Self-assembled monolayers (SAMs) provide simple, yet sophisticated surfaces to mimic the effect of proteins associated with the process of biomineralization. A careful selection of organic molecules with an appropriate surface chemistry (i.e. HS-(CH2)n-X− supported on a metal surface) allows the nucleation and growth of oriented calcite crystals and provides opportunities to study the formation of inorganic crystals assisted by organic molecules. We have successfully crystallized calcite crystals on different SAMs in the presence of additives such as proteins and/or ions in solution, and found correlations between the orientations of crystals to their final shapes, sizes and morphologies. We report here our experimental results demonstrating how underlying organic molecules along with inorganic additives can control and mold the final shape, size and morphology of calcium carbonate crystals

Silica-precipitating polypeptides were deposited onto an indium tin oxide (ITO) surface from bulk solution with and without the assistance of an externally applied electrostatic field. Exposure of the peptide-coated surface to an alkoxide precursor produced biosilica structures that were securely attached to the electrode surface. The silica morphologies resulting from the test cases using an externally applied electrostatic field during peptide deposition were distinct from the morphologies resulting from cases without an applied field. The silica morphologies observed on the ITO surface were different from the usual silica morphology resulting from static conditions. Peptide size was also shown to influence resulting biosilica morphology. The experimental results presented herein demonstrate the feasibility of creating biosilica nanostructures with controlled morphologies using polypeptides in vitro.

Reverse solid freeform (SFF) fabrication was used to create highly-controlled macroporous structures in nano-fibrous poly (L-lactic acid) (PLLA) scaffolds. By using a computer-aided design (CAD) program to create a negative template for the scaffold, the three-dimensional (3-D) mold was created on a 3-D printer using a wax. After the template was printed, a solution of PLLA in tetrahydrofuran (THF) was cast into the mold, and was subsequently phase separated at -70°C which gives the nano-fibrous morphology. This resulted in a 3-D nano-fibrous scaffold with a uniform fiber mesh throughout the entire matrix, and greatly increased the surface area within the scaffold. Fiber diameters in these scaffolds were 50-500 nm, similar to type I collagen, and the densities of the fiber meshes can be altered by changing the polymer concentration. To examine the scaffold's potential for tissue regeneration, MC3T3-E1 osteoblasts were seeded and cultured on the scaffolds. Results show that the osteoblasts attached and proliferated on the scaffolds. After 6 weeks in culture, bone-like tissue was evident within the nano-fibrous scaffolds. By having the ability to control the macroporous architecture, interconnectivity, orientation, and external shape of the scaffold, as well as the nanometer-scaled fibrous features in the pore walls, this SFF fabrication/phase separation technique has great potential to design and create ideal scaffolds for bone tissue engineering.

Cellular solids form the basis of many biological and engineering structures. Most models use the relative density and the mechanical properties of the bulk material as the main parameter for the prediction of the mechanical properties of such structures. In this work the influence of the architecture of periodic cellular solids on the mechanical properties is investigated numerically and experimentally.

Using computer aided design, structures with 8x8x8 base cells are designed and fabricated. The physical prototypes which are tested experimentally are made from thermosetting and thermoplastic polymers by employing Rapid Prototyping (RP) techniques. Various RP techniques are compared regarding their suitability for the fabrication of cellular materials.

For numerical simulation of the cellular structures, linear Finite Element analysis is employed. Three-dimensional models are set up using higher order beam elements. In a first step, the structure is treated as an infinite medium and homogenization via a 'periodic micro-field approach' is used. The entire elastic tensors for different relative densities are evaluated, from which the directional dependencies of the Young's moduli are derived. In a second step, simulations of finite structures are performed for direct comparison with experiments. Samples consisting of several basic cells are modeled which leads to a better correspondence to the experimental setup. Finite structures of different numbers of cells are modeled to study the influence of the sample size.

The experimental and numerical results correspond very well and form a consistent picture of the problem. The multi-disciplinary approach leads to a comprehensive view of effects which govern the mechanical behaviour of the investigated cellular structures.

The precipitation of calcium oxalate monohydrate (COM) was observed at biphasic phospholipid Langmuir monolayers with the aid of Brewster angle microscopy. COM appears preferentially at phase boundaries of a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a state of liquid expanded/liquid condensed coexistance. However, when the phase boundary is created by two different phospholipids that are phase segregated, such as DPPC and 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), crystal formation occurs away from the interface. It is suggested that COM growth at phase boundaries is preferred only when there is molecular exchange between the phases.

The observation of spectroscopic signals in response to mechanically induced changes in biological macromolecules can be enabled at an unprecedented level of resolution by coupling single-molecule manipulation/sensing using carbon nanotubes with single-molecule fluorescence imaging. Proteins, DNA and other biomolecules can be attached to nanotubes to give highly specific single-molecule probes for the investigation of intermolecular dynamics, the assembly of hybrid biological and nanoscale materials and the development of molecular electronics. Recent advances in nanotube fabrication and Atomic Force Microscope (AFM) imaging with nanotube tips have demonstrated the potential of these tools to achieve high-resolution images of single molecules. In addition, proof-of-principle demonstrations of nanotube functionalization and attachment of single molecules to these probes have been successfully made.

Improved techniques for the growth and attachment of single wall carbon nanotubes as robust and well-characterized tools for AFM imaging are being developed. This work serves as a foundation toward development of single-molecule sensors and manipulators on nanotube AFM tips for a hybrid atomic force microscope that also has single-molecule fluorescence imaging capability. An individual single wall carbon nanotube (SWNT) attached to an AFM tip can function as a structural scaffold for nanoscale device fabrication on a scanning probe. Such a probe can have a novel role, to trigger specific biochemical reactions or conformational changes in a biological system with nanometer precision. The consequences of these perturbations can be read out in real time by single-molecule fluorescence and/or AFM sensing. For example, electrical wiring of single redox enzymes to carbon nanotube scanning probes will allow for observation and electrochemical control of single enzymatic reactions, by monitoring fluorescence from a redox-active cofactor or the formation of fluorescent products. Enzymes “nanowired” to carbon nanotube tips may enable extremely sensitive probing of biological stimulus-response with high spatial resolution, including product-induced signal transduction.

The architectural design and surface properties of scaffolds are important aspects in tissue engineering. The porous scaffolds accommodate cells and guide their growth, while the surface nature of the scaffolds can directly affect cell attachment, proliferation, and ultimately neo tissue regeneration. In this work, a highly porous poly(L-lactic acid) (PLLA) scaffold with nano-fibrous pore wall architecture has been fabricated by mimicking the structure of natural collagen using a novel thermally induced phase separation method developed in our group. A universally effective surface modification method was developed, and gelatin was successfully grafted onto the surface of nano-fibrous PLLA scaffolds by entrapment procedure. The surface composition, morphology, and properties were examined using ATR-FTIR, XPS and SEM. The surface coverage of gelatin on the PLLA surface was as high as 39.4%. MC3T3-E1 osteoprogenitor cells were cultured for 6 weeks in solid-walled PLLA scaffolds, nano-fibrous PLLA scaffolds, and surface-modified nano-fibrous PLLA scaffolds, respectively. The osteoblasts proliferated in all three types of scaffolds, but the cell numbers were always significantly higher in the surface-modified nano-fibrous scaffolds than in the other two types of scaffolds, and the cell numbers in nano-fibrous scaffolds were higher than that in the solid-walled scaffolds. These results demonstrate that the surface-modified nano-fibrous architecture could serve as a superior scaffold for tissue engineering.

Surgeons and bioengineers have continuously been challenged by implant failure. Many of these engineers and surgeons trace implant failure to poor osseointegration (the bonding of an orthopedic implant to juxtaposed bone) and to the inability of implants to match the physical properties of surrounding bone. Researchers have recently shown that nanostructured materials (or materials with fundamental length scales less than 100 nm) enhance cell functions pertinent to effectively regenerating the tissue of numerous organs. Specifically, in a recent study, researchers demonstrated that metal surfaces utilizing low-micron to nanophase topography fostered increased adhesion of osteoblasts, the cells that create the matrix of bone. In this study, Ti, Ti6Al4V, and CoCrMo alloys were investigated, and these alloys were identical to current orthopedic implant alloys except for surface topography. The objective of this in vitro research was to determine whether these same nanophase metal surfaces not only foster osteoblast adhesion but also increase osteoblast metabolic activities leading to bone regeneration. Light microscopy and Energy Dispersion Spectroscopy (EDS) were used to verify the presence of calcium and phosphorous deposition by osteoblasts cultured on the metal substrates. Results indicated that both calcium and phosphorous are being deposited on several of the metal substrates. More importantly, compared to conventional metals, results provided the first evidence that more calcium and phosphorous was deposited by osteoblasts cultured on respective nanophase metals (Ti, Ti6Al4V, and CoCrMo). Nanophase CoCrMo had the most calcium and phosphorous minerals deposited by osteoblasts compared to any other metal substrate. Thus, the results of this study continue to provide evidence for the use of nanophase metals for the design of the next generation of more successful orthopedic implants.

Titanium was exposed to dilute solutions of hydrogen peroxide (H2O2) to better characterize the interaction at the interface between the solution and metal. The intensity of light passing through films of known thickness of titanium on quartz was measured as a function of time in contact with H2O2in concentrations of 0.3% and 1.0%. An atomic force microscope (AFM) was used to record deflection-distance (force) curves as a probe approached the interface of titanium in contact with solution containing 0.3% of H2O2. The interaction layer measured using AFM techniques was much greater than the thickness of the titanium films used in this study. Raman spectroscopy taken during interaction shows the emergence of a Ti-peroxy gel and titania after 2 hours in contact with 0.3% H2O2solution.

We have developed a means of producing thin, oriented lipid monolayers which are stable under repeated washing and which may be useful in biosensing or surface- coating applications. Phosphotidylcholine (PC) and the glycosphingolipid (GSL) GM1 were used as representative lipids for this process. Initially, a mixed self-assembled monolayer of octanethiol and hexadecanethiol was constructed on a clean gold surface. This hydrophobic surface was then brought into contact with a thin lipid layer that had been deposited at the air/liquid interface of a solution by evaporating a mixture of lipid in hexane on top of a layer of water. The lipid layer, now deposited on the gold surface, was then heated to cause intercalation of the fatty acid and alkanethiol chains, and cooled to form a highly stable film which withstood repeated rinsing and solution exposure. Presence and stability of the film was confirmed via ellipsometry, FTIR, and QCM, with an average overall thickness of ∼3.5 nm. These films may be potentially useful in biotoxin detection or as a protein resistant layer for the prevention of biofouling.

In the present work we are mainly interested in the use of the natural latex extract from the Hevea brasiliensis for medical applications. This presentation focuses on the influence of the fabrication parameters on the final elastic properties of the tubes obtained by dip-coating. The elastic properties of the prosthesis were investigated using tension-deformation experiments. The influence of important fabrication parameters were studied, such as: dip-coating velocity, final prosthesis thickness (which was varied as a function of the number of baths), and thermal treatment. Single and cycled tension-deformation experiments were performed with closed and opened samples considering longitudinal and axial directions. A model is proposed for the anisotropy of the elastic behavior, which involves the sulfur bonds between polymeric chains.

In situ atomic force microscopy (AFM) was used to investigate the kinetics of demineralization of human dentin collagen fibrils. Individual dentin collagen fibrils containing intrafibrillar mineral were isolated, transferred onto a glass slide and exposed to water for a day prior to demineralization studies. The fibrils then were exposed to trypsin for removal of non-collagenous proteins and subsequently demineralized in 10 vol % citric acid. Topographic images showed a gradual increase in gap-overlap depth of the fibril. The gap-overlap depth varied linearly with the square root of time before saturation at 7 nm in approximately sixty minutes, suggesting a diffusion process for demineralization of intrafibrillar mineral. Micro Raman studies of partially demineralized dentin revealed the existence of a phosphate peak at wave number close to 960 cm-1. The peak gradually disappeared in 60 minutes as the samples were exposed to 10% citric acid, supporting the notion that AFM topography may be correlated to the degree of intrafibrillar mineralization.

The adhesion of primary osteoblastic cells on top of biocompatible polyelectrolyte multilayer (PEM) films was investigated for native films and after changing the films properties either with a chemical stimulus (film functionalization), with a mechanical stimulus (film cross-linking), or with both stimuli combined. For the functionalization, a 15 amino acid peptide containing a –RGD- (-Arg-Gly-Asp) sequence was grafted to poly(L-glutamic) acid and deposited on top of poly(L-lysine)/poly(L-glutamic) (PLL/PGA), PLL/Poly(alginic), and PLL/Poly(galacturonic) films. The film buildup and the adsorption of the PGA-RGD was followed by Optical Waveguide Lightmode Spectroscopy and by Atomic Force Microscopy. The mechanical stimulus was achieved by crosslinking the films with a water soluble carbodiimide (EDC) in combination with N-hydroxysulfo-succinimide (sulfo-NHS) to induce amide formation. Fourier Transform Infrared Spectroscopy evidenced the conversion of amine and carboxylic groups into amide groups.

The alkaline phosphatase (ALP) activity test was used to assess osteoblast adhesion and proliferation on top of the different films over a period of eight days in culture. Whereas the native films are poorly adherent, the RGD-functionalized films exhibit an increased short time adhesion. The native films could also be successfully cross-linked thereby dramatically enhancing cell proliferation. The cells did not react similarly on the different types of films investigated : the cross-linked (PLL/Palg) and (PLL/Pgal) films were much more efficient than the native or functionalized films in terms of proliferation. On the other hand, for the (PLL/PGA) films, functionalization and film cross-linking had a similar long term effect. Very interestingly, for these latter films, both stimuli could be combined.

Porous metallic scaffolds have recently gained recognition as a promising avenue toward the regeneration of damaged bone structures. Interest in these materials resides in their ability to guide bone growth by presenting a favorable structure for cellular adhesion and three-dimensional proliferation. A powder metallurgy process to fabricate titanium foams with favorable microstructural parameters for applications in bone engineering has recently been developed. This study assesses the potential of this novel material for applications as an osteoconductive scaffold through in vitro characterization of early cellular interactions with titanium foams having pore sizes ranging from 167 to 500 µm. The foams exhibit no cytotoxic effects on J774 mouse macrophages while favoring adhesion and proliferation of MC3T3-E1 pre-osteoblasts. Three-dimensional morphology assumed by these cells on porous titanium suggests that the microstructure of the foams is biomimetic.

The growth of biogenic inorganic crystals demands much attention given the exceptional control of nucleation and growth that is evidenced in biological systems. In many cases the biological mineralization products are crystals engineered to express unique forms and habits; this being linked to the bio-function of minerals. It is acknowledged that the formation of these biogenic minerals is controlled by a complex directory of organic macromolecules which are chemically tailored to mediate the crystallization sequelae. In this study, an homologous series of sulphonated calix[4]arenes (further modified by the addition of an alkyl substituent (R = (CH2)nCH3; n = 0-7)) were selected as chemical and structural mimics for some of the active components in the acidic macromolecules known to be associated with many biogenic mineral phases. Here, these calix[4]arenes were assayed for their ability to control the growth of barium sulphate. Their presence in the crystallization liquor induced the twinning of the barium sulphate crystals; two forms of twinning were observed. Interpenetrant (2-31) twins were noted for substituted calix[4]arenes when R = (CH2)2CH3; and interpenetrant {610} twins when R = (CH2)nCH3; n = 0-7. When R = (CH2)nCH3; n = 0 or 1, the crystals also evidenced a novel acicular growth form. These effects were linked to the chemical properties of the calixarenes and structural recognition motifs created at the calix[4]arene/barium sulphate interface following the adsorption of these sulphonated moieties onto the nascent crystals. The results offer an insight into the mechanisms which may mediate such effects in vivo.

A bone-like composite coating of collagen protein and calcium phosphate minerals is considered to be bioactive and may enhance bone growth and fixation of metallic orthopedic implants. In this study, we have successfully prepared a uniform collagen fibril/octacalcium phosphate composite coating on silicon substrate by electrolytic deposition. Under a typical deposition condition, a thin (100 nm) layer of calcium phosphate coating would form on the cathode (Si) surface first, which was followed by a thick (∼100 μm) composite coating. The porous composite layer consists of a collagen fibril network on which clusters of octacalium phosphate crystals nucleate and grow. The results not only provide a novel bioactive coating for biomedical implants, but also establish a new experimental protocol for studying biomineralization mechanisms of collagen based biological tissues.

A biomimetic process has been developed to fabricate hydroxyapatite-gelatin (HAP-GEL) nanocomposites for bone regeneration (Chang and Ko et al. 2003). We hypothesize that this newly developed HAP-GEL is osteoconductive and is suitable for tissue engineered scaffolds. This preliminary study is aimed to characterize cell affinity and osseous regeneration of the HAP-GEL. The HAP-GEL was synthesized according to the procedures described in the previous publication. The attachment and proliferation of human fetal osteoblasts on HAP-GEL discs were evaluated using three different gelatin contents (2g, 3g, and 4g). The cells were seeded onto each disc and incubated at 34 degrees Celsius in 5% CO2 air atmosphere. At different time points of cultivation, cells were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) to determine their viability and morphology. To assess the cell proliferation, cells were detached at Days 1, 4, and 7 by trypsinzation and counted. For in vivo tests, HAP-GEL rods were implanted into the proximal femur of Sprague-Dawley rats. One month after the implantation, the femurs were harvested and the undecalcified HAP-GEL-bone sections were stained for histopathology. Four hours after attachment, most cells appeared round in all discs; cell spreading was observed after 24 hours. The highest gelatin content supported a significantly higher cell growth than the others at 7 days. Thus all compositions support satisfactory attachment, spreading and growth. In vivo results showed excellent interfacial bone regeneration. No necrotic tissues were found. In conclusion, the HAP-GEL not only mimics the biochemistry and nanostructures of bone but also supports the attachment, proliferation and differentiation (bone formation) of osteoblasts. The HAP-GEL we developed provides a suitable surface for regeneration.

Chemical action between cyclodextrins (CDs) and TOPO-(CdSe)ZnS quantum dots (QDs) generates a water-soluble solution of CD-QDs. Hydrophobic TOPO molecules on surface of the QDs are compatible to thread through the pockets of CDs and make the hydroxyl group on end of CDs to approach the ZnS surface, and then cause the interaction between ZnS and the hydroxyls. In this paper, Photoluminescence of the γ-CD-QD solution appeared about 15 nm of red movement compared with that of the QDs in hexane; 58% replacement of the crystal coordinate bond of Zn-S with that of Zn-O in the ZnS shell was demonstrated by using first-principles density functional theory and the red shift of the photoluminescence of CD-QDs; and –0.11eV of the energy gain of the exchange model was calculated by using an effective mass (EM) model. CD-QDs will provide water-soluble QDs with conjugational group for biology and molecule-device applications.

The crystallization of brushite has been investigated in the presence of typical ionic biological components, magnesium, fluoride and citrate and a natural protein, osteopontin, using a constant composition method capable of nano-mole sensitivity. While the simple ionic additives, magnesium and fluoride do not have a significant effect, citrate ions can adsorb on lateral faces due to electrostatic interactions involving the functional carboxylate groups, resulting in crystal morphology changes. Osteopontin was the most effective inhibitor influencing the growth of virtually all brushite crystal faces.

One of the major issues of joint implants is their loss (detachment) and subsequent failure after a limited number of years in application. Understanding and improving cell adhesion to implanting materials to extend lifespan, lubrication, and self-regeneration properties are therefore substantially important. In this research, we investigate effects of surface properties, such as wettabilty and texture on cell culture for the purpose of biotribological applications. Materials used are polyurethane, polyvinyl alcohol, and glass. Surface analysis indicates that surface structures have profound impact on cells growth.