Porcine Meniscus Research Articles

Human mesenchymal stem/stromal cell-derived extracellular vesicle transport in meniscus fibrocartilage.

Extracellular vesicles (EVs) derived from endometrial-derived mesenchymal stem/stromal cells (eMSC) play a crucial role in tissue repair due to their immunomodulatory and reparative properties. Given these properties, eMSC EVs may offer potential benefits for meniscal repair. The meniscus, being partly vascularized, relies on diffusivity for solute trafficking. This study focuses on EVs transport properties characterization within fibrocartilage that remains unknown. Specifically, EVs were isolated from Crude and CD146+ eMSC populations. Green fluorescence-labeled EVs transport properties were investigated in three structurally distinct layers (core, femoral, and tibial surfaces) of porcine meniscus. Diffusivity was measured via custom fluorescence recovery after photobleaching (FRAP) technique. Light spectrometry was used to determine EVs solubility. Both Crude and CD146+ eMSC EVs exhibited high purity (>90% CD63CD9 marker expression) and an average diffusivity of 10.924 (±4.065) µm²/s. Importantly, no significant difference was observed between Crude and CD146+ eMSC EV diffusivity on the meniscal layer (p > 0.05). The mean partitioning coefficient was 0.2118 (±0.1321), with Crude EVs demonstrating significantly higher solubility than CD146+ EVs (p < 0.05). In conclusion, this study underscores the potential of both Crude and CD146+ eMSC EVs to traverse all layers of the meniscus, supporting their capacity to enhance delivery of orthobiologics for cartilaginous tissue healing.

Development of a porcine decellularized extracellular matrix (DECM) bioink for 3D bioprinting of meniscus tissue engineering: formulation, characterisation and biological evaluation

ABSTRACT This study aims to present an easily scalable, cost-efficient process of dECM extraction from porcine meniscus, dedicated to bioink preparation and 3D bioprinting. Due to its cartilage like structure and mechanical robustness, the meniscus is an exceptionally demanding tissue for extraction and decellularisation of its ECM. Its processing poses a great difficulty and renders the methods previously developed for soft tissues useless. A process, combining homogenisation, hydrolysis, supercritical CO2 (scCO2) extraction and lyophilisation, was developed to meet this challenge. This protocol allows for retaining its native compounds and biocompatibility while offering good printability and providing a stimulatory environment for cell proliferation and differentiation towards a meniscus-like phenotype. Also, this process is economically and ecologically friendly since it doesn't require the use of high amounts of solvents, detergents or expensive enzymes (DNase). The decellularisation process has been meticulously studied, demonstrating a substantial reduction in DNA content but still exceeding accepted thresholds. The study further explores the biocompatibility of the dECM, demonstrating no detrimental effects of remnant DNA on cell survival during extended in vitro culture, indicating excellent biocompatibility. These findings challenge the current definition of decellularisation effectiveness based solely on DNA content, proposing a broader assessment of biological effects.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Age and anatomical region-related differences in vascularization of the porcine meniscus using microcomputed tomography imaging.

Meniscal lesions in vascularized regions are known to regenerate while lack of vascular supply leads to poor healing. Here, we developed and validated a novel methodology for three-dimensional structural analysis of meniscal vascular structures with high-resolution microcomputed tomography (µCT). We collected porcine medial menisci from 10 neonatal (not-developed meniscus, n-) and 10 adults (fully developed meniscus, a-). The menisci were cut into anatomical regions (anterior horn (n-AH anda-AH), central body (n-CB anda-CB), and posterior horn (n-PH anda-PH). Specimens were cut in half, fixed, and one specimen underwent critical point drying and µCT imaging, while other specimen underwent immunohistochemistry and vascularity biomarker CD31 staining for validation of µCT. Parameters describing vascular structures were calculated from µCT. The vascular network in neonatal spread throughout meniscus, while in adult was limited to a few vessels in outer region, mostly on femoral side. n-AH, n-CB, and n-PH had 20, 17, and 11 times greater vascular volume fraction than adult, respectively. Moreover, thickness of blood vessels, in three regions, was six times higher in adults than in neonatal. a-PH appeared to have higher vascular fraction, longer and thicker blood vessels than both a-AH and a-CB. Overall, neonatal regions had a higher number of blood vessels, more branching, and higher tortuosity compared to adult regions. For the first time, critical point drying-based µCT imaging allowed detailed three-dimensional visualization and quantitative analysis of vascularized meniscal structures. We showed more vascularity in neonatal menisci, while adult menisci had fewer and thicker vascularity especially limited to the femoral surface.

Assessing the role of surface layer and molecular probe size in diffusion within meniscus tissue.

Diffusion within extracellular matrix is essential to deliver nutrients and larger metabolites to the avascular region of the meniscus. It is well known that both structure and composition of the meniscus vary across its regions; therefore, it is crucial to fully understand how the heterogenous meniscal architecture affects its diffusive properties. The objective of this study was to investigate the effect of meniscal region (core tissue, femoral, and tibial surface layers) and molecular weight on the diffusivity of several molecules in porcine meniscus. Tissue samples were harvested from the central area of porcine lateral menisci. Diffusivity of fluorescein (MW 332 Da) and three fluorescence-labeled dextrans (MW 3k, 40k, and 150k Da) was measured via fluorescence recovery after photobleaching. Diffusivity was affected by molecular size, decreasing as the Stokes' radius of the solute increased. There was no significant effect of meniscal region on diffusivity for fluorescein, 3k and 40k dextrans (p>0.05). However, region did significantly affect the diffusivity of 150k Dextran, with that in the tibial surface layer being larger than in the core region (p = 0.001). Our findings contribute novel knowledge concerning the transport properties of the meniscus fibrocartilage. This data can be used to advance the understanding of tissue pathophysiology and explore effective approaches for tissue restoration.

High angular resolution diffusion imaging (HARDI) of porcine menisci: a comparison of diffusion tensor imaging and generalized q-sampling imaging.

Diffusion magnetic resonance imaging (MRI) allows for the quantification of water diffusion properties in soft tissues. The goal of this study was to characterize the 3D collagen fiber network in the porcine meniscus using high angular resolution diffusion imaging (HARDI) acquisition with both diffusion tensor imaging (DTI) and generalized q-sampling imaging (GQI). Porcine menisci (n=7) were scanned ex vivo using a three-dimensional (3D) HARDI spin-echo pulse sequence with an isotropic resolution of 500 µm at 7.0 Tesla. Both DTI and GQI reconstruction techniques were used to quantify the collagen fiber alignment and visualize the complex collagen network of the meniscus. The MRI findings were validated with conventional histology. DTI and GQI exhibited distinct fiber orientation maps in the meniscus using the same HARDI acquisition. We found that crossing fibers were only resolved with GQI, demonstrating the advantage of GQI over DTI to visualize the complex collagen fiber orientation in the meniscus. Furthermore, the MRI findings were consistent with conventional histology. HARDI acquisition with GQI reconstruction more accurately resolves the complex 3D collagen architecture of the meniscus compared to DTI reconstruction. In the future, these technologies have the potential to nondestructively assess both normal and abnormal meniscal structure.

Methods for Testing Meniscal Repair Using a 3D-Printed Meniscus

Torn and damaged menisci resulting from trauma are very common knee injuries, which can cause pain and mobility limitations and lead to osteoarthritis. Meniscal injuries can require surgery to repair the tissue damage and restore mobility. Here we describe the biomechanical testing of a 3D-printed meniscus to illustrate methods to determine if it has the strength and durability to effectively repair meniscal tears and restore knee biomechanics. This work was designed to demonstrate the steps needed to test novel meniscus repair devices prior to moving toward animal testing. The first testing step determined the ability of the 3D-printed meniscus to withstand surgical fixation by measuring the suture pull-out force. We show that vertical 2/0 silk or Fiberwire sutures need an average of 1.4 or 1.8 N, respectively, to pull through the meniscus, while horizontal sutures need only 0.7 and 1.2 N, respectively. The next step measured the compressive strength of normal, damaged, and repaired porcine meniscus tissue. Here, we show that meniscectomy decreased the stiffness of meniscus tissue from 26.7 ± 0.85 N to 7.43 ± 0.81 N at 25% strain. Menisci repaired with the 3D-printed tissue restored 66% of the measured force at 25% strain. The final step measured the contact pressures and areas in an ex vivo porcine knee before and after meniscal repair was made with the 3D-printed meniscus tissue. The example 3D-printed meniscus was successfully sutured into the porcine knee joint but failed to restore normal knee contact pressures. This work demonstrates the need for an iterative biomechanical testing process of biomaterial development, 3D-printing optimization, and knee kinematics to develop a durable and functional meniscus repair device. In summary, the methods described here serve as a guide for the functional evaluation of novel meniscus repair devices.

Inhibiting T-Cell-Mediated Rejection of the Porcine Meniscus Through Freeze-Thawing and Downregulating Porcine Xenoreactive Antigen Genes.

Immune rejection presents a significant challenge in xenogenic meniscal transplantation. Pigs are widely regarded as an advantageous tissue source for such transplants, with porcine GGTA1, CMAH, and B4GALNT2 being among the most common xenoreactive antigen (Ag) genes. While some studies have suggested that allogeneic meniscus (AM) transplants may exhibit immunoprivileged properties, our study observed slight immunological rejection has been observed following contact between human meniscal cells (HMCs) and human peripheral blood mononuclear cells (PBMCs). Given the limited systematic research on immune responses following xenograft meniscus transplantation, we established porcine meniscus transplantation (PMT) models to comprehensively assess the immunogenicity of porcine meniscus (PM) from both innate and adaptive immune perspectives. Our investigations confirmed that PMT beneath the epidermis led to innate cell infiltration into the xenografts and T-cell activation in local lymph nodes. T-cell activation upregulated the interleukin (IL)-17 signaling pathway, disrupting collagen organization and metabolic processes, thereby hindering PM regeneration. Using freeze-thaw treatment on PM alleviated T-cell activation post-transplantation by eliminating xenogenic DNA. In vitro findings demonstrated that gene editing in porcine meniscal cells (PMCs) suppressed human T-cell activation by downregulating the expression of xenoreactive Ag genes. These results suggest that GGTA1/CMAH/B4GALNT2 knockout (KO) pigs hold significant promise for advancing the field of meniscal transplantation.

Assessment of Surrogate Models for Research on Resistance and Deformation of Repairs of the Human Meniscal Roots: Porcine or Older Human Models?

Meniscal root repair is not routinely recommended for patients over 75 years old, yet surrogate age-unrestricted human or porcine models are used for its evaluation. This study assesses the suitability of older human or porcine meniscus models for in vitro testing of the sutured meniscal horn. Three groups of menisci underwent a load-to-failure test with continuous monitoring of the traction force and deformation around the suture: human &lt; 75 years, human ≥ 75 years, and porcine. Both surrogate models were compared to the younger group. The porcine group exhibited a 172.1%-higher traction force before tearing (p &lt; 0.001) and a 174.1%-higher ultimate force (p &lt; 0.001), without there being differences between the human groups. At tissue level, the older group had a 28.7%-lower cut-out stress (p = 0.012) and the porcine group had a 57.2%-higher stress (p &lt; 0.001). Regarding elasticity at the sutured area, a 48.1%-greater deformation rate was observed in the older group (p &lt; 0.001), without difference for the porcine group. In conclusion, neither the porcine nor the older human model demonstrated a clear advantage as a surrogate model for young human sutured meniscal horns. The older human meniscus is preferable for resistance at the specimen level, while the porcine model better represents deformation in the sutured zone.

Biomechanical properties of porcine meniscus as determined via AFM: Effect of region, compartment and anisotropy.

The meniscus is a fibrocartilaginous tissue that plays an essential role in load transmission, lubrication, and stabilization of the knee. Loss of meniscus function, through degeneration or trauma, can lead to osteoarthritis in the underlying articular cartilage. To perform its crucial function, the meniscus extracellular matrix has a particular organization, including collagen fiber bundles running circumferentially, allowing the tissue to withstand tensile hoop stresses developed during axial loading. Given its critical role in preserving the health of the knee, better understanding structure-function relations of the biomechanical properties of the meniscus is critical. The main objective of this study was to measure the compressive modulus of porcine meniscus using Atomic Force Microscopy (AFM); the effects of three key factors were investigated: direction (axial, circumferential), compartment (medial, lateral) and region (inner, outer). Porcine menisci were prepared in 8 groups (= 2 directions x 2 compartments x 2 regions) with n = 9 per group. A custom AFM was used to obtain force-indentation curves, which were then curve-fit with the Hertz model to determine the tissue's compressive modulus. The compressive modulus ranged from 0.75 to 4.00 MPa across the 8 groups, with an averaged value of 2.04±0.86MPa. Only direction had a significant effect on meniscus compressive modulus (circumferential > axial, p = 0.024), in agreement with earlier studies demonstrating that mechanical properties in the tissue are anisotropic. This behavior is likely the result of the particular collagen fiber arrangement in the tissue and plays a key role in load transmission capability. This study provides important information on the micromechanical properties of the meniscus, which is crucial for understanding tissue pathophysiology, as well as for developing novel treatments for tissue repair.

Comparison of adhesion of thawed and cultured synovial mesenchymal stem cells to the porcine meniscus and the relevance of cell surface microspikes

BackgroundPlacement of a cultured synovial mesenchymal stem cell (MSC) suspension on a repaired meniscus for 10 min accelerated meniscus repair. Upon placement of the MSC suspension on the meniscus, microspikes projecting from the MSC surface trap meniscus fibers and promote MSC adhesion. Thawed cryopreserved MSCs are preferred materials for meniscus repair, as they can be transplanted without additional culture. However, the adhesion ability of thawed cryopreserved MSCs is unknown. Here, we compared the proportion of cultured versus thawed MSCs adhering to a porcine meniscus immediately and 10 min after placement. We also investigated the relationship between adhesion and the number of microspikes on the synovial MSCs.MethodsSynovial MSCs were prepared from the knees of four donors with osteoarthritis. The “cultured MSCs” were thawed MSCs that were re-cultured and suspended in PBS for transplantation. A similarly prepared suspension was cryopreserved, thawed again, suspended in PBS, and used without further culture as the “thawed MSCs.” MSCs with at least three microspikes in SEM images were defined as microspike-positive MSCs. Porcine meniscus surfaces were abraded, cut into a cylindrical shape, and treated with MSC suspension. Non-adherent cells were counted immediately and again 10 min after placement to calculate the adhesion proportion.ResultsThe proportion of microspike-positive MSCs was significantly higher in thawed (53 ± 3%) than in cultured (28 ± 5%) MSC suspensions. MSC adhesion to the meniscus was significantly better for the thawed than for the cultured MSC suspensions immediately after placement on the meniscus, but no differences were detected after 10 min. The proportion of MSCs with microspikes in the cell suspension was significantly correlated with the proportion of adhered MSCs immediately after the placement, but not 10 min later. Addition of FBS to the cryopreservation medium promoted a concentration-dependent increase in the proportion of microspike-positive cells.ConclusionsThawed MSCs adhered better than cultured MSCs immediately after placement, but adhesion was similar for both MSC preparations after 10 min. Immediately after placement, the presence of microspikes was associated with better adhesion of synovial MSCs to the meniscus.

Cyclic freeze–thaw grinding to decellularize meniscus for fabricating porous, elastic scaffolds

Decellularized meniscus extracellular matrix (dmECM)-based biological scaffolds in the forms of sponge, hydrogel, nanofiber, and composite have gained increasing interest in meniscus tissue engineering and regeneration. A common shortcoming of those scaffolds is insufficient mechanical strength and poor elasticity. Herein, we report a practicable protocol for milder meniscus decellularization to prepare elastic, porous dmECM scaffolds. Porcine meniscus was pulverized by cyclic freeze-thaw grinding and then treated with DNase to obtain fine dmECM particles. Individual dmECM particles were condensed to bulk preparation by centrifuge, followed by lyophilization to form blocks, and finally crosslinked by dehydrothermal treatment to obtain porous dmECM scaffolds. Our results show that the freeze-thaw grinding method was effective in removing cellular DNA with good retention of meniscus-derived bioactive components. The dmECM scaffold had porous structure with interconnected mesopores and good mechanical properties. Primary articular chondrocytes proliferated robustly and maintained chondrogenic characteristics and produce abundant collagen on dmECM scaffolds. Evaluation of biocompatibility in a rat model shows that the dmECM scaffold elicited minor foreign body reactions, indicating effective antigen removal from dmECM. This study provides an alternative for preparing dmECM and fabricating porous scaffolds for meniscus repair and regeneration.

Real-time stereo reconstruction of intraoperative scene and registration to preoperative 3D models for augmenting surgeons' view during RAMIS.

During minimally invansive surgery (MIS) procedures, there exists an ever-growing/apparent need for providing computer generated visual feedback to the surgeon(s), through a visualization device. While multiple solutions have been proposed in the literature, there is limited evidence of such a system performing reliably in practice, and when it does, it is often tailored to a specific operation type. Another important aspect is regarding the usability of such systems, which typically include complicated and time-consuming steps, and often require the assistance of specialized personnel. In this study, we propose an auxiliary visualization system for surgeons, which includes streamlined process to use preoperative data of the patient, and apply it to two different MIS cases, namely, robot-assisted partial nephrectomy and robot-assisted partial lateral meniscectomy. The visualization and processing pipeline consists of an intraoperative 3D reconstruction of the surgical area, using an optimized version of the quasi-dense method, aimed to perform with good accuracy while maintaining real-time speed. A set of preprocessing and postprocessing techniques further contribute to the result by providing a smoother and more dense point cloud. DynamicFusion is used for the registration of the preoperative model to the intraoperative scene. Two silicon kidney phantoms and an ex-vivo porcine meniscus are used for evaluation, representing subjects for the examined surgicalcases. Performance is evaluated qualitatively using the two datasets. The preoperative model of the subject is projected on top of the actual 2D image and also in 3D space. The model is superimposed on top of the actual physical structure it represents, and remains in the correct position throughout the experiments, even when abrupt camera movements are taking place. Finally, when deformation is introduced, the model is deformed as well, resembling the real subject's structure. Results demonstrate and validate the use of the presented algorithms for each separate task of the pipeline. A complete methodology to provide surgeon(s) with visual information during surgery is presented. Its operation is evaluated over two different surgical scenarios, paving the way for a single visualization methodology that can adapt and perform robustly for multiple cases, with minimaleffort.

Experiments and hyperelastic modeling of porcine meniscus show heterogeneity at high strains.

Constitutive modeling of the meniscus is critical in areas like knee surgery and tissue engineering. At low strain rates, the meniscus can be described using a hyperelastic model. Calibration of hyperelastic material models of the meniscus is challenging on many fronts due to material variability and friction. In this study, we present a framework to determine the hyperelastic material parameters of porcine meniscus (and similar soft tissues) using no-slip uniaxial compression experiments. Because of the nonhomogeneous deformation in the specimens, a finite element solution is required at each step of the iterative calibration process. We employ a Bayesian calibration approach to account for the inherent material variability and a Bayesian optimization approach to minimize the resulting cost function in the material parameter space. Cylindrical specimens of porcine meniscus from the anterior, middle and posterior regions are tested up to 30% compressive strain and the Yeoh form of hyperelastic strain energy density function is used to describe the material response. The results show that the Yeoh form is able to accurately describe the compressive response of porcine meniscus and that the Bayesian calibration and optimization approaches are able to calibrate the model in a computationally efficient manner while taking into account the inherent material variability. The results also show that the shear modulus or the initial stiffness is roughly uniform across the different areas of the meniscus, but there is significant spatial heterogeneity in the response at high strains. In particular, the middle region is considerably stiffer at high strains. This heterogeneity is important to consider in modeling the response of the meniscus for clinical applications.

Tractography of Porcine Meniscus Microstructure Using High-Resolution Diffusion Magnetic Resonance Imaging.

To noninvasively evaluate the three-dimensional collagen fiber architecture of porcine meniscus using diffusion MRI, meniscal specimens were scanned using a 3D diffusion-weighted spin-echo pulse sequence at 7.0 T. The collagen fiber alignment was revealed in each voxel and the complex 3D collagen network was visualized for the entire meniscus using tractography. The proposed automatic segmentation methods divided the whole meniscus to different zones (Red-Red, Red-White, and White-White) and different parts (anterior, body, and posterior). The diffusion tensor imaging (DTI) metrics were quantified based on the segmentation results. The heatmap was generated to investigate the connections among different regions of meniscus. Strong zonal-dependent diffusion properties were demonstrated by DTI metrics. The fractional anisotropy (FA) value increased from 0.13 (White-White zone) to 0.26 (Red-Red zone) and the radial diffusivity (RD) value changed from 1.0 × 10-3 mm2/s (White-White zone) to 0.7 × 10-3 mm2/s (Red-Red zone). Coexistence of both radial and circumferential collagen fibers in the meniscus was evident by diffusion tractography. Weak connections were found between White-White zone and Red-Red zone in each part of the meniscus. The anterior part and posterior part were less connected, while the body part showed high connections to both anterior part and posterior part. The tractography based on diffusion MRI may provide a complementary method to study the integrity of meniscus and nondestructively visualize the 3D collagen fiber architecture.

Shock Response of Porcine Meniscus along with Axial and Radial Directions

The meniscus is a multifunctional fibrocartilage tissue in the knee joint which stables joint movement, bears load and absorbs impact. Improper collisions will cause damage to meniscus tissue and lose its original functionality. However, it is difficult to fully evaluate the mechanical properties of the meniscus based on static test results alone. In this study, Split Hopkinson Pressure Bar (SHPB) and hydraulic material testing system (MTS) were utilized to examine the quasi-static and dynamic properties of the porcine meniscus along with two different orientations. The results showed that the meniscus is a strain rate sensitive material and its mechanical properties mainly depend on the orientation of collagen fiber bundles in the peripheral direction. The meniscus tissue did not show obvious yield characteristics under quasi-static test conditions. However, the meniscus showed clear yield behavior under dynamic loading. When the strain rate increased, the elastic modulus of the radial meniscus remained around 35 MPa while the elastic modulus of the axial meniscus increased from 30 MPa to 80 MPa. This study demonstrates that the meniscus is sensitive to strain rate at both dynamic and quasi-static conditions, and the meniscus is an anisotropic biological tissue.

Fabrication of decellularized meniscus extracellular matrix according to inner cartilaginous, middle transitional, and outer fibrous zones result in zone-specific protein expression useful for precise replication of meniscus zones

Meniscus is a fibrocartilage composite tissue with three different microstructual zones, inner fibrocartilage, middle transitional, and outer fibrous zone. We hypothesized that decellularized meniscus extracellular matrix (DMECM) would have different characteristics according to zone of origin. We aimed to compare zone-specific DMECM in terms of biochemical characteristics and cellular interactions associated with tissue engineering. Micronized DMECM was fabricated from porcine meniscus divided into three microstructural zones. Characterization of DMECM was done by biochemical and proteomic analysis. Inner DMECM showed the highest glycosaminoglycan content, while middle DMECM showed the highest collagen content among groups. Proteomic analysis showed significant differences among DMECM groups. Inner DMECM showed better adhesion and migration potential to meniscus cells compared to other groups. DMECM resulted in expression of zone-specific differentiation markers when co-cultured with synovial mesenchymal stem cells (SMSCs). SMSCs combined with inner DMECM showed the highest glycosaminoglycan in vivo. Outer DMECM constructs, on the other hand, showed more fibrous tissue features, while middle DMECM constructs showed both inner and outer zone characteristics. In conclusion, DMECM showed different characteristics according to microstructural zones, and such material may be useful for zone-specific tissue engineering of meniscus.

High Resolution Micro-Computed Tomography Reveals a Network of Collagen Channels in the Body Region of the Knee Meniscus

The meniscus is an integral part of the human knee, preventing joint degradation by distributing load from the femoral condyles to the tibial plateau. Recent qualitative studies suggested that the meniscus is constituted by an intricate net of collagen channels inside which the fluid flows during loading. The aim of this study is to describe in detail the structure in which this fluid flows by quantifying the orientation and morphology of the collagen channels of the meniscal tissue. A 7 mm cylindrical sample, extracted vertically from the central part of a lateral porcine meniscus was freeze-dried and scanned using the highest-to-date resolution Microscopic Computed Tomography. The orientation of the collagen channels, their size and distribution was calculated. Comparisons with confocal multi-photon microscopy imaging performed on portions of fresh tissue have shown that the freeze-dried procedure adopted here ensures that the native architecture of the tissue is maintained. Sections of the probe at different heights were examined to determine differences in composition and structure along the sample from the superficial to the internal layers. Results reveal a different arrangement of the collagen channels in the superficial layers with respect to the internal layers with the internal layers showing a more ordered structure of the channels oriented at 30^{circ } with respect to the vertical, a porosity of 66.28% and the mean size of the channels of 22.14 mu {text {m}}.

Preparation and Characterization of an Optimized Meniscal Extracellular Matrix Scaffold for Meniscus Transplantation.

Many studies have sought to construct a substitute to partially replace irreparably damaged meniscus. Only the meniscus allograft has been used in clinical practice as a useful substitute, and there are concerns about its longevity and inherent limitations, including availability of donor tissue and possibility of disease transmission. To overcome these limitations, we developed an acellular xenograft from whole porcine meniscus. Samples were treated with 2% Triton X-100 for 10 days and 2% sodium dodecyl sulfate for 6 days. The DNA content of extracellular matrix (ECM) scaffolds was significantly decreased compared with that of normal porcine menisci (p < 0.001). Histological analysis confirmed the maintenance of ECM integrity and anisotropic architecture in the absence of nuclei. Biochemical and biomechanical assays of the scaffolds indicated the preservation of collagen (p = 0.806), glycosaminoglycan (p = 0.188), and biomechanical properties (elastic modulus and transition stress). The scaffolds possessed good biocompatibility and supported bone marrow mesenchymal stem cells (BMSCs) proliferation for 2 weeks in vitro, with excellent region-specific recellularization in vivo. The novel scaffold has potential value for application in recellularization and transplantation strategies.

Optimization of Meniscus Cell Transduction Using Lentivirus and Adeno-Associated Virus for Gene Editing and Tissue Engineering Applications.

The utilization of viral vectors to deliver genes of interest directly to meniscus cells and promote long-term modulation of gene expression may prove useful to enhance meniscus repair and regeneration. The objective of this study was to optimize and compare the potential of lentivirus (LV) and adeno-associated virus (AAV) to deliver transgenes to meniscus cells in both intact meniscus tissue and isolated primary cells in monolayer. Porcine meniscus tissue explants and primary meniscus cells in monolayer were transduced with LV or self-complementary AAV2 (scAAV2) encoding green fluorescent protein (GFP). Following transduction, explants were enzymatically digested to isolate meniscus cells, and monolayer cells were trypsinized. Isolated cells were analyzed by flow cytometry to determine percent transduction. LV and scAAV2 showed a high transduction efficiency in monolayer meniscus cells. scAAV2 was most effective at transducing cells within intact meniscus tissue but the efficiency was less than 20%. Outer zone meniscus cells were more readily transduced by both LV and scAAV2 than the inner zone cells. Higher virus titers and higher cell density resulted in improved transduction efficiency. Polybrene was necessary for the highest transduction efficiency with LV, but it reduced scAAV2 transduction. Both LV and scAAV2 efficiently transduce primary meniscus cells but only scAAV2 can modestly transduce cells embedded in meniscus tissue. This work lays the foundation for viral gene transfer to be utilized to deliver bioactive transgenes or gene editing machinery, which can induce long-term and tunable expression of therapeutic proteins from tissue-engineered constructs for meniscus repair and regeneration.