Alexander V. Zavgorodniy

Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2 g–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate.

Synthesis and characterization of hydroxyapatite with different crystallinity: Effects on protein adsorption and release†

Increasing demand exists for the development of a tissue-engineered alternative in the repair of nonunion and critical-sized bone defects. The delivery of osteoinductive proteins, such as bone morphogenetic proteins (BMPs), to replicate physiological bone-healing process appears a logical and promising option, but is currently limited in its clinical application due to lack of a suitable drug carrier. The study aimed to investigate the effects of the crystallinity of hydroxyapatite (HA) drug carrier on adsorption of proteins onto and their release from the carrier. HA samples with different crystallinities were synthesized under controlled conditions, that is, pH, temperature, and maturation time, and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and surface area analyzer. Results demonstrated that both bovine serum albumin (BSA) and cytochrome C had a greater tendency to bind onto amorphous calcium phosphate (ACP) than crystalline HA, and the adsorption rate was correlated oppositely with the HA crystallinity. For both BSA and cytochrome C, the release kinetics of protein from HA depended on the crystallinity of HA, in which ACP had the highest release rate at 74%, whereas only 15% of proteins were released from the highly crystalline HA over a 14-day period. Burst release within 12 h of incubation was observed for all groups.

A novel approach to enhance protein adsorption and cell proliferation on hydroxyapatite: citric acid treatment

Hydroxyapatite (HA) is a promising carrier for delivery of therapeutic proteins, especially bone morphogenetic proteins (BMPs), due to the affinity between the amino and carboxyl groups of proteins and the calcium and phosphate of HA. The main challenge of employing HA as a protein carrier is the burst release profile of protein. In this study, we developed a strategy to improve the protein loading capacity of HA and to sustain protein release from HA by immobilizing COO− groups onto the HA surface. HA particles were precipitated in presence of different amounts of citric acid (CA). The physico-chemical properties (crystallinity, surface area and surface charge) of CA-treated HA (CA–HA) were significantly changed compared to those of the unmodified HA. CA–HA demonstrated significantly higher affinity towards lysozyme, in which the lysozyme adsorption increased by increasing the concentration of CA during the HA preparation. Using the optimized parameters obtained from lysozyme adsorption, the results demonstrated that functionalizing HA with CA significantly increased the amount of BMP-2 loaded onto HA and prolonged its release profile. The in vitro results showed that the CA–HA is not toxic and indeed citric ions on HA shift the surface charge towards negative value, which promoted osteoblast-like cell proliferation on HA.

X-ray microdiffraction, TEM characterization and texture analysis of human dentin and enamel

Human tooth is a complex bioceramic composite, which consists of enamel, dentin and the interface, the dentin–enamel junction (DEJ). The crystal properties and ultrastructure of the inorganic phase through the thickness of healthy human molar teeth were investigated using X‐ray microdiffraction (μXRD), electron diffraction and transmission electron microscopy (TEM) techniques. The XRD data were analysed using the Le Bail profile fitting approach. The size and the texture of the crystallites forming enamel and dentin in the crown part of teeth were measured using both techniques and then compared. Results showed that the thickness of dentin crystallites was found to decrease towards the DEJ, whereas the thickness of the enamel crystallites increased from the DEJ towards the outer layers. It was demonstrated that enamel exhibited an increase of texture in 002 lattice planes from the DEJ towards the outer layers. Texture was also detected in 102 lattice planes. The texture effect in 002 planes at the scale of less than 1 μm was also demonstrated in dentin. The variation of lattice parameters as a function of the position within the thickness of dentin and enamel was also observed. The values of the nonuniform microstrain in the dentin and enamel crystallites were from 1.40 × 10−6% to 4.44 × 10−5%. The good correlation between XRD and TEM indicated that μXRD is a useful technique to study crystallography and microstructure of heterogeneous enamel and dentin. The observed gradient characteristics of texture and crystallite size in enamel and dentin maybe an evolutionary outcome to resist wear and fracture, thereby contributing to the excellent mechanical properties of teeth.

Mechanical stability of two-step chemically deposited hydroxyapatite coating on Ti substrate: Effects of various surface pretreatments†

The success of implants in orthopaedic and dental load-bearing applications crucially depends on the initial biological fixation of implants in surrounding bone tissues. Using hydroxyapatite (HA) coating on Ti implant as carrier for bone morphogenetic proteins (BMPs) may promote the osteointegration of implants; therefore, reduce the risk of implant failure. The goal of this study was to develop an HA coating method in conditions allowing the incorporation of protein-based drugs into the coating materials, while achieving a mechanical stable coating on Ti implant. HA coatings were deposited on six different groups of Ti surfaces: control (no pretreatment), pretreated with alkali, acid, heat at 800°C, grit blasted with Al₂O₃, and grit blasted followed by heat treatment. HA coating was prepared using a two-step procedure. First step was the chemical deposition of a monetite coating on Ti substrate in acidic condition at 75°C and the second step was the hydrolysis of the monetite coating to HA. Coatings were characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD). The roughness of substrates and coatings was measured using profilometry technique. The mechanical stability of the coatings deposited on the pretreated substrates was assessed using scratch test. The coatings deposited on the grit-blasted Ti surface demonstrated superior adhesive properties with critical shearing stress 131.6 ± 0.2 MPa.

Atomic-scale compositional mapping reveals Mg-rich amorphous calcium phosphate in human dental enamel

Atomic-scale study of human dental enamel reveals an intergranular amorphous phase thought to be responsible for tooth decay. Human dental enamel, the hardest tissue in the body, plays a vital role in protecting teeth from wear as a result of daily grinding and chewing as well as from chemical attack. It is well established that the mechanical strength and fatigue resistance of dental enamel are derived from its hierarchical structure, which consists of periodically arranged bundles of hydroxyapatite (HAP) nanowires. However, we do not yet have a full understanding of the in vivo HAP crystallization process that leads to this structure. Mg2+ ions, which are present in many biological systems, regulate HAP crystallization by stabilizing its precursor, amorphous calcium phosphate (ACP), but their atomic-scale distribution within HAP is unknown. We use atom probe tomography to provide the first direct observations of an intergranular Mg-rich ACP phase between the HAP nanowires in mature human dental enamel. We also observe Mg-rich elongated precipitates and pockets of organic material among the HAP nanowires. These observations support the postclassical theory of amelogenesis (that is, enamel formation) and suggest that decay occurs via dissolution of the intergranular phase. This information is also useful for the development of more accurate models to describe the mechanical behavior of teeth.

Regulating Protein Adsorption onto Hydroxyapatite: Amino Acid Treatment

Hydroxyapatite (HA) has been widely used as bone grafts due to its chemical and structural similarities to the mineral phase of hard tissues. Applying the combination of osteogenic proteins with HA materials can accelerate bone regeneration in defective areas. The aim of the study was investigating the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins in body. Results: The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2/g. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby positively charged Arg-HA had higher affinity towards BSA (0.269 mg/m2) compared to lysozyme (0.133 mg/m2). Alternatively, the binding affinity of lysozyme (0.2 mg/m2) onto the negatively charged Asp-HA was higher compared to BSA (0.129 mg/m2). The amino acids functionalized-HA particles that had higher proteins adsorption demonstrated a lower protein release rate.