The maintenance of cell volume is critical to health. Cell volume change reflects many biological and physiological processes. We have developed a lab-chip to measure cell volume change in real-time with high sensitivity and resolution, and applicable to both adherent and suspended cell populations. The volume change was detected by measuring the impedance of extra-cellular solution within a microfluidic chamber containing the cells. Using microfabrication to make precise chamber dimensions, volume change can be detected in response to an osmotic gradient <1mOsm. The sensor provides rapid screening of pharmaceutical agents affecting cell volume. We have screened for peptides that affect cell volume regulation and found one in spider venom that inhibits at ∼100pM.

DNA amplification is one of the most routine experiments carried out in biological laboratories. Continuous flow PCR chip releases biologists from their laborious exercises. The use of such chip is, however, hindered by costly expense of the syringe pump, which is used to maintain a constant flow rate. In this paper, we demonstrate a novel pressure indicator which makes up an in-line flow sensor in a continuous flow PCR chip. The pressure of PCR channel is achieved from pattern change in fabricated microstructures, and converted to volume flow rate. A polymeric PCR chip with such pressure indicators is presented. With a much less expense as compared to its conventional peers, this indicator has a wide potential for the use in the laboratories which runs daily activities like sequencing or mutagenesis.

We have designed a novel class of functional surfactants based on DNA and have explored their properties in relation to several possible applications. The DNA-surfactants consist of short chain DNA oligonucleotides covalently bound to a large hydrophobic group, which makes the DNA molecules amphiphilic. We demonstrate that these materials behave like common detergents and are surface-active at various fluid surfaces, e.g. air-water, oil-water interfaces, and lipid bilayers. We also show that once adsorbed the DNA-surfactants remain on the liquid surface upon hybridisation with a complementary DNA chain. We use complementary DNA-surfactants to functionalise fluid surfaces and to program the interactions between them based on Watson-Crick pairing. By selecting the appropriate DNA base sequences, the interaction between the fluid surfaces functionalised with DNA-surfactants can be programmed with a very high specificity. We have also developed a novel procedure for micro-patterning of solid surfaces with DNA by a microcontact printing with aqueous inks of DNA-surfactants which can be utilised for rapid fabrication of DNA assays and genetic biochips.

Rotating bioreactors have been used to overcome the limitations of passive nutrient diffusion in three-dimensional (3D) constructs for tissue engineering of bone. It is hypothesized that conventional scaffolds undergo repeated wall collisions in rotating bioreactors, which may disrupt bone tissue formation. In this study, we investigated the effects of wall collision on osteoblastic cells cultured on a microsphere based scaffold of varying densities in comparison to water. The conventional heavier than water (HTW; density > 1 g/cm3) scaffolds were fabricated by sintering HTW microspheres of 85:15 poly (lactide-co-glycolide) (PLAGA), and mixed scaffolds were designed by mixing lighter than water (LTW; density < 1 g/cm3) and HTW microspheres of PLAGA. We quantified average velocities of the two types of scaffolds using a particle tracking system, and no significant difference in average velocities was observed between the two types of scaffolds. However, HTW scaffolds have frequent wall collision and mixed scaffolds can avoid wall collision in bioreactors. When human Saos-2 osteoblast like cells were cultured on the scaffolds in bioreactors for 16 days, bone cell proliferation and cell differentiation on HTW scaffolds were significantly inhibited as compared to those cultured on mixed scaffolds in rotating bioreactors. These results indicate that collision between scaffolds and bioreactor wall is a confounding factor in osteoblastic cell proliferation and differentiation. These studies provide a foundation for development of 3D scaffolds for tissue engineering of bone in rotating bioreactors.

Engineering functional tissues and organs successfully depends on the ability to control cell orientation and distribution. Materials used for such purposes therefore have to be designed to facilitate cell distribution and eventually guide tissue regeneration in 3D.The field of tissue engineering hinges on developing degradable polymeric scaffolds that promote cell proliferation and expression of desired physiological behaviors through careful control of the polymer surface.

The development of materials for tissue engineering and guided tissue regeneration has accelerated over the last decade.[1] It has been demonstrated that non-patterned cells are effectively not tissue. “Tissues require that cells be placed and hold precise places often with precise orientations” [2–3]. Cell patterning is therefore very important for tissue engineering. We have developed a biocompatible, biostable chemically amplified bioresist, with which patterns are generated without involving harsh chemical treatment.

Titanium is broadly used in orthopedic and dental applications mainly because of its optimal mechanical properties in load-bearing applications. However, insufficient new bone formation is frequently observed on titanium which sometimes leads to implant loosening and failure. For this reason, the objective of the present in vitro study was to modify the surface of conventional titanium to include nanostructured surface features that promote the functions of osteoblasts (bone-forming cells). This study focused on creating nanostructured titanium surfaces since bone itself has a large degree of nanostructured roughness that bone cells are accustomed to interacting with. In this study, the surface of titanium was modified by anodic oxidation techniques. The electrolyte used for anodization was hydrofluoric acid. Depending on acid concentration and anodization time, two kinds of different nano-architectures, either particulate or tube-like structures, were formed on the titanium surface. X-ray diffraction results confirmed that the titanium oxide formed on the surface of titanium was amorphous. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the surface morphology. Cell adhesion studies showed that the anodized nanostructured titanium surface promoted osteoblast adhesion compared to non-anodized titanium. This result indicated that anodization may be a simple method to modify the surface of titanium implants to enhance bone-forming cell function thereby increasing orthopedic implant efficacy.

Direct printing of living cardiomyogenic stem cells and embryonic cortical neurons to generate complex cellular patterns and structures of such cells was demonstrated in the study. Furthermore, the immunostaining analyses and the whole-cell patch clamp recordings showed the cortical neurons grown in the printed cellular patterns and structures maintained their basic cellular functions, including neuronal phenotypes and electrophysiological properties. These results and findings may greatly prompt the inkjet printing method evolving into a viable and cost-effective approach for engineering human neural and cardiac tissues or even organs.

Present tissue engineering practice requires porous, bioresorbable scaffolds to serve as temporary 3D templates to guide cell attachment, differentiation, and proliferation. Recent research suggests that scaffold material and internal architecture significantly influence regenerate tissue structure and function. However, lack of versatile biomaterials processing methods have slowed progress towards fully testing these findings. Our research investigates using selective laser sintering (SLS) to fabricate bone tissue engineering scaffolds. Using SLS, we have fabricated polycaprolactone (PCL) and polycaprolactone/tri-calcium phosphate composite scaffolds. We report on scaffold design and fabrication, mechanical property measurements, and structural characterization via optical microscopy and micro-computed tomography.

The effect of grating textures on the alignment of cell shape and intracellular actin cytoskeleton has been investigated in bovine aortic endothelial cells (BAECs) cultured on a model cross-linked poly(dimethylsiloxane) (PDMS). Grating-textured PDMS substrates, having a variation in channel depths of 200 nm, 500 nm, 1 μm and 5 μm, were coated with fibronectin (Fn) to promote endothelial cell adhesion and cell orientation. As cells adhered to the Fn-coated surface, the underlying grating texture has shown to direct the alignment of cell shape, F-actin and focal contacts parallel to the channels. Cell alignment was observed to increase with increasing channel depths, reaching the maximum orientation where most cells aligned parallel to channels on 1-μm textured surface. Immunofluorescence studies showed that F-actin stress fibers and vinculin at focal contacts also aligned parallel to the channels. Cell proliferation was found to be independent of grating textures and the alignment of cell shape was maintained at confluence.

Adhesion of differentiated mammalian cells from various hard and soft tissues, including adult mesenchymal stem cells is different on nanophase than on microphase/conventional ceramics (such as alumina, titania and hydroxylapatite) as well as on composites of these ceramics with either poly(L-lactic) acid or poly(methyl) methacrylate. Most importantly, nanophase materials promote selective interactions, for example, of osteoblasts but not of fibroblasts. The type, amount and conformation of adsorbed proteins (such as fibronectin, collagen and vitronectin) are key aspects of the underlying mechanism(s) of subsequent cell interactions with nanophase materials. These cellular/molecular results provide evidence that nanophase biomaterials have the potential for improving the efficacy of implants and for promoting neotissue formation pertinent to tissue engineering, regenerative medicine and other clinical applications.

The overall goal of our work is to develop new methods and materials for the fabrication of hierarchically structured, three-dimensional (3D) tissue scaffolds. Conventional scaffolds commonly lack substantial mechanical strength, and there is difficulty in controlling porosity, pore distribution, and pore interconnectivity. Additionally, the chemical nature of these scaffolds is typically homogenous. The ability to chemically modify selected areas on a scaffold is one method to direct cell growth in deliberate patterns; which could aid in the engineering of complex, functioning tissues. The general aim of this work is to address these issues through the application of stereolithography (SL) to the fabrication of hierarchically structured scaffolds.

In order to achieve this goal, photopolymerizable materials must be developed that are both compatible with cell growth and with SL processing. SL methods are designed to produce arbitrary control over the physical structure of the part. In addition to physical structure control, control over the local surface chemistry of the scaffold is also desired. This would permit the use of both physical and chemical cues to control cell behavior in a tissue engineering construct. Chemical control could be achieved in SL methods by using photopolymerizable materials that can also be selectively chemically modified during the SL part building process. This paper provides an update on our work directed at using combined photoradical initiated polymerization and photoacid generator based chemical modification of a polymeric scaffold via multi-wavelength SL to produce hierarchically structured scaffolds.

Polymer/ceramic composite matrices for bone tissue engineering were constructed by synthesizing a poorly crystalline calcium phosphate within poly(lactide-co-glycolide) microspheres that were subsequently fused together to form 3-dimensional structures. Calcium ion dissolution from the composite matrices in simulated body fluid was examined over a 24 hour period. The initial 4 hour period showed an increase in calcium ion concentration, whereas, a decrease in calcium ion concentration was noted thereafter. This decrease in concentration coincided with the precipitation of calcium phosphate on the surface of the matrices. Osteoblast proliferation studies on composite matrices showed statistically significant increases in cell number throughout the 21 day time period. These data together suggest that the composite matrix acts as both a calcium ion donor for reprecipitation of calcium phosphate that may enhance osteointegration of the implant, and a suitable surface for osteoblast proliferation.

Titania submicron-scale rod arrays were fabricated on metallic titanium (α-Ti) surfaces by coating a layer of sodium tetraborate on titanium substrates and subsequent thermal treatment. Thin-film X-ray diffraction analysis indicated that the sodium tetraborate gave rutile (TiO2: PDF# 21-1276) submicron-scale rod arrays. The rods in the arrays are parallel to each other in the grain of metallic titanium surface. The titania submicron-scale rod arrays deposited apatite within 7 days after being soaked in a simulated body fluid, indicating that the rod arrays exhibit in vitro bioactivity.

Collagen is the principal tensile element of the extra-cellular matrix in animals and is the basic scaffold for cells and tissues. Abnormalities in its structure are known to result in a number of debilitating human diseases. Collagen mimetic peptides (CMPs) with repeat unit of (Pro-Hyp-Gly) are capable of forming right-handed triple-helical structures similar to that of the collagen triple helices. Recently, our group has shown that CMPs exhibit specific binding affinity to natural collagen under controlled thermal conditions. Using solid phase peptide synthesis, we have prepared a CMP cysteine derivative that was used to modify gold nanoparticles. Transmission electron microscopy (TEM) shows that the Cys-CMP functionalized gold nanoparticles have affinity to collagen fibers. We are investigating the interactions between Cys-CMP functionalized gold nanoparticles and collagen fibers. The Cys-CMP conjugated nanoparticles can potentially be used as a tool to visualize and understand unstable domains of collagen fibers which are related to a number of pathological conditions of extra cellular matrices.

Functionally graded Hydroxyapatite coating with graded Crystallinity across the thickness of the film has been processed and tested as a more effective orthopedic/ dental implant coating. The present study aims to increase the service-life of an orthopedic/ dental implant by creating materials that form a strong, long lasting, bond with the Ti substrate as well as juxtaposed bone. The health relatedness of the new material is to increase bonding between an implant and juxtaposed bone so that a patient who has received joint or dental replacement surgery may quickly return to a normal active lifestyle. Cross-sectional transmission electron microscopy analysis displayed that the films have a graded crystal structure with the crystalline layer near the substrate and the amorphous layer at the top surface. Compositional analysis was performed using SEM-EDX at the top surface as well as STEM-EDX at the cross section of the film. The average calcium to phosphorous ratio at the surface is 1.46, obtained by SEM-EDX. The Ca/P ratios in the crystalline and amorphous layers of the film are 1.6 to 1.7, close to the ratio of 1.67 for HA.

Three dimensional (3-D) scaffolds with appropriate mechanical properties play a significant role in scaffold-based tissue engineering. Chitosan, a natural polymer obtained from chitin, which forms a major component of crustacean exoskeleton, is a potential candidate for bone tissue engineering due to its excellent osteocompatibility and biodegradability. The aim of the present study is to develop 3-D porous chitosan scaffolds with mechanical properties in the range of trabecular bone as scaffolds for bone tissue engineering. Three dimensional scaffolds were prepared by sintering chitosan microspheres. Chitosan microspheres were prepared by ionotropic gelation of chitosan solution using sodium tripolyphosphate. It has been found that the microsphere size increased significantly with the increase of the concentration of chitosan solution. The microspheres were then sintered together using the synergetic effect of solvent and temperature. The compressive moduli of the 3-D sintered matrices were found to be in the mid range of trabecular bone. The osteocompatibility and osteoconductivity of the 3-D matrices were demonstrated by adhesion and proliferation of MC3T3-E1 osteoblast like cells on the matrices after 14 days in culture.

To develop a new class of in situ-forming, injectable, and biodegradable polymeric biomaterials based on time-dependent lower critical solution temperature (LCST) properties for localized delivery, copolymers of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacryl lactate (HEMA-lactate) and acrylic acid (AAc) were prepared with varying mole ratios of monomers. The copolymers showed LCST and gelation properties below body temperature in 0.1 N PBS solution of pH 7.4. The LCST and gelation temperature of the copolymers decreased as the HEMA-lactate content of the copolymers was increased. The copolymers also showed time-dependent LCST and gelation properties in 0.1 N PBS solution of pH 7.4 owing to hydrolysis of HEMA-lactate. Hydrolysis of HEMA-lactate caused the polymers to be more hydrophilic, resulting in an increase in LCST and gelation temperature. All the polymers with about 6 mol % AAc exhibited LCST and gelation temperature above body temperature after complete hydrolysis of HEMA-lactate.

Novel colloidosome microcapsules with aqueous gel cores and shells of different polymeric colloid particles have been prepared and characterized. Our preparation technique involves templating water-in-oil emulsions stabilized by either polymeric microrods or polystyrene (PS) latex particles and subsequent gelling of the aqueous phase with a suitable hydrocolloid. The obtained colloidosome microcapsules were transferred in water after dissolving the oil phase in ethanol and multiple centrifugation/washing cycles with ethanol and water. The presence of an aqueous gel core was found to enhance the structural integrity and mechanical stability of the obtained colloidosomes. In the case of latex particles forming the colloidosome membrane, the effect of the oil type on the final structure of the colloidosome shell was also studied. It was shown that by using appropriate oil, the latex particles within the colloidosome shell can be partially or completely swollen which not only binds them together but also allows direct control over the membrane pore size and its permeability with respect to entrapped species. Such microcapsules can find various applications for development of novel drug and vaccine delivery vehicles, slow release of cosmetic and food supplements.

The initial response of blood exposed to an artificial surface is the adsorption of blood proteins that triggers a number of biological reactions such as inflammation and blood coagulation. Competitive protein adsorption plays a key role in the hemocompatibility of the surface. The synthesis of nonfouling surfaces is therefore one of the major prerequisites for devices for biomedical applications. Polysaccharides are the main components of the endothelial cell glycocalyx and have the ability to reduce nonspecific protein adsorption and cell adhesion and, therefore, are generally coupled with a wide variety of surfaces to improve their biocompatibility. We have developed a procedure for covalently binding dextran and sodium hyaluronate (HA) on silicon wafers and we have been able to achieve a high level of control over the surface properties of the coatings. In the present research effort we focus on a detailed investigation of competitive bovine serum albumin (BSA) and bovine fibrinogen (Fg) adsorption on dextran- and HA-modified silicon surfaces. Polysaccharide based biomimetic layers preferentially adsorb BSA and, in general, strongly suppress protein adsorption with respect to bare silicon and APTES-activated silicon surfaces used as control.

Nanobiotechnology is a growing area of research, primarily due to the potentially numerous applications of new synthetic nanomaterials in engineering/science. Although various definitions have been given to the word “nanomaterials” by many different experts, the commonly accepted one refers nanomaterials as those materials which possess grains, particles, fibers, or other constituent components that have one dimension specifically less than 100 nm. In biological applications, most of the research to date has focused on the interactions between mammalian cells and synthetic nanophase surfaces for the creation of better tissue engineering materials. Although mammalian cells have shown a definite positive response to nanophase materials, the evidence for bacteria interactions with nanophase materials remains for the most part a mystery. For this reason, this study determined the capture of a model bacteria (Pseudomonas fluorescens) on nanophase compared to conventional grain size alumina. Results provided the first evidence of increased capture of Pseudomonas fluorescens on alumina with nanometer compared to conventional grain sizes. Although not measured at the atomic scale, similar chemistry, crystallinity, crystal phase, and porosity was observed between nanophase and conventional alumina. For this reason, a major material property difference between nanophase and conventional alumina was reduced grain size (and perhaps associated changes in charge density) which led to increased bacteria capture and the design of better environmental filters.