Hydroxyapatite Binding Peptide Research Articles

A Combination of Flexible Modified Plant Virus Nanoparticles Enables Additive Effects Resulting in Improved Osteogenesis.

Plant virus nanoparticles (VNPs) genetically engineered to present osteogenic cues provide a promising method for biofunctionalizing hydrogels in bone tissue engineering. Flexible Potato virus X (PVX) nanoparticles substantially enhance the attachment and differentiation of human mesenchymal stem cells (hMSCs) by presenting the RGD motif, hydroxyapatite-binding peptide (HABP), or consecutive polyglutamates (E8) in a concentration-dependent manner. Therefore, it is hypothesized that Tobacco mosaic virus nanoparticles, which present 1.6 times more functional peptides than PVX, will meliorate such an impact. This study hypothesizes that cultivating hMSCs on a surface coated with a combination of two VNPs presenting peptides for either cell attachment or mineralization can achieve additionally enhancing effects on osteogenesis. Calcium minerals deposited by differentiating hMSCs increases two to threefold for this combination, while the Alkaline Phosphatase activity of hMSCs grown on the PVX-RGD/PVX-HABP-coated surface significantly surpasses any other VNP combination. Superior additive effects are observed for the first time by employing a combination of VNPs with varying functionalities. It is found that the flexible VNP geometry plays a more critical role than the concentration of functional peptides. In conclusion, various peptide-presenting plant VNPs exhibit an additive enhancing effect offering significant potential for effectively functionalizing cell-containing hydrogels in bone tissue engineering.

Biomineralization of calcium phosphates functionalized with hydroxyapatite-binding peptide

Functionalization of calcium phosphates with biomimetic peptides is a promising strategy to increase cellular response for bone tissue repair. In this work, biphasic calcium phosphate pellets were functionalized with the hydroxyapatite-binding peptide pVTK by dropping a suspension of the peptide on the pellet surface. The bioactivity tests were performed in vitro by using McCoy culture medium. Cytotoxicity tests were also performed to assess cell viability. The material was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy with field emission gun (FEG-SEM). The results showed that functionalization with the biomimetic peptide was most effective in inducing precipitation of bone-like apatite on the pellets surface, when compared to the control groups (two positive control groups and one negative control group). Cytotoxicity tests showed that all samples are biocompatible but the pellets with peptide showed the highest values of cell viability.

Understanding the Adhesion Mechanism of Hydroxyapatite-Binding Peptide.

Understanding the interactions between the protein collagen and hydroxyapatite is of high importance for understanding biomineralization and bone formation. Here, we undertook a reductionist approach and studied the interactions between a short peptide and hydroxyapatite. The peptide was selected from a phage-display library for its high affinity to hydroxyapatite. To study its interactions with hydroxyapatite, we performed an alanine scan to determine the contribution of each residue. The interactions of the different peptide derivatives were studied using a quartz crystal microbalance with dissipation monitoring and with single-molecule force spectroscopy by atomic force microscopy. Our results suggest that the peptide binds via electrostatic interactions between cationic moieties of the peptide and the negatively charged groups on the crystal surface. Furthermore, our findings show that cationic residues have a crucial role in binding. Using molecular dynamics simulations, we show that the peptide structure is a contributing factor to the adhesion mechanism. These results suggest that even small conformational changes can have a significant effect on peptide adhesion. We suggest that a bent structure of the peptide allows it to strongly bind hydroxyapatite. The results presented in this study improve our understanding of peptide adhesion to hydroxyapatite. On top of physical interactions between the peptide and the surface, peptide structure contributes to adhesion. Unveiling these processes contributes to our understanding of more complex biological systems. Furthermore, it may help in the design of de novo peptides to be used as functional groups for modifying the surface of hydroxyapatite.

Reconfigurable Dual Peptide Tethered Polymer System Offers a Synergistic Solution for Next Generation Dental Adhesives.

Resin-based composite materials have been widely used in restorative dental materials due to their aesthetic, mechanical, and physical properties. However, they still encounter clinical shortcomings mainly due to recurrent decay that develops at the composite-tooth interface. The low-viscosity adhesive that bonds the composite to the tooth is intended to seal this interface, but the adhesive seal is inherently defective and readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite-tooth interface and bacterial by-products demineralize the tooth and erode the adhesive. These activities lead to wider and deeper gaps that provide an ideal environment for bacteria to proliferate. This complex degradation process mediated by several biological and environmental factors damages the tooth, destroys the adhesive seal, and ultimately, leads to failure of the composite restoration. This paper describes a co-tethered dual peptide-polymer system to address composite-tooth interface vulnerability. The adhesive system incorporates an antimicrobial peptide to inhibit bacterial attack and a hydroxyapatite-binding peptide to promote remineralization of damaged tooth structure. A designer spacer sequence was incorporated into each peptide sequence to not only provide a conjugation site for methacrylate (MA) monomer but also to retain active peptide conformations and enhance the display of the peptides in the material. The resulting MA-antimicrobial peptides and MA-remineralization peptides were copolymerized into dental adhesives formulations. The results on the adhesive system composed of co-tethered peptides demonstrated both strong metabolic inhibition of S. mutans and localized calcium phosphate remineralization. Overall, the result offers a reconfigurable and tunable peptide-polymer hybrid system as next-generation adhesives to address composite-tooth interface vulnerability.

Chemometrics-Assisted Raman Spectroscopy Characterization of Tunable Polymer-Peptide Hybrids for Dental Tissue Repair.

The interfaces that biological tissues form with biomaterials are invariably defective and frequently the location where failure initiates. Characterizing the phenomena that lead to failure is confounded by several factors including heterogeneous material/tissue interfaces. To seamlessly analyze across these diverse structures presents a wealth of analytical challenges. This study aims to develop a molecular-level understanding of a peptide-functionalized adhesive/collagen hybrid biomaterial using Raman spectroscopy combined with chemometrics approach. An engineered hydroxyapatite-binding peptide (HABP) was copolymerized in dentin adhesive and dentin was demineralized to provide collagen matrices that were partially infiltrated with the peptide-functionalized adhesive. Partial infiltration led to pockets of exposed collagen–a condition that simulates defects in adhesive/dentin interfaces. The spectroscopic results indicate that co-polymerizable HABP tethered to the adhesive promoted remineralization of the defects. The spatial distribution of collagen, adhesive, and mineral as well as crystallinity of the mineral across this heterogeneous material/tissue interface was determined using micro-Raman spectroscopy combined with chemometrics approach. The success of this combined approach in the characterization of material/tissue interfaces stems from its ability to extract quality parameters that are related to the essential and relevant portions of the spectral data, after filtering out noise and non-relevant information. This ability is critical when it is not possible to separate components for analysis such as investigations focused on, in situ chemical characterization of interfaces. Extracting essential information from complex bio/material interfaces using data driven approaches will improve our understanding of heterogeneous material/tissue interfaces. This understanding will allow us to identify key parameters within the interfacial micro-environment that should be harnessed to develop durable biomaterials.

Structure–Activity Relationships of Hydroxyapatite-Binding Peptides

Elucidating the structure-activity relationships between biomolecules and hydroxyapatite (HAP) is essential to understand bone mineralization mechanisms, develop HAP-based implants, and design drug delivery vectors. Here, four peptides identified by phage display were selected as model HAP-binding peptides (HBPs) to examine the effects of primary amino acid sequence, phosphorylation of serine, presence of charged amino acid residues, and net charge of the peptide on (1) HAP-binding affinity, (2) secondary conformation, and (3) HAP nucleation and crystal growth. Binding affinities were determined by obtaining adsorption isotherms by mass depletion, and the conformations of the peptides in solution and bound states were observed by circular dichroism. Results showed that the magnitude of the net charge primarily controlled binding affinity, with little dependence on the other HBP features. The binding affinity and conformation results were in good agreement with our previous molecular dynamics simulation results, thus providing an excellent benchmark for the simulations. Transmission electron microscopy was used to explore the effect of these HBPs on calcium phosphate (Ca-PO4) nucleation and growth. Results indicated that HBPs may inhibit nucleation of Ca-PO4 nanoparticles and their phase transition to crystalline HAP, as well as control crystal growth rates in specific crystallographic directions, thus changing the classical needle-like morphology of inorganically grown HAP crystals to a biomimetic plate-like morphology.

Improvement of physical properties of calcium phosphate cement by elastin-like polypeptide supplementation

Calcium phosphate cements (CPCs) are synthetic bioactive cements widely used as hard tissue substitutes. Critical limitations of use include their poor mechanical properties and poor anti-washout behaviour. To address those limitations, we combined CPC with genetically engineered elastin-like polypeptides (ELPs). We investigated the effect of the ELPs on the physical properties and biocompatibility of CPC by testing ELP/CPC composites with various liquid/powder ratios. Our results show that the addition of ELPs improved the mechanical properties of the CPC, including the microhardness, compressive strength, and washout resistance. The biocompatibility of ELP/CPC composites was also comparable to that of the CPC alone. However, supplementing CPC with ELPs functionalized with octaglutamate as a hydroxyapatite binding peptide increased the setting time of the cement. With further design and modification of our biomolecules and composites, our research will lead to products with diverse applications in biology and medicine.

Quantifying the efficiency of Hydroxyapatite Mineralising Peptides

We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0–100%) from pure components and multiple (N = 10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200 mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20 mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors.

Mass Spectrometry and Ion Mobility Characterization of Bioactive Peptide-Synthetic Polymer Conjugates.

The bioconjugate BMP2-(PEO-HA)2, composed of a dendron with two monodisperse poly(ethylene oxide) (PEO) branches terminated by a hydroxyapatite binding peptide (HA), and a focal point substituted with a bone growth stimulating peptide (BMP2), has been comprehensively characterized by mass spectrometry (MS) methods, encompassing matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), tandem mass spectrometry (MS2), and ion mobility mass spectrometry (IM-MS). MS2 experiments using different ion activation techniques validated the sequences of the synthetic, bioactive peptides HA and BMP2, which contained highly basic amino acid residues either at the N-terminus (BMP2) or C-terminus (HA). Application of MALDI-MS, ESI-MS, and IM-MS to the polymer-peptide biomaterial confirmed its composition. Collision cross-section measurements and molecular modeling indicated that BMP2-(PEO-HA)2 exists in several folded and extended conformations, depending on the degree of protonation. Protonation of all basic sites of the hybrid material nearly doubles its conformational space and accessible surface area.

Predicting the Structure–Activity Relationship of Hydroxyapatite-Binding Peptides by Enhanced-Sampling Molecular Simulation

Understanding the molecular structural and energetic basis of the interactions between peptides and inorganic surfaces is critical to their applications in tissue engineering and biomimetic material synthesis. Despite recent experimental progresses in the identification and functionalization of hydroxyapatite (HAP)-binding peptides, the molecular mechanisms of their interactions with HAP surfaces are yet to be explored. In particular, the traditional method of molecular dynamics (MD) simulation suffers from insufficient sampling at the peptide-inorganic interface that renders the molecular-level observation dubious. Here we demonstrate that an integrated approach combining bioinformatics, MD, and metadynamics provides a powerful tool for investigating the structure-activity relationship of HAP-binding peptides. Four low charge density peptides, previously identified by phage display, have been considered. As revealed by bioinformatics and MD, the binding conformation of the peptides is controlled by both the sequence and the amino acid composition. It was found that formation of hydrogen bonds between lysine residue and phosphate ions on the surface dictates the binding of positively charged peptide to HAP. The binding affinities of the peptides to the surface are estimated by free energy calculation using parallel-tempering metadynamics, and the results compare favorably to measurements reported in previous experimental studies. The calculation suggests that the charge density of the peptide primarily controls the binding affinity to the surface, while the backbone secondary structure that may restrain side chain orientation toward the surface plays a minor role. We also report that the application of enhanced-sampling metadynamics effects a major advantage over the steered MD method by significantly improving the reliability of binding free energy calculation. In general, our novel integration of diverse sampling techniques should contribute to the rational design of surface-recognition peptides in biomedical applications.

Ambulatory arterial stiffness index in adolescents with high ambulatory blood pressure

Although microcalcifications of hydroxyapatite can be found in both benign and malignant osteotropic tumors, they are mostly seen in proliferative lesions, including carcinoma. The aim of this present study is to develop a molecular imaging contrast agent for selective identification of hydroxyapatite calcification in human osteotropic tumor tissues ex vivo and in human osteosarcoma cells in vitro.A bioinspired biomarker, hydroxyapatite binding peptide (HABP), was designed to mimic natural protein osteocalcin property in vivo. A fluorescein isothiocyanate dye conjugated HABP (HABP-19) was utilized to characterize hydroxyapatite on human osteotropic tumor tissue sections ex vivo and to selectively image hydroxyapatite calcifications in human osteosarcoma cells in vitro.Using a HABP-19 molecular imaging probe, we have shown that it is possible to selectively image hydroxyapatite calcifications in osteotropic cancers ex vivo and in human SaOS-2 osteosarcoma cells in vitro.Hydroxyapatite calcifications were selectively detected in osteotropic tissues ex vivo and in the early stage of the calcification process of SaOS-2 human osteosarcoma in vitro using our HABP-19 molecular imaging probe. This new target-selective molecular imaging probe makes it possible to study the earliest events associated with hydroxyapatite deposition in various osteotropic cancers at the cellular and molecular levels.It potentially could be used to diagnose and treat osteotropic cancer or to anchor therapeutic agents directing the local distribution of desired therapy at calcified sites.

Protein-mediated hydroxyapatite composite layer formation on nanotubular titania

Realising controllable interactions at the bio-nanomaterial interfaces are vital in developing next-generation engineered implant materials. Titanium-based implants are key materials in biomedical engineering due to excellent bulk mechanical properties and biocompatibilities. Advanced bio-interfaces resolving nanostructured modulated surfaces that allow manipulation with the biological molecules is one of the keys to enhance favourable interactions with the surrounding biological species. Here, we developed a protein-mediated hydroxyapatite composite layer on nanotubular titania surface. Green fluorescence protein, engineered to contain hydroxyapatite binding peptides (GFPuv-HABP), was co-deposited with the hydroxyapatite precursors onto the titania nanotubes that are formed by anodisation. Ordered titanium dioxide nanotubular surfaces were coated with hydroxyapatite at physiological pH and temperature using simulated body fluid and pulsed electrochemical cathodisation. The hydroxyapatite deposit interdigitated into the nanotubes, producing a metal oxide-mineral composite. The engineered GFPuv-HABP protein was then self-assembled on the hydroxyapatite, forming a bio-modulated interface. Additionally, the engineered proteins were co-deposited with the precursor ions of hydroxyapatite mineral on the nanotubular titania plate. Bio-mediated assembly resulted in formation of a hybrid composite as an integrated interface on the nanotubular surface. Biomolecular assisted fabrication of hybrid composite interface on metal oxide substrate offers wide range of opportunities to design novel interfaces.

Layer-by-layer assembly of peptide based bioorganic-inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells.

In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration.

Osteotropic cancer diagnosis by an osteocalcin inspired molecular imaging mimetic

BackgroundAlthough microcalcifications of hydroxyapatite can be found in both benign and malignant osteotropic tumors, they are mostly seen in proliferative lesions, including carcinoma. The aim of this present study is to develop a molecular imaging contrast agent for selective identification of hydroxyapatite calcification in human osteotropic tumor tissues ex vivo and in human osteosarcoma cells in vitro. MethodsA bioinspired biomarker, hydroxyapatite binding peptide (HABP), was designed to mimic natural protein osteocalcin property in vivo. A fluorescein isothiocyanate dye conjugated HABP (HABP-19) was utilized to characterize hydroxyapatite on human osteotropic tumor tissue sections ex vivo and to selectively image hydroxyapatite calcifications in human osteosarcoma cells in vitro. ResultsUsing a HABP-19 molecular imaging probe, we have shown that it is possible to selectively image hydroxyapatite calcifications in osteotropic cancers ex vivo and in human SaOS-2 osteosarcoma cells in vitro. ConclusionHydroxyapatite calcifications were selectively detected in osteotropic tissues ex vivo and in the early stage of the calcification process of SaOS-2 human osteosarcoma in vitro using our HABP-19 molecular imaging probe. This new target-selective molecular imaging probe makes it possible to study the earliest events associated with hydroxyapatite deposition in various osteotropic cancers at the cellular and molecular levels. General significanceIt potentially could be used to diagnose and treat osteotropic cancer or to anchor therapeutic agents directing the local distribution of desired therapy at calcified sites.

Cementomimetics-constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides.

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein–mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure–function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

In vitro labeling of hydroxyapatite minerals by an engineered protein

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.

Identification of a Highly Specific Hydroxyapatite‐binding Peptide using Phage Display

Identification of a Highly Specific Hydroxyapatite‐binding Peptide using Phage Display

Regulation of in vitro Calcium Phosphate Mineralization by Combinatorially Selected Hydroxyapatite-Binding Peptides

We report selection and characterization of hydroxyapatite-binding heptapeptides from a peptide-phage library and demonstrate the effects of two peptides, with different binding affinities and structural properties, on the mineralization of calcium phosphate mineral. In vitro mineralization studies carried out using one strong- and one weak-binding peptide, HABP1 and HABP2, respectively, revealed that the former exhibited a drastic outcome on mineralization kinetics and particle morphology. Strong-binding peptide yielded significantly larger crystals, as observed by electron microscopy, in comparison to those formed in the presence of a weak-binding peptide or in the negative control. Molecular structural studies carried out by circular dichroism revealed that HABP1 and HABP2 differed in their secondary structure and conformational stability. The results indicate that sequence, structure, and molecular stability strongly influence the mineralization activity of these peptides. The implication of the research is that the combinatorially selected short-sequence peptides may be used in the restoration or regeneration of hard tissues through their control over of the formation of calcium phosphate biominerals.