INTRODUCTION

Anterior cruciate ligament (ACL) tears are common among athletes and may become functionally disabling knee injuries. Reconstruction of a torn ACL in order to restore function and limit injury to the menisci has become a common orthopaedic procedure. Despite advances in surgical techniques and the ability to implant an anatomic, isometric graft, ACL reconstruction is not a universally successful procedure. Rates of recurrent laxity 1 year postoperatively have been reported to be as high as 17%.⁷⁸

Failure of graft integration and tendon-to-bone healing may be an important cause of recurrent laxity. The healing of tendon to bone is the basic requirement for the long-term survival of the graft.^12,24 Whether an autograft or an allograft tendon is used, biomechanical testing has shown that the initial strength of the graft material is superior to that of the intact ACL.^12,24 Therefore, the weakest link after reconstruction is not the graft itself, but rather the fixation points until graft osteointegration occurs. The intra-articular portion of the graft must also undergo remodeling and a process of “ligamentization” to form a structure that resembles a native ligament.^3,5,28,36,38

Current techniques of ACL reconstruction require tendon-to-bone healing in a surgically created bone tunnel. There are no native sites in humans at which a tendon passes through a bone tunnel and, therefore, no analogous situation to the healing that is required after reconstruction. When a bone-tendon-bone graft is used for ACL reconstruction, graft fixation initially depends on bone-to-bone healing. However, tendon-to-bone healing still remains critical regardless of whether a soft tissue graft or a bone-tendon-bone graft is selected. The length of the tendinous portion of the bone-tendon-bone graft is greater than the intra-articular length of the native ACL, resulting in substantial tendon in the bone tunnel. Aperture fixation to minimize graft micromotion and tunnel widening requires tendon-to-bone healing with any graft.³⁸

Because all grafts depend on tendon-to-bone healing, this chapter focuses on graft osteointegration and the process by which structural and functional continuity between the graft and the bone is achieved. The biologic and biomechanical environment in the bone tunnel results in formation of an attachment site that differs from the native ligament-bone insertion. The biology of this healing remains incompletely understood and is subject to a number of biomechanical and biologic influences. The current understanding of the biology of graft reconstruction is reviewed and potential strategies to enhance early graft integration are discussed.

NATIVE TENDON-BONE INSERTION

The ACL is an intra-articular, extrasynovial structure that acts to control anterior translation and rotational movements of the femur on a fixed tibia. It is composed of multiple fascicular collagen bundles enveloped in a sheath that contains vascular and

neural elements.⁴ The ACL inserts to bone via a direct type of insertion, similar to the transition seen from tendon to bone. Microscopic examination of the sites of bony attachment show interdigitation of the collagen fibers with bone through four distinct transition zones: tendon, unmineralized fibrocartilage, mineralized fibrocartilage, and bone (Fig. 5-1).^4,17,57,70 This graduated change in stiffness allows for transmission of complex mechanical loads from soft tissue to bone while minimizing peak stresses at any single point along the ligament. Cartilage-specific collagens including types II, IX, X, and XI are found in the fibrocartilage insertion site. Collagen X plays a key role in maintaining the interface between the mineralized and the unmineralized zones.^4,17,57,70

FIGURE 5-1 Histology of native anterior cruciate ligament (ACL) insertion to bone. Examination shows interdigitation of the collagen fibers with bone through four distinct transition zones: tendon, fibrocartilage, mineralized fibrocartilage, and bone.

TENDON-BONE INSERTION AFTER ACL RECONSTRUCTION

The overall structure, composition, and organization of a native ACL insertion site are not reproduced after ACL reconstruction and reflect an inability to recapitulate the events that occur during embryonic development with current surgical techniques. Rather than regenerating the four organized zones of direct insertion, the graft heals with an interposed zone of vascular, highly cellular granulation tissue between the graft and the tunnel wall (Fig. 5-2).^20,21,65 After 3 to 4 weeks, this interface tissue undergoes a maturation process until its matrix consists of oriented, Sharpey-like collagen fibers that bridge the bone to the graft (Fig. 5-3). The number and size of these collagen fibers positively correlate with the pull-out strength of the graft (Fig. 5-4). Graft attachment strength further improves as bone grows into the interface tissue and outer portion of the graft.^20,21,65

FIGURE 5-2 Insertion site histology 1–2 weeks after ACL reconstruction. Vascular, highly cellular granulation tissue is interposed as a layer between the graft and the tunnel wall.

FIGURE 5-3 Insertion site histology 3–4 weeks after ACL reconstruction. The interposed tissue has matured with oriented, Sharpey-like collagen fibers bridging the tendon to bone.

FIGURE 5-4 Pull-out strength of graft correlates with the number and size of bridging, Sharpey-like collagen fibers.

(From Rodeo, S. A.; Arnoczky, S. P.; Torzilli, P. A.; et al.: Tendon-healing in a bone tunnel. A biomechanical and histological study in the dog. J Bone Joint Surg Am 75:1795–1803, 1993.)

The maturation process of a tendon graft in a bone tunnel was defined by Kanazawa and coworkers in a rabbit ACL model.³⁴ In the initial postoperative period, the graft-tunnel interface is filled with vascular granulation tissue containing type III collagen. Vascular endothelial growth factor (VEGF) and

fibroblast growth factor (FGF) are expressed, stimulating an influx of macrophages and enlarged fibroblasts. Chondroid cells accumulate along the walls of the bone tunnel and deposit type II collagen. The granulation tissue layer is degraded and becomes indistinct. The chondroid cells are gradually replaced with lamellar bone in a process similar to endochondral ossification.³⁴ The Sharpey-like fibers are composed of type III collagen and extend into the surrounding bone to resist shear stresses. The time interval for this process has been variably reported in the literature, ranging from 8 to 30 weeks.³⁴

CHALLENGES OF TENDON-BONE HEALING AFTER ACL RECONSTRUCTION

The biology of healing between a grafted tendon and a bone tunnel remains incompletely understood. The biologic and biomechanical environments result in the formation of an inferior attachment site that is different from the organized, direct-type ACL insertion. Current work suggests a number of fundamental challenges that are responsible for the suboptimal healing response between tendon and bone instead of regeneration of the insertion site.²³ These factors include

Strategies to promote tendon-to-bone healing in ACL reconstruction focus on overcoming these challenges through modification of the biologic and biomechanical environment.

MODULATION OF TENDON-BONE HEALING

Mechanical Factors

Relative graft-tunnel micromotion may preclude the formation of a firm attachment to the tunnel wall. Yu and Paessler⁸³ compared “aggressive” versus “conservative” rehabilitation protocols in patients after ACL reconstruction with quadrupled hamstring grafts. Significantly greater tunnel widening was reported in the aggressive rehabilitation group, supporting the view that graft-tunnel micromotion is a cause of tunnel widening and that the mechanical environment in the bone tunnel influences tendon-bone healing.

Animal studies have corroborated these clinical observations. Sakai and associates⁷¹ compared immediate motion with varying periods of immobilization for up to 6 weeks after ACL reconstruction in a rabbit model. Biomechanical analysis demonstrated a greater load-to-failure of the graft in immobilized animals, and histologic studies demonstrated closer tendon-bone apposition with less fibrovascular interface scar tissue. Rodeo and colleagues⁶⁶ evaluated the effect of graft-tunnel motion on tendon-to-bone healing in a rabbit ACL reconstruction model. ACL reconstruction was performed in five cadaveric rabbit limbs with aluminum beads fixed to the tendon and bone tunnel. Three-dimensional graft-tunnel motion was quantified using micro–computed tomography (CT) analysis. Motion was observed to be greatest at the tunnel apertures and least at the tunnel exits, adjacent to the graft fixation at the tunnel exit (Fig. 5-5). Histomorphometric analysis demonstrated healing to be slowest at the apertures, with a wider fibrovascular interface tissue present between tendon and bone. An inverse relationship between graft-tunnel motion and healing in the femoral tunnel was demonstrated. In addition, osteoclasts were preferentially observed at the tunnel aperture, supporting the hypothesis that graft-tunnel motion may stimulate osteoclast-mediated bone resorption and secondary tunnel widening.⁶⁶

FIGURE 5-5 Relative motion between the tendon and the bone tunnel in the femoral and tibial tunnels. ACL reconstruction was performed in five cadaveric rabbit limbs, and graft-tunnel motion was measured using micro–computed tomography. Graft-tunnel motion was greatest at the tunnel apertures and least at the tunnel exit in cadaveric testing. Healing of the graft was slowest at the tunnel apertures.

(From Rodeo, S. A.; Kawamura, S.; Kim, H. J.; et al.: Tendon healing in a bone tunnel differs at the tunnel entrance versus the tunnel exit: an effect of graft-tunnel motion? Am J Sports Med 34:1790–1800, 2006.)

Modulation of the mechanical environment may have profound effects on the cellular and molecular events at the tendon-bone interface. An improved understanding of these mechanisms may help to develop a postoperative rehabilitation program that provides an optimal environment for tissue regeneration and tendon-bone healing.

Biologic Factors

Modulation of the Inflammatory Response

Immediately after ACL reconstruction and release of the tourniquet, the knee is filled with blood from the drilled bone tunnels. This initiates an acute inflammatory response marked by influx of neutrophils, macrophages, and mesenchymal cells. Important lessons about the effect of this inflammatory response on tissue healing can be learned from the study of fetal wound healing. Wounds in the embryo and early fetus heal by tissue regeneration and result in “scarless healing.” These healing events are characterized by the absence of an acute inflammatory response. Inflammation after trauma, although essential for healing in adults, is also responsible for healing by scar rather than regeneration of native tissue. The rapid inflammatory response that ensues after ACL reconstruction may trigger a cascade of events that ultimately leads to fibrosis rather than tissue regeneration and an anatomic tendon-to-bone insertion.^19,39,72

The fibrin clot that forms after surgery allows for a controlled release of cytokines that drive the early response. Transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) act together to modulate tissue healing and matrix deposition by recruiting neutrophils and macrophages to the local tissue.^19,39,72 Neutrophil influx peaks at about 2 days after the operation and is followed by an influx of macrophages. These monocytes are essential to the early formation of granulation tissue from the clot, initiating the process of soft tissue adherence to bone.³⁵ They also drive the angiogenic phase, providing nutrients and oxygen for the prolonged period of matrix synthesis and remodeling that ensues over the next several weeks. TGF-β secreted by macrophages recruits and stimulates fibroblasts to degrade matrix through matrix metalloproteinases (MMPs). Simultaneously, these fibroblasts synthesize new matrix proteins to replace the early granulation tissue with scar tissue. Tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit MMPs and provide a check-and-balance regulation to this complex process of matrix degradation, synthesis, and remodeling.¹⁸

The accumulation of macrophages around the tendon graft in bone has been further characterized in a rat ACL reconstruction model.³⁵ Two distinct subpopulations of macrophages were identified at the tendon-bone interface. ED1+ macrophages derived from the circulation act in a proinflammatory fashion in the early response with neutrophils to migrate into the tissues and remove debris after surgery. This is followed by the accumulation of proregenerative ED2+ macrophages from the local tissues, which promote anabolic tissue healing and scar formation by fibroblasts via TGF-β secretion (Fig. 5-6).³⁵

FIGURE 5-6 Time course of inflammatory cell accumulation in the tendon-bone interface (A), inner tendon (B), and outer tendon (C). The number of positively stained cells for PCNA, PMN, ED1, ED2, and T-lymphocytes were counted in 15 randomly chosen high-power fields 50 μm x 50 μm at 400x for each area. A, There was a significant decrease in the number of neutrophils between 4 and 7 days (P < .001). ED2+ macrophages were not present until 11 days after surgery, with a significant increase in positive staining by 14 days after surgery (P < .001). B, There were significantly more ED1+ macrophages than PCNA+ cells at 28 days after surgery (P = .014). There were significantly more ED1+ macrophages than ED2+ macrophages at 14 days and 21 days after surgery (P < .001) and at 28 days after surgery (P = .004). There were significantly more ED2+ macrophages at 21 days than at 14 days after surgery (P < .001). C, There were significantly more ED1+ macrophages than ED2+ macrophages at 14 and 21 days after surgery in the outer tendon. There were no significant differences in the number of CD3-positive cells at all time points.

(A–C, Modified from Kawamura, S.; Ying, L.; Kim, H. J.; et al.: Macrophages accumulate in the early phase of tendon-bone healing. J Orthop Res 23:1425–1432, 2005.)

Given the critical role of macrophages in scar tissue formation, animal studies have investigated the role of macrophage depletion on tendon-bone healing. It has been theorized that depletion may promote formation of a “scarless,” native insertion site. Hays and coworkers²⁶ in the authors’ laboratory demonstrated less scar tissue formation, improved collagen organization, and superior biomechanical strength in a rat ACL reconstruction model when macrophages were depleted by administration of liposomal clodronate, a selective inducer of macrophage apoptosis. Other studies have identified gastric pentadecapeptide BPC 157, a novel anti-inflammatory used in the treatment of inflammatory bowel disease, to improve Achilles tendon-to-surface bone healing and tissue regeneration in a rat Achilles tendon model.³⁷

The impact of inflammatory response suppression, however, has not been uniformly favorable for tendon-bone healing. Cohen and associates¹¹ evaluated the effect of the anti-inflammatory medications indomethacin and celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on tendon-bone healing in a rat rotator cuff model. The treated group was found to have histologically and biomechanically inferior tendon-bone interfaces at both 4 and 8 weeks relative to controls (Fig. 5-7).

FIGURE 5-7 Photomicrographs demonstrate tendon-bone insertion site at 2 weeks (A, control group; B, celecoxib group; C, indomethacin group), 4 weeks (D, control group; E, celecoxib group; F, indomethacin group), and 8 weeks (G, control group; H, celecoxib group; I, indomethacin group) after surgery (hematoxylin and eosin, ×320).

(A–I, From Cohen, D. B.; Kawamura, S.; Ehteshami, J. R.; Rodeo, S. A.: Indomethacin and celecoxib impair rotator cuff tendon-to-bone healing. Am J Sports Med 34:362–369, 2006.)

These studies indicate that the acute inflammatory response that ensues after ACL reconstruction is highly complex and remains to be further defined. Selective reduction in inflammatory mediator expression, improved angiogenesis, and improved tissue regeneration may promote native tendon-bone insertion over scar tissue formation. However, a dichotomy exists because other elements of the inflammatory response are necessary in the postnatal organism to initiate the tissue healing response.

Modulation of Bone Ingrowth

Bone ingrowth plays a critical role in the later stages of tendon-bone healing after ACL reconstruction and is ultimately responsible for the improved biomechanical properties after healing is complete. Animal studies have provided strong evidence to support that increased bone ingrowth positively correlates with graft-bone fixation strength.⁶⁷ The primary strategies that have been employed include use of osteoinductive agents, osteoconductive agents, and modulation of osteoclast activity.

Osteoinductive agents, particularly from the bone morphogenic protein (BMP) family, have been shown to promote ingrowth of bone from the tunnel into the soft tissue graft. Rodeo and coworkers⁶⁷ delivered recombinant human BMP-2 (rhBMP-2) on an absorbable type I collagen sponge in a tendon-bone canine model. At all time points, the rhBMP-2–treated limbs healed with increased bone formation around the tendon. Biomechanical testing demonstrated higher tendon pull-out strength in the treated limbs relative to controls at 2 weeks. Ma and colleagues⁴¹ delivered rhBMP-2 in an injectable calcium phosphate matrix to the bone tunnel in a rabbit ACL reconstruction model. Histologic analysis revealed a dose-dependent increase in bone formation at the tendon-bone interface and significantly narrower tunnel diameters (15%–45%) relative to the control group. Increased construct stiffness was also seen in the treatment group at 8 weeks postoperatively.⁴¹ Martinek and associates⁴⁴ transected semitendinosus grafts in vitro with adenovirus-BMP-2 (Ad-BMP-2) and compared them with untreated controls in a rabbit ACL reconstruction model. These investigators demonstrated the formation of a fibrocartilaginous interface at the tendon-bone junction in the experimental group that was absent in the controls. Both stiffness and load-to-failure parameters were superior in the treatment group at 8 weeks. BMP-7 has also been shown to improve bone formation at the tendon-bone interface and load to failure at both 3 and 6 weeks postoperatively in an ovine reconstruction model.^1,45

Further support for the role of BMPs in bone ingrowth around tendon graft comes from studies of BMP inhibition. Ma and colleagues⁴¹ delivered noggin, a potent inhibitor of all BMP activity, to the healing tendon-bone interface using an injectable calcium phosphate matrix in a rabbit ACL reconstruction model. Noggin significantly inhibited new bone formation at the tendon-bone interface. Furthermore, a significant increase in the width of the fibrous tissue interface between tendon and bone was found in the noggin-treated animals (Fig. 5-8).

FIGURE 5-8 The width of new bone formation (mm ± SD) at the tunnel-graft interface after ACL reconstruction in a rabbit model. *Significant difference compared with rhBMP-2 group (P < .05). ^#Significant difference compared with the control group (P < .05). For each dosage of rhBMP-2 and noggin test, four rabbit limbs were used for histomorphometric analysis.

(Modified from Ma, C.; Kawamura, S.; Deng, X.; et al.: BMP-signaling plays a role in tendon-to-bone healing: A study of rhBMP-2 and noggin. Am J Sports Med 35:597–604, 2007.)

The favorable effect of osteoinductive agents on tendon-bone healing in a tunnel is further supported by studies that have used tendons wrapped in periosteum. Transplantation of a long digital extensor tendon wrapped in periosteum into a tunnel in the proximal tibia was compared with controls in a rabbit model.^10,61 Improved biomechanical strength and bone and fibrocartilage formation at the tendon-bone interface were shown in the treated animals at 8 and 12 weeks. Ohtera and colleagues⁶¹ completed further studies comparing fresh and frozen periosteum and demonstrated better histologic and biomechanical outcomes using fresh periosteal wraps. These findings support the hypothesis that the viable osteoinductive factors in the periosteum may contribute to the improved outcomes.

Osteoconductive agents have also been met with favorable outcomes in animal models. Calcium phosphate (CaP)–hybridized grafts used in a rabbit ACL reconstruction model demonstrated improved fibrocartilage and bone formation relative to animals treated with unhybridized grafts at 3 weeks postoperatively.⁵⁶ Furthermore, studies by Tien and coworkers⁷⁶ using injectable CaP cement in the femoral tunnel of a rabbit ACL reconstruction model showed improved bone formation and biomechanical strengths relative to controls at 1 and 2 weeks postoperatively.

Modulation of osteoclast activity in the bone tunnel is another technique of promoting bone formation at the tendon-bone interface. Furthermore, inhibition of osteoclast-mediated bone resorption offers a potential means by which to limit the bone tunnel expansion commonly seen after ACL reconstruction. One study demonstrated that increased knee laxity correlated with radiographic tunnel widening after ACL reconstruction using hamstring tendon.²⁵ The role of osteoclastic bone resorption on tendon-bone healing has been evaluated in a rabbit ACL reconstruction model.¹⁵ Osteoprotegerin (OPG), a potent inhibitor of osteoclast activity, or receptor activator of nuclear factor κB ligand (RANKL), a potent stimulant of osteoclast formation, were delivered to the bone tunnels around a tendon graft using a CaP carrier matrix. A significantly greater amount of bone surrounding the tendon at the interface was seen in the OPG-treated limbs relative to controls and RANKL-treated limbs at all time points (Fig. 5-9).¹⁵ Furthermore, biomechanical testing at 8 weeks demonstrated significantly increased stiffness of the femur-graft-tibia complex in the OPG-treated limbs compared with the RANKL-treated limbs.

FIGURE 5-9 Fifteen New Zealand white rabbits underwent unilateral ACL reconstruction using an autologous semitendinosis tendon graft. Animals treated with osteoprotegerin (OPG) 100 μg had a significant (P = .007) increase in newly formed bone around the graft compared with the control group (0.16 ± 0.01 mm²; 0.06 ± 0.02 mm²). RANKL, receptor activator of nuclear factor κB ligand.

(Modified from Rodeo, S. A.; Kawamura, S.; Ma, C. B.; et al.: The effect of osteoclastic activity on tendon-to-bone healing: an experimental study in rabbits. J Bone Joint Surg Am 89:2250–2259, 2007.)

Stem Cells

Undifferentiated, pluripotent mesenchymal cells, also termed stem cells, may be critical to stimulate tissue regeneration rather than scar formation at the tendon-bone interface. These cells retain the capacity to differentiate into various specialized cell types based on biologic signals in the local environment. Animal studies have tested the effects of local stem cell delivery on tendon-bone healing. Rabbit bone marrow stromal cells placed in a fibrin glue carrier were delivered to the tendon-bone interface of hallucis longus tendon in a calcaneal tunnel.⁶² Histologic analysis revealed improved healing with fibrocartilaginous attachment between tendon and bone in the experimental group. Lim and associates⁴⁰ performed bilateral ACL reconstructions in a rabbit model and evaluated the role of mesenchymal stem cells (MSC) on the tendon-bone interface. The grafts coated with MSC demonstrated cartilage at the tendon-bone interface, whereas only fibrous tissue was observed at the interface in the contralateral control limbs. The interface stained positively for type II collagen in the MSC-treated grafts, and was similar in organization to a native, direct ligament insertion. Furthermore, biomechanical testing at 8 weeks demonstrated higher loads to failure and stiffness relative to controls.⁴⁰

Further work is required to determine the mechanism by which pluripotent stem cells enhance tendon-bone healing. It is unknown whether these cells differentiate into fibrochondrocytes or if they produce cytokines that improve insertion site organization and tissue regeneration.

Modulation of Vascularity

Whereas vascularity is critical for the efficient delivery of oxygen and nutrients to support tissue healing, the precise role of local vascularity at the tendon-bone interface remains to be defined. Krivic and colleagues³⁷ demonstrated improved vascularity at the healing Achilles tendon–bone interface after treatment with gastric pentadecapeptide BPC 157 in a rat model. This improved vascularity correlated with favorable histologic and biomechanical properties of the tendon-bone interface. In contrast, however, a recent study examined the effect of VEGF on graft healing in an ovine ACL reconstruction model.⁸² Although vascularity and cellularity were increased in the VEGF-soaked grafts relative to controls, the stiffness of the femur-graft-tibia complex was significantly inferior to controls at 3 months. Although only a single concentration of VEGF was utilized, the results present the possibility that excessive vascularity may adversely affect healing and the biomechanical properties of the tendon-bone interface.⁸²

Modulation of MMPs

The MMPs are a family of zinc-dependent endoproteinases that play a critical role in tissue degradation, healing, and normal remodeling. They function both in an extracellular environment and through transmembrane and intracytoplasmic domains.¹⁴ Inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) initiate the transcription and activation of MMPs from their zymogen form. Their catabolic, destructive activity is balanced, however, by TIMPs. TIMPs provide a check-and-balance mechanism to control the activity of these degradative enzymes and thereby maintain homeostasis of extracellular matrix formation and degradation that occurs with tissue remodeling.¹⁸

Synovial fluid tracking between the graft and the tunnel walls after ACL reconstruction is known to contain large amounts of collagenase and stromelysin. It has been theorized that these MMPs may have adverse effects on the tendon-bone interface, perhaps by limiting the formation of Sharpey-like collagen fibers. Demirag and coworkers¹⁴ used α₂-macroglobulin, an endogenous inhibitor of MMPs, to study this hypothesis in a rabbit ACL reconstruction model. Each rabbit underwent bilateral ACL reconstruction with hamstring autograft. α₂-Macroglobulin was injected into one knee postoperatively and compared with the contralateral control limb. The interface tissue in treated specimens was more mature with significantly greater Sharpey-like fibers. Biomechanical studies demonstrated greater load to failure compared with controls at 2 and 5 weeks¹⁴ (Fig. 5-10). Further studies are necessary to characterize the precise mechanism of action of MMPs at the tendon-bone interface. Nonetheless, this work provides preliminary evidence that modulation of MMP activity can improve graft-bone healing in a bone tunnel.

FIGURE 5-10 The ultimate load to failure was significantly greater in the α₂-macroglobulin–treated specimens than in the untreated control specimens at both 2 and 5 weeks (P = .007 and P = .006, respectively).

(Modified from Demirag, B.; Sarisozen, B.; Durak, K.; et al:. The effect of alpha-2 macroglobulin on the healing of ruptured anterior cruciate ligament in rabbits. Connect Tissue Res 45:23–27, 2004.)

Modulation of Nitric Oxide

Nitric oxide is a free radical agent synthesized by nitric oxide synthase from l-arginine. It acts as a regulatory molecule both in cells and in the extracellular matrix. Studies have shown that it is induced during tendon healing in vitro, with a dose-dependent effect on fibroblast collagen production.^54,55 ACL ligament fibroblasts are uniquely able to produce more nitric oxide compared with other local fibroblasts, including those derived from the medial collateral ligament (MCL).⁴⁶ The influence of nitric oxide levels on tendon-bone healing after ACL reconstruction remains to be defined.

Effect of Hyperbaric Oxygen

Yeh and associates⁸¹ recently studied the influence of hyperbaric oxygen (HBO) therapy on the graft-bone tunnel interface in a rabbit model. The HBO group was exposed to 100% oxygen at 2.5 atm pressure for 2 hours daily, 5 consecutive days a week. The control group was exposed to normal air. The HBO group demonstrated increased neovascularization and an increased number of Sharpey fibers relative to controls. Furthermore, the HBO group achieved higher maximal pull-out strengths at 12 and 18 weeks relative to control specimens.⁸¹ Although the mechanism of action is unclear, these preliminary results suggest that HBO therapy may improve tendon-bone healing after ACL reconstruction.

Modulation of Other Biologic Mediators

Future techniques to enhance tendon-bone healing after ACL reconstruction will be directed by an improved understanding of the biology of this healing process. Use of cytokines to provide important signals for tissue formation and differentiation, gene therapy techniques to provide sustained cytokine delivery, stem cells, or transcription factors to modulate endogenous gene expression represent some of these possibilities. Scleraxis, a transcription factor expressed in mesenchymal tendon progenitor cells, and sox-9, a transcription factor critical for chondrogenesis, are two such proteins that may play key roles in native tendon insertion site formation.

BONE-TO-BONE HEALING IN ACL AUTOGRAFT RECONSTRUCTION

Healing of a bone plug to the osseous tunnel walls after autograft ACL reconstruction is unique and unlike anywhere else in the body. The biomechanical and biologic environments in the graft tunnels present significant challenges to union.

The sequence of bone-tendon-bone autograft incorporation has been defined in animal models. Tomita and colleagues⁷⁷ compared healing of a soft tissue graft to a bone–patellar tendon–bone autograft in a canine model. The bone plug undergoes osteonecrosis and is gradually replaced by a process of creeping substitution (Fig. 5-11). Newly formed bone surrounding the plug is seen at 3 weeks. These investigators found pull-out strength of the bone–patellar tendon–bone graft to be superior to the soft tissue graft at 3 weeks, but not significantly different at 6 weeks. At 6 weeks, a change in the point of failure was noted from the graft-tunnel interface to the tendon-bone plug interface. Papageorgiou and coworkers⁶⁴ directly compared tendon-bone with bone-bone healing in a goat model. Bone–patellar tendon autografts were harvested; the soft tissue graft was placed in the tibial tunnel while the bone plug was secured in the femoral tunnel. Biomechanical testing at 3 weeks demonstrated universal failure of the tendon-bone interface with

FIGURE 5-11 (A) The anterior portion of the bone plug–bony wall gap was filled with granulation tissue (white arrows), while the posterior aspect of the bone plug appeared to be in contact with the bony wall (black arrows) (T, patellar tendon; P, bone plug of the BPTB) (H&E, original magnification × 2). (B) In the granulation tissue around the intraosseous tendon portion, few collagen fibers are seen (B, bone wall; T, patellar tendon) (H&E, original magnification × 100). (C) In the bone plug of the BPTB graft, a number of empty lacunae (white arrows) that indicated bone necrosis were found, except for the superficial portion of the plug (B, bone wall; P, bone plug of the BPTB) (H&E, original magnification × 50). (D) At the tendon bone junction of the graft, both the noncalcified and calcified fibrocartilage layers appeared to be normal (T, patellar tendon; N, noncalcified fibrocartilage layer; C, calcified fibrocartilage layer; B, bone) (toluidine-blue, original magnification × 50).

(From Tomita, F.; Yasuda, K.; Mikami, S.; et al.: Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction. Arthroscopy 17:461–476, 2001.)

pull-out from the tibial tunnel. At 6 weeks, however, approximately 20% of cases demonstrated midsubstance failures. The remaining cases continued to be tibial tunnel graft pull-out. Histologic evaluation at 3 weeks confirmed creeping substitution with a necrotic bone plug surrounded by granulation tissue. Evaluation at 6 weeks revealed complete plug incorporation with bridging cancellous bone.⁶⁴

Synovial fluid flows from the joint into the tunnels and can interfere with healing at the tendon-bone interface. The high quantities of MMPs and other proteolytic enzymes in synovial fluid can slow bone-tendon-bone and bone-bone healing. Berg and associates⁹ evaluated the role of synovial fluid by drilling femoral and tibial tunnels and leaving them empty in a rabbit ACL model. These authors found healing to occur most rapidly at points farthest away from the joint, whereas the slowest and most incomplete regions of healing were at the tunnel apertures. These findings are suggestive of a possible inhibitory effect of synovial fluid on healing in the tunnel.

It is critical to remember that whether a soft tissue or a bone-tendon-bone graft is used, the critical aperture fixation will usually require tendon-bone healing. The tendon length of a bone–patellar tendon–bone graft exceeds the native ACL length, such that a substantial portion of the tunnel, including the aperture zone, will contain tendon. Tendon-bone healing at the aperture, regardless of graft choice, is essential to minimize graft micromotion and subsequent risk of tunnel widening.

INTRA-ARTICULAR GRAFT HEALING IN ACL AUTOGRAFT RECONSTRUCTION

Ligamentization refers to the complex process of biologic incorporation and remodeling that occurs to the tendon after ACL reconstruction. Animal studies have attempted to define the phases of intra-articular graft healing that occur postoperatively. The graft goes through an initial phase of avascular necrosis. At 2 weeks, the graft demonstrates patches of necrosis, although the collagen architecture and scaffold remain intact.^2,3,36,63 By 4 weeks, the graft is almost entirely avascular and acellular. This phase, however, is followed by cellular repopulation from the host synovial cells. Biopsy at 3 months reveals extensive vascular proliferation and cellular repopulation. By 9 months, the graft histologically resembles the native ligament.^2,3,36,63

Intra-articular healing has also been studied in other animal models. Oaks and colleagues⁶⁰ demonstrated remodeling to occur from the periphery toward the graft center. This was associated with a change from large-diameter to small-diameter collagen fibrils. This pattern of remodeling supports the hypothesis that healing is dependent on gradual revascularization and cellular repopulation from the host synovium. Arnoczky and coworkers³ evaluated patellar tendon graft revascularization in a canine model. Initially the grafts were avascular, but by 6 weeks, they were completely ensheathed in a vascular synovial envelope. The soft tissues of the infrapatellar fat pad, the tibial remnant of the ACL, and the posterior synovial tissues contributed to this synovial vasculature. Intrinsic revascularization of the patellar tendon graft progressed from the proximal and distal portions of the graft centrally.³ The tibial attachment of the patellar tendon graft did not contribute any vessels to the revascularization process. The contribution of the soft tissues of the knee to the revascularization process of the graft emphasized the importance of their preservation in maintaining the graft’s viability.

Although animal models have provided insight into the process of ligamentization, the process has not been fully characterized in humans. Jackson and associates²⁸ concluded that an incorporated graft never replicates the native ACL and rather functions like a checkrein of organized scar tissue. Delay and

colleagues¹³ described a case report of a bone–patellar tendon–bone autograft with avascular, acellular regions in the deep, distal graft 18 months after reconstruction. Rougraff and Shelbourne⁶⁹ performed patellar tendon graft biopsies on nine subjects 3 to 8 weeks after autogenous ACL reconstruction. Graft vascularity was present at 3 weeks and increased over the 8-week interval.

PRIMARY ACL HEALING AND REPAIR

Primary repair of the ACL after traumatic rupture was historically considered to be a viable surgical option. Outcome studies, however, have reported unacceptable rates of failure after primary surgical repair.¹⁶ In their classic study, Feagin and Curl¹⁶ reported a 94% rate of instability in patients at 5-year follow-up after primary suture and drill hole ACL repair. Marshall and coworkers⁴³ reported a 20% to 40% failure rate despite repair using a sophisticated, multiple-depth suture technique. Zysk and Refior⁸⁵ also reported modest outcomes in a study on middle-aged patients who underwent primary ACL repair and recommended that open primary repair be abandoned in favor of autogenous tissue reconstruction or augmentation. These results were recently corroborated by a Norwegian study that assessed long-term outcomes in a large series of patients who underwent primary ACL repair.⁷⁵ An open procedure using the original Palmer technique with nonabsorbable Bunnell sutures was performed in all cases. At 15 to 23 years postoperatively, 57% had greater than 3 mm of anterior translation on KT-1000 testing. The estimated rate of total failure was 27%. These results are in sharp contradistinction to the MCL, in which failure to primarily heal is the exception rather than the rule.

The poor rate of primary healing observed after ACL rupture is believed to be multifactorial in nature. One of the most salient factors is the intra-articular environment and synovial fluid that surrounds the ACL.47–53,⁷⁴ Studies by Murray and associates^47–53,74 elegantly demonstrated the differences in intra-articular (i.e., ACL) versus extra-articular (i.e., MCL) healing in a canine, central ACL wound model. An empty wound persists at the defect of an intra-articular ligament wound, whereas these wounds are rapidly filled with a fibrin-platelet scaffold in an extra-articular injury. This scaffold is critical to allow subsequent cellular repopulation, revascularization, and ligament remodeling into mature scar tissue. The lack of a scaffold in the intra-articular ligament wounds was associated with decreased inflammatory cytokines needed for the healing response, including fibrinogen, PDGF, TGF-β, and FGF.^47–53,74 Studies by Murray and associates^47–53,74 demonstrated that replacement of the central intra-articular ligament void with a collagen-platelet–rich plasma scaffold resulted in increased filling of the wound with repair tissue that

had similar profiles of protein expression to matched, extra-articular ligament wounds. Biomechanical studies of suture ACL repair augmented with a collagen-platelet–rich scaffold in a porcine model have shown significant improvement in load to failure and linear stiffness at 4 weeks compared with unaugmented, control repairs.^47–53,74

Other factors implicated in the poor ACL healing response include alterations in cellular metabolism after injury, cellular loss within the tissues after injury, and intrinsic deficiencies in ACL fibroblasts compared with fibroblasts in extra-articular ligaments. Murray and associates47–53,⁷⁴ defined the histologic changes that occur after ACL rupture. The human ACL undergoes four histologic phases: inflammation, epiligamentous regeneration, proliferation, and remodeling (Fig. 5-12). Although similar to the response to injury in other dense connective tissues, major exceptions include (1) formation of an alpha-smooth muscle actin-expressing synovial cell layer on the surface of the ruptured ends, (2) the lack of any tissue bridging the rupture site, and (3) the presence of an epiligamentous reparative phase that lasts 8 to 12 weeks.^47–53,74 The biology involved in intra-articular ligament healing will need to be further defined to overcome the obstacles to long-term success with primary ACL repair.

FIGURE 5-12 Schematic of the gross and histologic appearance of the four phases of the healing response in the human ACL. A, The inflammatory phase, showing mop-ends of the remnants (a), disruption of the epiligament and synovial covering of the ligament (b), intimal hyperplasia of the vessels (c), and loss of the regular crimp structure near the site of injury (d). B, The epiligamentous regeneration phase, involving a gradual recovering of the ligament remnant by vascularized, epiligamentous tissue and synovial tissue (e). C, The proliferative phase, with revascularization of the remnant with groups of capillaries (f). D, The remodeling and maturation phase, characterized by a decrease in cell number density and blood vessel density (g) and by retraction of the ligament remnant (h).

(A–D, Modified from Murray, M. M.; Martin, S. D.; Martin, T. L.; Spector, M.: Histological changes in the human anterior cruciate ligament after rupture. J Bone Joint Surg Am 82:1387–1397, 2000.)

ALLOGRAFT HEALING IN ACL RECONSTRUCTION

An increasing desire to avoid the morbidity of graft harvest and to reduce operative times has led to a dramatic increase in the use of allografts as an alternative graft source in primary ACL reconstruction.⁷ Allografts are also frequently used in revision procedures or multiligament reconstruction procedures. Good clinical outcomes have been reported with allograft reconstructions at 2 to 5 years follow-up, and multiple studies have found no significant subjective or objective difference in knee function after allograft versus autograft ACL reconstructions.⁸ However, other studies have reported less favorable results after allograft reconstruction, particularly in the setting of chronic or revision knee surgery.^58,59

Despite compatible clinical outcomes, animal studies have shown that allografts have a slower rate of incorporation, prolonged inflammatory response, and greater initial decrease in biomechanical properties compared with autografts.2–6,^29–33,84 The phases of healing, although delayed, resemble those of the autograft. The tendon-bone interface develops a fibrovascular granulation tissue interface that eventually undergoes bone

ingrowth and develops Sharpey-like anchoring fibers.^{2–6,32,33,84} The intra-articular graft undergoes ligamentization with a phase of avascular necrosis followed by cellular repopulation and vascular proliferation from host synovium. Donor DNA was entirely replaced by host DNA within 4 weeks in a goat reconstruction model. Revascularization starts at 3 weeks and progresses gradually over the next several weeks. Jackson and colleagues compared healing of patellar tendon autografts with fresh allografts in a goat ACL reconstruction model.^31–33 Although graft structural properties were similar at time zero, the allografts healed at a much slower rate. At 6 months, the autografts demonstrated a superior load to failure, larger increase in graft cross-sectional area, and better restraint to anteroposterior displacement. The allografts demonstrated a significantly greater decrease in their preimplantation structural properties.

Human studies have supported these findings of slower allograft incorporation. At 2 years postoperatively, biopsy studies have shown that the central portion of allografts can continue to be acellular.²⁷ Cellular repopulation of the entire graft was often seen only after 3 or more years after allograft reconstruction.⁴²

Because of the relative hypocellularity of tendon allografts and current sterilization techniques, the host immune response is relatively limited. Major histocompatibility complex (MHC) antigens that incite a potent immune response are largely depleted. Matrix antigens, however, persist and can elicit an immune response that may contribute to the delayed incorporation and the pronounced alteration in structural properties after surgery.^68,79 The specific influences of such an immune response on the biology of ACL graft healing remains to be defined.

CONCLUSION

This chapter presents the basic principles of the biology of ACL reconstruction and reviews potential techniques to improve graft healing. A sound understanding of the complex mechanism of tendon-to-bone healing offers new and exciting ways to manipulate the chemical and molecular mediators of the inflammatory response and to ultimately improve the quality of surgical and postoperative interventions.