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

Abstract

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.

Prognosis following meniscal injuries is highly variable depending on the size and location of the tear. Some reports state that if the lesion communicates with the peripheral one-third of the meniscus, increased vascularity may help it heal and therefore are more amenable for repairs.¹ Conversely, injuries in the “avascular” zone are almost always resected due to their low potential success rate. Until recently, it was believed that resecting a small percentage of meniscus would not significantly impact joint longevity. However, a direct relationship between the amount of meniscus resected and the presence/severity of chondral lesions in the ipsilateral knee compartment in prospective National Football League players with a previous medial and/or lateral meniscectomy has previously been reported.² Meniscectomies have been reported to significantly reduce the career lengths of professional athletes,³ whereas repairs carry high success rates at long-term follow-up.⁴ Yet, since there are no randomized controlled trials to compare meniscal repair with resection, it is not entirely clear whether repair or resection would be favorable on a case- by-case basis.⁵ The resident stromal progenitor cell population and the vascularization of the inner meniscus are not defined precisely in the literature and therefore strategies for repair might be better informed if a more consistent approach was used to characterize them.

Unlike highly vascularized bone tissue, fibrocartilaginous tissue has relatively limited self-repair capacity. Previous studies suggest healing of inner meniscal tears can be enhanced through progenitor cell mobilization⁶ and recruitment from the synovium, followed by formation of an intermediate fibrous integration and cartilaginous remodeling.⁷ Kobayashi et al.⁸ used an in vitro organ culture model of freshly prepared defects to investigate the healing potential of the rabbit meniscus without the influence of vascular supply. The authors found that grafts integrated better in the peripheral outer region of the meniscus, suggesting that the endogenous cellular composition of the meniscus may play a role in the local healing response. Progenitor cells have been identified in the menisci of goats, rabbits, and more recently in humans,⁹ which suggests an inherent healing capacity. Mauck et al.¹⁰ reported that resident meniscal fibrochondrocytes from all regions of the meniscus possess a multilineage differentiation capability, particularly toward chondrogenesis and adipogenesis in calf menisci. Since it is well established that mechanical cues affect the development and maturation of the cellular milieu,¹¹ it is important to better understand the cellular content of human menisci. The progenitor content between the different meniscal zones has not been investigated in adult human menisci, most likely because of the scarcity of fresh human grafts made available for research purposes.

The intrinsic healing capacity of the meniscus is considered limited due to a poor blood supply that only reaches the periphery of the meniscus.¹² King¹³ was the first to suggest that tears extending to the vascular periphery undergo spontaneous repair, whereas tears limited to the inner region do not. Seminal anatomical studies performed on human cadaveric menisci in the 1980s using injection techniques have established the current paradigm of meniscus vascularity.¹ However, there is controversy in the literature regarding the specific topology of meniscus vasculature as well as the specific timeline when the vasculature undergoes developmental changes.^12,14 Therefore, the purpose of this study was to identify, characterize, and compare the resident progenitor cell populations within the red‒red (RR), red‒white (RW) 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. We hypothesized that microvessels and resident progenitor cells in the inner zone of meniscus would be more prominent than previously reported.

Methods

Study Design

The cadaveric studies were conducted according to the approved institutional review board protocol (Pro00052234). Menisci from fresh human cadaveric knees (mean donor age: 21 ± 6.1 years) were donated by 3 tissue banks (Joint Restoration Foundation [JRF], Centennial, CO, Musculoskeletal Transplant Foundation [MTF], Edison, NJ, and BioSource Medical, Lakeland, FL) for medical research purposes, as they were deemed non-compatible with the recipient at the time of matching. Grafts were stored at 4°C at the tissue banks until shipment according to standard procedures and were shipped under sterile conditions on ice, with the same procedure followed for allografts used in the clinic. Allografts for meniscal trans-plantation have a 21-day window; thus, all donated grafts were between 1 and 21 days post-harvest.¹⁵ Specimens were assessed by at least 2 investigators (AP and DS). In our preliminary studies we have determined that the viability of mononuclear cells significantly declined after 7 days post-harvest (data not shown), thus grafts greater than 7 days post-harvest were allocated to histological analysis and uDisco experiments, whereas only fresh grafts were allocated to mononuclear cell isolations and characterization. A total of 34 allografts from 17 different donors (including medial and lateral) were used in this study. Tibial plateaus were dissected to harvest medial and lateral menisci along with their entire length, preserving 1 mm of their peripheral capsular attachments (Fig 1). Fourteen menisci from 7 different donors were used for cell isolation and characterization. Twenty meniscal allografts from 10 different donors were used for assessing vascularity using histology and the modified uDisco 3D staining approach.¹⁶ In this work, meniscal zones on cadaveric menisci were identified using the Cooper classification system.¹⁴ For the cell isolation/characterization experiments, zones were identified on cadavers and sectioned, whereas for the 2-dimensional (2D)/3D immunolabeling experiments, meniscal slices were sectioned, imaged as whole slide scans or 3D images, and software was used to establish the distance between each zone (Fig 1, available at www.arthroscopyjournal.org).¹⁷

Fig 1.

Research design. (1) The first aim of this study was to characterize and identify the resident stromal progenitor cell population in all 3 zones in freshly harvested human cadaveric menisci. (2) The second aim was to characterize the vascularity of the menisci, using histology, immunofluorescence (IF), and 3-dimensional (3D) light sheet fluorescence microscopy. (aSMA, alpha smooth muscle actin; CFU, colony-forming unit; H&E, hematoxylin and eosin; MSC, mesenchymal stromal cell; MTC, Masson’s trichrome.)

Mesenchymal Stromal Progenitor Cell Prevalence: Identification, Quantification, and Characterization

Cell Isolation From Fresh Meniscus Grafts

The RR, RW, and WW zones were dissected and sectioned into equal thirds as measured by a caliper from the inner aspect to the marginal border of the meniscus by cutting in the radial direction, to replicate the clinical setting and to aid in a more standardized sectioning technique (Fig 2A). A sterile gauge was used to remove excess media. Meniscal tissue was then placed in sterile tubes and wet weights were recorded. Tissue was manually minced to ~1-mm² pieces in a sterile environment and then enzymatically digested. Since it is well established that meniscus tissue cellular content varies between each zone,^18,19 several dissociation procedures were tested and the procedure yielding the greatest cellular content for all zones was chosen. Greatest yields were obtained from dissociation of meniscus tissue in 0.02% pronase (Millipore, Temecula, CA) for 1 hour at 37°C, followed by 18 hours 0.02% collagenase II (LS004205; Worthington Biochemical Corporation, Lakewood, NJ) at 37°C²⁰; therefore, this procedure was chosen for meniscal cell content characterizations. Isolated cells were plated in culture-treated plates in approximately 2 × 10³ cells/cm² density and incubated overnight at 37°C in 5% CO₂. Adherent cells were cultured at 37°C/5% CO₂ in growth medium containing 1 mM L-glutamine (Invitrogen, Carlsbad, CA), 1% antibiotic antimycotic solution (HyClone, Marlborough, MA) in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Carlsbad, CA), and 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA). Following the recommendations regarding nomenclature and mesenchymal stem/progenitor cell characterization from the International Society for Cell Therapy (ISCT), progenitor cells from the meniscus are referred to as mesenchymal stromal cells and cultured meniscal cells (CMCs) in this manuscript and they are clearly distinguished from bone marrow-derived mesenchymal stromal cells (BM-MSCs) that were used as controls for the various in vitro assays.^21,22 BM-MSCs were isolated from human bone marrow aspirate (Lonza, Benicia, CA) as previously described.^23–25

Fig 2.

Evidence of resident stromal progenitor cells in the WW of human meniscus. (A) Dissection of meniscal zones for cell isolation. (B) Box and whisker plots of cell yields of medial or lateral menisci normalized to tissue wet weight. Lines display median values. (C) Colony formation of isolated cells in vitro. Meniscal cells from all zones were clonogenic in culture. No significant differences were found between zones when assessed by colony-forming unit assays (P > .05). (LL, left lateral (meniscus); LM, left medial (meniscus); RR, red‒red; RW, red‒white; WW, white‒white.)

Self-Renewal Assessment (Colony Forming Unit-Fibroblast Assay)

Self-renewal of CMCs was assessed using a standard colony-forming unit-fibroblast (CFU-F) assay as previously described.²⁶ In summary, cells isolated from different zones of the meniscus were separately plated onto 6-well plates at 10⁴ cells/well in culture media, with media changed twice per week. Between 7 and 14 days, medium was removed, and cells were washed with phosphate-buffered saline (PBS). Afterward, cells were fixed with 4% formaldehyde, stained with hematoxylin, and aggregates of 50 cells or more were scored as CFUs.

Meniscus Stromal Progenitor Cell Characterization and Expansion (Surface Markers)

Meniscus stromal cells were characterized immediately after isolation using flow cytometry with antibodies against the following mesenchymal stromal cell (MSC) surface markers: CD105 (326–050; Ancell Corporation, Stillwater, MN), CD90 (MCA90F; Bio-Rad, Hercules, CA), CD44 (559942; BD Pharmigen, San Diego, CA), CD29 (PB-219-T100; Abcore, Ramona, CA), as well as respective isotype controls.^21,22 Afterward, meniscal cells were cultured, split twice when confluence was reached, characterized at passage 2, and compared with cultured BM-MSCs, (≤P5; Lonza) using the same markers.

In Vitro Differentiation to Mesenchymal Linages

Osteogenesis.

For assessment of osteogenic differentiation, CMCs and BM-MSCs at low passage were plated in 24-well plates (6 × 10⁴ per well) in triplicates and cultured in complete high-glucose DMEM (Life Technologies, Carlsbad, CA). Once confluence was reached, the medium was supplemented with 100 nM dexamethasone (D4902; Sigma, St. Louis, MO), 10 mM b-glycerophosphate (G9422; Sigma) and 50 µg/mL L-ascorbic acid (A4544; Sigma) for 7 days or 21 days, with media changes every other day. Alkaline phosphatase (ALP) activity assay was performed to determine osteogenic differentiation after 7 days according to the manufacturer’s protocol (ab83369; Abcam, Cambridge, MA). Production of pNP was determined by measuring absorbance at 405 nm using a microplate reader (Bio-Rad). ALP activity (U/mL) in the test samples was calculated based on the equation: ALP activity = (B/∆T *V)*D, where B = amount of pNP in sample wells calculated from standard curve (µmol), ∆T = reaction time (minutes), V = original sample volume added into the reaction well (mL), and D = sample dilution factor. ALP activity was normalized to total protein content quantified using BCA assay (Promega, San Luis Obispo, CA) performed on parallel wells. Each sample was run in technical triplicates. Lastly, cells were cultured in differentiation media for 3 weeks and gene expression of osteogenic marker collagen-1 was evaluated. To evaluate osteogenic gene expression, cells were harvested and RNA isolated. For RNA isolation, media were aspirated, and cells lysed with RLT buffer (Qiagen, Valencia, CA) containing β-mercaptoethanol (M3148; Sigma). Lysates were transferred to 1.5-mL tubes and a handheld pestle and mortar was used to fully homogenize the cells in the RLT buffer and RNA was isolated with the RNeasy mini kit (Qiagen) following the manufacturer’s recommendations. RNA yields were determined spectrophotometrically using a Nanodrop system (Thermo Fisher Scientific, Waltham, MA), and RNA was reverse-transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific). Gene expression analysis was conducted using Taqman gene expression assay for Collagen-1 (Hs00164004_m1; Thermo Fisher Scientific). Target gene mRNA levels were quantified using FAM-MBG technology (Bio-Rad). The threshold cycle (Ct) value of 18S rRNA was used as an internal control using the Taqman gene expression FAM/MGB probe system (4333760F; Thermo Fisher Scientific). The Livak method was used to calculate ∆∆Ct values and fold change was calculated as 2^−∆∆Ct as previously described.²⁷

Adipogenesis.

For assessment of adipogenic potential, CMCs and BM-MSCs at low passage were plated in 24-well plates (6 × 10⁴ cells per well) in triplicates and cultured in complete high-glucose DMEM (Life Technologies) until they were fully confluent. Adipogenic differentiation was induced as previously described.^24,28 To summarize, the medium was changed to “induction medium” composed of complete high-glucose DMEM and supplemented with 1 µm dexamethasone (D4902; Sigma), 10 µM insulin (I6634; Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (I7018; Sigma), and 200 µm indomethacin (I8280; Sigma) for 3 days, followed by 2 to 3 days of “maintenance medium,” composed of complete high-glucose DMEM and 10 µM insulin. Cells were inspected daily for presence of adipogenic vacuoles. Five full cycles of induction followed by maintenance were performed. Finally, wells were fixed in ice-cold formalin and washed and stained with Oil-Red-O (O0625; Sigma) for 15 minutes. Cells were then washed 3 times with double distilled water and microphotographs were taken at 20× magnification using an EVOS XL Core imaging system (Thermo Fisher Scientific).

Chondrogenesis.

The chondrogenic potential of CMCs was assessed as previously described.²⁸ Cells were trypsinized, neutralized with serum-containing low glucose DMEM, and counted. Cell aliquots of 5 × 10⁵ meniscal cells or BM-MSCs at low passage were span at 240g for 5 minutes and all media carefully removed. Pellets were resuspended in serum-free low glucose media and span again at 240g for 5 minutes to ensure complete removal of serum-containing medium. Then, cells were resuspended in 100 µL of chondrogenic differentiation medium composed of low-glucose DMEM, 1 × ITS (I2521; Sigma), 0.1 mM dexamethasone (D4902; Sigma), 40 µg/mL proline (Sigma), 50 µg/mL ascorbic acid (Sigma), and 10 ng/mL TGFβ1 (240B002; R&D Systems, Minneapolis, MN). The 100-µL cell suspension was placed in Transwells to induce disc-shaped 3D formation. The transwell plate was centrifuged at 200g for 5 minutes, and then filter inserts were transferred into a 24-well plate containing differentiation medium. Chondrogenic media were changed every 2 days. After 21 days, discs were fixed in formalin for 1 hour and dehydrated in passing through an increasing-grade series of ethanol baths. Afterward, discs were embedded in paraffin blocks, cut into 5-µm sections, and stained with Alcian blue to identify chondrocytes. Whole slide scans were attained and imaged using QuPath software.

Vascularity Assessment Using Standard Histology, immunofluorescent Labeling, and 3D Imaging

Vascular Tree Histology

Samples were fixed in 10% buffered formalin. Following fixation, samples were dehydrated by passing through an increasing-grade series of ethanol baths, paraffin embedded, sectioned (4-µm thick), deparaffinized, and histologic stains performed according to standard procedures. Hematoxylin and eosin (H&E) staining was used for morphologic evaluation. Masson’s trichrome (MTC) stain was performed to further evaluate structure of the extracellular matrix and visualization of the larger vessels. QuPath quantitative pathology and image analysis software was used for imaging H&E and MTC full-slide scans as well as quantifying the distance between zones.

Characterization of Microvasculature via Immunofluorescence

Immunofluorescence was performed on formalin-fixed (7 days in formalin), paraffin-embedded tissue sections. To summarize, sections were deparaffinized, rehydrated in PBS with 0.025% Triton-X (PBS-T), treated with antigen retrieval solution at 98°C for 1 hour (pH 6.1; Dako #S1699; Agilent Technologies, Carpinteria, CA), and blocked with 10% normal donkey serum in PBS-T. Endothelial cells were detected on meniscus cross sections by CD31 (1:50 dilution; ab28364; Abcam), alpha smooth muscle actin (1:250 dilution; Abcam, cat no. ab21027), and Alexa488-conjugated lectin antibody (1:200 dilution, Dylight; cat no. DL-1174, Vector Labs, Burlingame, CA) in blocking solution, overnight at 4°C. To control for un-specific background labeling, primary antibody was omitted on background labeling controls. Then, sections were washed 3 times in PBS-T, followed by incubation with Cy5 (donkey anti-rabbit), Cy3 (donkey anti-goat) secondary antibody (1:200 dilutions, all from Jackson ImmunoResearch, Westgrove, PA) for 2 hours at room temperature. Subsequently, sections were washed 3 times in PBS-T and mounted with Prolong Gold with DAPI (Life Technologies). Vascularity was assessed stereologically based on the morphology and topology of endothelial cell arrangement in the tissue. Vessels were considered structures with lumen and colocalization of all vascular markers. Microvessels with one to three endothelial cells (CD31-positive cells) spanning the vessel circumference were classified as capillaries as previously described.²⁹ Whole-slide scans were taken using a Leica DMi8 fluorescence microscope and imaged using Imaris Core 9.3 (Oxford Instruments, Concord, MA). Greater-magnification images (40×) of selected areas were taken with a Nikon Eclipse Ti-2 fluorescence microscope (Melville, NY).

Qualitative 3D Imaging Using Light Sheet Microscopy

Meniscal allografts from cadavers were fixed in formalin. Meniscus tissue was cleared using a modified uDisco passive clearing and staining procedure for whole organs.¹⁶ This multistep procedure allows for staining of entire pieces of tissue (or whole organs) with antibodies without the requirement for sectioning of the tissues. Tissues are made fully transparent, labeled using the same antibodies required for immunofluorescence, and then imaged using light sheet laser fluorescence microscopy. The ultimate 3D imaging of solvent-cleared organs (uDisco) protocol previously published in mice¹⁶ was optimized and adjusted for human cadaveric menisci. Meniscus tissue was segmented into quarters and 2-mm thin slices and fixed in formalin for up to 4 weeks, similar to the procedure followed in mouse tissues. Then tissue was incubated for 1 week in wash/permeabilization solution (0.4% v/v Triton-X, 0.3 M glycine w/v, 20% DMSO v/v all from Sigma), followed by 8 days in primary antibody (anti-CD31, 1:50 dilution; ab28364, Abcam) diluted in wash/permeabilization solution at 37°C. Tissue was washed overnight with permeabilization solution and incubated for 6 days with secondary antibody (Alexa Fluor 488-conjugated AffiniPure Donkey Anti-Rabbit IgG secondary antibody; Jackson ImmunoResearch), followed by a second wash with permeabilization solution, 37°C. Afterward, all cells were labeled with TO-PRO-3 nuclear stain (0.1% v/v, Thermo Fisher Scientific) for 4 days at 4°C^30,31 and gradient dehydrated in tert-butanol (Sigma; 360538). Specifically, tissue pieces were incubated in ascending grades of tert-butanol solution diluted in distilled water (30%, 50%, 70%, 90%, and 96%) for 1 to 3 days each and 100% tert-butanol at 37°C in the dark for 1 day, delipidated using dichloromethane (DCM, Sigma, 270997) for 1 day, and finally cleared with dibenzyl ether at RT in the dark for at least 3 days. A light sheet fluorescence microscope (LaVision Biotec Ultra-vision II; Miltenyi Biotec, Auburn, CA) was used for imaging and Imaris 9.3 was used for 3D reconstruction of the acquired images.

Statistical Analyses

All data are presented as mean ± standard deviation unless otherwise stated. Nonrepeated measures analysis of variance and Tukey‒Kramer post hoc analysis were performed on sample means for each analysis. For the changes in gene expression following 3 weeks of osteogenic induction one-way analysis of variance was performed using Sidak post hoc analysis for multiple comparisons between the induced and non-induced controls of each group (BM-MSCs, RR, RW, and WW respectively). For flow cytometry marker analysis, 2-way analysis of variance was performed using Dunnett’s post hoc analysis for comparisons between markers detected in different zones, using the WW zone as the control group. Statistical significance was set at P < .05. GraphPad Prism 8 software (GraphPad Software, San Diego, CA) was used to analyze the data.

Results

Progenitor Cell Prevalence

Cell Isolation From Meniscal Grafts, Cell Yields, and Self-Renewal Potential In Vitro

The enzymatic digestion protocol produced comparable results between medial or lateral menisci in all 3 zones (n = 6 donors, P > .05; Fig 2B). Therefore, data from medial and lateral menisci were pooled and analyzed per zone. Clonogenic potential of isolated cells from each zone was confirmed using low CFU-F assay. Colony counts showed that freshly isolated cells were clonogenic in culture. Further, there were no significant differences in clonogenicity of the cells isolated from the 3 meniscal zones (P > .05, Fig 2C).

Phenotypic Analysis; Cell Surface Marker Expression and Assessment of Differentiation Potential In Vitro

Flow cytometry analysis of cells from the 3 meniscal zones displayed presence of 2 distinct subpopulations of cells immediately after isolation. One subpopulation was CD44⁺CD105⁺CD29⁺CD90⁺ and the other one was CD44⁻CD105⁻CD29⁻CD90⁻ (Fig 3A, top panel). In addition, flow cytometry of CMCs at passage 2 displayed a shift of all 4 markers expression to the right (Fig 3A, bottom panel). Surface marker expression analysis showed differential marker expression patterns between different zones (Fig 3B). The WW zone contained a larger proportion of cells that express all 4 MSC markers (45.07 ± 0.36%) compared with RR and RW zones (17.75 ± 0.17% and 23.47 ± 3.62%, respectively, P < .05, Fig 3C).

Fig 3.

Identification of freshly isolated meniscus cells versus cultured controls using flow cytometry. (A) Flow cytometry analysis of cells from the 3 meniscal zones displayed presence of 2 distinct subpopulations of cells immediately after isolation (top panel). One subpopulation was positive to MSC surface markers and the other population was negative. Meniscal cells that were selected using plastic adherence and cultured to P2 were all positive for all MSC markers similarly to BM-MSCs (bottom panel). (B) Quantification of individual cell surface markers showing the proportion of each marker per zone. All 4 markers showed greater expression in the WW zone compared to RR and RW (P < .05). (C) Proportion of CD105⁺CD44⁺CD29⁺CD90⁺ cells per zone, P < .05. (BM-MSC, bone marrow-derived mesenchymal stem cell; MSC, mesenchymal stromal cell; P2, passage 2; RR, red‒red; RW, red‒white; WW, white‒white.)

CMCs were induced toward the 3 mesenchymal lineages (osteogenic, chondrogenic, and adipogenic) commonly used to assess MSC stem/progenitor cell potential.^21,22 After 1 week of induction with osteogenic media, CMCs from all zones displayed increased ALP activity compared with non-induced respective controls (Fig 4A). ALP activity of BM-MSCs that were treated under the same conditions were significantly greater (P < .05), although after 3 weeks of osteogenic induction all groups displayed increased collagen type I expression similar to BM-MSCs (P > .05 between groups, Fig 4B). CMCs from all zones were successfully induced toward the adipogenic lineage after 5 weeks of induction. CMCs from the RR zone displayed a greater prevalence of fully developed adipocytes with lipid droplets similar to those observed in induced BM-MSCs under the same conditions. CMCs from all zones were successfully induced to the chondrogenic lineage after 3 weeks in 3D culture in transwells, even though RW pellets showed less chondrogenic differentiation potential compared with BM-MSCs controls as can be observed in less Alcian blue‒stained extracellular matrix (Fig 4C).

Fig 4.

Multilineage differentiation potential of meniscal stromal cells. (A) ALP activity after 1 week of osteogenic induction. Controls were cultured for 1 week without osteogenic media (*P < .05, ***P < .001) (B) Col1 expression after 3 weeks of osteogenic induction (P < .05). (C) Adipogenesis was induced for 5 weeks with adipogenic supplements. BM-MSCs were treated under the same conditions and were used as assay positive controls. Red-Oil O stains lipid droplets in red. Bars represent 50 μm. (D) Chondrogenesis was induced in transwells for 3 weeks resulting in disk formation. Disks were processed histologically, sectioned and whole-slide scans attained. Alcian blue was used to determine presence of proteoglycans in the disks. BM-MSCs were treated under the same conditions and were used as assay controls. Bars represent 50 μm. (ALP, alkaline phosphatase; BM-MSC, bone marrow-derived mesenchymal stem cell; RR, red‒red; RW, red‒white; WW, white‒white.)

Vascularity Analysis

Histologic Features and Triple Immunofluorescence Colocalization Analysis

Histologic analysis and standard H&E staining confirmed the presence of larger vessels in the RR and RW zones (Fig 5 A and B). MTC staining, which can differentiate between smooth muscle and extracellular matrix, confirmed the presence of a network of arteries and veins in the RR and RW zones (Fig 5B). H&E and MTC staining use dyes to differentiate between cellular structures based on their generic physicochemical properties but cannot detect finer elements within a tissue including individual cells or smaller vessels, such as capillaries, that are composed of a single layer of endothelial cells and do not possess a smooth muscle actin lining (Fig 5C). Therefore, a more detailed immunofluorescence analysis was employed that uses antibodies that detect specific antigens within the tissue of interest. To this end, triple immunostaining with alpha smooth muscle actin and endothelial markers CD31 and lectin revealed the presence of endothelial cells in all 3 zones, including the WW zone, especially closer to the perimeter of the meniscus (Fig 6). The scan of entire immunofluorescence-stained slides showed that when dividing the meniscus into three zones using the most conservative approach, that is ≤1/3 of total area, blood vessels were found in the WW zone (Fig 1, available at www.arthroscopyjournal.org, presents the full slide scan of the images shown in Fig 6).

Fig 5.

Limitations of simple 2-dimensional histology approaches to visualize the vasculature of the human meniscus. (A) Schematic Illustration of the three meniscal zones: red‒red (RR), red‒white (RW), white‒white (WW). (B) Full-thickness cut of the meniscus stained with hematoxylin & eosin (H&E) (left) and Masson’s Trichome stain (MTC) (right), and greater magnification (bottom panels) of boxed areas shows relatively low cellularity throughout the RW and WW zones. Top bars represent 1 mm, in bottom panels bars represent 100 µm.

Fig 6.

Immunofluorescent triple staining with lectin (green), CD31 (orange), alpha smooth muscle actin (αSMA; magenta), and DAPI (blue) in all 3 zones using fluorescent microscopy and imaging with 2 different microscopes. Left images from whole-slide scans were taken (20× scan, Leica). Right, orange boxes from left images were identified using a Nikon Eclipse microscope and imaged at higher magnification (40×). Whole-slide scans allow visualization of the exact region imaged (left images, top right; yellow arrows). Areas were assessed using QuPath software to accurately quantify the distances between regions. The RW snapshot shown is at the bottom border of the RW zone of the whole-slide scan and was chosen in order to show the continuity of the scan. Bigger vessels with αSMA lining and lectin/CD31 can be found in both RR and RW zones. However, in WW zone only lectin/CD31/DAPI positive staining was found, suggesting presence of relatively smaller vessels, such as capillaries in this area. Scales bars represent 50µm. (RR, red‒red; RW, red‒white; WW, white‒white.)

3D Imaging of Vessels in the Meniscus

CD31⁺ staining demonstrated presence of endothelial cells, visualizing the vascular tree in RR toward RW and positive colocalization of green and red staining in the WW zone, especially on the periphery of the meniscus, confirming the findings in 2D immunofluorescence (Fig 7 and Video 1, available at www.arthroscopyjournal.org).

Fig 7.

Successful clearing of human meniscus with modified uDISCO procedure and nucleic staining with To-Pro-3 and the endothelial marker CD31. (A) Fully transparent slices of human meniscal tissue cleared with a modified uDisco approach. On the left, a quarter of fully cleared meniscus. On the right, a slice before and after clearing (B). 2D slice of cleared meniscus tissue stained with only with To-Pro-3, displaying cells distributed in all 3 zones (C). Three-dimensional reconstruction of meniscus slices after imaging with light sheet fluorescence microscopy. Double-labeling with To-Pro-3 (red, pan-nuclei marker) and CD-31 (green, endothelial marker) indicates presence of vessels of various sizes in all zones. Bigger vessels were found spanning the RR to RW zones, confirming 2D results. Blood vessels are present in the WW zone. Upper and bottom panels display different views of the same slice. Left; merged channels (red and green), middle; red channel (To-Pro-3), right; green channel (CD31). Arrows point to vessels. Bar represents 500 µm. (2D, 2-dimensional; RR, red‒red; RW, red‒white; WW, white‒white.)

Discussion

The main finding of this study was that multipotent mesenchymal stromal progenitor cells and blood vessels were observed in all 3 zones of the meniscus, including the WW zone. Isolated meniscus cells were clonogenic in vitro with no significant differences in the self-renewal potential between the 3 meniscal zones. Flow cytometry analysis demonstrated that these progenitor cells were expressing consensus MSC surface markers (CD44, CD105, CD29, and CD90). Meniscus stromal progenitor cells were enriched after in vitro culture, due to the plastic adherence that filters out nonadherent cells. Lastly, CMCs from all 3 meniscal zones were able to successfully differentiate into the 3 mesenchymal lineages (osteogenic, adipogenic, and chondrogenic), and found slightly inferior to BM-MSCs in their osteogenic and adipogenic potential. Bigger vessels were observed in the RR and RW zones, and smaller vessels were identified in the WW zone using 2D immunofluorescence co-localization of endothelial markers and alpha smooth muscle actin in combination with a modified uDISCO 3D imaging approach.

Biologic augmentation approaches are currently being investigated to promote chemotaxis, cellular proliferation, and/or matrix production at the site of meniscal repair. These approaches include mechanical stimulation, marrow-venting procedures, fibrin clots, injection of platelet-rich plasma, and stem cell-based therapies that involve injection of autologous MSCs.^32,33 These approaches may augment an inherent meniscus healing capacity if resident progenitor/stem cells are present, as demonstrated in rabbits and humans.^9,34,35 The main role of resident mesenchymal progenitor cells in connective tissues is to maintain homeostasis and contribute to tissue repair when needed.³⁶ Hennerbichler et al.³⁷ demonstrated that punch defects directly filled with the removed punches showed no significant difference in healing potential between the vascularized and avascular meniscus zone. Furthermore, Croutze et al.³⁸ reported equivalent differentiation potential toward chondrogenic phenotype and extracellular matrix production of human meniscus cells isolated from the inner and outer zones. Mauck et al.¹⁰ reported similar differentiation potential of cells isolated from different zones of calf meniscus.

MSCs are a heterogeneous cell population that consist of a mixture of multipotent and more committed progenitors. MSCs isolated from the bone marrow (BM-MSCs) have been shown to comply to the established stem cell criteria, that is, they are multipotent, clonogenic in vitro, and able to produce skeletal tissues via serial transplantations in vivo.²⁶ Stromal mesenchymal progenitors are found in multiple other tissues (adipose tissue, umbilical cord, etc), are clonogenic in culture, can be induced to differentiate into multiple lineages in vitro beyond skeletal tissues, and express similar markers with BM-MSCs.²⁶ Both terms are sometimes used interchangeably in the literature, resulting in ISCT issuing a set of criteria in a position statement to assist comparisons between different studies.^21,22 Based on ISCT criteria for mesenchymal stromal progenitor identification, we characterized CMCs on the basis of positive selection using plastic adherence and multipotentiality. We also assessed the expression of MSC consensus markers and compared them with freshly isolated and cultured BM-MSCs.

It is known that the cells of the meniscus differ in their morphology and their in vitro properties depending on their location.¹² At least 4 different meniscus cell types have been identified in the rabbit meniscus using fluorescent and scanning electron microscopy imaging.¹⁹ In the present study, we demonstrated a greater prevalence of isolated CD44⁺CD105⁺CD29⁺CD90⁺ meniscus cells in the WW zone compared with the other zones. Surface marker expression of cells freshly isolated from the WW zone was significantly greater than from the other 2 zones using standard flow cytometry analysis. The greater proportion of CD44⁺CD105⁺CD29⁺CD90⁺ cells in the WW zone suggests that the WW zone hosts a more homogeneous progenitor population compared with the other zones as defined by expression of those 4 markers, CFU-F clonogenicity and positive selection through plastic adherence. Interestingly, our results demonstrate that cells isolated from the RR zone have similar potential to be induced to the adipogenic lineage as BM-MSCs, whereas the cells from RW and WW could not be efficiently induced towards the adipogenic phenotype in vitro. In addition, RW cells might not be as potent toward induction to the chondrogenic phenotype compared to RR and WW. This suggests that some of the cells in the RW and WW zones cannot differentiate toward the chondrogenic adipogenic lineages, so they are more specialized toward the fibrocartilage phenotype of the meniscus. However, the presence of other cell types in the RR as well as the plasticity of the resident CD44⁺CD105⁺CD29⁺CD90⁺ CMCs identified in RR zone might be an important factor that contributes to the regenerative potential of that zone.⁸ Future research is needed to determine whether this phenomenon is age-dependent or related to other factors. It is unknown if the sole presence of progenitor cells can facilitate improved healing and therefore future studies are warranted. Also, recruitment mechanisms of these cells to the injury site needs to be addressed using in vivo models. Vascularization and progenitor cell availability will play an important role in recruitment of the cells and their contribution to the healing process.

Tears in the avascular zone have historically been treated with debridement, given the lower likelihood of successful healing of a repair in avascular tissue.³⁹ Although repair of meniscal tears in the avascular WW zone have recently been reported to yield satisfactory outcomes.^40,41 Cinque et al.⁴² reported low meniscal repair failure rates in avascular zone with only 3% of patients requiring a second surgery for a failed meniscus repair. Noyes et al.⁴³ reported that 62% of RW zone inside-out meniscal repairs had normal or nearly normal characteristics for pain, swelling, jumping, and their Cincinnati score. In theory, meniscal tears require vascularization to deliver the biologic factors necessary for tissue repair. However, some animal studies demonstrate that meniscal tissue may heal without significant vascular contributions.^37,44 In the present study, following identification of primary tissue features with standard histology (H&E and MTC stains), a comprehensive combined 2D and 3D immunofluorescence analysis was used to define vessels using cytological features and identify the presence of vessel-specific markers in cadaveric menisci. H&E and MTC staining revealed larger vessels in the RR zone of meniscus spanning toward the RW zone and sprouting to smaller arterioles. Small vessels and endothelial cells were found also in the WW zone, mainly in the periphery, suggesting that the WW zone might not be completely devoid of vasculature. However, it is possible that these are remnants of previously vascularized fetal tissues that regressed after birth, as has been shown previously.¹⁴ Thus, further research using functional imaging in vivo or cell tracking in animal models is needed to determine whether there is functional vasculature in the WW zone.^45,46

Large blood vessels were not found within the WW zone. However, around the perimeter of the tissue small vessels and endothelial cells were spotted, suggesting that they might be part of microvessels or remnants from vasculature that regressed in adulthood and that could potentially be revived with appropriate angiogenic stimulation. Schrepfer et al.⁴⁷ reported that MSCs injected into the lungs get trapped in the pulmonary capillary network because their mean size is greater than the pulmonary capillary lumen. The use of vasodilation agents has been proposed in combination with such treatments to improve perfusion and stem cell migration.⁴⁷ Importantly, recent evidence suggests that vasculature could increase in the case of meniscal tears, upregulating angiogenic factors, such as vascular endothelial growth factor.⁴⁸ This could modify the affluence of progenitor cells not only by chemotaxis but as a de novo source of pericytes.⁴⁹ The specific topology of the capillary network in the WW zone of healthy adult donors is yet to be mapped. The vessel distribution may be affected by age, health, or fitness level and other factors that are yet to be determined. In this study, we demonstrate that there is a small amount of vasculature and a significant population of progenitor cells in the WW zone of the meniscus. Future studies that investigate the healing potential of these resident stem/progenitor cells are warranted.

Limitations

This study is not without limitations. The sample size was limited and both medial and lateral meniscus were included. The donated cadaveric menisci were never frozen (received within 21 days) and stored at 4°C, increasing the potential of cell death immediately post-harvest compared with the in vivo setting. The mean age of the specimens was 21 years old, which is not representative of an older population. Thus, further studies exploring the cellular content and vascularity of the older population is warranted. Only 3 zones were selected for the analysis: inner, middle, and marginal, each representing a third of the width of the tissue, which might not represent a true division of the vascular tree density throughout the meniscus tissue. Vessel distribution might be different between medial and lateral menisci and also could be affected by age and/or health status. It is known that in every cell isolation procedure from a tissue cell yields inherently vary. Since the cellular composition of meniscus is heterogeneous, the current cell isolation approach was optimized for our work because it was shown as the most efficient for all 3 zones for our grafts. In addition, no significant differences were observed between medial and lateral menisci. However, it might not be pertinent for grafts with different characteristics, such as fetal and/or neonatal menisci, or menisci from aged and diseased populations. Finally, the presence of blood vessels does not necessarily indicate functional vascularization, which requires further analysis. These vessels might be remnants of retreating vasculature from the fetal meniscus.

Conclusions

In conclusion, our results demonstrate the presence of resident mesenchymal progenitors 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.