Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml−1 Dexloaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10−8 M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quanti!ed, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be bene!cial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes (SFRH/BD/21786/2005) and by the Canon Foundation in Europe. We wish to thank P.B. Malafaya for the technical support during the micro-CT analyses, and to Materialise for providing the Mimics software. This work was also supported by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283)

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/ poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml!1 Dexloaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes including project ProteoLight (PTDC/FIS/68517/2006). This work was also carried out with the support of the European Union funded STREP Project HIPPOCRATES (NMP3-CF-2003-505758) and European NOE EXPERTISSUES (NMP3-CT-2004-500283). The funding provided by Canon Foundation in Europe is gratefully acknowledged

Dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles enhances bone formation in vivo

[Excerpt] Dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles, CMC/PAMAM-Dex were successfully synthesized to find applications as a controlled system of relevant molecules in Bone Tissue Engineering. These are aimed at modulatingtheproliferation anddifferentiationofstem cells,both invitro and in vivo. In previous work, we have demonstrated that CMC/ PAMAM-Dex nanoparticles are internalized with high efficiency by different cell types, namely osteoblastic-cells, SaOs-2 and rat bone marrow stromal cells, RBMSCs. The biocompatibility of HA and SPCL scaffolds was also assessed by means of seeding RBMSCs onto the materials and performing a luminescent cell viability assay, after 24 and 72hrs. [...]info:eu-repo/semantics/publishedVersio

In vivo study of dendron-like nanoparticles for stem cells tune-up : from nano to tissues

The control of stem cell differentiation to obtain osteoblasts in vivo is still regarded as a challenge in stem-cell-based and bone-tissue engineering strategies. Biodegradable dexamethasone-loaded dendron-like nanoparticles (NPs) of carboxymethylchitosan/poly(amidoamine) dendrimer have been proposed as intracellular drug-delivery systems of bioactive molecules. In this study, combination of nanotechnology, stem-cell engineering and tissue engineering is proposed in pre-programming the fate of rat bone marrow stromal cells (RBMSCs) towards osteoblasts cells and development of new bone tissue, in vivo. This work demonstrated that the developed NPs were able to be taken up by RBMSCs, and exhibited a noncytotoxic behavior in vitro. The performance of the developed dendronlike NP system for the intracellular delivery of dexamethasone was investigated by seeding the engineered RBMSCs onto starch-polycaprolactone scaffolds ex vivo, and implanting subcutaneously in the back of Fischer 344/N rats (Syngeneic), in the absence of the typical osteogenic supplements. Favorable results were observed in vivo, thus suggesting that stem cell “tune-up” strategy can open up a new regenerative strategy for bone-tissue engineering.The authors would like to thank the financial support from Portuguese Foundation for Science and Technology (FCT, project SmartCarbo, ref. PTDC/QUI/68804/2006), through POCTI and FEDER programs. The funding provided by Canon Foundation in Europe is gratefully acknowledged. This work was also carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and HIPPOCRATES (NMP3-CT-2003-505758) projects

Surface engineered carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles for intracellular targeting

Novel highly branched biodegradable macromolecular systems have been developed by grafting carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers. Such structures organize into sphere-like nanoparticles that are proposed to be used as carriers to deliver bioactive molecules aimed at controlling the behavior of stem cells, namely their proliferation and differentiation. The nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 1 mg mL"1, and fluorescent probe labeled nanoparticles were found to be internalized with highly efficiency by both human osteoblast-like cells and rat bone marrow stromal cells, under fluorescence-activated cell sorting and fluorescence microscopy analyses. Dexamethasone (Dex) has been incorporated into CMCht/PAMAM dendrimer nanoparticles and release rates were determined by high performance liquid chromatography. Moreover, the biochemical data demonstrates that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles promote the osteogenic differentiation of rat bone marrow stromal cells, in vitro. The nanoparticles exhibit interesting physicochemical and biological properties and have great potential to be used in fundamental cell biology studies as well as in a variety of biomedical applications, including tissue engineering and regenerative medicine

Male-male marriage in Sinophone and Anglophone Harry Potter danmei and slash

The aim of this study is to compare Sinophone and Anglophone fan fiction consisting of female-oriented male-male romance: danmei and slash, respectively. To increase comparability, we analysed Harry Potter fan fiction in which the characters Harry and Draco are married. Male-male marriage was selected because our online Sinophone and Anglophone BL fandom surveys indicate this to be the most popular story element of the nine options we provided. We analysed five stories originally written in Chinese and five originally written in English which subsequently had been fan-translated into Chinese. Using Thematic Analysis (Braun & Clarke, 2006) we found some robust patterns. In contrast to the Anglophone fiction, the Sinophone tended to: stress the importance of family approval for the marriage; incorporate a wedding ceremony; employ clearly gendered roles between partners; utilise extended, as opposed to nuclear, families; and showed the couple to produce children, particularly boys. Hence, the stories mirror the relative social conservatism and social liberalism of their cultures of origin. However, in reading and writing such danmei young Chinese women are still pushing at the boundaries of the traditional family

Genetic linkage maps of Japanese and European pears aligned to the apple consensus map

Genetic linkage maps of the Japanese pear (Pyrus pyrifolia Nakai) cultivar `Housui¿ and the European pear (Pyrus communis L.) cultivar `Bartlett¿ were constructed based on Amplified Fragment Length Polymorphism markers (AFLPs), Simple Sequence Repeat markers (SSRs) (from pear, apple and Prunus), isozymes, and phenotypic traits by using their F1 progenies. The map of the female parent `Bartlett¿ consisted of 256 loci including 178 AFLPs, 76 SSRs (32 pear, 39 apple, 5 Prunus SSRs), 1 isozyme and a self-incompatibility locus on 19 linkage groups over a total length of 1020 cM. The map of `Housui¿ contained 180 loci including 110 AFLPs, 64 SSRs (29 pear, 29 apple, 6 Prunus SSRs), 2 phenotypic traits and 4 other markers on 20 linkage groups encompassing a genetic distance of 995 cM. These 2 pear maps were aligned using 37 co-dominant markers that showed segregating alleles in both parents. Out of 80 tested SSR markers developed from apple, more than four-fifth could produce discrete amplified fragments in pear. Thirty-eight apple SSR markers showed 39 segregating loci in the linkage map of `Bartlett¿, while 27 markers produced 29 loci in `Housui¿. All pear linkage groups could be successfully aligned to the apple consensus map by at least 1 apple SSRs, suggesting that positions and linkages of SSR loci were well conserved between pear and apple. The self-incompatibility locus (S locus) was mapped to linkage group 17 in Japanese and European pears as well as apple. Our results are the first major effort in comparative mapping of pear and appl

Macroporous hydroxyapatite scaffolds for bone tissue engineering applications : physicochemical characterization and assessment of rat bone marrow stromal cell viability

In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an Xray detector. The HA scaffolds obtained possess a porosity of !70%, and macropores diameter in the range of 50–600 lm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 6 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a ‘‘cauliflower- like’’ morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.Contract grant sponsor: European NoE EXPERTISSUES; contract grant number: NMP3-CT-2004-50028

A Tale of Two Sites: On Defining the Carrier Concentration in Garnet‐Based Ionic Conductors for Advanced Li Batteries

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111906/1/aenm201500096.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111906/2/aenm201500096-sup-0001-S1.pd