Anterior Cruciate Ligament Tissue Research Articles

The effect of melt electrospun writing fiber orientation onto cellular organization and mechanical properties for application in Anterior Cruciate Ligament tissue engineering

The effect of melt electrospun writing fiber arrangement on cellular behavior has not yet been thoroughly investigated. Cellular orientation is particularly important in the context of ligament tissue engineering for orthopedic applications whereby a high degree of cell alignment is present in the native tissue. The aim of this study was to investigate the response of human mesenchymal stem cells (hMSC) to three different patterned porous polycaprolactone scaffolds (aligned, crimped and random) fabricated by melt electrospinning writing, resulting in 20 μm diameter electrospun fibers. Cell orientation was investigated over 4 weeks in vitro and it was demonstrated that the aligned pattern was capable of orientating the hMSCs towards the main direction of the fibers and this feature was maintained over the entire culture period whereas the orientation was rapidly lost in the crimped pattern. In order to fabricate a functional scaffold for ligament tissue engineering, the scaffolds were rolled in three bundles, subsequently braided and combined with a bone compartment (consisting of a melt electrospun scaffold seeded with osteogenically induced hMSCs) for the development of a Bone-Ligament-Bone (BLB) construct. The mechanical properties of non-cellularized and cellularized BLB constructs were assessed under both quasi-static and cyclic conditions. This revealed that the in vitro maturation significantly softened the BLB constructs and that the mechanical properties were several fold lower than those of native tissue. The cyclic testing demonstrated that the presence of cell sheets resulted in increased resilience and elasticity, even though the global mechanical properties were decreased for the in vitro matured constructs (regardless of the pattern). In conclusion, we demonstrated that melt electrospinning writing fiber organization can induce spontaneous cell alignment and that large cellularized BLB constructs with complex geometry can achieve mechanical resilience under cyclic stretching.

Joint laxity varies in response to partial and complete anterior cruciate ligament injuries throughout skeletal growth

Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model. To do so, we applied anterior-posterior tibial loads and varus-valgus moments to stifle joints of female pigs ranging from early juvenile to late adolescent ages and assessed both kinematics and in-situ loads carried in the bundles of the ACL and other soft tissues including the collateral ligaments and the menisci. Partial ACL injury led to increased anterior tibial translation only in late adolescence and small increases in varus-valgus rotation at all ages. Complete ACL injury led to substantial increases in translation and rotation at all ages. At all ages, the medial collateral ligament and the medial meniscus combined to resist the majority of applied anterior tibial load following complete ACL transection. Across all ages and flexion angles, the contribution of the MCL ranged from 45 to 90% of the anterior load and the contribution of the medial meniscus ranged from 14 to 35% of the anterior load. These findings add to our current understanding of age-specific functional properties of both healthy and injured knees during skeletal growth.

Engagement of the Secondary Ligamentous and Meniscal Restraints Relative to the Anterior Cruciate Ligament Predicts Anterior Knee Laxity

Background: Patients with high-grade preoperative side-to-side differences in anterior laxity as assessed via the Lachman test after unilateral anterior cruciate ligament (ACL) rupture are at heightened risk of early ACL graft failure. Biomechanical factors that predict preoperative side-to-side differences in anterior laxity are poorly understood. Purpose: To assess, in a cadaveric model, whether the increase in anterior laxity caused by sectioning the ACL (a surrogate for preoperative side-to-side differences in anterior laxity) during a simulated Lachman test is associated with two biomechanical factors: (1) the tibial translation at which the secondary anterior stabilizers, including the remaining ligaments and the menisci, begin to carry force, or engage, relative to that of the ACL or (2) the forces carried by the ACL and secondary stabilizers at the peak applied anterior load. Study Design: Controlled laboratory study. Methods: Seventeen fresh-frozen human cadaveric knees underwent Lachman tests simulated through a robotic manipulator with the ACL intact and sectioned. The net forces carried by the ACL and secondary soft tissue stabilizers (the medial meniscus and all remaining ligaments, measured as a whole) were characterized as a function of anterior tibial translation. The engagement points of the ACL (with the ACL intact) and each secondary stabilizer (with the ACL sectioned) were defined as the anterior translation at which they began to carry force, or engaged, during a simulated Lachman test. Then, the relative engagement point of each secondary stabilizer was defined as the difference between the engagement point of each secondary stabilizer and that of the ACL. Linear regressions were performed to test each association (P < .05). Results: The increase in anterior laxity caused by ACL sectioning was associated with increased relative engagement points of both the secondary ligaments (β = 0.87; P < .001; R2 = 0.75) and the medial meniscus (β = 0.66; P < .001; R2 = 0.58). Smaller changes in anterior laxity were also associated with increased in situ medial meniscal force at the peak applied load when the ACL was intact (β = −0.06; P < .001; R2 = 0.53). Conclusion: The secondary ligaments and the medial meniscus require greater anterior tibial translation to engage (ie, begin to carry force) relative to the ACL in knees with greater changes in anterior laxity after ACL sectioning. Moreover, with the ACL intact, the medial meniscus carries more force in knees with smaller changes in anterior laxity after ACL sectioning. Clinical Relevance: Relative tissue engagement is a new biomechanical measure to characterize in situ function of the ligaments and menisci. This measure may aid in developing more personalized surgical approaches to reduce high rates of ACL graft revision in patients with high-grade laxity.

Effect of Remnant Tissues on Outcomes of Anterior Cruciate Ligament Reconstruction at Follow-up Arthroscopy.

Remnant-preserving anterior cruciate ligament reconstruction (ACLR) is sought to enhance vascularization and maturation of a graft and to produce positive clinical outcomes after reconstruction. The purpose of this study was to investigate an effect of remnant anterior cruciate ligament (ACL) tissues on outcomes of ACLR 1 year after reconstruction in younger populations. Retrospective chart review. Inpatient orthopedic surgical and rehabilitation clinic. The subjects were 644 patients who underwent primary ACLR. The independent variables were age, height, and body weight of the subjects, and the presence of remnant tissue. The outcome measure was the number of subjects with secondary infra-articular injuries at a follow-up arthroscopy after ACLR. The odds ratio was calculated and compared between the remnant-preserving ACLR (ACLR-P) and remnant-resecting (ACLR-R) groups to investigate the effect of remnant tissues on postoperative outcomes. A total of 416 cases met our inclusion criteria and were included in this study. There were 136 cases (49 males and 87 females) in the ACLR-P group with the mean age (±SD) of 17.86 ± 2.85 years. There were 280 cases (106 males and 174 females) in the ACLR-R group, and the mean age (±SD) was 18.13 ± 2.73 years. Of 136 cases in the ALCR-P group, 54 (39.7%) had abnormal arthroscopic findings that lead to a surgical intervention. Of 280 patients in the ACLR-R group, 128 (45.7%) had abnormal findings. Odds ratio was 0.78 [95% confidence interval (CI), 0.51-1.18; P = 0.24]. Four cases in the ACLR-P group and 8 in the ACLR-R group were diagnosed with ACL retear during follow-up arthroscopy. Odds ratio was 1.03 (95% CI, 0.30-3.48; P = 0.96). The presence of ACL remnant tissues did not have a significant effect on outcomes 1 year after reconstruction. However, there was a trend that lower percentage of patients with remnant-preserving ALCR had abnormal arthroscopic findings that lead to a surgical treatment at follow-up arthroscopy 1 year after initial reconstruction. The presence of remnant tissues did not have a significant effect on retear rate.

Viscoelastic Behavior of Embroidered Scaffolds for ACL Tissue Engineering Made of PLA and P(LA-CL) After In Vitro Degradation.

A rupture of the anterior cruciate ligament (ACL) is the most common knee ligament injury. Current applied reconstruction methods have limitations in terms of graft availability and mechanical properties. A new approach could be the use of a tissue engineering construct that temporarily reflects the mechanical properties of native ligament tissues and acts as a carrier structure for cell seeding. In this study, embroidered scaffolds composed of polylactic acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads were tested mechanically for their viscoelastic behavior under in vitro degradation. The relaxation behavior of both scaffold types (moco: mono-component scaffold made of PLA threads, bico: bi-component scaffold made of PLA and P(LA-CL) threads) was comparable to native lapine ACL. Most of the lapine ACL cells survived 32 days of cell culture and grew along the fibers. Cell vitality was comparable for moco and bico scaffolds. Lapine ACL cells were able to adhere to the polymer surfaces and spread along the threads throughout the scaffold. The mechanical behavior of degrading matrices with and without cells showed no significant differences. These results demonstrate the potential of embroidered scaffolds as an ACL tissue engineering approach.

An Anterior Cruciate Ligament Failure Mechanism

Background: Nearly three-quarters of anterior cruciate ligament (ACL) injuries occur as “noncontact” failures from routine athletic maneuvers. Recent in vitro studies revealed that repetitive strenuous submaximal knee loading known to especially strain the ACL can lead to its fatigue failure, often at the ACL femoral enthesis. Hypothesis: ACL failure can be caused by accumulated tissue fatigue damage: specifically, chemical and structural evidence of this fatigue process will be found at the femoral enthesis of ACLs from tested cadaveric knees, as well as in ACL explants removed from patients undergoing ACL reconstruction. Study Design: Controlled laboratory study. Methods: One knee from each of 7 pairs of adult cadaveric knees were repetitively loaded under 4 times–body weight simulated pivot landings known to strain the ACL submaximally while the contralateral, unloaded knee was used as a comparison. The chemical and structural changes associated with this repetitive loading were characterized at the ACL femoral enthesis at multiple hierarchical collagen levels by employing atomic force microscopy (AFM), AFM–infrared spectroscopy, molecular targeting with a fluorescently labeled collagen hybridizing peptide, and second harmonic imaging microscopy. Explants from ACL femoral entheses from the injured knee of 5 patients with noncontact ACL failure were also characterized via similar methods. Results: AFM–infrared spectroscopy and collagen hybridizing peptide binding indicate that the characteristic molecular damage was an unraveling of the collagen molecular triple helix. AFM detected disruption of collagen fibrils in the forms of reduced topographical surface thickness and the induction of ~30- to 100-nm voids in the collagen fibril matrix for mechanically tested samples. Second harmonic imaging microscopy detected the induction of ~10- to 100-µm regions where the noncentrosymmetric structure of collagen had been disrupted. These mechanically induced changes, ranging from molecular to microscale disruption of normal collagen structure, represent a previously unreported aspect of tissue fatigue damage in noncontact ACL failure. Confirmatory evidence came from the explants of 5 patients undergoing ACL reconstruction, which exhibited the same pattern of molecular, nanoscale, and microscale structural damage detected in the mechanically tested cadaveric samples. Conclusion: The authors found evidence of accumulated damage to collagen fibrils and fibers at the ACL femoral enthesis at the time of surgery for noncontact ACL failure. This tissue damage was similar to that found in donor knees subjected in vitro to repetitive 4 times–body weight impulsive 3-dimensional loading known to cause a fatigue failure of the ACL. Clinical Relevance: These findings suggest that some ACL injuries may be due to an exacerbation of preexisting hierarchical tissue damage from activities known to place larger-than-normal loads on the ACL. Too rapid an increase in these activities could cause ACL tissue damage to accumulate across length scales, thereby affecting ACL structural integrity before it has time to repair. Prevention necessitates an understanding of how ACL loading magnitude and frequency are anabolic, neutral, or catabolic to the ligament.

Migrating Myofibroblastic Iliotibial Band-Derived Fibroblasts Represent a Promising Cell Source for Ligament Reconstruction.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I–III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, β1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

Characterization of the structure of rabbit anterior cruciate ligament and its stem/progenitor cells.

It is known that anterior cruciate ligament (ACL) of the knee joint is prone to injuries with poor healing potential. The healing capacity of a tissue-like ACL is dependent on its structural components and the properties of the stem cells (SCs). Therefore, this study aimed to characterize the structure of ACL tissue and the properties of the SCs derived from the tissue components. The tissue structure of rabbit ACL was determined using a scanning electron microscope, hematoxylin and eosin, and immunohistochemical staining. The biological properties of SCs derived from the structural components of ACL were studied by colony formation, cell proliferation assay, SC marker expression and collagen exhibition, and multidifferentiation potential. The two distinct components of ACL are classified as sheath and core, which possess differential properties in terms of collagen type, organization, and presence of blood vessels. The sheath tissue contains vascular SCs and the core tissue contains ligamentous SCs, respectively. The two types of SCs differ in clonogenicity, proliferation, and multidifferentiation potential. This study shows that ACL consists of sheath and core tissues, which contain sheath and core SCs with distinctive biological properties. These findings highlight the need for use of both sheath and core SCs to promote the repair of the complex structure of injured ACL.

A novel method for continuous formation of cord-like collagen gels to fabricate durable fibers in which collagen fibrils are longitudinally aligned.

We developed a continuous formation method of cord-like collagen gels comprising fibrils preferentially aligned along the geometrical axes (CCGs). Collagen (2.5%) dissolved in a sodium phosphate buffer containing 280 mM of sodium chloride was introduced into a stainless cylinder (length 52 mm, diameter 2.0 mm) warmed at 38°C at a linear velocity of 2.5 mm/s. This process caused collagen fibril alignments under acute fibril formation in the cylinder, resulting in continuous formation of CCGs. Fibril formation rate, shear rate, and shear duration were substantial factors for successful CCG formation. Outstanding advantages of this method over conventional wet spinning include the capacity of this technique to form aligned fibrils in the entire gels and to control the diameter of cord-like gels over 1 mm. The air-drying of CCGs which were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-succinimide produced dry collagen fibers with cross-sectional areas of 0.0123-0.135 mm2 . Upon the rewetting of the fibers, they failed at a stress of 54.5 ± 7.8 MPa, which is higher than the mean failure stress of anterior cruciate ligament tissue (13.3-37.8 MPa). These findings indicate that the CCG formation method enables the fabrication of collagen fibers which are potential components of collagen-based artificial tendons. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1011-1023, 2019.

Mesenchymal Stem Cells Isolated from the Anterior Cruciate Ligament: Characterization and Comparison of Cells from Young and Old Donors.

PurposeMesenchymal stem cells (MSCs) isolated from the anterior cruciate ligament (ACL) share multiple characteristics of bone marrow-derived mesenchymal stem cells (BMSCs), allowing their use for regenerative therapies. Injuries to the ACL can affect people of all ages. This study assesses whether the regenerative potential of ACL-derived MSCs (ACL-MSCs) from old donors is as high as the potential of ACL-MSCs from young donors.Materials and MethodsACL-MSCs were isolated from ACL tissues obtained from young and old donors at the time of ACL reconstruction or arthroplasty. Proliferative capacity, multilineage differentiation potential (chondrogenic, osteogenic, and adipogenic lineages), and transcriptome-wide gene expression were assessed and compared between young and old donors. BMSCs of middle-aged donors served as an additional comparator.ResultsNo substantial differences between ACL-MSCs from young and old donors were observed in their proliferative capacity and multilineage differentiation potential. The latter did not substantially differ between both ACL-MSC groups and BMSCs. Differential expression of genes related to the cytoskeleton and to protein dephosphorylation amongst other pathways was detected between ACL-MSCs from young and old donors.ConclusionsRegenerative potential of ACL-MSCs from old donors was not substantially lower than that from young donors, suggesting that regenerative therapies of ACL tears are feasible in both age groups. In vivo studies of the effect of age on the efficacy of such therapies are needed.

Differences in expression of Mohawk transcription factors and collagen in anterior cruciate ligament grafts between two remodeling outcomes under arthroscopy

Objective To investigate the differences in expression of Mohawk (MKX) transcription factors and collagen of types Ⅰ and Ⅲ in anterior cruciate ligament (ACL) grafts between 2 remodeling outcomes under arthroscopy. Methods Enrolled for this study were 17 patients who had undergone arthroscopic single-bundle ACL reconstruction with autogenous hamstring tendons and secondary arthroscopic exploration 48 to 131 months (average, 83.1 months) after removal of tibial internal fixator at Department of Sports Medicine, The First Affiliated Hospital to Shenzhen University from March 2017 to December 2017. They were divided into a good remodeling group (11 cases) and a fair remodeling group (6 cases) according to the graft quality under arthroscopy (synovial and vascular coverage, and apparent tension, thickness and retear of the grafts). During the secondary arthroscopic procedures, biopsy of the central ACL grafts was performed. Moreover, normal ACL tissues were harvested from 8 contemporary controls of <60 years old who underwent total knee replacement. Immunohistochemical assay and quantificational real-time polymerase chain reaction were conducted to detect the expression of transcription factors and collagen of types Ⅰ and Ⅲ in all the samples. Results In the samples from good remodeling and control groups, there were abundant well-arranged collagen fibers of types Ⅰ and Ⅲ and MKX-positive cells; in the fair remolding group, the collagen fibers of types Ⅰ and Ⅲ and MKX-positive cells were much decreased in number and the fibers were not well arranged. The former 2 groups scored in immunohistochemical assay significantly higher than the latter one (P<0.05). qRT-PCR showed that the expression levels of MKX gene (0.44±0.30), COL1A1 gene (0.52±0.27) and COL3A1 gene (0.60±0.22) in the fair remolding group were significantly lower than in the control group (1.00±0.00, 1.00±0.00 and 1.00±0.00) and than in the good remolding group (0.97±0.67, 0.99±0.38 and 1.00±0.35) (P<0.05). Conclusion Good remodeling ACL grafts with histological maturation under arthroscopy are more similar to normal ACL than fair remodeling ACL grafts in expression of MKX transcription factors and collagen of types Ⅰ and Ⅲ. Key words: Anterior cruciate ligament; Transplantation, autologous; Ligaments, articular; Transcription factors; Collagen

Exogenous stromal derived factor-1 releasing silk scaffold combined with intra-articular injection of progenitor cells promotes bone-ligament-bone regeneration

Anterior cruciate ligament (ACL) is one of the most difficult tissues to heal once injured. Ligament regeneration and tendon-bone junction healing are two major goals of ACL reconstruction. This study aimed to investigate the synergistic therapeutic effects of Stromal cell-derived factor 1 (SDF-1)-releasing collagen-silk (CSF) scaffold combined with intra-articular injection of ligament-derived stem/progenitor cells (LSPCs) for ACL regeneration and the amelioration in the long-term complication of osteoarthritis (OA). The stem cell recruitment ability of CSF scaffold and the multipotency, particularly the tendon forming ability of LSPCs from rabbits were characterized in vitro, while the synergistic effect of the CSF scaffold and LSPCs for ACL regeneration and OA amelioration were investigated in vivo at 1, 3, and 6 months with a rabbit ACL reconstruction model. The CSF scaffold was used as a substitute for the ACL, and LSPCs were injected into the joint cavity after 7 days of the ACL reconstruction. CSF scaffold displayed a controlled release pattern for the encapsulated protein for up to 7 days with an increased stiffness in the mechanical property. LSPCs, which exhibited highly I Collagen and CXCR4 expression, were attracted by SDF-1 and successfully relocated into the CSF scaffold at 1 month in vivo. At 3 and 6 months post-treatment, the CSF scaffold combined with LSPCs (CSFL group) enhanced the regeneration of ACL tissue, and promoted bone tunnel healing. Furthermore, the OA progression was impeded efficiently. Our findings here provided a new strategy that using stem cell recruiting CSF scaffold with tissue-specific stem cells, could be a promising solution for ACL regeneration. Statement of SignificanceIn this study, we developed a silk scaffold with increased stiffness and SDF-1 controlled release capacity for ligament repair. This advanced scaffold transplantation combined with intra-articular injection of LSPCs (which was isolated from rabbit ligament for the first time in this study) promoted the regeneration of both the tendinous and bone tunnel portion of ACL. This therapeutic strategy also ameliorated cartilage degeneration and reduced the severity of arthrofibrosis. Hence, combining LSPCs injection with SDF-1-releasing silk scaffold is demonstrated as a therapeutic strategy for ACL regeneration and OA treatment in the clinic. [Display omitted]

The Influence of Ruptured Scar Pattern on the Healing Potential of Anterior Cruciate Ligament Remnant Cells

Background: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissues have a potential for high proliferation and multilineage differentiation, which can accelerate tendon-bone healing after ACL reconstruction. To predict outcomes of ACL reconstruction with remnant preservation or ruptured tissue incorporation, patient characteristics should be considered. However, the influence of ACL remnant morphologic pattern on healing potential remains unknown. Hypothesis: The healing potential of ACL remnants could differ among their morphologic patterns. Study Design: Descriptive laboratory study. Methods: ACL remnant tissues were harvested from patients aged <35 years who received primary ACL reconstruction within 3 months after injury. The tissues were evaluated according to the Crain classification (4 patterns). The patterns were divided into 2 groups: the reattachment group (Crain I-III) and the nonreattachment group (Crain IV). ACL remnant cells were characterized via fluorescence-activated cell sorting. The potential for proliferation and multilineage differentiation was assessed and compared between the groups. Results: The ratio of CD34+ cells was significantly higher in the nonreattachment group than in the reattachment group. In early passages, the nonreattachment group had a significantly higher expansion potential than the reattachment group. In the evaluation of osteogenic and endothelial differentiation potential, the nonreattachment group showed a higher potential in immunohistochemical/histochemical staining and quantitative real-time polymerase chain reaction analysis as compared with the reattachment group. Conclusion: In the subacute phase, ACL remnant tissue of the nonreattachment group possibly has a higher healing potential than that of the reattachment group. Clinical Relevance: If healing potential differs among the morphologic patterns of ACL remnants, surgeons may expect the healing potential when preserving remnants.

Suitability of a PLCL fibrous scaffold for soft tissue engineering applications: A combined biological and mechanical characterisation.

Poly(lactide-co-ε-caprolactone) (PLCL) has been reported to be a good candidate for tissue engineering because of its good biocompatibility. Particularly, a braided PLCL scaffold (PLL/PCL ratio = 85/15) has been recently designed and partially validated for ligament tissue engineering. In the present study, we assessed the in vivo biocompatibility of acellular and cellularised scaffolds in a rat model. We then determined its in vitro biocompatibility using stem cells issued from both bone marrow and Wharton Jelly. From a biological point of view, the scaffold was shown to be suitable for tissue engineering in all these cases. Secondly, while the initial mechanical properties of this scaffold have been previously reported to be adapted to load-bearing applications, we studied the evolution in time of the mechanical properties of PLCL fibres due to hydrolytic degradation. Results for isolated PLCL fibres were extrapolated to the fibrous scaffold using a previously developed numerical model. It was shown that no accumulation of plastic strain was to be expected for a load-bearing application such as anterior cruciate ligament tissue engineering. However, PLCL fibres exhibited a non-expected brittle behaviour after two months. This may involve a potential risk of premature failure of the scaffold, unless tissue growth compensates this change in mechanical properties. This combined study emphasises the need to characterise the properties of biomaterials in a pluridisciplinary approach, since biological and mechanical characterisations led in this case to different conclusions concerning the suitability of this scaffold for load-bearing applications.

Anterior cruciate ligament-derived mesenchymal stromal cells have a propensity to differentiate into the ligament lineage.

IntroductionThe anterior cruciate ligament (ACL) consists of various components, such as collagen, elastin fibres, and fibroblasts. Because ACL has a poor regenerative ability, ACL reconstruction need require the use of autologous tendons. In recent years, tissue-resident stem cells have been studied to promote ACL regeneration as an alternatively method. However, the existence of stem cells in ligaments has not been clearly defined. Here, we prospectively isolated stem cells from ACLs and characterized their properties.MethodsACLs from 11 donors and bone marrows (BM) from 8 donors were obtained with total knee arthroplasty. We used flow cytometry to screen the cell surface markers on ACL cells. Frozen sections were prepared from patient ACL tissues and stained with specific antibodies. Cultured ACL-derived and BM-derived cells at passage 3 were differentiated into adipocytes, osteoblasts and tendon/ligament cells.ResultsACL-derived mesenchymal stem/stromal cells (ACL-MSCs) expressed high levels of CD73 and CD90. Immunohistochemical analyses revealed that ACL-MSCs were located on the inner surface of ACL sinusoids. Furthermore, the expression of cell surface antigens was clearly different between ACL-MSCs and bone marrow (BM)-derived MSCs (BM-MSCs) at the time of isolation, but the two cell populations became indistinguishable after long-term culture. Interestingly, ACL-MSCs are markedly different from BM-MSCs in their differentiation ability and have a high propensity to differentiate into ligament-committed cells.ConclusionsOur findings suggest that ACL-MSCs express CD90 and CD73 markers, and their differentiation capacity is maintained even through culture. The cell population having tissue-specific properties is an important research target for investigating the ligament therapies.

Advantages of RNA‐seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018.

Identification of differentially expressed microRNAs in knee anterior cruciate ligament tissues surgically removed from patients with osteoarthritis.

The degradation of cruciate ligaments is frequently observed in degenerative joint diseases, such as osteoarthritis (OA). The present study aimed to identify the differentially expressed microRNAs (miRNAs or miRs) in knee anterior cruciate ligament (ACL) tissues derived from patients with OA and in health subjects (non-OA). By using Affymetrix miRNA 4.0 microarrays, a total of 22 miRNAs (including let-7f-5p, miR-26b-5p and miR-146a-5p) were found to be upregulated, while 17 (including miR-18a-3p, miR-138-5p and miR-485-3p) were downregulated in the osteoarthritic ACL tissues (fold change ≥2, P-value <0.05). The expression levels of 12 miRNAs were validated by quantitative PCR, and the corresponding results revealed an excellent correlation with the microarray data (R2=0.889). Genes (such as a disintegrin and metalloproteinase domain with thrombospondin type-1 motifs, bone morphogenetic protein-2, runt related transcription factor-2, collagen-1A1 and 2, interleukin-6 and transforming growth factor-β) involved in cartilage development and remodeling, collagen biosynthesis and degradation, inflammatory response and extracellular matrix homeostasis were predicted as potential targets of the dysregulated miRNAs. Moreover, a large set of putative genes were enriched in OA pathogenesis-associated pathways (such as mitogen-activated protein kinase and vascular endothelial growth factor signaling pathway). Collectively, the data from our study provides novel insight into the ligament injury-related miRNA dysregulation in patients with OA.

Immunohistochemical and immunocytochemical study of mechanoreceptors in anterior cruciate ligament reconstruction with the remnant-preserving technique using Achilles tendon allografts

BackgroundAttempts have been made to validate the significance of remnant preservation with anterior cruciate ligament (ACL) reconstruction using immunohistochemical and immunocytochemical techniques. The purpose of this study was to examine the expression of mechanoreceptors in the remnant tissue of ACL reconstruction performed with the remnant-preserving technique.MethodsTissue samples were obtained from 10 patients who underwent ACL reconstruction with the remnant-preserving technique. The specimens were obtained from remnant ACL tissue and Achilles allografts superficially and at the tibial attachment. The control group consisted of three normal ACLs procured from young males who underwent partial meniscectomy. Tissues and cells from the ACL remnants and Achilles allografts were characterized using hematoxylin and eosin (H&E) staining and immunohistochemical, immunocytochemical, and immunoblotting assays. In particular, the sensitivity of neural cell validation was improved using nerve growth factor (NGF) to stimulate the expression of neural cells.ResultsThe results are summarized as follows. (1) In H&E staining and immunohistochemical assays, no neural cells were detected in remnant or allograft tissue. (2) In the immunocytochemical study, neural cells were detected in remnant tissue. (3) The increased proliferation of remnant ACL cells with NGF treatment suggested their identity as neural cells. (4) NGF treatment also stimulated protein and RNA expression of Nestin (a specific marker for neural cells) in remnant ACL cells.ConclusionsThe improved immunocytochemical methodology proved useful. Although mechanoreceptors were detected relatively less frequently than expected, the authors consider that this finding does not negate the necessity of remnant-preserving ACL reconstruction.

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.