterior cruciate ligament. J Orthop Sci 2004;9:488–494.

Fig. 81-4 Histology of the anterior cruciate ligament (ACL) at 12 weeks after the in situ freeze-thaw treatment and cell therapy with transforming growth factor (TGF)-β application (A) and without TGF-β application (B).

From Okuizumi T, Tohyama H, Kondo E, et al. The effect of cell-based therapy with autologous synovial fibroblasts activated by exogenous TGF-beta1 on the in situ frozen-thawed anterior cruciate ligament. J Orthop Sci 2004;9:488–494.

Mesenchymal progenitor cells (MPCs) or mesenchymal stem cells (MSCs) also have a potential for cell therapy. For example, an autologous MSC–collagen graft could improve the quality as well as accelerate the rate of healing after the defect of the patellar tendon in rabbits.⁴⁹ Recently, Ouyang et al⁵⁰ reported the efficacy of using a large number of MSCs to enhance tendon–bone healing in a rabbit model. They found that introduction of a large number of MSCs to the bone tunnel improves the insertion healing of tendon to bone in a rabbit model through formation of fibrocartilaginous attachment at early time points. However, there are no reports on application of the cell-based therapy with MPCs or MSCs for ACL reconstruction at present.

Summary

Recent experimental studies have suggested that application of growth factors, in particular TGF-β, is a possible strategy to prevent graft deterioration in ACL reconstruction and that several types of BMPs and TGF-β enhances tendon–bone healing in animal models. 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. The recent advancements in ACL graft biology may bring new strategies and additional therapeutic options to accelerate the remodeling of the graft after ACL reconstruction.