Graft Remodeling and Ligamentization after Anterior Cruciate Ligament Reconstruction

Early Graft-Healing Phase

This phase is defined as the period from the time of ACL reconstruction until the fourth postoperative week. In comparison to studies of the subsequent proliferation and ligamentization phases, fewer studies have analyzed the biological events of this early graft-healing phase. Most authors agree, using different in vivo animal models, that increasing graft necrosis, mainly in its center, and hypocellularity mark this time period. Ultrastructural cell changes such as mitochondrial swelling, dilatation of the endoplasmatic reticulum, and intracytoplasmatic deposition of lipids, as well as macroscopic swelling and increased cross-sectional area, illustrate the increasing graft necrosis and degradation. During this time, no graft revascularization can be observed. Graft necrosis leads to a release of a number of cytokines, such as tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6, in addition to chemokines that trigger a cascade of growth factor expression, which in turn results in cell migration and proliferation, as well as extracellular matrix synthesis and revascularization. This remodeling activity becomes more pronounced during the latter proliferative phase. However, already between the first and second week, an influx of cells can be seen into the graft’s periphery. These cells originate from tissue other than the graft itself, and all original graft cells are completely replaced by 2–4 weeks. It is hypothesized that the source of cells is the synovial fluid, cells from the stump of the native ACL, or bone marrow elements originating from drilling maneuvers. Therefore some authors suggest that preservation of the ACL stump and the Hoffa fat pad might be beneficial, especially for the early healing period.

The lack of sufficient biological graft incorporation in the bone tunnels is the weak site of the reconstruction. Biomechanical testing of intra-articular ACL reconstructions between 2 and 4 weeks shows consistent failure by graft pullout from the tunnel, indicating insufficient anchorage of the graft to the tunnel wall. During the first postoperative weeks, the graft’s overall collagen structure and crimp pattern are still maintained, even though the beginning disintegration of the collagen fibrils and their orientation can be observed as early as 3 weeks after reconstruction. This explains the slow decrease in the mechanical properties of the graft at this early healing phase. The mechanical strength of the ACL reconstruction at this time is significantly lower than that at the time of implantation. However, it continues to decrease until around 6 weeks, when a further increase in graft remodeling activity can be found and the failure site shifts from the bone tunnels to the intra-articular graft region.

Proliferation Phase of Graft Healing

The proliferation phase is characterized by maximum cellular activity and changes of the extracellular matrix, which are paralleled by the lowest mechanical properties of the ACL reconstruction during healing. Because cellular proliferation has already begun during the early healing period, there is a continuous transition between these two phases. However, with the most characteristic changes occurring between the fourth and twelfth postoperative week, this phase is referred to as the proliferation phase of ACL graft healing.

During the proliferation phase, cellularity constantly increases and substantially surpasses that of the intact ACL. Cell clusters are found at the perimeter of the graft around 6 weeks, with large acellular areas remaining in the graft center in animal studies ( Fig. 85.1 ). These hypercellular regions consist of mesenchymal stem cells and activated fibroblasts that are actively secreting several growth factors, such as basic fibroblast growth factor, transforming growth factor beta-1, and isoforms of platelet-derived growth factor, to initiate and maintain graft remodeling. Release of these growth factors peaks between the third and sixth week and almost completely ceases at 12 weeks of healing, which lends further explanation for the maximum remodeling activity during this proliferation phase. A more even distribution of cells throughout the graft slowly develops thereafter. Cell numbers are still increased but recede toward the intact ACL cellularity at the end of the proliferation phase. An increased number of specific fibroblasts (so-called myofibroblasts) appear. These contractile fibroblasts are responsible for the restoration of the in situ tension that is required for the later ligamentization process.

At the same time of increased cellular proliferation of the graft tissue, upregulated expression of the vascular endothelial growth factor (VEGF) occurs at 2–3 weeks post reconstruction. VEGF is a potent stimulator of angiogenesis that is triggered by hypoxia during the avascular necrosis of the early healing phase. Significant revascularization starts between 4 and 8 weeks after ACL reconstruction and progresses from the periphery of the graft toward the entire graft diameter at the end of the proliferation phase, around 12 weeks of healing ( Fig. 85.2 ). Vascular density then returns to values of the intact ACL during the phase of ligamentization.

Animal studies have shown that the mechanical properties of the graft are at their weakest at 6–8 weeks. Graft failure at this time occurs either by midsubstance tear or graft pullout due to stripping of the graft out of the bone tunnels. This illustrates that the graft tissue has become the weak link in the reconstruction compared with the graft–bone interface (due to the lack of graft incorporation) during the early healing phase. Three factors contribute to the decline in the graft mechanical properties: (1) increased revascularization and extracellular infiltration, (2) loss of regular collagen orientation and crimp pattern, and (3) decrease in collagen fibril density, followed by increased collagen synthesis with a shift from large-diameter collagen fibrils to small-diameter fibrils. It is not until the next ligamentization phase that a slow restoration of the collagen orientation and crimp pattern progresses ( Fig. 85.3 ). Furthermore, increased collagen III synthesis (with lower mechanical strength than type I collagen) may further explain why a full restoration of the mechanical strength of the intact ACL has not been observed in any in vivo model, even after 2 years of healing.

The reduced mechanical properties of healing grafts in animal models seem to contradict the successful clinical outcomes after ACL reconstruction, with immediate aggressive rehabilitation in humans. Significant differences were found in biopsy studies between the remodeling activity of human ACL grafts during the first 3 months and the healing graft in animal models. The complete loss and replacement of all intrinsic grafts have not been observed in human biopsy studies. Loss of collagen organization was only detected in areas of neovascularization in human biopsies, which corresponds to the findings in animal studies. However, human biopsy studies confirm the remodeling cascade of (limited) graft necrosis, recellularization, revascularization, and changes in collagen crimp and composition during the early healing phase and proliferation phase, suggesting that the human ACL graft might also have its lowest mechanical strength around 6–8 weeks postoperatively. Loading of the graft must be high enough to stimulate graft cells to produce cellular and extracellular components for preservation of graft stability, but without compromising graft integrity, which might result in an early stretch-out of the ACL reconstruction.