High protein adsorptive capacity of amino acid-functionalized hydroxyapatite.

Charged functional groups present on the surface of biomaterials play an important role to regulate the affinity and attachment of macromolecules, including proteins, on the surface of biomaterials. In this study, the protein adsorptive capacity of hydroxyapatite (HA) was regulated by introducing different amino acids during the precipitation of HA. After incubation of HA samples in 5000 µg/mL lysozyme solution at pH 7.4 for 24 h, unmodified HA adsorbed 0.886 mg/m(2) of lysozyme while amino acid-functionalized HA (AA-HA) particles demonstrated higher adsorption capacity ranging from 1.090 to 1.680 mg/m(2). Incorporation of amino acids with longer side chain lengths decreased the crystallinity and increased the negative value of the surface charge of HA particles. The specific surface areas were significantly increased in the presence of amino acids. Protein loading capacity onto AA-HA was further enhanced by regulating the pH of working solution whereby the protein adsorption rate increased with decreasing the pH, while reverse trend obtained in unmodified HA. The study demonstrated that the amount of adsorbed lysozyme onto AA-HA particles was correlated with the particles' surface charges.

Hydroxyapatite nanoparticles as vectors for gene delivery.

The long-recognized promise of gene therapy to treat a broad range of currently incurable diseases remains largely unfulfilled, hindered by lack of a safe and efficient delivery vehicle. Hydroxyapatite nanoparticles are deemed a feasible candidate and possess many characteristics desired of an ideal gene vector. Current fabrication techniques can readily synthesize hydroxyapatite particles in the nanometer range; however, these particles suffer from extensive aggregation and heterogeneity, mainly in size, shape and surface charge, which render them inappropriate for gene-therapy application. There is thus a pertinent need to develop a method capable of fabricating homogenous and monodispersed hydroxyapatite nanoparticles in a rapid, efficient and cost-effective manner that can be easily upscaled. Cell transfection is impeded by several physical and biological barriers, with the vector's properties highly determinant of its ability to overcome these barriers. Fine-tuning hydroxyapatite nanoparticles' morphological and physicochemical properties, achievable through precise regulation of the reaction environment, can enhance transfection efficiencies of particles, in turn, generating safe and effective gene vectors.

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.

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 m(2) 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.

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.

Characterization of the chemically deposited hydroxyapatite coating on a titanium substrate.

Bioactive hydroxyapatite (HA) coating on titanium (Ti) implant can be used as a drug delivery device. A controlled release of drug around the implant requires the incorporation of drug into the coating material during the coating process. HA coating was prepared using a two-step procedure in conditions suitable for simultaneous incorporation of the protein-based drug into the coating material. Monetite coating was deposited on Ti substrate in acidic condition followed by the transformation of the monetite coating to HA. X-ray diffraction (XRD) confirmed the formation of the monetite phase at the first step of the coating preparation, which was transformed into HA at the second step. Fourier transform infrared spectroscopy demonstrated typical bands of a crystallized carbonated HA with A- and B-type substitution, which was confirmed by the XRD refinement of the structural parameters. Scanning electron microscope was used to observe the morphology of monetite and HA coatings. Adhesion of the coatings was measured using a scratch tester. The critical shearing stress was found to be 84.20 ± 1.27 MPa for the monetite coating, and 44.40 ± 2.39 MPa for the HA coating.

Ultrastructural observations and growth of occluding crystals in carious dentine.

The aim of the present study was to investigate the ultrastructural mechanisms involved in the formation of caries-induced intratubular dentine. Conventional, high resolution and scanning transmission electron microscopy, electron diffraction and energy-dispersive X-ray spectroscopy techniques were used to study the ultrastructure of the inorganic phase in the transparent zone of carious dentine. The results demonstrated that the bulk of the inorganic phase in caries-induced intratubular dentine had an apatite crystal structure with the presence of additional Mg-substituted beta-TCP (beta-tricalcium phosphate) phase in the carious region. Highly oriented apatite crystallites observed in intratubular dentine demonstrated a regulated biomineralization process during the formation of inorganic phase in this region, whereas Mg beta-TCP crystals were presumably formed purely via "dissolution/precipitation" mechanism. The study demonstrated the importance of "dissolution/precipitation" process and the growth kinetics of Mg-substituted beta-TCP crystals in understanding the process of formation of calcium-phosphate crystallites in carious intratubular dentine.

Ultrastructure of dentine carious lesions.

OBJECTIVE: The aim of the present study is to investigate the ultrastructural changes within the different zones of carious dentine and compare those changes with sound dentine. METHODS: Transmission electron microscopy and electron diffraction techniques were used to investigate the effect of caries on the inorganic phase of dentine. Areas of interest were identified with optical and scanning electron microscopes. RESULTS: The results demonstrated that the intertubular mineral crystallites decrease in size as caries lesion progresses. In the transparent zone of carious lesion, both intratubular and intertubular dentine consisted of nano-size apatetic crystallites with smaller size in the former. The intratubular mineral phase in transparent zone was found to be chemically similar to the intertubular dentin. CONCLUSIONS: The study suggests that a 'dissolution and precipitation' mechanism is important in understanding the process of formation of intratubular dentine within the transparent zone induced by caries attack. The observed partial demineralisation of intertubular dentine in transparent zone is discussed in terms of dissolution of tubule microbranches and exposure of intertubular dentin to acids.