Curcumin eluting nanofibers augment osteogenesis toward phytochemical based bone tissue engineering

Curcumin is a phenolic compound isolated from Curcuma longa that is known to exhibit wide ranging biological activity including potential benefits for bone growth. The aim of this work was to engineer curcumin eluting tissue scaffolds and investigate their potential use in bone tissue regeneration. We prepared curcumin loaded poly(e-caprolactone) (PCL) nanofibers by electrospinning. Morphological characterization of the nanofibers revealed that the average diameter of neat fibers and that of fibers with 1 wt% and 5 wt% curcumin is 840 +/- 130 nm, 827 +/- 129 nm and 680 +/- 110 nm, respectively. Fourier transformation infrared spectroscopy and H-1 nuclear magnetic resonance confirmed the successful loading of the drug in fibers. In aqueous medium, the fibers released approximate to 18% of the encapsulated drug in 3 d and approximate to 60% in 9 d. The cell response to the curcumin loaded nanofibers was assessed using MC3T3-E1 pre-osteoblasts. Cell proliferation was moderated with increased loading of curcumin and was 50% lower on the fibers containing 5% curcumin at day 10 than the control fibers. Osteogenesis was confirmed by assaying the expression of alkaline phosphatase and staining of mineral deposits by Alizarin red stain, which were both markedly higher for 1% curcumin compared to neat polymer but lower for 5% curcumin. Mineral deposition was also confirmed chemically by Fourier transform infrared spectroscopy. These results were corroborated by increased gene and protein expression of known osteogenic markers in 1% curcumin. Thus, controlled release of curcumin from polymer scaffolds is a promising strategy for bone tissue regeneration

Strontium eluting nanofibers augment stem cell osteogenesis for bone tissue regeneration

Strontium is known to offer a therapeutic benefit to osteoporotic patients by promoting bone formation. Thus, toward engineering scaffolds for bone tissue regeneration we have prepared polymer nanocomposite scaffolds by electrospinning. Strontium carbonate nanoparticles (nSrCO(3)) were added to poly(epsilon-caprolactone) (PCL) at 10 and 20 wt% to develop nanocomposite fibrous scaffolds (PCL/SrC10 and PCL/SrC20) with fiber diameter in the range of 300-500 nm. Incorporation of nSrCO(3) decreased crystallinity and the elastic modulus of PCL. The composite scaffolds released Sr2+ ions with up to 65 ppm in 4 days from the PCL/SrC20 scaffolds. Cell studies confirmed that the composite scaffold with 20% nSrCO(3) enhanced proliferation of human mesenchymal stem cells in vitro. There was marked increase in mineral deposition up to four folds in PCL/SrC20 suggesting enhanced osteogenesis. This was corroborated by increased mRNA and protein expression of various osteogenic markers such as BMP-2, Osterix and Runx2 in the PCL/SrC20 fibers. Thus, incorporation of nSrCO(3) in polymer scaffolds is a promising strategy for bone tissue engineering as an alternative to the use of labile growth factors to impart bioactivity to polymer scaffolds. (C) 2016 Elsevier B.V. All rights reserved

In situ preparation of multicomponent polymer composite nanofibrous scaffolds with enhanced osteogenic and angiogenic activities

Bioactive ceramics are extensively used for bone repair and regeneration, which release ions to initiate apatite formation and promote osteogenic differentiation eventually resulting in strong bonding to bone. Toward enhancing the bioactivity of polymeric nanofibrous scaffolds, this work presents a one-step in situ sol-gel method to fabricate electrospun composite nanofibrous scaffolds encapsulating well dispersed ceramic nanoparticles overcoming the limitations of current preparation techniques. Transmission electron micrographs revealed uniform distribution of ceramic nanoparticles within the polymer nanofibers. The multicomponent scaffolds were found to release calcium, silicon and phosphate ions that mimic the dissolution and bioactivity of conventional bioactive glasses. The scaffolds enhanced the bioactivity of PCL fibers as observed through enhanced apatite formation in simulated body fluid. The released ions markedly enhanced the proliferation and osteogenic differentiation of human mesenchymal stem cells and the angiogenic activity of human endothelial cells in vitro. This work has important implications for engineering the next-generation nanostructured scaffolds that exhibit multi-biofunctional activities for bone tissue regeneration

A self-assembling polycationic nanocarrier that exhibits exceptional gene transfection efficiency

The lack of an efficient and safe carrier is a major impediment in the field of gene therapy. Although gelatin (GT), a naturally derived polymer, is widely used in drug delivery applications, it is unable to bind DNA efficiently. In this study, a novel polycationic gene carrier was prepared by conjugation of low molecular weight polyethyleneimine (LPEI) with GT through 4-bromonaphthaleic anhydride as a coupling agent to avoid self crosslinking. Self-assembly of LPEI conjugated GT (GT-LPEI) with plasmid DNA (pDNA) yielded nanoparticles with high gene complexation ability to form similar to 250 nm cylindrical nanoparticles with a zeta potential of similar to 27 mV. GT-LPEI showed exceptionally high transfection efficiency (> 90%) in various mammalian cells including primary stem cells with minimal cytotoxicity. The transfection efficiency of GT-LPEI significantly surpassed that of many commercial reagents. The high gene transfection expression was confirmed in vivo. Thus, GT-LPEI is shown to be a promising nonviral carrier for potential use in gene therapy

Synthesis of a Block Copolymer Exhibiting Cell-Responsive Phytochemical Release for Cancer Therapy

Phytochemicals constitute a promising class of therapeutics for the treatment of various diseases, but, their delivery poses significant challenges. In this work, a nanoscale polyactive emulsion was designed for smart, cell-responsive delivery of a curcumin prodrug (curcumin dicarboxylate, CDA) that was chemically conjugated to enzymatically labile oligo-peptides with polycaprolactone (PCL) as the carrier. Matrix metalloproteinase (MMP)-sensitive (PLGLYAL) or nonsensitive (GPYYPLG) peptides were used as spacers for conjugating CDA and PCL. This CDA nanoemulsion incorporating the MMP-sensitive sequence exhibited markedly higher anti-cancer activity, cell internalization, and generation of reactive oxygen species in cancer cells in vitro than the control with the nonsensitive oligopeptide. Moreover, the nanopolyactives induced minimal cytotoxicity in noncancerous cell line. This work presents a unique strategy to engineer smart nano-polyactives for efficient and targeted delivery of phytochemicals

In Situ Silication of Polymer Nanofibers to Engineer Multi-Biofunctional Composites

The critical role of silica in bone homeostasis has motivated the development of silica-based biomaterials for orthopedic applications. Whereas polymer nanofibers have emerged as promising substrates for orthopedic applications, nanoparticle agglomeration precludes the preparation of silica containing composite nanofibers by electrospinning. This work presents a facile sol-gel process to fabricate electrospun nanocomposite fibers by insitu silica gelation in poly (epsilon-caprolactone) (PCL) solution. Citric acid is shown to be more effective than acetic acid as the pH catalyst for gelation by rapidly yielding near uniform nanoparticles (150 +/- 50 nm). The composite nanofibers exhibited increased water wettability than neat PCL with sustained release of silicon ions. The composite fibers induced early apatite formation in simulated body fluid. Quantitative characterization of the tubular networks formed by human umbilical cord vascular endothelial cells revealed that the eluted silicon ions and citric acid in fibers synergistically promoted angiogenic activity, which was corroborated by increased gene and protein expressions of several known angiogenic markers. Furthermore, silicate fibers augmented osteogenesis of human mesenchymal stem cells as measured by the increased mineral deposition and increased gene and protein expression of osteogenic markers. Thus, the insitu silicated fibers are promising multi-biofunctional materials for orthopedic applications

Engineering a Piperine Eluting Nanofibrous Patch for Cancer Treatment

The objective of this study was to engineer a biodegradable polymeric system for sustained release of piperine for cancer treatment. We prepared nanofibrous patches of poly­(ε-caprolactone) (PCL) and gelatin (GEL) blends of different ratios by electrospinning. The PCL/GEL nanofibers were loaded with up to 30 wt % piperine, a phytochemical derived from black pepper, which is believed to exhibit anticancer, antiarthritis, antibacterial, antioxidant, and anti-inflammatory properties. Scanning electron microscopy revealed that the fiber diameter was in the range of 300–400 nm. Fourier-transform infrared spectroscopy confirmed that the drug was successfully loaded into the nanofiber mats. In vitro release kinetics revealed the sustained release of the drug with 50% release in 3 days from the PCL/GEL (50:50 by weight) blend fibers. The reduced viability and growth of HeLa and MCF-7 cancer cells on the piperine eluting nanofibers demonstrated anticancer activity in vitro. The proliferation of noncancerous cells such as NIH3T3 cells and human mesenchymal stem cells was affected to a markedly lesser extent. Flow cytometry revealed that the released piperine induced the generation of reactive oxygen species (ROS) and cell cycle arrest in the G2/M phase, leading to cell death of cancer cells. The findings of this study suggest that piperine-loaded nanofiber mats could be developed into implantable biodegradable patches for use in postsurgical cancer treatment

Synthesis of a Block Copolymer Exhibiting Cell-Responsive Phytochemical Release for Cancer Therapy

Phytochemicals constitute a promising class of therapeutics for the treatment of various diseases, but their delivery poses significant challenges. In this work, a nanoscale polyactive emulsion was designed for smart, cell-responsive delivery of a curcumin prodrug (curcumin dicarboxylate, CDA) that was chemically conjugated to enzymatically labile oligo-peptides with polycaprolactone (PCL) as the carrier. Matrix metalloproteinase (MMP)-sensitive (PLGLYAL) or nonsensitive (GPYYPLG) peptides were used as spacers for conjugating CDA and PCL. This CDA nanoemulsion incorporating the MMP-sensitive sequence exhibited markedly higher anti-cancer activity, cell internalization, and generation of reactive oxygen species in cancer cells in vitro than the control with the nonsensitive oligopeptide. Moreover, the nanopolyactives induced minimal cytotoxicity in noncancerous cell line. This work presents a unique strategy to engineer smart nano-polyactives for efficient and targeted delivery of phytochemicals

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. d-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stem cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release

Role of Microtubules in Osteogenic Differentiation of Mesenchymal Stem Cells on 3D Nanofibrous Scaffolds

Human bone marrow mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) nanofibrous scaffolds are known to undergo osteogenic differentiation even in the absence of soluble osteoinductive factors. Although this process of differentiation has been attributed to the shape that cells assume on the fibrous scaffolds, it is unclear how constriction of cell shape would contribute to the differentiation phenotype. Here, we quantitatively compared cell and nuclear morphologies of cells cultured on 3D poly­(ε-caprolactone) (PCL) nanofibers (NF) and two-dimensional (2D) flat films using confocal fluorescence microscopy. We discovered that while cells on the 2D films exhibited cellular and nuclear morphologies similar to those cultured on tissue culture polystyrene, cells cultured on the 3D NF showed distinct cell and nuclear morphologies, with lower areas and perimeters, but higher aspect ratios. We next tested the effect of treatment of cells with actin-depolymerizing cytochalasin D and microtubule-depolymerizing nocodazole on these morphologies. In both 2D and 3D scaffolds, actin depolymerization brought about gross changes in cell and nuclear morphologies. Remarkably, microtubule depolymerization resulted in a phenotype similar to actin depolymerization in cells cultured on 3D NF alone, indicating a significant role for the microtubule cytoskeleton in the maintenance of cell shape and structure in 3D. The morphological changes of the nucleus that were apparent upon cytoskeletal perturbation were reflected in the organization of heterochromatin in the nucleus, with MSCs on 3D alone exhibiting a differentiation phenotype. Finally, we tested the effect of cytoskeletal depolymerization on mineralization of cells. Again, we observed higher mineralization in cells cultured on 3D NF, which was lost in cells treated with either cytochalasin D or nocodazole. Taken together, our results suggest that both the actin and microtubule cytoskeletons contribute significantly toward maintenance of cell and nuclear shape in cells cultured on 3D scaffolds, and consequently to their osteogenic differentiation