In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFß1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd.

Genetic diversity of the forage peanut in the Jequitinhonha, São Francisco, and Paranã River valleys of Brazil.

Arachis pintoi and A. repens are legumes with a high forage value that are used to feed ruminants in consortium systems. Not only do they increase the persistence and quality of pastures, they are also used for ornamental and green cover. The objective of this study was to analyze microsatellite markers in order to access the genetic diversity of 65 forage peanut germplasm accessions in the section Caulorrhizae of the genus Arachis in the Jequitinhonha, São Francisco and Paranã River valleys of Brazil. Fifty-seven accessions of A. pintoi and eight of A. repens were analyzed using 17 microsatellites, and the observed heterozygosity (HO), expected heterozygosity (HE), number of alleles per locus, discriminatory power, and polymorphism information content were all estimated. Ten loci (58.8%) were polymorphic, and 125 alleles were found in total. The HE ranged from 0.30 to 0.94, and HO values ranged from 0.03 to 0.88. By using Bayesian analysis, the accessions were genetically differentiated into three gene pools. Neither the unweighted pair group method with arithmetic mean nor a neighbor-joining analysis clustered samples into species, origin, or collection area. These results reveal a very weak genetic structure that does not form defined clusters, and that there is a high degree of similarity between the two species.

Fabrication of phospholipid-xanthan microcapsules by combining microfluidics with self-assembly.

We report the synthesis of an amphiphilic polysaccharide, a phospholipid (1,2-dioleoyl-sn-glycero-phosphoetilamine, DOPE) conjugated with the anionic xanthan gum, and its ability to spontaneously self-assemble under mild aqueous conditions. This work also aimed to apply a microfluidic platform that can precisely fabricate microsized and monodispersed capsules for cell encapsulation. Stable hollow capsular structures were obtained by the generation of homogeneous spherical droplets of the self-assembled polymer in the microfluidic device through the formation of a water-in-oil emulsion, followed by the stabilization of the polymer aggregates in a separate collection vessel containing phosphate-buffered saline (physiological ionic strength and pH). The properties (size, morphology, permeability) and performance (stability) of the obtained microcapsules were studied, as well their ability to support the viability, function and proliferation of encapsulated cells. ATDC5 cells were encapsulated within the capsules and shown to remain viable, evidencing increased cellular metabolic activity over 21 days of in vitro culture. By combining microfluidic droplet generation and self-assembly of xanthan-DOPE, we were able to fabricate microcapsules that provided an adequate environment for cells to survive and proliferate.

Injectable biodegradable starch/chitosan delivery system for the sustained release of gentamicin to treat bone infections.

Starch-conjugated chitosan microparticles were produced aimed to be used as a carrier for the long term sustained/controlled release of antibiotic drugs to control bone infection. The microparticles were prepared by a reductive alkylation crosslinking method. The obtained microparticles showed a spherical shape, with a slightly rough and porous surface, and a size range of 80-150µm. Gentamicin was entrapped into the starch-conjugated chitosan microparticles and its release profile was studied in vitro. Increasing concentrations of gentamicin (from 50 to 150mg/mL) led to a decrease in the encapsulation efficiency (from 67 to 55%), while drug loading increased from 4 to 27%. A sustained release of gentamicin was observed over a period of 30 days. The release kinetics could be controlled using an ionic crosslinker agent. In addition, a bacterial inhibition test on Staphylococcus aureus shows a diameter of the sample inhibition zone ranging from 12 to 17mm (70-100% of relative activity).

Silk fibroin microparticles as carriers for delivery of human recombinant BMPs. Physical characterization and drug release.

Bone morphogenetic proteins (BMPs) are cytokines with strong ability to promote new bone formation. Herein, we report the use of silk fibroin microparticles as carriers for the delivery of BMP-2, BMP-9 or BMP-14. BMP-containing fibroin microparticles were prepared by a mild methodology using dropwise addition of ethanol, exhibiting mean diameters of 2.7 +/- 0.3 microm. Encapsulation efficiencies varied between 67.9 +/- 6.1 % and 97.7 +/- 2.0 % depending on the type and the amount of BMP loaded. Release kinetics showed that BMP-2, BMP-9 and BMP-14 were released in two phases profile, with a burst release in the first two days followed by a slower release, for a period of 14 days. The release data were best explained by Korsmeyer's model and the Fickian model of drug diffusion. Silk fibroin microparticles can offer a promising approach for the sustained delivery of different BMPs in tissue engineering applications.

Starch-poly-epsilon-caprolactone microparticles reduce the needed amount of BMP-2.

BMP-2 is currently administered clinically using collagen matrices often requiring large amounts of BMP-2 due to burst release over a short period of time. We developed and tested a novel injectable drug delivery system consisting of starch-poly-epsilon-caprolactone microparticles for inducing osteogenesis and requiring smaller amounts of BMP-2. We evaluated BMP-2 encapsulation efficiency and the in vitro release profile by enzyme-linked immunosorbent assay. BMP-2 was rapidly released during the first 12 hours, followed by sustained release for up to 10 days. We then evaluated the osteogenic potential of dexamethasone (standard osteogenic induction agent) and BMP-2 after incorporation and during release using an osteo/myoblast cell line (C2C12). Alkaline phosphatase activity was increased by released BMP-2. Mineralization occurred after stimulation with BMP-2-loaded microparticles. A luciferase assay for osteocalcin promoter activity showed high levels of activity upon treatment with BMP-2-loaded microparticles. In contrast, no osteogenesis occurred in C2C12 cells using dexamethasone-loaded microparticles. However, human adipose stem cells exposed to the microparticles produced high amounts of alkaline phosphatase. The data suggest starch-poly-epsilon-caprolactone microparticles are suitable carriers for the incorporation and controlled release of glucocorticoids and growth factors. Specifically, they reduce the amount of BMP-2 needed and allow more sustained osteogenic effects.

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration.

We developed a novel injectable carrageenan/fibrin/hyaluronic acid-based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage-specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering.

Preparation and characterization of starch-poly-epsilon-caprolactone microparticles incorporating bioactive agents for drug delivery and tissue engineering applications.

One limitation associated with the delivery of bioactive agents concerns the short half-life of these molecules when administered intravenously, which results in their loss from the desired site. Incorporation of bioactive agents into depot vehicles provides a means to increase their persistence at the disease site. Major issues are involved in the development of a proper carrier system able to deliver the correct drug, at the desired dose, place and time. In this work, starch-poly-epsilon-caprolactone (SPCL) microparticles were developed for use in drug delivery and tissue engineering (TE) applications. SPCL microparticles were prepared by using an emulsion solvent extraction/evaporation technique, which was demonstrated to be a successful procedure to obtain particles with a spherical shape (particle size between 5 and 900 microm) and exhibiting different surface morphologies. Their chemical structure was confirmed by Fourier transform infrared spectroscopy. To evaluate the potential of the developed microparticles as a drug delivery system, dexamethasone (DEX) was used as model drug. DEX, a well-known component of osteogenic differentiation media, was entrapped into SPCL microparticles at different percentages up to 93%. The encapsulation efficiency was found to be dependent on the polymer concentration and drug-to-polymer ratio. The initial DEX release seems to be governed mainly by diffusion, and it is expected that the remaining DEX will be released when the polymeric matrix starts to degrade. In this work it was demonstrated that SPCL microparticles containing DEX can be successfully prepared and that these microparticular systems seem to be quite promising for controlled release applications, namely as carriers of important differentiation agents in TE.

Starch-poly(epsilon-caprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering applications: structure, mechanical properties and degradation behaviour.

In scaffold-based tissue engineering strategies, the successful regeneration of tissues from matrix-producing connective tissue cells or anchorage-dependent cells (e.g. osteoblasts) relies on the use of a suitable scaffold. This study describes the development and characterization of SPCL (starch with epsilon-polycaprolactone, 30:70%) and SPLA [starch with poly(lactic acid), 30:70%] fibre-meshes, aimed at application in bone tissue-engineering strategies. Scaffolds based on SPCL and SPLA were prepared from fibres obtained by melt-spinning by a fibre-bonding process. The porosity of the scaffolds was characterized by microcomputerized tomography (microCT) and scanning electron microscopy (SEM). Scaffold degradation behaviour was assessed in solutions containing hydrolytic enzymes (alpha-amylase and lipase) in physiological concentrations, in order to simulate in vivo conditions. Mechanical properties were also evaluated in compression tests. The results show that these scaffolds exhibit adequate porosity and mechanical properties to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. The results of the degradation studies showed that these starch-based scaffolds are susceptible to enzymatic degradation, as detected by increased weight loss (within 2 weeks, weight loss in the SPCL samples reached 20%). With increasing degradation time, the diameter of the SPCL and SPLA fibres decreases significantly, increasing the porosity and consequently the available space for cells and tissue ingrowth during implantation time. These results, in combination with previous cell culture studies showing the ability of these scaffolds to induce cell adhesion and proliferation, clearly demonstrate the potential of these scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects.

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends.

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.