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PubMed2005

Three-dimensional organization of helices: design principles for nucleobase-functionalized beta-peptides.

The construction and molecular recognition of various three-dimensional biomimetic structures is based on the predictable de novo design of artificial molecules. In this regard beta-peptides are especially interesting, since stable secondary structures are obtained already with short sequences; one of them is the 14-helix in which every third residue has the same orientation. The covalent functionalization of every third 14-helix side chain with nucleobases was used for a reversible organization of two helices based on nucleobase pairing. A series of beta-peptides with various nucleobase sequences was synthesized and the stability of double strand formation was investigated. As few as four nucleobases are sufficient for considerable duplex stability. The stability of base pairing was examined by temperature-dependent UV spectroscopy and the formation of the 14-helix was confirmed by circular dichroism (CD) spectroscopy. The preferred strand orientation of complementary-nucleobase-modified beta-peptide helices was investigated as well as the influence of helix content on the duplex stability. The preorganization of a 14-helix in regard to double-strand recognition was tuned by the sequential order of polar beta-amino acids or by the amount of 2-aminocyclohexanecarboxylic acid units incorporated, which are known to facilitate 14-helix formation, respectively.

Base Pairing↗

PubMed2006

Total synthesis of the cyclodepsipeptide apratoxin A and its analogues and assessment of their biological activities.

A novel total synthesis of apratoxin A is described, with key steps including the assembly of its ketide segment through a D-proline-catalyzed direct aldol reaction and Oppolzer's anti aldol reaction and the preparation of its thiazoline unit in a biomimetic synthesis. An oxazoline analogue of apratoxin A has also been elaborated by a similar approach. This compound has a potency against HeLa cell proliferation only slightly lower than that of apratoxin A, whilst a C(40)-demethylated oxazoline analogue of apratoxin A displays a much lower cytotoxicity and the C(37)-epimer and C(37) demethylation product of this new analogue are inactive. These results suggest that the two methyl groups at C(37) and C(40) and the stereochemistry at C(37) are essential for the potent cellular activity of the oxazoline analogue of apratoxin A. Further biological analysis revealed that both synthetic apratoxin A and its oxazoline analogue inhibited cell proliferation by causing cell cycle arrest in the G1 phase.

Antineoplastic Agents↗

PubMed2007

Reversible immobilization and direct electron transfer of cytochrome c on a pH-sensitive polymer interface.

A pH-sensitive polymer interface has been used as a matrix for reversible immobilization of cytochrome c (Cyt c) on an Au surface through a dip-coating process. The pH-sensitive behavior of the polymer brush interface has been demonstrated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The reversible immobilization and electron-transfer properties of Cyt c have been investigated by in situ UV/Vis spectrophotometry and CV. The results have shown that the poly(acrylic acid) (PAA) brush acted as an excellent adsorption matrix and a good accelerant for the direct electron transfer of Cyt c, which gave redox peaks with a formal potential of 40 mV versus Ag/AgCl in pH 7.6 phosphate buffer solution. The average surface coverage of Cyt c on the PAA film was about 1.7 x 10(-10) mol cm(-2), indicating a multilayer of Cyt c. The electron-transfer rate constant was calculated to be around 0.19 s(-1) according to the CV experiments. The interface was subjected to in situ attenuated total internal reflection Fourier-transform infrared (ATR-FTIR) spectroscopic analysis, in order to further confirm the immobilization of Cyt c on the surface. This polymer-protein system may have potential applications in the design of biosensors, protein separation, interfacial engineering, biomimetics, and so on.

Acrylic Resins↗

PubMed2006

True monolayer cell culture in a confined 3D microenvironment enables lineage informatics.

BACKGROUND: There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo-like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates "lineage informatics," a lineage-centric approach to cytomics. METHODS: We cultured cells in a confined monolayer using a novel "gap chamber" that produces images with confocal-like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. RESULTS: Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo-like than traditional culture systems. CONCLUSIONS: The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long-term cell imaging.

Animals↗

PubMed2004

Separation of biomolecules using electrophoresis and nanostructures.

A large number of nanostructures have the potential to be used together with electrophoresis as separation media or separation additive in capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other analytical techniques. Among those structures are nanotubes, nanocavities, nanowires, nanoposts, nanocones, nanospheres, molecular imprints, nanoparachutes (conical monodendrons), and general nanoparticles with random structures. This review is focused only on publications describing experimental works using molecular imprints, nanoposts, and nanospheres that are fabricated and applied for the purpose of separation media in electrophoresis-driven separations. The review follows an approximate chronological order in each section. As shown, the most popular are those resulting from molecular imprinting technologies. These biomimetic receptors are used in a great variety of fields, which includes electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other fields not reviewed in this work. A few examples of these other fields are, e.g., liquid chromatography, membranes, extractor or preconcentration techniques, immunosorbent assays, and sensing devices. The second topic scanned in the present work is the nanostructures that are used as obstacles to replace gels or polymers solutions in electrophoresis. Finally, the nascent field of nanospheres of gold and other materials as separation media is also reviewed.

Electrophoresis, Microchip↗

PubMed2002

Hydroxyapatite deposition by electrophoresis on titanium sheets with different surface finishing.

Hydroxyapatite coatings are commonly applied to metallic biomedical implants to accelerate osseointegration. These coatings, usually produced by plasma spray techniques, can be obtained by alternative processes, like biomimetic process, electrolytic deposition, or electrophoretic process as well. Electrophoretic deposition of hydroxyapatite exhibits several advantages like simplicity and low cost. In this article, titanium sheets with three different surface finishing were coated with hydroxyapatite by using electrophoresis. Surface treatments include: (1) abrading with SiC paper; (2) abrading with SiC paper plus electrolytic etch with H3PO4 solution; and (3) blasting with alumina powder followed by etch with a solution containing H2O2 and HF. Stoichiometric hydroxyapatite was used to coat titanium sheets. Blasted samples were also coated using a calcium-deficient hydroxyapatite. SEM, XRD, and FTIR were employed to characterize titanium substrates and coatings produced. Results show that electrophoretic process can produce a uniform thin layer, satisfactorily adhered, of hydroxyapatite on treated titanium samples. Furthermore, sintering at 800 degrees C do not promote the decomposition of calcium-deficient hydroxyapatite.

Biocompatible Materials↗

PubMed2003

Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals.

Replacement of bone tissue by graft materials and products of tissue engineering having composition, structure, and biological features that mimic natural tissue is a goal to be pursued. A biomimetic synthesis was performed to prepare new bone-like composites constituted of hydroxyapatite nanocrystals and self-assembled type I collagen fibers. We used a biological inspired approach that proved that the biological systems stored and processed information at the molecular level. Two different methodologies were used: dispersion of synthetic hydroxyapatite in telopeptides free collagen molecules solution and direct nucleation of hydroxyapatite into reconstituted collagen fibers during their assembling. The different preparation techniques were experimented then the composites thoroughly characterized and compared. Composite obtained by direct nucleation showed an intimated interaction of the inorganic and proteic components, which modified the apatitic phase and made its composition, morphology and structure similar to the mineral component of natural bone.

Bone Substitutes↗

PubMed2003

Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment.

Tissue engineering aims at resolving problems such as donor shortage and immune rejection faced by transplantation. Scaffolds (artificial extracellular matrices) have critical roles in tissue engineering. Recently, we developed nano-fibrous poly(L-lactic acid) scaffolds under the hypothesis that synthetic nano-fibrous scaffolding, mimicking the structure of natural collagen fibers, could create a more favorable microenvironment for cells. This is the first report that the nano-fibrous architecture built in three-dimensional scaffolds improved the features of protein adsorption, which mediates cell interactions with scaffolds. Scaffolds with nano-fibrous pore walls adsorbed four times more serum proteins than scaffolds with solid pore walls. More interestingly, the nano-fibrous architecture selectively enhanced protein adsorption including fibronectin and vitronectin, even though both scaffolds were made from the same poly(L-lactic acid) material. Furthermore, nano-fibrous scaffolds also allowed >1.7 times of osteoblastic cell attachment than scaffolds with solid pore walls. These results demonstrate that the biomimetic nano-fibrous architecture serves as superior scaffolding for tissue engineering.

Animals↗

PubMed2003

Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces. Contact angle goniometry and X-ray photoelectron spectroscopy (XPS) data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast (RCO) surfaces in serum-containing media. IPN modified titanium and negative control (RGE-IPN) surfaces inhibit cell adhesion and proliferation, while RGD-modified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants.

Amino Acid Sequence↗

PubMed2003

Modification of calcium-phosphate coatings on titanium by recombinant amelogenin.

Amelogenin proteins, the principal components of the developing dental enamel extracellular matrix, have been postulated to facilitate the elongated and oriented growth of the carbonated apatite crystals during enamel formation. We previously reported that amelogenin caused modulation of apatite crystals nucleated on a bioactive glass (Bioglass(R)) in vitro. Here, the effects of amelogenin on the growth morphology of calcium-phosphate crystals nucleated on a titanium surface were investigated in order to gain a better understanding of the role of amelogenins during enamel biomineralization and to explore their potential application in the design and development of novel biomaterials. The dose-dependent effects of a recombinant mouse amelogenin (rM179) were found to be different from those of bovine serum albumin, which significantly inhibited apatite crystal growth and caused the octacalcium phosphate (OCP) crystals to change from a plate-like shape to a curved shape, indicating a general inhibitory effect. The effects of rM179 on the crystal growth of OCP at 12.5-100 microg/mL and of apatite at 50 microg/mL were insignificant while the apatite crystals were remarkably elongated along their c-axes upon the use of 100 microg/mL of rM179. The unique modulation of the calcium-phosphate coatings on titanium by rM179 supports the view that amelogenins have a great potential for applications designed to develop novel biomimetic materials.

Amelogenin↗

PubMed2003

Osteogenecity of octacalcium phosphate coatings applied on porous metal implants.

The biomimetic route allows the homogeneous deposition of calcium phosphate (Ca-P) coatings on porous implants by immersion in simulated physiologic solution. In addition, various Ca-P phases, such as octacalcium phosphate (OCP) or bone-like carbonated apatite (BCA), which are stable only at low temperatures, can be deposited. In this pilot study, experiments were designed with a twofold-purpose: (1) to investigate the osteoinduction of OCP-coated and noncoated porous tantalum cylinders and of dense titanium alloy cylinders (5 mm in diameter and 10 mm in length) in the back muscle of goats at 12 and 24 weeks (n = 4); and (2) to compare the osteogenic potentials of BCA-coated, OCP-coated, and bare porous tantalum cylinders in a gap of 1 mm created in the femoral condyle of a goat at 12 weeks (n = 2). In the goat muscle, after 12 weeks the OCP-coated porous cylinder had induced ectopic bone as well as bone within the cavity of the OCP-coated dense titanium cylinder. In the femoral condyle, bone did not fill the gap in any of the porous implants. In contrast with the two other groups, OCP-coated porous cylinders exhibited bone formation in the center of the implant. The nature of the Ca-P coating, via its microstructure, its dissolution rate, and its specific interactions with body fluids, may influence the osteogenecity of the Ca-P biomaterial.

Animals↗

PubMed2003

Preparation and assessment of revised simulated body fluids.

A simulated body fluid (SBF) with ion concentrations approximately equal to those of human blood plasma has been used widely for in vitro assessment of the bioactivity of artificial materials and for the formation of bone-like apatite on various substrates. The ion concentrations of a conventional SBF (c-SBF) are, however, not exactly equal to those of blood plasma. In the present study, a revision of c-SBF was made to prepare new SBFs (r-SBF, i-SBF, and m-SBF) with ion concentrations equal to or closer to those of blood plasma. The ion concentrations of the r-SBF and i-SBF were designed to be equal to those of blood plasma in total and dissociated amounts, respectively. The m-SBF was designed to have a total ion concentration equal to that of blood plasma, except for the concentration of HCO(-) (3), which was set to the saturated level with respect to calcite. The ion concentrations and pH of the as-prepared new SBFs were found to be equal to those of the nominal values. Upon sealed storage, the r-SBF and i-SBF showed no change in ion concentrations for up to 4 weeks at 5 degrees C, and up to 2 weeks at 36.5 degrees C, but thereafter they showed a decrease in HCO(-) (3) concentration and an increase in pH. Under the same storage conditions, the c-SBF and m-SBF showed no change in ion concentrations and pH values over a period of up to 8 weeks. These results indicate that the r-SBF and i-SBF are less stable than the c-SBF and m-SBF in terms of changes in ion concentrations relative to storage period. The m-SBF is optimal for in vitro bioactivity assessment of artificial materials and for biomimetic production of bone-like apatite.

Blood Chemical Analysis↗

PubMed2004

The in vivo calcification capacity of a copolymer, based on methacryloyloxyethyl phosphate, does not favor osteoconduction.

Polymers can be interesting alternatives to bone grafts; they must present suitable mechanical and osteoconductive properties. Biomimetic properties may be a key factor for the recognition by bone cells. Methacryloyloxyethyl phosphate (MOEP) was found to enhance hydroxyapatite deposition. The copolymer containing MOEP and 1-vinyl-2-pyrrolidinone (50-50%) binds large amounts of calcium. Particles of the copolymer were used to fill large cranial bone defects in the rat. After a 12-week healing period, the animals were euthanized and the skulls examined by X-ray, histology, and electron microscopy (EM). The high phosphate content of the polymer conferred a marked calcium-binding capacity, and the particles were heavily calcified. They were embedded in a light fibrous stroma containing numerous capillaries and multinucleated giant cells. The osteoconductive properties were poor: only few trabeculae developed centripetally from the margins of the defects. There was no bone bonding and no osteoblast on the surface of the calcified material. Backscattered EM revealed that the degree of calcification was homogeneous in all particles. Calcium-phosphorus calcospherites were never observed. The material appeared to trap calcium but to impair nucleation because only small hydroxyapatite tablets were occasionally observed. Polyphosphated materials do not represent a suitable source of potentially usable bone substitutes.

Animals↗

PubMed2004

Effect of wollastonite ceramics and bioactive glass on the formation of a bonelike apatite layer on a cobalt base alloy.

A biomimetic method was used to promote a bioactive surface on a cobalt base alloy (ASTM F-75). The metallic substrates were alkali treated and some of the samples were subsequently heat treated. The treated samples were immersed in simulated body fluid (SBF) on granular particles of either bioactive glass or wollastonite. For comparative purposes, no bioactive system was used in some tests. Three different methods were used for the immersion of the samples in SBF: 1) 21 days in SBF, 2) 21 days in 1.5 SBF, and 3) 7 days in SBF followed by 14 days in 1.5 SBF (re-immersion method). A bonelike apatite layer was formed on all the samples placed on wollastonite and bioactive glass particles. The morphology of the apatite layer formed by using the re-immersion method and wollastonite closely resembled the existing bioactive systems. No apatite layer was observed on the samples treated without bioactive material and soaked for 21 days in SBF or 1.5 SBF, apart from the substrates treated by using the re-immersion method. The heat treatment delayed the apatite formation in all the cases studied.

Alloys↗

PubMed2005

Osteocalcin enhances bone remodeling around hydroxyapatite/collagen composites.

The effect of osteocalcin (OC), an extracellular bone matrix protein, on bone healing around hydroxyapatite/collagen composites was investigated. Cylindrical nanocrystalline hydroxyapatite implants of 2.5-mm diameter containing 2.5% biomimetically mineralized collagen type I were inserted press-fit into the tibial head of adult Wistar rats. To one implant group, 10 mug/g OC was added. Six specimens per group were analyzed at 2, 7, 14, 28, and 56 days. After 14 days, newly formed woven bone had reached the implant surface of the OC implants whereas a broad fibrous interface could still be observed around controls. Woven bone was formed directly around both implant groups after 28 days and had been replaced partially by lamellar bone around the OC implants only. No significant differences in total bone contact were seen between both groups after 56 days. The higher number of phagocytosing cells and osteoclasts characterized immunohistochemically with ED1, cathepsin D, and tartate-resistant alkaline phosphatase around the OC implants at the early stages of bone healing suggests an earlier onset of bone remodeling. The earlier and increased expression of bone-specific matrix proteins and multifunctional adhesion proteins (osteopontin, bone sialoprotein, CD44) at the interface around the OC implants indicates that OC may accelerate bone formation and regeneration. This study supports the observations from in vitro studies that OC activates both osteoclasts and osteoblasts during early bone formation.

Animals↗

PubMed2005

Long-term evaluation of porous poly(epsilon-caprolactone-co-L-lactide) as a bone-filling material.

Porous poly(epsilon-caprolactone-co-L-lactide) (P(CL-co-LA, wt % ca. 5/95) sponges were prepared, coated biomimetically with CaP/apatite, and implanted with noncoated control sponges into rat femur cortical defects and dorsal subcutaneous space. The implants were inspected histologically at 2, 4, and 33 weeks after the operation. All implants were filled with fibrovascular tissue within 4 weeks. The femur implants were partially ossified with compact bone, which in the CaP-coated sponges was less mature and more fragmented. Approximately equal amounts of bone were observed in both types of implants. The polymer induced a mild inflammatory reaction with foreign body giant cells but no accumulation of fluid. Degradation of the polymer was slow; most of it was found intact at 33 weeks in histological samples. Nondegraded polymer seems to prevent complete ossification. Cultured osteoblasts proliferated well on apatite-coated material, whereas only a few cells were seen on noncoated material. Thus CaP/apatite coating helped the attachment of osteoblasts in cell cultures but did not offer any advantage in bone formation over noncoated material in vivo. We conclude that a shorter degradation time of P(CL-co-LA) is needed to create an optimal implant. Furthermore, in vivo experiments seem to be necessary for the estimation of osteopromotive properties of a biomaterial.

Absorbable Implants↗

PubMed2006

Biodegradation and bioabsorption innovation of the functionally graded bovine bone-originated apatite with blood permeability.

Bioabsorbable and functionally graded apatite (fg-HAp) ceramics were designed using bovine bone by the calcination and partial dissolution-precipitation methods. The fg-HAp ceramics that were developed had gradual distributions of the degree of crystallinity and the grain size of single-phase hydroxyapatite from the surface layer of the pore wall to the bulk structure region. Calcination at 1073 K gave a specific surface area of 30 m2 x g-1 and porosities of 60-80%. The pore structure of the fg-HAp was classified into two regions: a macro-pore region (100-600 microm) originating from spongy bone and a micro-pore region (10-160 nm) related to body fluid permeation and blood permeability. By implantation in subcutaneous tissue of rat, it was confirmed that body fluid permeated the bulk region of the fg-HAp ceramics through the micro-pores. The volumetric populations occupied by body fluid were 60% at 4 weeks and 68% at 8 weeks in the ceramics explants, indicating drastic bioabsorption, although the body fluid was found to be immunopositive for an albumin as the main serum protein in blood. On the fg-HAp ceramics developed here, the bioabsorption rate could be controlled by careful selection of the calcination temperature. These ceramics can be applied as new biomimetic ceramics exhibiting surface and bulk degradations and cellular absorption by giant cells.

Adsorption↗

PubMed2006

Human neutrophils reaction to the biodegraded nano-hydroxyapatite/collagen and nano-hydroxyapatite/collagen/poly(L-lactic acid) composites.

The impact of biodegraded nano-hydroxyapatite/collagen (nHAC) composite and nano-hydroxyapatite/collagen/poly(L-lactic acid) (nHAC/PLA) scaffold composite on neutrophils reaction was evaluated in vitro. Neutrophils were separated from human peripheral blood of healthy subjects. The nHAC and nHAC/PLA materials were immersed in the D-Hanks' Balanced Salt Solution (D-HBSS) for 1 day, 7 days and 2, 4, 8 weeks (37 degrees C) as testing solution, which mixed with the neutrophils for 1 h. Both of the nHAC and nHAC/PLA materials were shown the same cell survival rate as blank control, but the lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) released from the neutrophils were increased significantly after the 2 weeks in nHAC sample. The possible reason relied on the high concentration of calcium due to the quick biodegradation of the nHAC material. Before 2 weeks, the LDH value of nHAC/PLA is higher than that of nHAC sample that corresponded to the initial PLA degradation in vitro. This study provided the biocompatibility test of neutrophils other than common methods, such as osteoblastic cells for biomimetic materials. Moreover, it demonstrated the calcium concentration stimulating effect for cytokine release from neutrophils.

Biocompatible Materials↗