Non-ionic CT contrast solutions rapidly alter bovine cartilage and meniscus mechanics.

OBJECTIVE: To evaluate effects of a common CT contrast agent (iohexol) on the mechanical behaviors of cartilage and meniscus. METHODS: Indentation responses of juvenile bovine cartilage and meniscus were monitored following exposure to undiluted contrast agent (100% CA), 50% CA/water, 50% CA/Phosphate Buffered Saline (PBS) or PBS alone, and during re-equilibration in PBS. The normalized peak force (Fpk¯), effective osmotic strain (εosm), and normalized effective contact modulus (Ec¯) were calculated for every cycle, with time constants determined for both exposure and recovery via mono- or biexponential fits to Fpk¯. RESULTS: All cartilage CA groups exhibited long-term increases in Fpk¯ following exposure, although the hyperosmolal 100% CA and 50% CA/PBS groups showed an initial transient decrease. Meniscus presented opposing trends, with decreasing Fpk¯ for all CA groups. Re-equilibration in PBS for 1hr after exposure to 100% CA produced recovery to baseline Fpk¯ in cartilage but not in meniscus, and extended tests indicated that meniscus required ∼2.5 h to recover halfway. Ec¯ increased with CA exposure time for cartilage but decreased for meniscus, suggesting an increased effective stiffness for cartilage and decreased stiffness for meniscus. Long-term changes to εosm in both tissues were consistent with changes in Ec¯. CONCLUSION: Exposure to iohexol solutions affected joint tissues differentially, with increased cartilage stiffness, likely relating to competing hyperosmotic and hypotonic interactions with tissue fixed charges, and decreased meniscus stiffness, likely dominated by hyperosmolarity. These altered tissue mechanics could allow non-physiological deformation during ambulatory weight-bearing, resulting in an increased risk of tissue or cell damage.

Rapid and durable photochemical bonding of cartilage using the porphyrin photosensitizer verteporfin.

OBJECTIVE: To evaluate the effectiveness of verteporfin as a photosensitizer to photochemically bond articular cartilage tissues and determine bond durability in vitro. DESIGN: Bond strength induced by verteporfin over a range of concentrations and light exposure conditions was investigated using a disk-annulus model and a pushout test. Exposure was parameterized by varying either irradiance or irradiation time. Bond robustness in a cell-mediated degeneration environment was examined by exposing newly bonded samples to interleukin-1 alpha for the first 4 days of a 7-day culture period, followed by mechanical testing and biochemical and cellular viability assays. RESULTS: Photochemical bonding using verteporfin produced high bonding shear strengths at relatively low photosensitizer concentrations. Low exposures produced by either low irradiance or short irradiation time were sufficient to produce shear strengths comparable to those previously produced with phthalocyanine photosensitizers with substantially higher light exposure. Photochemically produced bonds were resistant to cell-mediated degeneration in vitro with no evident differences in cell viability among treatments. CONCLUSIONS: Verteporfin offers distinct advantages as a photosensitizer for photochemical bonding of articular cartilage due to the production of strong, durable bonds at relatively low light exposures. Further exploration may lead to clinically feasible strategies to augment cartilage repair techniques.

Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines.

OBJECTIVE: This study compared the effects on cartilage and meniscus matrix catabolism and biosynthesis of several adipokines implicated in osteoarthritis (OA). DESIGN: Bovine cartilage and meniscus explants were cultured for 1 or 9 days in serum-free medium alone or with 0.02, 0.2, or 2 µg/ml of leptin, visfatin, adiponectin, or resistin. Media were supplemented with (3)H-proline or (35)S-sodium sulfate to evaluate protein and sulfated glycosaminoglycan (sGAG) accumulation on the last day of culture. Explants were assayed for radiolabel, sGAG, and DNA contents. Cultured media were assayed for sGAG, nitrite and lactate dehydrogenase. RESULTS: Cartilage tissue was minimally affected by adipokines, with only the highest resistin dose increasing sGAG release and nitrite production compared to controls. In sharp contrast, meniscus tissue was responsive to several adipokines, with elevated sGAG and nitrite release following treatment with resistin, leptin, or visfatin. Cartilage sGAG content was unaltered by adipokine treatment whereas meniscal sGAG content significantly decreased with resistin dosage. Protein ((3)H) incorporation was unaffected by adipokine treatment in both tissues. sGAG ((35)S) incorporation did not significantly vary with adipokine treatment in cartilage but was inhibited by treatment with leptin, visfatin, and resistin in meniscus. CONCLUSION: Our results indicate that meniscal tissue is more susceptible to adipokine-stimulated catabolism than is cartilage. Resistin had the strongest effect of the adipokines tested, inducing sGAG release in both tissues and depleting sGAG content in meniscus. These results suggest that increased adipokine levels due to obesity or joint injury may alter the mechanical integrity of the knee joint through biological pathways.

Fact versus artifact: avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs.

The 1,9-dimethylmethylene blue (DMMB) assay is widely used to quantify sulfated glycosaminoglycan (sGAG) contents of engineered tissues, culture media, tissue samples and bodily fluids, but the assay is subject to interference from polyanions such as hyaluronic acid (HA), DNA and RNA. We examined whether specific combinations of dye pH and absorbance wavelength could minimize non-sGAG artifacts without compromising DMMB assay sensitivity. HA and DNA solutions generated substantial signal at pH 3 but not at pH 1.5. Reducing dye pH did not significantly alter sGAG measurements for normal cartilage and meniscus tissues, but eliminated anomalously high apparent sGAG contents for enzymatically isolated chondrocytes, adipose-derived stem cell (ADSC)-agarose constructs and ADSC pellets. In a cartilage tissue-engineering case study, pH 3 dye indicated high apparent sGAG readings throughout culture in both basal and chondrogenic media, with a marked decline between day 14 and 21 for chondrogenic constructs. The pH 1.5 dye, however, indicated minimal sGAG accumulation in basal medium and stable sGAG content throughout culture in chondrogenic medium. As it is often difficult to know a priori whether all groups in a study will have sGAG contents high enough to overwhelm artifacts, we recommend modifying the standard DMMB assay to reduce the risk of spurious findings in tissue engineering and clinical research. Specifically, we recommend shifting to a pH 1.5 DMMB dye and basing quantification on the absorbance difference between 525 nm (µ peak) and 595 nm (ß peak) to compensate for the moderate loss of sensitivity associated with reducing the dye pH.

Regional variation in T1ρ and T2 times in osteoarthritic human menisci: correlation with mechanical properties and matrix composition.

OBJECTIVE: Changes in T1ρ and T2 magnetic resonance relaxation times have been associated with articular cartilage degeneration, but similar relationships for meniscal tissue have not been extensively investigated. This work examined relationships between T1ρ and T2 measurements and biochemical and mechanical properties across regions of degenerate human menisci. DESIGN: Average T1ρ and T2 relaxation times were determined for nine regions each of seven medial and 13 lateral menisci from 14 total knee replacement patients. Sulfated glycosaminoglycan (sGAG), collagen and water contents were measured for each region. Biomechanical measurements of equilibrium compressive, dynamic compressive and dynamic shear moduli were made for anterior, central and posterior regions. RESULTS: T1ρ and T2 times showed similar regional patterns, with longer relaxation times in the (radially) middle region compared to the inner and outer regions. Pooled over all regions, T1ρ and T2 times showed strong correlations both with one another and with water content. Correlations with biochemical content varied depending on normalization to wet or dry mass, and both imaging parameters showed stronger correlations with collagen compared to sGAG content. Mechanical properties displayed moderate inverse correlations with increasing T1ρ and T2 times and water content. CONCLUSION: Both T1ρ and T2 relaxation times correlated strongly with water content and moderately with mechanical properties in osteoarthritic menisci, but not as strongly with sGAG or collagen contents alone. While the ability of magnetic resonance imaging (MRI) to detect early osteoarthritic changes remains the subject of investigation, these results suggest that T1ρ and T2 relaxation times have limited ability to detect compositional variations in degenerate menisci.

Discrimination of meniscal cell phenotypes using gene expression profiles.

The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus). The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

Response of cartilage and meniscus tissue explants to in vitro compressive overload.

OBJECTIVE: To examine the relative susceptibility of cartilage and meniscus tissues to mechanical injury by applying a single, controlled overload and observing cellular, biochemical, and mechanical changes. DESIGN: Cartilage and meniscus tissue explants in radial confinement were subjected to a range of injury by indenting to 40% strain at three different strain rates: 0.5%/s (slow), 5%/s (medium), or 50%/s (fast). Following injury, samples were cultured for either 1 or 9 days. Explants were assayed for cell metabolic activity, water content, and sulfated glycosaminoglycan (sGAG) content. Mechanical properties of explants were determined in torsional shear and unconfined compression. Conditioned medium was assayed for sGAG and lactate dehydrogenase (LDH) release. RESULTS: Peak injury force increased with strain rate but both tissues displayed little to no macroscopic damage. Cell metabolism was lowest in medium and fast groups on day 1. Cell lysis increased with peak injury force and loading rate in both tissues. In contrast, sGAG content and release did not significantly vary with loading rate. Additionally, mechanical properties did not significantly vary with loading rate in either tissue. CONCLUSION: By use of a custom confinement chamber, large peak forces were obtained without macroscopic destruction of the explants. At the loads achieved in this studied, cell damage was induced without detectable physical or compositional changes. These results indicate that sub-failure injury can induce biologic damage that may not be readily detected and could be an early event in osteoarthritis genesis.

Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels.

Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression.

OBJECTIVE: To examine the functional behavior of the surface layer of the meniscus by investigating depth-varying compressive strains during unconfined compression. DESIGN: Pairs of meniscus and articular cartilage explants (n=12) site-matched at the tibial surfaces were subjected to equilibrium unconfined compression at 5, 10, 15, and 20% compression under fluorescence imaging. Two-dimensional (2D) deformations were tracked using digital image correlation (DIC). For each specimen, local compressive engineering strains were determined in 200 µm layers through the depth of the tissue. In samples with sharp strain transitions, bilinear regressions were used to characterize the surface and interior tissue compressive responses. RESULTS: Meniscus and cartilage exhibited distinct depth-dependent strain profiles during unconfined compression. All cartilage explants had elevated compressive engineering strains near the surface, consistent with previous reports. In contrast, half of the meniscus explants tested had substantially stiffer surface layers, as indicated by surface engineering strains that were ∼20% of the applied compression. In the remaining samples, surface and interior engineering strains were comparable. 2D Green's strain maps revealed highly heterogeneous compressive and shear strains throughout the meniscus explants. In cartilage, the maximum shear strain appeared to be localized at 100-250 µm beneath the articular surface. CONCLUSIONS: Meniscus was characterized by highly heterogeneous strains during compression. In contrast to cartilage, which consistently had a compliant surface region, meniscal explants were either substantially stiffer near the surface or had comparable compressive stiffness through the depth. The relatively compliant interior may allow the meniscus to maintain a consistent surface contour while deforming during physiologic loading.

Nondestructive assessment of sGAG content and distribution in normal and degraded rat articular cartilage via EPIC-microCT.

OBJECTIVE: The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-microCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models. DESIGN: After determination of an appropriate incubation time for the anionic contrast agent, EPIC-microCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 min of digestion with chondroitinase ABC. The EPIC-microCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora. RESULTS: Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 min with a 16.0% temporal increase (P<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4- to 8-weeks of age (P<0.01) and further increased by 2.5% from 8 to 16 weeks (P<0.05). After 60 min of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (P<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=-0.88, slope=-1.26, P<0.01, n=30) with EPIC-microCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation. CONCLUSIONS: EPIC-microCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

Photochemical approaches for bonding of cartilage tissues.

OBJECTIVE: The objective of this study was to evaluate photochemical bonding as an approach for adhering live cartilage tissues across a repair interface in a manner that may lead to enhanced integration. DESIGN: Photochemical bonding of both meniscal fibrocartilage and articular cartilage was explored using an anionic, hydrophilic phthalocyanine photosensitizer. Variations on surface preparations and irradiation parameters were explored using overlapped tissue strips and tested using a modified single-lap shear test. Durability of the photochemically induced bonds and cellular viability were examined in an in vitro cartilage defect model for up to 1 week in culture, with bond strength assessed via push-out test. RESULTS: Meniscal tissue strips bonded with no surface treatment, but cartilage strips required enzymatic treatment with chondroitinase-ABC to effectively bond. More aggressive removal of glycosaminoglycans at the interface led to increased bond strengths. Bond strength achieved with a 10min irradiation of treated tissue was on the order of that previously achieved through several weeks of culture. In the defect model, photochemical bonds between a tissue annulus and a press-fit tissue core were maintained for 1 week in culture without substantial increases in cell death near the bonded interface. CONCLUSIONS: With appropriate treatment parameters, photochemical bonding rapidly produced a stable structural interface between cartilage tissue samples and may be a promising strategy for enhancing initial attachment in cartilage repair strategies.

Composition-function relationships during IL-1-induced cartilage degradation and recovery.

OBJECTIVE: To examine the relationships between biochemical composition and mechanical properties of articular cartilage explants during interleukin-1 (IL-1)-induced degradation and post-exposure recovery. DESIGN: Bovine articular cartilage explants were cultured for up to 32 days with or without 20 ng/mL IL-1. The dynamic shear modulus |G*(dyn)| and equilibrium and dynamic unconfined compression moduli (E(equil) and |E*(dyn)|) were measured at intervals throughout the culture period. In a subsequent recovery study, explants were cultured for 4 days with or without 20ng/mL IL-1 and for an additional 16 days in control media. The dynamic moduli |E*(dyn)| and |G*(dyn)| were measured at intervals during degeneration and recovery. Conditioned media and explant digests were assayed for sulfated glycosaminoglycans (sGAG) and collagen content. RESULTS: Continuous IL-1 stimulation triggered progressive decreases in E(equil), |E*(dyn)|, and |G*(dyn)| concomitant with the sequential release of sGAG and collagen from the explants. Brief IL-1 exposure resulted in a short release of sGAG but not collagen, followed by a gradual and incomplete repopulation of sGAG. The temporary sGAG depletion was associated with decreases in both |E*(dyn)| and |G*(dyn)| which also recovered after removal of IL-1. During IL-1-induced degradation and post-exposure recovery, explant mechanical properties correlated well with tissue sGAG concentration. CONCLUSIONS: As previously shown for developing cartilages and engineered cartilage constructs, cytokine-induced changes in sGAG concentration (i.e., fixed charge density) are coincident with changes in compressive and shear properties of articular cartilage. Further, recovery of cartilage mechanical properties can be achieved by relief from proinflammatory stimuli and subsequent restoration of tissue sGAG concentration.

Central and peripheral region tibial plateau chondrocytes respond differently to in vitro dynamic compression.

OBJECTIVE: The objective of this study was to test the hypotheses that chondrocytes from distinct regions of the porcine tibial plateau: (1) display region-specific baseline gene expression, and (2) respond differently to in vitro mechanical loading. METHODS: Articular cartilage explants were obtained from central (not covered by meniscus) and peripheral (covered by meniscus) regions of porcine tibial plateaus. For baseline gene expression analysis, samples were snap frozen. To determine the effect of mechanical loading, central and peripheral region explants were exposed to equivalent dynamic compression (0-100 kPa) and compared to site-matched free-swelling controls (FSCs). mRNA levels for type II collagen (CII), aggrecan (AGGR), matrix metalloproteinase 1 (MMP-1), MMP-3, MMP-13, A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAM-TS4), ADAM-TS5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and tumor necrosis factor alpha (TNFalpha) were quantified using real time polymerase chain reaction (RT-PCR). RESULTS: At baseline, mRNA levels for the structural proteins CII and AGGR were approximately twofold greater in the central region compared with peripheral region explants. In vitro dynamic compression strongly affected expression levels for CII, AGGR, MMP-3, and TIMP-2 relative to FSCs. Response differed significantly by region, with greater upregulation of CII, AGGR, and MMP-3 in central region explants. CONCLUSIONS: Chondrocytes from different regions of the porcine tibial plateau express mRNA for structural proteins at different levels and respond to equivalent in vitro mechanical loading with distinctive changes in gene expression. These regional biological variations appear to be related to the local mechanical environment in the normal joint, and thus may indicate a sensitivity of the joint to conditions that alter joint loading such as anterior cruciate ligament (ACL) injury, meniscectomy, or joint instability.

Quantitative assessment of articular cartilage morphology via EPIC-microCT.

OBJECTIVE: The objective of the present study was to validate the ability of Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-microCT) to nondestructively assess cartilage morphology in the rat model. DESIGN: An appropriate contrast agent (Hexabrix) concentration and incubation time for equilibration were determined for reproducible segmentation of femoral articular cartilage from contrast-enhanced microCT scans. Reproducibility was evaluated by triplicate scans of six femora, and the measured articular cartilage thickness was independently compared to thickness determined from needle probe testing and histology. The validated technique was then applied to quantify age-related differences in articular cartilage morphology between 4, 8, and 16-week-old (n=5 each) male Wistar rats. RESULTS: A 40% Hexabrix/60% phosphate buffered saline (PBS) solution with 30 min incubation was optimal for segmenting cartilage from the underlying bone tissue and other soft tissues in the rat model. High reproducibility was indicated by the low coefficient of variation (1.7-2.5%) in cartilage volume, thickness and surface area. EPIC-microCT evaluation of thickness showed a strong linear relationship and good agreement with both needle probing (r(2)=0.95, slope=0.81, P<0.01, mean difference 11+/-22 microm, n=43) and histology (r(2)=0.99, slope=0.97, P<0.01, mean difference 12+/-10 microm, n=30). Cartilage volume and thickness significantly decreased with age while surface area significantly increased. CONCLUSION: EPIC-microCT imaging has the ability to nondestructively evaluate three-dimensional articular cartilage morphology with high precision and accuracy in a small animal model.

Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading.

OBJECTIVE: The cell morphology, gene expression, and matrix synthesis of articular chondrocytes are known to vary with depth from the tissue surface. The objective of this study was to investigate if chondrocytes from different zones respond to in vitro oscillatory tensile loading in distinct ways and whether tensile strain, which is most prevalent near the articular surface, would preferentially stimulate superficial zone chondrocytes. DESIGN: Chondrocytes were separately isolated from the superficial, middle, and deep zones of articular cartilage and seeded into three-dimensional fibrin hydrogel constructs. An intermittent protocol of oscillatory tensile loading was applied for 3 days, and the effects on extracellular matrix (ECM) synthesis were assessed by measuring the incorporation of radiolabed precursors, size exclusion gel chromatography, and western blotting. RESULTS: Tensile loading was found to be a potent stimulus for proteoglycan synthesis only in superficial zone chondrocytes. Although overall biosynthesis rates by deep zone chondrocytes were unaffected by tensile loading, the molecular characteristics of proteins and proteoglycans released to the culture medium were significantly altered so as to resemble those of superficial zone chondrocytes. CONCLUSIONS: Oscillatory tensile loading differentially affected subpopulations of articular chondrocytes in three-dimensional fibrin hydrogel constructs. Cells isolated from deeper regions of the tissue developed some characteristics of superficial zone chondrocytes after exposure to tensile loading, which may indicate an adaptive response to the new mechanical environment. Understanding how exogenous mechanical stimuli can differentially influence chondrocytes from distinct tissue zones will yield important insights into mechanobiological processes involved in cartilage tissue development, maintenance, disease, and repair.

Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs.

OBJECTIVE: The goal of this study was to characterize the proteoglycan (PG) production and processing by bone marrow stromal cells (BMSCs) within a tissue engineered construct. METHODS: Bovine BMSCs and articular chondrocytes (ACs) were isolated from an immature calf, seeded into agarose gels, and cultured up to 32 days in the presence of TGF-beta1. The localization of various PGs was examined by immunofluorescence and histological staining. The role of proteolytic enzymes in construct development was further investigated by examining the effects of aggrecanase and MMP inhibitors on PG accumulation, aggrecan processing, and construct mechanics. RESULTS: BMSCs developed a matrix rich in sulfated-glycosaminoglycans (sGAG) and full-length aggrecan, but had low levels of versican. The BMSC constructs had less collagen II and aggrecan compared to the AC constructs cultured under identical conditions. AC constructs also had high levels of pericellular collagen VI, while BMSCs had a pericellular matrix containing little collagen VI and greater levels of decorin, biglycan, and fibronectin. Treatment with the aggrecanase inhibitor did not affect sGAG accumulation or the dynamic moduli of the BMSC constructs. The MMP inhibitor slightly but significantly inhibited sGAG accumulation and lowered the dynamic moduli of BMSC constructs. CONCLUSIONS: The results of this preliminary study indicate that long-term culture of BMSCs with TGF-beta1 promotes the development of an aggrecan-rich matrix characteristic of native articular cartilage; however, BMSCs accumulate significantly lower levels of sGAG and assemble distinct pericellular microenvironments compared to ACs. PG turnover does not appear to play a major role in the development of tissue engineered cartilage constructs by BMSCs.

Ion-channel regulation of chondrocyte matrix synthesis in 3D culture under static and dynamic compression.

Inhibition of various ion channels alters chondrocyte mechanotransduction in monolayer, but the mechanisms involved in chondrocyte mechanotransduction in three- dimensional culture remain unclear. The objective of this study was to investigate the effects of inhibiting putative ion-channel influenced mechanotransduction mechanisms on the chondrocyte responses to static and dynamic compression in three-dimensional culture. Bovine articular cartilage explants were used to investigate the dose-dependent inhibition and recovery of protein and sulfated glycosaminoglycan (sGAG) syntheses by four ion-channel inhibitors: 4-Aminopyridine (4AP), a K(+) channel blocker; Nifedipine (Nf), a Ca(2+) channel blocker; Gadolinium (Gd), a stretch-activated channel blocker; and Thapsigargin (Tg), which releases intracellular Ca(2+) stores by inhibiting ATP-dependent Ca(2+) pumps. Chondrocyte-seeded agarose gels were used to examine the influence of 20 h of static and dynamic loading in the presence of each of the inhibitors. Overall, treatment with the ion-channel inhibitors had a greater effect on sGAG synthesis, with the exception of Nf, which more substantially affected protein synthesis. Treatment with Tg significantly impaired both overall protein and sGAG synthesis, with a drastic reduction in sGAG synthesis. The inhibitors differentially influenced the responses to mechanical stimuli. Dynamic compression significantly upregulated protein synthesis but did not significantly affect sGAG synthesis with Nf or Tg treatment. Dynamic compression significantly upregulated both protein and sGAG synthesis rates with Gd treatment. There was no significant stimulation of either protein or sGAG synthesis by dynamic compression with 4AP treatment. Interruption of many ion-channel signaling mechanisms affected sGAG synthesis, suggesting a complicated, multi-pathway signaling process. Also, Ca(2+) signaling may be critical for the transduction of mechanical stimulus in regulating sGAG synthesis. This modulation potentially occurs through direct interactions with the extracellular matrix.

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering.

OBJECTIVE: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. DESIGN: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. CONCLUSIONS: The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.

Chondrocyte phenotypes on different extracellular matrix monolayers.

Chondrocytes undergo a process of dedifferentiation in monolayer culture that is characterized by a transition to a fibroblast-like phenotype. This behavioral change poses a challenge for tissue-engineered cartilage constructs, as approaches using autologous cells require expansion in vitro. Because chondrocytes express a variety of integrin receptors specific to different adhesive proteins, we hypothesized that chondrocytes expanded on various underlying protein monolayers would have different phenotypic responses. Bovine articular chondrocytes were cultured for up to 2 weeks on tissue culture plastic, fibronectin, collagen type I or collagen type II substrate in the presence or absence of ascorbate. Contrary to our hypothesis, the extracellular matrix protein substrates used in this study did not significantly alter the changes in chondrocyte morphology, gene expression, matrix formation, or cytoskeletal organization. Cells on all substrates assembled equivalent matrices, which may have subsequently regulated cell behavior. In cultures with ascorbate, populations of round and spread cells emerged after 1 week, with round cells expressing collagen type II and the differentiated phenotype and spread cells dedifferentiating. In cultures without ascorbate, chondrocytes rapidly adhered and spread onto organized fibronectin matrices via the alpha5beta1 integrin, which has been associated with survival and proliferation of chondrocytes in vitro. These findings indicate that expanding chondrocytes on protein monolayers may not be an effective solution to preventing dedifferentiation and improving autologous chondrocyte transplantation.

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.