Organic nanotubes called helical rosette nanotubes (HRN) have been synthesized in this study for bone tissue engineering applications. They possess intriguing properties for various bionanotechnology applications since they can be designed to mimic the nanostructured constituent components in bone such as collagen fibers and hydroxyapatite (Ca5(PO4)3(OH)) which bone cells are naturally accustomed to interacting with. This is in contrast to currently used orthopaedic materials such as titanium which do not possess desirable nanometer surface roughness. The objective of this in vitro study was to determine bone-forming cell (osteoblasts) interactions on titanium coated with HRNs. Results of this study showed for the first time increased osteoblast adhesion on titanium coated with HRNs compared to those not coated with HRNs. In this manner, this study provided evidence that HRNs should be further considered for orthopaedic applications.

We have demonstrated deposition of dexamethasone thin films via matrix assisted pulsed laser evaporation (MAPLE). Infrared analysis revealed that dexamethasone thin films deposited by MAPLE and by drop casting had a similar absorbance spectra. AFM imaging of the MAPLE-deposited dexamethasone thin film revealed 2–10 μm punctuate ring-like structures. Deposited dexamethasone was tested for positive bioactivity by treating primary microglia cells with lipopolysacchride and measuring nitric oxide (NO) production. The successful deposition of dexamethasone thin films can be used on neural implants to prevent tissue injury and inflammatory response.

When bovine ligament fibroblast cells were cultured on parallel micro-grooved surfaces, they aligned their long axes parallel to the groove direction. This alignment was dependent on the groove depth, with increasing groove depth increasing guided cell alignment. When cells were cultured in a physiological dc electric field (EF) on non-grooved, flat surfaces, the cells aligned in response to the EF, with their long axes perpendicular to the EF vector. This response was EF strength dependent, increasing EF strength (from 20 to 200mV/mm) increased cell alignment, perpendicular to the EF vector. These two guidance cues were applied simultaneously, so that the EF vector was parallel to the groove direction. At high but still physiological EF strengths (200mV/mm) cells ignored the topography and were guided by the EF alone, aligning perpendicular to the EF vector, as on non-grooved surfaces. At low field strengths (20mV/mm) cells responded only to the topographic guidance cue, with cells aligning parallel to the grooves and therefore also to the EF vector. Intermediate field strengths (50 to 100mV/mm) produced a mixed response, with cells responding to both guidance cues. The effect of removing serum from the culture medium on the EF and topographical guidance of fibroblast cells was studied and the results were compared to cells on non-grooved surfaces. Removal of serum produced a small but significant decrease in the angle of cell alignment for cells on non-grooved surfaces, from 78 to 63 degrees, relative to the EF vector, but did not completely suppress the EF guidance cue. In contrast, the EF guidance of cells on grooved substrates was suppressed almost completely by the absence of serum, with cells responding only to the grooved topography, aligning their long axis parallel to the grooves and the EF vector. These results imply that alignment of fibroblasts by topography is serum-independent, but alignment by EFs is serum-dependent. In addition they demonstrate that the alignment of fibroblast cells can be tailored by the dual guidance cues of topography and electric fields.

Tissues and organs exhibit distinct shapes and functions nurtured by vascular connectivity. In order to mimic and examine these intricate structure-function relationships, it is necessary to develop efficient strategies for assembling tissue-like constructs. Many of the top-down fabrication techniques used to build microelectromechanical systems, including photolithography, are attractive due to the similar feature sizes, but are not suitable for delicate biological systems or aqueous environments. A layer-by layer approach has been proposed by us to pattern functional cell structures in three dimensions. Freeform cell structures are created by the inkjet method, in which cells are entrapped within hydrogels and crosslinked on demand. The cells are viable, functional and show potential for cell maturation as exemplified by the diversion of hematopoietic stem cells into multiple cell types. These results show promise for many tissue engineering applications.

Laser-fabricated joints of sub-millimeter widths between biocompatible, dissimilar materials have the potential for applications as encapsulation of miniature implant biomedical devices. In this work, we briefly describe the laser joining method of a very promising system, polyimide/titanium-coated borosilicate glass, and present and discuss results from characterization of such laser joints by means of mechanical failure (tensile) tests, optical microscopy, X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Our results suggest the formation of strong chemical bonds between Ti-containing species and certain polymeric functional groups. Mechanical tensile strength failure test showed that such joint experience only limited, disappearing with time degradation as a result of soaking in physiological solutions.

This work describes the integration of novel microfabrication techniques for vascular tissue engineering applications in the context of a novel biodegradable elastomer. The field of tissue engineering and organ regeneration has been borne out of the high demand for organ transplants. However, one of the critical limitations in regeneration of vital organs is the lack of an intrinsic blood supply. This work expands on the development of scaffolds for vascular tissue engineering applications by employing microfabrication techniques. Unlike previous efforts, this work focuses on fabricating single layer and three-dimensional scaffolds from poly(glycerol-sebacate) (PGS), a novel biodegradable elastomer with superior mechanical properties. The transport properties of oxygen and carbon dioxide in PGS were measured through a series of time-lag diffusion experiments. The results of these measurements were used to calculate a characteristic length scale for oxygen diffusion limits in solid PGS scaffolds. Single layer and three-dimensional microfluidic scaffolds were then produced using fabrication techniques specific for PGS. This work has resulted in the fabrication of solid PGS-based scaffolds with biomimetic fluid flow and capillary channels on the order of 10 microns in width. Fabrication of complex, three-dimensional microfluidic PGS scaffolds was also demonstrated by stacking and bonding multiple microfluidic layers.

This work is a part of an on-going research effort to develop an array of micro thermoelectric coolers (TECs) for highly localized control of temperature at the cellular level. Prefabrication experimentation and modeling were carried out to understand the behavior of the proposed device. Mathematical models were used to identify important device parameters and optimal device dimensions. Preliminary experiments have shown that it is feasible to produce the TECs through electrodeposition of bismuth and telluride on modules produced using a modified multistep LIGA (Lithographie, Galvanoformung and Abformung) technique. The development and characterization of the proposed TECs would enable the bioengineer highly localized control of temperature in a native or artificial tissue system. Thus enabling further usage of low temperatures in biological systems for both destructive (cryosurgical) and beneficial (cryopreservation) procedures.

A fresh approach with a novel process and a hybrid material is explored for developing designer 3-dimensional functional tissue scaffolds. The process of two photon-induced polymerization generally used for electronic and optical materials is introduced for developing biological scaffolds. Hybrid materials containing organic-inorganic units, ORMOCERs, fabricated with this process are tested for biocompatibility using various cell-types and compared with known standards such as polystyrene and ECM (Extracellular Matrix). Results show good adherence of different cells to these materials, and a growth rate comparable to bioactive materials. Structures with various surface topologies are developed and tested for preferential growth. The study is a first step towards developing bioactive and bioresorbable heterogeneous three-dimensional scaffolds.

The synthesis of poly(lactic-co-glycolic acid) (PLGA) by the ring opening copolymerization of D, L-lactide and glycolide was performed at 110 °C to 130 °C using Sn(Oct)2 as catalyst, 1, 10-decanediol as initiator in a supercritical sc-CO2 expanded medium at pressures of up to 3, 500 psi. Due to the limited monomer solubility in sc-CO2 at low temperatures (70 °C), only Mn = 2, 500 is typically obtained. However, molecular weight increases with both temperature and sc-CO2 pressure. Thus, Mn = 13, 000 (PDI = 1.28) was obtained at 110 °C - 130 °C even in the absence of fluorinated surfactants. Biodegradable drug delivery nanocomposites based on dexamethasone and poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) were prepared by the rapid expansion of the corresponding supercritical CHClF2 solutions (110 °C, 200-300 bar) in air (RESS) and in toluene (RESOLV). The RESS process leads to a broad particle size distribution (100-500 nm) while the RESOLV generates a narrower distribution centered around 100 nm and is accompanied by the formation of a few large particles, most likely due to aggregation.

Polycaprolactone is a bioresorbable polymer that has potential for tissue engineering of bone and cartilage. In this work, we report on the computational design and freeform fabrication of porous polycaprolactone scaffolds using selective laser sintering, a rapid prototyping technique. The microstructure and mechanical properties of the fabricated scaffolds were assessed and compared to designed porous architectures and computationally predicted properties. Compressive modulus and yield strength were within the lower range of reported properties for human trabecular bone. Finite element analysis showed that mechanical properties of scaffold designs and of fabricated scaffolds can be computationally predicted. Scaffolds were seeded with BMP-7 transduced fibroblasts and implanted subcutaneously in immunocompromised mice. Histological evaluation and micro-computed tomography (μCT) analysis confirmed that bone was generated in vivo. Finally, we have demonstrated the clinical application of this technology by producing a prototype mandibular condyle scaffold based on an actual pig condyle.

1. ABSTRACT: This study was performed to evaluate the in-vitro and in-vivo tumor-cellular uptake and biodistribution pattern of tamoxifen when administered intravenously as a simple solution and upon encapsulation into biodegradable, surface-modified poly(ε-caprolactone) (PCL) nanoparticles. PCL (MW ∼ 15, 000) nanoparticles were prepared by the solvent displacement method and characterized for particle size/charge and surface morphology (by scanning electron microscopy). We investigated the nanoparticle-surface modification potential of the hydrophilic stabilizer (Pluronic® F-68 and F-108) employed during the preparation by electron spectroscopy for chemical analysis (ESCA). Quantitative in-vitro cellular uptake of tritiated (3H) tamoxifen in solution form and as nanoparticulate formulation was assessed in MCF-7 breast cancer cells. In-vivo biodistribution studies for the same formulations were carried out in Nu/Nu mice bearing MDA-MB-231 human breast carcinoma xenograft. Spherical nanoparticles having positive zeta potential (∼25 mV) were obtained in the size range of 200-300 nm. Pluronics (both F-68 and F-108), the triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) induced surface hydrophilization of the nanoparticles via adsorption as evident by ESCA. Nanoparticulate formulations of tamoxifen achieved higher intracellular concentrations when exposed at therapeutic concentrations to tumor cells in-vitro compared to solutions. The in-vivo biodistribution studies carried out in nude mice bearing experimental breast tumor suggested increased tumor concentrations for the drug administered as nanoparticulate formulations besides longer retention times within tumor mass. This type of delivery system is expected to provide better therapeutic benefit by dual means: preferential concentration within the tumor mass via enhanced permeation and retention pathway, and; subsequent controlled release, thus maintaining the local drug concentration for longer periods of time to achieve maximal cell-kill.

Magnetic nanoparticle fluids have numerous biomedical applications, including magnetic imaging, drug delivery, and hyperthermia treatment for cancer. Ideal magnetic nanoparticle fluids have well-separated, biocompatible nanoparticles with a small size distribution that form a stable colloid. We have combined inert-gas condensation, which produces nanoparticles with low polydispersity, with deposition directly into a surfactant-laden fluid to prevent agglomeration. Iron, cobalt, and iron-nitride nanoparticle fluids fabricated using inert-gas condensation have with mean particle sizes from 5–50 nm and remain stable over long periods of time. Iron and cobalt nanoparticles oxidize on exposure to air, with oxidation rates dependent on surfactant type and concentration. Iron-nitride fluids are more oxidation and corrosion resistant, while retaining the same high degree of colloidal stability. Magnetic properties vary depending on the nanoparticle size and material, but can be varied from superparamagnetic to ferromagnetic with coercivities on the order of 1000 Oe. In addition to future biomedical applications, inertgas condensation into fluids offers the opportunity to study interparticle interactions over a broad range of intrinsic materials parameters and interparticle separations.

We studied the protein adsorption and subsequent macrophage adhesion on poly(ethylene glycol) [PEG] hydrogels crosslinked using a focused electron beam. Thin-film gels were patterned on silicon substrates and could be formed with swell ratios (hydrated height/ dry height) anywhere between fifteen and unity. We have shown that laminin does not adsorb onto highly swelling gels but that it does adsorb on heavily-crosslinked low-swelling gels. As part of ongoing research on patterning surfaces to control neurite growth in the context of the inflammatory environment of a spinal cord injury, we are interested in how these gel surfaces interact with macrophages. We show that the high-swelling PEG gels resist macrophage adhesion, but the macrophages adhere to low-swelling gels pre-exposed to laminin. By spatially patterning combinations of low and high swelling gels, we show that macrophage adhesion can be confined to specific locations on a surface.

Near infrared fluorescent dyes have a significant potential in biological applications. However, many of these organic dyes are hydrophobic and/or their fluorescence is quenched in aqueous media. Nanoparticle technology has been utilized to overcome the hydrophobic property of these dyes, but there remain problems in their fabrication. To overcome these problems, a novel fabrication technique for incorporating hydrophobic dyes in a biodegradable polymer nanoparticle has been developed. An infrared emitting dye (D1) and a two photon excitable fluorescent dye (BT101) were selected as prototypic dyes for incorporation. The D1 nanocrystal dye shows quenched emission in aqueous media. Upon encapsulation in a polymeric nanoparticle, its emission at 1.1 to 1.35 μm was recovered. BT101 dye doped nanoparticles demonstrated significant increase in fluorescent yield in aqueous media as compared to the free dye. In addition, several different sizes of BT101 doped polymer nanoparticles were fabricated (c.a. 35 nm to 100 nm in size). The fluorescence intensity was found to increase with the particle size. The investigation of uptake of BT101 nanocrystal and BT101 doped PLGA nanoparticles by culture cells revealed the importance of polymer coating of dye for efficient cell imaging.

In order to develop a safe and effective systemically-administered delivery system for solid tumors, the biodistribution of control gelatin and poly(ethylene-glycol) modified (PEGylated) gelatin nanoparticles was examined in Lewis lung carcinoma (LLC)-bearing female C57BL6 mice. Type B gelatin and PEGylated gelatin nanoparticles were radiolabeled (125I) for the in vivo biodistribution studies after intravenous (i.v.) administration through the tail vein in LLC-bearing mice. At various time intervals, the tumor-bearing mice were sacrificed and tumor, blood, and major organs were harvested for analysis of radioactivity corresponding to the localization of the nanoparticles. Percent recovered dose was determined and normalized to the weight of the tissue or fluid sample. Non-compartmental pharmacokinetic analysis was performed to determine the long-circulating property and preferential tumor targeting potential of PEGylated gelatin nanoparticles in vivo. From the radioactivity in plasma and various organs collected, it was evident that the majority of PEGylated nanoparticles were present either in the blood pool or taken up by the tumor mass and liver. For instance, after 3 hours, the PEGylated gelatin nanoparticles were almost 2-fold higher in the blood pool than the control gelatin nanoparticles. PEGylated gelatin nanoparticles remained in the blood pool for a longer period of time due to the steric repulsion effect of the PEG chains as compared to the control gelatin nanoparticles. In addition, approximately 4-5% of the recovered dose of PEGylated gelatin nanoparticles was present in the tumor mass for up to 12 hours. The plasma and the tumor half-lives, area-under-the-curve, and the mean residence time of the PEGylated gelatin nanoparticles were significantly greater than those of the control gelatin nanoparticles. The results of the study confirmed long-circulating property and preferential tumor targeting potential of PEGylated gelatin nanoparticles in a murine tumor model.

Much work is needed in the design of more effective bone tissue engineering materials to induce the growth of normal bone tissue. Nanotechnology offers exciting alternatives to traditional bone implants since bone itself is a nanostructured material composed of nanofibered hydroxyapatite well-dispersed in a mostly collagen matrix. For this purpose, poly-lactic-co-glycolic acid (PLGA) was dissolved in chloroform and nanometer grain size titania was dispersed by various sonication powers from 0 W to 166 W. Previous results demonstrated that the dispersion of titania in PLGA was enhanced by increasing the intensity of sonication and that greater osteoblast (bone-forming cells) adhesion correlated with improved nanophase titania dispersion in PLGA. However, adhesion of osteoblasts to material surfaces, alone, is not adequate to determine long-term functions of implant materials. For this reason, and as a next step to determine the efficacy of nanocomposites in bone applications, subsequent functions of osteoblasts on nanophase titania/PLGA composites were investigated in vitro in this study. For the first time, results correlated better osteoblast long-term functions, specifically the deposition of calcium-containing mineral, with improved nanophase titania dispersions in PLGA.In this manner, the present study demonstrated that PLGA composites with well-dispersed nanophase titania can improve osteoblast functions necessary for the further investigation of these materials in orthopedic applications.