Application of sodium triple-quantum coherence NMR spectroscopy for the study of growth dynamics in cartilage tissue engineering.

We studied the tissue growth dynamics of tissue-engineered cartilage at an early growth stage after cell seeding for four weeks using sodium triple-quantum coherence NMR spectroscopy. The following tissue-engineering constructs were studied: 1) bovine chondrocytes cultured in alginate beads; 2) bovine chondrocytes cultured as pellets (scaffold-free chondrocyte pellets); and 3) human marrow stromal cells (HMSCs) seeded in collagen/chitosan based biomimetic scaffolds. We found that the sodium triple-quantum coherence spectroscopy could differentiate between different tissue-engineered constructs and native tissues based on the fast and slow components of relaxation rate as well as on the average quadrupolar coupling. Both fast (Tf ) and slow (Ts ) relaxation times were found to be longer in chondrocyte pellets and biomimetic scaffolds compared to chondrocytes suspended in alginate beads and human articular cartilage tissues. In all cases, it was found that relaxation rates and motion of sodium ions measured from correlation times were dependent on the amount of macromolecules, high cell density and anisotropy of the cartilage tissue-engineered constructs. Average quadrupolar couplings were found to be lower in the engineered tissue compared to native tissue, presumably due to the lack of order in collagen accumulated in the engineered tissue. These results support the use of sodium triple-quantum coherence spectroscopy as a tool to investigate anisotropy and growth dynamics of cartilage tissue-engineered constructs in a simple and reliable way.

Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage.

The appearance of a high molecular weight gelatinolytic enzyme (230 kDa) correlated with cartilage collagen loss in chick embryonic tibias cultured with lipopolysaccharide. This 230 kDa enzyme was purified and its activity was measured on synthetic and natural substrates. The enzyme was activated by aminophenylmercuric acetate and inhibited by ethylenediaminetetraacetic acid, phenanthroline, marimastat or tissue inhibitors of metalloproteinases. Amino acid sequences of peptides derived from the purified enzyme showed identity with avian MMP-9. Digestion of the intact enzyme with chondroitinase decreased the size of the molecule to 80 kDa on SDS-PAGE. When chick embryonic tibia cultures were radiolabeled with (35)S-sulfate, the radiolabel co-purified with the 230 kDa gelatinase. Chondroitinase treated 230 kDa gelatinase also reacted with specific anti-chondroitin sulfate antibodies and FACE analysis revealed a predominance of chondroitin-4-sulfate. These results demonstrate this avian matrix metalloproteinase contained glycosaminoglycan chains. To our knowledge, this is the first report of a matrix metalloproteinase in a proteoglycan form.

Extracorporeal shockwave-induced expression of lubricin in tendons and septa.

Lubricin, a lubricating glycoprotein that facilitates tendon gliding, is upregulated by mechanical as well as biochemical stimuli, prompting this study of its induction by extracorporeal shockwave therapy (ESWT). The objective of this study was to characterize and quantify the effect of ESWT on lubricin expression in tendons and septa in a rat model. Hindlimbs of six rats were treated with low-dose ESWT and those of another six with high-dose ESWT, using contralateral limbs as controls. After 4 days, resected samples were processed for immunolocalization of lubricin using a purified monoclonal antibody. ESWT was found to increase lubricin expression in both low-dose and high-dose ESWT-treated tendons and also in septa. Lubricin expression generally increased with increasing dose of ESWT. Increased lubricin expression may contribute to the beneficial effects of ESWT in providing pain and symptom relief in musculoskeletal disorders by decreasing erosive wear.

Presence and distribution of the lubricating protein, lubricin, in the meibomian gland in rabbits.

PURPOSE: Lubricin is a principal boundary lubricating and anti-adhesion protein found in synovial fluid and several musculoskeletal tissues. This study investigates the presence of lubricin in the meibomian gland, lacrimal gland and ocular surface of healthy rabbits; prompted by the hypothesis that lubricin acts as boundary lubricant and anti-adhesive protein in the eye. METHODS: Thirty six eyelids were resected from ten cadaveric New Zealand White rabbits and two eyeballs and two lacrimal glands from two of them. Thirty two samples from 8 animals were processed for immunohistochemical localization of lubricin using a purified monoclonal antibody and quantification of the lubricin-containing meibocytes. Confirmatory western blot analysis was performed on four eyelids from 2 animals. RESULTS: Lubricin-positive meibomian cells were seen in the glands in all eight animals evaluated immunohistochemically. The percentage of lubricin-positive cells ranged from was 8%-50% in the upper and 3%-50% in the lower eyelid, with no significant difference between the upper and lower eyelid. Western blot analysis confirmed the presence of lubricin ranging from 10 to 40 ng in four eyelids from the other two rabbits. Occasional staining was seen in the epithelium of the hair follicles of the eyelid. No lubricin was evident on the ocular surface or in the lacrimal gland. CONCLUSIONS: Lubricin is secreted by the meibomian gland. The results provide a basis for the hypothesis that lubricin plays a role in boundary lubrication and in preventing adhesions in the eye, as well as in contributing to other functions of the meibomian gland. Moreover, if lubricin functions to decrease the friction between the eyelid and ocular surface, this study provides a rationale to supplement the amount of lubricin in cases of compromised meibomian gland function and other conditions.

A MOVING CONTACT OF ARTICULATION ENHANCES THE BIOSYNTHETIC AND FUNCTIONAL RESPONSES OF ARTICULAR CARTILAGE.

Biomechanical influences play a fundamental role in the structural, functional, and biosynthetic properties of articular cartilage. During physiologic joint loading, the contact area between two surfaces migrates due to the primary and secondary motions of the joint. It has been demonstrated that a migratory contact area plays a critical role in reducing the coefficient of friction at the cartilage surface. However, a detailed analysis of the influences that a migratory contact area plays on the structural, functional, and biosynthetic properties remain to be explored. In this study, bovine cartilage explants were placed in a biotribometer. Explants were subjected to compression and shear forces of migratory contact area, namely moving contact (MC) articulation, or stationary contact area, namely stationary contact (SC) articulation. Free swelling explants were used as control. In a separate study, bovine cartilage-bone grafts were used for frictional testing. On histologic analysis, the SC group had evidence of surface fibrillations, which was not evident in the MC group. Compared to the SC group, the MC group cartilage explants had increased chondrocyte viability, increased lubricin synthesis, and comparable proteoglycan synthesis and release. MC articulation had reduced coefficient of friction as compared to SC articulation. MC articulation led to reduced surface roughness as compared to SC articulation. In conclusion, a migratory contact area can play an important role in maintaining the structural, function, and biosynthetic properties of articular cartilage. This study provides further evidence of the importance of migratory contact area and in vitro assessment of natural joint movement, which can be further evaluated in the context of cartilage homeostasis and disease.

Distribution of lubricin in the ruptured human rotator cuff and biceps tendon: a pilot study.

BACKGROUND: Lubricin is a lubricant for diarthrodial joint tissues and has antiadhesion properties; its presence in the (caprine) rotator cuff suggests it may have a role in intrafascicular lubrication. QUESTIONS/PURPOSES: To preliminarily address this role, we asked: (1) What is the distribution of lubricin in human ruptured supraspinatus and biceps tendons? (2) What are the potential cellular sources of lubricin? METHODS: We obtained seven torn rotator cuff samples and four torn biceps tendon samples from 10 patients; as control tissues, we obtained the right and left supraspinatus tendons from each of six cadavers. Specimens were fixed in formalin and processed for immunohistochemical evaluation using a monoclonal antibody for lubricin. RESULTS: We found lubricin as a discrete layer on the torn edges of all of the ruptured supraspinatus and biceps tendon samples. None of the transected edges of the tissues produced during excision of the tissues showed the presence of lubricin. Lubricin was found in 3% to 10% of the tendon cells in the cadaveric controls and in 1% to 29% of the tendon cells in the torn supraspinatus and biceps tendon samples. CLINICAL RELEVANCE: The lubricin layer on the torn edges of ruptured human supraspinatus and biceps tendons may interfere with the integrative bonding of the torn edges necessary for repair.

Competitive Binding of Bilirubin and Fatty Acid on Serum Albumin Affects Wear of UHMWPE.

Total Joint Replacement (TJR) devices undergo standardized wear testing in mechanical simulators while submerged in a proteinaceous testing solution to mimic the environmental conditions of artificial joints in the human body. Typically, bovine calf serum is used to provide the required protein content. However, due to lot-to-lot variability, an undesirable variance in testing outcome is observed. Based on an earlier finding that yellowish-orange serum color saturation is associated with wear rate, we examined potential sources of this variability, by running a comparative wear test with bilirubin; hemin; and a fatty acid, oleic acid, in the lubricant. All these compounds readily bind to albumin, the most abundant protein in bovine serum. Ultrahigh molecular weight polyethylene (UHMWPE) pins were articulated against CoCrMo discs in a pin-on-disc tribometer, and the UHMWPE wear rates were compared between lubricants. We found that the addition of bilirubin increased wear by 121%, while hemin had a much weaker, insignificant effect. When added at the same molar ratio as bilirubin, the fatty acid tended to reduce wear. Additionally, there was a significant interaction with respect to bilirubin and hemin in that UHMWPE wear rate decreased with increasing fatty acid concentration. We believe the conformational change in albumin by binding bilirubin makes it more likely to form molecular bridges between UHMWPE and the metal counterface, thus increasing adhesive wear. However, fatty acids compete for binding sites on albumin, and can prevent this conformational change. Hence, the protein is stabilized, and the chance for albumin to form bridges is lowered. Ultimately, UHMWPE wear rate is driven by the competitive binding of bilirubin and fatty acid to albumin.

Correction: Nanoscale insight into the degradation mechanisms of the cartilage articulating surface preceding OA.

Correction for 'Nanoscale insight into the degradation mechanisms of the cartilage articulating surface preceding OA' by Tooba Shoaib, et al., Biomater. Sci., 2020, 8, 3944-3955, DOI: 10.1039/D0BM00496K.

Nanoscale insight into the degradation mechanisms of the cartilage articulating surface preceding OA.

Osteoarthritis (OA) is a degenerative joint disease and a leading cause of disability globally. In OA, the articulating surface of cartilage is compromised by fissures and cracks, and sometimes even worn away completely. Due to its avascular nature, articular cartilage has a poor self-healing ability, and therefore, understanding the mechanisms underlying degradation is key for OA prevention and for optimal design of replacements. In this work, the articulating surface of bovine cartilage was investigated in an environment with enhanced calcium concentration -as often found in cartilage in relation to OA- by combining atomic force microscopy, spectroscopy and an extended surface forces apparatus for the first time. The experimental results reveal that increased calcium concentration irreversibly weakens the cartilage's surface layer, and promotes stiction and high friction. The synergistic effect of calcium on altering the cartilage surface's structural, mechanical and frictional properties is proposed to compromise cartilage integrity at the onset of OA. Furthermore, two mechanisms at the molecular level based on the influence of calcium on lubricin and on the aggregation of the cartilage's matrix, respectively, are identified. The results of this work might not only help prevent OA but also help design better cartilage replacements.

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage.

INTRODUCTION: Pre-clinical testing of hemiarthroplasty devices requires that the tribological conditions present in vivo with live cartilage be closely duplicated. A current limitation in the tribological testing of live cartilage involves the use of cell-culture media as lubricant. STUDY AIM: to develop and test a new hyaluronan-phospholipid based medium (HA-phospholipid medium) that combines the rheological and frictional properties of synovial fluid with the nourishing properties of culture media to keep cells alive. MATERIALS AND METHODS: The HA-phospholipid medium consisted of culture medium with added phospholipid dipalmitoylphosphatidylcholine (0.3 mg/mL), and hyaluronic acid (2.42 mg/mL). A standard cell culture medium was used as the control. The rheology of each medium was determined using a flat plate configuration. Bovine calf cartilage was used to assess cell viability and friction in each medium. For friction measurements, a cobalt-chrome alloy ball was articulated against cartilage disks immersed in medium. RESULTS: Lipid vesicles 0.1 to 50 µm in diameter were identified in the HA-phospholipid medium. Cartilage cell viability was significantly higher in the HA-phospholipid medium (62% ± 8%, 95% CI) than in control medium (49.5% ± 5%) (p = 0.009). The HA-phospholipid medium exhibited strong shear-thinning behavior, similar to synovial fluid, with viscosities ~100-fold higher at 10 s-1 and 5-fold higher at 20,000 s-1 than the approximately Newtonian control medium. The HA-phospholipid medium also yielded 20% lower friction values than the control medium after one hour of testing. CONCLUSIONS: The rheological and friction results indicate that the HA-phospholipid medium is superior to the control cell culture medium in emulating the shear thinning and lubricative properties of natural synovial fluid, making it more clinically relevant for in vitro wear and friction testing with live cartilage.

Increased accumulation of superficial zone protein (SZP) in articular cartilage in response to bone morphogenetic protein-7 and growth factors.

The purpose of this study was to investigate the role of bone morphogenetic proteins (BMPs), such as BMP-7, growth factors, and cytokines, in the accumulation of superficial zone protein (SZP) in bovine articular cartilage. Calf superficial articular cartilage discs and chondrocytes were obtained for explant and monolayer culture systems, respectively. Dose- and time-dependent actions of BMP-7 on SZP accumulation were investigated in both explant and monolayer culture systems. In addition, actions of various morphogens and growth factors [BMP-2, BMP-4, fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-beta1)], and cytokines [interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor (TNF-alpha)] alone, and in combination with BMP-7, on SZP accumulation were investigated in monolayer culture systems. SZP accumulation was quantified in both the cartilage and the medium using SDS-PAGE and subsequent immunoblotting. In both explant and monolayer cultures, BMP-7 increased SZP accumulation in a dose- and time-dependent fashion (p < 0.05). Furthermore, SZP accumulation was significantly increased in monolayer cultures by FGF-2, IGF-1, PDGF, and TGF-beta1 (p < 0.05). Both IL-1alpha and TNF-alpha significantly reduced SZP accumulation (p < 0.05). The inhibition of SZP accumulation by TNF-alpha was partially alleviated by concurrent treatment with BMP-7. The results of this investigation provide novel insights into the role of morphogens, especially BMP-7, growth factors, and cytokines in the accumulation of SZP in articular cartilage. This information has clinical implications because stimulation of SZP may ameliorate the pathology of joint function in arthritis. Furthermore, tissue engineering approaches to articular cartilage may depend on the optimal synthesis and assembly of SZP in the superficial zone to ensure functional tissue architecture.

Albumin Protein Cleavage Affects the Wear and Friction of Ultra-High Molecular Weight Polyethylene.

It is well established that the total protein concentration and albumin-to-globulin ratio influence the wear of ultra-high molecular polyethylene (UHMWPE, "polyethylene") in joint prostheses. A factor on wear not yet studied, but of possible clinical relevance, is protein cleavage. Such cleavage is expected in the presence of an inflammatory response and as a result of wear processes at the articular interface. The aim of this study was to compare the tribological behavior of polyethylene articulated against an orthopedic wrought CoCrMo alloy for three lubricants: cleaved albumin, uncleaved albumin, and newborn calf serum (control). We hypothesized that the cleavage of albumin will increase the friction and wear rate of polyethylene, with a concomitant roughening of the polymer surface and the generation of larger wear debris particles. Cleavage of the bovine albumin into five fragments was performed by digestion with cyanogen bromide. In pin-on-flat (POF) wear tests of polyethylene pins made of Ticona GUR® 1020/1050 against CoCrMo alloy discs, the cleaved albumin led to the lowest polyethylene wear and highest friction coefficients, whereas albumin led to the highest wear rates. In knee simulator tests, the albumin lubricant also led to a 2.7-fold increase in the tibial insert wear rate compared to the regular bovine serum lubricant (a wear rate for the cleaved albumin could not be obtained). The generated polyethylene wear particles were of increasing size and fibrillar shape in going from serum to albumin to cleaved albumin, although only the shape achieved statistical significance. Unlike bovine serum, cleaved albumin led to wear scars for both the POF and simulator wear tests that closely emulated the morphological features observed on explanted polyethylene tibial inserts from total knee replacements. We posit that the smaller protein fragments can more efficiently adsorb on the surfaces of both the polyethylene and the metal, thus offering protection against wear, while at the same time leading to an increase in friction, particle size, and particle elongation, as the protein fragment films interact adhesively during sliding. The results of this study have implications for pre-clinical wear testing methodology as they suggest that albumin concentration may be more pertinent than total protein concentration for wear testing polyethylene.

Implications of trauma and subsequent articulation on the release of Proteoglycan-4 and tissue response in adult human ankle cartilage.

The purpose of this study was to investigate the effects of trauma and subsequent articulation on adult human ankle cartilage subjected to an injurious impact. Trauma was initiated through impaction on talar cartilage explants. Articulation and loading were applied in a joint bioreactor over 5 consecutive days. The early (24 h) effects of impaction included a reduced chondrocytes viability (51% vs. 81% for non-impacted; p = 0.03), increased levels of apoptosis (43% vs. 27%; p = 0.03), and an increase in the histopathology score (4.4 vs. 1.7; p = 0.02) as compared to non-impacted cartilage explants. One of the key findings was that damage also stimulated the PRG4 release (2.2 vs. 1.5 µg/ml). Subsequent articulation for 5 days did not lead to further changes in tissue histopathology and cell viability, neither for injured nor non-injured samples. However, articulation led to an increased apoptosis in the injured samples (p = 0.03 for the interaction term). Articulation also caused a significant increase of PG/GAG release into the culture medium (p = 0.04) for both injured and non-injured samples; however, the synthesis of PG was not affected by articulation (p = 0.45) though the PG synthesis was higher in injured samples (p < 0.01). With regard to the PRG4 release, impacted samples continued to show higher amounts (p = 0.01), adding articulation led to a reduction (p = 0.02). The current study demonstrated that adult human talar cartilage increases both the PRG4 release and biosynthetic activity as an immediate cellular response to injury. Articulation played a less contributing role to biosynthesis and remodeling, behaving mostly neutral, in that no further damage emerged. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:667-676, 2017.

Sialidase Unmasks Mucin Domain Epitopes of Lubricin.

Lubricin is a secreted, mucin-like glycoprotein and proteoglycan abundant in synovial fluid that provides boundary lubrication and prevents cell adhesion in synovial joints. The antilubricin S6.79 monoclonal antibody recognizes an O-linked glycopeptide epitope in lubricin's mucin domain. The central, long mucin domain of lubricin is extensively O-glycosylated with Gal(ß1-3)GalNAc-O-Ser/Thr, and about two thirds of the O-glycosylated sites are capped with sialic acid. Our aim was to determine whether removal of sialic acid by sialidase could improve the detection of lubricin in a number of human tissues using the S6.79 monoclonal antibody. Sialidase treatment caused a dramatic increase in antibody reactivity in human pericardium, splenic capsule and trabeculae, plasma, serum, eye sleep extract, and liver sinusoids. Sialidase had minimal effect on S6.79 antibody reactivity with lubricin in synovial fluid and synovial tissue. These observations suggest that the origin of lubricin in blood may be different from that in synovial fluid and that desialylation of lubricin is essential for unmasking epitopes within the mucin domain.

Abundant lubricin expression suggests a link between synoviocytes, synovial tumors, and myxomas.

Progenitor cell differentiation into fibroblast-like synoviocytes (FLSs) and their ensuing phenotypic changes are incompletely explored. Synovial lining is composed of intimal macrophages and FLSs. FLSs have epithelioid morphology and directionally secrete components of synovial fluid, including lubricin. We stained human tissues and tumors using two anti-lubricin antibodies. Lubricin was found in FLSs in synovium and in tenosynovial giant cell tumors (TSGCTs) and not in the associated monocyte/macrophage cells, which were identified by double immunostaining for CD163. In TSGCTs, giant cells, known to form by fusion of mononuclear cells, were negative for both lubricin and CD163. Occasional mononuclear cells with the same phenotype were also seen, suggesting that the precursors of the giant cells are derived from the minor CD163-negative monocyte subset. Lubricin was also detected in intramuscular myxomas, in early myxoid changes of ganglion cysts, and in one of five low-grade myxofibrosarcomas, but not in other fibroconnective tissues, epithelial tissues, or other tumors tested. This suggests that lubricin expression may typify adaptive and neoplastic changes along a pathway toward FLSs. Further support for this concept comes from ganglion cysts and juxta-articular myxoma tumors, which show a spectrum of myxoid, cystic and synovial differentiation, and in which moderate lubricin staining of myxoid stroma was seen.

Lubricin in human achilles tendon: The evidence of intratendinous sliding motion and shear force in achilles tendon.

Achilles tendon is one of the most commonly injured tendons. Mechanical force is regarded as a major causative factor. However, the biomechanics of Achilles tendon and mechanical mechanism of the injuries are unclear. Lubricin expresses at regions exposed to sliding motion and shear force in a number of tissues. This study investigated the distribution and concentration of lubricin in human Achilles tendons for better understanding the biomechanics of Achilles tendon. Achilles tendons were harvested from nine cadavers. Lubricin was extracted from various locations proximal to the calcaneal insertion and quantified with ELISA. The distribution of lubricin was investigated with immunohistochemistry. Lubricin was mainly identified at the interfaces of tendon fascicles, especially in the mid-portion of the tendon. The concentration of lubricin in Achilles tendons varied by individual and the distance from its calcaneal insertion. The distal portion of the tendon had a higher concentration of lubricin than the proximal regions of the tendon. This study suggests the presence of intratendinous sliding motion of fascicles and shear force at interfaces of fascicles in human Achilles tendon. Shear force could be an important mechanical factor for the development of Achilles tendinopathy and rupture.

Effects of trypsinization and mineralization on intrasynovial tendon allograft healing to bone.

The purpose of the current study was to develop a novel technology to enhance tendon-to-bone interface healing by trypsinizing and mineralizing (TM) an intrasynovial tendon allograft in a rabbit bone tunnel model. Eight rabbit flexor digitorum profundus (FDP) tendons were used to optimize the trypsinization process. An additional 24 FDP tendons were stratified into control and TM groups; in each group, 4 tendons were used for in vitro evaluation of TM and 8 were transplanted into proximal tibial bone tunnels in rabbits. The samples were evaluated histologically and with mechanical testing at postoperative week 8. Maximum failure strength and linear stiffness were not significantly different between the control and TM tendons. A thin fibrous band of scar tissue formed at the graft-to-bone interface in the control group. However, only the TM group showed obvious new bone formation inside the tendon graft and a visible fibrocartilage layer at the bone tunnel entrance. This study is the first to explore effects of TM on the intrasynovial allograft healing to a bone tunnel. TM showed beneficial effects on chondrogenesis, osteogenesis, and integration of the intrasynovial tendon graft, but mechanical strength was the same as the control tendons in this short-term in vivo study.

Mechanistic and stereochemical aspects of the asymmetric cyclocarbonylation of 1,6-enynes with rhodium catalysts.

NMR and kinetic investigations of the cyclocarbonylation of 1,6-enynes with cationic rhodium(i) catalysts, modified with atropisomeric diphosphines, disprove the involvement of carbonyl species for 1,6-enyne activation; low-temperature catalysis, with molecular sieves as the carbon monoxide reservoir, is highly enantioselective (ee up to 97%).

Reduction of water diffusion coefficient with increased engineered cartilage matrix growth observed using MRI.

Non-destructive monitoring of tissue-engineered cartilage growth is needed to optimize growth conditions, but extracting quantitative biomarkers of extracellular matrix development remains a technical challenge. MRI provides a non-invasive way to obtain a three dimensional map of growing tissue where the image contrast is based on tissue water relaxation times and the apparent diffusion coefficient (ADC). In this study, bovine chondrocytes were seeded in alginate beads (0, 1, 2, and 4 million cells/ml) and the ADC was measured weekly using diffusion-weighted MRI at 14.1 T over a one-month incubation period. Two groups of tissue-engineering constructs were created: one with ascorbic acid (vitamin C) added as a vitamin cofactor to increase collagen synthesis, and another with no added ascorbic acid. When normalized to the control beads without chondrocytes, the ADC was found to monotonically fall with incubation time (decreasing by up to 40% at 4 weeks), and with the administration of vitamin C. These results reflect the expected development of the extracellular matrix in the tissue-engineered constructs. We conclude that the normalized ADC is a potential biomarker for characterizing engineered cartilage tissue growth.

Mechanical characterization of tissue-engineered cartilage using microscopic magnetic resonance elastography.

Knowledge of mechanical properties of tissue-engineered cartilage is essential for the optimization of cartilage tissue engineering strategies. Microscopic magnetic resonance elastography (µMRE) is a recently developed MR-based technique that can nondestructively visualize shear wave motion. From the observed wave pattern in MR phase images the tissue mechanical properties (e.g., shear modulus or stiffness) can be extracted. For quantification of the dynamic shear properties of small and stiff tissue-engineered cartilage, µMRE needs to be performed at frequencies in the kilohertz range. However, at frequencies greater than 1 kHz shear waves are rapidly attenuated in soft tissues. In this study µMRE, with geometric focusing, was used to overcome the rapid wave attenuation at high frequencies, enabling the measurement of the shear modulus of tissue-engineered cartilage. This methodology was first tested at a frequency of 5 kHz using a model system composed of alginate beads embedded in agarose, and then applied to evaluate extracellular matrix development in a chondrocyte pellet over a 3-week culture period. The shear stiffness in the pellet was found to increase over time (from 6.4 to 16.4 kPa), and the increase was correlated with both the proteoglycan content and the collagen content of the chondrocyte pellets (R(2)=0.776 and 0.724, respectively). Our study demonstrates that µMRE when performed with geometric focusing can be used to calculate and map the shear properties within tissue-engineered cartilage during its development.