The osteogenic potential of autogenous bone grafts is superior to that of allografts and xenografts because of their ability to release osteoinductive growth factors and provide a natural osteoconductive surface for cell attachment and growth. In this in vitro study, autogenous bone particles were harvested by four commonly used techniques and compared for their ability to promote an osteogenic response. Primary osteoblasts were isolated and seeded on autogenous bone grafts prepared from the mandibles of miniature pigs with a bone mill, piezo-surgery, bone scraper, and bone drill (bone slurry). The osteoblast cultures were compared for their ability to promote cell attachment, proliferation, and differentiation. After 4 and 8 hrs, significantly higher cell numbers were associated with bone mill and bone scraper samples compared with those acquired by bone slurry and piezo-surgery. Similar patterns were consistently observed up to 5 days. Furthermore, osteoblasts seeded on bone mill and scraper samples expressed significantly elevated mRNA levels of collagen, osteocalcin, and osterix at 3 and 14 days and produced more mineralized tissue as assessed by alizarin red staining. These results suggest that the larger bone graft particles produced by bone mill and bone scraper techniques have a higher osteogenic potential than bone slurry and piezo-surgery.

Objective: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system.Methods: MSCs were obtained by digesting the knee-joint synovial membranes of ostecarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4 x 10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-betal (TGF-beta 1), TGF-beta 2, TGF-beta 3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry.Results: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta 1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta 1-induced expression of these chondrogenic markers was not obviously affected.Conclusions: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process. (C) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Connective tissues, with few exceptions, contain easily distinguishable large and small proteoglycans with chondroitin sulphate or dermatan sulphate side-chains. One group consists of the large aggregating proteoglycans that have the capacity to interact specifically with hyaluronate, thereby forming very large aggregates. These proteoglycans can be divided into two families which can be separated by electrophoresis. Preliminary results indicate that one of these may be derived from the other by processing in the extracellular matrix. Although most prominent in cartilage, similar proteoglycans are present in many types of tissue, such as aorta, sclera and tendon. Another population are the large non-aggregating proteoglycans, identified in cartilage. These proteoglycans show structural features partially different from any of the others. They may represent a distinct population of molecules present in many connective tissues. Many tissues contain major populations of small, non-aggregating proteoglycans. These can be divided into two major groups, differing in the composition of their core proteins, while having similar types of side-chain constituents. One group is represented by proteoglycans from nasal cartilage and aorta, while the other is represented by proteoglycans from tendon, bone, sclera and cornea.

To characterize cartilage regeneration in partial-thickness chondral injuries, medial femoral condyles of 22 sheep were given 4 mm chondral defects not extending to subchondral bone. The sheep were sacrificed at 4, 6, 8, 12, 16, 26, and 52 weeks post-injury and injuries were examined macroscopically, radiologically and histologically. No cases of spontaneous healing were observed. Early cases showed decreased defect size, secondary to inflow of healthy cartilage from the surrounding edges. Later cases showed increased degenerative changes leading to re-expansion of the defect. MR showed subchondral edema, but there was no histological,evidence of chondral regeneration arising from subchondral bone. In humans, partial-thickness chondral injuries are more commonly sustained than full-thickness ones. Although previous studies have reported spontaneous healing of small full-thickness defects in animals, this appears to be secondary to mesenchymal cells released from subchondral bone. Healing of partial-thickness injuries does not display this phenomenon.

Die primäre Arthrose, deren Pathogenese nur unvollständig geklärt ist, stellt die häufigste Ursache für Schäden des Gelenkknorpels dar. Seltener treten Gelenkknorpelläsionen durch traumatisch bedingte Verletzungen des Knorpels auf. Ebenfalls selten sind primäre Läsionen des Gelenkknorpels als Folge von genetischen, entwicklungsbedingten oder metabolischen Ursachen, wie z.B. bei der Osteochondrosis dissecans. Alle diese Störungen resultieren sehr häufig in sekundären Arthrosen. Adulter artikulärer Knorpel hat nur eine sehr beschränkte Möglichkeit, auf chronische Verletzungen und akute traumatische Läsionen zu reagieren. Das Knorpelgewebe ist unfähig, eine dauerhafte, biomechanisch resistente Regeneration wie z.B. die Haut in Form einer Narbe zu erzielen [1]. Die Pathogenese sowohl der primären wie sekundären Arthrose wird durch Risikofaktoren unterschiedlich stark beeinflusst. Die Risikofaktoren können aufgrund ihres pathogenetischen Mechanismus in zwei Gruppen aufgeteilt werden (Tab. 1). Die initialen pathologischen Veränderungen des Gelenkes bestehen in einer hypermetabolen Phase der kompensatorischen Bildung von Matrixbestandteilen (hauptsächlich Proteoglykane und Kollagen Typ II), die verloren gegangen sind und zu einer Knorpelerweichung geführt haben. Diese Veränderung ist einfach zu verstehen, wenn man sich einen Ballon vorstellt, der Luft verloren hat. Das Problem der kompensatorischen Überproduktion von Baustoffen im Sinne einer versuchten Reparatur besteht darin, dass dies überstürzt und ungeordnet passiert. Anstelle des differenzierten hyalinen Gelenkknorpels entsteht nur unreifer Faserknorpel, der weitaus weniger resistent gegenüber den biomechanischen Stressoren wie Druck-, Zugund Scherkräfte ist. Als zweites folgt dann nach unterschiedlich langer Zeit die Dekompensation der Reparationsmechanismen mit schnellem Nettoverlust der Knorpelbestandteile und Entstehen von typischerweise regionalen Knorpelfissuren und Rhagaden bis hin zum vollständigen Verlust des Knorpels (Abb. 1). Neben dem Knorpel reagiert aber auch der subchondrale Knochen, der durch vermehrte Druckbelastung sklerosiert und die synoviale Schleimhaut, die zeitweise entzündet ist und wahrscheinlich dem klinischen Bild der aktivierten Arthrose entspricht. Auch die allenfalls vorhandenen Menisken, Bursen und ligamentären Strukturen sowie die Muskulatur und die Subkutis reagieren sekundär auf die veränderten Verhältnisse, insbesondere auf die initial auftretende Inkongruenz der Gelenkflächen Von der Pathogenese der Arthrose zu therapeutischen Empfehlungen und Knorpelersatz

We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL-1beta-induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized. Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL-1beta-induced chondrocyte products (nitric oxide and prostaglandin E(2)) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A-D proteins were detected by immunoblotting. The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL-1beta-induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL-1beta-induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E(2) (PGE(2)) enabled PDE4 inhibitors to reduce IL-1beta-induced NO in this experimental setting. Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL-1beta and cyclic AMP-mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein. Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.

L’arthrose primitive, dont la pathogenèse n’est que partiellement élucidée, est la cause la plus fréquente de lésions du cartilage articulaire. Les lésions résultent plus rarement de traumatismes. Et les lésions primitives du cartilage articulaire sont rarement le fait d’étiologies génétiques, développementales ou métaboliques, comme c’est le cas dans l’ostéochondrite disséquante p.ex. Tous ces problèmes sont très souvent à l’origine d’arthroses secondaires. Le cartilage articulaire adulte n’a qu’une possibilité très limitée de réagir à des traumatismes aigus et chroniques. Le tissu cartilagineux est incapable de régénération biomécanique durable, comme l’est p.ex. la peau sous forme de cicatrice [1]. La pathogenèse de l’arthrose primitive et secondaire est diversement influencée par plusieurs facteurs de risque. Ces facteurs se répartissent en deux groupes selon le mécanisme pathogénétique (tabl. 1). Les lésions pathologiques initiales de l’articulation consistent en une phase hypermétabolique, avec production compensatoire d’éléments matriciels (surtout protéoglycanes et collagène de type II) détruits et ayant provoqué un ramollissement du cartilage. Cette pathologie est facile à comprendre en s’imaginant un ballon perdant de l’air. Le problème de l’hyperproduction compensatoire de substance dans le sens d’une tentative de réparation est la production excessive et désordonnée. Au lieu de cartilage articulaire hyalin différencié, il s’agit de cartilage fibreux immature, nettement moins résistant aux contraintes biomécaniques telles que pression, traction et cisaillement. Puis vient après un délai variable la décompensation des mécanismes réparateurs, avec perte nette rapide des constituants cartilagineux et apparition de fissures et rhagades cartilagineuses régionales typiques, pouvant aller jusqu’à la disparition complète du cartilage (fig. 1). En plus du cartilage, l’os sous-chondral réagit lui aussi à la pression accentuée par une sclérose, et la muqueuse synoviale s’enflamme de temps en temps, ce qui correspond probablement au tableau clinique de l’arthrose dite activée. Les ménisques, s’ils sont encore présents, les bourses, les structures ligamentaires, la musculature et le tissu sous-cutané réagissent secondairement aux changements de contrainte, surtout à l’incongruence initiale des structures articulaires, ou à l’instabilité de l’articulation au stade ultime. A la différence de l’arthrite rhumatoïde, inflammatoire au départ, les lésions inflammatoires ou dégénératives de ces tissus sont d’importants phénomènes d’accompagnement secondaires. De la pathogenèse de l’arthrose aux recommandations thérapeutiques et à la substitution de cartilage

Arthritic diseases cause enormous burdens in terms of pain, crippling, and disability. Osteoarthritis (OA), the most common form of arthritis, is characterized by a slow progressive degeneration of articular cartilage. The exact etiology of OA is not known, but the degradation of cartilage matrix components is generally agreed to be due to an increased synthesis and activation of extracellular proteinases, mainly matrix metalloproteinases. Insufficient synthesis of new matrix macromolecules is also thought to be involved, possibly as a consequence of deficient stimulation by growth factors. Although OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of nitric oxide and prostaglandin E2, two factors that have been shown to induce a number of the cellular changes associated with OA. The generation of these key signal molecules depends on inducible enzymes and can be suppressed by pharmacological inhibitors.

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcoreTM2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K D ) of ∼30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the α2(VI) chain probably plays a role in the interaction.

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.

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.

Local repair of acute or chronic cartilage lesions has not been successful so far. An attempt has been made to use synthetic materials to improve the quality of the repair tissue, but no method has achieved reliable regrowth of normal hyaline cartilage with adequate biomechanical properties and bonding to surrounding tissue. After publication of the first short-term results of chondrocyte transplantation in patients with localized cartilage lesions of the knee joints by a Swedish group in 1994 [1], the situation seems to have changed. Even though the advantages of this method of chondrocyte transplantation is a matter of controversy, the interest in the so-called "Carticel" approach has grown steadily. Indeed, the technique was recently approved by the FDA, on condition of a randomized, "placebo"-controlled trial. In view of this rapid development, we feel that independent experimental studies are urgently needed. In this article we present our own results in synthesizing de novo cartilage from cultured and phenotypically stable chondrocytes in a truly three-dimensional cartilage-like polyanionic matrix. With the experience gained in animals, we expect to set the stage for future experimental therapy in young human patients with early cartilage lesions.

Cartilage fibrils contain collagen II as well as smaller amounts of collagens IX and XI, The three collagens are thought to co-assemble into cartilage-specific arrays, The precise role of collagen IX in cartilage has been addressed previously by generating mice harboring an inactivated Col9a1 gene encoding the alpha 1(IX) chain, i.e. one of the three constituent chains of collagen IX (Fassler, R., Schnegelsberg, P. N. J., Dausman, J., Shinya, T,, Muragaki, Y,, McCarthy, M, T,, Olsen, B, R,, and Jaenisch, R, (1994) Proc. Natl, Acad. Sci, U, S, A. 91, 5070-5074), The animals did not produce alpha 1(M) mRNA or polypeptides and were born with no conspicuous skeletal abnormality but post-natally developed early onset osteoarthritis, Here we show that the deficiency in alpha 1(M) chains leads to a functional knock-out of all polypeptides of collagen IX, whereas the Col9a2 and Col9a3 genes were normally transcribed, Therefore, synthesis of alpha 1(M) polypeptides is essential for the assembly of heterotrimeric collagen IX molecules, Surprisingly, cartilage fibrils of all shapes and banding patterns found in normal newborn, adolescent, or adult mice were formed in transgenic animals, although they lacked collagen IX. Therefore, collagen IX is not essen tial, and may be functionally redundant, in fibrillogenesis in cartilage in vivo, The protein is required, however, for long term tissue stability, presumably by mediating interactions between fibrillar and extrafibrillar macromolecules.

Decorin, a member of a family of proteins with leucine-rich repeat motifs, is a widely distributed extracellular matrix proteoglycan that is thought to be responsible for the structure, tissue organization, and surface properties of fibrils. In mammals, decorin carries one chondroitin/dermatan sulfate chain as a distinction from its homologue, biglycan, which contains two glycosaminoglycan chains. With the aim to study decorin-collagen interactions in chicken, where the fibrillar organization of cartilage collagens is best understood, we have isolated decorin-related proteoglycans from sternal cartilage of 40-day-old broiler chickens. Small chondroitin/dermatan sulfate proteoglycans were resolved by hydrophobic interaction chromatography into two fractions, DCN I and DCN II. Both forms contained dermatan sulfate and, in addition, keratan sulfate chains. Tryptic fingerprinting revealed that the core proteins of DCN I and DCN II were identical. The protein was identified as decorin by amino-terminal sequencing. DCN II was found to contain two dermatan sulfate chains, whereas DCN I had a single dermatan sulfate chain. The dermatan sulfate attachment sites are located near the NH2 terminus of the core protein, i.e. at Ser-4 and Ser-16 in DCN II and at Ser-4 in DCN I. The keratan sulfate attachment sites are located in the central portion of the core protein, at Asn-179 and Asn-230. The presence of two dermatan sulfate chains renders the chicken proteoglycan DCN II structurally similar to mammalian biglycan. Interestingly, biglycan has not been detected in chicken. Therefore, in birds, DCN II may function as a biglycan substitute.

The small proteoglycans, decorin, fibromodulin, bi-glycan, and lumican, represent a family of structurally related but genetically distinct molecules present in many types of connective tissues. Fibromodulin and decorin interact with collagens I and II (Hedbom, E., and Heinegard, D. (1989) J. Biol. Chem. 264, 6898-6905). These interactions have been characterized further by using native radiolabeled components from fibroblast cultures and nonlabeled proteoglycans purified from guanidine hydrochloride extracts of bovine tendon. Binding of metabolically labeled macromolecules to collagen I was measured in an assay based on precipitation of collagen fibrils formed in vitro. Among a large number of secreted fibroblast products, decorin and fibromodulin represented the vast majority of the collagen binding components. These molecules showed poor binding to denatured collagen, in contrast to fibronectin, which was also present in the medium. Decorin and fibromodulin bind to different sites on collagen I fibrils, since the binding of either radiolabeled component could be competed for only by the corresponding nonlabeled proteoglycan. Similarly, these proteoglycans showed binding to separate sites on collagen II. Binding of isolated fibromodulin and decorin to collagens in solution was measured in a solid-phase inhibition assay. Each of the proteoglycans interacted with triple helical molecules, but not with denatured collagen chain constituents or fragments. For fibromodulin, the data indicated an average of one binding site per collagen I molecule (K(d) = 9.9 nM). The data on decorin indicated additional interactions, some apparently mediated by the dermatan sulfate side chain. The results suggest that the small proteoglycans bind to distinct triple helical sites, apparently differing from several other similar structures within each collagen molecule.

An M(r) = 524,000 oligomeric protein was isolated from bovine cartilage and designated COMP (Cartilage Oligomeric Matrix Protein). The protein is composed of disulfide-bonded subunits with an apparent M(r) of 100,000 each. It is markedly anionic, probably due to its high contents of aspartic acid and glutamic acid, as well as to its substitution with negatively charged carbohydrates. COMP was found in all cartilages analyzed, but could not be detected in other tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. Within a given cartilage, COMP shows a preferential localization to the territorial matrix surrounding the chondrocytes.

A non-collagenous quantitatively prominent protein was purified from guanidine hydrochloride extracts of bovine tracheal cartilage. Purification was achieved by cesium chloride density gradient centrifugation and chromatography on DEAE-cellulose at pH 7.0 followed by CM-cellulose at pH 5.0. The protein has a marked tendency to form aggregates in denaturing solutions of high ionic strength, e.g. 6 M guanidine hydrochloride. The purified protein contains a single, M(r) 36,000 polypeptide chain, with a particularly high content of leucine. It contains about 1% carbohydrate with a remarkable absence of hexosamines and sialic acid, whereas xylose, galactose, mannose, and fucose were identified in the preparation.The protein was identified in extracts of cartilage and bone and could be shown to be primarily extracellular. Tendon may contain trace amounts of the protein, whereas extracts of several other tissues showed no immunoreactivity in enzyme-linked immunosorbent assay.