The present study demonstrated that the use of TW (titanium-web) in ACL reconstruction surgery promoted formation and maturation of collagen cross-links in the grafted tendon, and also directly binding the bone and tendon tissue through the TW.
Rodeo et al.[22, 23] reported that tendon healing occurred by bone ingrowth into the fibrovascular interface tissue that initially formed between bone and tendon in a dog model. There was progressive mineralization of the interface tissue, with subsequent bone ingrowth into the outer tendon and incorporation of the tendon graft into surrounding bone. As fibrovascular organization matured, the collagen fibers that attached the tendon to bone resembled Sharpey fibers. Biomechanical testing demonstrated a progressive increase in tendon pull-out strength that correlated with the amount of bone ingrowth, mineralization, and maturation of the healing tissue over 12 weeks. Similarly, on the control limbs of this study, the interface tissue between the bone and the tendon was formed 4 weeks after surgery, and the formation of Sharpey-like fibers was observed 15 weeks after surgery suggesting indirect insertion. Other hand, on the TW limbs of this experiment, ingrowth of tendon-like soft tissue was found to be similar to tendon tissue by collagen analysis. Furthermore, calcified bone tissue began to form in the TW 4 weeks after surgery, and connected the bone and the transplanted tendon directly within 15 weeks after surgery. Although this insertion is different from direct insertions which consists of a fibrocartilage layer, it is histologically closer to the normal insertion compared to indirect insertions.
Process of “ligamentization” was proposed by Amiel et al in 1986 [24]. Both tendons and ligaments are composed of connective tissue primarily containing types I and III collagen, proteoglycans, and cells. However, their precise composition and arrangement of matrix macromolecules differ to provide the specific mechanical properties required [25]. For example, compared with tendons, ligaments contain cells with rounded nuclei, and have more type III collagen and proteoglycans, less total collagen, different collagen cross-link composition, and different distribution of collagen fibril diameters [25]. Amiel et al demonstrated that a patellar tendon transplanted into the rabbit knee to replace an excised ACL continuously developed into a substance similar to a normal ACL [24]. They reported that spindle-shaped fibroblasts and coarse fibrillar crimp in PT autografts converted to rounded fibroblasts and fine fibrillar crimp similar to the normal ACL under histological examination. Also, collagen cross-links analysis revealed that the tissue changed from the low DHLNL PT pattern to the high DHLNL pattern observed in normal ACL [24].
Collagen cross-links of collagen fibers in tendon, ligament and bone can be divided into two types: lysine hydroxylase and lysyl oxidase-controlled cross-links (enzymatic cross-links) and glycation or oxidation-induced advanced glycation end products (AGEs) cross-links [26]. Among collagen cross-links, enzyme-dependent collagen cross-links play an important role in the strength of collagen fibers and acts as a scaffold for cell differentiation [10, 21, 26–29]. Enzyme-dependent collagen cross-links also can be divided into reducible immature cross-links anchoring two collagen molecules (lysinonorleucine cross-links) and nonreducible mature cross-links anchoring three collagen molecules (pyridinoline cross-links). The total number of enzyme-dependent cross-links (immature cross-links + mature cross-links) is controlled by lysyl oxidase and will not be formed to exceed its required biomechanical strength [10, 26, 28, 29]. Three structures (dihydroxy-lysinonorleucine: DHLNL, hydroxy-lysinonorleucine: HLNL, and lysinonorleucine: LNL) have been identified by the difference of lysine hydroxylation rate in the immature cross-links. Identically, in the mature cross-links, two structures (pyridinoline: PYD, and deoxypyridinoline: DPD) have been identified. The component ratio of the five enzyme dependent cross-links (cross-links pattern) is considered to be tissue-specific. Skin, tendon, ligament, and bone types were identified by cross-link patterns using the ratio of high hydroxide to low hydroxide cross-links (a DHLNL/HLNL ratio or a PYD/DPD ratio) [10, 21, 26–29]. The change of collagen cross-links after ACL reconstruction in human has been considered. The autologous tendon implanted in the joint is initially subjected to a temporary lack of blood flow, reducing cellular synthesis of collagen. Thereafter, blood circulation is restored, promoting collagen synthesis, leading to collagen content increase, which results in increased immature cross-links and collagen fiber formation. Notably, the collagen fibers formed are arranged correctly. As mechanical load is applied, immature cross-links are converted to mature cross-links. Finally, collagen cross-links pattern converted from the tendon type (DHLNL/HLNL ratio < 1) seen in semitendinosus, gracilis and patellar tendons into the ligament type (DHLNL/HLNL ratio > 1) in the native ACL, is observed within one year postoperatively [10].
In this study we examined how the TW affects the biochemical ligamentization process from the early to middle stage. The total number of collagen cross-links of transplanted tendon in TW limbs and the tendon-like tissue within the TW itself were, although low compared with that of the original patellar tendon, significantly greater than that of the transplanted tendon in control limbs (Fig. 7a). Likewise, the maturation of collagen cross-links (the ratio of mature to immature cross-links) of the transplanted tendon and the tendon-like tissue within the TW were significantly greater than that of the transplanted tendon in the control limbs (Fig. 7b). These results suggested that the use of TW can promote the maturation and formation of collagen cross-links in the tendon 15 weeks after ACL reconstruction. For tissue specificity analysis, the DHLNL/HLNL ratio in the tendon parenchyma of control limbs increased significantly. In contrast, the ratio in the tendon parenchyma and tendon-like tissue in the TW maintained its tendon type pattern, similar to that of the original patellar tendon (Fig. 7c). This result suggested that TW does not adversely affect the collagen pattern.
There are some limitations to this study that must be addressed. First, the exact mechanism that promotes the formation of bone tissue and the maturation of the tendon tissue by the TW is still unclear, and warrants further consideration. Secondly, the formation of a fibrocartilaginous layer was not seen during the observed period. It is possible that a more natural structural regeneration of the tendon-bone junction may occur with more time. Adequate mechanical stresses and strong fixation are required to regenerate a firm tendon-bone junction, and it is possible that a stronger connection may be achieved by altering the shape and position of the TW. Thirdly, we did not perform biomechanical tests in the present study due to the limited number of samples. Further studies are needed to determine the breaking load and stiffness. Lastly, the procedure requires a relatively large bone tunnel compared to the graft tendon alone, because it is necessary to pass the transplanted tendon with the TW complex through the tunnel. When considering clinical application, damage to the meniscus or the transverse ligament can occur when porting a clinically sufficient thickness of the tendon, or bone tunnels can overlap in two-root reconstruction. These problems may be solved by improving the installation position of the TW.