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Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formedin vitrofrom collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly,in vitrofibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.

Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.

Objective real-time acoustic feedback of complex movements arises in applications ranging from assisted training in sport to medical rehabilitation. This so called interactive movement sonification enables continuous feedback. Multiple studies have shown the benefit of sonification in movement relearning. Ubiquitous feedback requires a mobile and wearable hardware platform independent from any application. Therefore, in this paper a design space exploration regarding the design objectives power dissipation and latency is performed. Especially in sports or medical rehabilitation a low-latency feedback is crucial. Furthermore, low power dissipation mobile devices enable the utilization of the sonification technique within new applications. The most computational intensive task within the data-flow is real-time interactive sound synthesis. In contrast to commercial processors, customizable processors enable execution time reduction of critical sections by integration of application specific instruction set extensions. This paper aims to show the benefits from processor customization, as well as the location of consumer electronics devices within the design space.

Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.

Fully mobile and wireless motion capturing is a mandatory requirement for undisturbed and non-reactive analysis of human movements. Inertial sensor platforms are used in applications like training session analysis in sports or rehabilitation, and allow non-restricted motion capturing. The computation of the required reliable orientation estimation based on the inertial sensor RAW data is a demanding computational task. Therefore, an analysis of the computational costs and achievable accuracy of a Kalman filter and a complementary filter algorithm is provided. Highly customized and thus low-power, wearable computation platforms require low-level, platform independent communication protocols and connectivity. State-of-the-art small sized commercial inertial sensors either lack the availability of an open, platform independent protocol, wireless connectivity or extension interfaces for additional sensors. Therefore, an extensible, wireless inertial sensor called Institute of Microelectronic Systems Inertial Measurement Unit (IM)2SU, featuring onboard inertial sensor fusion, for use in home based stroke rehabilitation is presented. Furthermore, a Quaternion based, singularity free orientation estimation accuracy error measure is proposed and applied. To evaluate orientation estimation accuracy an optical system is used as golden reference. Orientation estimation based on a Kalman filter and a complementary filter algorithm is evaluated. The proposed IMU provides high orientation estimation accuracy, is platform independent, offers wireless connection and extensibility and is low cost.

ObjectiveInterterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor β (TGFβ), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFβ in decorin-deficient (Dcn−/−) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA).MethodsUnchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor β-1 (TGFβ1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFβ1 treatment. In addition, OA was induced inDcn−/−and wild-type (WT) mice via forced exercise on a treadmill.ResultsAFM analysis revealed a strikingly higher compressive stiffness inDcn−/−than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFβ1. Increased TGFβ signalling led to enhancedChst11sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice.ConclusionsOur study demonstrates that the disruption of decorin-restricted TGFβ signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.

Objective Mutations in human cartilage oligomeric matrix protein (COMP) cause multiple epiphyseal dysplasia or pseudoachondroplasia. Electron microscopic analyses of patient biopsy tissue have shown that, in most cases, mutated COMP is retained in granular or lamellar inclusions in the endoplasmic reticulum of chondrocytes. However, some mutations that do not interfere with protein trafficking, resulting in normal secretion of the mutated protein, have been identified. These mutations are likely to cause the chondrodysplasia phenotype, via events that occur after secretion. The aim of the present study was to identify such extracellular mechanisms associated with the pathogenesis of chondrodysplasias. Methods A mutated but secreted COMP variant, p.H587R, as well as wild-type COMP were recombinantly expressed and purified from cell culture supernatants. Since recent studies have shown that COMP can facilitate collagen fibrillogenesis in vitro, the effect of the p.H587R mutation on this process was determined by analyzing the kinetics of fibrillogenesis in vitro and determining the structure of the collagen fibrils formed by immunogold electron microscopy. Results Mutated p.H587R COMP accelerated fibril formation by type I collagen in vitro to a slightly greater extent than that with wild-type COMP. However, p.H587R COMP induced aggregation and disorganization of fibril intermediates and end products. Mixtures of cartilage collagens or of type XI collagen alone produced similar results. The addition of p.H587R COMP to preformed fibrils induced aggregation and fusion of the fibrils, whereas wild-type COMP had little effect. Conclusion The mutant COMP variant p.H587R generally interferes with normal collagen organization during fibrillogenesis. This constitutes a novel pathogenetic mechanism of COMP-associated chondrodysplasias.

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MS(n)). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MS(n). MS(2) on bisulfated 4,5-Delta-HexAGalNAc revealed an additional sulfate ester group at 4,5-Delta-HexA. MS(2) data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Delta-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS(1) mode indicated direct correlation between the sulfate distribution and HexA epimerization. MS(n) performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)], trisulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)] with IdoA-derived 4,5-Delta-HexA at the nonreducing end, tetrasulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] and monosulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] with GlcA-derived 4,5-Delta-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.

For a large part, skeletal development, growth, and repair occur by endochondral ossification which comprises an orderly sequence of consecutive steps of proliferation and late differentiation of chondrocytes. After vascular invasion into hypertrophic cartilage, the tissue is remodelled into bone. At all stages, the process is under tight environmental control exerted by a combination of regulators, including nutritional supply and signalling through growth factors, hormones, and cell-matrix-interactions. Therefore, genetic elimination of collagen IX, a stabilizing component of the periphery of thin cartilage fibrils, is expected to compromise extracellular matrix properties and, hence, the chondrocyte environment required for normal cartilage development and homeostasis. Here, we have shown that growth plate cartilage morphology is markedly disturbed in mice lacking collagen IX. Abnormalities were most prominent in late proliferative, pre-hypertrophic, and hypertrophic zones whereas resting and early proliferative zones were less affected. In central epiphyseal regions of long bones, newborn animals show grossly abnormal areas with strongly reduced cell numbers, irregular distribution of glycosaminoglycans in the extracellular matrix, and a profoundly disturbed columnar arrangement of chondrocytes with an irregular β 1 integrin immunostaining. As a result, all long bones are shorter and broader in newborn Col9a1−/− mice. Remarkably, these abnormalities are attenuated in adult mice, but the number of cells per area still is too low due to reduced cell proliferation.

Endochondral ossification comprises a cascade of cell differentiation culminating in chondrocyte hypertrophy and is negatively controlled by soluble environmental mediators at several checkpoints. Proteinases modulate this control by processing protein signals and/or their receptors. Here, we show that insulin-like growth factor I can trigger hypertrophic development by stimulating production and/or activation of proteinases in some populations of chick embryo chondrocytes. Cell surface targets of the enzymes include 1,25-dihydroxyvitamin D3 membrane-associated rapid response steroid receptor (1,25 D3 MARRS receptor), also known as ERp57/GRp58/ERp60. This protein is anchored to the outer surface of plasma membranes and inhibits late chondrocyte differentiation after binding of 1,25-dihydroxyvitamin D3. Upon treatment with insulin-like growth factor I, 1,25 D3 MARRS receptor is cleaved into two fragments of approximately 30 and 22 kDa. This process is abrogated along with hypertrophic development by E-64 or cystatin C, inhibitors of cysteine proteinases. Cell differentiation is enhanced by treatment with antibodies to 1,25 D3 MARRS receptor that either block binding of the inhibitory ligand 1,25-dihydroxyvitamin D3 or inactivate 1,25 D3 MARRS receptor left intact after treatment with proteinase inhibitors. Therefore, proteolytic shedding of 1,25 D3 MARRS receptor constitutes a molecular mechanism eliminating the 1,25-dihydroxyvitamin D3-induced barrier against late cartilage differentiation and is a potentially important step during endochondral ossification or cartilage degeneration in osteoarthritis.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.

Decorin is a multifunctional small leucine-rich proteoglycan involved in the regulation of collagen fibrillogenesis. In patients with a variant of Ehlers-Danlos syndrome, about half of the secreted decorin lacks the single glycosaminoglycan side chain. Notably, these patients have a skin-fragility phenotype that resembles that of decorin null mice. In this study, we investigated the role of glycanated and unglycanated decorin on collagen fibrillogenesis. Glycosaminoglycan-free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN-S4A), showed reduced inhibition of fibrillogenesis compared with the decorin proteoglycan. Interestingly, using a 3D matrix generated by decorin-null fibroblasts, an increase in fibril diameter was found after the addition of decorin, and even greater effects were observed with DCN-S4A. To avoid potential side effects of artificial tags, adenoviruses containing decorin and DCN-S4A were used to transduce decorin-null fibroblasts prior to matrix formation. Both molecules were efficiently incorporated into the matrix, with no changes in collagen composition and network formation, or altered expression of the related proteoglycan biglycan. Both decorin and DCN-S4A mutants increased the collagen fibril diameter, with the latter showing the most prominent effects. These data show that at early stages of fibrillogenesis, the glycosaminoglycan chain of decorin has a reducing effect on collagen fibril diameter.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1 −/− calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.

Various applications demand mobile, accurate and reliable motion capturing. Wireless inertial measurement units provide non-reactive and undisturbed real-time tracking of human movements. The use of customized sensor fusion methods is crucial for achieving the highest orientation accuracy. Due to the high dependency of movement data on orientation estimation accuracy based on inertial measurement data a comparison of the various algorithms presented in literature is hampered. Furthermore, the execution time and energy efficiency of the algorithms on programmable, heterogeneous and ASIC platforms is an important design space parameter. Therefore, this chapter compares the orientation estimation accuracy of inertial sensor fusion algorithms based on a single data set using a highly accurate optical motion capturing system as reference. The data set comprises of RAW data of from a commercial and from a custom developed wireless measurement unit. The provided energy consumption, execution time and achievable accuracy analysis of eleven different sensor fusion algorithms enables the selection of the optimal orientation estimation algorithm with regard to application demands and processing architecture. Furthermore, a customized processor core with the presented Kalman filter based orientation estimation hardware accelerator demonstrates techniques for increasing energy efficiency in respect of achievable throughput rate.

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with K(D) values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of "permanent" cartilage structures during development and in mature cartilages.

Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J.G. (1980) Biochemistry, 19, 4065-4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one-dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33 degrees C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple-helical domain of type IV collagen (326-328 nm). A specific cleavage site was located at a distance of 215 nm from the 7-S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7-S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization[alpha 1(IV)]2 alpha2(IV); (c) the order of the pepsin-resistant fragments within a type IV molecule is 7S-F3-(F1)2F2.

Objective To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1β (IL-1β). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-κB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor β (TGFβ) and TGFβ receptors, and production of nitric oxide (NO) and prostaglandin E2 (PGE2). Methods Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O2 (hypoxia) or 21% O2 (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1β. For reoxygenation experiments, cells were treated in the same way in 5% O2, except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction. Results In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1β-induced DNA binding of NF-κB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1β treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1β in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1β in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1β in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1β only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1β in reoxygenated cultures. Stimulation of TGFβ1 expression by IL-1β was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1β on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1β–induced PGE2 production. Conclusion Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1β. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1β on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1β–induced NO production is associated with a significant decrease in PGE2 synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The α3′ chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type α3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, β3′and γ2′, produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the α3′ chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble α3β1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with α3β1 integrin. The N-glycosylation sites in wild-type α3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor α3β1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the α3′LG1–3 chain showed an increased binding affinity for α3β1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with α3β1 integrin.

Suprastructures of the extracellular matrix, such as banded collagen fibrils, microfibrils, filaments, or networks, are composites comprising more than one type of macromolecule. The suprastructural diversity reflects tissue-specific requirements and is achieved by formation of macromolecular composites that often share their main molecular components alloyed with minor components. Both, the mechanisms of formation and the final macromolecular organizations depend on the identity of the components and their quantitative contribution. Collagen I is the predominant matrix constituent in many tissues and aggregates with other collagens and/or fibril-associated macromolecules into distinct types of banded fibrils. Here, we studied co-assembly of collagens I and XI, which co-exist in fibrils of several normal and pathologically altered tissues, including fibrous cartilage and bone, or osteoarthritic joints. Immediately upon initiation of fibrillogenesis, the proteins co-assembled into alloy-like stubby aggregates that represented efficient nucleation sites for the formation of composite fibrils. Propagation of fibrillogenesis occurred by exclusive accretion of collagen I to yield composite fibrils of highly variable diameters. Therefore, collagen I/XI fibrils strikingly differed from the homogeneous fibrillar alloy generated by collagens II and XI, although the constituent polypeptides of collagens I and II are highly homologous. Thus, the mode of aggregation of collagens into vastly diverse fibrillar composites is finely tuned by subtle differences in molecular structures through formation of macromolecular alloys.

The structural and functional diversity of extracellular matrices is determined, not only by individual macromolecules, but even more decisively, by the alloyed aggregates they form. Although quantitatively major matrix molecules can occur ubiquitously, their organization varies from one tissue to another due to their amalgamation with specific sets of minor components. Here, we show that the fibril-associated collagen with interrupted triple helices collagen XVI is unique in that, depending on the tissue context, it can be incorporated into distinct suprastructural aggregates. In papillary dermis, the protein unexpectedly does not occur in banded collagen fibrils, but rather, is a component of specialized fibrillin-1-containing microfibrils. In territorial cartilage matrix, however, collagen XVI is not a component of aggregates containing fibrillin-1. Instead, the protein resides in a discrete population of thin, weakly banded collagen fibrils also containing collagens II and XI. Collagen IX also occurs in this population of fibrils, but at longitudinal locations discrete from those of collagen XVI. This suprastructural versatility of a collagen is without precedent and highlights pivotal differences in the tissue-specific organization of matrix aggregate structures.

SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.

The pleiotropic effects of Calloselasma rhodostoma venom is caused by various toxins, among them kistrin and ancrod, which block platelet activation triggered by RGD-dependent integrins and the blood clotting cascade, respectively. Here, we demonstrate that rhodocetin, another component of this venom, acts as alpha2beta1 integrin inhibiting disintegrin and antagonizes important cellular responses to type I collagen. Cell adhesion, migration, and collagen lattice contraction in vitro were specifically inhibited by rhodocetin, whereas expression of collagen-degrading matrix metalloproteases was differently modulated. Moreover, cell invasion of HT1080 fibrosarcoma cells into a type I collagen matrix, but not into a fibrin gel or a basement membrane-extracted matrigel was efficiently blocked by rhodocetin. Unlike its natural ligand collagen, rhodocetin failed to cluster alpha2beta1 integrin, despite similar binding affinities. Hence, in the absence of focal adhesions cells do not attach firmly to rhodocetin and do not respond with any of alpha2beta1-triggered cell reactions, except for MMP-1 production. Therefore, this disintegrin may be a valuable tool to specifically target stromal tumor invasion and to manipulate other alpha2beta1 integrin-mediated functions, such as excessive scar contraction and fibrosis. Rhodocetin might be therapeutically useful because of its lack of interference with RGD-dependent integrins, low molecular mass, high solubility, and biochemical stability.

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.

Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.

Background and Purpose— The chronological development and natural history of cerebral aneurysms (CAs) remain incompletely understood. We used 14 C birth dating of a main constituent of CAs, that is, collagen type I, as an indicator for biosynthesis and turnover of collagen in CAs in relation to human cerebral arteries to investigate this further. Methods— Forty-six ruptured and unruptured CA samples from 43 patients and 10 cadaveric human cerebral arteries were obtained. The age of collagen, extracted and purified from excised CAs, was estimated using 14 C birth dating and correlated with CA and patient characteristics, including the history of risk factors associated with atherosclerosis and potentially aneurysm growth and rupture. Results— Nearly all CA samples contained collagen type I, which was P =0.012). CAs and cerebral arteries did not share a dominant structural protein, such as collagen type I, which would allow comparison of their collagen turnover. Conclusions— The abundant amount of relatively young collagen type I in CAs suggests that there is an ongoing collagen remodeling in aneurysms, which is significantly more rapid in patients with risk factors. These findings challenge the concept that CAs are present for decades and that they undergo only sporadic episodes of structural change.

Introduction: The time required for intracranial aneurysms (IAs) to develop until their diagnosis is unknown. We used a novel method of vascular radiocarbon birth dating of collagen extracted from aneurysms of patients undergoing surgical repair of ruptured and unruptured IAs to estimate their ages. Methods: Between 03/2009 and 04/2013, 46 aneurysm samples from 43 patients were obtained. Collagen was extracted and purified after pepsin digestion of IA tissue. and accelerator mass spectrometry was used to measure 14 C levels in the collagen samples. The age of the aneurysm collagen was estimated and then correlated with patient age, incidence of modifiable risk factors for aneurysm progression, IA rupture status and morphological measurements. Findings: Nearly all collagen in cerebral aneurysms was five or less years old, irrespective of patient age, aneurysm size, morphology, or rupture status. However, IAs from patients with a history of risk factors for aneurysm formation (smoking and hypertension) contained significantly younger collagen, as compared to patients with no risk factors (mean 1·6 ± 1·2 years vs. 3·9 ± 3·3 years, respectively, p= 0·012). Interpretation: We show that chronological development of IAs can be consistently estimated, that they are relatively young when detected, and/or that there is constant collagen remodelling within them. In either case, the results suggest there are no cerebral aneurysms that are stable and old. Furthermore, aneurysm age or collagen turnover is significantly more rapid in patients with modifiable risk factors for aneurysm progression and rupture. These results have important implications for the understanding of the biology of cerebral aneurysms as well as for recommendations for follow-up of patients with unruptured cerebral aneurysms.

Knochen und Knorpel sind die beiden Gewebskomponenten des Skeletts. Die Funktion dieser Gewebe wird besonders stark durch die extrazellulare Matrix definiert, die im Knochen besonders auch von anorganischem Mineral gekennzeichnet ist. Dieses verleiht den Geweben die charakteristische Harte und Druckbelastbarkeit. Daneben dient die Mineralphase des Knochens als wichtiger Speicher von Calcium und Phosphat im gesamten Korper. Der Auf- und Abbau des Mineralspeichers im Knochen unterliegt einer strengen Kontrolle sowohl durch hormonal systemische als auch durch lokale Gewebsfaktoren.

Mobile hardware platforms are a major requirement for interactive movement sonification. Emerging applications for the computationally demanding auditory feedback technique are stroke rehabilitation and assisted training in sports. This paper presents and evaluates a smartphone based hardware platform for interactive, low latency movement sonification based on inertial measurement units. In contrast to existing hardware platforms the proposed platform enables low latency and battery powered long-term training sessions and improved usability in stroke rehabilitation by an intuitive graphical user interface and wearability. The evaluation focuses on real-time operation performance and overall latency. Furthermore, the overall latency of the smartphone-based platform are compared to results of a heterogeneous RISC/DSP and a PC-based processing platform.

Schon ab dem vierzelligen Entwicklungsstadium besitzen Vielzeller, wie z. B. der Mensch, eine extrazellulare Matrix (EZM), d. h. eine auserhalb der Zellen angesiedelte und organisierte Substanz, die an der Homoodynamik aller biologischen Vorgange beteiligt ist. Zum Beispiel ermoglicht die EZM auf makroskopischer Ebene eine stabile Form der Organe oder einen funktionellen Bewegungsapparat. Entwicklungsbiologisch kontrolliert sie Gewebsfunktionen auf allen Ebenen und/oder entscheidet uber Vitalitat oder programmierten Zelltod. Diese Wirkungen erzielt die EZM uber rezeptorvermittelte Zell-Matrix-Wechselwirkungen im Konzert mit anderen, loslichen Signalen.

Real-time orientation estimation based on mobile, low-power hardware platforms requires customized processor cores or dedicated hardware accelerators to achieve the throughput requirements demanded by applications like monitoring training sessions in sports or medical rehabilitation. In this paper, instruction-set extensions for a basic ASIP core and a dedicated hardware accelerator for sensor fusion of inertial measurement unit RAW data are presented. The results show the minimization of the execution time due to the instructionset extensions and the dedicated hardware accelerator. The use of an optimized ASIP increases the throughput up to a factor of 1.7. Compared to the base core, the hardware accelerator achieves an increase by a factor of 2.5. Utilizing the hardware accelerator increases energy efficiency by a factor of 17.0.

Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.

Objective Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation. This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions. We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes. Design Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources. The culture media either contained or lacked FBS. Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype). Results The cells often retained their differentiated phenotype only if cultured with serum. Under serum-free conditions, cartilage characteristics were lost. The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens. Shape changes were influenced by β1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns. Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability. Conclusions Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment. Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype. However, other suprastructures, e.g. in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype. The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage. If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further.

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. The subchondral vascular system is essential for this process and late chondrocyte differentiation is subject to negative control at several checkpoints. Endothelial cells of subchondral blood vessels not only are the source of vascular invasion accompanying the transition of hypertrophic cartilage to bone but also produce factors overruling autocrine barriers against late chondrocyte differentiation. Here, we have determined that the action of proteases secreted by endothelial cells were sufficient to derepress the production of the hypertrophy-markers collagen X and alkaline phosphatase in arrested populations of chicken chondrocytes. Signalling by thyroid hormones was also necessary but endothelial factors other than proteinases were not. Negative signalling by PTH/PTHrP- or TGF-beta-receptors remained unaffected by the endothelial proteases whereas signalling by FGF-2 did not suppress, but rather activated late chondrocyte differentiation under these conditions. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.

Energy efficiency is a major design goal for mobile and wearable devices. These kind of devices most often comprise System-on-Chip processor cores and further hardware accelerators. A novel heterogeneous hardware architecture introduced by various FPGA manufacturers consists of a programmable FPGA like structure and a common RISC processor core. For system designers this commercial architecture enables enhanced flexibility in partitioning of algorithmic tasks. The hardware demonstrator for auditory feedback of movements (sonification) captured by multiple inertial measurement units proposed in this paper bases on a heterogeneous Xilinx Zynq System-on-Chip processing core and a custom hardware accelerator. Energy efficiency is enhanced by utilizing the hardware accelerator for orientation estimation based on a Kalman filter algorithm. The evaluation furthermore explores the usability of High Level Synthesis tools based on a fixed-point software implementation. Moreover, the area and power consumption of hardware accelerator ASIC implementations based on a 40 nm TSMC library are evaluated.

Pluripotent cells from the periosteal layer adjacent to cortical bone attain an osteoblast-like phenotype in culture when reaching confluence in monolayer. It is unknown whether such newly differentiated osteoblast-like cells preserve the chondrogenic potential characteristics for stem cells derived from the periosteum. Primary osteoprogenitor cells derived from bovine metacarpal periosteum were differentiated into alkaline phosphatase-positive osteoblast-like cells by an established monolayer culture protocol. After transfer into suspension culture in agarose gels, the cells differentiated into chondrocytes demonstrated by the production of collagen II, but not of collagen I, as well as alkaline phosphatase activity was abated. Contrarily, with continuation of monolayer culture, the cells maintained their osteoblast-like phenotype and secreted large amounts of collagen I and a minor quantity of collagen III and V. The alkaline phosphatase activity steadily increased during the entire culture period of 2 weeks. Thus, our culture techniques can serve as useful tools to study mechanisms of differentiation by modulating the phenotypic potential of osteogenic cells. The results presented here support the notion that the extracellular environment strongly influences the cell type and its metabolism.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo–epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo–epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-β2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-β2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo . Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-β in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.