Modulation of collagen synthesis in normal and osteoarthritic cartilage

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.     

The structure and mechanical properties of the proteins of lamprey cartilage

The cyanogen bromide‐resistant proteins of lamprey cartilage are biochemically related to the mammalian elastic protein, elastin. This study investigates their mechanical properties and enquires whether, like elastin, long‐range elasticity arises in them from a combination of entropic and hydrophobic mechanisms. Branchial and pericardial proteins resembled elastin mechanically, with elastic moduli of 0.13–0.35 MPa, breaking strains of 50%, and low hysteresis. Annular and piston proteins had higher elastic moduli (0.27–0.75 MPa) and larger hysteresis. Exchanging solvent water for trifluoroethanol increased the elastic moduli, whereas increasing temperature lowered the elastic moduli. Raman microspectrometry showed small differences in side‐chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were like those of elastin, preponderantly unordered, which probably confer the conformational flexibility necessary for entropy elasticity. Piston and annular proteins showed the strongest interactions with water, suggesting, together with the mechanical testing data, a greater role of hydrophobic interactions in their mechanics. Two‐photon imaging of intrinsic fluorescence and dye injection experiments showed that annular and piston proteins formed closed‐cell honeycomb structures, whereas the branchial and pericardial proteins formed open‐cell structures, which may account for the differences in mechanical properties. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 187–202, 2015.     

Mechanical stress promotes matrix synthesis of mandibular condylar cartilage via the RKIP-ERK pathway

Mandibular hypoplasia is a common jaw deformity that affects breathing, occlusal function and facial aesthetics. Stimulating mandibular condylar growing with functional appliances is an ordinary but controversial treatment method in orthodontics. Therefore, it is vital to clarify how functional appliances affect condylar growing. Raf-1 kinase inhibitor protein (RKIP), as an endogenous inhibitory molecule of the ERK signaling, is postulated to involve in stress-induced response to articular cartilage. This study was to reveal the role of RKIP in regulating cartilage matrix synthesis with functional appliance treatment. Here, position rat mandibular forward simulating functional appliance effect to examine the stress-induced modification of mandibular condylar in vivo, meanwhile rat mandibular condylar chondrocytes (Mccs) were subjected to cyclic tensile stress (CTS, 16%, 1 HZ). The results showed that mandibular forward therapy enhanced condylar cartilage growth. The thicknesses of all layers of condylar cartilage were increased significantly. RKIP expression was also increased in the mature cartilage layer. In addition, CTS could enhance extracellular matrix formation and cartilage marker expression (aggrecan and collagen II), which shared a similar expression pattern with RKIP in Mccs. However, CTS induced up-regulation of collagen II and aggrecan was blocked by RKIP knockdown. Nuclear p-ERK, targeting downstream of RKIP, showed a decrease after CTS,which was disappeared in RKIP-knockdown Mccs. Taken together, physiological mechanical stimulation promotes cartilage growth modification by up-regulating RKIP through inhibiting ERK signaling pathway.     

An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage

Summary The synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.Supported by grants CA 05196-04S1 and GM-K3-13, 979-C1-A from the United States Public Health Service.The results reported in this paper were presented at the second annual meeting of the American Society for Cell Biology, November 6, 1962.     

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.     

Effect of specimen thickness on fracture toughness of bovine patellar cartilage

Fracture toughness and crack tip opening angle were measured for bovine patellar cartilage using modified single-edged notch specimens of two thicknesses. There was no difference in fracture toughness between thin (0.7 mm) versus relatively thick (2.7 mm) specimens, but the crack tip opening angle at initiation of crack propagation was larger for the thin specimens (106 deg) than for the thick specimens (70 deg). Fracture toughness of the bovine patellar cartilage (1.03 kJ/m2) was not statistically different than that reported previously for canine patellar cartilage (1.07 kJ/m2) employing the same methods. Large variation in measurements for both bovine and canine cartilage are in part attributable to variation between individual animals, and are consistent with variation in other mechanical property measurements for articular cartilage. The observed reduction in crack tip opening angle with increased specimen thickness is consistent with behavior of some engineering materials, and demonstrates that specimen thickness influences fracture behavior for bovine patellar cartilage.     

Effect of rooperol on collagen synthesis and cell growth

The effect of rooperol on type I collagen synthesis in normal skin and lung fibroblasts and cell growth in normal and transformed fibroblasts was investigated. Low concentrations of rooperol selectively inhibited the growth of transformed cells while stimulating collagen synthesis in normal fibroblasts. Elevated collagen synthesis and deposition could impede tumour cell invasion and metastasis, implying that rooperol may be useful as an antimetastatic agent in the treatment of cancer.     

Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints. Understanding the detailed effects of the mechanical environment of chondrocytes on cell behavior is therefore essential for the study of the development, adaptation, and degeneration of articular cartilage. Recent progress in macroscopic models has improved our understanding of depth-dependent properties of cartilage. However, none of the previous works considered the effect of realistic collagen orientation or depth-dependent negative charges in microscopic models of chondrocyte mechanics. The aim of this study was to investigate the effects of the collagen network and fixed charge densities of cartilage on the mechanical environment of the chondrocytes in a depth-dependent manner. We developed an anisotropic, inhomogeneous, microstructural fibril-reinforced finite element model of articular cartilage for application in unconfined compression. The model consisted of the extracellular matrix and chondrocytes located in the superficial, middle, and deep zones. Chondrocytes were surrounded by a pericellular matrix and were assumed spherical prior to tissue swelling and load application. Material properties of the chondrocytes, pericellular matrix, and extracellular matrix were obtained from the literature. The loading protocol included a free swelling step followed by a stress-relaxation step. Results from traditional isotropic and transversely isotropic biphasic models were used for comparison with predictions from the current model. In the superficial zone, cell shapes changed from rounded to elliptic after free swelling. The stresses and strains as well as fluid flow in cells were greatly affected by the modulus of the collagen network. The fixed charge density of the chondrocytes, pericellular matrix, and extracellular matrix primarily affected the aspect ratios (height/width) and the solid matrix stresses of cells. The mechanical responses of the cells were strongly location and time dependent. The current model highlights that the collagen orientation and the depth-dependent negative fixed charge densities of articular cartilage have a great effect in modulating the mechanical environment in the vicinity of chondrocytes, and it provides an important improvement over earlier models in describing the possible pathways from loading of articular cartilage to the mechanical and biological responses of chondrocytes.     

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional ²H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo. This paper is presented in part in ISMRM 11th Scientific Meeting, p. 55, 2003 and ISMRM 14th Scientific Meeting, p. 59, 2006. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

The effect of silica-treated macrophages on the synthesis of collagen and other proteins in vitro

Treatment with SiO₂ releases from peritoneal macrophages a soluble factor which stimulates the synthesis of collagen and other proteins in incubated slices of experimental granulation tissue. This factor can also be obtained by SiO₂-treatment from certain subcellular particles of intact macrophages. A similar agent is released from the macrophages by incubation with rheumatoid synovialtissue extract. Macrophages induced by paraffin or thioglycollate medium cannot be stimulated further by SiO₂. The SiO₂-treated macrophages have no effect on detached matrix-free cells from embryonic-chick tendon or granulation tissue. Another factor from macrophages, present in the 100000 g-supernatant of the homogenate, inhibits the synthesis of collagen in granuloma slices. The synthesis of DNA and RNA in slices is suppressed by the extract from intact macrophages but not affected by preparations obtained with SiO₂. The possible relevance of these findings to lysosomal actions, to the regulation of granuloma formation and to inflammation are discussed.     

Tensile properties of the mandibular condylar cartilage

Mandibular condylar cartilage plays a crucial role in temporomandibular joint (TMJ) function, which includes facilitating articulation with the temporomandibular joint disc and reducing loads on the underlying bone. The cartilage experiences considerable tensile forces due to direct compression and shear. However, only scarce information is available about its tensile properties. The present study aims to quantify the biomechanical characteristics of the mandibular condylar cartilage to aid future three-dimensional finite element modeling and tissue engineering studies. Porcine condylar cartilage was tested under uniaxial tension in two directions, anteroposterior and mediolateral, with three regions per direction. Stress relaxation behavior was modeled using the Kelvin model and a second-order generalized Kelvin model, and collagen fiber orientation was determined by polarized light microscopy. The stress relaxation behavior of the tissue was biexponential in nature. The tissue exhibited greater stiffness in the anteroposterior direction than in the mediolateral direction as reflected by higher Young's (2.4 times), instantaneous (1.9 times), and relaxed (1.9 times) moduli. No significant differences were observed among the regional properties in either direction. The predominantly anteroposterior macroscopic fiber orientation in the fibrous zone of condylar cartilage correlated well with the biomechanical findings. The condylar cartilage appears to be less stiff and less anisotropic under tension than the anatomically and functionally related TMJ disc. The anisotropy of the condylar cartilage, as evidenced by tensile behavior and collagen fiber orientation, suggests that the shear environment of the TMJ exposes the condylar cartilage to predominantly but not exclusively anteroposterior loading.     

Biosynthesis of collagen and other matrix proteins by articular cartilage in experimental osteoarthrosis.

Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.