Meniscal Explants Research Articles

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

A human meniscus explant model for studying early events in osteoarthritis development by proteomics.

Degenerative meniscus lesions have been associated with both osteoarthritis etiology and its progression. We, therefore, sought to establish a human meniscus ex vivo model to study the meniscal response to cytokine treatment using a proteomics approach. Lateral menisci were obtained from five knee-healthy donors. The meniscal body was cut into vertical slicesand further divided into an inner (avascular) and outer region. Explants were either left untreated (controls) or stimulated with cytokines. Medium changes were conducted every 3 days up to Day 21 and liquid chromatography-mass spectrometry was performed at all the time points for the identification and quantification of proteins. Mixed-effect linear regression models were used for statistical analysis to estimate the effect of treatments versus control on protein abundance. Treatment by IL1ß increased release of cytokines such as interleukins, chemokines, and matrix metalloproteinases but a limited catabolic effect in healthy human menisci explants. Further, we observed an increased release of matrix proteins (collagens, integrins, prolargin, tenascin) in response to oncostatin M (OSM) + tumor necrosis factor (TNF) and TNF+interleukin-6 (IL6) + sIL6R treatments, and analysis of semitryptic peptides provided additional evidence of increased catabolic effects in response to these treatments. The induced activation of catabolic processes may play a role in osteoarthritis development.

Pneumatospinning Biomimetic Scaffolds for Meniscus Tissue Engineering.

Nanofibrous scaffolds fabricated via electrospinning have been proposed for meniscus tissue regeneration. However, the electrospinning process is slow, and can only generate scaffolds of limited thickness with densely packed fibers, which limits cell distribution within the scaffold. In this study, we explored whether pneumatospinning could produce thicker collagen type I fibrous scaffolds with higher porosity, that can support cell infiltration and neo-fibrocartilage tissue formation for meniscus tissue engineering. We pneumatospun scaffolds with solutions of collagen type I with thicknesses of approximately 1 mm in 2 h. Scanning electron microscopy revealed a mix of fiber sizes with diameters ranging from 1 to 30 µm. The collagen scaffold porosity was approximately 48% with pores ranging from 7.4 to 100.7 µm. The elastic modulus of glutaraldehyde crosslinked collagen scaffolds was approximately 45 MPa, when dry, which reduced after hydration to 0.1 MPa. Mesenchymal stem cells obtained from the infrapatellar fat pad were seeded in the scaffold with high viability (>70%). Scaffolds seeded with adipose-derived stem cells and cultured for 3 weeks exhibited a fibrocartilage meniscus-like phenotype (expressing COL1A1, COL2A1 and COMP). Ex vivo implantation in healthy bovine and arthritic human meniscal explants resulted in the development of fibrocartilage-like neotissues that integrated with the host tissue with deposition of glycosaminoglycans and collagens type I and II. Our proof-of-concept study indicates that pneumatospinning is a promising approach to produce thicker biomimetic scaffolds more efficiently that electrospinning, and with a porosity that supports cell growth and neo-tissue formation using a clinically relevant cell source.

Proteomic and biochemical analysis of R399E, a recombinant and mutant form of GDF5 on the secretome of human articular cartilage and meniscal explants from osteoarthritic joints

Purpose: Growth differentiation factor 5 (GDF5) is a growth factor that increases extracellular matrix (ECM) production and decreases expression of MMP13 and ADAMTS4. However, GDF5 also stimulates hypertrophy and osteophyte formation. Therefore, to mitigate the hypertrophic properties of GDF5, a mutant variant of the molecule codenamed R399E was genetically engineered. R399E has been shown to stimulate chondrogenesis in MSCs similarly to GDF5 but exhibits reduced hypertrophic and osteogenic properties.

A single dose of P188 prevents cell death in meniscal explants following impact injury

ObjectiveTo determine the efficacy of single and multiple administrations of Poloxamer 188 (P188) in saving meniscal cells following an injurious impact. MethodsMeniscal explants were harvested from both the lateral and medial menisci of Flemish Giant rabbits. After a 24-h incubation period, explants were subjected to 50% impact strain to simulate traumatic joint injury, and the explants were then placed in media with or without supplemented P188. Temporal administrations of P188 over a 14-day period were given based on one of 6 different treatments regimes. Over the 14-day period, explants were cyclically loaded to 10% strain at 1 Hz for 1 h per day, five days a week. Cell viability was assessed on day 14, with the remainder of the tissue being fixed to determine cell apoptosis levels and proteoglycan changes via histology. ResultsThe injurious impact proved to produce significant levels of cell death in meniscal explants. The ability of P188 to prevent cell death was not affected by the number of P188 doses (single versus multiple). P188 treatment proved to maintain cell viability levels comparable to those from unimpacted explants. There were no significant changes in cell apoptosis or proteoglycan coverage in the tissues over a 14-day period for any group, all treatment groups were statistically similar to the unimpacted explants. ConclusionA single dose of P188 following impact is all that is necessary to inhibit cell death in the meniscus following a traumatic impact. Thus, orthopaedic surgeons may choose to administer P188 in addition to treating any other acute damage due to a traumatic load to the knee, such as anterior cruciate ligament rupture, although more in depth in vivo studies are necessary.

Use of a Hyperosmolar Saline Solution to Mitigate Proinflammatory and Degradative Responses of Articular Cartilage and Meniscus for Application to Arthroscopic Surgery

This study was designed to evaluate differences in proinflammatory and degradative mediator production and extracellular matrix degradation from osteoarthritic knee articular cartilage and meniscus explants treated with either hyperosmolar saline or isotonic saline. 6 mm-diameter full-thickness explants were created from articular cartilage and menisci recovered after patients underwent knee surgery. One explant half was treated for 3 hours with hyperosmolar saline (600 mOsm/L) and the corresponding half with isotonic saline (300 mOsm/L). Explants were subsequently cultured for 3 days in tissue culture media. On day 3, media were collected for biomarker analyses. Results were normalized to tissue wet weight and analyzed statistically. Articular cartilage was collected from 10 patients (5male, 5 female; mean age= 66.9 years) and menisci were collected from 8 patients (2 male, 6 female; mean age= 66 years). Articular cartilage media concentrations of monocyte chemoattractant protein-1 (P= .001) and interleukin (IL)-6 (P= .049) were significantly lower in explants treated with hyperosmolar saline. Meniscus media concentrations of prostaglandin E2 (P= .008), monocyte chemoattractant protein-1 (P= .011), IL-6 (P= .029), IL-8 (P= .012), matrix metalloproteinase-2 (P= .011), and glycosaminoglycan (P= .008) were significantly lower in explants treated with hyperosmolar saline. Treatment of cartilage and meniscus explants with hyperosmolar saline effectively mitigated key proinflammatory mediator production, as well as degradative mediator production and glycosaminoglycan loss from meniscus, with no detrimental effects noted compared to isotonic saline. These results suggest that hyperosmolar saline irrigation fluid may provide a safe alternative to standard isotonic saline irrigation fluid, and could mitigate untoward effects associated with inflammatory responses after standard-of-care knee arthroscopy.

AB0069 LORECIVIVINT (SM04690), AN INTRA-ARTICULAR, SMALL-MOLECULE CLK/DYRK1A INHIBITOR THAT MODULATES THE WNT PATHWAY, AS A POTENTIAL TREATMENT FOR MENISCAL INJURIES

Background:Meniscal injuries, associated with pain, stiffness, and localized swelling, are the most common pathology of the knee with a prevalence of 61 per 100,000.1Meniscal damage is a frequent finding on MRI images of knee osteoarthritis (OA)2; while a meniscal tear can lead to knee OA, knee OA can also lead to a spontaneous meniscal tear.3Efforts to repair meniscal damage have been largely unsuccessful and do not prevent the progression of degenerative changes that lead to knee OA.4The Wnt signaling pathway has been shown to be regulated during meniscal development,5,6suggesting that manipulation of this pathway may influence the regenerative capacity of the meniscus. Lorecivivint (LOR; SM04690) is an intra-articular (IA), small-molecule CLK/DYRK1A inhibitor that modulates the Wnt pathway.Objectives:LOR was evaluated in preclinical studies to determine its protective and anabolic effects in ex vivo explants and in a rat model of chemically induced inflammatory meniscus degeneration.Methods:Effects of LOR (30 nM) on expression of matrix metalloproteinases (MMPs) in cultured rat menisci treated with IL-1B were measured by qPCR. In vivo, LOR activity was evaluated in a rat model of monosodium iodoacetate (MIA) injection-induced inflammatory meniscus degeneration. A single IA injection of MIA was immediately followed by a single IA injection of LOR (0.3 ug) or vehicle. Knees were harvested on Days 1, 4, and 11 and menisci were isolated. Anti-inflammatory effects were evaluated by measuringTNFAandIL6expression by qPCR. Meniscus protection was evaluated by qPCR for MMPs and aggrecanase and anabolic effects by qPCR for collagens.Results:In ex vivo meniscal explants, LOR inhibited expression ofMMP1,MMP3, andMMP13compared to DMSO (P<0.01). In vivo, LOR significantly decreased expression of these MMPs and aggrecanase (P<0.05) compared to vehicle in the rat model of inflammatory meniscus degeneration at Day 4 after MIA injection. In addition, LOR reduced expression of inflammatory cytokinesTNFAandIL6at Day 4 compared to vehicle. Finally, LOR increased expression of collagen types I, II, and III at Day 11 after MIA injection.Conclusion:LOR exhibited protective effects in the meniscus ex vivo and in vivo by reducing the expression of catabolic enzymes compared to control. Anti-inflammatory effects of LOR were demonstrated by inhibition of inflammatory cytokine expression. Compared to vehicle, LOR increased expression of collagens in vivo, indicating potential meniscal anabolic effects. These data support further investigation of LOR as a potential disease-modifying therapy for meniscal injuries.

Metabolic responses of meniscal tissue to focal collagenase degeneration

ABSTRACTPurpose: The objective of this study was to determine the responses of normal meniscus to collagenase activity. It was hypothesized that meniscal explants exposed to collagenase would significantly increase release of pro-inflammatory cytokines and degradative enzymes, in a dose-dependent manner, compared to control.Methods: Menisci were harvested from adult dogs (n = 6) euthanized for reasons unrelated to this study. Meniscal explants were created from the central portion of lateral and medial meniscus. Explants were injected with 100 µl collagenase at a concentration of 50 µg/ml, 5 µg/ml, or 0 µg/ml of collagenase. Explants were cultured for 12 days, and media were changed and collected every 3 days for biomarker analyses. Differences among collagenase concentrations were determined by a three factor ANOVA with adjustment for multiple comparisons, with pre-adjustment statistical significance set at p < 0.05.Results: When data from all explants were compared, the 50 µg group released significantly higher IL-6 and PGE2, and the 5 µg group released significantly higher levels of MMP-3 and CTX-II compared to the 0 µg group. Explants from the medial meniscus released significantly more MMP-1, MMP-2, MMP-3, and MMP-13 in response to stimulation with 5 µg/ml of collagenase compared to explants from the lateral meniscus.Discussion: The data from this study indicate that in response to localized degradative enzyme activity, the meniscus increases the release of pro-inflammatory and degradative biomarkers in a dose-dependent manner. Further, these data indicate potential differences in metabolic responses of lateral versus medial menisci to collagenase insult.

Core-Shell Nanofibrous Scaffolds for Repair of Meniscus Tears.

Electrospinning is an attractive method of fabricating nanofibers that replicate the ultrastructure of the human meniscus. However, it is challenging to approximate the mechanical properties of meniscal tissue while maintaining the biocompatibility of collagen fibers. Our objective was to determine if functionalizing polylactic acid (PLA) nanofibers with collagen would enhance their biocompatibility. We therefore used coaxial electrospinning to generate core-shell nanofibers with a core of PLA for mechanical strength and a shell of collagen to enhance cell attachment and matrix synthesis. We characterized the nanostructure of the engineered scaffolds and measured the hydrophilic and mechanical properties. We assessed the performance of human meniscal cells seeded on coaxial electrospun scaffolds to produce meniscal tissue by gene expression and histology. Finally, we investigated whether these cell-seeded scaffolds could repair surgical tears created ex vivo in avascular meniscal explants. Histology, immunohistochemistry, and mechanical testing of ex vivo repair provided evidence of neotissue that was significantly better integrated with the native tissue than with the acellular coaxial electrospun scaffolds. Human meniscal cell-seeded coaxial electrospun scaffolds may have potential in enhancing repair of avascular meniscus tears. Impact Statement The success of any tissue-engineered meniscus graft relies on its ability to mimic native three-dimensional microstructure, support cell growth, produce tissue-specific matrix, and enhance graft integration into the repair site. Polylactic acid scaffolds possess the desired mechanical properties, whereas collagen scaffolds induce better cell attachment and enhanced tissue regeneration. We therefore fabricated nanofibrous scaffolds that combined the properties of two biomaterials. These novel coaxial scaffolds more closely emulated the structure, mechanical properties, and biochemical composition of native meniscal tissue. Our findings of meniscogenic tissue generation and integration in meniscus defects have the potential to be translated to clinical use.

Effects of tissue culture on the biomechanical properties of porcine meniscus explants

BackgroundThe meniscus is critical for the normal functioning of the knee joint. The specific aim of this study was to validate an in vitro culture model of meniscus explants for testing the impact of culture conditions on meniscus biomechanical properties. We hypothesized that culturing menisci in the presence of intermediate and high concentration of serum would have a positive effect on the compressive stiffness of the meniscus. MethodsUnconstrained microindentation testing was performed on porcine meniscus explants cultured with varying concentrations 1%, 5%, or 10% of fetal bovine serum media. Meniscus explants that were not cultured were used as a control. These tests quantified the Young's Modulus of the listed groups of cultured and uncultured explant tissues. FindingsThe Young's modulus for 10% cultured explants were significantly higher compared to the control, 1%, and 5% cultured meniscus explants. There was no statistical significance when the Young's modulus between control, 1%, and 5% cultured explants were compared. InterpretationThese results suggest that low concentrations of serum do not impart an anabolic effect on meniscus tissue explant biomechanical properties.

Inlammatory response of a human meniscal in vitro model studied by proteomics

Purpose: There is likely an inflammatory phenotype of OA where pro-inflammatory cytokines contribute to the pathogenesis. Degenerative meniscus lesions have been associated with both OA etiology and its progression. Thus, we sought to establish a human meniscus in vitro model to explore the meniscal tissue’s response to cytokine treatment where we used a discovery proteomics approach to determine and follow the release of matrix proteins. Methods: One lateral meniscus was harvested within 40 hours postmortem from a human donor (male, age 79) without known chronic joint disease. The procedure was approved by the local ethics committee at Lund University. The meniscus was visually inspected to be macroscopically intact. We then obtained 12 slices (1mm wide) cut radially from the meniscal body. These slices were further divided in two by the inner 1/3 (inner zone) or the peripheral 2/3 (outer zone). The explants were either left untreated (controls, n=6+6) or with interleukin-1ß (IL-1ß) (n=6+6) at 5 ng/ml, and cultured in serum-free DMEM media (supplemented with ITS and BSA) for 21 days. Medium changes were carried out every 3 days, and used medium was collected and stored at -20°C for analysis. The glycosaminoglycan (GAG) release was quantified by the 1,9-dimethylmethylene blue (DMMB) assay for all samples and viability was checked using AlamarBlue at day 0,6,12 and 21. Tissue explants were frozen (-20°C) at the end of the 21 day culture. Liquid chromatography-mass spectrometry (LC-MS) runs using a Q-Exactive orbitrap were performed for medium samples at all the time points for the identification and quantification of proteins using pooled samples of the six replicates for each zone and treatment. Proteome Discoverer 2.2 software was used for the discovery analysis and the quantification was made at the MS1 level using label-free quantification with feature detection. The total treatment effect was estimated by summing up all the time points and calculating the fold change. Results: Explant viability was mainly stable during the whole cultivation period and that IL-1ß had moderate effect on glycosaminoglycan release with a significant increased release only at day 6 and 9 (see Figure 1). The proteomics data, however, revealed a distinct elevation in the levels of multiple cytokines and proteins related to inflammation in response to IL-1ß. The LC-MS experiment from different samples (n=28) resulted in a total of 479 proteins identified in at least two samples at a protein confidence <0.01. The principal component analysis (PCA) plot for the four sample groups are shown in Figure 2, illustrating a clear separation between control and IL-1ß treated groups, and also a time dependent response in all groups. The cumulative release until day 21 was used to pick up overall treatment effects such as up-regulation of several cytokines and proteins involved in the inflammatory response, including C-X-C motif chemokines, interleukins, and complement components. We found differences in outer vs inner zones, particularly the more cartilage specific proteins such as collagen II and aggrecan were prominent in the inner avascular zone, which could be expected as the different zones contain different cell populations. Interestingly we also found release of MMP-13 only in the inner zone. Proteins with an increased release upon IL-1 treatment are listed in Table 1 for both inner and outer zones with a major overlap seen. Conclusions: This pilot proteomics approach allowed us to unambiguously identify and quantify multiple proteins in the media of human meniscus explant cultures upon IL-1ß exposure that may mimic the early events in the meniscus phenotype of OA development. We followed the quantitative release of more than 200 proteins including cytokines, proteases as well as other extracellular proteins throughout the 21-day culture period. Several of the upregulated proteins belonged to the cytokine family.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Effect of dose and release rate of CTGF and TGFβ3 on avascular meniscus healing.

Meniscus tears in the avascular region rarely functionally heal due to poor intrinsic healing capacity, frequently resulting in tear propagation, followed by meniscus deterioration. Recently, we have reported that time-controlled application of connective tissue growth factor (CTGF) and transforming tissue growth factor β3 (TGFβ3) significantly improved healing of avascular meniscus tears by inducing recruitment and step-wise fibrocartilaginous differentiation of mesenchymal stem/progenitor cells (MSCs). In this study, we investigated effects of the dose of CTGF and the release rate of TGFβ3 on avascular meniscus healing in our existing explant model. Our hypothesis was that dose and release rate of CTGF and TGFβ3 are contributing factors for functional outcome in avascular meniscus healing by stem cell recruitment. Low (100 ng/ml) and high (1,000 ng/ml) doses of CTGF as well as fast (0.46 ± 0.2 ng/day) and slow (0.29 ± 0.1 ng/day) release rates of TGFβ3 were applied to our established meniscus explant model for meniscus tears in the inner-third avascular region. The release rate of TGFβ3 was controlled by varying compositions of poly(lactic-co-glycolic acids) (PLGA) microspheres. The meniscus explants were then cultured for 8 weeks on top of mesenchymal stem/progenitor cells (MSCs). Among the tested combinations, we found that a high CTGF dose and slow TGFβ3 release are most effective for integrated healing of avascular meniscus, demonstrating improvements in alignment of collagen fibers, fibrocartilaginous matrix elaboration and mechanical properties. This study may represent an important step toward the development of a regenerative therapy to improve healing of avascular meniscus tears by stem cell recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1555-1562, 2019.

Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration.Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-β (PDGFRβ) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks.Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRβ expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF.In conclusion, PDGF-BB-coated scaffold increased PDGFRβ expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears. Statement of SignificanceMeniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use.We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells.The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties.This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

Metabolic responses of meniscal explants to injury and inflammation ex vivo.

This study was designed to characterize metabolic responses of meniscal tissue explants to injury and inflammation. We hypothesized that impact injury and interleukin (IL-1β) stimulation of meniscal explants would result in significant increases in matrix metalloproteinase (MMP) activity and relevant cytokine production compared to controls. Mature canine meniscal explants (n = 9/group) were randomly assigned to: (i) IL-1β (0.1 ng/ml) treated (IL); (ii) 25% strain (25); (iii) 75% strain (75); (iv) 25% + IL-1β (25IL); (v) 75% + IL-1β (75IL); or (vi) 0% + no IL-1β control (NC). Explants were impacted at 100 mm/s to 0%, 25%, or 75% strain and then cultured for 12 days with or without 0.1 ng/ml rcIL-1β. Media were refreshed every 3 days and analyzed for MMP activity, ADAMTS-4 activity, MMP-1, MMP-2, MMP-3, GAG, NO, PGE2 , IL-6, IL-8, MCP-1, and KC concentrations. Treatment with IL-1β alone significantly increased NO, PGE2, general MMP activity, IL-6, IL-8, KC, and MCP-1 media concentrations compared to negative controls. Impact at 75% significantly increased PGE2, IL-6, IL-8, and KC media concentrations compared to negative controls. The combination of IL-1β and 75% strain significantly increased production of PGE2 compared to IL-1β or 75% strain alone. Impact injury to meniscal explants ex vivo is associated with increased production of pro-inflammatory mediators and degradative enzyme activity, which are exacerbated by stimulation with IL-1β. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2657-2663, 2018.

Metabolic Responses of Meniscus to IL-1β.

This article identifies the potential mechanisms of action for meniscal degeneration in response to joint inflammation and potential contributions of the meniscus to the development and progression of osteoarthritis (OA). It was hypothesized that interleukin-1β (IL-1β) stimulation of meniscal explants would result in significant increases in nitric oxide (NO), matrix metalloproteinase (MMP) production and activity, and relevant cytokine production compared with controls. Canine meniscal explants (4 mm) were cultured for 21 days with (IL-1) or without (negative control [NC]) 50 ng/mL rcIL-1β (n = 6/group). Media were changed every 3 days and analyzed for MMP activity, ADAMTS-4 activity, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, NO, prostaglandin E2 (PGE2), IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and keratinocyte-derived chemokine (KC) concentrations. Media NO and PGE2 concentrations were significantly higher in the IL-1 group at all time points except for days 9 and 12. The concentrations of MMP-13 were significantly higher in the IL-1 group at days 3, 6, 9, and 12. The production of MMP-2 was significantly lower in the IL-1 group on days 3 through 15. ADAMTS4 activity was significantly higher in the IL-1 group on days 6 through 18. MMP-3 concentrations and general MMP activity were significantly higher in the IL-1 group at all time points. Concentrations of IL-6, IL-8, MCP-1, and KC were significantly higher in the IL-1 group at most time points. Glycosaminoglycans (GAG) content decreased significantly (p = 0.009) in the IL-1 group compared with the NC group. Proinflammatory mediators appear to directly influence degradative processes in the meniscus, which in turn contribute to development and progression of OA by production of proinflammatory and degradative mediators. These findings have important clinical implications for the management of the degenerative meniscus and the osteoarthritic knee.

Chondrocyte-based intraoperative processing strategies for the biological augmentation of a polyurethane meniscus replacement

ABSTRACTPurpose/aim of study: Menisectomies account for over 1.5 million surgical interventions in Europe annually, and there is a growing interest in regenerative strategies to improve outcomes in meniscal replacement. The overall objective of this study was to evaluate the role of intraoperatively applied fresh chondrocyte (FC) isolates compared to minced cartilage (MC) fragments, used without cell isolation, to improve bioactivity and tissue integration when combined with a polyurethane replacement.Materials and methods: First, to optimize the intraoperative cell isolation protocol, caprine articular cartilage biopsies were digested with 750 U/ml or 3000 U/ml collagenase type II (ratio of 10 ml per g of tissue) for 30 min, 1 h or 12 h with constant agitation and compared to culture-expanded chondrocytes in terms of matrix deposition when cultured on polyurethane scaffolds. Finally, FCs and MC-augmented polyurethane scaffolds were evaluated in a caprine meniscal explant model to assess the potential enhancements on tissue integration strength.Results: Adequate numbers of FCs were harvested using a 30 min chondrocyte isolation protocol and were found to demonstrate improved matrix deposition compared to standard culture-expanded cells in vitro. Upon evaluation in a meniscus explant defect model, both FCs and MC showed improved matrix deposition at the tissue-scaffold interface and enhanced push-out strength, fourfold and 2.5-fold, respectively, compared with the acellular implant.Conclusions: Herein, we have demonstrated a novel approach that could be applied intraoperatively, using FCs or MC for improved tissue integration with a polyurethane meniscal replacement.

Stem cell delivery in tissue-specific hydrogel enabled meniscal repair in an orthotopic rat model.

Interest in non-invasive injectable therapies has rapidly risen due to their excellent safety profile and ease of use in clinical settings. Injectable hydrogels can be derived from the extracellular matrix (ECM) of specific tissues to provide a biomimetic environment for cell delivery and enable seamless regeneration of tissue defects. We investigated the in situ delivery of human mesenchymal stem cells (hMSCs) in decellularized meniscus ECM hydrogel to a meniscal defect in a nude rat model. First, decellularized meniscus ECM hydrogel retained tissue-specific proteoglycans and collagens, and significantly upregulated expression of fibrochondrogenic markers by hMSCs versus collagen hydrogel alone invitro. The meniscus ECM hydrogel in turn supported delivery of hMSCs for integrative repair of a full-thickness defect model in meniscal explants after invitro culture and invivo subcutaneous implantation. When applied to an orthotopic model of meniscal injury in nude rat, hMSCs in meniscus ECM hydrogel were retained out to eight weeks post-injection, contributing to tissue regeneration and protection from joint space narrowing, pathologic mineralization, and osteoarthritis development, as evidenced by macroscopic and microscopic image analysis. Based on these findings, we propose the use of tissue-specific meniscus ECM-derived hydrogel for the delivery of therapeutic hMSCs to treat meniscal injury.

The Effects of Bone Morphogenetic Protein‐7 and Interleukin‐1 on Biomechanical Properties of Meniscus Explants

IntroductionThe meniscus is a wedge, C‐shape fibrocartilaginous tissue that is essential for the normal biomechanical function of the knee. Damage to the meniscus leads to degenerative changes and loss of function mechanical function of the knee. Cytokines like interleukin‐1 (IL‐1) can inhibit repair process. However, anabolic factors like bone morphogenetic protein‐7 (BMP‐7) may help aid in the repair process. The goal of this project is to observe the effects of BMP‐7 and IL‐1 on the biomechanical properties of the meniscus using microindentation test.MethodSix‐millimeter porcine meniscus explants were cut using a biopsy punch and cultured in 1% fetal bovine serum media for seven days. Explants were cultured without treatment (control group) or treated with BMP‐7, IL‐1 alpha, and or combined with BMP‐7 and IL‐1 alpha. Microindentation test was used to test the elasticity of the explant tissues.ResultsThe elasticity for the explants treated with BMP‐7 (2.480 MPa) were significant compared to explants treated with either IL‐1 alpha (1.501 MPa; p= .001) or both BMP‐7 and IL‐1 alpha (.866; p ≤ .000). This indicates BMP‐7 and IL‐1 have a significant effect on the meniscus biomechanical properties. BMP‐7 increase the elasticity (but not significant) compared to the control explants (2.072 MPa; p= .065), while IL‐1significantly decrease the elasticity (p= .017).ConclusionThis data shows that BMP‐7 has the potential to exert an anabolic effect on cultured meniscus explants reflected by the presence of increased mechanical compression properties. As a result, the meniscus explant tissues that were cultured with BMP‐7 became more elastic after being cultured for seven days. In addition, meniscus cultured with IL‐1 alpha or a combination of BMP‐7 and IL‐1 alpha became less elastic. Future experiments will continue testing the effect of these two treatments.

Evaluation of novel biodegradable three-armed- and hyper-branched tissue adhesives in a meniscus explant model.

Current treatment methods to repair meniscal tears do not bring fully satisfactory results. Tissue adhesives are considered promising alternatives, since they are easy to apply and cause minimal tissue trauma. The first aim of this study was to analyze the adhesive properties of and tissue response to two recently developed biodegradable block copolymeric three-armed- and hyper-branched tissue adhesives. The second aim was to investigate if tissue surface modification with collagenase improves the attachment of the adhesives and increases the healing potential of the tissue. Cylindrical explants were harvested from bovine menisci. The central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing, using one of the novel glues, Dermabond® or fibrin glue. The repair constructs were cultured in vitro for 1 and 28 days. Adhesion tests and histology were performed to analyze the effects of the glue in combination with the additional treatment. The adhesive strength of the novel glues was 40-50 kPa, which was significantly higher than that of fibrin glue (15 kPa). Cells were present in direct contact with the glues, and the tissue remained vital during the whole culture period. Increased cellularity around the tear in the collagenase treated explants was observed after 1 day. The two newly developed tissue adhesives are attractive materials to be used for repair of meniscal tears. The beneficial influence of collagenase treatment in treating meniscal tears with glues still needs to be confirmed in more clinical relevant studies. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1405-1411, 2017.

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues

ABSTRACTPurpose: Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Materials and Methods: Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Results: Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. Conclusions: This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.