Otto C. Wilson

Embedding of magnetic nanoparticles in polycaprolactone nanofiber scaffolds to facilitate bone healing and regeneration

Scaffolds used for tissue engineering are made to mimic natural surroundings of tissues, the extracellular matrix (ECM). The ECM plays a large part in maintaining the structural integrity of the connective tissue. When producing a tissue in the laboratory, structural integrity of the cells is ensured only when a biomimetic ECM is present. Nanofibrous polymer fibers have been chosen for their resemblance to natural fibers of the ECM and their capability to provide the support necessary for cells to grow and differentiate into tissue. Polycaprolactone based nanofibrous scaffolds for tissue engineering have been fabricated through the electrospinning process. Electrospinning is a simple and cost-effective method for producing nanofibers which involves applying a high voltage to a falling polymer solution to form a fluid jet producing nanofibers. Magnetic nanoparticles (MNPs) have been incorporated within the nanofibers by addition of MNPs to the polymer solution to increase the rate of bone cell growth, prol...

Development of a custom biological scaffold for investigating ultrasound-mediated intracellular delivery

In vitro investigations of ultrasound mediated, intracellular drug and gene delivery (i.e. sonoporation) are typically carried out in cells cultured in standard plastic well plates. This creates conditions that poorly resemble in vivo conditions, as well as generating unwanted ultrasound phenomena that may confound the interpretation of results. Here, we present our results in the development of a biological scaffold for sonoporation studies. The scaffolds were comprised of cellulose fibers coated with chitosan and gelatin. Scaffold formulation was optimized for adherence and proliferation of mouse fibroblasts in terms of the ratio and relative concentration of the two constituents. The scaffolds were also shown to significantly reduce ultrasound reflections compared to the plastic well plates. A custom treatment chamber was designed and built, and the occurrence of acoustic cavitation in the chamber during the ultrasound treatments was detected; a requirement for the process of sonoporation. Finally, experiments were carried out to optimize the ultrasound exposures to minimize cellular damage. Ultrasound exposure was then shown to enable the uptake of 100nm fluorescently labeled polystyrene nanoparticles in suspension into the cells seeded on scaffolds, compared to incubation of cell-seeded scaffolds with nanoparticles alone. These preliminary results set the basis for further development of this platform. They also provide motivation for the development of similar platforms for the controlled investigation of other ultrasound mediated cell and tissue therapies.

Identification of Bacteria and Sterilization of Crustacean Exoskeleton Used as a Biomaterial

Derivatives of the crustacean exoskeleton like chitin have a long history of being used as biomaterials. In the BONE/CRAB lab, the blue claw crab exoskeleton is our biomaterial of choice for a possible bone implant material. The blue claw crustacean, Callinectes sapidus, is found in the Chesapeake Bay. Chitinolytic bacteria, such as those belonging to the Vibrio and Bacillus genera, are common to marine crustaceans. Previous in vitro studies in our lab indicated that bacterial contamination is a major concern. One of the fundamental considerations with the use of an implant biomaterial is sterilization. Materials implanted into the human body must be sterile to avoid subsequent infection or other more serious consequences. An effective sterilization method strikes a balance between the required sterility level and minimum detrimental effect on the properties of the biomaterial while being cost-effective, simple, and readily available. The objective of this study was to isolate, identify bacterial contaminants and develop the best sterilization method for those bacteria found on blue claw crab exoskeleton. Bacteria belonging to the genera Bacillus were identified based on bacterial growth morphologies of dry, dull, raised, rough, and white-grey appearance on LB agar. Bacillus members form endospores which are difficult to eliminate and poses a significant concern for implantable materials. There was no bacterial growth on the TCBS agar plates which is a differential and selective media for Vibrio species. Antimicrobial susceptibility tests were conducted to measure the effectiveness of 70% isopropyl alcohol, povidone-iodine, and household bleach against the bacteria found. The susceptibility tests revealed sensitivities towards the compounds studied. Bacterial identification and susceptibility provide vital guidance to the best method to sterilize while maintaining biological performance. Further studies will evaluate the effect the sterilization protocol has on the physical, chemical, and biological properties of the implant material.

Ties That Bind: Evaluation of Collagen I and α -Chitin

Collagen and chitin are two fascinating unique matrix framework molecules that play a key role in the hierarchical development of hard tissue. Of the 28 identified types, collagen I accounts for more 90% of the total collagen found in nature and is the main constituent of bone. Of the three identified types, α-Chitin is the most abundant form found in nature and is the main component in the exoskeletons of crustaceans like crabs. α-chitin is a plentiful natural material, similar to collagen I in composition, structure, and function. Both collagen I and α-chitin have extensive use in the biomedical field. In an effort to obtain processing strategies and microstructure design criteria for the development of new bone substitute materials with novel properties, biomimetics was used to establish relationships between the properties of collagen I and α-chitin. The objective of this study was to compare collagen I and α-chitin to illuminate notable similarities and differences in chemical, physical, structural and functional properties. The more intriguing similarities involve hierarchical structuring, liquid crystal characteristics and self assembly. One of the more conspicuous differences is the biochemistry of collagen I, a protein, and α-chitin, a polysaccharide. Collagen I and α-chitin were studied using electron microscopy, elemental and thermal analysis. Bouligand or a twisting plywood system was observed on both collagen and chitin micrographs. One major difference is that the SEM micrographs of collagen fibers demonstrated a banding pattern while chitin fibers did not. The elemental analysis revealed that chitin’s carbon/nitrogen of 3 was doubled in collagen. Thermal analysis revealed that chitin had two thermal transitions while collagen had three. Both collagen and chitin have a thermal event associated with the evolution of freely bound water. These are among the similarities and differences that will be addressed in this paper.

Magnetic nanoparticle-loaded alginate beads for local micro-actuation of in vitro tissue constructs

Magnetic nanoparticles (MNPs) self-align and transduce magnetic force, two properties which lead to promising applications in cell and tissue engineering. However, the toxicity of MNPs to cells which uptake them is a major impediment to applications in engineered tissue constructs. To address this problem, MNPs were embedded in millimeter-scale alginate beads, coated with glutaraldehyde cross-linked chitosan, and loaded in acellular and MDA-MB-231 cancer cell-seeded collagen hydrogels, providing local micro-actuation under an external magnetic field. Brightfield microscopy was used to assess nanoparticle diffusion from the bead. Phase contrast microscopy and digital image correlation were used to track collagen matrix displacement and estimate intratissue strain under magnetic actuation. Coating the magnetic alginate beads with glutaraldehyde-chitosan prevents bulk diffusion of nanoparticles into the surrounding microenvironment. Further, the beads exert force on the surrounding collagen gel and cells, resulting in intratissue strains of 0-10% tunable with bead dimensions, collagen density, and distance from the bead. Cells seeded adjacent to the embedded beads are subjected to strain gradients without loss of cell viability over two days culture. This study describes a simple way to fabricate crosslinked magnetic alginate beads to load in a collagen tissue construct without direct exposure of the construct to nanoparticles. The findings are significant to in vitro studies of mechanobiology in enabling precise control over dynamic mechanical loading of tissue constructs.

Extraction and Characterization of a Soluble Chicken Bone Collagen

Our understanding of collagen has increased considerably. Collagen is a fibrous protein that plays a key role in the framework and development of hard and connective tissue. Collagen has vast structural possibilities for modifications to generate novel properties, functions, and applications especially in the bone implant arena. Collagen is found throughout nature in skin, tendon, bone, cartilage, etc. and in numerous different species. Of the 28 identified types, collagen I is the most abundant in nature and the main component of bone. Despite its abundance, the utilization and characterization has been restricted by its insolubility. Collagen solutions are usually viscous and difficult to study using techniques such as zeta potential analysis, chromatography and electrophoresis. The specific aim of this study was to develop a protocol to extract and solubilize collagen from chicken femur bone. In the thermogram of our chicken femur bone collagen (CBC), there was only a 35% reduction in weight. This signifies that the CBC collagen sample contained 65% inorganic material. Elemental analysis revealed that CBC consisted of 11% Carbon (C), 2% Hydrogen (H), and 3% Nitrogen (N), totaling 16%. This is substantially lower than 77%, the calculated theoretical value of CHN% in collagen. Our CBC shared the theoretical Carbon/Nitrogen value of 3.4. Elemental analysis confirms that the CBC contained elements other than carbon, nitrogen, and hydrogen. This suggests that the CBC sample was not fully demineralized and adjustments need to be made to the existing collagen extraction protocol. Further studies are needed to confirm chemical composition of the CBC in solution. This work is part of a larger study to compare collagen I, derived from bone, and α-chitin, derived from crab exoskeleton.

Optical properties of a new inorganic liquid crystal

We measured the optical properties of a new inorganic liquid crystal material. By changing the synthesization process AlFe colloidal rods approximately 200-400 nm in length and ellipsoidal platelets 2000-5000 nm in diameter can be created. In this paper we show that the optical properties of this new material change with its exposure to an external magnetic field making it particularly suitable for medical diagnostic applications.