Human Anterior Cruciate Ligament Research Articles

Commitment of human mesenchymal stromal cells towards ACL fibroblast differentiation upon rAAV-mediated FGF-2 and TGF-β overexpression using pNaSS-grafted PCL films.

Despite various clinical options, human anterior cruciate ligament (ACL) lesions do not fully heal. Biomaterial-guided gene therapy using recombinant adeno-associated virus (rAAV) vectors may improve the intrinsic mechanisms of ACL repair. Here, we examined whether poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can deliver rAAV vectors coding for the reparative basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-β) in human mesenchymal stromal cells (hMSCs) as a source of implantable cells in ACL lesions. Efficient and sustained rAAV-mediated reporter (red fluorescent protein) and therapeutic (FGF-2andTGF-β) gene overexpression was achieved in the cells for at least 21 days in particular with pNaSS-grafted PCL films relative to all other conditions (up to 5.2-fold difference). Expression of FGF-2 and TGF-β mediated by rAAV using PCL films increased the levels of cell proliferation, the DNA contents, and the deposition of proteoglycans and of type-I and -III collagen (up to 2.9-fold difference) over time in the cells with higher levels of transcription factor expression (Mohawk, Scleraxis) (up to 1.9-fold difference), without activation of inflammatorytumor necrosis alpha especially when using pNaSS-grafted PCL films compared with the controls. Overall, the effects mediated by TGF-β were higher than those promoted by FGF-2, possibly due to higher levels of gene expression achieved upon rAAV gene transfer. This study shows the potential of using functionalized PCL films to apply rAAV vectors for ACL repair.

High-speed spinning of collagen microfibers comprising aligned fibrils for creating artificial tendons

Bundles of engineered collagen microfibers are promising synthetic tendons as substitutes for autogenous grafts. The purpose of this study was to develop high-speed and continuous spinning of collagen microfibers that involves stretching of collagen stream. Our study revealed the ‘critical fibrillogenesis concentration (CFC)’ of neutralized collagen solutions, which is defined as the upper limit of the collagen concentration at which neutralized collagen molecules remain stable as long as they are cooled ( ⩽ 10 °C). Neutralized collagen solutions at collagen concentrations slightly below the CFC formed cord-like collagen gels comprising longitudinally aligned fibrils when extruded from nozzles into an ethanol bath. Dry collagen microfibers with a controlled diameter ranging from 122 ± 2–31.2 ± 1.7 μm can be spun from the cord-like gels using nozzles of various sizes. The spinning process was improved by including stretching of collagen stream to further reduce diameter and increase linear velocity. We extruded a collagen solution through a 182 μm diameter nozzle while simultaneously stretching it in an ethanol bath during gelation and fiber formation. This process resembles the stretching of a melted thermoplastic resin because it solidifies during melt spinning. The mechanical properties of the stretched collagen microfibers were comparable to the highest literature values obtained using microfluidic wet spinning, as they exhibited longitudinally aligned fibrils both on their surface and in their core. Previous wet spinning methods were unable to generate collagen microfibers with a consistent tendon-like fibrillar arrangement throughout the samples. Although the tangent modulus (137 ± 7 MPa) and stress at break of the swollen bundles of stretched microfibers (13.8 ± 1.9 MPa) were lower than those of human anterior cruciate ligament, they were within the same order of magnitude. We developed a spinning technique that produces narrow collagen microfibers with a tendon-like arrangement that can serve as artificial fiber units for collagen-based synthetic tendons.

Nanosurgical and Bioengineering Treatment of Human Anterior Cruciate Ligament Tears with Ultrasound-Guided Injection of Modified Platelet-Rich Plasma Using Human Cell Memory Based on Clinical, Ultrasound, MRI, and Nanoscope Analyses: A Double-Blind Randomized Trial.

Background: Anterior cruciate ligament (ACL) tears account for 40% to 50% of all ligamentous knee injuries. Most patients with ACL ruptures undergo surgical treatment. There is currently no objective, well-documented, repeatable, and standardized nonsurgical method for ACL tear treatment. This study aimed to investigate ACL outcomes in patients who underwent a novel nanosurgery and bioengineering treatment (NSBT) for an ACL tear. Methods: This was a double-blind randomized trial including 44 patients with a history of traumatic knee injury and a confirmed ACL tear. The final sample comprised 40 patients who met all the eligibility criteria. The patients were divided into two groups: the treatment group (n = 30) and the control group (n = 10). The treatment group underwent nanosurgery with an ultrasound-guided injection of modified platelet-rich plasma (PRP) using human cell memory (RP-hCM). The control group was treated with an ultrasound-guided PRP injection into the joint capsule. At baseline and post-treatment, all patients underwent both ultrasonography and magnetic resonance imaging (MRI), and the following clinical variables were assessed: the WOMAC score, the Lysholm knee score, the visual analog scale score, and knee instability. In most patients, the clinical outcome was verified using nanoscopy. Results: The median WOMAC, VAS, and LKS scores, as well as knee instability, improved significantly 12 weeks after the procedure in the treatment group (p < 0.001). We found a significantly larger improvement in the assessed parameters in the treatment group compared to the control group (p < 0.001). In the treatment group, all the patients had good and very good clinical outcomes, while 90% of the patients had a normal ACL signal in a follow-up MRI scan. In the control group, a physical examination revealed no changes in knee stability after treatment. Conclusions: This study showed that there is a significant difference in patient experience and the duration of recovery for patients with ACL tears treated with NSBT. The novel nonsurgical method was shown to be repeatable, objective, well documented, standardized, and highly effective.

Topographically and Chemically Enhanced Textile Polycaprolactone Scaffolds for Tendon and Ligament Tissue Engineering.

The use of tissue engineering to address the shortcomings of current procedures for tendons and ligaments is promising, but it requires a suitable scaffold that meets various mechanical, degradation-related, scalability-related, and biological requirements. Macroporous textile scaffolds made from appropriate fiber material have the potential to fulfill the first three requirements. This study aimed to investigate the biocompatibility, sterilizability, and functionalizability of a multilayer braided scaffold. These macroporous scaffolds with dimensions similar to those of the human anterior cruciate ligament consist of fibers with appropriate tensile strength and degradation behavior melt-spun from Polycaprolactone (PCL). Two different cross-sectional geometries resulting in significantly different specific surface areas and morphologies were used at the fiber level, and a Chitosan-graft-PCL (CS-g-PCL) surface modification was applied to the melt-spun substrates for the first time. All scaffolds elicited a positive cell response, and the CS-g-PCL modification provided a platform for incorporating functionalization agents such as drug delivery systems for growth factors, which were successfully released in therapeutically effective quantities. The fiber geometry was found to be a variable that could be manipulated to control the amount released. Therefore, scaled, surface-modified textile scaffolds are a versatile technology that can successfully address the complex requirements of tissue engineering for ligaments and tendons, as well as other structures.

Do the Differences in the Epiligament of the Proximal and Distal Parts of the Anterior Cruciate Ligament Explain Their Different Healing Capacities? Quantitative and Immunohistochemical Analysis of CD34 and α-SMA Expression in Relation to the Epiligament Theory.

The aim of this study was to assess the epiligament theory by determining the normal epiligament morphology of the proximal and distal parts of the anterior cruciate ligament in humans and analyzing the differences between them and the midportion of the ligament in terms of cell numbers and expression of CD34 and α-SMA. Samples were obtained from the anterior cruciate ligaments of 12 fresh knee joints. Monoclonal antibodies against CD34 and α-SMA were used for immunohistochemistry. Photomicrographs were analyzed using ImageJ software, version 1.53f. The cell density was higher in the epiligament than in the ligament connective tissue. Cell counts were higher in the proximal and distal thirds than in the midsubstance of the epiligament. CD34 was expressed similarly in the proximal and distal thirds, although it seemed slightly more pronounced in the distal third. α-SMA expression was more robust in the proximal than the distal part. The results revealed that CD34 and α-SMA are expressed in the human epiligament. The differences between the numbers of cells in the proximal and distal parts of the epiligament and the expression of CD34 and α-SMA enhance epiligament theory. Future investigations into improving the quality of ligament healing should not overlook the epiligament theory.

A single-cell atlas depicting the cellular and molecular features in human anterior cruciate ligamental degeneration: A single cell combined spatial transcriptomics study.

To systematically identify cell types in the human ligament, investigate how ligamental cell identities, functions, and interactions participated in the process of ligamental degeneration, and explore the changes of ligamental microenvironment homeostasis in the disease progression. Using single-cell RNA sequencing and spatial RNA sequencing of approximately 49,356 cells, we created a comprehensive cell atlas of healthy and degenerated human anterior cruciate ligaments. We explored the variations of the cell subtypes' spatial distributions and the different processes involved in the disease progression, linked them with the ligamental degeneration process using computational analysis, and verified findings with immunohistochemical and immunofluorescent staining. We identified new fibroblast subgroups that contributed to the disease, mapped out their spatial distribution in the tissue and revealed two dynamic trajectories in the process of the degenerative process. We compared the cellular interactions between different tissue states and identified important signaling pathways that may contribute to the disease. This cell atlas provides the molecular foundation for investigating how ligamental cell identities, biochemical functions, and interactions contributed to the ligamental degeneration process. The discoveries revealed the pathogenesis of ligamental degeneration at the single-cell and spatial level, which is characterized by extracellular matrix remodeling. Our results provide new insights into the control of ligamental degeneration and potential clues to developing novel diagnostic and therapeutic strategies. This study was funded by the National Natural Science Foundation of China (81972123, 82172508, 82372490) and 1.3.5 Project for Disciplines of Excellence of West China Hospital Sichuan University (ZYJC21030, ZY2017301).

Stabilization and Gap Formation of Adjustable Versus Fixed Primary ACL Repair With Internal Brace: An in Vitro Full-Construct Biomechanical Cadaveric Study.

A knotless, tensionable primary anterior cruciate ligament (ACL) repair system preloaded with an internal brace has been released. Currently, there is no biomechanical data on the stabilization and gap formation behavior of the adjustable system when compared with fixed repairs in human ACL tissue. That knotless adjustable suture repair with an internal brace would provide overall higher construct stability and greater load share on the ACL with less gap formation compared with fixed repair. Controlled laboratory study. Human cadaveric knees were utilized for internal braced ACL repair constructs (each group n = 16). Two fixed groups consisting of a single-cinch loop (SCL), cortical button (SCL group), and knotless suture-anchor (anchor group) were compared with an SCL-adjustable loop device (SCL-ALD) group. Testing was performed at 4 different peak loads (50, 150, 250, 350 N) over 4000 cycles at 0.75 Hz including suture repair preconditioning (10 cycles at 0.5 Hz) for SCL-ALD. Specimens were ultimately pulled to failure with a cut internal brace. The final loading situation of the construct and ACL repair with gap formation and ultimate strength were evaluated. Peak elongation at various peak loads showed a significantly higher (P < .001) stabilization of SCL-ALD when compared with both fixed groups. There was a significantly higher (P < .001) load share of SCL-ALD, especially at lower loads (48% of 50 N), and the gap formation remained restricted up to 250 N. With only a little load share on the fixed constructs (<6%) at lower loads (50, 150 N), gap formation in these groups started at a load of 150 N, leading to significantly higher gaps (P < .001). The ultimate failure load for SCL-ALD and anchor groups was significantly increased (P < .001) as compared with SCL. The stiffness of SCL-ALD (62.9 ± 10.6 N/mm) was significantly increased (P < .001). Internal braced knotless adjustable fixation for ACL repair with preconditioning of the suture repaired ligament increased the overall stabilization with higher load share on the ACL and restricted gap formation (<0.5 mm up to 350 N) compared with fixed suture repair. All internal braced repairs restored stability according to native ACL function. Adjustable ACL repair improved the mechanical characteristics and reduced gap formation, but the overall clinical significance on healing remains unclear.

Multiphasic bone-ligament-bone integrated scaffold enhances ligamentization and graft-bone integration after anterior cruciate ligament reconstruction

The escalating prevalence of anterior cruciate ligament (ACL) injuries in sports necessitates innovative strategies for ACL reconstruction. In this study, we propose a multiphasic bone-ligament-bone (BLB) integrated scaffold as a potential solution. The BLB scaffold comprised two polylactic acid (PLA)/deferoxamine (DFO)@mesoporous hydroxyapatite (MHA) thermally induced phase separation (TIPS) scaffolds bridged by silk fibroin (SF)/connective tissue growth factor (CTGF)@Poly(l-lactide-co-ε-caprolactone) (PLCL) nanofiber yarn braided scaffold. This combination mimics the native architecture of the ACL tissue. The mechanical properties of the BLB scaffolds were determined to be compatible with the human ACL. In vitro experiments demonstrated that CTGF induced the expression of ligament-related genes, while TIPS scaffolds loaded with MHA and DFO enhanced the osteogenic-related gene expression of bone marrow stem cells (BMSCs) and promoted the migration and tubular formation of human umbilical vein endothelial cells (HUVECs). In rabbit models, the BLB scaffold efficiently facilitated ligamentization and graft-bone integration processes by providing bioactive substances. The double delivery of DFO and calcium ions by the BLB scaffold synergistically promoted bone regeneration, while CTGF improved collagen formation and ligament healing. Collectively, the findings indicate that the BLB scaffold exhibits substantial promise for ACL reconstruction. Additional investigation and advancement of this scaffold may yield enhanced results in the management of ACL injuries.

Human anterior cruciate ligament-derived mesenchymal stem cells for regenerative medicine applications

Background: The anterior cruciate ligament (ACL) has poor healing capabilities and is the most commonly injured knee ligament. Although ACL repair is being highly studied, the current treatment involves reconstructive surgery utilizing autografts or allografts, which have limitations. The use of Mesenchymal Stem Cells (MSCs) as a possible therapeutic option has grown. ACL-derived MSCs are likely to be the best source because studies have shown that target tissue derived stem cells will better differentiate into the target tissue than the stem cells derived from non-target ones. However, the existing literature discusses only the isolation of a mixed population of MSCs. Here we present the isolation, differentiation and characterization of human ACL-derived MSCs according to the International Society for Cellular Therapy (ISCT) criteria.The ACL tissue was enzymatically digested. Separation of MSCs from the crude mixture of cells was then performed by fluorescence activated cell sorting (FACS) analysis. The isolated population were passaged in specific induction medium to differentiate them into adipocyte, osteocytes and chondrocytes. The cells were then further characterized with respect to their growth curve, population doubling time, colony forming ability, anchorage independent growth, and cell surface markers. The cells were finally examined for their tumorigenic potential by cell cycle analysis.Immunoprofiling via FACSs showed an average isolation rate for cells carrying MSCs markers of 5.5%. Cells exhibited spindle-shaped morphology, and immunocytochemistry confirmed the expression of appropriate cell surface markers. The growth curve showed distinct lag and log phase. Over agar assay demonstrated no anchorage independent growth, but clonogenic potential was observed post-culture on plastic Petri dishes. The cells showed a population doubling time of about 1.5 days. Oil Red O, Alizarin Red S, and Alcian Blue staining confirmed adipogenic, osteogenic and chondrogenic differentiation, respectively. Cell cycle analysis displayed more ACL-derived MSCs in G/G phase compared to BMSCs, showing that the isolates were non-tumorigenic. The presence of MSCs within the human ACL was confirmed via ISCT criteria, paving the way for their potential use for future ACL reconstructions. Although BMSCs have been the choice for regenerative purposes, making use of MSCs derived from ACL ligament will cut down the burden of trauma one has to undergo to obtain the Bone Marrow. Moreover, it is more convenient to harvest MSCs from otherwise discarded ACL. Finally, MSCs derived from the target tissue are believed to better differentiate to the ligament tissue than the bone derived MSCs.

Primary Human Ligament Fibroblast Adhesion and Growth on 3D-Printed Scaffolds for Tissue Engineering Applications

The current gold standard technique for the treatment of anterior cruciate ligament (ACL) injury is reconstruction with a tendon autograft. These treatments have a relatively high failure and re-rupture rate and are associated with early-onset osteoarthritis, developing within two decades of injury. Furthermore, both autografting and allografting come with several drawbacks. Tissue engineering and additive manufacturing present exciting new opportunities to explore 3D scaffolds as graft substitutes. We previously showed that 3D-printed scaffolds using low-cost equipment are suitable for tissue engineering approaches to regenerative medicine. Here, we hypothesize that Lay-Fomm 60, a commercially available nanoporous elastomer, may be a viable tissue engineering candidate for an ACL graft substitute. We first printed nanoporous thermoplastic elastomer scaffolds using low-cost desktop 3D printers and determined the mechanical and morphological properties. We then tested the impact of different surface coatings on primary human ACL fibroblast adhesion, growth, and ligamentous matrix deposition in vitro. Our data suggest that poly-L-lysine-coated Lay-Fomm 60 scaffolds increased ligament fibroblast activity and matrix formation when compared to uncoated scaffolds but did not have a significant effect on cell attachment and proliferation. Therefore, uncoated 3D printed Lay-Fomm 60 scaffolds may be viable standalone scaffolds and warrant further research as ligament tissue engineering and reconstruction grafts.

BMP-2/TGF-β1 gene insertion into ligament-derived stem cells sheet promotes tendon-bone healing in a mouse.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)-derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP-2/TGF-β1 on the differentiation of LDSC. We developed a BMP-2/TGF-β1 gene insertion into an LDSC cell sheet that promotes tendon-bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP-2 or TGF-β1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP-2-, TGF-β1-, and BMP-2/TGF-β1-transduced. At 4 and 8weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-β1-transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon-bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP-2/TGF-β1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-β1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon-bone healing after ACLR using autografts wrapped with cell sheets in a mouse model.

Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

Comparison of the morphology of the anterior cruciate ligament and related bony structures between pigs and humans.

Pigs are widely used for clinical research on the anterior cruciate ligament (ACL) because of the similarity of the knee structure to the human knee. But evidence to support the suitability of using porcine samples to guide clinical practices is limited. This study aims to explore the qualitative and quantitative morphological features of the porcine knee and ACL, and to compare these with data on humans reported in literature. Nineteen porcine knees were used for this study. The bone structures were measured on coronal X-ray images. The length of the ACL was measured using a caliper. The ACL bone insertion sites were marked and measured on a digital photograph. The lengths of the long and short axis of the ACL isthmus were measured on the X-ray microscopy reconstructed images. The outcomes were compared with previously reported data on humans using an abstract independent-samples T test. Qualitative observation indicated a similar location, orientation and general morphology of the porcine ACL to human ACLs. The major difference was the location of the ACL tibial insertion with respect to the anterior horn of the lateral meniscus (AHLM). The porcine ACL was split into AM and PL bundles by the AHLM, while the AHLM was adjacent to the anterolateral border of the ACL tibial insertion in human knees. The quantitative comparison showed no significant difference between the human and porcine ACL in terms of the length of the ACL, the width of the femoral condyle and tibial plateau, and the tibial interspinal width. However, the CSA, the lengths of the long and short axis of the ACL isthmus, and the femoral and tibial insertion areas of the porcine ACL were all significantly larger than the reported features in human knees. The location, orientation and basic morphology of the porcine ACL and knee are similar to humans. However, the two-bundle structure is more distinct in a porcine ACL, and the dimensions of the porcine ACL are generally larger. This study may provide useful information to researchers when assessing the feasibility and limitations of using porcine samples for research on the human ACL and knee.

Continuous Two-Zone In Vitro Co-culture Model of the Enthesis

The enthesis is the interfacial tissue between ligament or tendon, and bone, which connects tissues of distinctly different mechanical properties. Although ligament and enthesis injury is commonplace, the development and healing mechanisms of these tissues are yet unclear. Current models for investigating these mechanisms are primarily in vivo animal models as in vitro models have been limited in their structural and mechanical biomimicry of the native enthesis. In this study, an in vitro enthesis model was developed using a modified gel aspiration-ejection method. Continuous two-zone aligned dense collagen (ADC) hydrogels with an overlapping interface were fabricated within 2 h. The mechanical properties of acellular two-zone ADC confirmed the continuous nature of this model, as the mechanical properties showed no significant difference compared to single-zone ADC and maintained comparable structural and mechanical characteristics of immature ligaments and unmineralized bone. Human anterior cruciate ligament fibroblasts and human spine vertebral osteoblasts were isolated from donor tissues and were seeded to form the enthesis model. These were cultured for 14 days, at which the viability and proliferation was observed to be 85 ± 7.5% and 230 ± 52%, respectively. Histological and immunofluorescence analyses at day 14 revealed extensive cell-driven matrix remodelling, and the seeded fibroblasts and osteoblasts maintained their phenotype within their compartments of the layered co-culture model. These results demonstrate the rapid fabrication of a two-zone co-culture system that can be utilized as an in vitro model to further understand the degenerative and regenerative mechanisms within the enthesis.

An off-the-shelf decellularized and sterilized human bone-ACL-bone allograft for anterior cruciate ligament reconstruction

Approximately 1% of active individuals participating in sports rupture their anterior cruciate ligaments (ACL) every year, which is currently reconstructed using tendon autografts. Upon reconstruction, clinical issues of concern are ACL graft rupture, persistent knee instability, limited return to sports, and early onset of osteoarthritis (OA). This happens because tendon autografts do not have the same compositional, structural, and mechanical properties as a native ACL. To overcome these problems, we propose to use decellularized bone-ACL-bone allografts in ACL reconstruction (ACLR) as a mechanically robust, biocompatible, and immunologically safe alternative to autografts. Here, a decellularization protocol combined with sterilization using supercritical carbon dioxide (scCO2) was used to thoroughly decellularize porcine and human ACLs attached to tibial and femoral bone blocks. The specimens were named ultrACLean and their compositional, structural, and mechanical properties were determined. Our results indicate that: 1) decellularization of ultrACLean allografts leads to the removal of nearly 97% of donor cells, 2) ultrACLean has mechanical properties which are not different to native ACL, 3) ultrACLean maintained similar collagen content and decreased GAG content compared to native ACL, and 4) ultrACLean is not cytotoxic to seeded tendon-derived cells in vitro. Results from an in vivo pilot experiment showed that ultrACLean is biocompatible and elicits a moderate immunological response. In summary, ultrACLean has proven to be a mechanically competent and biocompatible graft with the potential to be used in ACLR surgery.

Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ

PurposeCertain types of repetitive sub-maximal knee loading cause microfatigue damage in the human anterior cruciate ligament (ACL) that can accumulate to produce macroscopic tissue failure. However, monitoring the progression of that ACL microfatigue damage as a function of loading cycles has not been reported. To explore the fatigue process, a confocal laser endomicroscope (CLEM) was employed to capture sub-micron resolution fluorescence images of the tissue in situ. The goal of this study was to quantify the in situ changes in ACL autofluorescence (AF) signal intensity and collagen microstructure as a function of the number of loading cycles.MethodsThree paired and four single cadaveric knees were subjected to a repeated 4 times bodyweight landing maneuver known to strain the ACL. The paired knees were used to compare the development of ACL microfatigue damage on the loaded knee after 100 consecutive loading cycles, relative to the contralateral unloaded control knee, through second harmonic generation (SHG) and AF imaging using confocal microscopy (CM). The four single knees were used for monitoring progressive ACL microfatigue damage development by AF imaging using CLEM.ResultsThe loaded knees from each pair exhibited a statistically significant increase in AF signal intensity and decrease in SHG signal intensity as compared to the contralateral control knees. Additionally, the anisotropy of the collagen fibers in the loaded knees increased as indicated by the reduced coherency coefficient. Two out of the four single knee ACLs failed during fatigue loading, and they exhibited an order of magnitude higher increase in autofluorescence intensity per loading cycle as compared to the intact knees. Of the three regions of the ACL - proximal, midsubstance and distal - the proximal region of ACL fibers exhibited the highest AF intensity change and anisotropy of fibers.ConclusionsCLEM can capture changes in ACL AF and collagen microstructures in situ during and after microfatigue damage development. Results suggest a large increase in AF may occur in the final few cycles immediately prior to or at failure, representing a greater plastic deformation of the tissue. This reinforces the argument that existing microfatigue damage can accumulate to induce bulk mechanical failure in ACL injuries. The variation in fiber organization changes in the ACL regions with application of load is consistent with the known differences in loading distribution at the ACL femoral enthesis.

First detection of primary cilia in injured human anterior cruciate ligament: A pilot study with pathophysiological reflections

The anterior cruciate ligament (ACL) plays a significant role in knee stability, protects the joint under multiple loading conditions and shows complex biomechanics. Beside mechanical stability, the ACL seems to play a crucial role in proprioception, and it is well known, that ACL injuries can cause functional deficits due to decreased proprioception. However, the mechanism of proprioception is not completely understood yet. In this context, primary cilia (PC), which play a significant role in the signaling between the intra- and extracellular space, could be of interest. However, until today, primary cilia are not yet described in human ACL. In total, seven human ACL’s underwent transmission electron microscopical examination. Three cadaveric ACL’s and four freshly injured ACL’s were examined. Single cells of each ACL were examined regarding the presence of axonemes or basal bodies, which represent components of a PC. In total, 276 cells of the cadaveric ACL’s and 180 cells of the injured ACL’s were examined. Basal bodies could be detected in three of the four specimens of the injured ACL’s as well as in one of the three cadaveric ACL’s, resulting in a mean positivity of 2.54% in the cadaveric group and 2.78% in the injured group. In case of PC-presence, only one PC per cell could be detected. No statistically significant difference regarding the frequency could be detected between both groups. In this pilot-study, we present for the first time an ultrastructural study of human ACLs with respect to the occurrence of PC and any structural and morphological features of these complex and dynamic cell organelles. PCs are present in almost all non-hematopoietic tissues of the human body. However, there are different reports on the number, incidence, orientation, and morphology of these cell organelles in the respective tissues. Compared to other tissues and ligaments of other species, we found a significantly lower rate of PC positive cells. This observation might represent a tissue-specific characteristic of ACL tissue. However, our observations need to be explored in more detail in further studies.

The influence of ruptured scar pattern of human anterior cruciate ligament remnant tissue on tendon-bone healing in vivo.

The purpose of this study was to determine whether the transplantation of human cells from a non-reattached injured anterior cruciate ligament (ACL) remnant could enhance tendon-bone healing. Human ACL remnant tissue was classified into two groups based on the morphologic pattern as per Crain's classification: (1) non-reattachment group (Crain Ⅳ) and (2) reattachment group (Crain Ⅰ-Ⅲ). Seventy-five 10-week-old immunodeficient rats underwent ACL reconstruction followed by intracapsular administration of one of the following: (1) ACL-derived cells from the non-reattached remnant (non-reattachment group) (n = 5), (2) ACL-derived cells from the reattached tissue (reattachment group) (n = 5), or (3) phosphate-buffered saline(PBS) only (PBS group) (n = 5). Histological (Weeks 2, 4, and 8), immunohistochemical (Week 2), radiographic (Weeks 0, 2, 4, and 8), and biomechanical (Week 8) assessments were performed. Histological evaluation showed high and early healing, induction of endochondral ossification-like integration, and mature bone ingrowth at Week 4 in the non-reattachment group. Microcomputed tomography at Week 4 showed that the tibial bone tunnels in the non-reattachment group were significantly reduced compared to those in the reattachment and PBS groups. Moreover, biomechanical testing showed that ultimate load-to-failure in the non-reattachment group tended to be larger than that in the reattachment group, though not statistically significant. The enhanced healing potential in the non-reattachment group was explained by the increase in intrinsic angiogenesis/osteogenesis. In the subacute phase, the ACL-derived cells with the non-reattached morphologic pattern showed greater and earlier tendon bone healing compared with the cells obtained from the reattached morphologic pattern.

Differential regional perfusion of the human anterior cruciate ligament: quantitative magnetic resonance imaging assessment

PurposeSurgical reconstruction is the current standard for ACL rupture treatment in active individuals. Recently, there is renewed interest in primary repair of proximal ACL tears. Despite this, ACL biology and healing potential are currently not well understood. Vascularity is paramount in ACL healing; however, previous ACL vascularity studies have been limited to qualitative histological and dissection-based techniques. The study objective was to use contrast-enhanced quantitative-MRI to compare relative perfusion of proximal, middle, and distal thirds of the in situ ACL. We hypothesized perfusion would be greatest in the proximal third.MethodsFourteen cadaveric knees were studied (8 females, 6 males), age 25–61 years. Superficial femoral, anterior tibial, and posterior tibial arteries were cannulated; without intraarticular dissection. Contrast-enhanced quantitative-MRI was performed using a previously established protocol. ACL regions corresponding to proximal, middle, and distal thirds were identified on sagittal-oblique pre-contrast images. Signal enhancement (normalized to tibial plateau cartilage) was quantified to represent regional perfusion as a percentage of total ACL perfusion. Comparative statistics were computed using repeated measures ANOVA, and pairwise comparisons performed using the Bonferroni method.ResultsRelative perfusion to proximal, middle, and distal ACL zones were 56.0% ±17.4%, 28.2% ±14.6%, and 15.8% ±16.3%, respectively (p = 0.002). Relative perfusion to the proximal third was significantly greater than middle (p = 0.007) and distal (p = 0.001). No statistically relevant difference in relative perfusion was found to middle and distal thirds (p = 0.281). Post-hoc subgroup analysis demonstrated greater proximal perfusion in males (66.9% ± 17.3%) than females (47.8% ± 13.0%), p = 0.036.ConclusionUsing quantitative-MRI, in situ adult ACL demonstrated greatest relative perfusion to the proximal third, nearly 2 times greater than the middle third and 3 times greater than the distal third. Knowledge of differential ACL vascular supply is important for understanding pathogenesis of ACL injury and the process of biological healing following various forms of surgical treatment.

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways

Exceeded mechanical stress leads to a sublethal injury to anterior cruciate ligament (ACL) fibroblasts, and it will hinder cell mobility and ACL regeneration, and even induce osteoarthritis. The mechano growth factor (MGF) could be responsible for mechanical stress and weakening its negative effects on cell physiological behaviors. In this study, effects of MGF on cell mobility and relevant molecules expression in injured ACL fibroblasts were detected. After an injurious mechanical stretch, the analysis carried out, at 0 and 24 h, respectively, showed that the cell area, roundness, migration, and adhesion of ACL fibroblasts were reduced. MGF (10, 100 ng/mL) treatment could improve cell area, roundness and promote cell migration and adhesion capacity compared with the injured group without MGF. Further study indicated that cell mobility-relevant molecules (PAK1/2, Cdc42, Rac1, RhoA, and ROCK1) expression in ACL fibroblasts was down-regulated at 0 or 24 h after injurious stretch (except Rac1 and RhoA at 0 h). Similarly, MGF improved cell mobility-relevant molecule expression, especially the ROCK1 expression level in ACL fibroblasts at 0 or 24 h after injurious stretch. Protein expression of ROCK1 in injured ACL fibroblasts was also reduced and could be recovered by MGF treatment. In a rabbit partial ACL transection (ACLT) model, ACL exhibited poor regenerative capacity in collagen and extracellular matrix (ECM) synthesis after partial ACLT for 2 or 4 weeks, and MGF remarkably accelerated ACL regeneration and restored its mechanical loading capacity after partial ACLT for four weeks. Our findings suggest that MGF weakens the effects of pathological stress on cell mobility of ACL fibroblasts and accelerates ACL repair, and might be applied as a future treatment approach to ACL rupture in the clinic.