Modeling costal cartilage using local material properties with consideration for gross heterogeneities

Contemporary computer models of the thorax designed to predict injury in automobile collisions model the costal cartilage as a homogeneous material using properties derived from local material characterization tests. No studies have validated the accuracy of these models in predicting the structural mechanics of costal cartilage. Two heterogeneities - the perichondrium and calcified regions - may affect the behavior of costal cartilage in a manner not accounted for by current models. This study sought to investigate the predictive ability of subject-specific models of whole costal cartilage segments, with the calcified regions modeled distinctly and with the perichondrium removed (from the physical specimens as well as from the simulations). Finite element models were developed in the case of five cadaveric costal cartilage segments. The properties of the cartilage were derived from indentation testing of each specimen, where the characteristic average instantaneous elastic moduli ranged from 8.7 to 12.6 MPa. Matched simulations and experiments were then performed, subjecting each specimen to cantilever-like loading with a dynamic posterior displacement of the sternal boundary (all other boundary degrees-of-freedom fixed). The models predicted the resulting peak anterior-posterior forces generated on the costal boundary with a minimum error of 1% and a maximum error of 36%. These results provide support to the previous implicit assumption that insight can be gained into the structural behavior of costal cartilage by observing the local material properties (when calcified regions are included and the perichondrium is removed). Future work includes the addition of the perichondrium, so as to model the whole costal cartilage composite structure.     

Computational model of rib movement and its application in studying the effects of age-related thoracic cage calcification on respiratory system

A 3D finite element model of rib cage movement is developed and used to study the role of age-related costal cartilage and sternocostal joint calcification, as well as respiratory muscle weakness on the ‘bucket-handle’ movement of human rib. The volume displacement of the rib cage is related to changes in its circumference using an empirical equation presented by Agostoni et al. (1965, J Appl Physiol, 20:1179–1186). A systematic study is carried out to quantify the role of costal cartilage, sternocostal joint calcification and muscle weakness on the volume displacement of the rib cage. The results provide insight into some of the mechanisms underlying age-related changes in the respiratory system.     

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.     

Costal cartilages--a clue for determination of sex

Mineralization and ossification in the human costal cartilages were studied radiologically. The aim of our study was to evaluate differences between males and females with respect to patterns of costal cartilage calcification and also with respect to ageing. Material for this study consists of 1044 chest and abdominal radiograms of the Czech population from the Department of Radiology (537 males and 507 females). Further radiograms of 18 chest plates were obtained at routine necropsy of cadavers. The radiograms were examined for pattern of ossification of the costal cartilage. The first rib cartilages were not considered because there are no sex differences. The lower ribs exhibit sexual dimorphism. Mineralization and ossification changes appear at the end of puberty and their occurrence increases with age. The sexual difference in pattern of human costal cartilages is statistically significant and thus highly predictive of sex determination.     

The dual role of perichondrium in cartilage wound healing

Cartilage structures from the head and neck possess a certain but limited capacity to heal after injury. This capacity is accredited to the perichondrium. In this study, the role of the inner (cambium) and the outer (fibrous) layers of the perichondrium in cartilage wound healing in vitro is investigated. For the first time, the possibility of selectively removing the outer perichondrium layer is presented. Using rabbit ears, three different conditions were created: cartilage explants with both perichondrium layers intact, cartilage explants with only the outer perichondrium layer dissected, and cartilage explants with both perichondrium layers removed. The explants were studied after 0, 3, 7, 14, and 21 days of in vitro culturing using histochemistry and immunohistochemistry for Ki-67, collagen type II, transforming growth factor beta 1 (TGFbeta1), and fibroblast growth factor 2 (FGF2). When both perichondrium layers were not disturbed, fibrous cells grew over the cut edges of the explants from day 3 of culture on. New cartilage formation was never observed in this condition. When only the outer perichondrium layer was dissected from the cartilage explants, new cartilage formation was observed around the whole explant at day 21. When both perichondrium layers were removed, no alterations were observed at the wound surfaces. The growth factors TGFbeta1 and FGF2 were expressed in the entire perichondrium immediately after explantation. The expression gradually decreased with time in culture. However, the expression of TGFbeta1 remained high in the outer perichondrium layer and the layer of cells growing over the explant. This indicates a role for TGFbeta1 in the enhancement of fibrous overgrowth during the cartilage wound-healing process. The results of this experimental in vitro study demonstrate the dual role of perichondrium in cartilage wound healing. On the one hand, the inner layer of the perichondrium, adjacent to the cartilage, provides (in time) cells for new cartilage formation. On the other hand, the outer layer rapidly produces fibrous overgrowth, preventing the good cartilage-to-cartilage connection necessary to restore the mechanical function of the structure.     

Histochemistry of the extracellular matrix of aging hyaline cartilage

Cartilage proteoglycans (PGs) exhibit marked structural changes with increasing age. There is an increase in small PGs rich in KS as compared to larger PGs rich in chondroitin sulfate (CS) with increasing age. In the present study investigations have been performed to obtain more detailed information about the distribution of different glycosaminoglycans (GAGs). Changes were observed in the interterritorial matrix by means of ultrastructural visualization of PGs with acridin orange. The changes in the ultrastructural organization of the interterritorial matrix of costal cartilage are followed by significant changes in its mechanical properties.     

Knee cartilage loss in symptomatic knee osteoarthritis over 4.5 years

The objective of this study was to describe the rate of change in knee cartilage volume over 4.5 years in subjects with symptomatic knee osteoarthritis (OA) and to determine factors associated with cartilage loss. One hundred and five subjects were eligible for this longitudinal study. Subjects' tibial cartilage volume was assessed by magnetic resonance imaging (MRI) at baseline, at 2 years and at 4.5 years. Of 105 subjects, 78 (74%) completed the study. The annual percentage losses of medial and lateral tibial cartilage over 4.5 years were 3.7 ± 4.7% (mean ± SD; 95% confidence interval 2.7 to 4.8%) and 4.4 ± 4.7% (mean ± SD; 95% confidence interval 3.4 to 5.5%), respectively. Cartilage volume in each individual seemed to track over the study period, relative to other study participants. After multivariate adjustment, annual medial tibial cartilage loss was predicted by lesser severity of baseline knee pain but was independent of age, body mass index and structural factors. No factors specified a priori were associated with lateral cartilage volume rates of change. Tibial cartilage declines at an average rate of 4% per year in subjects with symptomatic knee OA. There was evidence to support the concept that tracking occurs in OA. This may enable the prediction of cartilage change in an individual. The only significant factor affecting the loss of medial tibial cartilage was baseline knee pain, possibly through altered joint loading.     

Amianthoid (asbestoid) transformation: electron microscopical studies on aging human costal cartilage

The present study reports on the fine structure of human costal cartilage at different ages in order to obtain information on the morphogenesis of amianthoid fibers. Our results reveal an overall increase of collagen fibril diameter with increasing age, even in areas with no signs of amianthoid transformation. Ultrastructural evidence is presented that this increase in diameter is due to a gathering of the preexisting collagen fibrils. The age-related change in collagen fibril diameter is paralleled by changes in the composition and ultrastructural appearance of cartilage proteoglycans (as revealed by acridine orange staining). Acridine-orange-positive filaments indicative for proteoglycans are markedly reduced in size with advancing age in centrally located regions of costal cartilage. Treatment with testicular hyaluronidase previous to acridine-orange staining leaves these small proteoglycan filaments unaffected. By contrast, the filaments visible after acridine-orange staining in the extracellular matrix near to the perichondrium are susceptible to hyaluronidase treatment. Infrequently, a sharp increase in collagen fibril diameter can be observed in territorial matrix areas of degenerating chondrocytes. This observation is conspicuous at ages of 10 and 20 years. Amianthoid transformation is characterized by the appearance of collagen fibrils strictly arranged in parallel. These amianthoid fibers are embedded in a matrix rich in small acridine-orange-positive filaments similar to the proteoglycan filaments observed in centrally located matrix regions. It can be concluded that extensive remodelling not only of the collagen fibrils but also of the cartilage proteoglycans is involved in the development of amianthoid transformation.     

Depth-dependent analysis of the role of collagen fibrils, fixed charges and fluid in the pericellular matrix of articular cartilage on chondrocyte mechanics

The pericellular matrix of articular cartilage has been shown to regulate the mechanical environment of chondrocytes. However, little is known about the mechanical role of collagen fibrils in the pericellular matrix, and how fibrils might help modulate strains acting on chondrocytes when cartilage is loaded. The primary objective was to clarify the effect of pericellular collagen fibrils on cell volume changes and strains during cartilage loading. Secondary objectives were to investigate the effects of pericellular fixed charges and fluid on cell responses. A microstructural model of articular cartilage, in which chondrocytes and pericellular matrices were represented with depth-dependent structural and morphological properties, was created. The extracellular matrix and pericellular matrices were modeled as fibril-reinforced, biphasic materials with swelling capabilities, while chondrocytes were assumed to be isotropic and biphasic with swelling properties. Collagen fibrils in the extracellular matrix were represented with an arcade-like architecture, whereas pericellular fibrils were assumed to run tangential to the cell surface. In the early stages of a stress-relaxation test, pericellular fibrils were found to sensitively affect cell volume changes, even producing a reversal from increasing to decreasing cell volume with increasing fibril stiffness in the superficial zone. Consequently, steady-state volume of the superficial zone cell decreased with increasing pericellular fibril stiffness. Volume changes in the middle and deep zone chondrocytes were smaller and opposite to those observed in the superficial zone chondrocyte. An increase in the pericellular fixed charge density reduced cell volumes substantially in every zone. The sensitivity of cell volume changes to pericellular fibril stiffness suggests that pericellular fibrils play an important, and as of yet largely neglected, role in regulating the mechanical environment of chondrocytes, possibly affecting matrix synthesis during cartilage development and degeneration, and affecting biosynthetic responses associated with articular cartilage loading.     

A model for studying the initiation of normal calcification in vivo

1. Rat costal cartilage was found to begin to calcify normally when the rats weigh 35-45g. 2. The cartilage is suggested as a model for the study in vivo of mechanisms concerned with normal calcification. 3. The model was tested by studying the incorporation of fluoride into the mineral deposited in the tissue. 4. The percentage of inorganic material in cartilage rose from approx. 3% of the dry weight in the uncalcified tissue to 62% in the tissue from rats weighing 300g. 5. Mineral deposited had a calcium/phosphorus molar ratio of 1.65. 6. After the oral administration of sodium fluoride to rats, fluoride was incorporated into cartilage mineral. 7. The concentration of fluoride in cartilage ash increased rapidly with calcification and the mineral became more highly fluoridated than the corresponding rib bone. 8. Fluoridated mineral showed a marked decrease in citrate concentration.     

Zonal and directional variations in tensile properties of bovine articular cartilage with special reference to strain rate variation

THE AIMS of this study were: (i) to investigate the variation in the tensile properties of articular cartilage with depth through cartilage thickness and fibre orientation; (ii) to determine the effect of strain rate on tensile properties of articular cartilage. MATERIALS AND METHOD: All experimental work was performed on cartilage specimens taken from two bovine knee joints. Osteochondral plugs 12 mm in diameter were harvested with a special reamer from the femur and the tibial plateaux of each knee. Slices (0.2 mm thick), of articular cartilage were cut from the plug with a microtome. The predominant orientation of the collagen fibres on the cartilage surface was determined using the pinpricking technique. Each specimen used for the tensile test was cut, so as to produce a dumbbell shape, with a gauge length of 6 mm. Uniaxial tensile tests were performed on each specimen in order to determine the tensile Young's modulus, and ultimate tensile strength (UTS). In this investigation, these tensile tests were carried out at different strain rate: 1, 20, 50 and 70%/sec. RESULTS: As regards the zonal properties, it was found that tensile stiffness was greater in the superficial layer than in deep layer. However, a few specimens from the deep layer displayed similar or greater stiffness compared to the superficial layer. With respect to the directional properties, the specimens oriented parallel to the predominant alignment of collagen, were stiffer than those, which were perpendicular to it in each layer. However, only the results regarding the deep layer can be considered statistically significant. In regard to the variation of modulus with the strain-rate, the results showed that there is no significant increase of the modulus with increasing strain rate from 20 to 50% per second. However, at 70% per second, articular cartilage stiffness considerably increased by up to one order of magnitude greater than that determined at lower strain rates in both the superficial and deep layer. Moreover, the UTS of cartilage specimens tested at 70% per second showed a significant rise, reaching values of four to five times that of those measured at 1, 20 or 50% per second. CONCLUSION: The steep increases in both the stiffness and ultimate tensile strength of cartilage at high strain rates point to the existence in cartilage of a mechanism for its protection from damage by stresses arising in trauma, which are usually applied at high rates. This mechanism needs to be elucidated. The reduced anisotropy found in the present study pointed out that collagen is likely to be less organized in bovine cartilage than in the human and therefore, a study of its ultra-structure would be appropriate.     

A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

Soft-tissue bone interface: How do attachments of muscles,tendons, and ligaments change during growth? A light microscopic study

This study addressed the problem of how soft structures maintain approximately the same relative positional relationships during long bone growth. Attachments of the popliteus muscle, semitendinosus tendon, medial collateral knee ligament, and extensor retinaculum were examined histologically in rabbits, aged 2-60 days, to determine the manner in which soft structures attached to long bones during growth. Soft structures inserted principally into fibrous periosteum or perichondrium in the age range studied. However, an extensive collagen fiber framework within the cellular periosteum and perichondrium, present by at least 2 days of age, linked the fibrous periosteum or perichondrium to subjacent bone or cartilage. Maturation of soft tissue-bone interfaces was viewed from two related perspectives. The first stressed temporal patterning of cartilage and bone differentiation. The second emphasized incorporation of attachments of soft structures into bone and cartilage matrices during growth and remodeling. Differentiation and remodeling of bone and cartilage varied not only with age, but also between regions of attachment of single muscles and ligaments. Insertion regions were characterized by the presence of coarse-fibered periosteal bone and chondroid bone, both morphologically intermediate between fibrocartilage and lamellar bone. These results provide evidence that periosteal attachments, characterizing the soft-tissue bone interface, are a necessary structural prerequisite for compensatory movement and invariance of the relative positions of muscles, tendons, and ligaments during long bone growth.     

Double-network acrylamide hydrogel compositions adapted to achieve cartilage-like dynamic stiffness

Since articular cartilage has a limited potential for spontaneous healing, various techniques are employed to repair cartilage lesions. Acrylate-based double-network (DN) hydrogels containing ~90% water have shown promising properties as repair materials for skeletal system soft tissues. Although their mechanical properties approach those of native cartilage, the critical factor—stiffness—of DN-gels does not equal the stiffness of articular cartilage. This study investigated whether revised PAMPS/PAAm compositions with lower water content result in stiffness parameters closer to cartilage. DN-gels containing 61, 86 and 90% water were evaluated using two non-destructive, mm-scale indentation test modes: fast-impact (FI) and slow-sinusoidal (SS) deformation. Deformation resistance (dynamic modulus) and energy handling (loss angle) were determined. The dynamic modulus increased with decreasing water content in both testing modes. In the 61% water DN-gel, the modulus resembled that of cartilage (FI-mode: DN-gel = 12, cartilage = 17; SS-mode: DN-gel = 4, cartilage = 1.7 MPa). Loss angle increased with decreasing water content in fast-impact, but not in slow-sinusoidal deformation. However, loss angle was still much lower than cartilage (FI: DN-gel = 5, cartilage = 11; SS: DN-gel = 10, cartilage = 32°), indicating somewhat less ability to dissipate energy. Overall, results show that it is possible to adapt DN-gel composition to produce dynamic stiffness properties close to normal articular cartilage.     

A comparative microscopic and ultrastructural study of perichondrial tissue in cartilage of Octopus vulgaris (Cephalopoda, Mollusca)

The microscopic and submicroscopic structures of perichondrial tissues in the head cartilages of Octopus vulgaris were studied by polarized light and transmission electron microscopy. The orbital cartilages possess a birefringent layer parallel to the surface of the cartilage; ultrastructurally, this layer, which may be considered perichondrial tissue, has the typical organisation of connective tissue but does not possess the stratification of collagen laminae found in vertebrate perichondria. Perichondrial extracellular matrix is clearly distinct from that of cartilage because its collagen fibrils are of a larger diameter than collagen fibrils from cartilage. In addition, perichondrial fibroblasts are characteristically located at the center of collagen fibers. In the cerebral cartilage, the perichondrium is absent or discontinuous in relation to complex interconnections between cartilage and connective fibres, muscle fibres, blood vessels and nerve. Distinctive cartilage-lining cells, rich in electron dense cytoplasmatic granules, are stratified either along the cartilage surface or along vessels and muscle fibres that penetrate within the cartilage. The perichondrium of cephalopod cartilage, whose structure varies according to the location and function of its skeletal segments, mimics that of vertebrate perichondrium, exemplifying the high level of tissue differentiation attained by cephalopods.     

Comparison of carbohydrate-containing substances from non-calcified and calcified portions of bovine costal cartilage.

Carbohydrate-containing substances were extracted from non-calcified (NCC) and calcified (CC) portions of bovine costal cartilage with 0.5 M LaCl3 by the method of Mason and his co-workers, followed by dilution of the extract with 9 volumes of water. The precipitate formed on dilution yielded Fr. P, while Fr. S was obtained from the supernatant. Fr. P was separated into two subfractions by gel filtration on Sepharose 2B. The experimental results showed that Fr. P contained proteoglycans with different molecular sizes and compositions, while Fr. S contained proteoglycan, hyaluronic acid, glycoproteins, and glycogen. The present data suggest that in the proteoglycan of Fr. P, the relative content of chondroitin sulfate decreases with a concomitant increment in that of keratan sulfate on calcification. In addition, elevation of the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate, together with a small increment of non-sulfated disaccharide units in the chondroitin sulfate chains appear to occur on calcification. The glycogen content in Fr. S diminished on calcification. The present observations suggest therefore that the remodeling of proteoglycan consumption of glycogen in bovine costal cartilage occur on calcification.     

Age-related changes in the tensile properties of human articular cartilage: a comparative study between the femoral head of the hip joint and the talus of the ankle joint

Specimens of articular cartilage from the superficial and mid-depth zones of the human femoral head and the talus of the ankle joint were tested in tension in planes parallel to the articular surface and parallel to the predominant orientation of the superficial collagen fibrils. The tensile fracture stress of cartilage from both the superficial and mid-depth zones of the femoral head decreased considerably with age. The superficial zone decreased from 33 MPa at 7 years to 10 MPa by the age of 90 years, while the mid-depth zone decreased from 32 MPa at 7 years to 2 MPa by the age of 85 years. In contrast the fracture stress of both levels of cartilage from the talus of the ankle did not decrease significantly with increasing age. The tensile stiffness at 10 MPa of both the superficial and mid-depth zones of the femoral head decreased with age. That of the superficial zone decreased from 150 MPa at 7 years to 80 MPa at 90 years, while the mid-depth zone decreased from 60 MPa at 7 years to 10 MPa at 60 years. The stiffness of talar cartilage from the superficial zone decreased by 20%, while that of the mid-depth zone showed a slight increase in stiffness at 10 MPa with increasing age. There was no significant decrease in the tensile stiffness at 1 MPa with age for either the femoral head or talar cartilage. Based on the results of previous studies it is possible to conclude that the decrease in tensile properties seen in the femoral head results from a deterioration in the tensile properties of the network of collagen fibrils. It is suggested that progressive fatigue failure, perhaps with associated changes in the structure of cartilage due to altered chondrocyte metabolism, causes the reduction in tensile properties with age. The results offer a potential explanation for the observation that osteoarthritis commonly occurs in the hip and knee joints at an increasing incidence as age increases, while the condition only rarely occurs in the ankle joint except as a secondary event to trauma.     

Decreased collagen stability as a response to the loss of structural integrity of thyroid cartilage

The thermal behavior, birefringence properties, and the biochemical composition of thyroid cartilage tissues have been studied. The hyaline cartilage, which was visualized as a quasi-isotropic medium, was composed of type II collagen, which did not denature at temperatures up to 100 degrees C. However, in hyaline cartilage digested by trypsin, the denaturation of collagen occured at 60 degrees C. Collagen fibers in the perichondrium were composed of type I and II collagen and formed a highly organized anisotropic structure (birefringence about 4.75 x 10(-3)) with a melting temperature of about 65 degrees C. The temperature of collagen denaturation in perichondrium in the whole system perichondrium-hyaline cartilage increased up to 75 degrees C, indicating the immobilization of perichondrium collagen by the extracellular matrix of the hyaline constituent.     

Age changes in the level of pyridinoline cross-linking and ratio of soluble and insoluble collagen in human rib collagen]

The changes in the content of mature crosslinks with pyridinoline structure and soluble/insoluble collagen ratio in the costal cartilage tissue of human beings aged from 1 month to 57 years were found to be age-dependent. The effect of the pyridinoline crosslink content on the soluble/insoluble collagen ratio in human costal cartilage tissue may constitute no less than 67% of the total influence of the sum of all factors. The pronounced nonlinearity of the studied dependencies points to a possible involvement of a factor(s) other than the pyridinoline crosslink content.     

Characterization of growth factors in human cartilage

Growth factor activity has been identified in the chondrocytes and extracellular matrix (ECM) fractions of human costal cartilage. There was about five times more growth factor activity in the ECM than was found to be associated with the chondrocytes. The growth factor activity in chondrocytes was found to be associated with chromatin. Both the chromatin-associated growth factor (CAGF) activity and extracellular matrix growth factor (EMGF) activity were characterized for molecular weight, charge, and the effect of reduction by sulfhydryl reducing reagents. Biorex cation exchange chromatography showed that both CAGF and EMGF were cationic. CAGF and EMGF have molecular weights between 15,000 and 18,000 as determined by size exclusion chromatography on HPLC TSK 3000 columns equilibrated with guanidine-HCl and dithiothreitol.