Morten Østergaard Andersen

siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles.

Surface functionalisation of PLGA nanoparticles for gene silencing

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFalpha in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

MicroRNA functionalized microporous titanium oxide surface by lyophilization with enhanced osteogenic activity.

Developing biomedical titanium (Ti) implants with high osteogenic ability and consequent rigid osseointegration is a constant requirement from the clinic. In this study, we fabricate novel miRNA functionalized microporous Ti implants by lyophilizing miRNA lipoplexes onto a microporous titanium oxide surface formed by microarc oxidation (MAO). The microporous titanium oxide surface provides a larger surface area for miRNA loading and enables spatial retention of the miRNAs within the pores until cellular delivery. The loading of lipoplexes into the micropores on the MAO Ti surface is facilitated by the superhydrophilicity and Ti-OH groups gathering of the MAO surface after UV irradiation followed by lyophilization. A high miRNA transfection efficiency was observed in mesenchymal stem cells (MSCs) seeded onto the miRNA functionalized surface with no apparent cytotoxicity. When functionalizing the Ti surface with miR-29b that enhances osteogenic activity and antimiR-138 that inhibits miR-138 inhibition of endogenous osteogenesis, clear stimulation of MSC osteogenic differentiation was observed, in terms of up-regulating osteogenic expression and enhancing alkaline phosphatase production, collagen secretion and ECM mineralization. The novel miRNA functionalized Ti implants with enhanced osteogenic activity promisingly lead to more rapid and robust osseointegration of a clinical bone implant interface. Our study implies that lyophilization may constitute a versatile method for miRNA loading to other biomaterials with the aim of controlling cellular function.

RNAi Using a Chitosan/siRNA Nanoparticle System: In Vitro and In Vivo Applications

Delivery is a key issue in development of clinically relevant RNAi therapeutics. Polymeric nanoparticles formed by self-assembly of polycations with siRNA can be used for extracellular delivery, cellular uptake and intracellular trafficking as a strategy to improve the therapeutic potential of siRNA. This chapter describes a chitosan-based nanoparticle system for in vitro and in vivo transfection of siRNA into cells. The method exploits the mucoadhesive and mucopermeable properties of this cationic polysaccharide to deliver siRNA across mucosal epithelium and provides a platform for targeting human diseases with RNAi therapeutics.

Investigation of particle-functionalized tissue engineering scaffolds using X-ray tomographic microscopy

A low-density, porous chitosan/poly-(dl-lactide-co-glycolide) (PLGA) microparticle composite scaffold was produced by thermally induced phase separation followed by lyophilization, to provide a bicontinuous microstructure potentially suitable for tissue engineering and locally controlled drug release. PLGA particles were mixed into the chitosan solution and subsequent phase separation during chitosan solidification forced PLGA particles into chitosan phase (Plateau borders). The distributions of volume, surface area, and elongation of 15,422 inclusions of agglomerated PLGA particles were calculated and approximated with log-normal distribution functions from nanotomography reconstructions. Cluster analysis revealed a homogenous inclusion distribution throughout the scaffold. The spatial location and orientation of individual inclusions within the Plateau borders of the scaffold were determined and from these the nearest-neighbor inter-inclusion distance distribution calculated, showing a mean of 2.5 microm. The depth of the inclusions in Plateau borders peaks at 700 or 125 nm, respectively, indicating a step-wise drug release from inclusions successively exposed during scaffold decomposition. Particle diameter ranged from 400 nm to 3 microm and inclusion Feret lengths ranged from 800 nm to 12 microm. These findings on composite morphology and distribution of inclusions are fundamental for predicting scaffold deterioration and particle-mediated drug release during ex vivo and in vivo cell cultivation.

Simple additive manufacturing of an osteoconductive ceramic using suspension melt extrusion

OBJECTIVE Craniofacial bone trauma is a leading reason for surgery at most hospitals. Large pieces of destroyed or resected bone are often replaced with non-resorbable and stock implants, and these are associated with a variety of problems. This paper explores the use of a novel fatty acid/calcium phosphate suspension melt for simple additive manufacturing of ceramic tricalcium phosphate implants. METHODS A wide variety of non-aqueous liquids were tested to determine the formulation of a storable 3D printable tricalcium phosphate suspension ink, and only fatty acid-based inks were found to work. A heated stearic acid-tricalcium phosphate suspension melt was then 3D printed, carbonized and sintered, yielding implants with controllable macroporosities. Their microstructure, compressive strength and chemical purity were analyzed with electron microscopy, mechanical testing and Raman spectroscopy, respectively. Mesenchymal stem cell culture was used to assess their osteoconductivity as defined by collagen deposition, alkaline phosphatase secretion and de-novo mineralization. RESULTS After a rapid sintering process, the implants retained their pre-sintering shape with open pores. They possessed clinically relevant mechanical strength and were chemically pure. They supported adhesion of mesenchymal stem cells, and these were able to deposit collagen onto the implants, secrete alkaline phosphatase and further mineralize the ceramic. SIGNIFICANCE The tricalcium phosphate/fatty acid ink described here and its 3D printing may be sufficiently simple and effective to enable rapid, on-demand and in-hospital fabrication of individualized ceramic implants that allow clinicians to use them for treatment of bone trauma.

Co-delivery of siRNA and doxorubicin to cancer cells from additively manufactured implants

Tumors in load bearing bone tissue are a major clinical problem, in part because surgical resection invokes a dilemma whether to resect aggressively, risking mechanical failure, or to resect conservatively, risking cancer recurrence due to residual malignant cells. A chemo-functionalized implant, capable of physically supporting the void while killing residual cancer cells, would be an attractive solution. Here we describe a novel additively manufactured implant that can be functionalized with chitosan/siRNA nanoparticles. These induce long term gene silencing in adjacent cancer cells without showing toxicity to normal cells. When scaffolds are functionalized with siRNA/chitosan nanoparticles and doxorubicin in combination, their effects synergized leading to cancer cell death. This technology may be used to target resistance genes by RNA interference and thereby re-sensitizing the cancer cells to co-delivered chemotherapy.

RNA Interference Enhanced Implants

RNA interference (RNAi) has in the last decade seen ever increasing use in cell biology as both a tool with which most cell functions can be modulated and as a natural mechanism through which cells regulate their gene expression. As RNAi can be used to direct stem cell differentiation, enhance tissue development, modulate inflammation and control other cellular phenomenon relevant to implant medicine the potential for using RNAi to enhance regenerative medicine is huge. Unfortunately, there are a number of obstacles and special concerns that need to be addressed before the use of RNAs on scaffolds and implants can go into clinical use. A number of studies have recently started to address these problems and demonstrate both the current limitations and the great promise of the technique. In this chapter we will discuss the basic principles of RNAi, it’s applications in regenerative medicine and approaches to functionalize implants. We will describe various delivery methods for the inducer of RNAi, small interfering RNAs, in conjunction with scaffolds and how the application determines the delivery strategy. Finally, we describe how natural RNAi, mediated by microRNAs, can be manipulated by similar delivery techniques.

The Role of MicroRNAs in Natural Tissue Development and Application in Regenerative Medicine

Many cellular functions rely on the coordinated expression and repression of a large number of messenger RNAs; these are tightly controlled in part by microRNAs (miRNAs) at the posttranscriptional level. The number of characterised miRNAs that are involved in tissue development and repair is steadily increasing, and our understanding of their functions is starting to merge. Modulating miRNA levels through externally applied stimuli enables us to control the translation of numerous mRNAs giving us unprecedented control over cellular events; therefore, we predict that such techniques will revolutionise regenerative medicine. This chapter will introduce miRNA biology and their role in controlling pluripotency, stem cell differentiation, proliferation, senescence, survival, inflammation and angiogenesis. There are several strategies by which miRNA-modulating technologies can be used to specifically target tissue engineering and repair, either in culture or in association with implanted cells and/or implants. We will here summarise these methods providing examples from present literature. Based on previous results, we will also predict more advanced technologies that may deliver miRNA in a spatial/temporally regulated manner that may imitate natural miRNA expression. Furthermore, exogenous miRNAs, carried between cells in secreted vesicles, have recently been characterised and may further increase the role and potential of miRNA in relation to regenerative medicine.

The Application of Nanotechnology for Implant Drug Release

The use of medical implants is a cornerstone of modern medicine. All implants face, however, a number of challenges including infection and inflammation which cause many of them to fail. In addition, tissue engineering implants must also direct stem cell differentiation and tissue regeneration in order to work properly. These problems may be overcome using drugs that are delivered directly from the implant. For this to work the drugs have to be protected until they have performed their function, their release must be timed with when they are needed, they may have to affect specific regions of an implant only and some drugs must be delivered to specific sub-cellular locations in certain cells types. This chapter explores how various forms of nanotechnology may be employed to reach these goals and reviews many of the studies that have used nanotechnology for different implant mediated drug release applications.