Microfabrication and soft lithographic techniques are combined to develop three-dimensional (3D) polydimethylsiloxane (PDMS) scaffolds comprising multiple levels of meandering pore geometry textured with 10 μm posts. Both micro-architecture and surface micro-textures have been shown to selectively stimulate cell and tissue behavior. To achieve a 3D scaffold with precise micro-architecture and surface micro-textures, 100 μm thick PDMS films were manufactured using a stacking technique to realize a 66% porous 3D structure with 200 × 400 μm horizontal through holes, 300 μm diameter vertical through holes and 71% surface coverage with 10 μm diameter and 10 μm high posts. Each PDMS porous film level was manufactured by the dual-sided molding of uncured PDMS between a three level SU-8 photoresist mold (of 200, 10, and 100 μm thick features) and a PDMS mold with 10 μm deep micro-textures. Dual-sided molding was achieved using a custom motion control mechanical jig that allowed relative mold alignment to within ∼ ±10 μm.

Rational design and selection of candidate porous biomaterials to serve as tissue engineering constructs rests on our ability to understand the influence of the porous microarchitecture on the transport of chemical species (e.g., nutrients and signaling compounds), fluid flow, and cellular locomotion and growth. We have begun to study the behavior of chemotactically mobile cells in response to unsteady signaling molecule concentration fields using a computational simulation-based model. The model couples fully time-dependent finite-difference solution of a reaction-diffusion equation for the concentration field of a generic chemoattractant to biased random walks representing individual moving cells. This model is a first step in building a quantitative, pore-level model of mass and cellular transport in porous tissue-engineered constructs. In these proceedings, we focus on our recent findings regarding the influence of flux-reactive boundary conditions in heterogeneous 2D domains on the chemotactic response of otherwise randomly moving cells. In particular, we find that, when cells are forced to “crawl” around obstacles in order to approach a point source of chemoattractant, the reactivity of the obstacle surface with respect to the chemoattractant strongly determines the morphology of the cells' paths of locomotion. Cells crawl along non-reactive surfaces and strongly avoid reactive surfaces, due to the nature of the chemoattractant concentration gradients near the surface. We show further that tuning the reactivity of the surfaces of two obstacles defining a gap can control the passage of cells through the gap. From our work, we infer the importance of a proper treatment of boundary conditions in any future pore-level quantitatve modeling of mass transport and cellular response in porous media.

Hydroxyapatite formed from low temperature setting calcium phosphate cements (CPC) are currently been used for various orthopaedic applications. CPCs are attractive candidates for the development of scaffolds for bone tissue engineering, since they are moldable, resorbable, set at physiological temperature without the use of toxic chemicals, and can be processed in an operating room setting. However they may have mechanical disadvantages which seriously limit them to non-load bearing orthopaedic applications. The aim of the present study was to develop composites from polyphosphazenes and calcium deficient hydroxyapatite precursors to form poorly crystalline hydroxyapatite-polymer composites. Composites were formed from calcium deficient hydroxyapatite precursors (Ca/P – 1.5, 1.6) and biodegradable polyphosphazenes, poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(50%ethyl alanato) (50%methyl phenoxy)phosphazene] (PNEA50mPh50) at physiological temperature. The results demonstrated that poorly crystalline hydroxyapatite that resembled the mineral component of bone was formed in the presence of biodegradable polyphosphazenes. The surface morphology of all the four composites was identical with a porous microstructure. The composites supported the adhesion and proliferation of osteoblast like MC3T3-E1 cells making them potential candidates for bone tissue engineering.

Lanthanide-ion doped oxide (YVO4: Eu) nanoparticles were synthesized as aqueous colloids and functionalized by a bioactive silane shell to be used as fluorescent biological labels. Nanoparticles functionalized with guanidinium groups were able to act as artificial toxins which specifically target Na+ channels. They were individually detectable in live cardiac myocytes. Functionalized oxide nanoparticles appear as a new interesting tool, especially attractive for long-term single-molecule tracking due to their photo-stability and long luminescence lifetime.

To increase the targeting potential, circulation time, and the flexibility of surface-attached biomedically-relevant ligands on gold nanoparticles, hetero-bifunctional poly(ethylene glycol) (PEG, MW 1, 500) was synthesized having a thiol group on one terminus and a reactive functional group on the other. Coumarin, a model fluorescent dye, was conjugated to the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreases the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within one hour of incubation.

Cellulose acetate (CA) thin, porous membranes were produced by electrospinning precursor polymer solutions in acetone at room temperature. During this process, CA nanofibers were produced when a high electric field of 12 kV was applied to the precursor solution. The diameters of fibers obtained varied from 100 nm to 1.2 μm while the average diameter was approximately 500 nm. The electrospinning parameters used to control the morphology of the fibers and their membranes are flow rate, the distance between the syringe needle that ejects fluid and the collector, and the voltage applied. These membranes were used as scaffolds for microvascular cells growth. The structure of the membranes that were produced mimic the topography and porosity of extracellular matrix (ECM) in two key ways. The fiber diameter mimics extracellular protein fiber diameter, thus enabling cellular attachment and facilitating cellular migration. The porosity mimics that of extracellular matrix such that microvascular capillary tube formation is enhanced. The non-woven fiber mats were examined by means of electron microscopy and the nanofibers were seen to be oriented randomly. The issue of strengthening the CA scaffold is currently studied by adding ceramic nano-structured component (carbon nanotubes) in the polymer membranes.

Nanocrystalline hydroxyapatite (HAp) exhibits better bioactivity and biocompatibility with enhanced mechanical properties compared to the microcrystalline counterpart. In the present work, nanocrystalline hydroxyapatite was synthesized by wet chemical method. Sintering was carried out with nanocrystalline alumina as additive, the content of alumina being varied from 10 to 30 wt% in the composite. For 20 and 30 wt % Al2O3, hydroxyapatite decomposed into tricalcium phosphate (TCP) above the sintering temperature of 1100°C. The fracture toughness of nano HAp-nano Al2O3 composite is anisotropic in nature and reached a maximum value of 6.9 MPa m1/2.

A review of the ways in which cells react to nanofeatured surfaces is given. One of the prime reactions is of adhesion or otherwise to such surfaces. Topography appears to be of considerable importance and a wide range of cell properties are affected by the type, scale and regularity of topography. Chemistry can be combined with topography to fine- tune effects. Mechanical forces are also of importance but in practice it is hard to control these.

Examples will be given of methods of controlling adhesion, morphology, orientation, movement, phagocytic activity and activation and gene expression of cells, Effects vary according cell type and also the spacing and size of nanofeatures. A discussion of the application of these findings to the medical devices concludes this short review.

We have developed water-soluble, biocompatible, fluorescent, and photostable silver/dendrimer nanocomposites that have a potential to be used for in vitro cell labeling. A PAMAM_E5.NH2 dendrimer was used as a template to prepare first a silver-dendrimer complex in an aqueous solution at biologic pH=7.4. Conversion into nanocomposite was achieved by irradiating the solution of the [(Ag+)25-PAMAM_E5.NH2] complex by UV light to reduce the bound Ag+ to zero-valent Ag0 atoms, which were simultaneously trapped in the dendrimer network. Results indicate that the {(Ag0)25-PAMAM_E5.NH2} silver/dendrimer nanocomposite forms positively charged single particles of 4–5 nm under the experimental conditions used. The dendrimer nanocomposite proved to be fluorescent. Toxicity testing of {(Ag0)25-PAMAM_E5.NH2} nanocomposite revealed a behavior similar to the template dendrimer. Intracellular internalization of the silver nanocomposite and cell labeling capabilities was confirmed by confocal microscopy.

We present a new synthesis method based on laser generation and processing of aerosol particles that can produce calcium phosphate coatings in a porous nanostructured configuration. The process uses laser ablation of crystalline hydroxyapatite targets to produce a calcium phosphate aerosol comprising micro- and nanoparticles that are processed and deposited on metallic substrates under well-controlled temperature and ambient conditions, creating a microporous calcium phosphate network suitable for growth of biogenic calcium phosphate materials. Laser ablation is carried out using a KrF excimer laser at fluences between 0.4 J/cm2 and 2.8 J/cm2 and temperatures ranging from 500°C to 760°C. X-ray diffraction and scanning electron microscopy measurements on samples deposited above 750°C show that the obtained material is crystalline hydroxyapatite with good mechanical stability. Its microstructure features a porous framework of partially sintered microparticles surrounded by nanoparticulate material.

Induction of angiogenesis is necessary for the success of engineered implantable tissues in order to meet oxygen and nutrient requirements of cells during tissue repair. Insufficient vascularization in bone graft reconstruction may impede healing and initiate hypoxic cell death at the interior of the implant. As a result, endogenous growth factors have been studied to enhance angiogenesis during wound repair. However, these peptide-based molecules are highly sensitive to processing that occurs during scaffold biomaterial fabrication and treatment for tissue engineering purposes. We report here the development of new small molecule regulators of angiogenesis that may circumvent the impediments associated with protein-based growth factor delivery. In this study, we report the design and evaluation of SC–3–143 as a regulator of endothelial function. We show that the compound significantly increases the formation of microvascular networks in vitro, and selectively enhances endothelial survivability by reducing endothelial cell death under serum deprived culture conditions.

Membrane protein study critically depends on detergents, which are amphilhilic molecules containing a hydrophilic “head” and a hydrophobic “tail” to mimic biological lipid bilayers to stabilize membrane proteins. However, detergents are not fully equivalent to lipid bilayers and in fact they only partly mimic lipid bilayers function. Consequently, membrane proteins in detergent solution are more or less denatured because detergents can not effectively stabilize membrane protein structures. Therefore, it is urgent to develop new types of detergents for more effectively stabilizing membrane proteins. Previously, we have reported a new type of self-assembly peptide detergents containing a hydrophilic head composed of either a negatively charged aspartic acid or a positively charged lysine and a tail of hydrophobic amino acids of six connective alanines. This new peptide detergent has been shown to be more effective for protecting membrane protein PS I structure than that the conventional detergent does. However, what type of physical structures peptide detergent can form is unclear yet. Here we presented our AFM and DSL analysis of the peptide detergent A6D, which not only form mixed micelles with n-Octyl-beta-D-Glucoside (OG) to solubilize membrane protein rhodopsin, but also can mimic lipid bilayers to keep rhodopsin in lipid-like vesicles for its structure preservation.

Current methods for analysis of unknown powders in suspicious packages involve sending samples to laboratory facilities where a variety of time-consuming tests are performed. We have developed and investigated the use of a lead zirconate titanate - titanium (PZT-Ti) microcantilever for in situ detection of the common food- and water-born pathogen, Salmonella typhimurium. Using a bifunctional linking molecule to immobilize antibody on the titanium surface of the microcantilever, we can directly detect salmonella cells in suspensions of differing concentration. This novel surface functionalization technique along with the sub-nanogram sensitivity of the cantilever has allowed for direct quantification of S. typhimurium cells in suspension.

A laser-based approach, Matrix Assisted Pulsed Laser Direct Write (MAPLE) technique is used to demonstrate two-dimensional direct writing of ceramics such as hydroxyl-apatite and zirconia, for developing inorganic scaffolds. We also demonstrate the patterning of live MG63 osteoblast cells onto various substrates. Our results show successful direct writing of ceramics and live cells concurrently, with a growth profile similar to that of as-deposited cells. After several days of growth, a live/dead assay shows live cells suggesting the biocompatibility of the ceramic and the viability of the process. This investigation demonstrates a novel method of developing heterogeneous tissue scaffolds, such as a cell-ceramic composite by CAD/CAM patterns.

RGD (arginine-glycine-aspartic acid) containing peptide sequences, common cell attachment sites present in many extracellular matrix (ECM) proteins, mediate many important cellular processes. The role of nanoscale organization of RGD peptides in the regulation of the adhesion, proliferation and differentiation of both preosteoblasts (MC3T3-E1) and multipotential (D1) cell lines in vitro was investigated in this study. Alginate polymer chains with varying RGD peptide degree of substitution were mixed with unmodified polymer chains at different ratios to allow variation of RGD peptide spacing in the nanometer scale, independently of the overall bulk density of peptides presented from the material. Proliferation of both cell types was observed to be closely correlated to RGD island (defined as a cluster of RGD peptides) spacing, independently of overall bulk ligand density, following cell adhesion to alginate hydrogels. Increased RGD island spacing was observed to promote spreading of MC3T3-E1 cells while simultaneously suppressing their proliferation. However, increased RGD island spacing decreased spreading of D1 cells while also decreasing proliferation. Moreover, differentiation of preosteoblasts was significantly upregulated in response to decreased RGD island spacing, whereas differentiation of multipotential cells was modestly regulated by this variable. These results demonstrate that the nanoscale organization of adhesion ligands may be an important variable in controlling cell phenotype and function. In addition, cellular responses to nanoscale ligand organization differ depending on the cell type, and this may be related to the differentiation stage of the cells.

A piezoelectric drop on demand printer has been used to print primary human osteoblast and bovine chondrocyte cells. After deposition the cells were incubated at 37°C and characterised using optical microscopy, SEM and cell viability assays. Cells showed a robust response to printing exhibiting signs of proliferation and spreading. Increasing the drop velocity results in a reduced cell survival and proliferation rates but both cell types grew to confluence after printing under all conditions studied.

The present study was motivated by the need for accurately-controlled and well-characterized novel biomaterial formulations for the study of cell-protein-material interactions. For this purpose, the current research has focused on the design, fabrication and characterization of model native oxide-coated silicon surfaces decorated with silica nanoparticles of select sizes, and has examined the adhesion of osteoblasts and fibroblasts on these nanoparticle-decorated surfaces. The results demonstrate the capability to deposit nanoparticles of select diameters and substrate surface coverage onto native silicon oxide-coated silicon, the firm attachment of these nanoparticles to the underlying native silicon oxide, and that nanoparticle size and coverage modulate adhesion of osteoblasts and fibroblasts to these substrates. The material formulations tested provide a well-controlled and well-characterized set of model substrates needed to study the effects of nanoscale features on the functions of cells that are critical to the clinical fate of implantable biomaterials.

Biodegradable polymeric nanofibers are of great interest as scaffolds for tissue engineering and drug delivery due to their extremely high surface area, high aspect ratio and similarity in structure to the extracellular matrix (ECM). Polyphosphazenes due to their synthetic flexibility, wide range of physico-chemical properties, non-toxic and neutral degradation products and excellent biocompatibility are suitable candidates for biomedical applications. The objective of the present study was to develop and evaluate composite nanofibers of a biodegradable polyphosphazene, poly[bis(ethyl alanato)phosphazene] (PNEA) and nanocrystals of hydroxyapatite (nHAp) via electrospinning. A suspension of nHAp in dimethyl formamide (DMF) sonicated with PNEA solution in tetrahydrofuran (THF) was used to develop composite nanofiber matrices via electrospinning at ambient conditions. In the present study the theoretical loading of nHAp was varied from 50%-90% (w/w) to PNEA. The nHAp content (actual loading of nHAp) of the composite nanofibers was determined by gravimetric estimation. The composite nanofibers were characterized by transmission electron microscopy (TEM), gravimetry and energy dispersive X-ray mapping. This study demonstrated the feasibility of developing novel composite nanofibers of biodegradable polyphosphazenes with more than 50% (w/w) loading of nHAp on and within the nanofibers.

Gram positive (Lactoccocus lactis) and Gram negative bacteria (Escherichia coli) were used to study the interaction of water-dispersible muti-walled carbon nanotubes (CNTs) with the bacterial cell envelope during microwave (MW) energy exposure. It was observed that the addition of a tiny amount of CNTs to a medium containing bacteria and subsequent exposure of the samples to MW, leads to an intimate contact between the CNT tips and the cell envelope. This phenomenon can be explained in terms of attractive forces between opposite charges of polar structures. Since CNTs under MW irradiation behave like electric dipoles, this would make it possible for the CNTs to target the cell surface without inducing changes in the cell shape and viability. Thus, the electrochemical properties of CNTs and their capillarity make them useful tools for cell manipulation, and therefore for the intracellular transport of drugs, dyes or biomolecules.

We have deposited poly (D, L lactic acid) (PDLLA) thin films using matrix assisted pulsed laser evaporation (MAPLE). FTIR spectroscopy revealed that the PDLLA had similar absorption bands to the dropcast material. X-ray photoelectron spectroscopy has shown that peaks corresponding to C-H, C-O and C=O represented 38.4, 30.1 and 31.4% of the C1s spectrum, respectively. XPS O1s analysis revealed that the O=C and O-C components make up 52 and 48 % of the O1s content. Atomic force microscopy revealed that MAPLE deposition provides smooth, continuous thin biomaterial films. These matrix assisted pulsed laser evaporation-deposited biomaterial thin films may serve to improve the implant/tissue interface.