Due to their bioresorbable characteristics biodegradable polyesters are attractive materials for sutures, fracture fixation devices, and drug delivery systems. These polymers degrade primarily through a bulk erosion mechanism. In many instances including drug delivery and fabrication of orthopaedic devices, surface erosion is desired as it confers linearity with respect to degradation and changes in modulus. We hypothesized that surface erosion could be achieved in poly(αΏ-hydroxy acids) by tuning the lipophilicity of the system and hence the water uptake. Toward this end, we used a modular design strategy to manipulate the hydrophilic-lipophilic balance (HLB) of a polymer chain at various stages by judicious choice of building blocks. Using this novel synthetic strategy we have synthesized a library of degradable polyesters derived from poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) that exhibit surface erosion behavior. Surface erosion was further verified by ultrastructure analysis of degraded polymer pellets and thin films by Scanning Electron Microscopy and tapping mode Atomic Force Microscopy respectively.

Dental enamel is a unique composite bioceramic material that is the hardest tissue in the vertebrate body, containing long-, thin-crystallites of substituted hydroxyapatite. Enamel functions under immense loads in a bacterial-laden environment, and generally without catastrophic failure over a lifetime for the organism. Unlike all other biogenerated hard tissues of mesodermal origin, such as bone and dentin, enamel is produced by ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches have been coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principle protein of forming enamel, two domains have been identified that are required for the proper assembly of multimeric units of amelogenin to form nanospheres. One domain is at the amino-terminus and the other domain in the carboxyl-terminal region. Amelogenin nanospheres are believed to influence the hydroxyapatite crystal habit. Both the yeast two-hybrid assay and surface plasmon resonance have been used to examine the assembly properties of engineered amelogenin proteins. Amelogenin protein was engineered using recombinant DNA techniques to contain deletions to either of the two self-assembly domains. Amelogenin protein was also engineered to contain single amino-acid mutations/substitutions in the amino-terminal self-assembly domain; and these amino-acid changes are based upon point mutations observed in humans affected with a hereditary disturbance of enamel formation. All of these alterations reveal significant defects in amelogenin self-assembly into nanospheres in vitro. Transgenic animals containing these same amelogenin deletions illustrate the importance of a physiologically correct bio-fabrication of the enamel protein extracellular matrix to allow for the organization of the enamel prismatic structure.

Protein microtubules (MTs) have been used as templates for the biomimetic synthesis of metal oxide, metal sulfide, and metallic nanomaterials. These materials were coated onto MTs via three distinct synthetic pathways: metal ion hydrolysis which yielded iron oxide or zinc oxide-coated microtubules, metal ion/sulfide co-precipitation which yielded zinc sulfide coated MTs, and metal ion reduction which yielded gold-coated MTs. The growth process of metal oxide coating involves heterogeneous nucleation on the MT surface and produces even, microcrystalline films. Metal sulfide and metal coating initially involves the formation of nanoparticle arrays that decorate the MT surface and can eventually lead to either semi- or fully continuous coatings.

Fast relaxation of stresses lower than the yield stress is demonstrated in Bombyx mori(silkworm) cocoon silk and Nephila clavipes (spider) major ampullate silk (MAS; dragline). Stress relaxation and creep make natural silk unsuitable as a long-term load-bearing material. Instead, silk-like materials are better suited to applications in which energy dissipation is important, and in which high loads need to be withstood on a once-off basis for only very short periods of time. Examples might include use as a ballistic material that arrests the penetration of fragments from the explosion of a pressure vessel, an aircraft luggage container, or a tyre. Treatment in a domestic microwave oven is shown to significantly reduce the rate of stress relaxation in both silkworm cocoon and spider MAS. Except for ductility, the tensile properties of cocoon silk measured in constant strain rate experiments are enhanced by this treatment. Initial experiments on MAS suggest that the tensile properties of this material also are enhanced by exposure to microwaves, in this case with the exception of initial modulus.

In spite of under performance, metals and metal alloys are currently being used in orthopedic implantable devices. Poor osseointegration, severe stress shielding, bone cell death and eventual necrotic bone resulting from the generation of wear debris are known to be some of the reasons responsible for their under performance. In addition, metallic corrosion products may also initiate cancer. Steady growth in the use of metals in orthopedic applications inspired researchers to deal with these problems in an integrated way. When conventional Ti, Ti6Al4V, and CoCrMo surfaces were modified to the nano-range, this study showed increased percentages of osteoblast (bone forming cell) adhesion on nanophase metals. Moreover, larger amounts of osteoblast adhesion was related to quantitative increases in the total length of particle boundaries per unit area and the total number of pores between surface particles per unit area, and the surface particle boundary index (SPBI) of nanophase metals. Additionally, we have developed a novel anticarcinogenic orthopedic metalloid, selenium (Se). When micron range surface particles of Se compacts were modified to the nano-range by chemical etching, we found positive relationships between directed osteoblast adhesion and various particle boundary parameters mentioned above under in vitro conditions. These results provided the first evidence to utilize nanosurface Se as an anticarcinogenic and bio-inspiring material for future applications in orthopedic metallic devices.

Inorganic and organic double templates were used to fabricate silica nanospheres and nanotubes with nanochannels perpendicular to the shells. Sphere and needle like CaCO3 nanoparticles, synthesized by a high gravity reactive precipitation method, were used as inorganic templates and C16H33N(CH3)3Br (C16-CTAB) was used as an organic surfactant template. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were employed to characterize the nanoporous structure. The nanochannels were found perpendicular to the surface of nanospheres and nanotubes. The size of nanochannels is about 3~5 nm. The size of hollow nanosphers and nanotubes can be controlled by the inorganic CaCO3 nanoparticle templates and the nanochannels in the shells can be tuned by different surfactant micelles. The nanospheres and nanotubes with nanochannels perpendicular to the shells have a potential application in chemical bio-catalyst, bio-separation, and drug delivery.

Nanoindentation was used to assess the mechanical properties of lamellar and interlamellar tissue in dehydrated rabbit cancellous bone. The effects of surface roughness and maximum nanoindentation load on the measured mechanical properties were examined in two samples of differing surface roughness using maximum loads ranging from 250-3000 μN. As the ratio of indentation depth to surface roughness decreased below approximately 3:1, the variability in material properties increased substantially. At low loads, the indentation modulus of the lamellar bone was approximately 20% greater than that of the interlamellar bone, while at high loads the measured properties of both layers converged to an intermediate value. Relatively shallow indentations made on smooth surfaces revealed significant differences in the properties of lamellar and interlamellar bone that are consistent with microstructural observations of lamellar bone as more mineralized than interlamellar bone.

We designed and fabricated a class of self-assembling peptides into nanofiber scaffolds. KLDL-12 has been shown to be a permissible nanofiber scaffold for chondrocytes in cartilage 3-D cell cultures. However, the biochemical, structural, and biophysical properties of KLDL-12 remain unclear. We show that KLDL-12 peptides form stable β-sheet structures at different pH values and that KLDL-12 and RIDI-12 self-assemble into nanofibers. The nanofiber length, though, is sensitive to pH changes. These results not only suggest the importance of electrostatic attraction or repulsion affecting the fiber lengths but also provide us with useful information for rational design and fabrication of peptide scaffolds.

There is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell- based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment–e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

Block copolymer-based membranes can be functionalized with energy transducing proteins to reveal a versatile family of nanoscale materials. Our work has demonstrated the fabrication of protein-functionalized ABA triblock copolymer nanovesicles that possess a broad applicability towards areas like biosensing and energy production. ABA triblock copolymers possess certain advantages over lipid systems. For example, they can mimic biomembrane environments necessary for membrane protein refolding in a single chain (hydrophilic(A)- hydrophobic(B)-hydrophilic(A)), enabling large-area membrane fabrication using methods like Langmuir-Blodgett (LB) deposition. Furthermore, the robustness of the polymer molecules/structure result in spontaneous and rapid protein-functionalized nano-vesicle formation that retains structure as well as protein functionality for up to several months, compared to one to two weeks for the lipid systems (e.g. POPC). The membrane protein, Bacteriorhodopsin (BR), found in Halobacterium Halobium, is a light-actuated proton pump that develops gradients towards the demonstration of coupled functionality with other membrane proteins, such as the production of electricity through Bacteriorhodopsin activity-dependent reversal of Cytochrome C Oxidase (COX), found in Rhodobacter Sphaeroides. Protein-functionalized materials have the exciting potential of serving as the core technology behind a series of fieldable devices that are driven completely by biomolecules.

Calcium phosphate is sparingly soluble at neutral pH but the milk of nearly all species contains many times the concentrations of calcium and phosphate that this solubility allows. The explanation for this apparent paradox is that milk calcium is mainly present in the form of thermodynamically stable nanoclusters formed from casein phosphoproteins and amorphous calcium phosphate. A simplified model system comprising the main milk salts and a hydrophilic N-terminal phosphopeptide from β-casein, at or about neutral pH, forms complexes called calcium phosphate nanoclusters. It is shown that two constants, KS and y, together with the empirical chemical formula of the nanoclusters allow the concentrations of all the important chemical species in this model system to be calculated. The method has been extended successfully to the more complex problem of calculating all the important chemical species in milk. It is suggested, on the basis of a simple analysis of the thermodynamics of the formation of nanoclusters, that their size and stability is determined principally by three factors. These are the relatively low free energy of formation of the amorphous core material, the particular pattern of phosphorylation of seryl residues in the caseins that gives a large chelate effect during the sequestration of the calcium phosphate and the unfolded conformation of the caseins that allows a high density of phosphate centres on the core surface.

Recent progress in microfabrication of biodegradable materials has resulted in the development of a three-dimensional construct suitable for use as a scaffold for engineering blood vessel networks. These networks are designed to replicate the critical fluid dynamic properties of physiological systems such as the microcirculation within a vital organ. Ultimately, these 3D microvascular constructs will serve as a framework for population with organ-specific cells for applications in organ assist and organ replacement. This approach for tissue engineering utilizes highly engineered designs and microfabrication technology to assemble cells in three-dimensional constructs which have physiological values for properties such as mechanical strength, oxygen, nutrient and waste transport, and fluidic parameters such as flow volume and pressure.

Three-dimensional networks with appropriate values for blood flow velocity, pressure drop and hematocrit distribution have been designed and fabricated using replica molding techniques, and populated with endothelial cells for long-term microfluidic cell culture. One critical aspect of the fluid dynamics of these systems is the shear stress exerted by blood flow at the walls of the vessel; a key parameter because of well-known mechanotransduction phenomena from mechanical shear forces which govern endothelial cell behavior. In this work, we report the design and construction of three-dimensional microfluidic constructs for tissue engineering which have uniform wall shear stress throughout the network. This type of control over the shear stress offers several advantages over earlier approaches, including more uniform seeding, more rapid achievement of confluent coatings, and better control over endothelial cell behavior for in vitro and in vivo studies.

: Inspired by the water-repellent behavior of the micro- and nano-structured plant surfaces, superhydrophobic materials, with a water contact larger than 150° superhydrophobic surfaces using a combination of nanosphere lithography and plasma etching. It has been found that the water contact angle on these surfaces can be systematically tuned from 132° to 168° by trimming the diameters of polystyrene nanospheres using oxygen plasma. The water contact angles measured on these surfaces can be modeled by the Cassie's formulation without any adjustable parameter.

Tissue engineering scaffolds with precisely controlled geometries, particularly with surface features smaller than typical cell dimensions (1-10μm), can improve cellular adhesion and functionality. In this paper, soft lithography was used to fabricate polydimethylsiloxane (PDMS) stamps of arrays of parallel 5μm wide, 5μm deep grooves separated by 45 μm ridges, and an orthogonal grid of lines with the same geometry. Several methods were compared for the fabrication of 3-D multi-layer polycaprolactone (PCL) scaffolds with precise features. First, micromolding in capillaries (MIMIC) was used to deliver the polymer into the small grooves by capillarity; however the resultant lines were discontinuous and not able to form complete lines. Second, spin coating and oxygen plasma were combined to build 3-D scaffolds with the line pattern. The resultant scaffolds had good alignment and adhesion between layers; however, the upper layer collapsed due to the poor mechanical rigidity. Finally, a new multi-layer micromolding (MMM) method was developed and successfully applied with the grid pattern to fabricate 3-D scaffolds. Scanning electron microscopy (SEM) characterization showed that the multi-layered scaffolds had high porosity and precisely controlled 3-D structures.

Micrometer-sized spheres have been found to assemble from homopolymer electrolytes and small, multivalent counterions in water. In contrast to previous efforts, these vesicles do not use preformed templates, do not require block copolymers, and do not necessarily employ nanoparticles. We have investigated the requirements for vesicle formation with regards to both components of the assembly. Self-assembly occurs with a variety of poly-amino acids and counterions, all of which require a minimum number of charged groups to promote non-covalent crosslinking. We show how the assembly process is controlled by pH and how, in consequence, the pKa's of the reactants can be used to reliably predict sphere formation. By varying the nature of the small counterions, we have determined the requirements for assemblies. The assemblies have been further investigated using confocal microscopy and fluorescent labeling of the different components.

Carbon nanotubes are known for their exceptional mechanical and unique electronic properties. The size dependant properties of nanomaterials have made them attractive to develop highly sensitive sensors and detection systems. This is especially true in biological sciences, where the efficiency of detection systems reflect on the size of the detector and the sample required for detection. At approximately 1.5 to 10nm wide, and approximately 1.5 to 2μm long, the use of carbon nanotubes as sensors in biological systems would greatly increase the sensitivity of detection and diagnostics, for a reduced sample size consisting of few individual proteins and antibodies. Since all the atoms in carbon nanotubes are surface atoms, binding proteins or antibodies to the surfaces can greatly affect their surface states, and thus their electrical and optical properties. This effect can be exploited as a basis for detecting biological surface reactions in a single protein or antibody attached to carbon nanotube surfaces.

In this paper, we show the binding of fluorescently tagged antibodies in phosphate buffered saline on the surfaces of carbon nanotubes. Investigations using a confocal microscope suggest a significant interaction of the antibodies with the surfaces of the nanotubes, the intensity depending on incubation time. Since the surface area to volume ratio of CNTs is high, the use of surfactant to separate the nanotubes creates a greater surface area for antibody attachment. The interaction between CNTs and antibodies is seen to be primarily due to adsorptive surface phenomenon, between the nanotube sidewalls and antibody molecule clusters.

As microcomponents in engineered systems, biological muscles have unique advantages such as large force transduction, utilization of biochemical fuel, and self-assembly from single cells, over other inorganic actuators for biomedical engineering applications. Successful integration of muscles with inorganic fabricated structures and electronics promises the capability of precisely characterizing muscles' mechanical properties and fabricating self-assembled controllable autonomous structures powered by ubiquitous glucose. However, the use of extracted muscle tissue from animals on these devices is impractical and inefficient, as the tissues must be dissected and incorporated into each device by hand with crude interfaces between the biological tissue and inorganic materials. Integration of muscle with fabricated structures would be optimally achieved through self-assembling muscle cells on MEMS. The construction of self-assembled muscle-powered MEMS structures is complicated by the stringent requirements to spatially direct the cell growth, control the tight connection of these differentiated structures with MEMS structures, and enable the cells and the integrated hybrid freedom to move. Conventional and soft photolithography techniques have been extensively employed to pattern the growth of a variety of cell types and investigate their interaction with substrate in the micrometer level. However, all studies are only suitable for patterning static cells on an immobile surface, so a novel system of spatially patterning the contractible cells must be developed to enable the cells and the integrated hybrid devices to be free to move.

We present a novel system of self-assembling myocytes on MEMS devices. This system has shown its capability of spatially and selectively directed growth and differentiation of myocytes into single muscle bundles, attachment of these functional bundles to MEMS structures, and the controlled partial release of the resultant hybrid devices. Two groups of self- assembled muscle-MEMS devices, force-measuring cantilevers and muscle-powered microrobots have been created. Here the further detailed studies of this system are discussed, especially the concept of the self-assembly and the material interfacial problems in this system.

There has been growing interest of late in the fracture properties of human bone. As understanding such properties in the context of the hierarchical microstructure of bone is of obvious importance, this study addresses the evolution of the in vitro fracture toughness with crack extension (Resistance-curve behavior) in terms of the salient mechanisms involved. Fracture-mechanics based measurements were performed on compact-tension specimens hydrated in Hanks' Balanced Salt Solution using cortical bone from mid-diaphyses of 34-41 year-old human humeri. Post-test observations of the crack path were made by optical microscopy and three-dimensional X-ray computed tomography. The fracture toughness was found to rise linearly with crack extension with a mean crack-initiation toughness of Ko ∼ 2.0 MPa√m for crack growth in the proximal-distal direction. The increasing cracking resistance had its origins in several toughening mechanisms, most notably crack bridging by uncracked ligaments. Uncracked-ligament bridging, which was observed by tomography in the wake of the crack, was identified as the dominant toughening mechanism responsible for the observed Rcurve behavior through compliance-based experiments. The extent and nature of the bridging zone was examined quantitatively using multi-cutting compliance experiments in order to assess the bridging stress distribution. The results obtained in this study provide an improved understanding of the mechanisms associated with the failure of cortical bone, and as such are of importance from the perspective of developing a realistic framework for fracture risk assessment, and for determining how the increasing propensity for fracture with age can be prevented.

Amelogenins are hydrophobic proteins that constitute more than 90% of the secretory stage enamel matrix. The assembly of amelogenin into nanospheres has been postulated to be a key factor in controlling the structural organization of the enamel extracellular matrix framework, which provides the scaffolding for the elongated and oriented growth of enamel apatite crystals. To get insight into the structure and function of amelogenin in controlling the process of crystal growth we have utilized two different approaches to investigate adsorption of amelogenin nanospheres onto charged surfaces: A) analysis of adsorption of amelogenin onto hydroxyapatite crystals by means of Langmuir Model for protein adsorption. B) analysis of amelogenin mono or multi-layer formation by sequential adsorption process onto auto-assembled polyelctrolytes films. Our data indicate that amelogenin nanospheres adsorb onto the surface of apatite crystals as binding units with defined adsorption sites. We found that amelogenin nanospheres are negatively charged and a monolayer of these nanospheres adsorbed in an irreversible way on positively ending polyelectrolyte multilayers most likely through electrostatic interactions.