Uropathogenic Escherichia coli can cause cystitis at extremely low inocula in a pig model.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. Experimental models that accurately reflect the high susceptibility to UTI in humans have, however, been lacking. This situation has limited detailed research into the early bladder colonization by uropathogens and the early innate defence mechanisms elicited to prevent this. We recently presented a model of urinary tract infection in pigs, animals that are naturally susceptible to UTI and have greater similarity to the physiology and anatomy of the human urinary tract than traditional rodent UTI models. In the current study, we used the pig model to investigate the minimal infectious inoculum of uropathogenic Escherichia coli, the most common cause of urinary tract infection. We show that in this animal a few individual bacteria that come into contact with the urothelium can give rise to fulminant cystitis, indicating the high infectious potential of uropathogenic E. coli.

In vivo non-invasive monitoring of tissue development in 3D printed subcutaneous bone scaffolds using fibre-optic Raman spectroscopy.

The development of novel biomaterials for regenerative therapy relies on the ability to assess tissue development, quality, and similarity with native tissue types in in vivo experiments. Non-invasive imaging modalities such as X-ray computed tomography offer high spatial resolution but limited biochemical information while histology and biochemical assays are destructive. Raman spectroscopy is a non-invasive, label-free and non-destructive technique widely applied for biochemical characterization. Here we demonstrate the use of fibre-optic Raman spectroscopy for in vivo quantitative monitoring of tissue development in subcutaneous calcium phosphate scaffolds in mice over 16 weeks. Raman spectroscopy was able to quantify the time dependency of different tissue components related to the presence, absence, and quantity of mesenchymal stem cells. Scaffolds seeded with stem cells produced 3-5 times higher amount of collagen-rich extracellular matrix after 16 weeks implantation compared to scaffolds without. These however, showed a 2.5 times higher amount of lipid-rich tissue compared to implants with stem cells. Ex vivo micro-computed tomography and histology showed stem cell mediated collagen and bone development. Histological measures of collagen correlated well with Raman derived quantifications (correlation coefficient in vivo 0.74, ex vivo 0.93). In the absence of stem cells, the scaffolds were largely occupied by adipocytes. The technique developed here could potentially be adapted for a range of small animal experiments for assessing tissue engineering strategies at the biochemical level.

Treating mouse skull defects with 3D-printed fatty acid and tricalcium phosphate implants.

Skull surgery, also known as craniectomy, is done to treat trauma or brain diseases and may require the use of an implant to reestablish skull integrity. This study investigates the performance of 3D printed bone implants in a mouse model of craniectomy with the aim of making biodegradable porous implants that can ultimately be fitted to a patient's anatomy. A nonpolymeric thermoplastic bioink composed of fatty acids and ß-tricalcium phosphate was used to 3D print the skull implants. Some of these were sintered to yield pure ß-tricalcium phosphate implants. The performance of nonsintered and sintered implants was then compared in two semi-quantitative murine calvarial defect models using computed tomography, histology, and luciferase activity. Both types of implants were biocompatible, but only sintered implants promoted defect healing, with osseointegration to adjacent bone and the formation of new bone and bone marrow tissue in the implant pores. Luciferase scanning and histology showed that mesenchymal stem cells seeded onto the implants engraft and proliferate on the implants after implantation and contribute to forming bone. The experiments indicate that fatty acid-based 3D printing enables the creation of biocompatible and bone-forming ß-tricalcium phosphate implants.

Falcarindiol Purified From Carrots Leads to Elevated Levels of Lipid Droplets and Upregulation of Peroxisome Proliferator-Activated Receptor-γ Gene Expression in Cellular Models.

Falcarindiol (FaDOH) is a cytotoxic and anti-inflammatory polyacetylenic oxylipin found in food plants of the carrot family (Apiaceae). FaDOH has been shown to activate PPARγ and to increase the expression of the cholesterol transporter ABCA1 in cells, both of which play an important role in lipid metabolism. Thus, a common mechanism of action of the anticancer and antidiabetic properties of FaDOH may be due to a possible effect on lipid metabolism. In this study, the effect of sub-toxic concentration (5 µM) of FaDOH inside human mesenchymal stem cells (hMSCs) was studied using white light microscopy and Raman imaging. Our results show that FaDOH increases lipid content in the hMSCs cells as well as the number of lipid droplets (LDs) and that this can be explained by increased expression of PPARγ2 as shown in human colon adenocarcinoma cells. Activation of PPARγ can lead to increased expression of ABCA1. We demonstrate that ABCA1 is upregulated in colorectal neoplastic rat tissue, which indicates a possible role of this transporter in the redistribution of lipids and increased formation of LDs in cancer cells that may lead to endoplasmic reticulum stress and cancer cell death.

Simple Defocused Fiber Optic Volume Probe for Subsurface Raman Spectroscopy in Turbid Media.

We investigated the ability to perform deep subsurface Raman spectroscopy in turbid media using a simple fiber optic volume probe. Being able to collect Raman signals from regions deep within a biological sample provides the ability to noninvasively study underlying living tissue and tissue engineered constructs with high chemical specificity. Spatially offset Raman spectroscopy has shown great potential for obtaining subsurface Raman signals in biological samples. The applicability of the method for in vivo studies depends on the system complexity and small size probes are often desirable. Most real-time studies on human patients utilizing Raman spectroscopy have been performed with easy-to-handle miniaturized probes. Here we show both experimentally and theoretically that the sampling depth from a simple volume probe can be controlled by changing the probe to sample distance effectively suppressing Raman and fluorescence contributions from shallow sample layers while favoring the collection of signals from deeper layers. Relative spectral intensities as function of probe to sample distance were investigated for layered phantoms of poly(methyl methacrylate) and trans-stilbene and compared with theory. The volume probe was then utilized for the collection of spectra from phantoms mimicking in vivo transcutaneous measurement configurations of bone and engineered scaffold as well as from an ex vivo sample of bone and soft tissue. Together the results show that Raman fiber optic volume probes can be utilized for subsurface Raman spectroscopy in turbid media, providing a simple alternative to spatially offset Raman systems for, e.g., noninvasive monitoring and studying mineralized tissue and implanted scaffolds in vivo.

Patient-specific 3D printed plates improve stability of Le Fort 1 osteotomies in vitro.

PURPOSE: Selective laser melting used to manufacture patient-specific 3D-printed (PSP) plates is a delicate process, which may introduce weakened areas in the plates, with risk of fracture. This in vitro study's purpose was to test the ability of PSP plates to stabilize Le Fort I osteotomies compared with manually adapted stock plates. The study's objectives were to measure the force needed to compress the osteotomy and evaluate whether the PSP plates would break during compression. MATERIALS AND METHODS: This controlled in vitro study evaluated the maxillary stability using the clinical data from 7 patients. The virtually planned maxillary reposition was 3D-printed in 2 copies, and the osteotomy gap was fixated by either PSP plates or stock plates. The models were compressed until the Le Fort I osteotomy gap was eliminated. The primary outcome was the force needed to compress the model. The primary predictor variable was a comparison between PSP and stock plates. Secondary outcome measurements were the slope of elastic modulus, yield point, and force needed for 2 mm compression. Statistical testing was performed by Wilcoxon signed-rank test with significance level at P ≤ 0.05. RESULTS: The PSP plates performed better than stock plates in all outcome measurements. None of the plates broke during compression despite forces of more than 4000 N. The first point of failure in PSP plates was the first screw cranial to the osteotomy. In comparison, the first point of failure in stock plates was in the plates' bend at the osteotomy. CONCLUSION: In this in vitro setup, the Le Fort I osteotomies fixated with PSP plates were more stable than the osteotomies fixated with conventional stock plates. No adverse effects occurred during testing of PSP plates; thus, PSP plates seem to be a safe alternative to stock plates and may even be preferable.

Co-delivery of siRNA and etoposide to cancer cells using an MDEA esterquat based drug delivery system.

Cancer has become the leading cause of death in many countries. Chemotherapy is a key component in the treatment of most cancers but has limited efficacy if the cancer develops resistance to the treatment over time and recur. RNA interference may be used to reduce the production of the proteins responsible for chemotherapeutic resistance. Small interfering RNAs (siRNA) may be used to induce RNA interference but the application of these to cancer cells is hampered by poor serum stability and delivery to their cytoplasmic site of activity. This work introduces a novel nanoparticle delivery system for siRNA and hydrophobic anticancer drugs. The system is based on a cationic MDEA esterquat, which is widely and safely used in personal care products but has never been assessed for drug delivery applications. We show that MDEA forms spherical compact nanoparticles when combined with siRNA that delivers the siRNA to cancer cells where it induces gene silencing. By combining DOPE and MDEA in ratios of 2:1 and 3:1, even higher gene silencing levels (>90%) may be achieved. The system is capable of combinational therapy by co-delivering siRNA and the chemotherapeutic drug etoposide to cancer cells and these particles both induce gene silencing and chemotherapy induced cell death. We believe the present system may be used for intra-tumoral injection of chemotherapy in solid chemotherapy resistant tumors and for systemic delivery with further development.

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.

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.