Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury.

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor ß1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. We hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

The role of bioactive nanofibers in enamel regeneration mediated through integrin signals acting upon C/EBPα and c-Jun.

Enamel formation involves highly orchestrated intracellular and extracellular events; following development, the tissue is unable to regenerate, making it a challenging target for tissue engineering. We previously demonstrated the ability to trigger enamel differentiation and regeneration in the embryonic mouse incisor using a self-assembling matrix that displayed the integrin-binding epitope RGDS (Arg-Gly-Asp-Ser). To further elucidate the intracellular signaling pathways responsible for this phenomenon, we explore here the coupling response of integrin receptors to the biomaterial and subsequent downstream gene expression profiles. We demonstrate that the artificial matrix activates focal adhesion kinase (FAK) to increase phosphorylation of both c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Jun (c-Jun). Inhibition of FAK blocked activation of the identified matrix-mediated pathways, while independent inhibition of JNK nearly abolished phosphorylated-c-Jun (p-c-Jun) and attenuated the pathways identified to promote enamel regeneration. Cognate binding sites in the amelogenin promoter were identified to be transcriptionally up-regulated in response to p-c-Jun. Furthermore, the artificial matrix induced gene expression as evidenced by an increased abundance of amelogenin, the main protein expressed during enamel formation, and the CCAAT enhancer binding protein alpha (C/EBPα), which is the known activator of amelogenin expression. Elucidating these cues not only provides guidelines for the design of synthetic regenerative strategies and opportunities to manipulate pathways to regulate enamel regeneration, but can provide insight into the molecular mechanisms involved in tissue formation.

Functional supramolecular polymers.

Supramolecular polymers can be random and entangled coils with the mechanical properties of plastics and elastomers, but with great capacity for processability, recycling, and self-healing due to their reversible monomer-to-polymer transitions. At the other extreme, supramolecular polymers can be formed by self-assembly among designed subunits to yield shape-persistent and highly ordered filaments. The use of strong and directional interactions among molecular subunits can achieve not only rich dynamic behavior but also high degrees of internal order that are not known in ordinary polymers. They can resemble, for example, the ordered and dynamic one-dimensional supramolecular assemblies of the cell cytoskeleton and possess useful biological and electronic functions.

Emerging peptide nanomedicine to regenerate tissues and organs.

Peptide nanostructures containing bioactive signals offer exciting novel therapies of broad potential impact in regenerative medicine. These nanostructures can be designed through self-assembly strategies and supramolecular chemistry, and have the potential to combine bioactivity for multiple targets with biocompatibility. It is also possible to multiplex their functions by using them to deliver proteins, nucleic acids, drugs and cells. In this review, we illustrate progress made in this new field by our group and others using peptide-based nanotechnology. Specifically, we highlight the use of self-assembling peptide amphiphiles towards applications in the regeneration of the central nervous system, vasculature and hard tissue along with the transplant of islets and the controlled release of nitric oxide to prevent neointimal hyperplasia. Also, we discuss other self-assembling oligopeptide technology and the progress made with these materials towards the development of potential therapies.

Porous NiTi for bone implants: a review.

NiTi foams are unique among biocompatible porous metals because of their high recovery strain (due to the shape-memory or superelastic effects) and their low stiffness facilitating integration with bone structures. To optimize NiTi foams for bone implant applications, two key areas are under active study: synthesis of foams with optimal architectures, microstructure and mechanical properties; and tailoring of biological interactions through modifications of pore surfaces. This article reviews recent research on NiTi foams for bone replacement, focusing on three specific topics: (i) surface modifications designed to create bio-inert porous NiTi surfaces with low Ni release and corrosion, as well as bioactive surfaces to enhance and accelerate biological activity; (ii) in vitro and in vivo biocompatibility studies to confirm the long-term safety of porous NiTi implants; and (iii) biological evaluations for specific applications, such as in intervertebral fusion devices and bone tissue scaffolds. Possible future directions for bio-performance and processing studies are discussed that could lead to optimized porous NiTi implants.

Imaging and nanomedicine for diagnosis and therapy in the central nervous system: report of the eleventh annual Blood-Brain Barrier Disruption Consortium meeting.

The blood-brain barrier (BBB) presents a major obstacle to the treatment of malignant brain tumors and other central nervous system (CNS) diseases. The Eleventh Annual Blood-Brain Barrier Disruption Consortium Meeting was convened to discuss recent advances and future directions in imaging and nanomedicine. Two sessions, one on Cell and Molecular Imaging in the CNS and another on Nanotechnology, Nanobiology, and Nanomedicine, were held March 17-18, 2005, in Portland, Ore. CNS imaging presentations targeted differentiating tumor, neural lesions, and necrosis from healthy brain tissue; methods of delivery of imaging agents across the BBB; and new iron oxide-based nanoparticle contrast agents for MR imaging. Nanobiology presentations covered the development of new nanotechnology and its use in imaging, diagnosis, and therapy in the CNS. Discussions at this meeting stressed the role of biotechnology in the convergence of CNS imaging and nanomedicine and are summarized in this article.

Charge induced pattern formation on surfaces: segregation in cylindrical micelles of cationic-anionic peptide-amphiphiles.

Micelles, vesicles, and films composed of two species of incompatible heterogeneous molecules exhibit full internal segregation of the component species. This macroscopic segregation can be inhibited by oppositely charging the two different molecular species. The degree of compatibility achieved by the charges leads to either fully homogenous mixtures or to local segregation and the possible formation of regular patterns. We investigate the induction of periodic surface patterns by the presence of opposite charges in flat films and cylindrical micelles. In the strong segregation limit the incompatibility between species can be described by a line tension parameter gamma. The size of the patterns formed is of the order of a characteristic size L approximately (gamma/sigma(2))(1/2), where sigma is the surface charge density. The pattern symmetry on flat surfaces is function only of the fraction of area covered by the components, f: lamellar for 0.34

Self-assembly and mineralization of peptide-amphiphile nanofibers.

We have used the pH-induced self-assembly of a peptide-amphiphile to make a nanostructured fibrous scaffold reminiscent of extracellular matrix. The design of this peptide-amphiphile allows the nanofibers to be reversibly cross-linked to enhance or decrease their structural integrity. After cross-linking, the fibers are able to direct mineralization of hydroxyapatite to form a composite material in which the crystallographic c axes of hydroxyapatite are aligned with the long axes of the fibers. This alignment is the same as that observed between collagen fibrils and hydroxyapatite crystals in bone.

Poly(amino acid) bioadhesives for tissue repair.

We report here on poly(amino acid)-based bioadhesives with potential to bond to soft tissues. The systems investigated included homopolymer poly(amino acids), mixtures of poly(amino acids) and amino acids, and blends of different poly(amino acids). Adhesive performance was tested in tension on glass surfaces, chondroitin sulfate surfaces, as well as bovine cartilage surfaces. The amino acid structural units contained acidic, basic, or polar side chains and were found to adhere reasonably well to the surfaces of glass and chondroitin sulfate. The formation of polymer-monomer complexes with the addition of a basic monomer such as L-lysine to negatively charged polymers such as poly(L-aspartic acid) and poly(L-glutamic acid) was found to result in greater adhesive strength relative to homopolymers. Further improvement in adhesion was observed in blends of poly(L-lysine) with polar poly(amino acids) such as poly(L-asparagine). Adhesion on wet cartilage surfaces was the weakest measured but a priming approach designed to form electrostatic or hydrogen bonds appears promising. We believe the strength of the adhesives studied here is based on the ability of their constituent polymers and monomers to form molecular entanglements and crosslinks for load transfer. We also believe hydrogen bonds and electrostatic forces play a role in the adhesion of the systems to the substrates probed in this work.

Organoapatite growth on an orthopedic alloy surface.

We report here a method to coat orthopedic metals with the artificial bone material organoapatite. The growth of organoapatite on titanium alloy surfaces of foils and porous cylinders involves sequential preadsorption of poly(L-lysine) and poly(L-glutamic acid) on metal, followed by exposure to organoapatite-precipitating solutions. The organoapatite characterization of the coating was carried out by transmission electron microscopy, electron diffraction, scanning electron microscopy, energy-dispersive X-ray scattering, powder X-ray diffraction, FT-IR, and elemental analysis. The preadsorbed poly(amino acids) in the form of a self-assembled bilayer of oppositely charged macromolecules can lead to a surface coverage of titanium alloy in the range of 70-90%. The deposition mechanisms could involve the surface capture of embryonic crystals and the nucleation of apatite on the bilayer. Bioabsorbable organoapatite could serve as a tissue-engineering scaffold for bone regeneration into porous implants.

Molecular manipulation of microstructures: biomaterials, ceramics, and semiconductors.

Organic molecules can alter inorganic microstructures, offering a very powerful tool for the design of novel materials. In biological systems, this tool is often used to create microstructures in which the organic manipulators are a minority component. Three groups of materials-biomaterials, ceramics, and semiconductors-have been selected to illustrate this concept as used by nature and by synthetic laboratories exploring its potential in materials technology. In some of nature's biomaterials, macromolecules such as proteins, glycoproteins, and polysaccharides are used to control nucleation and growth of mineral phases and thus manipulate microstructure and physical properties. This concept has been used synthetically to generate apatite-based materials that can function as artificial bone in humans. Synthetic polymers and surfactants can also drastically change the morphology of ceramic particles, impart new functional properties, and provide new processing methods for the formation of useful objects. Interesting opportunities also exist in creating semiconducting materials in which molecular manipulators connect quantum dots or template cavities, which change their electronic properties and functionality.

Nanoapatite and organoapatite implants in bone: histology and ultrastructure of the interface.

This article reports on the reaction of bone to a new family of nanocrystalline hydroxyapatite biomaterials with crystal sizes similar to those of human bone. Pure nanoapatite cylinders and organoapatite cylinders containing a synthetic nanopeptide were analyzed 28 days after implantation into the spongy bone of Chinchilla rabbits. The experimental techniques used for analysis were light microscopy, scanning electron microscopy, and transmission electron microscopy. Both implant types were well incorporated, and interface events were found to be similar to those observed on human bone surfaces with regard to resorption by osteoclast-like cells and bone formation by osteoblasts. Different types of giant cells were observed resorbing the outermost surfaces of implants. There seemed to be both dissolution of the implant and particulate biodegradation leading to less dense implant regions near the interface, whereas the bulk of the implants remained denser. Transmission electron micrographs revealed that bone bonding occurred with and without an afibrillar intervening layer. Given the biologic reaction observed, these implant materials should be suitable for bone replacement and the organoapatite form could be useful for additional functions such as the release of drugs and optimized release of antibiotics, growth factors, or other substances. The organic component can also be used to control physical properties in a bony implantation bed.

Organoapatites: materials for artificial bone. II. Hardening reactions and properties.

This article reports on chemical reactions and the properties they generated in artificial bone materials termed "organoapatites." These materials are synthesized using methodology we reported in the previous article of this series. Two different processes were studied here for the transition from organoapatite particles to implants suitable for the restoration of the skeletal system. One process involved the hardening of powder compacts by beams of blue light derived from a lamp or a laser and the other involved pressure-induced interdiffusion of polymers. In both cases, the hardening reaction involved the formation of a polyion complex between two polyelectrolytes. In the photo-induced reaction an anionic electrolyte polymerizes to form the coulombic network and in the pressure-induced one, pressure forms the complex by interdiffusion of two polyions. Model reactions were studied using various polycations. Based on these results the organoapatite selected for the study was that containing dispersed poly(L-lysine) and sodium acrylate as the anionic monomer. The organomineral particles can be pressed at room temperature into objects of great physical integrity and hydrolytic stability relative to anorganic controls. The remarkable fact about these objects is that intimate molecular dispersion of only 2-3% by weight organic material provides integrity to the mineral network in an aqueous medium and also doubles its tensile strength. This integrity is essentially nonexistent in "anorganic" samples prepared by the same methodology used in organoapatite synthesis. The improvement in properties was most effectively produced by molecular bridges formed by photopolymerization. The photopolymerization leads to the "hardening" of pellets prepared by pressing of organoapatite powders. The reaction was found to be more facile in the microstructure of the organomineral, and it is potentially useful in the surgical application of organoapatites as artificial bone.

Organoapatites: materials for artificial bone. III. Biological testing.

This article reports on the in vivo testing of new artificial bone materials we have termed "organoapatites." These materials consist of mineral networks in which organic polymers are intimately dispersed by nucleation and growth of apatite crystals from a mother liquor containing the organic substances. Organoapatites were tested as implants in adult canine cortical bone for periods in the range from 12-35 weeks and fluorochromes were used in the model to investigate the kinetics of bone growth or repair. The analysis of histological samples was carried out using histomorphometric methods as well as fluorescence microscopy. Results showed excellent apposition of poly(amino acid) organoapatites with mineralized bone and fibrous encapsulation when a synthetic polyelectrolyte was the only organic component. This observation suggests that the molecularly dispersed organic dopant amounting to only 2-3% by weight of the microstructure can play a critical role in the tissue response to the implant. Relative to apatite controls, organoapatites were also found to have greater resistance to fragmentation in vivo and those containing amino acid units revealed interfacial bioerosion accompanied by regeneration of mineralized tissue. Design of organoapatite compositions and microstructures may therefore be useful in achieving the specific rate of biological response which is clinically desired.

Synthesis of two-dimensional polymers.

A synthetic pathway is described to construct "in bulk" two-dimensional (2D) polymers shaped as molecular sheets. A chiral oligomeric precursor is used that contains two reactive sites, a polymerizable group at one terminus and a reactive stereogenic center near the middle of the molecule. The bulk reaction yields bilayer 2D polymers of molecular weight in the order of millions and a monodisperse thickness of 50.2 angstroms. The 2D molecular objects form through molecular recognition by the oligomers, which self-organize into layers that place the reactive groups within specific planes. The oligomers become catenated by two different stitching reactions involving the reactive sites. At room temperature, stacks of these molecular objects can organize as single crystals and at higher temperatures melt into smectic liquid crystals. Nonlinear optical experiments reveal that solid films containing the 2D polymers form structures that are thermally and temporally more stable than those containing analogous 1D polymers. This observation suggests that the transformation of common polymers from a 1D to a 2D architecture may produce generations of organic materials with improved properties.

Organoapatites: materials for artificial bone. I. Synthesis and microstructure.

We have synthesized a new family of materials we termed organoapatites which may be useful in the formulation of artificial bone. These materials are synthesized by nucleation and growth of apatite crystals in media containing poly(amino acids) or synthetic organic polyelectrolytes using strict atmospheric, temperature, and pH control. The macromolecules used to synthesize the organoapatites include poly(L-lysine), poly(L-glutamic acid), and poly(sodium acrylate). The products were characterized by x-ray diffraction, scanning electron microscopy, surface area measurements, elemental analysis, and spectroscopic techniques. Organoapatites were found to contain large surface area morphologies with small crystallites which mature slowly based on analysis of Ca/P ratios. The organic macromolecules are thought to induce nucleation of crystals but also to quench their growth, thus becoming intimately dispersed in a mineral network. The organomineral particles harvested from the reaction medium contain polymer-netted microcrystals, and for this reason the synthetic approach can be used to modulate crystal maturation and biological response. It is likely that the preparative approach mimics some aspects of natural bone matrix synthesis and could be specially useful in the preparation of mineral implants containing intimate dispersions of small amounts of biomolecules such as growth factors, special drugs, or bioadhesives.

Surface charge on polymeric implants and calcified tissues: interfacial implications.

Samples of human bone, dentin, and enamel were analyzed through the technique known as thermally stimulated discharge (TSD). It is possible through this technique to detect current flow resulting from appearance or loss of net polarization in a material. Samples of freshly extracted tissue give rise to well-defined TSD current maximal without having been exposed to external electrical potentials. Calculation of activation energies for these currents and their thermal range suggests the involvement of collagen denaturation in the loss of appearance of a net surface charge on bone and dentin surfaces. In the case of enamel samples, TSD current maxima are possibly the result of dipolar alignment in water or biopolymers by surface charges in the mineral phase. Interfacial implications of surface charge were studied through the measurement of adhesive strength in dentin/acrylic polymer joints. Enhancement of joint strength by a factor of two or higher was observed when powder particles of the experimental adhesive carried externally induced surface charge. It is hypothesized that electrostatic coupling between polarization domains on the tissue surface and the setting implant improves wetting and produces stronger interfaces.

Mechanical strength of poly(methyl methacrylate) cement-human bone interfaces.

A device was constructed to test the interfacial strength of PMMA-based bone cement and human cancellous bone under pure tension. Two types of tissue were used in the investigation: (1) formalin-fixed vertebral bone as an in vitro model for weak cancellous bone, and (2) freshly removed metatarsal bone. Tissue--cement joints were allowed to solidify under two different pressures (0.11 and 0.47 MPa), and cement placement time on tissue surfaces was also controlled as a variable. The higher curing pressure only seemed to enhance the strength of interfaces formed with mechanically weak fixed bone but had no significant effect for joints formed with the stronger, freshly extracted tissue. Cement placement time did not have a discernible effect on interfacial strength regardless of the tissue used or the pressure applied during setting. An analysis of fracture morphology by optical microscopy revealed largely cement cohesive failure in some cases and bone or mixed fractures in others. Joints exhibiting mainly cement fracture had the highest interfacial tensile strengths (in the order of 7.5 MPa). Once measured values of tissue porosity were taken into account, the observed joint strength correlated well with cement tensile strength. Based on experimental findings, better stress-dissipating qualities and higher tensile strength are suggested as two important necessary improvements of bone cements based on poly(methyl methacrylate).