Computational model of a synovial joint morphogenesis.

Joints enable the relative movement between the connected bones. The shape of the joint is important for the joint movements since they facilitate and smooth the relative displacement of the joint's parts. The process of how the joints obtain their final shape is yet not well understood. Former models have been developed in order to understand the joint morphogenesis leaning only on the mechanical environment; however, the obtained final anatomical shape does not match entirely with a realistic geometry. In this study, a computational model was developed with the aim of explaining how the morphogenesis of joints and shaping of ossification structures are achieved. For this model, both the mechanical and biochemical environments were considered. It was assumed that cartilage growth was controlled by cyclic hydrostatic stress and inhibited by octahedral shear stress. In addition, molecules such as PTHrP and Wnt promote chondrocyte proliferation and therefore cartilage growth. Moreover, the appearance of the primary and secondary ossification centers was also modeled, for which the osteogenic index and PTHrP-Ihh concentrations were taken into account. The obtained results from this model show a coherent final shape of an interphalangeal joint, which suggest that the mechanical and biochemical environments are crucial for the joint morphogenesis process.

Effect of estradiol on fibroblasts from postmenopausal idiopathic carpal tunnel syndrome patients.

Fibrosis of the subsynovial connective tissue (SSCT) is a characteristic finding in patients with idiopathic carpal tunnel syndrome (CTS). Idiopathic CTS frequently occurs in postmenopausal women; therefore, female steroid hormones, especially estrogens, may be involved in its development. In this study, we evaluated the effect of the estradiol on the expression of genes and proteins related to fibrosis of SSCT fibroblasts from patients with idiopathic CTS. This study included 10 postmenopausal women (mean age 76 years). Fibroblasts derived from SSCT were treated with estradiol (10-4 -10-12 M), and the expression levels of TGF-ß-responsive genes were evaluated. The relationships between the expression of untreated estrogen receptor α (ERα) and ERß and changes in gene expression due to estradiol treatment were examined by quantitative real-time polymerase chain reaction. The effects of 10-4 M estradiol on collagen type I (Col1) and collagen type III (Col3) protein expression levels were also evaluated by fluorescent staining. The relationships between ERα/ß and Col1/3 expression were evaluated by immunohistochemical staining. The reduction in Col1A1 mRNA expression due to estradiol treatment was positively correlated with ERα expression (r = 0.903, p < 0.01). At the protein level, expression of Col1 and Col3 were down-regulated. These results indicated that ERα-mediated signaling may be involved in the regulation of Col1A1, and its regulatory effect may be dependent on the ERα expression level. The accurate evaluation of ERα expression level in the SSCT of individual patients with idiopathic CTS might guide the effective use of new estrogen replacement therapy.

Comparative study of membranes induced by PMMA or silicone in rats, and influence of external radiotherapy.

The induced membrane technique has been used for long bone defect reconstruction after traumatism. One of the major drawbacks of this method is the difficult removal of the polymethyl methacrylate spacer after membrane formation. We therefore replaced the stiff PMMA spacer with a semi-flexible medical grade silicone spacer. This study aimed to compare subcutaneously formed membranes, induced by PMMA and silicone, in the irradiated or not irradiated areas within 28 rats that received the spacers. Histological analysis was performed to evaluate the composition of the membrane and to quantify the amount of vessels. Histomorphometric measurements were used to evaluate membranes' thickness, while fibrosis and inflammation were scored. The expression of VEGF and BMP-2 in lysates of the crushed membranes was determined by Western blotting. ALP expression was analyzed in HBMSC cultures in contact with the same lysates. Non-irradiated membranes induced by the two spacer types were non-inflammatory, fibrous and organized in layers. Irradiation did not change the macroscopic properties of membranes that were induced by silicone, while PMMA induced membranes were sensitive to the radiotherapy, resulting in thicker, strongly inflammatory membranes. Irradiated membranes showed an overall reduced osteogenic potential. Medical grade silicone is safe for the use in radiotherapy and might therefore be of great advantage for patients in need of cancer treatment.

Enamel matrix derivative inhibits proteoglycan production and articular cartilage repair, delays the restoration of the subchondral bone and induces changes of the synovial membrane in a lapine osteochondral defect model in vivo.

Improving the structural qualities of the new tissue that fills osteochondral defects is critical to enhance articular cartilage repair. Enamel matrix derivative (EMD) modulates chondrocyte proliferation and differentiation. In the present study, we assessed the effect of EMD on early chondrogenesis and bone repair in an osteochondral defect model in vivo. Standardized osteochondral defects were established in the trochlear groove of rabbits. EMD or the carrier substance without EMD activity was applied to the blood clot that was forming within the defect. After 3 weeks in vivo, the quality of articular cartilage repair was evaluated using a semiquantitative scoring system and biochemical assays for proteoglycan and DNA contents. The extent of formation of the subchondral bone within the original osteochondral defect was measured. Application of EMD resulted in an inferior histological articular cartilage repair. The total proteoglycan content of the repair tissue as well as the proteoglycan production standardized to the cell proliferative activities within the defects were reduced following treatment with EMD. Restoration of the subchondral bone within the osteochondral defect was delayed when EMD was applied. Significant changes of the synovial membrane were present, reflected in an increased villus thickening and changes in villus architecture. These data suggest that EMD inhibits the early repair of the osteochondral unit in vivo.

Etiology of osteoarthritis: genetics and synovial joint development.

Osteoarthritis (OA) has a considerable hereditary component and is considered to be a polygenic disease. Data derived from genetic analyses and genome-wide screening of individuals with this disease have revealed a surprising trend: genes associated with OA tend to be related to the process of synovial joint development. Mutations in these genes might directly cause OA. In addition, they could also determine the age at which OA becomes apparent, the joint sites involved, the severity of the disease and how rapidly it progresses. In this Review, I propose that genetic mutations associated with OA can be placed on a continuum. Early-onset OA is caused by mutations in matrix molecules often associated with chondrodysplasias, whereas less destructive structural abnormalities or mutations confer increased susceptibility to injury or malalignment that can result in middle-age onset. Finally, mutations in molecules that regulate subtle aspects of joint development and structure lead to late-onset OA. In this Review, I discuss the genetics of OA in general, but focus on the potential effect of genetic mutations associated with OA on joint structure, the role of joint structure in the development of OA--using hip abnormalities as a model--and how understanding the etiology of the disease could influence treatment.

Canonical Wnt signaling activity during synovial joint development.

Wnt signaling plays important roles in skeletal development. However, the activation and function of canonical Wnt signaling in joint development remains unclear. We analyzed the lineage identity and developmental changes of the Wnt-responsive cells during synovial joint formation as well as adulthood in the Wnt signaling reporter TOPgal transgenic mice. At embryonic day (E) 12.5, we found that the TOPgal was inactivated in the presumptive joint forming interzone, but it was intensively activated in the cartilage anlage of developing long bones and digits. At E14.5, the TOPgal activity was found in a subgroup of the articular chondrocyte lineage cells, which were co-immunolabeled with Doublecortin intensively and with Vinculin weakly. At E18.5, the TOPgal/Doublecortin co-immunolabeled cells were found in the superficial layer of the developing articular cartilage. During postnatal development, the TOPgal(+) articular chondrocytes were abundant at P7 and decreased from P10. A small number of TOPgal(+) articular chondrocytes were also found in adult joints. Our study suggests an age- and lineage-specific role of canonical Wnt signaling in joint development and maintenance.

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Immunocytochemical localization of caveolin-3 in the synoviocytes of the rat temporomandibular joint during development.

Caveolins -- caveolin-1, -2, -3 (Cav1, 2, 3) -- are major components of caveolae, which have diverse functions. Our recent study on the temporomandibular joint (TMJ) revealed expressions of Cav1 and muscle-specific Cav3 in some synovial fibroblast-like type B cells with well-developed caveolae. However, the involvement of Cav3 expression in the differentiation and maturation of type B cells remains unclear. The present study therefore examined the chronological alterations in the localization of Cav3 in the synovial lining cells of the rat TMJ during postnatal development by immunocytochemical techniques. Observations showed immature type B cells possessed a few caveolae with Cav1 but lacked Cav3 protein at postnatal day 5 (P5). At P14, Cav3-immunopositive type B cells first appeared in the synovial lining layer. They increased in number and immunointensity from P14 to P21 as occlusion became active. In immunoelectron microscopy and double immunolabeling with heat shock protein 25 (Hsp25) and Cav3, coexpressed type B cells developed rough endoplasmic reticulum and numerous caveolae, while the Cav3-immunonegative type B cell with Hsp25 immunoreaction possessed few of these. Results suggest that Cav3 expression, which is closely related to added functional stimuli, reflects the differentiation of the type B synoviocytes.

Synovial joint formation during mouse limb skeletogenesis: roles of Indian hedgehog signaling.

Indian hedgehog (Ihh) has been previously found to regulate synovial joint formation. To analyze mechanisms, we carried out morphological, molecular, and cell fate map analyses of interzone and joint development in wild-type and Ihh(-/-) mouse embryo long bones. We found that Ihh(-/-) cartilaginous digit anlagen remained fused and lacked interzones or mature joints, whereas wrist skeletal elements were not fused but their joints were morphologically abnormal. E14.5 and E17.5 wild-type digit and ankle prospective joints expressed hedgehog target genes including Gli1 and Gli2 and interzone-associated genes including Gdf5, Erg, and tenascin-C, but expression of all these genes was barely detectable in mutant joints. For cell fate map analysis of joint progenitor cells, we mated Gdf5-Cre(+/-)/Rosa R26R(+/-) double transgenic mice with heterozygous Ihh(+/-) mice and monitored reporter beta-galactosidase activity and gene expression in triple-transgenic progeny. In control Gdf5-Cre(+/-)/R26R(+/-)/Ihh(+/-) limbs, reporter-positive cells were present in developing interzones, articulating layers, and synovial lining tissue and absent from underlying growth plates. In mutant Gdf5-Cre(+/-)/R26R(+/-)/Ihh(-/-) specimens, reporter-positive cells were present also. However, the cells were mostly located around the prospective and uninterrupted digit joint sites and, interestingly, still expressed Erg, tenascin-C, and Gdf5. Topographical analysis revealed that interzone and associated cells were not uniformly distributed, but were much more numerous ventrally. A similar topographical bias was seen for cavitation process and capsule primordia formation. In sum, Ihh is a critical and possibly direct regulator of joint development. In its absence, distribution and function of Gdf5-expressing interzone-associated cells are abnormal, but their patterning at prospective joint sites still occurs. The joint-forming functions of the cells appear to normally involve a previously unsuspected asymmetric distribution along the ventral-to-dorsal plane of the developing joint.

Lineage tracing using matrilin-1 gene expression reveals that articular chondrocytes exist as the joint interzone forms.

We have developed a mouse in which the Cre recombinase gene has been targeted to exon 1 of the matrilin-1 gene (Matn1) to investigate the origins of articular chondrocytes and the development of the knee joint. Analysis of joints from offspring of Matn1-Cre/R26R crosses demonstrated that articular chondrocytes are derived from cells that have never expressed matrilin-1 whereas the remainder of the chondrocytes in the cartilage anlagen expresses matrilin-1. A band of chondrocytes adjacent to the developing interzone in the E13.5 day knee joint became apparent because these chondrocytes did not turn on expression of matrilin-1 in contrast to the other chondrocytes of the anlagen. The chondrocytes of the presumptive articular surface therefore appear to arise directly from a subpopulation of early chondrocytes that do not activate matrilin-1 expression rather than by redifferentiation from the flattened cells of the interzone. In addition, lineage tracing using both Matn1-Cre/R26R and Col2a1-Cre/R26R lines indicated that non-cartilaginous structures in the knee such as cruciate ligament, synovium and some blood vessels are formed by cells derived from the early chondrocytes of the anlagen.

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri-joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri-joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF-5, Wnt-14, and CD-44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C-1-1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C-1-1 (ERGp55Delta81) and conditionally expressed it in transgenic mice under cartilage-specific Col2 gene promotor-enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular-like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild-type littermate. Together, our studies reveal that peri-joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

N-Glycosyl-thiophene-2-carboxamides: synthesis, structure and effects on the growth of diverse cell types.

A range of N-glycosyl-thiophene-2-carboxamides, including a 6H-thieno[2,3-c]pyridin-7-one and a bivalent compound, have been synthesised and assayed for their effects on DNA synthesis in bovine aortic endothelial cells or on the growth of synoviocytes. Per-O-acetylated analogues of the glycoconjugates were significantly more effective inhibitors when compared to their corresponding non-acetylated analogues, indicating that the lower potency observed for hydroxylated derivatives is due to less efficient transport of these compounds across the cell membrane. Thiophene-2-carboxamide was inactive as an inhibitor of bFGF induced proliferation, confirming the requirement of the carbohydrate residue for the observed biological properties. Glucose, mannose, galactose and 2-amino-2-deoxy-glucose analogues were active as were a variety of substituted thiophene derivatives; the 6H-thieno[2,3-c]pyridin-7-one conjugate was inactive. Conformational analysis of the title compounds was investigated. X-ray crystal structural analysis of four N-glucosyl-thiophene-2-carboxamides showed that the pyranose rings adopted the expected 4C1 conformations and that Z-anti structures were predominant (H1-C1-N-H anomeric torsion angle varied from -168.2 degrees to -175.0 degrees ) and that the carbonyl oxygen and sulfur of the thiophene adopted an s-cis conformation in three of the isomers. In a crystal structure of a 3-alkynyl derivative, the hydrogen atom of the NH group was directed toward the acetylene group. The distance between the hydrogen atom and acetylene carbons and angles between nitrogen, hydrogen and carbon atoms were consistent with hydrogen bonding and this was supported by IR and NMR spectroscopic studies. The geometries of thiophene-2-carboxamides were explored by density functional theory (DFT) and Møller-Plesset (MP2) calculations and the s-cis conformer of thiophene-2-carboxamide was found to be more stable than its s-trans isomer by 0.83 kcal mol(-1). The s-cis conformer of 3-ethynyl-thiophene-2-carboxamide was 5.32 kcal mol(-1) more stable than the s-trans isomer. The larger stabilisation for the s-cis conformer in the 3-alkynyl derivatives is explained to be due to a moderate hydrogen bonding interaction between the alkyne and NH group.

Development of the synovial membrane in the rat temporomandibular joint as demonstrated by immunocytochemistry for heat shock protein 25.

The synovial lining layer of the temporomandibular joint (TMJ) consists of macrophage-like type A cells and fibroblast-like type B cells. Until now, little information has been available on the development of the synovial membrane in TMJ. In the present study we examined the development of the synovial lining layer in the rat TMJ by light- and electron-microscopic immunocytochemistry for heat shock protein (Hsp) 25, which is a useful marker for type B cells. At embryonic day 19 (E19), a few Hsp25-positive cells first appeared in the upper portion of the developing condyle. During the formation of the upper articular cavity (E21 to postnatal day 1 (P1)), a few positive cells were arranged on its surface. Immunoelectron microscopy demonstrated that these cells had ultrastructural features of fibroblast-like type B cells. In addition, some Hsp25-positive cells moved to the deep portion by extending their cytoplasmic processes toward the articular cavity at P3. At that time, the presence of typical macrophage-like type A cells in the lining layer was confirmed by immunoelectron microscopy. The slender processes of Hsp25-positive cells showed a continuous covering with the synovial surface at P7, followed by a drastic increase in the Hsp25-positive cells at P15 and later, when active jaw movement occurred. These findings suggested that the arrangement and morphological maturation of type B cells are closely related to the formation of the articular cavity in the embryonic period and the commencement of active jaw movement after birth, respectively.

Expression of notch homologues in the synovium of rheumatoid arthritis and osteoarthritis patients.

Notch is known as a receptor that controls differentiation or proliferation in various cells and is associated with several diseases. The objective of the present study was to clarify whether human Notch homologues Notch-1, -2, -3, and -4 are expressed in synovium and synoviocytes from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Immunohistochemical staining showed that Notch-1, -2, and -3 were clearly expressed in the synovium from both RA and OA, whereas Notch-4 was only slightly detected. We further performed Western blotting with the same antibodies used in immunohistochemical staining. Notch-1 and -2 were strongly detected in both RA and OA, and the expression of Notch-3 was slightly detected, while there was no Notch-4 expression in both RA and OA synoviocytes. In contrast, all Notch homologues were strongly expressed in the synovium at the developmental stage obtained from the infant. These results indicate that the expression pattern of Notch among synovium from OA and RA patients differed from that of normal subjects.

Which morphologies of synovial folds result from degeneration and/or aging of the radiohumeral joint: an anatomic study with cadavers and embryos.

The synovial folds of the radiohumeral joints in cadaveric elbows from 179 elderly subjects and 40 embryos were investigated macroscopically and histologically to determine any morphologic changes caused by aging or degeneration. The anterior and posterior folds found in the elderly population shared characteristics of folds seen in embryos, with some modifications, and were thought to originate from the primitive septum. Proportionally, the length, width, and thickness of these folds were consistent between adults and embryos. However, the embryonic folds showed a homogenous morphology. In contrast, in the adult the anterior fold was characterized by a shorter and narrower villous pattern, and the posterior fold tended to be wider. Lateral extension of the anterior or posterior folds was also observed. Moreover, the lateral fold, never seen in embryos, was present and characterized by a hard plicate pattern in the adult. These derived or specific morphologies in adults probably result from alterations in the movement of the radial head caused by aging.

Fibroblast biology. Signals targeting the synovial fibroblast in arthritis.

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.

The infrapatellar adipose body in humans of various age groups.

Sections were taken from the infrapatellar adipose body of 74 males and females of various ages. In subjects exposed to long-lasting burden of the lower limbs and in older persons the subsynovial lymphocytic infiltration was found.

A developmental study of the synovial membrane of the rat temporomandibular joint: changes in the three-dimensional configuration during postnatal development.

The development of synovial membranes in the posterior synovial portion of the rat temporomandibular joint was studied and the three-dimensional structure of the posterior synovial portion reconstructed from sagittal semithin sections. Reconstructions showed that the synovial membrane expanded and that synovial folds increased in number and became complicated in shape with the growth of the joint. Using transmission-electron microscopy, it was observed that the synovial lining cells degenerated, that the synovial membrane split to make further synovial folds, and that the folded-end structures consisted of synovial lining cells that extended into the subsynovial connective tissue. It is suggested that in the development of the three-dimensional configuration of the synovial membrane, several processes proceed simultaneously to form the synovial folds: a splitting of the synovial membrane, infolding of the synovial membrane into the subsynovial connective tissue, and outgrowth of the synovial folds towards the synovial cavity.

[Synovial fluid on non-synovial tendon: experimental study].

The non-synovial tendon was placed in the synovial cavity of rabbit knee joint to carry out tissue culture in vivo. The surface of the cultured tendon was covered with a smooth membrane-like tissue which possessed the morphologic characteristics of the synovial membrane identified by histological and electron microscopic examinations. The tendon segments were kept free in the cavity of knee joint and adhesions were not observed. It is suggested that non-synovial tendon could transform into a synovial tendon in the environment filled with synovial fluid.

[The cellular aspects of the development of synovial joints and articular cartilage]. / Kletochnye aspekty razvitiia sinovial'nykh sustavov i sustavnogo khriashcha.

There have been many reports on the histological development of mammalian diarthrodial or synovial joints. While these are useful for comparative purposes, they tell us little of the cellular basis of joint morphogenesis which must underlie a number of morphogenetic defects. The process of joint morphogenesis is complex and can be subdivided into a number of facets and this report will focus on 2 of them. First, the process of joint cavitation in the chick metatarsophalangeal joint, where we propose that the selective secretion of hyaluronan into the presumptive cavity plays a central role. Secondly, the development of articular cartilage where we have used the South American opossum Monodelphis domestica as a model for mammalian development. Like most marsupials, the young are born at a much earlier developmental stage than eutherian mammals. Using antibodies which detect proliferating chondrocytes and those synthesizing insulin-like growth factors 1 and 2 and insulin-like growth factor 1 binding protein, we report that the majority of growth (as assessed by these indicators) appears appositional.