Short-term response of primary human meniscus cells to simulated microgravity.

BACKGROUND: Mechanical unloading of the knee articular cartilage results in cartilage matrix atrophy, signifying the osteoarthritic-inductive potential of mechanical unloading. In contrast, mechanical loading stimulates cartilage matrix production. However, little is known about the response of meniscal fibrocartilage, a major mechanical load-bearing tissue of the knee joint, and its functional matrix-forming fibrochondrocytes to mechanical unloading events. METHODS: In this study, primary meniscus fibrochondrocytes isolated from the inner avascular region of human menisci from both male and female donors were seeded into porous collagen scaffolds to generate 3D meniscus models. These models were subjected to both normal gravity and mechanical unloading via simulated microgravity (SMG) for 7 days, with samples collected at various time points during the culture. RESULTS: RNA sequencing unveiled significant transcriptome changes during the 7-day SMG culture, including the notable upregulation of key osteoarthritis markers such as COL10A1, MMP13, and SPP1, along with pathways related to inflammation and calcification. Crucially, sex-specific variations in transcriptional responses were observed. Meniscus models derived from female donors exhibited heightened cell proliferation activities, with the JUN protein involved in several potentially osteoarthritis-related signaling pathways. In contrast, meniscus models from male donors primarily regulated extracellular matrix components and matrix remodeling enzymes. CONCLUSION: These findings advance our understanding of sex disparities in knee osteoarthritis by developing a novel in vitro model using cell-seeded meniscus constructs and simulated microgravity, revealing significant sex-specific molecular mechanisms and therapeutic targets.

Definition of Synovial Mesenchymal Stem Cells for Meniscus Regeneration by the Mechanism of Action and General Amp1200 Gene Expression.

The quality control (QC) of pharmaceutical-grade cell-therapy products, such as mesenchymal stem cells (MSCs), is challenging. Attempts to develop such products have been hampered by difficulties defining cell-type-specific characteristics and therapeutic mechanisms of action (MoAs). Although we have developed a cell therapy product, FF-31501, consisting of human synovial MSCs (SyMSCs), it was difficult to find specific markers for SyMSCs and to define the cells separately from other MSCs. The purpose of this study was to create a method for identifying and defining SyMSCs from other tissue-derived MSCs and to delve deeper into the mechanism of action of SyMSC-induced meniscus regeneration. Specifically, as a cell-type-dependent approach, we constructed a set of 1143 genes (Amp1200) reported to be associated with MSCs and established a method to evaluate them by correlating gene expression patterns. As a result, it was possible to define SyMSCs separately from other tissue-derived MSCs and non-MSCs. In addition, the gene expression analysis also highlighted TNSF-15. The in vivo rat model of meniscus injury found TNSF-15 to be an essential molecule for meniscus regeneration via SyMSC administration. This molecule and previously reported MoA molecules allowed an MoA-dependent approach to define the mechanism of action for SyMSCs. Therefore, SyMSCs for meniscus regeneration were defined by means of two approaches: the method to separate them from other MSCs and the identification of the MoA molecules. These approaches would be useful for the QC of cell therapy products.

Effect of cell seeding density on matrix-forming capacity of meniscus fibrochondrocytes and nasal chondrocytes in meniscus tissue engineering.

The presence of intact menisci is imperative for the proper function of the knee joint. Meniscus injuries are often treated by the surgical removal of the damaged tissue, which increases the likelihood of post-traumatic osteoarthritis. Tissue engineering holds great promise in producing viable engineered meniscal tissue for implantation using the patient's own cells; however, the cell source for producing the engineered tissue is unclear. Nasal chondrocytes (NC) possess many attractive features for engineering meniscus. However, in order to validate the use of NC for engineering meniscus fibrocartilage, a thorough comparison of NC and meniscus fibrochondrocytes (MFC) must be considered. Our study presents an analysis of the relative features of NC and MFC and their respective chondrogenic potential in a pellet culture model. We showed considerable differences in the cartilage tissue formed by the two different cell types. Our data showed that NC were more proliferative in culture, deposited more extracellular matrix, and showed higher expression of chondrogenic genes than MFC. Overall, our data suggest that NC produce superior cartilage tissue to MFC in a pellet culture model. In addition, NCs produce higher quality cartilage tissue at higher cell seeding densities during cell expansion.

Precision 3D printed meniscus scaffolds to facilitate hMSCs proliferation and chondrogenic differentiation for tissue regeneration.

BACKGROUND: The poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue. RESULTS: Herein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility. CONCLUSIONS: Overall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration.

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.

Potential of Melt Electrowritten Scaffolds Seeded with Meniscus Cells and Mesenchymal Stromal Cells.

Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants® (CMI®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young's modulus when compared to the CMI® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment.

Hypoxia as a Stimulus for the Maturation of Meniscal Cells: Highway to Novel Tissue Engineering Strategies?

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.

Selection of Highly Proliferative and Multipotent Meniscus Progenitors through Differential Adhesion to Fibronectin: A Novel Approach in Meniscus Tissue Engineering.

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.

Degeneration of the articular disc in the human triangular fibrocartilage complex.

INTRODUCTION: Traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent reasons for ulnar wrist pain. The assessment of the extent of articular disc (AD) degeneration is important for the differentiation of acute injuries versus chronic lesions. MATERIALS AND METHODS: The AD of the TFCC of eleven human cadaver wrists was dissected. Degeneration was analyzed according to the grading of Krenn et al. Hematoxylin-eosin was used to determine the tissue morphology. Degeneration was evaluated using the staining intensity of alcian blue, the immunohistochemistry of the proteoglycan versican and the immunoreactivity of NITEGE, an aggrecan fragment. RESULTS: The staining homogeneity of HE decreased with higher degeneration of the AD and basophilic tissue areas were more frequently seen. Two specimens were characterized as degeneration grade 1, five specimens as grade 2, and four specimens as grade 3, respectively. Staining intensity of alcian blue increased with higher degeneration grade of the specimens. Immunoreactivity for NITEGE was detected around tissue fissures and perforations as well as matrix splits. Immunoreactivity for versican was found concentrated in the tissue around matrix fissures and lesions as well as loose connective tissue at the ulnar border of the AD. Specimens with degeneration grade 2 had the strongest immunoreactivity of NITEGE and versican. Cell clusters were observed in specimens with degeneration grade 2 and 3, which were stained by alcian blue and immunoreactive for NITEGE and versican. Increasing age of the cadaver wrists correlated with a higher degree of degeneration (p < 0.0001, r = 0.68). CONCLUSIONS: The fibrocartilage of degenerated ADs contains NITEGE and versican. The amount of the immunoreactivity of these markers allows the differentiation of degenerative changes into three grades. The degeneration of the AD increases with age and emphasizes its important mechanical function.

Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration.

OBJECTIVES: The heterogeneity of meniscus cells and the mechanism of meniscus degeneration is not well understood. Here, single-cell RNA sequencing (scRNA-seq) was used to identify various meniscus cell subsets and investigate the mechanism of meniscus degeneration. METHODS: scRNA-seq was used to identify cell subsets and their gene signatures in healthy human and degenerated meniscus cells to determine their differentiation relationships and characterise the diversity within specific cell types. Colony-forming, multi-differentiation assays and a mice meniscus injury model were used to identify meniscus progenitor cells. We investigated the role of degenerated meniscus progenitor (DegP) cell clusters during meniscus degeneration using computational analysis and experimental verification. RESULTS: We identified seven clusters in healthy human meniscus, including five empirically defined populations and two novel populations. Pseudotime analysis showed endothelial cells and fibrochondrocyte progenitors (FCP) existed at the pseudospace trajectory start. Melanoma cell adhesion molecule ((MCAM)/CD146) was highly expressed in two clusters. CD146+ meniscus cells differentiated into osteoblasts and adipocytes and formed colonies. We identified changes in the proportions of degenerated meniscus cell clusters and found a cluster specific to degenerative meniscus with progenitor cell characteristics. The reconstruction of four progenitor cell clusters indicated that FCP differentiation into DegP was an aberrant process. Interleukin 1ß stimulation in healthy human meniscus cells increased CD318+ cells, while TGFß1 attenuated the increase in CD318+ cells in degenerated meniscus cells. CONCLUSIONS: The identification of meniscus progenitor cells provided new insights into cell-based meniscus tissue engineering, demonstrating a novel mechanism of meniscus degeneration, which contributes to the development of a novel therapeutic strategy.

Meniscus regeneration combining meniscus and mesenchymal stromal cells in a degradable meniscus implant: an in vitro study.

Meniscus regeneration is an unmet clinical need as damage to the meniscus is common and causes early osteoarthritis. The aim of the present study was to investigate the feasibility of a one-stage cell-based treatment for meniscus regeneration by augmenting a resorbable collagen-based implant with a combination of recycled meniscus cells and mesenchymal stromal cells (MSCs). Cell communication and fate of the different cell types over time in co-culture were evaluated by connexin 43 staining for gap junctions and polymerase chain reaction (PCR) to discriminate between meniscus cells and MSCs, based on a Y-chromosome gene. To define optimal ratios, human meniscus cells and bone-marrow-derived MSCs were cultured in different ratios in cell pellets and type I collagen hydrogels. In addition, cells were seeded on the implant in fibrin glue by static seeding or injection. Cellular communication by gap junctions was shown in co-culture and a decrease in the amount of MSCs over time was demonstrated by PCR. 20 : 80 and 10 : 90 ratios showed significantly highest glycosaminoglycan and collagen content in collagen hydrogels. The same statistical trend was found in pellet cultures. Significantly more cells were present in the injected implant and cell distribution was more homogenous as compared to the statically seeded implant. The study demonstrated the feasibility of a new one-stage cell-based procedure for meniscus regeneration, using 20 % meniscus cells and 80 % MSCs seeded statically on the implant. In addition, the stimulatory effect of MSCs towards meniscus cells was demonstrated by communication through gap junctions.

Hyaluronic acid promotes proliferation and migration of human meniscus cells via a CD44-dependent mechanism.

PURPOSE: Treatment of meniscal injury is important for osteoarthritis (OA) prevention. Meniscus cells are divided between inner and outer cells, which have different characteristics and vascularity. We evaluated the effects of hyaluronic acid (HA) on the proliferation and migration of human inner and outer meniscus cells, and investigated the underlying healing mechanisms. MATERIALS AND METHODS: Lateral menisci from 18 patients who underwent total knee arthroplasty were used. Meniscus cells were harvested from the outer and inner menisci and evaluated using migration and proliferation assays after treatment with HA or chondroitin sulfate (CS). The effects of HA on prostaglandin E2 (PGE2)-induced apoptosis and gene expression were evaluated. RESULTS: Cell migration and proliferation were increased by HA in a concentration-dependent manner, in both inner and outer meniscus cells. PGE2-induced apoptosis and caspase-3/7 activity were suppressed by HA in both inner and outer meniscus cells, and these effects were blocked by an anti-CD44 antibody. COL2A1 and ACAN mRNA levels were upregulated following HA treatment of inner meniscus cells. MMP13 mRNA was downregulated following CS stimulation of both inner and outer meniscus cells. These results suggest that CS treatment suppresses the inflammatory reaction rather than providing meniscal restoration. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways were activated by HA in both types of meniscus cells; these effects were blocked by treatment with an anti-CD44 antibody. CONCLUSIONS: HA promoted human meniscus regeneration by inhibiting apoptosis, promoting cell migration, and accelerating cell proliferation, potentially through the PI3K/MAPK pathway via the CD44 receptor.

Suturable regenerated silk fibroin scaffold reinforced with 3D-printed polycaprolactone mesh: biomechanical performance and subcutaneous implantation.

The menisci have crucial roles in the knee, chondroprotection being the primary. Meniscus repair or substitution is favored in the clinical management of the meniscus lesions with given indications. The outstanding challenges with the meniscal scaffolds include the required biomechanical behavior and features. Suturability is one of the prerequisites for both implantation and implant survival. Therefore, we proposed herein a novel highly interconnected suturable porous scaffolds from regenerated silk fibroin that is reinforced with 3D-printed polycaprolactone (PCL) mesh in the middle, on the transverse plane to enhance the suture-holding capacity. Results showed that the reinforcement of the silk fibroin scaffolds with the PCL mesh increased the suture retention strength up to 400%, with a decrease in the mean porosity and an increase in crystallinity from 51.9 to 55.6%. The wet compression modulus values were significantly different for silk fibroin, and silk fibroin + PCL mesh by being 0.16 ± 0.02, and 0.40 ± 0.06 MPa, respectively. Both scaffolds had excellent interconnectivity (>99%), and a high water uptake feature (>500%). The tissue's infiltration and formation of new blood vessels were assessed by means of performing an in vivo subcutaneous implantation of the silk fibroin + PCL mesh scaffolds that were seeded with primary human meniscocytes or stem cells. Regarding suturability and in vivo biocompatibility, the findings of this study indicate that the silk fibroin + PCL mesh scaffolds are suitable for further studies to be carried out for meniscus tissue engineering applications such as the studies involving orthotopic meniscal models and fabrication of patient-specific implants.

Meniscus-Derived Matrix Bioscaffolds: Effects of Concentration and Cross-Linking on Meniscus Cellular Responses and Tissue Repair.

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing.

Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy.

Carbon nanofiber amalgamated 3D poly-ε-caprolactone scaffold functionalized porous-nanoarchitectures for human meniscal tissue engineering: In vitro and in vivo biocompatibility studies.

We developed customizable biomolecule functionalized 3D poly-ε-caprolactone (PCL) scaffolds reinforced with carbon nanofibers (CNF) for human meniscal tissue engineering. 3D nanocomposite scaffolds exhibited commendable mechanical integrity and electrical properties with augmented cytocompatibility. Especially, the functionalized 3D (10wt% CNF) scaffolds showed ~363% increase in compressive moduli compared to the pristine PCL. In dynamic mechanical analysis, these scaffolds achieved highest value (~42 MPa at 10 Hz) among all tested scaffolds including pristine PCL and human menisci (33, 41, 56 years). In vitro results were well supported by the outcomes of cell proliferation analysis, microscopic images, Hoechst staining and extracellular-matrix estimation. Further, in vivo rabbit bio toxicity studies revealed scaffold's non-toxicity and its future potential as a meniscus scaffold. This study also indicates that the incorporation of CNF in polymer matrix may be optimized based on mechanical properties of patient meniscus and it may help in developing the customized patient specific 3D constructs with improved multifunctional properties.

Current advances in the development of natural meniscus scaffolds: innovative approaches to decellularization and recellularization.

The increasing rate of injuries to the meniscus indicates the urgent need to develop effective repair strategies. Irreparably damaged menisci can be replaced and meniscus allografts represent the treatment of choice; however, they have several limitations, including availability and compatibility. Another approach is the use of artificial implants but their chondroprotective activities are still not proved clinically. In this situation, tissue engineering offers alternative natural decellularized extracellular matrix (ECM) scaffolds, which have shown biomechanical properties comparable to those of native menisci and are characterized by low immunogenicity and promising regenerative potential. In this article, we present an overview of meniscus decellularization methods and discuss their relative merits. In addition, we comparatively evaluate cell types used to repopulate decellularized scaffolds and analyze the biocompatibility of the existing experimental models. At present, acellular ECM hydrogels, as well as slices and powders, have been explored, which seems to be promising for partial meniscus regeneration. However, their inferior biomechanical properties (compressive and tensile stiffness) compared to natural menisci should be improved. Although an optimal decellularized meniscus scaffold still needs to be developed and thoroughly validated for its regenerative potential in vivo, we believe that decellularized ECM scaffolds are the future biomaterials for successful structural and functional replacement of menisci.

Identification and characterization of adult mouse meniscus stem/progenitor cells.

Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

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 origin and distribution of CD68, CD163, and αSMA+ cells in the early phase after meniscal resection in a parabiotic rat model.

We previously reported that circulating peripheral blood-borne cells (PBCs) contribute to early-phase meniscal reparative change. Because macrophages and myofibroblasts are important contributors of tissue regeneration, we examined their origin and distribution in the reparative meniscus. Reparative menisci were evaluated at 1, 2, and 4 weeks post-meniscectomy by immunohistochemistry to locate monocytes and macrophages (stained positive for CD68 and CD163), and myofibroblasts (stained positive for αSMA). Of the total number of cells, 13% were CD68+ at 1 week post-meniscectomy, which decreased to 1% by 4 weeks post-meniscectomy; of these, almost half of CD68+ cells (49.4%: 98.8% as PBCs) were green fluorescent protein (GFP)-positive post-meniscectomy (1, 2, and 4 weeks), indicating that the majority of CD68+ cells were derived from PBCs. Of the total cells, 6% were CD163+ at 1 week post-meniscectomy, which decreased to 1% by week 4. Of the CD163+ cells, the majority were GFP-positive (42.5%: 85.0% as PBCs) after 1 week; however, this decreased significantly over time, which indicates that the majority of CD163+ cells are derived from PBCs during the early phase of meniscal reparative change, but are derived from resident cells at later time points. Of the total cells, 38% were αSMA+ at 1 week post-meniscectomy, which decreased to 3% by 4 weeks. The proportion of GFP-positive αSMA+ cells was 2.8% after 1 week, with no significant change over time, which indicates that the majority of αSMA+ cells originated from resident cells. Here, we describe the origin and distribution of macrophages and myofibroblasts during meniscal reparative change.