The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Differences between the root and horn cells of the human medial meniscus from the osteoarthritic knee in cellular characteristics and responses to mechanical stress.

BACKGROUND: Many histological, mechanical, and clinical studies have been performed on the medial meniscus posterior root attachment, as it often tears in patients with osteoarthritic knee. Medial meniscal root repair is recommended in clinical situations; however, to date, no studies have examined the differences between meniscal root and horn cells. The aim of this study was, therefore, to investigate the morphology, reaction to cyclic tensile strain, and gene expression levels of medial meniscal root and horn cells. METHODS: Meniscal samples were obtained from the medial knee compartments of 10 patients with osteoarthritis who underwent total knee arthroplasty. Root and horn cells were cultured in Dulbecco's modified Eagle's medium without enzymes. The morphology, distribution, and proliferation of medial meniscal root and horn cells, as well as the gene and protein expression levels of Sry-type HMG box 9 and type II collagen, were determined after cyclic tensile strain treatment. RESULTS: Horn cells had a triangular morphology, whereas root cells were fibroblast-like. The number of horn cells positive for Sry-type HMG box 9 and type II collagen was considerably higher than that of root cells. Although root and horn cells showed similar levels of proliferation after 48, 72, or 96 h of culture, more horn cells than root cells were lost following a 2-h treatment with 5% and 10% cyclic tensile. Sry-type HMG box 9 and α1(II) collagen mRNA expression levels were significantly enhanced in both cells after 2- and 4-h cyclic tensile strain (5%) treatment. CONCLUSIONS: Medial meniscal root and horn cells have distinct morphologies, reactions to mechanical stress, and cellular phenotypes. Our results suggest that physiological tensile strain is important to activate extracellular matrix production in horn cells.

Mouse Models of Osteoarthritis: Surgical Model of Post-traumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus.

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of post-traumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.

Intra-Articular Injection of Stromal Cell-Derived Factor 1α Promotes Meniscal Healing via Macrophage and Mesenchymal Stem Cell Accumulation in a Rat Meniscal Defect Model.

The stromal-cell-derived factor-1α (SDF-1) is well-known for playing important roles in the regeneration of tissue by enhancing cell migration. However, the effect of SDF-1 in meniscal healing remains unknown. The purpose of this study is to investigate the effects of intra-articular injection of SDF-1 on meniscus healing in a rat meniscal defect model. The intra-articular SDF-1 injection was performed at meniscectomy and one week later. Macroscopic and histological assessments of the reparative meniscus were conducted at one, two and six weeks after meniscectomy in rats. In the macroscopic evaluation, the SDF-1 group showed an increase in the size of the reparative meniscus at six weeks after meniscectomy compared to the phosphate-buffered saline (PBS) injection (no-treatment) group. Histological findings showed that intra-articular injection of SDF-1 enhanced the migration of macrophages to the site of the regenerative meniscus at one and two weeks after meniscectomy. CD68- and CD163-positive cells in the SDF-1 group at one week after meniscectomy were significantly higher than in the no-treatment group. CD163-positive cells in the SDF-1 group at two weeks were significantly higher than in the no-treatment group. At one week after meniscectomy, there were cells expressing mesenchymal-stem-cell-related markers in the SDF-1 group. These results indicate the potential of regenerative healing of the meniscus by SDF-1 injection via macrophage and mesenchymal stem cell accumulation. In the present study, intra-articular administration of SDF-1 contributed to meniscal healing via macrophage, CD90-positive cell and CD105-positive cell accumulation in a rat meniscal defect model. The SDF-1-CXCR4 pathway plays an important role in the meniscal healing process. For potential clinical translation, SDF-1 injection therapy seems to be a promising approach for the biological augmentation in meniscal injury areas to enhance healing capacity.

Distinct degenerative phenotype of articular cartilage from knees with meniscus tear compared to knees with osteoarthritis.

OBJECTIVE: To compare the transcriptome of articular cartilage from knees with meniscus tears to knees with end-stage osteoarthritis (OA). DESIGN: Articular cartilage was collected from the non-weight bearing medial intercondylar notch of knees undergoing arthroscopic partial meniscectomy (APM; N = 10, 49.7 ± 10.8 years, 50% females) for isolated medial meniscus tears and knees undergoing total knee arthroplasty (TKA; N = 10, 66.0 ± 7.6 years, 70% females) due to end-stage OA. Ribonucleic acid (RNA) preparation was subjected to SurePrint G3 human 8 × 60K RNA microarrays to probe differentially expressed transcripts followed by computational exploration of underlying biological processes. Real-time polymerase chain reaction amplification was performed on selected transcripts to validate microarray data. RESULTS: We observed that 81 transcripts were significantly differentially expressed (45 elevated, 36 repressed) between APM and TKA samples (≥ 2 fold) at a false discovery rate of ≤ 0.05. Among these, CFD, CSN1S1, TSPAN11, CSF1R and CD14 were elevated in the TKA group, while CHI3L2, HILPDA, COL3A1, COL27A1 and FGF2 were highly expressed in APM group. A few long intergenic non-coding RNAs (lincRNAs), small nuclear RNAs (snoRNAs) and antisense RNAs were also differentially expressed between the two groups. Transcripts up-regulated in TKA cartilage were enriched for protein localization and activation, chemical stimulus, immune response, and toll-like receptor signaling pathway. Transcripts up-regulated in APM cartilage were enriched for mesenchymal cell apoptosis, epithelial morphogenesis, canonical glycolysis, extracellular matrix organization, cartilage development, and glucose catabolic process. CONCLUSIONS: This study suggests that APM and TKA cartilage express distinct sets of OA transcripts. The gene profile in cartilage from TKA knees represents an end-stage OA whereas in APM knees it is clearly earlier in the degenerative process.

Microarray analysis of the molecular mechanisms associated with age and body mass index in human meniscal injury.

The aim of the present study was to identify genes and functional pathways associated with meniscal injuries affected by age or body mass index (BMI) using microarray analysis. The GSE45233 gene expression dataset with 12 injured meniscus samples associated with age and BMI and GSE66635 dataset with 12 injured and 12 normal meniscus samples were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified based on age or BMI in GSE45233. DEGs between injured and normal meniscus samples in GSE66635 were also identified. Common DEGs between GSE45233 and GSE66635 were identified as feature genes associated with age or BMI, followed by protein­protein interaction (PPI) network and functional pathway enrichment analyses for the feature genes. Finally, the GSE51588 genome­wide expression profile was then downloaded from the GEO database to validate the results. A total of 1,328 DEGs were identified. Of these, 28 age­associated and 20 BMI­associated meniscal injury genes were obtained. B­cell lymphoma­2 (Bcl­2) and matrix metalloproteinase­14 were identified as hub genes in the PPI networks. Functional pathway enrichment analysis revealed that vascular endothelial growth factor A (VEGFA), transferrin (TF) and Bcl­2 were involved in the hypoxia­inducible factor 1 signaling pathway. TF was involved in the mineral absorption function pathway associated with BMI. Additionally, TF and VEGFA were identified to be overlapping candidate genes of GSE45233 and GSE66635, and DEGs in GSE51588. Therefore, VEGFA, TF, and Bcl­2 may be important genes for human meniscal injuries. Additional evaluations of these results are required.

Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis.

OBJECTIVE: To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. DESIGN: Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. RESULTS: Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. CONCLUSION: These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

Evidence for Genetic Contribution to Variation in Posttraumatic Osteoarthritis in Mice.

OBJECTIVE: Recombinant inbred mouse strains generated from an LG/J and SM/J intercross offer a unique resource to study complex genetic traits such as osteoarthritis (OA). We undertook this study to determine the susceptibility of 14 strains to various phenotypes characteristic of posttraumatic OA. We hypothesized that phenotypic variability is associated with genetic variability. METHODS: Ten-week-old male mice underwent surgical destabilization of the medial meniscus (DMM) to induce posttraumatic OA. Mice were killed 8 weeks after surgery, and knee joints were processed for histology to score cartilage degeneration and synovitis. Micro-computed tomography was used to analyze trabecular bone parameters including subchondral bone plate thickness and synovial ectopic calcifications. Gene expression in the knees was assessed using a QuantiGene Plex assay. RESULTS: Broad-sense heritability ranged from 0.18 to 0.58, which suggested that the responses to surgery were moderately heritable. The LGXSM-33, LGXSM-5, LGXSM-46, and SM/J strains were highly susceptible to OA, while the LGXSM-131b, LGXSM-163, LGXSM-35, LGXSM-128a, LGXSM-6, and LG/J strains were relatively OA resistant. This study was the first to accomplish measurement of genetic correlations of phenotypes that are characteristic of posttraumatic OA. Cartilage degeneration was significantly positively associated with synovitis (r = 0.83-0.92), and subchondral bone plate thickness was negatively correlated with ectopic calcifications (r = -0.59). Moreover, we showed that 40 of the 78 genes tested were significantly correlated with various OA phenotypes. However, unlike the OA phenotypes, there was no evidence for genetic variation in differences in gene expression levels between DMM-operated and sham-operated knees. CONCLUSION: For these mouse strains, various characteristics of posttraumatic OA varied with genetic composition, which demonstrated a genetic basis for susceptibility to posttraumatic OA. The heritability of posttraumatic OA was established. Phenotypes exhibited various degrees of correlations; cartilage degeneration was positively correlated with synovitis, but not with the formation of ectopic calcifications. Further investigation of the genome regions that contain genes implicated in OA, as well as further investigation of gene expression data, will be useful for studying mechanisms of OA and identifying therapeutic targets.

Delivery of epidermal growth factor receptor inhibitor via a customized collagen scaffold promotes meniscal defect regeneration in a rabbit model.

Meniscal injury is one of the most common knee joint injuries, which remains an intractable challenge in clinical practice to date. Aberrant epidermal growth factor receptor (EGFR) activation levels in both human and mice menisci following injury, prompted us to investigate the functional role of EGFR by utilizing an inducible cartilage-specific EGFR-deficient mouse model. We demonstrated that conditional EGFR deletion in mice resulted in increased partial meniscectomy-induced ECM production within the meniscus, which is comparable to utilization of the small molecule EGFR inhibitor, gefitinib, to block EGFR activity. Here, we combined intra-articular delivery of gefitinib with an implanted customized collagen scaffold to substitute for lost meniscal tissue, as well as to promote meniscal regeneration and prevent osteoarthritis (OA) progression in a rabbit meniscectomy model. STATEMENT OF SIGNIFICANCE: The main novelty of this study is the finding of a new application for small molecule EGFR inhibitor in meniscal injury therapy. This study also highlights the importance of using a customized collagen scaffold to provide robust mechanical strength and effectively promote meniscus regeneration. In summary, our study finds that intra-articular delivery of gefitinib together with implantation of a customized, multi-layer collagen scaffold not only enhanced meniscal regeneration, but also protected articular cartilage from degeneration in rabbit model. These results provide valuable insight for meniscal tissue engineering studies and clinical practice.

Traumatic and Degenerative Meniscus Tears Have Different Gene Expression Signatures.

BACKGROUND: Meniscus tears are classified as traumatic or degenerative based on the tear pattern. There is little evidence demonstrating biological differences between the 2 tear types. HYPOTHESIS: Gene expression signatures in the injured meniscus are different between traumatic (vertical) and degenerative (complex, horizontal, or flap) tears. STUDY DESIGN: Controlled laboratory study. METHODS: Samples of the torn meniscus from the white-white zone were removed at the time of clinically indicated partial meniscectomy from 48 patients (37 with degenerative tears and 11 with traumatic tears). mRNA expression in the injured menisci was measured by quantitative real-time polymerase chain reaction for selected molecular markers of osteoarthritis, inflammation, and cartilage homeostasis (eg, cytokines/chemokines, aggrecanases/metalloproteinases, transcription factors, cartilage matrix genes, and adipokines). The tear pattern (traumatic or degenerative) and location (medial or lateral) were recorded for each patient. Gene expression differences between degenerative and traumatic tears were computed after adjusting for patients' age, sex, and body mass index and for location of the resected meniscus (medial/lateral). RESULTS: Gene expression in meniscus tears varied by pattern. Chemokines ( IL8 [ P < .001] and CXCL6 [ P < .001]) and matrix metalloproteinases ( MMP1 [ P = .011] and MMP3 [ P = .016]) were expressed at a significantly higher level in traumatic tears compared with degenerative tears. In contrast, COL1A1 was expressed at a lower level in traumatic tears compared with degenerative tears ( P = .058). None of the genes tested demonstrated significant differences between medial and lateral meniscus tears. CONCLUSION: Traumatic meniscus tears overall exhibited a higher inflammatory/catabolic response as evidenced by higher levels of chemokine and matrix metalloproteinase expression than degenerative tears. These findings suggest that there is a (molecular) biological distinction between traumatic and degenerative tears. CLINICAL RELEVANCE: The catabolic/inflammatory differences between traumatic and degenerative tears may be relevant to treatment decisions regarding the meniscus as well as advance our understanding of how meniscus tears relate to the development of knee osteoarthritis.