Intact Cartilage Samples Research Articles

MicroRNA Signatures in Cartilage Ageing and Osteoarthritis.

Osteoarthritis is the most common degenerative joint disorder. MicroRNAs are gene expression regulators that act post-transcriptionally to control tissue homeostasis. Microarray analysis was undertaken in osteoarthritic intact, lesioned and young intact cartilage. Principal component analysis showed that young intact cartilage samples were clustered together; osteoarthritic samples had a wider distribution; and osteoarthritic intact samples were separated into two subgroups, osteoarthritic-Intact-1 and osteoarthritic-Intact-2. We identified 318 differentially expressed microRNAs between young intact and osteoarthritic lesioned cartilage, 477 between young intact and osteoarthritic-Intact-1 cartilage and 332 between young intact and osteoarthritic-Intact-2 cartilage samples. For a selected list of differentially expressed microRNAs, results were verified in additional cartilage samples using qPCR. Of the validated DE microRNAs, four-miR-107, miR-143-3p, miR-361-5p and miR-379-5p-were selected for further experiments in human primary chondrocytes treated with IL-1β. Expression of these microRNAs decreased in human primary chondrocytes treated with IL-1β. For miR-107 and miR-143-3p, gain- and loss-of-function approaches were undertaken and associated target genes and molecular pathways were investigated using qPCR and mass spectrometry proteomics. Analyses showed that WNT4 and IHH, predicted targets of miR-107, had increased expression in osteoarthritic cartilage compared to young intact cartilage and in primary chondrocytes treated with miR-107 inhibitor, and decreased expression in primary chondrocytes treated with miR-107 mimic, suggesting a role of miR-107 in chondrocyte survival and proliferation. In addition, we identified an association between miR-143-3p and EIF2 signalling and cell survival. Our work supports the role of miR-107 and miR-143-3p in important chondrocyte mechanisms regulating proliferation, hypertrophy and protein translation.

Resolving the Near-Infrared Spectrum of Articular Cartilage.

Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by (a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and (b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples. As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models. Spectral peaks in the regions 5500 to 6250 cm-1 and 8100 to 8600 cm-1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

CircHYBID regulates hyaluronan metabolism in chondrocytes via hsa-miR-29b-3p/TGF-\u03b21 axis

BackgroundHyaluronan (HA) metabolism by chondrocytes is important for cartilage development and homeostasis. However, information about the function of circular RNAs (circRNAs) in HA metabolism is limited. We therefore profiled the role of the novel HA-related circRNA circHYBID in the progression of osteoarthritis (OA).MethodsCircHYBID function in HA metabolism in chondrocytes was investigated using gain-of-function experiments, and circHYBID mechanism was confirmed via bioinformatics analysis and luciferase assays. The expression of circHYBID–hsa-miR-29b-3p–transforming growth factor (TGF)-β1 axis was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. CircHYBID, TGF-β1, and HA levels in cartilage samples were evaluated using qRT-PCR and pathological examination. Enzyme-linked immunosorbent assay was used to assess HA accumulation in chondrocyte supernatant.ResultsCircHYBID expression was significantly downregulated in damaged cartilage samples compared with that in the corresponding intact cartilage samples. CircHYBID expression was positively correlated with alcian blue score. Interleukin-1β stimulation in chondrocytes downregulated circHYBID expression and decreased HA accumulation. Gain-of-function experiments revealed that circHYBID overexpression in chondrocytes increased HA accumulation by regulating HA synthase 2 and HYBID expression. Further mechanism analysis showed that circHYBID upregulated TGF-β1 expression by sponging hsa-miR-29b-3p.ConclusionsOur results describe a novel HA-related circRNA that could promote HA synthesis and accumulation. The circHYBID–hsa-miR-29b-3p–TGF-β1 axis may play a powerful regulatory role in HA metabolism and OA progression. Thus, these findings will provide new perspectives for studies on OA pathogenesis, and circHYBID may serve as a potential target for OA therapy.

No pressure, no diamonds? - Static vs. dynamic compressive in-situ loading to evaluate human articular cartilage functionality by functional MRI

Biomechanical Magnetic Resonance Imaging (MRI) of articular cartilage, i.e. its imaging under loading, is a promising diagnostic tool to assess the tissue's functionality in health and disease. This study aimed to assess the response to static and dynamic loading of histologically intact cartilage samples by functional MRI and pressure-controlled in-situ loading. To this end, 47 cartilage samples were obtained from the medial femoral condyles of total knee arthroplasties (from 24 patients), prepared to standard thickness, and placed in a standard knee joint in a pressure-controlled whole knee-joint compressive loading device. Cartilage samples' responses to static (i.e. constant), dynamic (i.e. alternating), and no loading, i.e. free-swelling conditions, were assessed before (δ0), and after 30 min (δ1) and 60 min (δ2) of loading using serial T1ρ maps acquired on a 3.0T clinical MRI scanner (Achieva, Philips). Alongside texture features, relative changes in T1ρ (Δ1, Δ2) were determined for the upper and lower sample halves and the entire sample, analyzed using appropriate statistical tests, and referenced to histological (Mankin scoring) and biomechanical reference measures (tangent stiffness).Histological, biomechanical, and T1ρ sample characteristics at δ0 were relatively homogenous in all samples. In response to loading, relative increases in T1ρ were strong and significant after dynamic loading (Δ1 = 10.3 ± 17.0%, Δ2 = 21.6 ± 21.8%, p = 0.002), while relative increases in T1ρ after static loading and in controls were moderate and not significant. Generally, texture features did not demonstrate clear loading-related associations underlying the spatial relationships of T1ρ.When realizing the clinical translation, this in-situ study suggests that serial T1ρ mapping is best combined with dynamic loading to assess cartilage functionality in humans based on advanced MRI techniques.

Longitudinal T2 Mapping and Texture Feature Analysis in the Detection and Monitoring of Experimental Post-Traumatic Cartilage Degeneration.

Background: Traumatic cartilage injuries predispose articulating joints to focal cartilage defects and, eventually, posttraumatic osteoarthritis. Current clinical-standard imaging modalities such as morphologic MRI fail to reliably detect cartilage trauma and to monitor associated posttraumatic degenerative changes with oftentimes severe prognostic implications. Quantitative MRI techniques such as T2 mapping are promising in detecting and monitoring such changes yet lack sufficient validation in controlled basic research contexts. Material and Methods: 35 macroscopically intact cartilage samples obtained from total joint replacements were exposed to standardized injurious impaction with low (0.49 J, n = 14) or high (0.98 J, n = 14) energy levels and imaged before and immediately, 24 h, and 72 h after impaction by T2 mapping. Contrast, homogeneity, energy, and variance were quantified as features of texture on each T2 map. Unimpacted controls (n = 7) and histologic assessment served as reference. Results: As a function of impaction energy and time, absolute T2 values, contrast, and variance were significantly increased, while homogeneity and energy were significantly decreased. Conclusion: T2 mapping and texture feature analysis are sensitive diagnostic means to detect and monitor traumatic impaction injuries of cartilage and associated posttraumatic degenerative changes and may be used to assess cartilage after trauma to identify “cartilage at risk”.

Identification of Cartilage Microbial DNA Signatures and Associations With Knee and Hip Osteoarthritis.

Alterations of the gut microbiota have been implicated in many forms of arthritis, but an examination of cartilage microbial patterns has not been performed. This study was undertaken to characterize the microbial DNA profile of articular cartilage and determine changes associated with osteoarthritis (OA). We performed 16S ribosomal RNA gene deep sequencing on eroded and intact cartilage samples from knee OA patients (n = 21 eroded and 21 intact samples) and hip OA patients (n = 34 eroded and 33 intact samples) and cadaver controls (n = 10 knee samples and 10 hip samples). Microbial DNA diversity was assessed, groups were compared, and metagenomic profiles were reconstructed. Confirmation was performed in an independent cohort by clade-specific quantitative polymerase chain reaction. Findings in human cartilage were compared to those in cartilage from OA-susceptible C57BL/6 (B6) mice and OA-resistant MRL/MpJ (MRL) mice. Germ-free B6 mouse cartilage was analyzed as a methodologic control. Alpha diversity was reduced in human OA versus control samples (P < 0.0001), and in hip versus knee samples (P < 0.0001). Numerous clades were different in human OA versus control samples, and similar findings were noted in comparisons of murine B6 versus MRL mice. Hip samples were microbiologically distinct from knee samples. OA microbial DNA demonstrated increased gram-negative constituents (P = 0.02). Functional analysis demonstrated increases in lipopolysaccharide production (P = 9.9 × 10-3 ), phosphatidylinositol signaling (P = 4.2 × 10-4 ), and nitrogen metabolism (P = 8 × 10-3 ) and decreases in sphingolipid metabolism (P = 7.7 × 10-4 ) associated with OA. Our study reveals a microbial DNA signature in human and mouse cartilage. Alterations in this signature, including increases in gram-negative constituents, occur during the development and progression of human OA. Furthermore, our findings indicate that strain-specific signatures exist within mouse cartilage that mirror human patterns. Further study of the establishment and potential pathogenic role of these DNA signatures is needed.

STUDY ON MECHANICAL PROPERTIES OF TOTAL KNEE ARTICULAR CARTILAGE UNDER DIFFERENT LOADING RATES

Knee joint is the main weight bearing tissue of human body, also it is one of the prone parts of the clinical disease. Under different sports conditions, knee joint was loaded at different forms. In this study, the changes of average contact pressure, peak contact pressure, contact area and pressure-sharing regions were researched using the intact and defect pig knee joints under different loading rates and loads, including fast rates and large loads. These data were measured and recorded by usage of the sensor plate that placed between the unilateral meniscus and the femur cartilage during loading process. As for the intact cartilage samples, the average contact pressure and peak contact pressure of the femur cartilage increase with the loading rate, while the contact area is contrast to it. As for defect cartilage samples, it not only emerged stress concentration on the edge of the defect and pressure distribution in joint cavity was different with intact cartilage samples, but also the main bearing region was transferred from the femur cartilage-meniscus contact area to the femur cartilage-tibial cartilage contact area at different loading forms. In different loading stages, the pressure-sharing regions between the cartilage and the meniscus also changes. Different loading rates, different loads and defects will change the mechanical states of the knee joint. In loading forms, the mechanical condition may cause or aggravate damnification of the knee joint cartilage. Therefore, this study is beneficial for promoting and perfecting the research of mechanical properties of knee joint cartilage and provides a theoretical basis for the prevention and treatment of knee cartilage injury.

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)

Cartilage surface integrity influences contrast enhanced ct imaging of osteoarthritis

Purpose: To assess the utility of contrast-enhanced uCT (CE-uCT) as an imaging biomarker for preclinical assessment of osteoarthritis in a sheep model using IA injection of iodinated contrast medium and short exposure times that can be obtained in vivo. Methods: An in-vitro pilot study was conducted to assess differences in the apparent density between damaged (fibrillated/incised or shaved/superficial zone removed) and intact bovine osteochondral blocks after exposure to an anionic contrast medium (Conray 60) for 10 minutes (n=22). The sidewalls of blocks were wrapped in Parafilm™ to limit contrast contact to the cartilage surface only. Fibrillated (n=6), shaved (n=7), and intact (n=4) blocks were immersed in the Conray 60 and intact control (N=5) blocks were immersed in lactated ringer’s solution (LRS) for 10 minutes prior to uCT imaging at 45 micron resolution. Uptake of contrast was measured by calculation of the average apparent density in Hounsfield Units (HU). Twenty-four sheep were enrolled in a follow-up, ex-vivo, study. Osteoarthritis was induced by arthroscopic meniscal destabilization in n=20 sheep and n=4 remained as naive controls. The x-ray attenuation of cartilage was measured in animals with early (3 month) and late stage (12 month) OA after a 10-minute exposure to anionic contrast media (Conray 60). Concentration (ug/mg) of sulfated-glycosaminoglycan (sGAG) and histological OARSI scores for proteoglycan (PG) staining were also collected and recorded from the medial tibial plateau (MTP) of the operated leg. ANOVA was used to analyze variance in sGAG and HU between sites on the MTP or between groups. Correlation coefficients between sGAG and HU were analyzed using Pearson’s correlation coefficients and simple linear regression analysis. Spearmann-rank coefficients were used to examine the association between histological PG score and either HU or sGAG. We hypothesized apparent density (HU) would vary with respect to concentration of local sGAG, and that HU would be higher in regions where sGAG is depleted. Results: In the in-vitro CE-uCT study we observed a significant increase in apparent density of intact cartilage samples exposed to Conray 60 for 10 minutes compared to LRS controls (p=0.03). HU was also significantly higher in fibrillated (p=0.030) and shaved (p=0.0007) blocks compared to non-contrasted samples. In ex-vivo CE-uCT imaging of the MTP location accounted the variance in HU for both 3 month (p=0.09) and 12 month (p=0.0074) groups but not controls (p=0.32). Early 3 month OA sheep had elevated levels of sGAG that were significantly higher than the late phase 12 month group group (Fig 1, p =0.0032). A significant, negative correlation between sGAG concentration and HU of the same location was observed (r= -0.71, p = 0.047) in the early stage (3 month) sheep. Linear regression analysis revealed that every one unit (ug CSC/mg cartilage) increase in sGAG resulted in a reduction in HU by 8.62 units (R2 = 0.43, p< 0.05). Conclusions: Ten minutes of exposure to contrast media was sufficient to clearly outline cartilage borders due to the increase in apparent density and measured HU. Differences in cartilage HU of CE-uCT images can be used to estimate differences in local sGAG concentrations in animals with thin cartilage during early and late OA but variance in local sGAG concentration cannot exclusively explain the resulting variance in HU, suggesting that other factors may play a role such as an the integrity of the superficial cartilage zone. Elevated sGAG cartilage concentrations at three months returned to near normal levels at 12 months suggesting the hypertrophic phase of OA may be longer than expected. During this time matrix depletion as measured by CE-CT may not be a reliable imaging biomarker.

T2 MR imaging vs. computational modeling of human articular cartilage tissue functionality

The detection of early stages of cartilage degeneration remains diagnostically challenging. One promising non-invasive approach is to functionally assess the tissue response to loading by serial magnetic resonance (MR) imaging in terms of T2 mapping under simultaneous mechanical loading. As yet, however, it is not clear which cartilage component contributes to the tissue functionality as assessed by quantitative T2 mapping. To this end, quantitative T2 maps of histologically intact cartilage samples (n=8) were generated using a clinical 3.0-T MR imaging system. Using displacement-controlled quasi-static indentation loading, serial T2 mapping was performed at three defined strain levels and loading-induced relative changes were determined in distinct regions-of-interest. Samples underwent conventional biomechanical testing (by unconfined compression) as well as histological assessment (by Mankin scoring) for reference purposes. Moreover, an anisotropic hyperelastic constitutive model of cartilage was implemented into a finite element (FE) code for cross-referencing. In efforts to simulate the evolution of compositional and structural intra-tissue changes under quasi-static loading, the indentation-induced changes in quantitative T2 maps were referenced to underlying changes in cartilage composition and structure. These changes were parameterized as cartilage fluid, proteoglycan and collagen content as well as collagen orientation. On a pixel-wise basis, each individual component correlation with T2 relaxation times was determined by Spearman's ρs and significant correlations were found between T2 relaxation times and all four tissue parameters for all indentation strain levels. Thus, the biological changes in functional MR Imaging parameters such as T2 can further be characterized to strengthen the scientific basis of functional MRI techniques with regards to their perspective clinical applications.

The In Vitro Chondrotoxicity of Single-Dose Local Anesthetics

Background: The administration of amide-type local anesthetics to cartilaginous tissues has revealed potential chondrotoxicity. Purpose: This study evaluated whether administration of single doses of 1% lidocaine, 0.25% bupivacaine, and 0.5% ropivacaine resulted in decreased chondrocyte viability or cartilage matrix degradation in vitro. Study Design: Controlled laboratory study. Methods: Monolayer human chondrocytes and intact cartilage samples were cultured for 1 week in media. Each drug was delivered in a custom bioreactor over its clinical duration of action. A Live/Dead Viability/Cytotoxicity Assay was used to determine the ratio of dead to live cells for monolayer chondrocyte cultures compared with controls. Damage to the cartilage extracellular matrix (ECM) in en bloc cartilage samples was evaluated by analysis of DNA, glycosaminoglycan (GAG), and collagen content. Results: Chondrocytes treated for 3 hours with a single dose of 1% lidocaine exhibited significantly more cell death (7.9%) compared with control media (2.9%; P < .001). No significant difference in cell death was observed in chondrocytes treated for 6 hours with 0.25% bupivacaine (2.7%) versus controls (2.8%; P = .856) or cells treated for 12 hours in 0.5% ropivacaine (2.9%) versus controls (2.4%; P = .084). There was no significant difference in GAG expression (P = .627) or DNA-normalized GAG expression (P = .065) between the intact cartilage treatment groups; however, the DNA-normalized GAG expression was markedly lower in cartilage cultures treated with 1% lidocaine (3.36 ± 1.15) compared with those in control media (7.61 ± 3.83). Conclusion: The results of this in vitro study indicate that a single-dose administration of 1% lidocaine resulted in a significant decrease in chondrocyte viability when compared with control cultures. Clinical Relevance: Single-dose injections of 1% lidocaine may be significantly chondrotoxic, and further investigation regarding in vivo chondrotoxicity appears warranted.

Hypotonic challenge modulates cell volumes differently in the superficial zone of intact articular cartilage and cartilage explant

The objective of this study was to evaluate the effect of sample preparation on the biomechanical behaviour of chondrocytes. We compared the volumetric and dimensional changes of chondrocytes in the superficial zone (SZ) of intact articular cartilage and cartilage explant before and after a hypotonic challenge. Calcein-AM labelled SZ chondrocytes were imaged with confocal laser scanning microscopy through intact cartilage surfaces and through cut surfaces of cartilage explants. In order to clarify the effect of tissue composition on cell volume changes, Fourier Transform Infrared microspectroscopy was used for estimating the proteoglycan and collagen contents of the samples. In the isotonic medium (300 mOsm), there was a significant difference (p < 0.05) in the SZ cell volumes and aspect ratios between intact cartilage samples and cartilage explants. Changes in cell volumes at both short-term (2 min) and long-term (2 h) time points after the hypotonic challenge (180 mOsm) were significantly different (p < 0.05) between the groups. Further, proteoglycan content was found to correlate significantly (r(2) = 0.63, p < 0.05) with the cell volume changes in cartilage samples with intact surfaces. Collagen content did not correlate with cell volume changes. The results suggest that the biomechanical behaviour of chondrocytes following osmotic challenge is different in intact cartilage and in cartilage explant. This indicates that the mechanobiological responses of cartilage and cell signalling may be significantly dependent on the integrity of the mechanical environment of chondrocytes.

Proliferative remodeling of the spatial organization of human superficial chondrocytes distant from focal early osteoarthritis

Human superficial chondrocytes show distinct spatial organizations, and they commonly aggregate near osteoarthritic (OA) fissures. The aim of this study was to determine whether remodeling or destruction of the spatial chondrocyte organization might occur at a distance from focal (early) lesions in patients with OA. Samples of intact cartilage (condyles, patellofemoral groove, and proximal tibia) lying distant from focal lesions of OA in grade 2 joints were compared with location-matched nondegenerative (grade 0-1) cartilage samples. Chondrocyte nuclei were stained with propidium iodide, examined by fluorescence microscopy, and the findings were recorded in a top-down view. Chondrocyte arrangements were tested for randomness or significant grouping via point pattern analyses (Clark and Evans Aggregation Index) and were correlated with the OA grade and the surface cell densities. In grade 2 cartilage samples, superficial chondrocytes were situated in horizontal patterns, such as strings, clusters, pairs, and singles, comparable to the patterns in nondegenerative cartilage. In intact cartilage samples from grade 2 joints, the spatial organization included a novel pattern, consisting of chondrocytes that were aligned in 2 parallel lines, building double strings. These double strings correlated significantly with an increased number of chondrocytes per group and an increased corresponding superficial zone cell density. They were observed in all grade 2 condyles and some grade 2 tibiae, but never in grade 0-1 cartilage. This study is the first to identify a distinct spatial reorganization of human superficial chondrocytes in response to distant early OA lesions, suggesting that proliferation had occurred distant from focal early OA lesions. This spatial reorganization may serve to recruit metabolically active units as an attempt to repair focal damage.

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.

Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies.

The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 +/- 130 microM), the maximal velocity (36 +/- 3.2 x 10(-5)microMsec(-1)) and the velocity constant (1.27 +/- 0.2 x 10(-7)sec(-1)) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 microg/ml propidium iodide (PI) and 4 microM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative.