Whole-Transcriptome Sequence of Degenerative Meniscus Cells Unveiling Diagnostic Markers and Therapeutic Targets for Osteoarthritis

Meniscus plays an important role in joint homeostasis. Tear or degeneration of meniscus might facilitate the process of knee osteoarthritis (OA). Hence, to investigate the transcriptome change during meniscus degeneration, we reveal the alterations of messenger RNA (mRNA), microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) in meniscus during OA by whole-transcriptome sequence. A total of 375 mRNAs, 15 miRNAs, 56 lncRNAs, and 90 circRNAs were significantly altered in the degenerative meniscus treated with interleukin-1β (IL-1β). More importantly, highly specific co-expression RNA (ceRNA) networks regulated by lncRNA LOC107986251-miR-212-5p-SESN3 and hsa_circ_0018069-miR-147b-3p-TJP2 were screened out during IL-induced meniscus degeneration, unveiling potential therapeutic targets for meniscus degeneration during the OA process. Furthermore, lipocalin-2 (LCN2) and RAB27B were identified as potential biomarkers in meniscus degeneration by overlapping three previously constructed databases of OA menisci. LCN2 and RAB27B were both upregulated in osteoarthritic menisci and IL-1β-treated menisci and were highly associated with the severity of OA. This could introduce potential novel molecules into the database of clinical diagnostic biomarkers and possible therapeutic targets for early-stage OA treatment.

Collagen orientation probed by polarized Raman spectra can serve as differential diagnosis indicator between different grades of meniscus degeneration

The purpose of the present study was to analyze normal and degenerated menisci with Raman methodology on thin sections of formalin fixed paraffin embedding tissues and to correlate the Raman findings with the grade of meniscus degeneration. Menisci (n = 27) were removed from human knee joints after total knee replacement or meniscectomy. Following routine histopathological analysis to determine the grade of meniscal lesions obtained from healthy and degenerated formaline fixed paraffin embedded (FFPE) meniscal sections, Raman polarization approach was applied to evaluate the orientation of collagen fibrils in different levels of the same 5 μm thick FFPE meniscal tissue sections, used for histopathological assessment. We collected Raman spectra in two different polarization geometries, v-HH and v-VV, and calculated the mean value of the v-HH/v-VV intensity ratio of two Raman bands, sensitive and non-sensitive to the molecular orientation. The collagen specific amide I band at 1665 cm−1, has the higher sensitivity dependence on the Raman polarization. The mean values of ratio v-HH/v-VV of the 1665 cm−1 peak intensity was significantly higher in healthy, mean ± SD: 2.56 ± 0.46, compared to degenerated menisci, mean ± SD: 1.85 ± 0.42 (p = 0.0014). The mean values of v-HH/v-VV intensity ratio were 2.18 and 1.50 for low and high degenerated menisci, respectively (p < 0.0001). The difference of peak intensities in the two laser polarizations is decreased in the degenerated meniscus; this difference is diminishing as the degeneration increases. The v-HH/v-VV ratio was also of significant difference in low as compared to control and high grade meniscus lesions (p = 0.036 and p < 0.0001, respectively) offering valuable information for the approach of its biology and function. In the present study we showed that the 5 μm thick sections can be used for Raman analysis of meniscal tissue with great reliability, in terms of sensitivity, specificity, false-negative and false-positive results. Our data introduce the interesting hypothesis that compact portable Raman microscopy on tissue sections can be used intra-operatively for fast diagnosis and hence, accurate procedure design in the operating room.

Identification and validation of pivotal genes related to age-related meniscus degeneration based on gene expression profiling analysis and in vivo and in vitro models detection

Background The componential and structural change in the meniscus with aging would increase the tissue vulnerability of the meniscus, which would induce meniscus tearing. Here, we investigated the molecular mechanism of age-related meniscus degeneration with gene expression profiling analysis, and validate pivotal genes in vivo and in vitro models. Methods The GSE45233 dataset, including 6 elderly meniscus samples and 6 younger meniscus samples, was downloaded from the Gene Expression Omnibus (GEO) database. To screen the differential expression of mRNAs and identify the miRNAs targeting hub genes, we completed a series of bioinformatics analyses, including functional and pathway enrichment, protein–protein interaction network, hub genes screening, and construction of a lncRNA–miRNA–mRNA network. Furthermore, crucial genes were examined in human senescent menisci, mouse senescent meniscus tissues and mouse meniscus cells stimulated by IL-1β. Results In total, the most significant 4 hub genes (RRM2, AURKB, CDK1, and TIMP1) and 5 miRNAs (hsa-miR-6810-5p, hsa-miR-4676-5p, hsa-miR-6877-5p, hsa-miR-8085, and hsa-miR-6133) that regulated such 4 hub genes, were finally identified. Moreover, these hub genes were decreased in meniscus cells in vitro and meniscus tissues in vivo, which indicated that hub genes were related to meniscus senescence and could serve as potential biomarkers for age-related meniscus tearing. Conclusions In short, the integrated analysis of gene expression profile, co-expression network, and models detection identified pivotal genes, which elucidated the possible molecular basis underlying the senescence meniscus and also provided prognosis clues for early-onset age-related meniscus tearing.

Ultrasound-Guided Meniscal Injection of Autologous Growth Factors: A Brief Report

Meniscal degeneration is a common finding even in young patients’ knees, and it is regarded as a predictor for the onset of early osteoarthritis (OA). When symptomatic, it represents a challenge since arthroscopic surgery provides unpredictable results: recent evidence has shown that partial meniscectomy is not better than conservative management up to 2 years of follow-up, and the removal of meniscal tissue may accelerate OA progression toward OA. Intra-articular injection of corticosteroids or hyaluronic acid may help in providing temporary symptomatic relief, but no influence should be expected on the quality of the meniscal tissue. Biologic agents have been adopted to treat a variety of degenerative musculoskeletal pathologies, and the use of platelet-derived growth factors (GFs) has become routine. Preclinical studies have documented that platelet-derived GFs may play a beneficial role in stimulating meniscal repair and regeneration by triggering anabolic pathways and stimulating local mesenchymal stem cells from synovium. Furthermore, also mechanical stimulation (e.g., arthroscopic trephination or percutaneous needling) in the red-red or red-white zone may further promote tissue healing. The purpose of the present brief report is to describe the clinical outcomes at 18 months’ follow-up in a cohort of patients affected by symptomatic medial meniscus degeneration and treated by percutaneous needling plus intra- and perimeniscal injection of autologous conditioned plasma (ACP). The procedure was shown to be safe and provided significant pain reduction and improvement in subjective scores. This treatment option deserves further investigation in a comparative setting, to establish whether it could offer advantage over isolated intra-articular injections.

Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model

Objective: Joint instability and meniscal dysfunction contribute to the onset and progression of knee osteoarthritis (OA). In the destabilization of the medial meniscus (DMM) model, secondary OA occurs due to the rotational instability and increases compressive stress resulting from the meniscal dysfunction. We created a new controlled abnormal tibial rotation (CATR) model that reduces the rotational instability that occurs in the DMM model. So, we aimed to investigate whether rotational instability affects articular cartilage degeneration using the DMM and CATR models, as confirmed using histology and immunohistochemistry. Design: Twelve-week-old male mice were randomized into 3 groups: DMM group, CATR group, and INTACT group (right knee of the DMM group). After 8 and 12 weeks, we performed the tibial rotational test, safranin-O/fast green staining, and immunohistochemical staining for TNF-α and MMP-13. Results: The rotational instability in the DMM group was significantly higher than that of the other groups. And articular cartilage degeneration was higher in the DMM group than in the other groups. However, meniscal degeneration was observed in both DMM and CATR groups. The TNF-α and MMP-13 positive cell rates in the articular cartilage of the CATR group were lower than those in the DMM group. Conclusions: We found that the articular cartilage degeneration was effectively suppressed by controlling the rotational instability caused by meniscal dysfunction. These findings suggest that suppression of rotational instability in the knee joint is an effective therapeutic measure for preventing OA progression.

Identifcation of Pathways and Genes Associated With Meniscus Degeneration Using Bioinformatics Analyses

There are few studies on the genetic changes of meniscus degeneration. We used anterior cruciate ligament resection of Wuzhishan pig to prepare a meniscus degeneration model, and applied gene chip technology to detect differentially expressed genes in degenerative meniscus tissue. Then we applied GO analysis, Pathway analysis, Core gene network analysis and Relevant miRNAs analysis to discover relevant regulatory networks of meniscus degeneration. As a result, we detected 893 differentially expressed genes, mainly involving hormone, apoptosis, inflammation and other mechanisms, and obtained MUC13, Inflammatory mediator regulation of TRP channels, MDFI, mir-335-5p and so on that may play a key role. In summary, we have established a reliable animal model of meniscus degeneration and found that meniscus degeneration involves several possible molecular mechanisms, which will provide molecular targets for further research of the disease in the future.

Characterization of regional meniscal cell and chondrocyte phenotypes and chondrogenic differentiation with histological analysis in osteoarthritic donor-matched tissues

Meniscus degeneration is closely related to the progression of knee osteoarthritis (OA). However, there is currently a lack of quantitative and objective metrics to assess OA meniscal cell phenotypes. In this study we investigated the phenotypic markers and chondrogenic potency of avascular and vascular meniscal cells and chondrocytes from medial OA knee joints (n = 10). Flow cytometry results showed that a significantly greater percentage of meniscal cells were positive for CD49b, CD49c and CD166 compared to donor-matched chondrocytes after 14 days in monolayer culture. The integrins, CD49b and CD29, were expressed at a significantly higher level on avascular meniscal cells derived from tissues with a more degenerated inner border than non-degenerate menisci, suggesting that the integrin family may play an important role in meniscus OA pathology. Collagen fibres arranged in a “tree-like” formation within the meniscus appeared to have less blood vessels associated with them in the vascular region of the most degenerate menisci, which may indicate that such structures are involved in the pathological process. We have demonstrated that meniscal cells derived from the lateral meniscus in medial OA patients have chondrogenic capacity in vitro and hence could represent a potential cell source to consider for meniscus tissue engineering.

In vitro Method to Quantify and Visualize Volumetric Wear in Meniscus Subjected to Joint Loading Using a 3D Optical Scanner

The menisci are fibrocartilaginous soft tissues that act to absorb and distribute load across the surface of the knee joint. As a result of mechanical wear and large repetitive loading, meniscus tissue can begin to breakdown, or degenerate. Meniscus degeneration increases the risk of tearing, weakened tissue integrity, and the progression of osteoarthritis. Therefore, it is imperative to understand the wear behavior of whole human meniscus to identify conditions that may significantly increase the risk of degeneration. The objective of this study is to develop and validate an in vitro methodology for characterizing volumetric wear behavior in whole human meniscus using a 3D optical scanning system. This study was done in three parts. Part I and II consisted of assessing the accuracy and repeatability of the proposed methodology for meniscus tissue. Two surrogate models were developed for this purpose: (1) Simple Surrogate: Geometric Blocks and (2) Complex Surrogate: Menisci & Tibia Replicas. Part III utilized the method to quantify wear in whole human meniscus subjected to physiological loading conditions. One fresh-frozen cadaveric knee joint was potted in a custom designed and built knee simulator and subjected to four loading stages of 250,000 cycles. A 3D optical scanner was used to generate 3D renderings for pre- and post-wear conditions for both surrogates and human meniscus. An open-source software, CloudCompare, was then used to computationally evaluate volume loss. For the surrogate models, the process was repeated at varying wear depths, and the percentage error between real-life measured volumes and CloudCompare calculated volumes was determined. The human meniscus followed the same scanning procedure for pre- and post-wear; however, post-wear volume was recorded following each loading stage. Results from the simple surrogate model showed that the method was capable of measuring wear with < 2% error when detecting volumetric changes of 1.08 cm3 ; however, as defect depth decreased, the absolute mean percentage error increased (p < 0.001). The complex surrogate model showed significant difference when measuring wear in the lateral and medial meniscus (p < 0.05) with percentage errors of less than 7.9% when detecting volumetric changes of 0.4 cm3. The results obtained from whole human meniscus testing indicate that with an increase in loading cycles, a higher degree of meniscal wear and deformation is present. For the first time, this study provides a methodology to identify volumetric loss due to wear behavior in whole human meniscus. This is also the first study to provide comprehensive visualization and identification of global defects within the meniscus tissue. Results of this study have the potential to help identify the physical and biochemical factors that lead to meniscus degeneration thereby advancing fundamental knowledge of the etiology of degenerative wear within articulating soft tissue.

The underlying mechanism of partial anterior cruciate ligament injuries to the meniscus degeneration of knee joint in rabbit models

Background The diagnosis, treatment, and efficacy evaluation of anterior cruciate ligament (ACL) partial rupture remains controversial. This research aims to investigate the underlying mechanism of partial ACL injuries to the meniscus degeneration in the rabbit knee. Methods Sixty New Zealand white rabbits were randomly divided into three groups including an experimental group, a sham group (n = 6), and a blank control group (n = 6). The experimental group is composed of an anteromedial bundle (AMB) rupture group (n = 24) and a posterolateral bundle (PLB) rupture group (n = 24). Rabbits in the experimental group were subjected to right hind limbs knee surgery to induce ACL part injury under the arthroscopy. Finally, eight rabbits including 6 in the model group and 2 in the control group were sampled randomly on the 2nd, 4th, and 8th weeks respectively. We observed the typical form of the meniscus through HE staining. Expressions of inflammatory factors including interleukin-1β (IL-1β) and IL-17 in the knee joint fluid were determined by means of an ELISA. Analysis of the mRNA expressions of matrix metalloproteinases-13(MMP-13) was performed to evaluate the inflammatory mediators in the pathogenesis of the meniscus. Results HE staining results showed that the surface was rough and the tissues were loose displaying collagen fibers of varying thickness. Both IL-1β and IL-17 in the synovial fluid and the positive rate of MMP-13 in addition to MMP-13 mRNA showed a demonstrable increase treads from the 2nd to the 8th week. The significant difference was found (P < 0.05) compared to the control group. Conclusion We conclude that the elevated levels of IL-1β and IL-17, along with increased MMP13 expression, resulted in meniscus degradation in the rabbit knee joint model with partial ACL injury.