Human Anterior Cruciate Ligament Research Articles

Different expression profiles of the lysyl oxidases and matrix metalloproteinases in human ACL fibroblasts after co-culture with synovial cells

ABSTRACTPurposes The anterior cruciate ligament (ACL) has poor functional healing response. The synovial tissue surrounding ACL ligament might be a major regulator of the microenvironment in the joint cavity after ACL injury, thus affecting the repair process. Using transwell co-culture, this study explored the direct influence of human synovial cells (HSCs) on ACL fibroblasts (ACLfs) by characterizing the differential expression of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, −2, −3), which facilitate extracellular matrix (ECM) repair and degradation, respectively. Methods The mRNA expression levels of LOXs and MMP-1, −2, −3 were analyzed by semi-quantitative PCR and quantitative real-time PCR. The protein expression levels of LOXs and MMP-1, −2, −3 were detected by western blot. Results We found that co-culture resulted in an increase in the mRNAs of LOXs in normal ACLfs and differentially regulated the expression of MMPs. Then we applied 12% mechanical stretch on ACLfs to induce injury and found the mRNA expression levels of LOXs in injured ACLfs were decreased in the co-culture group relative to the mono-culture group. Conversely, the mRNA expression levels of MMPs in injured ACLfs were promoted in the co-culture group compared with the mono-culture group. At translational level, we found that LOXs were lower while MMPs were highly expressed in the co-culture group compared to the mono-culture group. Conclusions The co-culture of ACLfs and HSCs, which mimicked the cell-to-cell contact in a micro-environment, could contribute to protein modulators for wound healing, inferring the potential reason for the poor self-healing of injured ACL.

Induced Remodeling of Porcine Tendons to Human Anterior Cruciate Ligaments by α-GAL Epitope Removal and Partial Cross-Linking.

This review describes a novel method developed for processing porcine tendon and other ligament implants that enables in situ remodeling into autologous ligaments in humans. The method differs from methods using extracellular matrices (ECMs) that provide postoperative orthobiological support (i.e., augmentation grafts) for healing of injured ligaments, in that the porcine bone-patellar-tendon-bone itself serves as the graft replacing ruptured anterior cruciate ligament (ACL). The method allows for gradual remodeling of porcine tendon into autologous human ACL while maintaining the biomechanical integrity. The method was first evaluated in a preclinical model of monkeys and subsequently in patients. The method overcomes detrimental effects of the natural anti-Gal antibody and harnesses anti-non-gal antibodies for the remodeling process in two steps: Step 1. Elimination of α-gal epitopes—this epitope that is abundant in pigs (as in other nonprimate mammals) binds the natural anti-Gal antibody, which is the most abundant natural antibody in humans. This interaction, which can induce fast resorption of the porcine implant, is avoided by enzymatic elimination of α-gal epitopes from the implant with recombinant α-galactosidase. Step 2. Partial cross-linking of porcine tendon with glutaraldehyde—this cross-linking generates covalent bonds in the ECM, which slow infiltration of macrophages into the implant. Anti-non-gal antibodies are produced in recipients against the multiple porcine antigenic proteins and proteoglycans because of sequence differences between human and porcine homologous proteins. Anti-non-gal antibodies bind to the implant ECM, recruit macrophages, and induce the implant destruction by directing proteolytic activity of macrophages. Partial cross-linking of the tendon ECM decreases the extent of macrophage infiltration and degradation of the implant and enables concomitant infiltration of fibroblasts that follow the infiltrating macrophages. These fibroblasts align with the implant collagen fibers and secrete their own collagen and other ECM proteins, which gradually remodel the porcine tendon into human ACL. This ligamentization process lasts ∼2 years and the biomechanical integrity of the graft is maintained throughout the whole period. These studies are the first, and so far the only, to demonstrate remodeling of porcine tendon implants into permanently functional autologous ACL in humans.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Design and characterization of three-dimensional twist-braid scaffolds for anterior cruciate ligament regeneration

The anterior cruciate ligament (ACL) is the most commonly injured ligament in the knee, with more than 350,000 ACL injuries reported annually in the US. Current treatments include the use of autografts and allografts, which have a number of disadvantages. Previous attempts to use synthetic materials in ligament replacement have been unsuccessful due to their inability to replicate the long-term mechanical properties of the native ligament. The focus of this study was to develop twist-braid poly(L-lactic acid) (PLLA) scaffolds for ACL regeneration. Poly(ethylene glycol) diacrylate (PEGDA) was incorporated into the twist-braid scaffolds to evaluate its impact on their mechanical behavior. The twist-braid scaffolds were also compared with braided scaffolds. Scaffold mechanical properties were evaluated based on stress-relaxation, tensile and fatigue properties of the braided-only, twist-braid, and the twist-braid scaffolds with PEGDA. All the scaffolds exhibited properties comparable to the native human ACL with the twist-braid scaffolds displaying resistance to fatigue. Scaffolds were seeded with rat patellar tendon fibroblasts. Cell viability and the amount of protein released were studied over a course of 8 weeks. The scaffolds were stained with Picrosirius red after 8 weeks to show the deposition of extracellular matrix by the cells. The results from this study showed that the twist-braid scaffolds have properties most suitable for ligament regeneration.

Microstructural properties of the anterior cruciate ligament

The anterior cruciate ligament (ACL) of the knee is one of the most commonly injured structures in musculoskeletal medicine. Although the ligament is widely researched and studied for its macroscopic biomechanical role in knee stability, surprisingly few studies have analyzed the microscopic material properties of this important ligament. Several recent studies, however, have evaluated the microstructural and fiber alignment properties of the ACL. The anteromedial (AM) region of the ACL has a different cellular composition and exhibits greater fiber alignment and failure strength compared to the posterolateral (PL) regions. Furthermore, these properties vary in a linear fashion across the ligament moving from anterior to posterior, rather than just differing in discrete anatomical bundles. This information provides a greater understanding of the native properties of the human ACL and serves as a guide for surgical techniques.

Microstructure Variations in the Soft-Hard Tissue Junction of the Human Anterior Cruciate Ligament.

The role of the sub-bundles in the anterior cruciate ligament (ACL) has been defined, such that the anterior-medial bundle directly resists anterior tibial translation while the posterior lateral bundle is involved in rotational stability. With regards to this biomechanical function, much of the previous work on bundle-specific morphology has been carried out on the macroscale, with much less attention given to the micro-to-ultrastructural scalar levels. This is especially true of the enthesis and its microstructure, a biomechanically significant region that has been largely neglected in the published literature dealing with ACL sub-bundle anatomy. In this study, the human ACL tibial enthesis was investigated at multiple scalar levels using differential interference contrast and scanning electron microscopies with the aim of determining whether the sub-bundle ligament structure, and its known macroscale function, is consistent with its micro-architecture at the ligament-bone junction. The investigation found that different ligament insertion morphologies exist between the two bundles, where the AM bundle has more intense interdigitation with the bone matrix than that of the PL bundle. The results suggest that such structure-function relationships, especially across scalar-levels, provide new insight into the significance of the sub-bundle anatomy of the ACL. Anat Rec, 300:1547-1559, 2017. © 2017 Wiley Periodicals, Inc.

Influence of the Injury-to-Surgery Interval on the Healing Potential of Human Anterior Cruciate Ligament–Derived Cells

Background: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissue have the potential for high proliferation and multilineage differentiation that can accelerate tendon-bone healing. While patient characteristics, such as age, can affect tendon-bone healing, the influence of elapsed time after injury on the healing process is unclear. Hypothesis: Cells obtained during the early phase after injury will exhibit a greater tendon-bone healing potential compared with chronic phase counterparts when applied to an immunodeficient rat model of ACL reconstruction. Study Design: Controlled laboratory study. Methods: Adult human ACL-ruptured tissue was harvested from patients undergoing arthroscopic primary ACL reconstruction and classified into 2 groups based on the time elapsed between injury and surgery: (1) early group (≤3 months from injury) and (2) chronic group (>3 months from injury). In addition, 76 ten-week-old female immunodeficient rats underwent ACL reconstruction, followed by intracapsular administration of one of the following: (1) ACL-derived cells from the early group (n = 5), (2) ACL-derived cells from the chronic group (n = 5), or (3) phosphate-buffered saline (PBS) only (n = 5). During the 8 weeks after surgery, histological (weeks 2, 4, 8), immunohistochemical (week 2), radiographic (weeks 0, 2, 4, 8), and biomechanical (week 8) analyses were performed to evaluate tendon-bone healing. Results: In the early group, the histological evaluation showed early healing, induction of endochondral ossification–like integration, and mature bone ingrowth. Micro–computed tomography showed that the tibial bone tunnels at week 4 and week 8 were significantly reduced in the early group compared with those in the chronic group and PBS group (P < .05). Moreover, biomechanical tensile strength was significantly greater in the early group than in the other groups (P < .05). An accelerated healing potential in the early group was further demonstrated by the enhancement of intrinsic angiogenesis/osteogenesis and human-derived vasculogenesis/osteogenesis. Conclusion: Compared with human ACL-derived cells obtained during the chronic phase, cells obtained during the early phase after injury have a greater tendon-bone healing potential when used in an immunodeficient rat model of ACL reconstruction. Clinical Relevance: During ACL reconstruction surgery, transplanting ACL remnant tissue in the early phase after injury could accelerate and enhance tendon-bone healing.

* Hierarchically Structured Electrospun Scaffolds with Chemically Conjugated Growth Factor for Ligament Tissue Engineering.

The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue-engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macroscale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF)-conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF were assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles), or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles), and ligament-specific tissue formation was assessed in comparison to non-CTGF-conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament-specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

Bony Morphology: Comparative Anatomy and its Importance for the Anterior Cruciate Ligament

Bone morphology is related to static and dynamic parameters during anterior cruciate ligament (ACL) kinematics and can affect injury mechanism patterns, gait analysis, biomechanical properties, and surgical references for ligament reconstructions. It does not only depend on mechanical factors but also reflects a developmental program with many other factor involved, including nutrition and hormones, especially those involved in the calcium metabolism. Although Wolff׳s law continues to be a dominant paradigm in Orthopaedics, in the past 15 years enormous advances have been made that have improved our understanding of how individual bones achieve their internal and external structure. Transition from the quadrupedal mode of locomotion to bipedalism compelled changes in the femur morphology and the modern human knee has an extensive history of modifications produced by natural selection acting on its function in habitually upright walking and running. The human ACL is a band-like structure of dense connective tissues that binds the femur to the tibia. The femoral ACL attachment is located on the posterior aspect of the medial surface of the lateral femoral condyle and is composed of both direct and indirect fibers; the central part of the insertion is constructed in the manner described by the classical 4-phase architecture (direct insertion), whereas the posterior margin of the insertion is composed of a 2-phase architecture more typical of indirect insertions. The knowledge of anatomy, comparative anatomy, evolution, and natural selection allows us to have a more profound understanding of the interaction between form and function; after all, as Theodosius Dobzhansky has stated, Nothing in biology makes sense except in the light of Evolution.

A morphologic and quantitative comparison of mechanoreceptors in the tibial remnants of the ruptured human anterior cruciate ligament.

Reconstruction of the ruptured anterior cruciate ligament (ACL) does not always result in expected successful outcome. A satisfactory outcome depends not only on the tightness or strength of the graft but also on the quality of proprioceptive restoration. Mechanoreceptors of ACL are supposed to play considerable roles in the proprioceptive feedback system of knee. This study aimed to observe the condition and number of the surviving mechanoreceptors in the tibial remnant of ruptured ACL in human knees.From April 2009 to January 2012, 60 patients with existing free tibial remnants who had undergone arthroscopic ACL reconstruction were enrolled and divided into 4 groups according to the time duration of injury to surgery (Group I: no more than 3 months; Group II: 3 to 6 months; Group III, 6 months to 1 year; Group IV: more than 1 year). Six normal ACL specimens were taken as controls. Specimens were obtained from ACL tibial remnant and stained by the immunohistochemical staining method. The type, size, and quantity of mechanoreceptors were observed under the light microscope. A total of 92 Ruffini-like corpuscles, 9 Pacini-like corpuscles, 5 unclassified neural endings, and free nerve endings were identified via immunohistochemical staining.There were no significant differences in the number of mechanoreceptors in the 5 groups (P = 0.238). Some degenerative changes were observed in Group IV. The results suggest that the residual mechanoreceptors in the ruptured ACL exhibit long-term survival and showed no obvious signs of withering within 1 year.Residual mechanoreceptors do exist in the tibial remnants of ruptured anterior cruciate ligament in human knees and identified clearly by using immunohistochemistry staining. No significant difference was found regarding quantitative variation of the residual mechanoreceptors about the injury duration.

MGF E peptide pretreatment improves collagen synthesis and cell proliferation of injured human ACL fibroblasts via MEK-ERK1/2 signaling pathway

Injured anterior cruciate ligament (ACL) is hard to heal due to the poor proliferative potential of ACL fibroblasts. To verify whether mechano-growth factor (MGF) E peptide can restore the cell proliferation of injured ACL fibroblasts, ACL fibroblasts pretreated with MGF E peptide were subjected to injurious stretch and the outcomes were evaluated at 0 and 24 h. After injured, the type III collagen synthesis was increased at 0 h while inhibited at 24 h. The matrix metalloproteinase-2 (MMP-2) activity/expression was up-regulated, but the cell proliferation was inhibited. Fortunately, exogenous MGF E peptide decreased the type I/III collagen synthesis at 0 h but improved the type III collagen synthesis at 24 h. It decreased the MMP-2 activity/expression of injured ACL fibroblasts. Besides, MGF E peptide accelerated the cell proliferation via MEK-ERK1/2 signaling pathway. Our results implied that MGF E peptide pretreatment could provide a new efficient approach for ACL regeneration.

Bone contusion progression from traumatic knee injury: association of rate of contusion resolution with injury severity.

BackgroundBone contusions are frequently encountered in magnetic resonance imaging (MRI) evaluation of knee anterior cruciate ligament (ACL) injuries. Their role as indicators of injury severity remains unclear, primarily due to indeterminate levels of joint injury forces and to a lack of preinjury imaging.PurposeThe purpose of this study was to 1) quantify bone contusion pathogenesis following traumatic joint injuries using fixed imaging follow-ups, and 2) assess the feasibility of using longitudinal bone contusion volumes as an indicator of knee injury severity.Study designProspective sequential MRI follow-ups of a goat cohort exposed to controlled stifle trauma in vivo were compared to parallel clinical MRI follow-ups of a human ACL tear patient series.MethodsReproducible cartilage impact damage of various energy magnitudes was applied in a survival goat model, coupled with partial resection of anterior portions of medial menisci. Both emulate injury patterns to the knee osteochondral structures commonly encountered in human ACL injury imaging as well as instability from resultant ligament laxity. Longitudinal clinical MRI sequences portrayed stifle bone contusion evolution through 6 months after the inciting event.ResultsIn the first 2 weeks, biological response variability dominated the whole-joint response with no apparent correlation to trauma severity. Control goats subjected to partial meniscectomy alone exhibited minimal bone response. Thereafter, 0.6 J impact bone contusions portrayed a faster rate of resolution than those induced by 1.2 J cartilage impacts.ConclusionBone contusion sizes combined with time of persistence are likely better measures of joint injury severity than isolated bone contusion volume.

Strain behavior of the human anterior cruciate ligament near the femoral attachment region

Strain behavior of the human anterior cruciate ligament near the femoral attachment region

Differentiation of Human Amniotic Mesenchymal Stem Cells into Human Anterior Cruciate Ligament Fibroblast Cells by In Vitro Coculture.

Anterior cruciate ligament injuries are common in humans, though cellular components of the knee have little regenerative or proliferation potential. This study investigated the differentiation of human amnion-derived mesenchymal stem cells (hAMSCs) into human anterior cruciate ligament fibroblasts (hACLFs) in vitro through induction with bFGF and TGF-β1 with coculture systems. Groups A and B comprised hAMSCs at the 3rd passage cultured with and without bFGF and TGF-β1, respectively; Groups C and D consisted of hAMSCs and hACLFs in monolayer coculture with and without bFGF and TGF-β1, respectively; Groups E and F were composed of hAMSCs and hACLFs in Transwell coculture with and without bFGF and TGF-β1, respectively. Cell morphology and proliferation were recorded. Protein expression and relative mRNA expression were evaluated in each group. Cell proliferation was significantly higher in the induced groups than in the noninduced groups. Protein expression increased over time with the highest expression observed in Group E. mRNA levels were significantly higher in Group E than in other groups. This study is the first to demonstrate the use of the Transwell coculture system for this purpose, and hAMSCs were successfully differentiated into hACLFs. Thus, hAMSCs may be a superior choice for hACLF differentiation via Transwell coculture.

Anterior Cruciate Ligament–Derived Stem Cells Transduced With BMP2 Accelerate Graft-Bone Integration After ACL Reconstruction

Background: Strong graft-bone integration is a prerequisite for successful graft remodeling after reconstruction of the anterior cruciate ligament (ACL) using soft tissue grafts. Novel strategies to accelerate soft tissue graft-bone integration are needed to reduce the need for bone-tendon-bone graft harvest, reduce patient convalescence, facilitate rehabilitation, and reduce total recovery time after ACL reconstruction. Hypothesis: The application of ACL-derived stem cells with enhanced expression of bone morphogenetic protein 2 (BMP2) onto soft tissue grafts in the form of cell sheets will both accelerate and improve the quality of graft-bone integration after ACL reconstruction in a rat model. Study Design: Controlled laboratory study. Methods: ACL-derived CD34+ cells were isolated from remnant human ACL tissues, virally transduced to express BMP2, and embedded within cell sheets. In a rat model of ACL injury, bilateral single-bundle ACL reconstructions were performed, in which cell sheets were wrapped around tendon autografts before reconstruction. Four groups containing a total of 48 rats (96 knees) were established (n = 12 rats; 24 knees per group): CD34+BMP2 (100%), CD34+BMP2 (25%), CD34+ (untransduced), and a control group containing no cells. Six rats from each group were euthanized 2 and 4 weeks after surgery, and each graft was harvested for immunohistochemical and histological analyses. The remaining 6 rats in each group were euthanized at 4 and 8 weeks to evaluate in situ tensile load to failure in each femur-graft-tibia complex. Results: In vitro, BMP2 transduction promoted the osteogenic differentiation of ACL-derived CD34+ cells while retaining their intrinsic multipotent capabilities. Osteoblast densities were greatest in the BMP2 (100%) and BMP2 (25%) groups. Bone tunnels in the CD34+BMP2 (100%) and CD34+BMP2 (25%) groups had the smallest cross-sectional areas according to micro–computed tomography analyses. Graft-bone integration occurred most rapidly in the CD34+BMP2 (25%) group. Tensile load to failure was significantly greater in the groups containing stem cells at 4 and 8 weeks after surgery. Tensile strength was greatest in the CD34+BMP2 (100%) group at 4 weeks, and in the CD34+BMP2 (25%) group at 8 weeks. Conclusion: ACL-derived CD34+ cells transduced with BMP2 accelerated graft-bone integration after ACL reconstruction using soft tissue autografts in a rat model, as evidenced by improved histological appearance and graft-bone interface biology along with tensile load to failure at each time point up to 8 weeks after surgery. Clinical Relevance: A primary disadvantage of using soft tissue grafts for ACL reconstruction is the prolonged time required for bony ingrowth, which delays the initiation of midsubstance graft remodeling. The lack of consistent correlation between the appearance of a “healed” ACL on postoperative magnetic resonance imaging and readiness to return to sport results in athletes being released to sport before the graft is ready to handle high-intensity loading. Therefore, it is desirable to identify strategies that accelerate graft-bone integration, which would reduce the time to biologic fixation, improve the reliability of biologic fixation, allow for accelerated rehabilitation, and potentially reduce the incidence of early graft pullout and late midsubstance failure.

MRI depiction and 3D visualization of three anterior cruciate ligament bundles.

The anterior cruciate ligament (ACL) is divided into three fiber bundles (AM-M: anteromedial-medial, AM-L: anteromedial-lateral, PL: posterolateral). We attempted to depict the three bundles of the human ACL on MRI images and to obtain 3-dimensional visualization of them. Twenty-four knees of healthy volunteers (14 males, 10 females) were scanned by 3T-MRI using the fat suppression 3D coherent oscillatory state acquisition for the manipulation of imaging contrast (FS 3D-COSMIC). The scanned images were reconstructed after the isotropic voxel data, which allows the images to be reconstructed in any plane, was acquired. We conducted statistical examination on the identification rate of the three ACL bundles by 2D planes. Segmentation and 3D visualization of the fiber bundles using volume rendering were performed. The triple-bundle ACL was best depicted in the oblique axial plane. While the AM-M and AM-L bundles were clearly depicted in all cases, the PL bundle was not clearly visualized in two knees (8%). Therefore, the three ACL bundles were depicted in 22 knees (92%). The results of 3D visualization of the fiber arrangement agreed well with macroscopic findings of previous anatomical studies. 3T-MRI and the isotropic voxel data from FS 3D-COSMIC made it possible to demonstrate the identifiable depiction of three ACL bundles in nearly all cases. 3D visualization of the bundles could be a useful tool to understand the ACL fiber arrangement. Clin. Anat. 30:276-283, 2017. 2016 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts

ABSTRACTPurposes: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2 to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. Materials and methods: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. Results: The real-time PCR results show that mechanical stretch or CoCl2 treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2 treatment group alone. Western blot analysis and ELISA also confirmed these results. Conclusions: Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.

Overlap Between Anterior Cruciate Ligament and Anterolateral Meniscal Root Insertions: A Scanning Electron Microscopy Study

Background: The anterolateral meniscal root (ALMR) has been reported to intricately insert underneath the tibial insertion of the anterior cruciate ligament (ACL). Previous studies have begun to evaluate the relationship between the insertion areas and the risk of iatrogenic injuries; however, the overlap of the insertions has yet to be quantified in the sagittal and coronal planes. Purpose: To investigate the insertions of the human tibial ACL and ALMR using scanning electron microscopy (SEM) and to quantify the overlap of the ALMR insertion in the coronal and sagittal planes. Study Design: Descriptive laboratory study. Methods: Ten cadaveric knees were dissected to isolate the tibial ACL and ALMR insertions. Specimens were prepared and imaged in the coronal and sagittal planes. After imaging, fiber directions were examined to identify the insertions and used to calculate the percentage of the ACL that overlaps with the ALMR instead of inserting into bone. Results: Four-phase insertion fibers of the tibial ACL were identified directly medial to the ALMR insertion as they attached onto the tibial plateau. The mean percentage of ACL fibers overlapping the ALMR insertion instead of inserting into subchondral bone in the coronal and sagittal planes was 41.0% ± 8.9% and 53.9% ± 4.3%, respectively. The percentage of insertion overlap in the sagittal plane was significantly higher than in the coronal plane (P = .02). Conclusion: This study is the first to quantify the ACL insertion overlap of the ALMR insertion in the coronal and sagittal planes, which supplements previous literature on the insertion area overlap and iatrogenic injuries of the ALMR insertion. Future studies should determine how much damage to the ALMR insertion is acceptable to properly restore ACL function without increasing the risk for tears of the ALMR. Clinical Relevance: Overlap of the insertion areas on the tibial plateau has been previously reported; however, the results of this study demonstrate significant overlap of the insertions superior to the insertion sites on the tibial plateau as well. These findings need to be considered when positioning for tibial tunnel creation in ACL reconstruction to avoid damage to the ALMR insertion.

New perspectives on ACL injury: On the role of repetitive sub-maximal knee loading in causing ACL fatigue failure.

ABSTRACTIn this paper, we review a series of studies that we initiated to examine mechanisms of anterior cruciate ligament (ACL) injury in the hope that these injuries, and their sequelae, can be better prevented. First, using the earliest in vitro model of a simulated single‐leg jump landing or pivot cut with realistic knee loading rates and trans‐knee muscle forces, we identified the worst‐case dynamic knee loading that causes the greatest peak ACL strain: Combined knee compression, flexion, and internal tibial rotation. We also identified morphologic factors that help explain individual susceptibility to ACL injury. Second, using the above knee loading, we introduced a possible paradigm shift in ACL research by demonstrating that the human ACL can fail by a sudden rupture in response to repeated sub‐maximal knee loading. If that load is repeated often enough over a short time interval, the failure tended to occur proximally, as observed clinically. Third, we emphasize the value of a physical exam of the hip by demonstrating how limited internal axial rotation at the hip both increases the susceptibility to ACL injury in professional athletes, and also increases peak ACL strain during simulated pivot landings, thereby further increasing the risk of ACL fatigue failure. When training at‐risk athletes, particularly females with their smaller ACL cross‐sections, rationing the number and intensity of worst‐case knee loading cycles, such that ligament degradation is within the ACL's ability to remodel, should decrease the risk for ACL rupture due to ligament fatigue failure.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2059–2068, 2016.