Identification of adsorbed molecules via STM tip manipulation: CO, H₂O, and O₂ on TiO₂ anatase (101)

While Scanning Tunneling Microscopy (STM) has evolved as an ideal tool to study surface chemistry at the atomic scale, the identification of adsorbed species is often not straightforward. This paper describes a way to reliably identify H2O, CO and O2 on the TiO2 anatase (101) surface with STM. These molecules are of a key importance in the surface chemistry of this and many other (photo-) catalytic materials. They exhibit a wide variety of contrasts in STM images, depending on the tip condition. With clean, metallic tips the molecules appear very similar, i.e., as bright, dimer-like features located in the proximity of surface Ti5c atoms. However, each species exhibits a specific response to the electric field applied by the STM tip. It is shown that this tip–adsorbate interaction can be used to reliably ascertain the identity of such species. The tip–adsorbate interactions, together with comparison of experimental and calculated STM images, are used to analyse and revisit the assignments of molecular adsorbed species reported in recent studies.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Triple innominate osteotomy for Legg-Calvé-Perthes disease in children: does the lateral coverage change with time?

Triple innominate osteotomy (TIO) is one of the modalities of surgical containment in Legg-Calvé-Perthes disease (LCPD). However, overcoverage with TIO can lead to pincer impingement.

Endothelin antagonism prevents diabetic retinopathy in NOD mice: a potential role of the angiogenic factor adrenomedullin

Altered activity of retinal endothelin-1 (ET-1) and nitric oxide may play a causal role in the hemodynamic and histopathological changes of diabetic retinopathy. This study evaluated the therapeutic potential of long-term selective blockade of the ET-1(A) receptor (ETRA) to prevent the development of retinopathy in a genetic mouse model of nonobese type 1 diabetes (NOD). Mice with NOD that received subcutaneous implantation of insulin pellets and wild-type control mice were treated for 4 months with the selective ETRA antagonist LU208075 (30 mg/kg/day) via drinking water. At the end of the study, blood glucose levels were evaluated, and animals were anesthetized and perfused intracardially with FITC-labeled dextran. Retinas were removed and either fixed in formalin for confocal microscope evaluation of retinal vascular filling or transferred to RNALater for quantitative reverse transcriptase-polymerase chain reaction to evaluate expression of NOS-3, NOS-1, ET-1, ETRA, ETRB, and the angiogenic factor adrenomedullin. Compared with wild-type controls, expression of ET-1, ETRA, ETRB, and adrenomedullin in mice with NOD were markedly upregulated in the retinas of nontreated mice (cycle time values relative to GAPDH [deltaCt], 14.8 vs. 13.7, 18.57 vs. 17.5, 10.76 vs. 9.9, and 11.7 vs. 9.1, respectively). Mean integral fluorescence intensity (MIFI) of retinal vascular filling was reduced from normal values of 24 to 12.5 in nontreated animals. LU208075 treatment normalized the upregulated expression of ET-1 and adrenomedullin, as well as the deficit in MIFI, but did not affect the increased ETRA and ETRB expression or the elevated plasma glucose levels found in nontreated animals. NOS isoform expression was essentially unchanged. ETRA antagonists may provide a novel therapeutic strategy to slow or prevent progression of retinal microvascular damage and proliferation in patients for whom there is clear evidence of activation of the ET-1 system.

Visual histological grading system for the evaluation of in vitro-generated neocartilage

Here we present the development of a visual evaluation system for routine assessment of in vitro-engineered cartilaginous tissue. Neocartilage was produced by culturing human articular chondrocytes in pellet culture systems or in a scaffold-free bioreactor system. All engineered tissues were embedded in paraffin and were sectioned and stained with Safranin O-fast green. The evaluation of each sample was broken into 3 categories (uniformity and intensity of Safranin O stain, distance between cells/amount of matrix produced, and cell morphology), and each category had 4 components with a score ranging from 0 to 3. Three observers evaluated each sample, and the new system was independently tested against an objective computer-based histomorphometry system. Pellets were also assessed biochemically for glycosaminoglycan (GAG) content. Pellet histology scores correlated significantly with GAG contents and were in agreement with the computer-based histomorphometry system. This system allows a valid and rapid assessment of in vitro-generated cartilaginous tissue that has a relevant association with objective parameters indicative of cartilage quality.

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.