Bovine Cartilage Research Articles

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

Biomimetic proteoglycans diffuse throughout articular cartilage and localize within the pericellular matrix.

Biomimetic proteoglycan (BPG) diffusion into articular cartilage has the potential to restore the lost proteoglycan content in osteoarthritic cartilage given these molecules mimic the structure and properties of natural proteoglycans. We examined the diffusion characteristics of BPGs through cartilage with the use of a custom-made in vitro cartilage diffusion model in both normal bovine and human osteoarthritic cartilage explants. BPGs were introduced into the cartilage through essentially one-dimensional diffusion using osteochondral plugs. The molecular diffusion was shown to be size and concentration dependent. Diffusion profiles were found over different diffusion time intervals and the profiles were fit to a nonlinear Fickian diffusion model. Steady state 011012-7diffusion coefficients for BPGs were found to be 4.01 and 3.53 μm2 /s for 180 and 1600 kDa BPGs, respectfully, and these values are similar to other large molecule diffusion in cartilage. In both bovine and osteoarthritic human cartilage, BPGs were found localized around the chondrocytes. BPG localization was examined by labeling collagen type VI and soaking 5 μm thick sections of cartilage with BPG solutions demonstrating that the BPGs diffused into the cartilage and preferentially localized alongside collagen type VI in the pericellular matrix.

A Synthetic Bottle-brush Polyelectrolyte Reduces Friction and Wear of Intact and Previously Worn Cartilage.

A poly(7-oxanorbornene-2-carboxylate) polymer containing pendent triethyleneglycol (TEG) chains of 2.8 MDa ("2.8M TEG") was synthesized and evaluated for long-term lubrication and wear reduction of ex vivo bovine cartilage as well as for synovitis in rats and dogs after intra-articular administration. Bovine cartilage surfaces were tested under torsional friction for 10,080 rotations while immersed in either saline, bovine synovial fluid (BSF), or 2.8M TEG. For each solution, coefficient of friction (μ), changes in surface roughness, and lost cartilage glycosaminoglycan were compared. To directly compare 2.8M TEG and BSF, additional samples were tested sequentially in BSF, BSF, 2.8M TEG, and then BSF. Finally, another set of samples were tested twice in saline to induce surface roughness and then tested in BSF, Synvisc, or 2.8M TEG to determine each treatment's effect on worn cartilage. Next, male Lewis rats were injected in one knee with 2.8M TEG or saline and evaluated for effects on gait, and female beagles were injected with either 2.8M TEG or saline in one knee, and their synovial tissues analyzed for inflammation by H&E staining. Treatment with 2.8M TEG lowers μ, lessens surface roughness, and minimizes glycosaminoglycan loss compared to saline. The 2.8M TEG also reduces μ compared to BSF in pairwise testing and on worn cartilage surfaces. Injection of 2.8M TEG in rat or beagle knees gives comparable effects to treatment with saline, and does not cause significant synovitis.

The investigation of fluid flow in cartilage contact gap

Synovial fluid flow in articular joint capsule plays an important role during mixed mode lubrication. However, the actual fluid flow behaviour during cartilage contact has not been fully understood so far. This is due to the difficulties in measuring the gap permeability using conventional experimental techniques. The problem becomes further complicated with consideration of the cartilage surface roughness. Here a validated numerical study was developed to quantify the gap permeability of lateral synovial fluid flow. Both macro- and micro-scale gap flow models were created based on Darcy's law at the macro-scale and the Navier-stokes equation at the micro-scale. To generate model inputs, the cartilage topography was numerically synthesised based on the experimental measurements of bovine medial tibia cartilage surface roughness using Dektak Stylus Profilers. The experimental results show that the average roughness height Ra is 1.97 μm and root-mean-square roughness height Rq is 2.44 μm, while the correlation lengths of the secondary and tertiary undulations are round 100 μm and 20 μm, respectively. The numerical results indicate that the contact gap height and fluid pressure gradient are two critical parameters which significantly affect the gap permeability. As the contact gap closes, there is a decrease in gap permeability, and most importantly, the gap permeability is also very sensitive to the fluid pressure gradient. Furthermore, with gap closure, the permeability of the contact gap gradually approaches that of the cartilage tissue, at which point the contact gap is functional closed. This occurs at a contact gap height around 1 μm and fluid pressure gradient below 5 × 105 Pa/m in this study.

Guggulipid ameliorates adjuvant-induced arthritis and liver oxidative damage by suppressing inflammatory and oxidative stress mediators

BackgroundArthritis is a common degenerative joint disease characterized by deterioration of articular cartilage, subchondral bone, and associated with immobility, pain and inflammation. The incessant action of reactive oxygen species (ROS) during progressive arthritis causes severe oxidative damage to vital organs and circulatory system. PurposeIn this study we investigated the ability of guggulipid (GL), a lipid rich extract from the gum resin of the plant Commiphora whighitii to suppress the progressive arthritis and associated liver oxidative stress both in vivo and in vitro. Study design/MethodsThe anti-arthritic ability of GL was demonstrated in vitro using IL-1β stimulated bovine nasal cartilage model and in vivo Freund's complete adjuvant-induced arthritic rat model. Collagen/proteoglycan degradation and pro-inflammatory mediators were monitored in the harvested culture medium of nasal cartilage by estimating the levels of matrix metalloproteinases (MMPs), hydroxy proline, glycosaminoglycans and inflammatory mediators. Further, anti-arthritic ability of GL was evaluated in vivo by measuring enzymatic and non-enzymatic mediators of cartilage degradation, inflammation and oxidative stress markers. ResultsGL significantly inhibited the IL-1β stimulated cartilage degradation in vitro by mitigating the MMPs activity, collagen degradation and secretion of pro-inflammatory mediators. Further, GL significantly reduced the adjuvant-induced paw swelling and body weight loss in vivo. GL remarkably reduced the MMPs and hyaluronidases activities in serum and bone homogenate along with altered hematological parameters. GL also mitigated the elevated bone resorbing enzymes cathepsins, exoglycosidases and phosphatases. Additionally, GL effectively mitigated ROS and oxidative stress-mediators recuperating the altered serum/liver oxidative stress and liver damage incurred during arthritic progression. ConclusionIn summary, the study clearly demonstrates the protective efficacy of GL against arthritis and its associated oxidative stress, particularly, liver oxidative damage. Hence, GL could be a potential alternative and complementary medicine to treat inflammatory joint diseases.

The fate of nanoscale drug delivery systems for cartilage is influenced by osteoarthritis progression and tissue targeting strategy

Purpose: Advancing therapeutics for osteoarthritis (OA) is a pressing clinical need. Many emerging therapies act on chondrocytes, but drug delivery to cartilage has prohibitively low efficiency due to its dense, avascular extracellular matrix. Accordingly, there is significant interest in engineering targeted nanoscale delivery vehicles to facilitate retention in cartilage for localized drug release, thereby improving therapeutic impact. Here, a polymeric nanoparticle (NP) system was developed with amine functionalization for passive cartilage targeting via electrostatic interactions, and conjugation of a type 2 collagen (col2) binding peptide for active targeting. We have shown previously that cationic NPs indeed exhibit greater particle retention in healthy cartilage, likely due to electrostatic interactions with glycosaminoglycans (GAGs), but this observation is diminished in diseased tissue as GAGs are depleted. It is conceivable that the targeting approach may need to be tuned for specific diseases states or clinical applications; therefore, the purpose of this study was to directly compare targeting methods at different stages of cartilage degeneration. We hypothesize that active targeting to the col2 matrix will provide superior tissue retention relative to passive targeting via electrostatic interactions in diseased cartilage, as the tissue becomes less anionic and the porosity increases. Methods: NPs were synthesized with a poly(lactide-co-glycolide) core and polymers on the surface: polyvinyl alcohol for stabilization and/or polyallylamine (PAA) for amine decoration and surface charge modification. For passive cartilage targeting, PAA was increased to yield a cationic NP, which is designed to target the anionic cartilage matrix. For untargeted NP controls, polyallylamine concentration was reduced until the NPs were neutrally charged. For active targeting, NPs were functionalized with col2 binding peptides via conjugation. AlexaFluor dyes were conjugated to the NPs for fluorescence labelling. NP accumulation (loading) within bovine cartilage was assessed after exposing NP suspensions to the articular surface of fresh and enzymatically digested (“OA” mimic) cartilage explants for 1hr. NP release from the explants was assessed after incubation in phosphate buffered saline (PBS) at 37°C for 24hrs. Quantification of the NP loading into and release from cartilage was determined by fluorescence measurements of the tissue homogenate and surrounding PBS media, respectively, and calculated from standard curves. In vivo distribution was performed in healthy and collagenase-induced OA knees by injecting 20uL of NPs intra-articularly. Biodistribution was assessed 48hrs post injection by isolating the major knee tissues and imaging via an In Vivo Imaging System (IVIS). Results: All NP formulations were statistically the same size by dynamic light scattering (Fig 1A). Passive and active NPs were positively charged while untargeted NPs were neutral (Fig 1B). When loading NPs into cartilage, significant improvements were observed in OA tissue relative to healthy tissue in untargeted and active targeted NPs, likely because of increased pore size and access to the col2 network (Fig 1C). Passive targeted NPs demonstrated similar loading in healthy and OA tissue, likely because the increased porosity was counteracted by the weakened electrostatic gradient associated with GAG loss. After 24 hours, most of the untargeted NPs were released from the cartilage in both healthy and OA conditions (Fig 1D). In contrast, the majority of targeted NPs were retained in healthy and OA cartilage, with active targeting resulting in greater retention than passive targeting in OA tissue (Fig 1D). After intra-articular injection, whole joint distribution of both actively and passively targeted NPs was impacted by disease state, with greater cartilage accumulation in OA relative to healthy cartilage (Fig 2). Conclusions: In vitro data suggest that the tissue condition is the factor driving NP loading into cartilage, while NP retention within the cartilage is more heavily influenced by NP targeting strategy. This trend regarding NP loading was confirmed in vivo, with considerable differences in joint tissue biodistribution between healthy and OA joints. Future work will assess the NP-cartilage interactions at longer time scales for improved clinical relevance, as well as the interplay between NP retention, release of emerging OA drugs from the NP system, and therapeutic impact in vitro and in vivo. Overall, these studies highlight the importance of evaluating NP systems across degrees of disease progression. Accordingly, patient stage of disease may inform vehicle design for optimal delivery of emerging OA therapeutics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Chondroitin Sulfate Pharmacological Activity from Four Different Sources in Human Osteoarthritic Cartilage

ObjectiveChondroitin sulfate (CS) is a glycosaminoglycan and a well‐known component of cartilage extracellular matrix, which has been shown to have clinical benefit in symptomatic osteoarthritis (OA) and data suggest to be a modulating agent of joint inflammation. CS is generally produced by means of extraction from different animal tissues (bovine, porcine and marine cartilage). Because of differences in CS in terms of source, purity, quality and the production/purification process, we compared the effects of CS from four different sources on the function of human OA chondrocytes.MethodsExperiments were carried out on human OA chondrocytes obtained from knee patients undergoing total joint arthroplasty (n=8). Four different CS products were tested: CS#1, which is a high purity CS (98–100%) of bovine origin (Sioux Pharm Inc., Sioux Center, IA, US) and CS#2, CS#3 and CS#4, which are of lower purity and from other animal sources, CS#2 and CS#4, porcine; CS#3, avian. Determination of protein production of metalloproteinase (MMP)‐1 and MMP‐3, interleukin (IL)‐6 and prostaglandin E2 (PGE2) was performed on the conditioned medium using specific ELISAs. All determinations were performed in duplicate.ResultsCS#2 and CS#3 upregulated MMP‐1 (p=0.002, p=0.004) and CS#2 and CS#4, MMP‐3 (p=0.008, p=0.037). IL‐6 was also increased by CS#2, CS#3 and CS#4 (p=0.008, p=0.007, p=0.014). For PGE2 production, as the values were too low and unreliable without an inducer, the experiment was done only in the presence of IL‐1b (100 pg/ml), which greatly increased the value (absence of IL‐1b, 0.98±0.08 pg/mg protein; presence of IL‐1b, 42.18±10.80 pg/mg protein; p=0.007). CS#1 was the only one to significantly reduce PGE2 levels (p=0.035). In the presence of IL‐1b, CS#1 was also the only compound to show a trend toward significance (p=0.062) in reducing IL‐6 production.ConclusionsData showed that the three CS from non‐bovine origins increased an inflammatory factor in human OA chondrocytes, as well as upregulated the production of two catabolic factors. In contrast, CS of higher purity and coming from bovine source showed anti‐inflammatory properties as it reduced PGE2 and IL‐6 levels, even in the presence of IL‐1b. Therefore, the source/nature of these products could contribute to the overall biological and pharmacological effects of these agents in the symptomatic treatment of OA, compromising the safety and efficacy of the final products.Support or Funding InformationSupported by BIOIBÉRICA, S.A.U., Barcelona, Spain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Numerical Study on Electromechanics in Cartilage Tissue with Respect to Its Electrical Properties.

Hyaline cartilage undergoes many substantial age-related physiochemical and biomechanical changes that reduce its ability to overcome the effects of mechanical stress and injury. In quest of therapeutic options, magnetic stimulation and electrical stimulation (ES) have been proposed for improving tissue engineering approaches for the repair of articular cartilage. The aim of this study is to summarize in silico investigations involving induced electrical properties of cartilage tissue due to various biophysical stimuli along their respective mathematical descriptions. Based on these, a preliminary numerical study involving electromechanical transduction in bovine cartilage tissue has been carried out using an open source finite element computational software. The simulation results have been compared to experimental results from the literature. This study serves as a basis for further in silico studies to better understand the behavior of hyaline cartilage tissue due to ES and to find an optimal stimulation protocol for the cartilage regeneration. Moreover, it provides an overview of the basic models along with mathematical description and scope for future research regarding electrical behavior of the cartilage tissue using open source software.Impact StatementThe presented research summarizes the basic models with mathematical description regarding electrical behavior of the cartilage tissue. A preliminary numerical study involving electromechanical transduction in bovine cartilage tissue sample has been carried out using an open source finite element software. This research will provide scope for future research regarding electrical behavior of the cartilage tissue using open source software.

The superficial zone in cartilage adapts with stiffening when challenged tribologically

Purpose: In osteoarthritis, the superficial zone of articular cartilage is compromised by fissures and cracks and, thus, may lose its intended function. While there is agreement that the superficial zone is important for the load bearing capacity of the tissue, its mechanical response upon articulation is less investigated. In previous studies, using fully intact cartilage samples, we have shown that the cartilage surface undergoes a transient stiffening response upon articulation. Here, we hypothesize that this response is lost when the surface is removed. In order to address this question we used a tribological bioreactor and articulated on cartilage samples with and without superficial zone. Further, we started to study structural changes at the surface with an atomic force microscope (AFM) before and after the application of tribological stress. Methods: Twenty-four 14x20x3 mm oval bovine cartilage explants that had undergone one freeze-thaw cycle were distributed into four groups per animal: (1) intact surface and articular loading; (2) removed surface and articular loading; (3) intact free-swelling-control (FSC); 4) removed surface FSC. Thus, each group had n=6 samples. A vibratome was used to remove the superficial zone of the explants (at least 20% of full sample thickness). Microindentation stress-relaxation tests were performed in the center of explants using a Hysitron TI 950 with a 20 μm spherical indenter and an 8 μm indent depth. All indentation on explants was performed submerged in 1× PBS to maintain tissue hydration. Following initial characterization, cartilage explants were removed from the indentation sample holder and placed into the bioreactor. Cartilage explants were confined in porous polyethylene scaffold rings and fixed in a well. Using a 32-mm ceramic ball (Ra ∼10 nm), a 40 N static confined compression was applied that lead to approx. 2 MPa contact stress. The ball oscillated at 0.5 Hz, and was migrated at a speed of 1mm/s across the cartilage surface generating a wear scar length of 10 mm. Following 1-hr of bioreactor testing, microindentation was repeated in the center/wear scar. The reduced modulus was obtained using the Oliver-Pharr method on 80-95% of the unloading curve. Cartilage surfaces removed by vibratome were prepared for AFM analysis (Nanowizard Ultra, JPK Instruments, Germany). All measurements were conducted in 1× PBS. Using QI imaging, a force sampling mode, an initial image of 256 x 256 pixels (approx. 10 x 10 μm2) was obtained. The AFM was then put into contact mode, and an 80 nm silicon tip was repeatedly (>850 times) slid linearly across the cartilage surface. Following these sliding experiments, the tissue was reimaged in QI mode. For data analysis, the ratio of post- to pre-bioreactor reduced modulus was calculated and analyzed using Shapiro-Wilks tests for normality and Paired Student’s T-tests. Results: Our results show that intact surface groups exhibited significantly increased stiffening following articular loading compared to the FSC (p=0.006). For the removed surface groups, no significant difference between articulated and FSC explants was detected (p=0.927) (Fig 1). Also in the AFM the cartilage surface demonstrated topographical and stiffness related changes upon sliding that are shown in Figure 2. Conclusions: Surface stiffening following tribological loading may be an important biomechanical function of load transfer during articulation. Surface removal leads to a loss of this adaptive response. The stiffening response may be related to a reorganization of matrix molecules that are unique to the superficial zone. In the future we plan to investigate the influencing parameters of this stiffening response and identify the matrix molecules that are involved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Manganese dioxide nanoparticles modulate oxidative stress and protect cartilage from interleukin-1β induced degradation

Purpose: Osteoarthritis (OA) is associated with chronic joint inflammation, whereby pro-inflammatory cytokines such as IL-1β upregulate reactive oxygen species (ROS) production while downregulating anti-oxidants in cells. The resulting oxidative stress leads to extracellular matrix degradation, joint inflammation, and chondrocyte death and senescence. The overall goal of this research is to develop a bioactive nanoparticle (NP) system that scavenges ROS in cartilage to modulate the impact of joint inflammation. Manganese dioxide (MnO2) catalyzes the breakdown of hydrogen peroxide (H2O2) and is currently being evaluated for scavenging ROS in inflammatory diseases such as atherosclerosis. We previously reported engineered MnO2 NPs with properties that enable them to penetrate cartilage and become endocytosed by chondrocytes. The objective of this study was to evaluate the chondroprotective mechanisms of MnO2 NPs and their in vivo retention and biocompatibility. Methods: MnO2 NPs were synthesized and characterized as previously described. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2D culture and cartilage explants were confirmed by fluorescent imaging after 24 hours of incubation. Gene expression analysis of both catabolic and ROS mediator genes was conducted on a monolayer of primary bovine chondrocytes challenged by human recombinant IL-1β in the presence of MnO2 NPs after 24-hour culture. Fresh bovine cartilage biopsies were evaluated for loss of glycosaminoglycans (GAG), the production of nitric oxide (NO) and cell viability over a 14-day period when challenged by IL-1β in the presence of MnO2NPs. Articular joint retention of Alexa Fluor 750 and 594 conjugated NPs in Lewis rats was monitored using an in vivo IVIS Imaging System. Long-term cytocompatibility of the particles 6 weeks after intraarticular injection in healthy rat joints was assessed histologically using GEKO©, a tool for quantitative grading of toluidine stained knees. Results: The PEG stabilized MnO2 NPs had a hydrodynamic diameter of 15 nm, TEM size of 8 nm and had a cationic surface charge. The NPs effectively scavenged H2O2, with 5μg/mL MnO2 NPs neutralizing 55% of 100uM H2O2 in PBS. Chondrocytes in monolayer showed uptake of MnO2 NPs without cytotoxicity. Furthermore, the NPs reduced the gene expression of catabolic mediator, heme oxygenase-1, catabolic factor matrix metalloproteinase 3, antioxidant mediator nuclear factor erythroid 2-related factor 2, antioxidants catalase, glutathione peroxidase and manganese superoxide dismutase by chondrocytes (Fig. 1A) indicating protection of the particles against oxidative stress. When incubated with cartilage explants, the MnO2 NPs penetrated through the cartilage depth and were found in the extracellular matrix as well as intracellularly in chondrocytes. In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased GAG loss by 50% and NO production by 40% compared to controls (Fig. 1B and C). Importantly, treatment with MnO2 NPs improved viability of chondrocytes in cartilage explants over the 2-week study. Upon in vivo injection, the MnO2 NPs persisted in the joint space over one week, with 20% of the initial fluorescent signal still observed by day 13 (Fig. 2A and B). Biodistribution and histological analysis (Fig. 2C) revealed accumulation of particles at the chondral surfaces, and colocalization of the NPs with the lacunae of chondrocytes, indicating particle penetration into the cartilage and uptake by chondrocytes following intraarticular injection. A gradient of NPs existed in cartilage with the highest levels of NPs on the articular surface which decreased with tissue depth. Histological analysis also revealed no adverse effects of intra-articular MnO2 NP injection into healthy animals. Histological parameters such as synovium thickness, total cartilage degeneration width and cartilage matrix loss width were comparable in knees injected with the NPs and those injected with saline after 6 weeks, indicating biocompatibility of the particles in vivo (Fig. 2D). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. In vitro, the NPs protected chondrocytes in cartilage explants from IL-1β mediated oxidative stress and apoptosis. Further analysis of the joint confirmed penetration of MnO2 NPs into cartilage in vivo. Given their joint retention time and ROS scavenging capacity, these NPs could target oxidative stress to treat or prevent OA. As the NPs show intracellular localization in chondrocytes, they could also deliver other chondroprotective agents including nucleic acids to target multiple pathways in the OA pathology. Further studies will focus on the therapeutic impact of MnO2 NPs in OA disease models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

GPDPLQ1237\u2014A Type II Collagen Neo-Epitope Biomarker of Osteoclast- and Inflammation-Derived Cartilage Degradation in vitro

C-telopeptide of type II collagen (CTX-II) has been shown to be a highly relevant biomarker of cartilage degradation in human rheumatic diseases, if measured in synovial fluid or urine. However, serum or plasma CTX-II have not been demonstrated to have any clinical utility to date. Here, we describe the GPDPLQ1237 ELISA which targets the EKGPDPLQ↓ neo-epitope, an elongated version of the CTX-II neo-epitope (EKGPDP↓), speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. Human osteoclast cartilage resorption cultures as well as oncostatin M and tumour necrosis factor α-stimulated bovine cartilage explant cultures were used to validate GPDPLQ1237 biologically by treating the cultures with the cysteine protease inhibitor E-64 and/or the matrix metalloproteinase (MMP) inhibitor GM6001 to assess the potential contributions of these two protease classes to GPDPLQ1237 release. Cartilage resorption-derived GPDPLQ1237 release was inhibited by E-64 (72.1% inhibition), GM6001 (75.5%), and E-64/GM6001 (91.5%), whereas CTX-II release was inhibited by GM6001 (87.0%) but not by E-64 (5.5%). Cartilage explant GPDPLQ1237 and CTX-II release were both fully inhibited by GM6001 but were not inhibited by E-64. No clinically relevant GPDPLQ1237 reactivity was identified in human serum, plasma, or urine from healthy donors or arthritis patients. In conclusion, the GPDPLQ1237 biomarker is released during osteoclast-derived cysteine protease- and MMP-mediated cartilage degradation in vitro, whereas CTX-II release is mediated by MMPs and not by cysteine proteases, as well as from MMP-mediated cartilage degradation under a pro-inflammatory stimulus. These findings suggest that GPDPLQ1237 may be relevant in diseases with pathological osteoclast activity and cartilage degradation. Further studies are required to validate the neo-epitope in human samples.

Effects of Antibiotics on α-Toxin Levels during Staphylococcus aureus Culture: Implications for the Protection of Chondrocytes in a Model of Septic Arthritis.

Septic arthritis results from joint infection by Staphylococcus aureus, which produces potent α-toxin causing cell death, potentially leading to permanent cartilage damage. Treatment is by joint irrigation and antibiotics, although it is unclear if, following treatment with antibiotics which cause bacterial lysis, there is release of additional stored α-toxin. A rabbit erythrocyte hemolysis assay was optimised to assess biologically-active α-toxin from cultured S. aureus α-toxin strain DU5946. Hemoglobin release was measured spectrophotometrically following addition of a bacteriostatic antibiotic (linezolid) or a bacteriolytic antibiotic (penicillin). A bovine cartilage model of septic arthritis was used to test the protective effects of antibiotics against S. aureus infection. During S. aureus culture, α-toxin levels increased rapidly but the rate of rise was quickly (within 20 minutes) suppressed by linezolid (25 μg/mL). Penicillin also reduced the increase in α-toxin levels; however, the time course was relatively slow compared to linezolid even at high concentrations (50,000 U/mL). The efficacy of penicillin (250,000 U/mL) at reducing the rise in α-toxin was approximately 8% less than that of linezolid (P < 0.05) suggesting the presence of additional toxin. This could be due to a delayed action of penicillin, and/or release of a small pool of stored α-toxin from dying bacteria. In a bovine cartilage model, however, there was no difference between the protection of in situ chondrocytes against S. aureus by penicillin or linezolid (P > 0.05). The results suggested that equally effective protection of chondrocytes against S. aureus septic arthritis may be obtained by the bacteriostatic or bacteriolytic antibiotics tested.

Physicobiochemical Characteristics and Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) in Biodegradable Porous Sponge Bovine Cartilage Scaffold.

Tissue engineering had been believed to overcome the limitation of cartilage lesions treatment. Nowadays the studies focus on using mesenchymal stem cells in scaffold. A biodegradable porous sponge bovine cartilage scaffold is expected to have the physicobiochemical characterization to promote chondrogenic differentiation of hBM-MSCs. Scaffold from bovine cartilage was printed in 5 mm diameter sponge, categorized into nondecellularized (SBCS) and decellularized (DSBCS). Physical characteristics (pore diameter and interconnectivity) were done using a Scanning Electron Microscope (SEM). Biodegradability assessment used Phosphate Buffered Saline in 15, 30, 60 minutes, 6, 24, 48, 72 hours, and 1, 2 weeks. The swelling ratios were counted in 5, 10, 15, 30, 60, and 360 minutes. Biochemical characteristics were obtained by enzyme-linked immunosorbent assay for type II collagen, aggrecan, and Transforming Growth Factors-β (TGF-β). Data were statistically compared. hBM-MSCs were seeded on both scaffolds. Histological examination used hematoxylin-eosin taken at the 2nd and 4th weeks after seeding. There was no significant difference (p=0.473; p=0.142) on mean porosity 90.07 ± 4.64% vs. 88.93 ± 4.18% and pore diameter 111.83 ± 14.23 μm vs. 105.29 ± 11.14 μm assessment between SBCS and DSBCS groups. Scaffolds from both groups showed pore interconnectivity. DSBCS group had faster biodegradability. SBCS group sweals better. SBCS group contains type II collagen, aggrecan, and TGF-β with mean values 380.78 ± 18.63 ng/ml, 30.71 ± 4.50 ng/ml, and 130.12 ± 7.73 ng/ml, respectively, while DSBCS contained type II collagen, aggrecan, and TGF-β with mean values 64.83 ± 13.54 ng/ml, 8.41 ± 2.38 ng/ml, and 16.39 ± 4.49 ng/ml, respectively. The results were statistically different (p<0.001). Chondrocytes were found within scaffold on the 2nd and 4th weeks. Physicobiochemical characteristic of biodegradable sponge bovine cartilage scaffold promotes chondrogenic differentiation of hBM-MSCs.

Laser perforation and cell seeding improve bacterial nanocellulose as a potential cartilage implant in the in vitro cartilage punch model

Bacterial nanocellulose (BNC) shows high biocompatibility as wound dressing or dura mater, blood vessel, and cartilage implant. Three-dimensional perforation (3-D-∅) favors migration of chondrocytes into the BNC and cartilage matrix formation. Thus, the regenerative capacity of 3-D-∅ BNC implants was tested in a standardized bovine cartilage punch model. Cartilage rings containing a central defect with an outer diameter of 6 mm and an inner diameter of 2 mm were prepared from the trochlear groove (femur-patellar articulation site). Three-D-∅ BNC implants (cell-free or cell-loaded) were cultured inside the cartilage rings for up to 12 weeks. Cartilage-BNC-constructs were then investigated by histology (hematoxylin/eosin; safranin O) and immunohistology (aggrecan, collagens 1 and 2), as well as for protein content, RNA expression, and implant push-out force. Cartilage-BNC-constructs remained vital with preserved matrix integrity during culture and almost no loss of matrix-bound proteoglycan (aggrecan) or collagen 2 from ‘host’ cartilage (with very limited quantities of collagen 1). Interestingly, 3-D-∅ BNC implants displayed: (1) significantly increased superficial, but also 3-D cell seeding over time (cell-loaded significantly earlier than cell-free); (2) progressively increased aggrecan/collagen 1 and collagen 2/collagen 1 mRNA ratios, as well as aggrecan and collagen 2 protein levels; and (3) significantly increased push-out forces over time (cell-loaded). Progressively increasing cell seeding and chondrogenic differentiation suggest beginning cartilage regeneration of the 3-D-∅ BNC in this model system, and indicate an excellent potential of 3-D-∅ BNC as a cartilage replacement material. Cell-loading may favor implant performance by accelerating cell colonization.

In vitro analysis of the potential cartilage implant bacterial nanocellulose using the bovine cartilage punch model

Biocompatible bacterial nanocellulose (BNC) shows high potential as wound dressing and dura mater replacement, and even for the development of blood vessel or cartilage implants. Thus, the regenerative capacity of BNC implants was analyzed using a standardized bovine cartilage punch model. Cartilage rings with an outer diameter of 6 mm and an inner defect diameter of 2 mm were derived from the trochlear groove (femur-patellar articulation site). BNC implants were cultured inside the cartilage rings for up to 12 weeks. Cartilage-BNC-constructs were then evaluated by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, mRNA expression, and push-out force of the implants. Cartilage-BNC-constructs displayed vital chondrocytes (≥ 90% until week 9; > 80% until 12 weeks), preserved matrix integrity during culture, limited loss of matrix-bound proteoglycan from ‘host’ cartilage or cartilage-BNC-interface, and constant release of proteoglycans into the culture supernatant. In addition, the content of the matrix protein collagen 2 in cartilage and cartilage-BNC-interface was approximately constant over time (with very limited quantities of collagen 1). Interestingly, BNC implants showed: (1) cell colonization of the implant; (2) progressively increasing mRNA levels for the proteoglycan aggrecan and collagen 2 (max. fivefold); and (3) significantly increasing push-out forces during culture (max. 1.6-fold). Retained tissue integrity and progressively increasing chondrogenic differentiation in implant and cartilage-implant-interface suggest beginning cartilage regeneration in the BNC in the present model and indicate a high potential of BNC as a cartilage replacement material. Thus, the present model appears suitable to predict the in vivo performance of cartilage replacement materials (e.g., BNC) for tissue engineering.

Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of “AuriScaff”, an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control.Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo.With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. Statement of SignificanceAfter two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

Detection of glycosaminoglycan loss in articular cartilage by fluorescence lifetime imaging.

Glycosaminoglycan (GAG) loss is an early marker of osteoarthritis, which is a clinical late stage disease that affects millions of people worldwide. The goal of our study was to evaluate the ability of a fiber-based fluorescence lifetime imaging (FLIm) technique to detect GAG loss in articular cartilage. Native bovine cartilage explants (n = 20) were exposed to 0 (control), 0.5 (low), or 1 U / mL (high) concentrations of chondroitinase ABC (cABC) to create samples with different levels of GAG loss. FLIm assessment (excitation: 355nm; detection: channel 1: 375 to 410nm, channel 2: 450 to 485nm, channel 3: 530 to 565nm) was conducted on depth-resolved cross-sections of the cartilage sample. FLIm images, validated with histology, revealed that loss of GAG resulted in a decrease of fluorescence lifetime values in channel 2 (Δ = 0.44 ns, p < 0.05) and channel 3 (Δ = 0.75 ns, p < 0.01) compared to control samples (channel 2: 6.34ns; channel 3: 5.22ns). Fluorescence intensity ratio values were lower in channel 1 (37%, p < 0.0001) and channel 2 (31% decrease, p < 0.0001) and higher in channel 3 (23%, p < 0.0001) relative to control samples. These results show that FLIm can detect the loss of GAG in articular cartilage and support further investigation into the feasibility of in vivo FLIm arthroscopy.

Functional bioreactor characterization to assess potentials of nanocomposites based on different alginate types and silver nanoparticles for use as cartilage tissue implants.

In this work, functional characterization of biomaterials concerning potential application as articular cartilage implants was performed by using a biomimetic bioreactor with dynamic compression in the physiological regime (10% strain, 0.84 Hz frequency, 1 h on/1 h off). Specifically, two alginate types with low (LG) and high (HG) guluronic/mannuronic residue ratios with electrochemically synthesized silver nanoparticles (AgNPs) were evaluated. HG Ag/alginate hydrogels were clearly indicated as potential candidates due to better initial mechanical properties as compared to LG hydrogels (dynamic compression modulus of ~60 vs. ~40 kPa) as well as the mechanical stability displayed during 7 days of dynamic compression. Cytotoxicity studies in 3D bovine cartilage explant cultures under dynamic compression have shown negligible effects as compared to standard 2D monolayers of bovine chondrocytes where moderate cytotoxicity was observed. Finally, experimental and mathematical modeling studies revealed different mechanisms of AgNP release under physiological-like bioreactor conditions as compared to static conditions. Overall, the results clearly demonstrate bioreactor advantages in characterization and selection of candidate biomaterials as well as potentials to bridge the in vitro-in vivo gap. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 755-768, 2019.

Physiologic Medium Maintains the Homeostasis of Immature Bovine Articular Cartilage Explants in Long-Term Culture.

The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.

Viscoelasticity measurement of ex vivo bovine cartilage using Lamb wave method

Cartilage viscoelasticity changes with cartilage degeneration. Therefore, measurement of cartilage viscoelasticity could offer an alternative to traditional imaging methods for early diagnosis of osteoarthritis. In previous studies, we confirmed the feasibility of using the Lamb wave method (LWM) to assess the elasticity within a simulated cartilage–bone structures. The objective of the present study was to test the suitability of the LWM for measuring viscoelastic properties of ex vivo bovine tibial plateau cartilage. Theoretical and numerical analyses were performed to understand the viscoelastic Lamb wave propagation in cartilage and a suitable measurement range and loading frequency for the LWM were determined. The viscoelasticity of bovine cartilage was experimentally estimated to be 32.8 ± 0.50, 30.9 ± 0.50, and 31.9 ± 1.85 Pa · s for cartilage samples with thicknesses of 2.84 ± 0.02, 2.32 ± 0.03, and 2.01 ± 0.08 mm, respectively, which showed good repeatability during LWM measurements. However, cartilage elasticity was not sensitive to the viscoelastic Lamb wave speed. Indentation tests were also performed and cartilage elasticities were 0.88 ± 0.09, 1.03 ± 0.12, and 0.93 ± 0.09 MPa, respectively. This study validates the use of the LWM for measuring the viscoelasticity of ex vivo cartilage and provides a critical step toward the ultimate goal of measuring the viscoelasticity of cartilage in vivo.