Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α-TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4-37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α-TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

Nanocomposite-coated porous templates for engineered bone scaffolds: a parametric study of layer-by-layer assembly conditions.

Using the layer-by-layer (LbL) assembly technique to deposit mechanically reinforcing coatings onto porous templates is a route for fabricating engineered bone scaffold materials with a combination of high porosity, strength, and stiffness. LbL assembly involves the sequential deposition of nano- to micro-scale multilayer coatings from aqueous solutions. Here, a design of experiments (DOE) approach was used to evaluate LbL assembly of polyethyleneimine (PEI), polyacrylic acid (PAA), and nanoclay coatings onto open-cell polyurethane foam templates. The thickness of the coatings, and the porosity, elastic modulus and collapse stress of coated foam templates were most strongly affected by the pH of PAA solutions, salt concentration, and interactions between these factors. The mechanical properties of coated foams correlated with the thickness of the coatings, but were also ascribed to changes in the coating properties due to the different assembly conditions. A DOE optimization aimed to balance the trade-off between higher mechanical properties but lower porosity of foam templates with increasing coating thickness. Micromechanical modeling predicted that deposition of 116 QLs would achieve mechanical properties of cancellous bone (>0.05 GPa stiffness and >2 MPa strength) at a suitable porosity of >70%. When capped with a final layer of PAA and cross-linked via thermal treatment, the PEI/PAA/PEI/nanoclay coatings exhibited good indirect cytotoxicity with mesenchymal stem cells. The ability of LbL assembly to deposit a wide range of functional constituents within multilayer-structured coatings makes the general strategy of templated LbL assembly a powerful route for fabricating engineered tissue scaffolds that can be applied onto various porous template materials to achieve a wide range of properties, pore structures, and multifunctionality.

Simple Radical Polymerization of Poly(Alginate-Graft-N-Isopropylacrylamide) Injectable Thermoresponsive Hydrogel with the Potential for Localized and Sustained Delivery of Stem Cells and Bioactive Molecules.

In this study, thermoresponsive copolymers that are fully injectable, biocompatible, and biodegradable and are synthesized via graft copolymerization of poly(N-isopropylacrylamide) onto alginate using a free-radical reaction are presented. This new synthesis method does not involve multisteps or associated toxicity issues, and has the potential to reduce scale-up difficulties. Chemical and physical analyses verify the resultant graft copolymer structure. The lower critical solution temperature, which is a characteristic of sol-gel transition, is observed at 32 °C. The degradation properties indicate suitable degradation kinetics for drug delivery and bone tissue engineering applications. The synthesized P(Alg-g-NIPAAm) hydrogel is noncytotoxic with both human osteosarcoma (MG63) cells and porcine bone marrow derived mesenchymal stem cells (pBMSCs). pBMSCs encapsulated in the P(Alg-g-NIPAAm) hydrogel remain viable, show uniform distribution within the injected hydrogel, and undergo osteogenic and chondrogenic differentiation under appropriate culture conditions. Furthermore, for the first time, this work will explore the influence of alginate viscosity on the viscoelastic properties of the resulting copolymer hydrogels, which influences the rate of medical device formation and subsequent drug release. Together the results of this study indicate that the newly synthesized P(Alg-g-NIPAAm) hydrogel has potential to serve as a versatile and improved injectable platform for drug delivery and bone tissue engineering applications.

MiRNA 34a: a therapeutic target for castration-resistant prostate cancer.

INTRODUCTION: Development of a therapy for bone metastases is of paramount importance for castration-resistant prostate cancer (CRPC). The osteomimetic properties of CRPC confer a propensity to metastasize to osseous sites. Micro-ribonucleic acid (miRNA) is non-coding RNA that acts as a post-transcriptional regulator of multiple proteins and associated pathways. Therefore identification of miRNAs could reveal a valid third generation therapy for CRPC. AREAS COVERED: miR34a has been found to play an integral role in the progression of prostate cancer, particularly in the regulation of metastatic genes involved in migration, intravasation, extravasation, bone attachment and bone homeostasis. The correlation between miR34a down-regulation and metastatic progression has generated substantial interest in this field. EXPERT OPINION: Examination of the evidence reveals that miR34a is an ideal target for gene therapy for metastatic CRPC. We also conclude that future studies should focus on the effects of miR34a upregulation in CRPC with respect to migration, translocation to bone micro-environment and osteomimetic phenotype development. The success of miR34a as a therapeutic is reliant on the development of appropriate delivery systems and targeting to the bone micro-environment. In tandem with any therapeutic studies, biomarker serum levels should also be ascertained as an indicator of successful miR34a delivery.