Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer-lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin-fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin-fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles.

We have developed a 3D nanofibrous spiral scaffold for bone tissue engineering which has shown enhanced cell attachment, proliferation and differentiation compared to traditional cylindrical scaffolds due to the spiral structures and the nanofiber incorporation. Some important parameters of these spiral scaffolds including gap distance, wall thickness and especially fiber thickness are crucial to the performance of the spiral structured scaffolds. In this study, we investigated the fiber thickness, gap distance and wall thickness of the spiral structure on the behavior of osteoblast cells. The human osteoblast cells are seeded on spiral structured scaffolds with various fiber thickness, gap distance and wall thickness and cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds are evaluated. It was found that increasing the thickness of nanofiber layer not only limited the cell infiltration into the scaffolds, but also restrained the osteoblastic cell phenotype development. Moreover, the geometric effect studies indicated that scaffolds with the thinner wall and gap distance 0.2mm show the best bioactivity for osteoblasts.

1Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ 07030, USA 2Xylos Corporation, Langhorne, PA 19047, USA 3Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China 4RainDance Technologies Inc., Lexington, MA 02421, USA 5McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA

Polymeric nanofiber matrices have already been widely used in tissue engineering. However, the fabrication of nanofibers into complex three-dimensional (3D) structures is restricted due to current manufacturing techniques. To overcome this limitation, we have incorporated nanofibers onto spiral-structured 3D scaffolds made of poly (epsilon-caprolactone) (PCL). The spiral structure with open geometries, large surface areas, and porosity will be helpful for improving nutrient transport and cell penetration into the scaffolds, which are otherwise limited in conventional tissue-engineered scaffolds for large bone defects repair. To investigate the effect of structure and fiber coating on the performance of the scaffolds, three groups of scaffolds including cylindrical PCL scaffolds, spiral PCL scaffolds (without fiber coating), and spiral-structured fibrous PCL scaffolds (with fiber coating) have been prepared. The morphology, porosity, and mechanical properties of the scaffolds have been characterized. Furthermore, human osteoblast cells are seeded on these scaffolds, and the cell attachment, proliferation, differentiation, and mineralized matrix deposition on the scaffolds are evaluated. The results indicated that the spiral scaffolds possess porosities within the range of human trabecular bone and an appropriate pore structure for cell growth, and significantly lower compressive modulus and strength than cylindrical scaffolds. When compared with the cylindrical scaffolds, the spiral-structured scaffolds demonstrated enhanced cell proliferation, differentiation, and mineralization and allowed better cellular growth and penetration. The incorporation of nanofibers onto spiral scaffolds further enhanced cell attachment, proliferation, and differentiation. These studies suggest that spiral-structured nanofibrous scaffolds may serve as promising alternatives for bone tissue engineering applications.

Many next-generation biomaterials will need the ability to not only promote healthy tissue integration but to simultaneously resist bacterial colonization and resulting biomaterials-associated infection. For this purpose, antimicrobial nanofibers of polycaprolactone (PCL) were fabricated by incorporating calcium peroxide. PCL nanofibers containing different ratios of calcium peroxide (1%, 5% and 10% (w/w)) with or without ascorbic acid were fabricated using an electrospinning technique. Antimicrobial evaluations confirmed the inhibitory properties of the nanofibers on the growth of E. coli and S. epidemidis because of a significant burst release of calcium peroxide from the nanofibers. Analysis of tissue cell response showed that despite an initial toxic effect over the first 24 h, after 4 days of culture, osteoblast viability and morphology were both healthy. These results demonstrate that oxygen-generating nanofibers can be designed and developed to provide a short-term peroxide-based antimicrobial response while still maintaining attractive tissue-integration properties.

The Blood Brain Barrier (BBB) represents a fundamental challenge towards development of therapy for disease in the brain such as brain tumor, Parkinsons disease and Alzheimers disease. The passage across the BBB is vital to the success of a therapeutic platform and nanotechnology based systems provide great potential for solving the problem. This chapter discusses the structures and functions of the BBB, the transport mechanisms at the BBB, the strategies for crossing the BBB, and nanostructures for delivering drugs and imaging in the brain.

Peripheral nerve regeneration can be significantly enhanced by the distribution of the extracellular matrix (ECM) proteins at an increasing concentration along the length of a scaffold. In this study, we have created a gradient of an extracellular matrix protein, laminin, on nanofibrous scaffolds using an external magnetic field. The laminin was crosslinked to ferritin, a biocompatible protein with functional amino and carboxylic acid groups on the surface and a magnetically inducible iron core. The presence of laminin gradients on the scaffolds was demonstrated through immunofluorescent staining with antibodies against laminin. When culturing Schwann cells on the nanofibrous scaffolds, the number of cells increased along the gradients with increasing laminin concentrations. The method developed in this study allows for precise control over the gradient formation in terms of distances and concentrations and thus provides a platform for high throughput screening of cell materials interactions. The nanofibrous scaffolds with laminin gradients can be potentially used in neural tissue engineering.

In the previous studies, we have successfully developed a novel spiral structured nanofibrous scaffolds with improved osteoconductivity for bone tissue engineering. The spiral structure design facilitates the nutrient transport and waste removal, and allows uniform cellular growth and distribution within the scaffolds, thus enhanced the bioactivity of the scaffolds. In this chapter, HAP and BMP-2 were incorporated within the nanofibrous spiral scaffolds in order to enhance the osteoinductivity of the established system. The effect of the blending materials was evaluated through cell proliferation, cell differentiation of human osteoblast cells seeded on the scaffolds and cultured for 4 and 8 days. The results has demonstrated that the functionalization of PCL nanofibrous spiral scaffolds leads to higher ALP expression level and increased amount of mineralization level however lower cell proliferation rate.

Due to several drawbacks associated with autografts and allografts, tissue-engineering approaches have been widely used to repair peripheral nerve injuries. Most of the traditional tissue-engineered scaffolds in use are either tubular (single or multi-lumen) or hydrogel-based cylindrical grafts, which provide limited surface area for cell attachment and regeneration. Here, we show a novel poly(lactide-co-glycotide) (PLGA) microsphere-based spiral scaffold design with a nanofibrous Surface that has enhanced surface areas and possesses sufficient mechanical properties and porosities to support the nerve regeneration process. These scaffolds have an open architecture that goes evenly throughout the scaffolds hence leaving enough volume for media influx and deeper cell penetration into the scaffolds. The in vitro tests conducted using Schwann cells show that the nanofibrous spiral scaffolds promote higher cell attachment and proliferation when compared to contemporary tubular scaffolds or nanofiber-based tubular scaffolds. Also, the nanofiber coating on the surfaces enhances the surface area, mimics the extracellular matrix and provides unidirectional alignment of cells along its direction. Hence, we propose that these scaffolds Could alleviate some drawbacks in current nerve grafts and could potentially be used in nerve regeneration.