MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. Thus, MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. Conversely, MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. Interestingly, MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. Moreover, we observed a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development.     

Bone morphogenetic proteins are involved in the pathobiology of synovial chondromatosis

Synovial chondromatosis is characterized by the formation of osteocartilaginous nodules (free bodies) under the surface of the synovial membrane in joints. Free bodies and synovium isolated from synovial chondromatosis patients expressed high levels of BMP-2 and BMP-4 mRNAs. BMP-2 stimulated the expression of Sox9, Col2a1, and Aggrecan mRNAs in free-body and synovial cells and that of Runx2, Col1a1, and Osteocalcin mRNAs in the free-body cells only. BMP-2 increased the number of alcian blue-positive colonies in the free-body cell culture but not in the synovial cell culture. Noggin suppressed the expression of Sox9, Col2a1, Aggrecan, and Runx2 mRNAs in both the free-body and synovial cells. Further, it inhibited Osteocalcin expression in the synovial cells. These results suggest that BMPs are involved in the pathobiology of cartilaginous and osteogenic metaplasia observed in synovial chondromatosis.     

碱性成纤维细胞生长因子对胚胎神经细胞存活与生长的影响

本文利用大鼠胚胎中脑神经细胞作原代微团培养,培养物经不同浓度碱性成纤维细胞生长因子（ｂａｓｉｃ Ｆｉｂｒｏｂｌａｓｔ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｂＥＧＦ）,研究ｂＦＧＦ对细胞生长和分化的影响,并利用图象分析细胞形态的变化。结果表明ｂＥＧＦ可促进微团中的集落形成率明显增加,并显示量效应关系。图象显示ｂＦＧＦ可促进细胞神经突起增多,而且有丰富的神经纤维连结成网络状。认为ｂＥＧＦ能促进中脑细胞生长和分     

ATF-2 cooperates with Smad3 to mediate TGF-beta effects on chondrocyte maturation

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.     

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 μM, 200 μM, and 400 μM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.     

1,25(OH)2D3 attenuates pulmonary arterial hypertension via microRNA-204 mediated Tgfbr2/Smad signaling

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH)₂D₃] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH)₂D₃ in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH)₂D₃ on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH)₂D₃ was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH)₂D₃, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH)₂D₃ administration. Findings outlined in the current study demonstrated that 1,25(OH)₂D₃ was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.     

Direct chitosan-mediated gene delivery to the rabbit knee joints in vitro and in vivo

Chitosan vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, chitosan-DNA nanoparticles containing IL-1Ra or IL-10 gene were injected directly into the knee joint cavities of osteoarthritis rabbits to clarify the in vivo transfer availability of the chitosan vectors. Clear expression of IL-1Ra was detected in the knee joint synovial fluid of the chitosan IL-1Ra-injected group. While no expression was detected in the chitosan IL-10-injected group, this demonstrates that the transfection efficiency of chitosan-DNA nanoparticles was closely related to the type of the gene product. A significant reduction was also noted in the severity of histologic cartilage lesions in the group that received the chitosan IL-1Ra injection. This avenue may therefore represent a promising future treatment for osteoarthritis.     

Cytokine profile in the synovial fluid of patients with temporomandibular joint disorders: A systematic review

The aim of this study was to review the cytokine profiles in the synovial fluid (SF) of patients with temporomandibular joint disorders (TMJD). Databases were searched from 1965 till September 2015 using different combinations of the following key words: “Temporomandibular joint”; “Cytokine”; “disorder”; and “synovial fluid” and “inflammation”. Titles and abstracts of studies identified using the above-described protocol were screened and checked for agreement. Full-texts of articles judged by title and abstract to be relevant were read and independently evaluated. Hand-searching of the reference lists of potentially relevant original and review articles was also performed. The pattern of the present systematic review was customized to mainly summarize the relevant data. Fifteen studies were included. In 12 studies, cytokine profile of patients with TMJD was assessed using enzyme linked immunosorbent assay; and in 2 studies, histological analysis was performed to assess the cytokine profile of patients with TMJD. Patients with TMJD presented raised levels of interleukin (IL)-6 in 8 studies, IL-1beta (1β) in 5 studies and tumor necrosis factor-alpha (TNF-α) in 5 studies. Two studies showed no significant difference in TNF-α levels in patients with and without TMJD; and IL-1β levels were comparable in patients with and without TMJD in 2 studies. Raised levels of IL-6, TNF-α, IL-1β, IL-8, and IFN-γ in the SF have been associated with inflammation in patients with TMJD. Cytokines IL-10, osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG), and VEGF found in the SF of TMJs could have an anti-inflammatory effect.     

Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.     

Chondrocyte acting as phagocyte to internalize polyethylene wear particles and leads to the elevations of osteoarthritis associated NO and PGE2

It remains a mystery about the role of chondrocyte or cartilage on the co-existence of ultra-high molecular weight polyethylene (UHMWPE) wear particles from partial joint arthroplasty. An inverted co-culture system was performed to investigate the interactions between chondrocytes and UHMWPE wear particles. It was first time observed that chondrocytes can engulf UHMWPE particles and release osteoarthritis associated pro-inflammatory factors. TEM observation and flow cytometric analysis demonstrated the phagocytosis of particles by chondrocytes. It was found that polyethylene particles may reduce the viability of chondrocytes, and enhance the secretion of nitric oxide (NO) and PGE₂. In conclusion, all these phenomena may contribute to further cartilage degeneration after partial joint arthroplasty surgery. It is first identified in this study that the chondrocyte acts as phagocyte to internalize wear particles and leads to the elevations of precursor mediators of osteoarthritis.     

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri-Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2. Shox is expressed in central regions of early chick limb buds and proximal two thirds of later limbs, whereas Shox2 is expressed more posteriorly in the proximal third of the limb bud. Shox expression is inhibited distally by signals from the apical ectodermal ridge, both Fgfs and Bmps, and proximally by retinoic acid signaling. We tested Shox functions by overexpression in embryos and micromass cultures. Shox-infected chick limbs had normal proximo-distal patterning but the length of skeletal elements was consistently increased. Primary chick limb bud cell cultures infected with Shox showed an initial increase in cartilage nodules but these did not enlarge. These results fit well with the proposed role of Shox in cartilage and bone differentiation and suggest chick embryos as a useful model to study further the role of Shox in limb development.     

Chemical nature of implant-derived titanium(IV) ions in synovial fluid

Previous investigations have indicated a deleterious leakage of Ti(III) and/or Ti(IV) species from Ti-Al-V alloy joint prostheses into adjacent tissue, synovium or synovial fluid (SF) in vivo. In view of the importance of the particular chemical nature of such complexes in determining their biological activity, we have employed high field proton (1H) NMR spectroscopy to speciate Ti(IV) in inflammatory SF. Treatment of osteoarthritic SF samples with increasing concentrations of Ti(IV) (0.10-1.03 mM [TiO(C2O4)2]2-) gave rise to a specific broadening of the citrate proton resonances, indicating that this bioavailable oxygen-donor ligand plays an important role in complexing implant-derived Ti(IV). 1H NMR analysis of Ti(IV)-loaded SF samples subsequently treated with a large excess of ascorbate (0.05 M) showed that this added Ti(IV) chelator was only poorly effective in removing this metal ion from Ti(IV)-citrate/Ti(IV)-oxycitrate complexes. The results obtained here provide evidence for complexation of the low-molecular-mass (non-protein-bound) fraction of implant-derived Ti(IV) by citrate in vivo.     

Effect of surface roughness on squeeze film poroelastic bearings with special reference to synovial joints

A simplified mathematical model has been developed for understanding the combined effects of surface roughness and couple stresses on lubrication aspects of synovial joints. The modified Reynolds equation which incorporates the elastic as well as randomized surface roughness structure of cartilage with couple-stress fluid as lubricant is derived. The mean pressure, load carrying capacity and time of approach as functions of film thickness during normal articulation of joints are obtained by using Christensen stochastic theory with the assumption that the roughness asperity heights are to be small compared to the film thickness. The effects of surface roughness and elasticity are considerably pronounced for the poroelastic bearings with couple-stress fluid as lubricant compared with classical case.     

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire^−/− mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2.     

郑州孙庄遗址仰韶文化居民的颅骨形态

孙庄遗址位于河南省郑州市中原区孙庄村南,是分布在黄河中下游地区的一处仰韶文化晚期遗存。通过对遗址出土的10例保存基本完整的颅骨进行测量与观察后,得出以下结论:孙庄组的颅面部特征为高颅型与狭颅型相结合的颅型特点、中等偏大的面部扁平度、狭额型、中鼻型、低眶型、中面角属平颌型、犬齿窝和鼻根凹欠发达、颅顶缝简单等。孙庄组古代居民颅骨的形态特征归属于亚洲蒙古人种范围。颅骨形态学特征分析结果显示,孙庄组与亚洲蒙古人种近代华南组(R=1.26)关系最为密切,与近代蒙古组(R=1.80)和通古斯组(R=2.06)关系疏远;在与各新石器时代组的对比中,孙庄男性组与仰韶合并组(R=1.00)、庙子沟组(R=1.00)、西山组(R=1.07)、大汶口组(R=1.13)关系接近,孙庄女性组最接近于大汶口组(D_ij=3.10)、徐堡组(D_ij=4.58)和西山组(D_ij=4.60)。综上,我们可知分布在黄河中下游地区的仰韶时代中晚期人群在颅面部特征上颇为一致,具有较高的同源性,应同属于\"古中原类型\"居民。