Frank N. Unterhauser

Biomechanical properties and vascularity of an anterior cruciate ligament graft can be predicted by contrast-enhanced magnetic resonance imaging. A two-year study in sheep.

Magnetic resonance imaging has been used to determine graft integrity and study the remodeling process of anterior cruciate ligament grafts morphologically in humans. The goal of the present study was to compare graft signal intensity and morphologic characteristics on magnetic resonance imaging with biomechanical and histologic parameters in a long-term animal model. Thirty sheep underwent anterior cruciate ligament reconstruction with an autologous Achilles tendon split graft and were sacrificed after 6, 12, 24, 52, or 104 weeks. Before sacrifice, all animals underwent plain and contrast-enhanced (gadolinium-diethylenetriamine pentacetic acid) magnetic resonance imaging (1.5 T, proton density weighted, 2-mm sections) of their operated knees. The signal/noise quotient was calculated and data were correlated to the maximum load to failure, tensile strength, and stiffness of the grafts. The vascularity of the grafts was determined immunohistochemically by staining for endothelial cells (factor VIII). We found that high signal intensity on magnetic resonance imaging reflects a decrease of mechanical properties of the graft during early remodeling. Correlation analyses revealed significant negative linear correlations between the signal/noise quotient and the load to failure, stiffness, and tensile strength. In general, correlations for contrast-enhanced measurements of signal intensity were stronger than those for plain magnetic resonance imaging. Immunohistochemistry confirmed that contrast medium enhancement reflects the vascular status of the graft tissue during remodeling. We conclude that quantitatively determined magnetic resonance imaging signal intensity may be a useful tool for following the graft remodeling process in a noninvasive manner.

The Influence of Locally Applied Platelet-Derived Growth Factor–BB on Free Tendon Graft Remodeling After Anterior Cruciate Ligament Reconstruction

Background Ligaments and tendons do not gain mechanical properties of the native tissue after injury or grafting. Purpose To determine the influence of platelet-derived growth factor on tendon graft remodeling. Study Design Laboratory animal study. Methods Forty-eight sheep underwent anterior cruciate ligament reconstruction and were sacrificed after 3, 6, 12, and 24 weeks. In 6 animals at each time point, platelet-derived growth factor was locally delivered via coated sutures. After mechanical testing, tissue samples were taken for histologic, immunohistochemical, and electron microscopy evaluations. Results With platelet-derived growth factor treatment, cross-sectional area was significantly lower at 3 and 12 weeks. Load to failure was significantly higher at 6 weeks. Tensile stress was significantly higher at 3 and 12 weeks. Crimp length was significantly higher at 3 and 6 weeks. Vascular density was significantly higher at 6 weeks. Electron microscopy showed a significantly higher collagen fibril amount at 12 weeks. Differences in these parameters at other time points were not significant. Conclusions There were alterations in several but not all time points. The local application of platelet-derived growth factor alters the tissues mechanical properties during free tendon graft remodeling after anterior cruciate ligament reconstruction. Growth factors present a promising tool toward the complete mechanical restitution of a healing ligament substitute.

α-Smooth muscle actin containing contractile fibroblastic cells in human knee arthrofibrosis tissue

IntroductionPrimary arthrofibrosis is of major concern after joint trauma or knee ligament surgery. The underlying mechanism in detail remains unclear. Highly differentiated fibroblastic cells, so-called myofibroblasts, express the actin isoform α-smooth muscle actin (ASMA) and have been found to play a major role in tissue contraction during wound healing and organ fibrosis. We therefore studied the expression of myofibroblasts in human primary knee arthrofibrosis tissue.Materials and methodsTissue samples were taken from the infrapatellar fat pad and intercondylar region of nine patients who underwent revision surgery due to arthrofibrosis after anterior cruciate ligament (ACL) reconstruction (study group). Control tissue was taken from five patients who underwent primary ACL reconstruction (control group I) and from eight patients, who underwent second-look arthroscopy after primary ACL reconstruction (control group II). ASMA containing fibroblasts were immunostained with a monoclonal antibody. Histomorphometry was performed for total cell amount, ASMA containing fibroblasts, and vessel cross-sections.ResultsThe arthrofibrosis group showed a tenfold higher amount of ASMA containing myofibroblasts (23.4% vs. 2.3%) than in control group I. There was a significantly higher total cell count and lower vessel density than in control group I. Control group II showed an upregulation of myofibroblasts almost five times that in control group I; nevertheless there was no evidence of scar formation or tissue fibrosis.ConclusionsMyofibroblasts are responsible for scar tissue contraction during wound healing. In arthrofibrosis tissue fibroblast contraction may be involved in tissue fibrosis and contraction with consecutive loss of motion. We found that myofibroblasts are upregulated in arthrofibrosis tissue. ACL reconstruction itself caused an up regulation of myofibroblast content. Nevertheless these patients did not show any clinical or histological signs of arthrofibrosis. Thus it is reasonable to assume that the ratio of myofibroblasts and total cell amount in connective tissue are responsible for the onset of arthrofibrosis. Address the expression of this highly differentiated cell type may therefore present a target for future therapeutic interventions.

α‐Smooth muscle actin is expressed by fibroblastic cells of the ovine anterior cruciate ligament and its free tendon graft during remodeling

Contractile fibroblastic cells expressing the α‐smooth muscle actin isoform, so‐called myofibroblasts, have been identified to play a possible role during the healing of the medial collateral ligament by means of restoring the tissues in situ strain via extracellular matrix contraction. Recently, these cells have also been identified to be a normal part of the human anterior cruciate ligament. It has been hypothesized that myofibroblasts play a role in the wrinkling of the extracellular matrix. The goal of the present study was to identify myofibroblasts in the intact ovine anterior cruciate ligament and a free autologous tendon graft during remodeling after anterior cruciate ligament reconstruction. In 36 mature merino sheep the anterior cruciate ligament was replaced with an ipsilateral Achilles tendon split graft. Midsubstance tissue samples were immunostained for α‐smooth muscle actin at 6, 9, 12, 24, 52, and 104 weeks. Myofibroblasts were identified in the intact ovine anterior cruciate ligament as well as in the Achilles tendon graft prior to implantation. During remodeling the first myofibroblasts were found at six weeks within newly formed fiber bundles. At 24, 52, and 104 weeks myofibroblast distribution and cell density were similar to those of the intact ovine anterior cruciate ligament. These findings indicate that α‐smooth muscle actin containing fibroblastic cells are a regular part of the intact as well as the remodeled anterior cruciate ligament. There is evidence that myofibroblasts may be involved in maintaining tissue homeostasis in the mature ligament e.g., by means of crimp formation. The presence of these cells during the early remodeling may further indicate that α‐smooth muscle actin containing fibroblastic cells are involved in the earliest stages of fiber bundle formation. The role and function of this special cell type for the anterior cruciate ligament needs to be further clarified.

Endoligamentous revascularization of an anterior cruciate ligament graft

After replacement of the anterior cruciate ligament with a free tendon graft, the substitute initially is avascular and without a synovial surface. To ensure long-term survival, the graft must become revascularized. Despite numerous studies on the topic, there still is discussion regarding revascularization. The goal of the current study was to investigate the endoligamentous microcapillary revascularization of the free tendon graft after anterior cruciate ligament replacement with time. Thirty-six mature sheep had an anterior cruciate ligament reconstruction with an ipsilateral flexor tendon split graft. Midsubstance tissue samples were immunostained for von Willebrand factor (Factor VIII) at 6, 9, 12, 24, 52, and 104 weeks to detect the endothelial cells of capillaries. Cross sections of vessels were determined in three zones (subsynovial, intermediate, and center of the graft) and were evaluated by means of histomorphometry using a digital imaging analysis system. The observations showed that capillary vessels, which originate from the synovial envelope, invaded the avascular graft tissue from the surface toward the center zone. The highest level of vascular density was found after 6 weeks, reaching the vascular status of the native anterior cruciate ligament after 24 weeks. These findings are in contrast to the findings of previous studies in animals. The current study showed, for the first time, the kinetics of an endoligamentous revascularization of a free tendon graft at the capillary level. In the current model, the process of revascularization terminated earlier than previously described.

Crimp frequency is strongly correlated to myofibroblast density in the human anterior cruciate ligament and its autologous tendon grafts

PurposeCollagen crimp is essential for maintaining viscoelastic properties of normal ligament and tendon tissue. The actin isoform α-smooth muscle actin (ASMA) has been identified in fibroblastic cells of these tissues. These highly differentiated cells, so-called myofibroblasts may transmit tensile forces to the extracellular matrix, thus it has been suggested that they are responsible for the wrinkling of the extracellular matrix and the formation of crimp. During anterior cruciate ligament (ACL) graft remodeling, crimp formation plays an integral role. Thus, it was our purpose to determine the relationship between myofibroblast density and crimp frequency in human tendon graft tissue and the ACL.MethodsDifferent tendon grafts and ACLs were harvested from young human multi-organ donors immediately after death. Myofibroblasts were immunostained with a monoclonal antibody, and histomorphometry was performed using a digital imaging system. Crimp length was measured, and data were correlated.ResultsAll tendons and ACLs showed a significant correlation of myofibroblast density and crimp frequency (R2 0.81–0.43). The strongest correlation was found for the patellar tendon, the poorest for the gracilis tendon. There is also evidence that the phenotype respectively the shape of myofibroblasts might be responsible for different stages of crimp formation.ConclusionWith the present investigation, we found that myofibroblasts might be involved in crimp formation and should be viewed as an integral part of normal tendon and ligament tissue. Furthermore, the shape of myofibroblasts may further indicate the contractile potency of the extracellular matrix, thus presenting a dynamic and variable crimp rather than a static situation. This study is an experimental study. In terms of clinical relevance all the mentioned tendons can be used as auto- or allografts for ACL reconstruction, nevertheless their microscopic structure and cellular population have yet not been adequately investigated and compared.