Enzymic heterogeneity of normal canine articular cartilage

Articular cartilage is generally considered to be an homogeneous tissue. It has now been shown that, although different regions of the medial tibial cartilage of the dog have very similar oxidative enzymic activities, each region is heterogeneous with respect to these activities. The conventional histological delineation of this cartilage has been modified, to take into account a narrow band (designated zone 2a), just below the most superficial spindle-shaped cells, that has higher oxidative enzymic activity than any other. Changes in the activity in this zone might be diluted by the lack of change in other zones if measured by conventional biochemical procedures which could not measure the activities of the different zones separately.     

Plasmin degradation of cartilage proteoglycan

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen or plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.     

Deformation of nasal septal cartilage during mastication

The cartilaginous nasal septum plays a major role in structural integrity and growth of the face, but its internal location has made physiologic study difficult. By surgically implanting transducers in 10 miniature pigs (Sus scrofa), we recorded in vivo strains generated in the nasal septum during mastication and masseter stimulation. The goals were (1) to determine whether the cartilage should be considered as a vertical strut supporting the nasal cavity and preventing its collapse, or as a damper of stresses generated during mastication and (2) to shed light on the overall pattern of snout deformation during mastication. Strains were recorded simultaneously at the septo‐ethmoid junction and nasofrontal suture during mastication. A third location in the anterior part of the cartilage was added during masseter stimulation and manipulation. Contraction of jaw closing muscles during mastication was accompanied by anteroposterior compressive strains (around ?1,000 με) in the septo‐ethmoid junction. Both the orientation and the magnitude of the strain suggest that the septum does not act as a vertical strut but may act in absorbing loads generated during mastication. The results from masseter stimulation and manipulation further suggest that the masticatory strain pattern arises from a combination of dorsal bending and/or shearing and anteroposterior compression of the snout. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.     

Improved cartilage integration and interfacial strength after enzymatic treatment in a cartilage transplantation model

The objective of the present study was to investigate whether treatment of articular cartilage with hyaluronidase and collagenase enhances histological and mechanical integration of a cartilage graft into a defect. Discs of 3 mm diameter were taken from 8-mm diameter bovine cartilage explants. Both discs and annulus were either treated for 24 hours with 0.1% hyaluronidase followed by 24 hours with 10 U/ml collagenase or left untreated (controls). Discs and annulus were reassembled and implanted subcutaneously in nude mice for 5 weeks. Integration of disc with surrounding cartilage was assessed histologically and tested biomechanically by performing a push-out test. After 5 weeks a significant increase in viable cell counts was seen in wound edges of the enzyme-treated group as compared with controls. Furthermore, matrix integration (expressed as a percentage of the total interface length that was connected; mean ± standard error) was 83 ± 15% in the treated samples versus 44 ± 40% in the untreated controls. In the enzyme-treated group only, picro-Sirius Red staining revealed collagen crossing the interface perpendicular to the wound surface. Immunohistochemical analyses demonstrated that the interface tissue contained cartilage-specific collagen type II. Collagen type I was found only in a small region of fibrous tissue at the level of the superficial layer, and collagen type III was completely absent in both groups. A significant difference in interfacial strength was found using the push-out test: 1.32 ± 0.15 MPa in the enzyme-treated group versus 0.84 ± 0.14 MPa in the untreated controls. The study shows that enzyme treatment of cartilage wounds increases histological integration and improves biomechanical bonding strength. Enzymatic treatment may represent a promising addition to current techniques for articular cartilage repair.     

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions

Salmon nasal cartilage was micronized in ethanol using a rotor–stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.     

Intrahissian Anisotropy and Second Degree Type I Block

A 79 year old female presents for evaluation of multiple episodes of witnessed syncope. Invasive electrophysiologic evaluation revealed evidence of both intrahissian Wenckebach and anisotropy. This is the first report documenting both phenomena in the same patient. The patient underwent a dual-chamber pacemaker implantation without complication.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

低切应力作用下体外培养动脉内皮细胞内皮素的表达

研究了切应力对完整血管的生物学作用以及应力引起血管重建过程中内皮素(ET)的变化．采用血管体外应力培养系统,将一段完整的猪颈总动脉在体外进行培养,设切应力分别为2Pa(％组)和0．5Pa(S5组),设置2、4、6．8．10．12、14．16和18h共9个时相观察点,非平衡法放射免疫检测灌流液中的ET含量．通过Logistic曲线方程拟合,分析切应力作用下完整动脉ET表达变化规律。结果显示：S20组ET总体变化不明显;S5组分泌速率在前12．37h内明显上升,而后又逐渐下降趋于稳定,且始终高于S20组。说明低切应力作用下ET的表达及分泌增高．结果提示,在低切应力引起的血管重建中,ET可能起着重要作用。     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection

The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 ± 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.     

In situ measurement of articular cartilage deformation in intact femoropatellar joints under static loading

The deformational behavior of articular cartilage has been investigated in confined and unconfined compression experiments and indentation tests, but to date there exist no reliable data on the in situ deformation of the cartilage during static loading. The objective of the current study was to perform a systematic study into cartilage compression of intact human femoro-patellar joints under short- and long-term static loading with MR imaging. A non-metallic pneumatic pressure device was used to apply loads of 150% body weight to six joints within the extremity coil of an MRI scanner. The cartilage was delineated during the compression experiment with previously validated 2D and 3D fat-suppressed gradient echo sequences. We observed a mean (maximal) in situ deformation of 44% (57%) in patellar cartilage after 32 h of loading (mean contact pressure 3.6 MPa), the femoral cartilage showing a smaller amount of deformation than the patella. However, only around 7% of the final deformation (3% absolute deformation) occurred during the first minute of loading. A 43% fluid loss from the interstitial patellar matrix was recorded, the initial fluid flux being 0.217 +/- 0.083 microm/s, and a high inter-individual variability of the deformational behavior (coefficients of variation 11-38%). In conjunction with finite-element analyses, these data may be used to compute the load partitioning between the solid matrix and fluid phase, and to elucidate the etiologic factors relevant in mechanically induced osteoarthritis. They can also provide direct estimates of the mechanical strain to be encountered by cartilage transplants.     

Articular cartilage and changes in Arthritis: Noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage

Cartilage contains numerous noncollagenous proteins in its extracellular matrix, including proteoglycans. At least 40 such molecules have been identified, differing greatly in structure, distribution, and function. Some are present in only selected cartilages or cartilage zones, some vary in their presence with a person's development and age, and others are more universal in their expression. Some may not even be made by the chondrocytes, but may arise by absorption from the synovial fluid. In many cases, the molecules' function is unclear, but the importance of others is illustrated by their involvement in genetic disorders. This review provides a selective survey of these molecules and discusses their structure, function, and involvement in inherited and arthritic disorders.     

On the anisotropy and inhomogeneity of permeability in articular cartilage

Articular cartilage is known to be anisotropic and inhomogeneous because of its microstructure. In particular, its elastic properties are influenced by the arrangement of the collagen fibres, which are orthogonal to the bone-cartilage interface in the deep zone, randomly oriented in the middle zone, and parallel to the surface in the superficial zone. In past studies, cartilage permeability has been related directly to the orientation of the glycosaminoglycan chains attached to the proteoglycans which constitute the tissue matrix. These studies predicted permeability to be isotropic in the undeformed configuration, and anisotropic under compression. They neglected tissue anisotropy caused by the collagen network. However, magnetic resonance studies suggest that fluid flow is "directed" by collagen fibres in biological tissues. Therefore, the aim of this study was to express the permeability of cartilage accounting for the microstructural anisotropy and inhomogeneity caused by the collagen fibres. Permeability is predicted to be anisotropic and inhomogeneous, independent of the state of strain, which is consistent with the morphology of the tissue. Looking at the local anisotropy of permeability, we may infer that the arrangement of the collagen fibre network plays an important role in directing fluid flow to optimise tissue functioning.     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

Measurement of local strains in intervertebral disc anulus fibrosus tissue under dynamic shear: Contributions of matrix fiber orientation and elastin content

Shear strain has been implicated as an initiator of intervertebral disc anulus failure, however a clear, multi-scale picture of how shear strain affects the tissue microstructure has been lacking. The purposes of this study were to measure microscale deformations in anulus tissue under dynamic shear in two orie ntations, and to determine the role of elastin in regulating these deformations. Bovine AF tissue was simultaneously shear loaded and imaged using confocal microscopy following either a buffer or elastase treatment. Digital image analysis was used to track through time local shear strains in specimens sheared transversely, and stretch and rotation of collagen fiber bundles in specimens sheared circumferentially. The results of this study suggest that sliding does not occur between AF plies under shear, and that interlamellar connections are governed by collagen and fibrilin rather than elastin. The transverse shear modulus was found to be approximately 1.6 times as high in plies the direction of the collagen fibers as in plies across them. Under physiological levels of in-plane shear, fiber bundles stretched and re-oriented linearly. Elastin was found to primarily stiffen plies transversely. We conclude that alterations in the elastic fiber network, as found with IVD herniation and degeneration, can therefore be expected to significantly influence the AF response to shear making it more susceptible to micro failure under bending or torsion loading.     

Molecular heterogeneity of tumor endothelium

Tumors depend on a vascular supply for their growth. Tumor blood vessels, which are derived from normal tissue vasculature, display a markedly abnormal phenotype. Tumor endothelial properties are highly varied in space and time. An important goal is to delineate the range of phenotypes in tumor endothelium and to identify tumor endothelial-specific molecular signatures. This information should lead to a more complete understanding of the mechanisms of tumor growth, the discovery of new therapeutic targets, and the development of biomarkers for diagnosis and surveillance. The goals of this review are to outline recent advances in dissecting tumor endothelial-cell-specific gene expression, to address mechanisms of phenotypic heterogeneity in tumor vascular beds, and to discuss the therapeutic implications of these findings.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.