Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus.

PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

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.

Engineered Healing of Avascular Meniscus Tears by Stem Cell Recruitment.

Meniscus injuries are extremely common with approximately one million patients undergoing surgical treatment annually in the U.S. alone. Upon injury, the outer zone of the meniscus can be repaired and expected to functionally heal but tears in the inner avascular region are unlikely to heal. To date, no regenerative therapy has been proven successful for consistently promoting healing in inner-zone meniscus tears. Here, we show that controlled applications of connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFß3) can induce seamless healing of avascular meniscus tears by inducing recruitment and step-wise differentiation of synovial mesenchymal stem/progenitor cells (syMSCs). A short-term release of CTGF, a selected chemotactic and profibrogenic cue, successfully recruited syMSCs into the incision site and formed an integrated fibrous matrix. Sustain-released TGFß3 then led to a remodeling of the intermediate fibrous matrix into fibrocartilaginous matrix, fully integrating incised meniscal tissues with improved functional properties. Our data may represent a novel clinically relevant strategy to improve healing of avascular meniscus tears by recruiting endogenous stem/progenitor cells.

The nutrition of the human meniscus: A computational analysis investigating the effect of vascular recession on tissue homeostasis.

The meniscus is essential to the functioning of the knee, offering load support, congruency, lubrication, and protection to the underlying cartilage. Meniscus degeneration affects ∼35% of the population, and potentially leads to knee osteoarthritis. The etiology of meniscal degeneration remains to be elucidated, although many factors have been considered. However, the role of nutritional supply to meniscus cells in the pathogenesis of meniscus degeneration has been so far overlooked. Nutrients are delivered to meniscal cells through the surrounding synovial fluid and the blood vessels present in the outer region of the meniscus. During maturation, vascularization progressively recedes up to the outer 10% of the tissue, leaving the majority avascular. It has been hypothesized that vascular recession might significantly reduce the nutrient supply to cells, thus contributing to meniscus degeneration. The objective of this study was to evaluate the effect of vascular recession on nutrient levels available to meniscus cells. This was done by developing a novel computational model for meniscus homeostasis based on mixture theory. It was found that transvascular transport of nutrients in the vascularized region of the meniscus contributes to more than 40% of the glucose content in the core of the tissue. However, vascular recession does not significantly alter nutrient levels in the meniscus, reducing at most 5% of the nutrient content in the central portion of the tissue. Therefore, our analysis suggests that reduced vascularity is not likely a primary initiating source in tissue degeneration. However, it does feasibly play a key role in inability for self-repair, as seen clinically.

Use of intravoxel incoherent motion diffusion-weighted imaging in identifying the vascular and avascular zones of human meniscus.

PURPOSE: To investigate the hypothesis that the intravoxel incoherent motion (IVIM) diffusion-weighted imaging may depict microcirculation of meniscus and the perfusion changes in meniscal disorder. MATERIALS AND METHODS: Fifty patients received diffusion-weighted MRI with multiple b-values ranging from 0 to 400 s/mm2 . The four horns of the menisci were divided into normal, degenerated, and torn groups. IVIM parameters including perfusion fraction (f), pseudo-diffusion coefficient (D*), true diffusion coefficient (D), and the product of f and D* (f D*) of normal meniscal red zone and white zone were derived and compared for microcirculation changes of normal, degenerated, and torn posterior horn of the medial meniscus (PMM). The parameters between red and white zones among the groups were compared. Significant differences were considered when P < 0.05. RESULTS: Mean f and fD* were significantly higher in the red zone than those in the white zone for the normal four meniscal horns (P < 0.05), whereas D* (P = 0.882, 0.011, 0.593, and 0.33) and D (P = 0.186, 0.099, 0.767, and 0.041) did not significantly differ between the two zones. Among the normal, degenerated, and torn PMM, f was observed to be lower in the red zone of torn horns as compared to the normal horns (P = 0.013). D*, fD*, and D did not exhibit statistically significant difference among different groups (P = 0.353, 0.661, and 0.327, respectively). CONCLUSION: This hypothesis driven work shows that IVIM imaging is able to depict microcirculation of meniscus and the perfusion changes in meniscal disorder. LEVEL OF EVIDENCE: 3 J. Magn. Reson. Imaging 2017;45:1090-1096.

Relevance of meniscal cell regional phenotype to tissue engineering.

PURPOSE: Meniscus contains heterogeneous populations of cells that have not been fully characterized. Cell phenotype is often lost during culture; however, culture expansion is typically required for tissue engineering. We examined and compared cell-surface molecule expression levels on human meniscus cells from the vascular and avascular regions and articular chondrocytes while documenting changes during culture-induced dedifferentiation. MATERIALS AND METHODS: Expressions of 16 different surface molecules were examined by flow cytometry after monolayer culture for 24 h, 1 week, and 2 weeks. Menisci were also immunostained to document the spatial distributions of selected surface molecules. RESULTS: Meniscus cells and chondrocytes exhibited several similarities in surface molecule profiles with dynamic changes during culture. A greater percentage of meniscal cells were positive for CD14, CD26, CD49c, and CD49f compared to articular chondrocytes. Initially, more meniscal cells from the vascular region were positive for CD90 compared to cells from the avascular region or chondrocytes. Cells from the vascular region also expressed higher levels of CD166 and CD271 compared to cells from the avascular region. CD90, CD166, and CD271-positive cells were predominately perivascular in location. However, CD166-positive cells were also located in the superficial layer and in the adjacent synovial and adipose tissue. CONCLUSIONS: These surface marker profiles provide a target phenotype for differentiation of progenitors in tissue engineering. The spatial location of progenitor cells in meniscus is consistent with higher regenerative capacity of the vascular region, while the surface progenitor subpopulations have potential to be utilized in tears created in the avascular region.

The meniscus vascularization: the direct correlation with tissue composition for tissue engineering purposes.

Meniscal lesions still represent an unsolved problem in clinical practice. Like the articular cartilage, meniscus has a scarce healing potential. Thus, when this tissue is damaged, the joint biomechanics is completely altered, leading to the development and progression of premature osteoarthritis. Therefore, in the last years, several tissue-engineering strategies have been developed to regenerate the meniscus with debated results. The comprehension of complex processes underlying meniscus maturation and structure is essential for a correct approach for the generation of a biomimetic meniscal substitute. In this chapter, we will first review the morphology of the meniscus during growth, focusing on the unique pattern of vascularization, and then we will discuss the most common tissue engineering strategies for meniscus repair.