Zone Of Bovine Articular Cartilage Research Articles

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

Purpose: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states.Methods: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly.Results: Our results revealed differences in SHG forward–backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.Conclusions: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Distinct patterns of gene expression in the superficial, middle and deep zones of bovine articular cartilage

Hyaline articular cartilage will not heal spontaneously, and lesions in hyaline articular cartilage often result in degenerative joint disease. Considerable progress has been made with respect to the responsive stem cells, inductive signals and extracellular scaffolding required for the optimal regeneration of cartilage. However, many challenges remain, such as topographic differences in the functional zones of articular cartilage. We hypothesized that a distinct set of differentially expressed genes define the surface, middle and deep zones of hyaline articular cartilage. Microarray analysis of bovine articular cartilage from the superficial and middle zones revealed 52 genes differentially expressed ≥ 10-fold and 114 additional genes differentially expressed ≥ five-fold. However, no genes were identified with a ≥ five-fold difference in expression when comparing articular cartilage from the middle and deep zones. There are distinct, differential gene expression patterns in the superficial and middle zones of hyaline articular cartilage that highlight the functional differences between these zones. This investigation has implications for the tissue engineering and regeneration of hyaline articular cartilage.

Gene expression of alginate-embedded chondrocyte subpopulations and their response to exogenous IGF-1 delivery

The delivery of growth factors to aid in cartilage engineering has received considerable attention. However, phenotypical differences between chondrocyte cell populations and their distinct responses to growth factors are not fully understood. To address this issue, we have investigated the gene expression of chondrocytes isolated from the superficial, middle, and deep zones of bovine articular cartilage. A three-dimensional (3D) alginate bead model was used to encapsulate zonal chondrocytes and culture with or without exogenous insulin-like growth factor-1(IFG-1) delivery. Following culture, mRNA expression of type I collagen, type II collagen, aggregan, IGF-1 and IGF-1 binding protein (IGF-BP3) were analysed at 1, 4 and 8 days. To the best of our knowledge, this is the first study to investigate gene expression of IGF-1 and IGF-BP3 by zone, and among the first studies to investigate growth factor delivery to chondrocytes in a 3D culture environment. Histological images and cell count data confirm the isolation of chondrocyte subpopulations, and gene expression data show distinct profiles for each zone, both with and without IGF-1 delivery. The data also show similar gene expression for the middle and deep zone cells, while the superficial zone group displays unique activity. Deep zone cells appear the most robust in their phenotype retention and most responsive to IGF-1 delivery. The results highlight differences in metabolic activity and varying responses to delivered growth factors between zonal chondrocyte populations.

Differential response of cartilage oligomeric matrix protein (COMP) to morphogens of bone morphogenetic protein/transforming growth factor-β family in the surface, middle and deep zones of articular cartilage

Cartilage oligomeric matrix protein (COMP) is a prominent non-collagenous component of the extracellular matrix (ECM) in articular cartilage. The regulation of COMP synthesis and secretion is critical for the understanding of cartilage homeostasis in health and disease. We therefore investigated the role of bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily members on COMP. Articular chondrocytes were isolated from three distinct zones (surface, middle and deep) and cultured as monolayers in serum-free chemically defined medium. Protein levels of COMP were determined in the medium by enzyme-linked immunosorbent assay. The mRNA expression was determined by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). TGFβ1 significantly stimulated the expression of COMP at the mRNA and protein levels in the superficial zone in a time-dependent manner. An unexpected discovery was that surface chondrocytes were more responsive to TGFβ isoforms than those in the deep layer. However, BMP-7 and growth differentiation factor-5 (GDF-5) also upregulate COMP expression; the effects were not as potent as those of TGFβ1. Activins A, B and AB demonstrated no effects on COMP in any of the zones. In conclusion, COMP synthesis is differentially regulated by TGFβ1 in the surface and middle zones of bovine articular cartilage.

Bicarbonate‐dependent pHi regulation by chondrocytes within the superficial zone of bovine articular cartilage

Control of chondrocyte pH (pH(i)) determines articular cartilage matrix metabolism. However, the transporters of chondrocytes in situ throughout cartilage zones are unclear, and we tested the hypothesis that chondocytes within the superficial zone (SZ) utilise a HCO(3) (-)-dependent system absent from other zones. Imaging of single BCECF-labelled cells was used to monitor the pH(i) of in situ chondrocytes within the cartilage zones, and also that of cells isolated from the SZ or full depth (FD) explants. Resting pH(i) and intrinsic buffering power (beta(i)) in HEPES-buffered saline was not different between SZ and DZ cells, however the pH(i) of SZ chondrocytes was lower in HCO(3) (-) saline. Ammonium pre-pulse was used to acid-load cells and pH(i) recovery by in situ or isolated SZ chondrocytes shown to be totally dependent on HCO(3) (-). pH(i) recovery rate was significantly (P < 0.05) greater for in situ cells, suggesting that isolation damaged the HCO(3) (-)-dependent system. Recovery of pH(i) by in situ cells was blocked by the anion transport inhibitor DIDS, and partially inhibited by EIPA probably non-specifically. Recovery of pH(i) by acidified MZ or DZ cells or those isolated from FD explants was not affected by HCO(3) (-) (P > 0.05). Na(+)-dependent HCO(3) (-)-(NBC) transporters were identified in SZ chondrocytes by fluorescence immunohistochemistry suggesting that this system might account for the HCO(3) (-)-dependent recovery of pH(i). Bovine articular cartilage chondrocytes possess a HCO(3) (-)-dependent transporter which plays a key role in pH(i) regulation in cells in the SZ, but not in chondrocytes within deeper cartilage zones.

Formation of Biphasic Constructs Containing Cartilage with a Calcified Zone Interface

The zone of calcified cartilage is the mineralized region of articular cartilage that anchors the hyaline cartilage to the subchondral bone and serves to disperse mechanical forces across this interface. In an attempt to mimic this zonal organization, we have developed the methodology to form biphasic constructs composed of cartilaginous tissue anchored to the top surface of a bone substitute (porous calcium polyphosphate, CPP) with a calcified interface. To accomplish this, chondrocytes were selectively isolated from the deep zone of bovine articular cartilage, placed on top of the CPP substrate, and grown in the presence of beta-glycerophosphate (10 mM, beta-GP). By 8 weeks, cartilage tissue had formed with two zones: a calcified region adjacent to the CPP substrate and a hyaline-like zone above. Little or no mineralization occurred in the absence of beta-GP. The mineral that formed in vitro was identified as hydroxyapatite, similar in composition and crystal size to that found in vivo. The tissue stiffness was seven times greater, and the interfacial shear properties at the cartilage-CPP interface were at least two times greater in the presence of this mineralized zone within the in vitro-formed cartilage than in tissue lacking a mineral zone. In conclusion, developing a biphasic construct with a calcified zone at the tissue-biomaterial interface resulted in significantly better cartilage load-bearing (compressive) properties and interfacial shear strength, emphasizing the importance of the presence of a mineralized zone in bioengineered cartilage. Because failure due to shear occurred at the cartilage-CPP interface instead of the tidemark, as occurs with osteochondral tissue, further study is required to optimize this system so that it more closely mimics the native tissue.

Removal of the superficial zone of bovine articular cartilage does not increase its frictional coefficient

To investigate the role of the superficial zone in regulating the frictional response of articular cartilage. This zone contains the superficial protein (SZP), a proteoglycan synthesized exclusively by superficial zone chondrocytes and implicated in reducing the friction coefficient of cartilage. Unconfined compression creep tests with sliding of cartilage against glass in saline were carried out on fresh bovine cylindrical plugs (slashed circle Ø6 mm, n=35) obtained from 16 bovine shoulder joints (ages 1-3 months). In the first two experiments, friction tests were carried out before and after removal of the superficial zone ( approximately 100 microm), in a control and treatment group, using two different applied load magnitudes (4.4 N and 22.2 N). In the third experiment, friction tests were conducted on intact surfaces and the corresponding microtomed deep zone of the same specimen. In all tests the friction coefficient exhibited a transient response, increasing from a minimum value (mu(min)) to a near-equilibrium final value (micro(eq)). No statistical change (P>0.5) was found in micro(min) before and after removal of the superficial zone in both experiments 1 and 2. However, micro(eq) was observed to decrease significantly (P<0.001) after removal of the surface zone. Results from the third experiment confirm that micro(eq) is even lower at the deep zone. Surface roughness measurements with atomic force microscopy (AFM) revealed an increase in surface roughness after microtoming. Immunohistochemical staining confirmed the presence of SZP in intact specimens and its removal in microtomed specimens. The topmost ( approximately 100 microm) superficial zone of articular cartilage does not have special properties which enhances its frictional response.

Experimental Model for Cartilage Tissue Engineering to Regenerate the Zonal Organization of Articular Cartilage

Objective: Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. Design: Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. Results: Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. Conclusion: We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Expression of clusterin in the superficial zone of bovine articular cartilage.

To investigate the differences between chondrocytes of the superficial and underlying zones of articular cartilage at the level of gene expression. Messenger RNA (mRNA) was isolated from chondrocytes harvested from the superficial and deep zones of immature bovine articular cartilage. This mRNA was reverse transcribed, radiolabeled, and then each complementary DNA (cDNA) sample was used to screen duplicate filters of a bovine chondrocyte cDNA library. By comparing autoradiographic signals on matching filter sets, clones exclusively expressed in the superficial zone of articular cartilage were isolated and characterized further. Of the superficial-specific gene clones isolated, 25% were found to be a single gene product, clusterin. Northern hybridization was used to show that clusterin is expressed specifically in the superficial zone of articular cartilage and that its expression is up-regulated in mature cartilage. In situ hybridization was used to precisely localize clusterin transcripts in articular cartilage, where it was found that clusterin expression was confined to the articular surface in both immature and mature samples. The discovery of clusterin expression at the articular cartilage surface extends previous observations that superficial articular chondrocytes are highly specialized cells. Clusterin is a multifunctional, secreted glycoprotein that has been shown to be expressed in diverse locations that have in common a tissue-fluid boundary. Additionally, clusterin has been implicated in regulating complement activation and cell death in injured and degenerating tissues.

Characterization of the mineral in calcified articular cartilagenous tissue formed in vitro.

Cartilagenous tissue with mineralized and nonmineralized layers was generated in vitro using bovine chondrocytes isolated from the deep zone of articular cartilage. Mineralization was induced by addition of either beta-glycerophosphate (beta-GP), phosphoethanolamine (PEA), or adenosine triphosphate (ATP). As this tissue might be suitable for use in joint resurfacing, the mineral of the calcified layer was characterized and compared to that present in the in vivo mineralized zone of bovine articular cartilage. Von Kossa staining demonstrated the presence of mineralization in the lower half of the tissue. The calcium content in the tissue varied from 4.9% to 7.8% of dry weight. Electron diffraction demonstrated a pattern consistent with hydroxyapatite. Brightfield transmission electron microscopy showed that the crystals were acicular and when measured under electron diffraction dark field imaging were 16.6 +/- 3.8 (beta-GP), 16.4 +/- 3.8 nm (ATP), and 17.0 +/- 6.3 nm (PEA) in length. The crystals were similar in size (16.0 +/- 5.5 nm) and appearance to the crystals in the in vivo calcified cartilage. This data suggests that the mineralization that occurs in vitro is similar to the in vivo cartilage.

Composition of cartilagenous tissue with mineralized and non-mineralized zones formed in vitro

We have previously shown that cartilagenous tissue with both non-mineralized and mineralized zones can be formed by chondrocytes which have been selectively isolated from the deep zone of bovine articular cartilage. In this study, we quantitate proteoglycan and collagen content, calcification, tissue thickness and cellularity over a 10 week culture period in order to study matrix accumulation and tissue formation. The cartilagenous tissue cellularity and proteoglycan and collagen accumulation continued up to 8 weeks and this was paralleled by an increase in tissue thickness. The amount of mineral in the tissue as well as the amount of collagen, in contrast to proteoglycan, was still increasing at 10 weeks. At the end of week 10, the amount of glycosaminoglycan and collagen as a percentage of dry weight of the tissue were 11.0 ± 0.6% and 14.8 ± 0.1%, respectively, compared with 10.5 ± 1.2% and 35.1 ± 5.8% for the in vitro deep articular cartilage. The amount of calcium as a percentage of dry weight of the cartilagenous tissue was 8.1 ± 0.7% which was similar to the in vivo cartilage (9.1 ± 1.6%). This data suggests that 8 weeks of culture may be necessary before the cartilagenous tissue is suitable for use as a transplant.

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes.

Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CMR filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when beta-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.

Effects of Misoprostol with Interleukin-1 on Proteoglycan Metabolism of Cultured Articular Chondrocytes.

Bovine and normal human articular chondrocytes in suspension culture were treated with misoprostol (an analog of prostaglandin E(1) [PGE(1)]), alone and in combination with interleukin-1 (IL-1) and/or diclofenac sodium, to study effects on proteoglycan metabolism. A concentrations of 50 ng ml(minus sign1) and above, misoprostol suppressed synthesis of proteoglycans but did not affect their rate of catabolism. The mild inhibitory effect of misoprostol on proteoglycan synthesis was additive to that of IL-1, especially in chondrocytes from the superficial zone of bovine articular cartilage. In IL-1-treated cultures, diclofenac NA caused a modest improvement in proteoglycan synthesis, but this beneficial effect was diminished by the simultaneous addition of misoprostol. Cartilage or chondrocyte cultures treated with IL-1, in which proteoglycan synthesis is suppressed, serve as model systems in which to study metabolic responses of chondrocytes to potential therapeutic agents, but in these experiments, no chondroprotective effects of misoprostol were observed in IL-1-activated chondrocytes.

A Novel Proteoglycan Synthesized and Secreted by Chondrocytes of the Superficial Zone of Articular Cartilage

A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4°C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.

Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.