Revealing the complexity of meniscus microvasculature through 3D visualization and analysis.

Three-dimensional information is essential for a proper understanding of the healing potential of the menisci and their overall role in the knee joint. However, to date, the study of meniscal vascularity has relied primarily on two-dimensional imaging techniques. Here we present a method to elucidate the intricate 3D meniscal vascular network, revealing its spatial arrangement, connectivity and density. A polymerizing contrast agent was injected into the femoral artery of human cadaver legs, and the meniscal microvasculature was examined using micro-computed tomography at different levels of detail and resolution. The 3D vascular network was quantitatively assessed in a zone-base analysis using parameters such as diameter, length, tortuosity, and branching patterns. The results of this study revealed distinct vascular patterns within the meniscus, with the highest vascular volume found in the outer perimeniscal zone. Variations in vascular parameters were found between the different circumferential and radial meniscal zones. Moreover, through state-of-the-art 3D visualization using micro-CT, this study highlighted the importance of spatial resolution in accurately characterizing the vascular network. These findings, both from this study and from future research using this technique, improve our understanding of microvascular distribution, which may lead to improved therapeutic strategies.

Meniscal Injuries: Mechanism and Classification.

Meniscal tears may be managed through conservative physical therapy and nonsteroidal anti-inflammatory medications or operative intervention. Meniscal repair is superior to partial meniscectomy with better functional outcomes and less severe degenerative changes over time. Surgical advances in operative techniques, modern instrumentation and biological enhancements collectively improve healing rates of meniscal repair. However, failed repair is not without consequences and can negative impact patient outcomes. Therefore, it is imperative for surgeons to have a thorough understanding of the vascular zones and biomechanical classifications of meniscal tears in order to best determine the most appropriate treatment.

Updates and Advances in the Management of Lateral Meniscal Radial Tears: A Critical Analysis Review.

Because of their increased mobility, lack of resistance to hoop stresses, and decreased blood supply, radial tears of the lateral meniscus are more troublesome to heal than vertical longitudinal tears. Given the success of meniscal root repairs, radial tears of the lateral meniscal body should be given strong consideration for repair because of a more reproducible ability to heal such lesions in young, active patients. Technique options that should be considered for the less common anterior radial tears of the lateral meniscus include outside-in repair, self-capturing suture-passing devices, and orthobiologic treatments to stimulate healing. Although a variety of suture techniques, including the double horizontal mattress and horizontal butterfly patterns, have demonstrated improvements in patient outcomes, evidence is still limited with regard to the ideal suture pattern for radial tears.

Microvascular Anatomy and Intrinsic Gene Expression of Menisci From Young Adults.

BACKGROUND: Meniscal vascular supply is an important determinant of its healing potential. It has been reported that only the peripheral 30% of the meniscus is vascularized in cadavers aged 53 to 94 years; however, the vascularity in young patients, in whom meniscal repair is more often performed, is unknown. PURPOSE: The primary objective was to analyze and measure the microvascular anatomy of the meniscus in adult cadaveric specimens <35 years old. The secondary objective was to assess angiogenic potential by quantifying regional gene expression in a meniscal allograft cohort <45 years old. STUDY DESIGN: Descriptive laboratory study. METHODS: In part 1 of this study, 13 fresh-frozen cadaveric knees (age range, 22-34 years; mean, 28.5 years) underwent popliteal artery India ink injection and tissue clearing using a Spalteholz technique, followed by microvascular vascular measurement. In part 2, mRNA was isolated from 13 meniscal allografts (age range, 17-43 years; mean, 27.2 years), and expression of angiogenic genes, vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 1 (FLT1) was quantified using real-time polymerase chain reaction. RESULTS: The maximal depth of vascular penetration into the periphery of the medial and lateral menisci ranged from 0% to 42% and 0% to 48%, respectively. There was variation in the degree of vascular penetration within the medial meniscus, with the posterior horn having a significantly smaller depth of penetration (median, 8.7%) than that of the anterior horn (median, 17.4%; P < .0001) or midbody (median, 17.5%; P = .0003). There were no differences in angiogenesis gene expression (VEGF/FLT1) based on circumferential or radial meniscal locations. CONCLUSION: The vascular supply of the medial and lateral menisci in specimens from adults <35 years of age extended farther than what was reported in specimens from older individuals; however, median values remained consistent. Gene expression of the angiogenic marker VEGF was low throughout all regions of uninjured menisci from young adults, which is consistent with reports in older specimens. CLINICAL RELEVANCE: Improved understanding of meniscal vascular supply in young adults is critical to informing clinical treatment decisions.

Current concepts in the techniques, indications and outcomes of meniscal repairs.

Knee arthroscopy for meniscal tears is one of the most commonly performed orthopaedic procedures. In recent years, there has been an increasing incidence of meniscal repairs, as there are concerns that meniscectomy predisposes patients to early osteoarthritis. Indications for meniscal repair are increasing and can now be performed in older patients who are active, even if the tear is in the avascular zone. Options for meniscal tear management broadly fall into three categories: non-operative management, meniscal repair or meniscectomy. With limited evidence directly comparing each of these options optimal management strategies can be difficult. Decision making requires thorough assessment of patient factors (e.g. age and comorbidities) and tear characteristics (e.g. location and reducibility). The purpose of this paper is, therefore, to review the management options of meniscal tears and summarize the evidence for meniscal tear repair.

Intercondylar and central regions of complete discoid lateral meniscus have different cell and matrix organizations.

BACKGROUND: A complete discoid lateral meniscus (DLM) has a high risk of horizontal tear. However, cellular phenotypes and extracellular matrix organizations in complete DLMs are still unclear. The aim of this study was to investigate histological and cellular biological characteristics in both the intercondylar and central regions of complete DLM. MATERIALS AND METHODS: Meniscal samples were obtained from the intercondylar and central regions of complete DLM (n = 6). Blood vessels and aggregated cell ratio were measured in each region. Depositions of type I/II collagens and safranin O-stained proteoglycans in the extracellular matrix were assessed. Experiments in gene expression, morphology, proliferation, and effect of mechanical stretch were performed using cultured cells derived from each region. RESULTS: Blood vessel counts were significantly higher in the intercondylar region than in the central region. The ratio of aggregated cells was lower in the intercondylar region than in the central region. Deposition of type I collagen was comparable for both regions. The central region contained a larger quantity of type II collagen and safranin O staining density compared with the intercondylar region. Proliferation of the fibroblastic intercondylar cells was not affected by 5%-stretching. However, stretching treatments decreased relative proliferation of the chondrocytic central cells. CONCLUSIONS: This study demonstrated that the central region of complete DLM had different cellular properties and collagen components compared with the intercondylar region. Our results suggest that the central region of complete DLM may have a low healing potential like the inner avascular region of the meniscus.

Quantitative analysis of the perimeniscal position of the inferior lateral genicular artery (ILGA): magnetic resonance imaging study.

PURPOSE: The inferior lateral genicular artery (ILGA) passes around the lateral knee joint, adjacent to the lateral meniscus (LM). ILGA injuries in total knee arthroplasty or arthroscopic surgery can result in recurrent hemarthrosis or painful pseudoaneurysms. Detailed information about the perimeniscal position of the ILGA relative to the LM is necessary to avoid these complications. METHODS: 3-T MR images of 100 knees (mean age 36.3 ± 11.2 years) were retrospectively reviewed. The perimeniscal area was divided into four regions: the anterior, middle, popliteal hiatus, and posterior zones. In each zone, the ILGA diameter, superoinferior position (assessed as the height of the ILGA from the LM base), and distance between the meniscocapsular junction and the ILGA were measured. RESULTS: The distance between the ILGA and meniscocapsular junction was significantly smaller in the middle zone than in the other three zones (anterior 5.3 ± 0.8 mm, middle 1.4 ± 0.4 mm, popliteal hiatus 6.1 ± 1.0 mm, and posterior 5.6 ± 1.5 mm, p < 0.05). In the superoinferior position, the height of the ILGA was 3.4 ± 0.9 mm in the anterior zone, 0.4 ± 1.3 mm in the middle zone, - 1.9 ± 1.8 mm in the popliteal hiatus zone, and - 1.3 ± 4.3 mm in the posterior zone. When the LM bottom is the base, the ILGA was located superiorly in the anterior zone, close to the base in the middle zone, and inferiorly in the popliteal hiatus zone. CONCLUSIONS: To avoid ILGA injury, close attention is necessary during surgical procedures involving the meniscocapsular junction of the LM, especially at the meniscal base in the middle zone.

Angiogenic approaches to meniscal healing.

Meniscal injuries commonly result in osteoarthritis causing long term morbidity, lifelong treatment, joint replacement and significant financial burden to the Canadian healthcare system. Injuries to the outer third of the meniscus often heal well due to adequate blood supply. Healing of injuries in the inner two thirds of the meniscus are often critically retarded due to a lack of blood flow necessitating partial meniscectomy in many instances. Localized angiogenesis in the inner meniscus has yet to be achieved despite a belief that vascularization of these lesions corresponds with meniscal healing. This review briefly summarizes the growth factors that have been assessed for a role in meniscal healing and points to a significant knowledge gap in our understanding of meniscal healing.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

Histological and biological comparisons between complete and incomplete discoid lateral meniscus.

The discoid lateral meniscus (DLM) is an anatomically abnormal meniscus that covers a greater area of the tibial plateau than the normal meniscus. The DLM is classified into two types: complete (CDLM) and incomplete (ICDLM) types. In this study, we investigated the histological and cell biological characteristics of CDLM and ICDLM. The number of blood vessels, proteoglycan deposition, and collagen distribution were assessed using meniscal tissues. Collagen production was also investigated in CDLM and ICDLM cells. The intercondylar region of the CDLM had a higher number of blood vessels than the inner region of the ICDLM. Safranin O staining density and type II collagen deposition in ICDLM were higher than those in CDLM. Type II collagen-positive cells were higher in ICLDM than in CDLM. CDLM cells showed slender fibroblastic morphology, while ICDLM cells were triangular chondrocytic in shape. This study demonstrated that the intercondylar region of the CDLM showed similar properties to the outer region of the meniscus. The inner region of the ICDLM, on the other hand, differed from the intercondylar region of the CDLM. Our results suggest that the intercondylar region of the CDLM may have a high healing potential like the outer meniscus.

Characterization of Meniscal Pathology Using Molecular and Proteomic Analyses.

The meniscus is a complex tissue and is integral to knee joint health and function. Although the meniscus has been studied for years, a relatively large amount of basic science data on meniscal health and disease are unavailable. Genomic and proteomic analyses of meniscal pathology could greatly improve our understanding of etiopathogenesis and the progression of meniscal disease, yet these analyses are lacking in the current literature. Therefore, the objective of this study was to use microarray and proteomic analyses to compare aged-normal and pathologic meniscal tissues. Meniscal tissue was collected from the knees of five patient groups (n = 3/group). Cohorts included patients undergoing meniscectomy with or without articular cartilage pathology, patients undergoing total knee arthroplasty with mild or moderate-severe osteoarthritis, and aged-normal controls from organ donors. Tissue sections were collected from the white/white and white/red zones of posterior medial menisci. Expression levels were compared between pathologic and control menisci to identify genes of interest (at least a ×1.5 fold change in expression levels between two or more groups) using microarray analysis. Proteomics analysis was performed using mass spectrometry to identify proteins of interest (those with possible trends identified between the aged-normal and pathologic groups). The microarray identified 157 genes of interest. Genes were categorized into the following subgroups: (1) synthesis, (2) vascularity, (3) degradation and antidegradation, and (4) signaling pathways. Mass spectrometry identified 173 proteins of interest. Proteins were further divided into the following categories: (1) extracellular matrix (ECM) proteins; (2) proteins associated with vascularity; (3) degradation and antidegradation proteins; (4) cytoskeleton proteins; (5) glycolysis pathway proteins; and (6) signaling proteins. These data provide novel molecular and biochemical information for the investigation of meniscal pathology. Further evaluation of these disease indicators will help researchers develop algorithms for diagnostic, therapeutic, and prognostic strategies related to meniscal disorders.

Radiofrequency stimulation for potential healing of meniscal injuries in the avascular zone.

We conducted this study to evaluate the effect of radiofrequency (RF) stimulation with suture repair on the healing of tears in the meniscal white-white zone. Fifty-four New Zealand white rabbits underwent surgically induced meniscal injuries within the white-white region. RF was applied using a 0.8-mm TOPAZ MicroDebrider RF wand (ArthroCare) at level 4 for 500 milliseconds. Rabbits were sacrificed at 28 and 84 days for gross and histologic analysis by 3 blinded observers and at 9, 28, and 84 days for biochemical examination. Biochemical analyses included evaluation of cell proliferation (3H-thymidine), as well as mitogenic (IGF-1, bFGF) and angiogenic (VEGF, αV) factors. Of specimens repaired with RF combined with suture, 19 (58%) showed a degree of gross morphologic and histologic healing. No significant healing was seen in specimens with either no repair or repair with suture alone. We observed a 40% increase in cellular proliferation when RF supplementation was used (P<.05). With regards to mitogenic and angiogenic markers (IGF-1, bFGF, VEGF, and αV), there was a significant increase in groups treated with RF at 9 and 28 days (P>0.05). RF supplementation of avascular zone meniscal repairs may lead to an increased healing response.

Robust revascularization, despite impaired VEGF production, after meniscus allograft transplantation in rabbits.

BACKGROUND: Little is known about vascularization restoration and vascular circulation after allogenic graft transplantation, which are both important prerequisites for optimal use of allograft meniscus transplantation. PURPOSE: To study vascularization restoration through autograft and allograft meniscus models in Oryctolagus cuniculus. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-eight rabbits at mature bone age were randomized to receive either an autograft or allograft after the meniscus of the left knee was completely resected. Vascularization, blood circulation, histological characteristics of the grafted meniscus and surrounding tissues, and vascular endothelial growth factor (VEGF) expression in the meniscus were assessed at 4, 8, and 12 weeks after allograft or autologous transplantation. RESULTS: The grafted meniscus was in good condition and was well connected to the surrounding joint capsule, and no obvious damage of the joint cartilage at the tibial plateau was observed. Even though the revascularization pattern was similar in the 2 groups, the meniscus body showed vessel growth mainly at the adhesion margin for less than one-third of the meniscus transverse diameter, and no significant vascular distribution was found at the free margin. Blood circulation peaked after 8 weeks at the anterior and posterior horns and declined thereafter. This was mimicked by VEGF expression, which showed a progressive decrease with time, even though the vascular endothelial cells gradually increased over time. There were no statistical differences in the various assessments between the allograft and autograft groups. CONCLUSION: At 12 weeks after meniscus allografting, the vascular circulation had almost recovered and gradual reconstruction of cells and fibers had begun, mimicking similar observations in the autograft group. CLINICAL RELEVANCE: Our data provide test reference for clinical rehabilitation after meniscus autograft.

An evaluation of meniscal collagenous structure using optical projection tomography.

BACKGROUND: The collagenous structure of menisci is a complex network of circumferentially oriented fascicles and interwoven radially oriented tie-fibres. To date, examination of this micro- architecture has been limited to two-dimensional imaging techniques. The purpose of this study was to evaluate the ability of the three-dimensional imaging technique; optical projection tomography (OPT), to visualize the collagenous structure of the meniscus. If successful, this technique would be the first to visualize the macroscopic orientation of collagen fascicles in 3-D in the meniscus and could further refine load bearing mechanisms in the tissue. OPT is an imaging technique capable of imaging samples on the meso-scale (1-10 mm) at a micro-scale resolution. The technique, similar to computed tomography, takes two-dimensional images of objects from incremental angles around the object and reconstructs them using a back projection algorithm to determine three-dimensional structure. METHODS: Bovine meniscal samples were imaged from four locations (outer main body, femoral surface, tibial surface and inner main body) to determine the variation in collagen orientation throughout the tissue. Bovine stifles (n = 2) were obtained from a local abattoir and the menisci carefully dissected. Menisci were fixed in methanol and subsequently cut using a custom cutting jig (n = 4 samples per meniscus). Samples were then mounted in agarose, dehydrated in methanol and subsequently cleared using benzyl alcohol benzyl benzoate (BABB) and imaged using OPT. RESULTS: Results indicate circumferential, radial and oblique collagenous orientations at the contact surfaces and in the inner third of the main body of the meniscus. Imaging identified fascicles ranging from 80-420 µm in diameter. Transition zones where fascicles were found to have a woven or braided appearance were also identified. The outer-third of the main body was composed of fascicles oriented predominantly in the circumferential direction. Blood vessels were also visualized using this technique, as their elastin content fluoresces more brightly than collagen at the 425 nm wavelength used by the OPT scanner. CONCLUSIONS: OPT was capable of imaging the collagenous structure, as well as blood vessels in the bovine meniscus. Collagenous structure variability, including transition zones between structural regions not previously described in the meniscus, was identified using this novel technique.

A novel hypothesis: the application of platelet-rich plasma can promote the clinical healing of white-white meniscal tears.

The white-white tears (meniscus lesion completely in the avascular zone) are without blood supply and theoretically cannot heal. Basal research has demonstrated that menisci are unquestionably important in load bearing, load redistribution, shock absorption, joint lubrication and the stabilization of the knee joint. It has been proven that partial or all-meniscusectomy results in an accelerated degeneration of cartilage and an increased rate of early osteoarthritis. Knee surgeons must face the difficult decision of removing or, if possible, retaining the meniscus; if it is possible to retain the meniscus, surgeons must address the difficulties of meniscal healing. Some preliminary approaches have progressed to improve meniscal healing. However, the problem of promoting meniscal healing in the avascular area has not yet been resolved. The demanding nature of the approach as well as its low utility and efficacy has impeded the progress of these enhancement techniques. Platelet-rich plasma (PRP) is a platelet concentration derived from autologous blood. In recent years, PRP has been used widely in preclinical and clinical applications for bone regeneration and wound healing. Therefore, we hypothesize that the application of platelet-rich plasma for white-white meniscal tears will be a simple and novel technique of high utility in knee surgery.

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis upon destabilization of the medial meniscus.

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.

Biological enhancement of meniscus repair and replacement.

When a meniscus injury occurs, it is generally accepted that preserving the meniscus is important for life-long joint preservation. Traditional suture repair of the meniscus has good results; however, the healing potential of meniscus tissue remains as a biological challenge because it is not a completely vascularized structure. For this reason, investigators have continued to search for adjuncts to improve clinical results. Mechanical adjuncts, local factor enhancement, scaffolds, gene therapy, and cell therapy have all been examined as options for improvement of biology and structure. This study reviews the basic science and clinical application of these modalities and provides an assessment of techniques on the horizon.

Cell study of the three areas of the meniscus: effect of growth factors in an experimental model in sheep.

Meniscus had two areas with different vascular supply. Cells of the two areas and the synovium were monolayer cultivated. We analyzed the expression of genes of Col1, Col 2A, MMP-2, MMP-13, and aggrecan in a baseline state and after incubation with VEGF, TGF-ß, FGF, and IGF. We found that the growth factors used produced a major increase in the MMP-13 in all three areas. In the vascular area, the stimulation of MMP-3 was produced by FGF, while in the synovial and avascular areas, it was caused by TGF-ß. MMP-2 was only stimulated in the synovial area by IGF. Col 2A was stimulated in the synovial area by VEGF, and in the avascular area by TGF-ß, FGF, and IGF, whereas Col 1 was stimulated in the avascular area by IGF, FGF, and VEGF. The vascular or avascular areas of the meniscus, behave differently in terms of repair, and their cells express different factors. The growth factors act in a different way in each meniscal area.

Increased vascular penetration and nerve growth in the meniscus: a potential source of pain in osteoarthritis.

OBJECTIVES: Meniscal damage is a recognised feature of knee osteoarthritis (OA), although its clinical relevance remains uncertain. This study describes vascular penetration and nerve growth in human menisci, providing a potential mechanism for the genesis of pain in knee OA. METHODS: Menisci obtained post mortem were screened on the basis of high or low macroscopic tibiofemoral chondropathy as a measure of the presence and degree of OA. Forty cases (20 per group) were selected for the study of meniscal vascularity, and 16 (eight per group) for the study of meniscal innervation. Antibodies directed against α-actin and calcitonin gene-related peptide (CGRP) were used to localise blood vessels and nerves by histochemistry. Image analysis was used to compare vascular and nerve densities between groups. Data are presented as median (IQR). RESULTS: Menisci from knees with high chondropathy displayed degeneration of collagen bundles in their outer regions, which were more vascular than the inner regions, with an abrupt decrease in vascularity at the fibrocartilage junction. Vascular densities were increased in menisci from the high compared with low chondropathy group both in the synovium (3.8% (IQR 2.6-5.2), 2.0% (IQR 1.4-2.9), p=0.002) and at the fibrocartilage junction (2.3% (IQR 1.7-3.1), 1.1% (IQR 0.8-1.9), p=0.003), with a greater density of perivascular sensory nerve profiles in the outer region (high chondropathy group, 144 nerve profiles/mm(2) (IQR 134-189); low chondropathy group, 119 nerve profiles/mm(2) (IQR 104-144), p=0.049). CONCLUSION: Tibiofemoral chondropathy is associated with altered matrix structure, increased vascular penetration, and increased sensory nerve densities in the medial meniscus. The authors suggest therefore that angiogenesis and associated sensory nerve growth in menisci may contribute to pain in knee OA.