Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome.

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS.

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

Generation of composites for bone tissue-engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means.

Mineralization of hydrogels, desirable for bone regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). ALP-loaded gellan gum (GG) hydrogels were mineralized by incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP, MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all values mol/dm3 , denoted A, B, C, D and E, respectively) were compared. Mineral formation was confirmed by IR and Raman, SEM, ICP-OES, XRD, TEM, SAED, TGA and increases in the the mass fraction of the hydrogel not consisting of water. Ca was incorporated into mineral to a greater extent than Mg in samples mineralized in media A-D. Mg content and amorphicity of mineral formed increased in the order A < B < C < D. Mineral formed in media A and B was calcium-deficient hydroxyapatite (CDHA). Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed in medium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline and amorphous MgP. Young's moduli and storage moduli decreased in dependence of mineralization medium in the order A > B > C > D, but were significantly higher for samples mineralized in medium E. The attachment and vitality of osteoblastic MC3T3-E1 cells were higher on samples mineralized in media B-E (containing Mg) than in those mineralized in medium A (not containing Mg). All samples underwent degradation and supported the adhesion of RAW 264.7 monocytic cells, and samples mineralized in media A and B supported osteoclast-like cell formation. Copyright © 2014 John Wiley & Sons, Ltd.

Optimization of the time efficient calcium phosphate coating on electrospun poly(d,l-lactide).

The coating of fibrous polyester constructs with a layer of bioactive calcium phosphate (CP) is efficient to improve the potential use as bone tissue engineering scaffold. In this study, a fast procedure for the coating of electrospun poly(d,l-lactide) (PDLLA) fibers with a CP layer was optimized. The fiber surface was activated by immersion in demineralized water under ultrasonication. The resulting reactive groups served as nucleation points for CP precipitation, induced by alternate dipping of the samples in Ca(2+) and PO4 (3-) rich solutions. Variations in the conditions of the alternate dipping procedure, in particular the number of cycles, concentration and immersion time of both solutions, not only affected the degree of surface mineralization but also the type of deposited CP. For the current experimental conditions, in about 30 minutes either a slightly carbonated calcium deficient apatite (CDAp; Ca10-x-y (PO4 )6-x-y (HPO4 )y (CO3 )x (OH)2-x-y ) or a combination of apatite and dicalcium phosphate dihydrate (DCPD; CaHPO4 .2H2 O) was formed. The cell viability, adhesion, and proliferation of MC3T3-E1 cells on untreated samples were compared with samples coated with either an adequate amount of CDAp, an excess of CDAp or an excess of a combination of apatite and DCDP. After 7 days of culture the number of attached cells was significantly higher on all CP coated samples compared to the untreated PDLLA. In particular, the samples coated with an adequate amount of CDAp showed an exceedingly enhanced cell response with similar cell morphologies as the ones found on the positive control.

Optimization of the activation and nucleation steps in the precipitation of a calcium phosphate primer layer on electrospun poly(ɛ-caprolactone).

The present study aimed to optimize the procedure for coating electrospun poly(ε-caprolactone) (PCL) fibers with a calcium phosphate (CP) layer in order to improve their potential as bone tissue engineering scaffold. In particular, attention was paid to the reproducibility of the procedure, the morphology of the coating, and the preservation of the porous structure of the scaffold. Ethanol dipping followed by an ultrasonic assisted hydrolysis of the fiber surface with sodium hydroxide solution efficiently activated the surface. The resulting reactive groups served as nucleation points for CP precipitation, induced by alternate dipping of the samples in calcium and phosphate rich solutions. By controlling the deposition, a reproducible thin layer of CP was grown onto the fiber surface. The deposited CP was identified as calcium-deficient apatite (CDHAp). Analysis of the cell viability, adhesion, and proliferation of MC3T3-E1 cells on untreated and CDHAp coated PCL scaffolds showed that the CDHAp coating enhanced the cell response, as the number of attached cells was higher in comparison to the untreated PCL and cells on the CDHAp coated samples showed similar morphologies as the ones found in the positive control.

The effect of a photopolymerizable poly(ε-caprolactone-co-glycolide) matrix on the cement reactions of tetracalcium phosphate and tetracalcium phosphate-monocalcium phosphate monohydrate mixtures.

In this study, the influence of a biodegradable polymer matrix on the conversion of tetracalcium phosphate (TTCP) or TTCP-monocalcium phosphate monohydrate (MCPM) powders was investigated. As a reference, the properties of three calcium phosphate cements (CPCs) based on TTCP or TTCP-MCPM mixtures were discussed. Additionally, the influence of these calcium phosphate (CP) reacting powders on the polymer degradation was studied. Composites were formulated by mixing cross-linkable dimethacrylates of the ε-caprolactone/glycolide co-polymer with hydroxyethylmethacrylate, a photo-initiator and TTCP or TTCP-MCPM. The composite samples were set by visible light irradiation. CPC and composite samples were immersed in HEPES at 37 °C. The CPC based on TTCP converted to a carbonated apatite. Adding MCPM to the TTCP powder improved the conversion of TTCP. By varying the MCPM/TTCP ratio it was possible to tailor the conversion reactions so that an apatitic phase could be formed via intermediate products like DPC, DCPD and OCP. In the composites, a mutual interaction between the CP reacting powders and the polymer was observed. The co-polymer and its degradation products influenced the conversion reactions of the CP reacting powders. The degradation products tend to enhance the TTCP conversion after a long immersion time. The conversion of the TTCP-MCPM mixtures was retarded by the polymer matrix although the intermediate products were not altered. The basicity or acidity of the CP reacting powders and their conversion reactions were the main cause for the retarded polymer degradation, which was more pronounced when the basicity of the CP reacting powders increased.

Carbonated apatites obtained by the hydrolysis of monetite: influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells.

The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO(4), DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l(-1). Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (+ or - 50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

Characterization of calcium phosphate cements modified by addition of amorphous calcium phosphate.

In this study the influence of amorphous calcium phosphate (ACP) on the setting of, and the formed apatite crystallite size in, a calcium phosphate cement (CPC) based on alpha-tricalcium phosphate (alpha-TCP) or tetracalcium phosphate (TTCP)/monocalcium phosphate monohydrate (MCPM) was investigated. Setting times at 22 degrees C were measured in air atmosphere; those at 37 degrees C were measured at 100% relative humidity. The phase composition of the set cements was investigated after 1 week using X-ray diffractometry and infrared spectroscopy and the morphology was investigated using scanning electron microscopy. The compressive strength (CS) of the set CPCs was measured after 1 day. Viability of MC3T3-E1 cells on the CPCs was analyzed after 7, 14 and 21 days of incubation using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay. The alpha-TCP-based cement exhibited long setting times, a high CS and was converted to a calcium-deficient hydroxyapatite (CDHAp). The TTCP/MCPM-based CPC was only partly converted to CDHAp, produced acceptable setting times and had a low CS. Addition of ACP to these two CPCs resulted in cements that exhibited good setting times, CS suitable for non-load-bearing applications and a full conversion to nanocrystalline CDHAp. Moreover, the ACP containing CPCs demonstrated good cell viability, making them suitable candidates for bone substitute materials.

Apatite formation in composites of alpha-TCP and degradable polyesters.

The objective of this study was to investigate the conversion of alpha-Ca3(PO4)2 (alpha-TCP) in composite bone cements based on a water-degradable polyester matrix as a function of the polymer formulation and the alpha-TCP filler content. Cross-linkable dimethacrylates of epsilon-caprolactone/ D,L-lactide co-polymer or of epsilon-caprolactone/glycolide co-polymer were mixed with hydroxyethylmethacrylate, a photo-initiator and alpha-TCP to obtain composites with a filler content of 80 or 40 wt% alpha-TCP. The disk shaped composite samples were set by visible light irradiation and immersed in HEPES at 37 degrees C. At selected times the samples were removed from the solution and analysed with X-ray diffractometry and infrared spectroscopy. Conversion of alpha-TCP into calcium-deficient hydroxyapatite (CDHAp) was observed for all composites, but the reaction was not completed after 8 weeks immersion. The conversion rate of alpha-TCP and the crystallinity of the formed apatite apparently were not affected by the type of polyester used, but significantly depended on the alpha-TCP content of the composites. An increase of the amount of alpha-TCP in the composite resulted in a slower formation of CDHAp with a higher crystallinity.

Polyphasic characterisation of Burkholderia cepacia-like isolates leading to the emended description of Burkholderia pyrrocinia.

Twenty-five Burkholderia cepacia-like isolates of human and environmental origin, comprising five different recA RFLP types, were examined by using a polyphasic taxonomic approach, including recA gene sequence analysis, 16S rRNA gene sequence analysis, DNA:DNA hybridisation studies, tDNA-PCR, fatty acid analysis and biochemical analysis. The results of the present study demonstrated that twenty-three of these strains belong to Burkholderia pyrrocinia, a B. cepacia complex species thus far comprising one single soil isolate only. An emended description of Burkholderia pyrrocinia is proposed. The taxonomic status of the remaining two isolates requires further analysis.

Isolation, proliferation and differentiation of osteoblastic cells to study cell/biomaterial interactions: comparison of different isolation techniques and source.

A sufficient amount of easily obtained and well-characterized osteoblastic cells is a useful tool to study biomaterial/cell interactions essential for bone tissue engineering. Osteoblastic cells were derived from adult and fetal rat via different isolation techniques. The isolation and in vitro proliferation of primary cultures were compared. The osteogenic potential of subcultures was studied by culturing them in osteogenic medium and compared with respect to alkaline phosphatase activity, nodule formation and mineralization potential. Calvaria cells were easier to obtain and the amount of cells released by enzymatic isolation was higher than for the long bone cells. The expansion of the cells in primary culture was highest for fetal calvaria cells compared to fetal and adult long bone cells. All cultures expressed high alkaline phosphatase activity except for calvaria cells obtained by spontaneous outgrowth. Enzymatic isolation of fetal calvaria and long bone cells favoured the osteogenic differentiation. Enzymatically isolated calvaria cells formed well-defined three-dimensional nodules which mineralized restricted to this area. On the contrary, cultures derived from fetal as well as adult long bones mineralized in ill-defined deposits throughout the culture and only formed occasionally nodular-like structures. The mineral phase of all osteoblastic cultures was identified as a carbonate-containing apatite. The present study demonstrates that considering the isolation method, proliferation capacity and the osteogenic potential, the enzymatically released fetal calvaria cells are most satisfactory to study cell/biomaterial interactions.

Evaluation of tRNA intergenic length polymorphism (tDNA-pCR) for the differentiation of the members of the Burkholderia cepacia complex.

Nowadays, tentative identification of B. cepacia complex bacteria in routine diagnostic laboratories is based on a combination of selective media, conventional biochemical reactions, commercial test systems and PCR-based assays. Some of these assays have the capacity to discriminate reliably among several members of the B. cepacia complex, however one single method differentiating all B. cepacia-like organisms is not available. In this study, the applicability of tDNA-PCR for the differentiation and rapid identification of the different members of the B. cepacia complex was evaluated. For B. gladioli and most of the B. cepacia genomovars, differentiable patterns were obtained. For some of the members of the B. cepacia complex however, the tDNA-PCR patterns were very similar and sometimes multiple patterns existed within in a single genomovar. No distinction could be made between the tDNA-PCR patterns of B. vietnamiensis and B. pyrrocinia and of B. cepacia genomovars I and VIII respectively. We could conclude that, although tDNA-PCR is not sufficient as a single method to identify all the members of the B. cepacia complex unambiguously or to replace the currently used methods, it is a very fast and easily applicable method that could be a very useful tool for the differentiation and identification of B. cepacia-like organisms.