Biological Apatite Research Articles

Effect of carbonate incorporation on the hydroxyl content of hydroxylapatite

AbstractMechanisms for the incorporation of carbonate into minerals of the apatite group have been explored in both the geological and medical literature. An important problem with respect to biological apatite, which requires further clarification, is the hydroxyl content of the carbonated apatite of bone. Recent studies reveal bone apatite to contain only ∼20 mol.% of the hydroxyl content of stoichiometric hydroxylapatite, with negligible chloride or fluoride. We investigated the hypothesis that the development of vacancies in the hydroxyl channel sites is a charge-balancing mechanism for the substitution of carbonate ions into hydroxylapatite. Raman spectroscopic analyses of synthetic carbonated apatites (containing 1 to >15 wt.% carbonate) show that their hydroxyl ion concentration correlates inversely with carbonate concentration. The specific relationship between carbonate and hydroxyl concentration in these samples closely follows the theoretical relationship defined by type-B substitution of carbonate for phosphate in the apatite structure. However, the 6–8 wt.% carbonate concentration in bone apatite falls far short of accounting for all of the hydroxyl depletion that occurs in bone apatite. Some of the additional hydroxyl depletion in bone apatite might result from substitution of Na+ for Ca2+, but further mechanism(s), perhaps (HPO4)2– substitution, must also play a significant role.

Degree of biological apatite c-axis orientation rather than bone mineral density controls mechanical function in bone regenerated using recombinant bone morphogenetic protein-2

The aim of the present study was to assess the bone regeneration process in defects introduced into rabbit long bones, which were regenerated with controlled release of recombinant bone morphogenetic protein-2 (rBMP-2). The orientation of the biological apatite (BAp) c-axis and bone mineral density (BMD) were compared as predictors of bone mechanical function. A 20-mm-long defect was introduced in rabbit ulnas, and 17 µg of rBMP-2 was controlled-released into the defect using a biodegradable gelatin hydrogel as the carrier. In the bone regeneration process, two characteristic phases may have been governed by different factors. First, new bone formation actively occurred, filling the bone defect with newly formed bone tissue and increasing the BMD. This process was regulated by the strong osteoinductive capacity of rBMP-2. Second, after filling of the defect and moderate BMD restoration, preferential BAp c-axis orientation began to increase, coincident with initiation of remodeling. In addition, the BAp c-axis orientation, rather than BMD, was strongly correlated with Young's modulus, an important index of bone mechanical function, particularly in the later stage of bone regeneration. Thus, preferential BAp c-axis orientation is a strong determinant and predictor of the mechanical function of tissue-engineered bone. Therefore, analysis of BAp preferential c-axis orientation in addition to measurement of BMD is crucial in assessment of bone mechanical function.

Design and optimization of the oriented groove on the hip implant surface to promote bone microstructure integrity

We proposed a novel surface modification for an artificial hip joint stem from the viewpoint of maintenance and establishment of appropriate bone function and microstructure, represented by the preferred alignment of biological apatite (BAp) and collagen (Col). Oriented grooves were introduced into the proximal medial region of the femoral stem to control the principal stress applied to the bone inside the grooves, which is a dominant factor contributing to the promotion of Col/BAp alignment. The groove angle and the stem material were optimized based on the stress inside the grooves through a finite element analysis (FEA). Only the groove oriented proximally by 60° from the normal direction of the stem surface generated the healthy maximum principal stress distribution. The magnitude of the maximum principal stress inside the groove decreased with increasing the stem Young's modulus, while the direction of the stress did not largely changed. An in vivo implantation experiment showed that this groove was effective in inducing the new bone with preferential Col/BAp alignment along the groove depth direction which corresponded to the direction of maximum principal stress inside the groove. The anisotropic principal stress distribution and the oriented microstructure inside the groove are similar to those found in the femoral trabeculae; therefore, the creation of the oriented groove is a potent surface modification for optimizing implant design for a long-term fixation.

Monolithic and assembled polymer–ceramic composites for bone regeneration

The rationale for the use of polymer–ceramic composites for bone regeneration stems from the natural composition of bone, with collagen type I and biological apatite as the main organic and inorganic constituents, respectively. In the present study composite materials of PolyActive™ (PA), a poly(ethylene oxide terephthalate)/poly(butylene terephtalate) co-polymer, and hydroxyapatite (HA) at a weight ratio of 85:15 were prepared by rapid prototyping (RP) using two routes. In the first approach pre-extruded composite filaments of PA–HA were processed using three-dimensional fibre deposition (3DF) (conventional composite scaffolds). In the second approach PA scaffolds were fabricated using 3DF and combined with HA pillars produced inside stereolithographic moulds that fitted inside the pores of the PA three-dimensional structure (assembled composite scaffolds). Analysis of calcium and phosphate release in a simulated physiological solution, not containing calcium or phosphate, revealed significantly higher values for the HA pillars compared with other scaffolds. Release in simulated body fluid saturated with respect to HA did not show significant differences among the different scaffolds. Human mesenchymal stromal cells were cultured on polymer (3DF), conventional composite (3DF-HA) and assembled composite (HA assembled in 3DF) scaffolds and assessed for morphology, metabolic activity, DNA amount and gene expression of osteogenic markers using real time quantitative polymerase chain reaction (PCR). Scanning electron microscopy images showed that the cells attached to and infiltrated all the scaffolds. Assembled composites had a higher metabolic activity compared with 3DF-HA scaffolds while no significant differences were observed in DNA amounts. Gene expression of osteopontin in the assembled composite was significantly higher compared with the conventional composites. The strategy of composite fabrication by assembly appears to be a promising alternative to the conventional composite fabrication route for scaffolds for bone regeneration.

Pathological Biominerals: Raman and Infrared Studies of Bioapatite Deposits in Human Heart Valves

We studied pathological bioapatite from patients undergoing valvular replacement due to severe aortic and mitral stenosis. Three different types of mineralized human cardiac valves were analyzed. We used infrared and Raman spectroscopy to infer the presence of the carbonate group and evaluate the carbonate substitution in bioapatite structure. The Raman spectra showed that the pathological bioapatite is a B-type "carbonate-apatite" (CO(3)(2-) for PO(4)(3-)) similar to the major mineralized products derived from normal biomineralization processes occurring in the human body. Fourier transform infrared spectra (FT-IR) confirmed the B-type carbonate substitution (CO(3)(2-) for PO(4)(3-)) and showed evidence for the partial replacement of [OH] by [CO(3)] (A-type substitution). The carbonate content of the samples inferred by the spectroscopic measurements is in good agreement with the range of values estimated for biological apatite. On the contrary, the crystal size of the pathological apatite estimated using the percentage area of the component at 1059 cm(-1) of the infrared spectrum is in the nanometer range and it is significantly smaller than the crystal size of normal mineralized tissues.

Investigation of Apatite Mineralization on Antioxidant Polyphosphazenes for Bone Tissue Engineering

Synthetic bone grafts that promote the natural mineralization process would be excellent candidates for the repair or replacement of bone defects. In this study, a series of antioxidant-containing polyphosphazenes were evaluated for their ability to mineralize apatite during exposure to a solution of simulated body fluid (SBF). All polymers contained ferulic acid (antioxidant), cosubstituted with different amino acid esters linked to the polyphosphazene backbone. Differences in the side groups determined the hydrophobicity or hydrophilicity of the resulting polymers. All of the polymers mineralized monocalcium phosphate monohydrate, a type of biological apatite. However, the mineralization process (the amount of deposition and length of time) was dependent on the hydrophilicity or hydrophobicity of the polymers. The polymer–apatite composites were examined by electron scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and thermogravametric analysis. Weight gain data were also obtained. To verify that the nucleation process was due to the presence of calcium and phosphate, two standard solutions were prepared: one solution (NaCl solution) contained only sodium chloride, and the second solution (mSBF) was similar to SBF except without known crystal growth inhibitors such as Mg2+ and HCO3–. No mineralization occurred when the polymers were exposed to the NaCl solution, but mineralization took place upon exposure to mSBF. The apatite phase produced was hydroxyapatite (HAp). The mineralization process in mSBF was much more extensive, with all samples gaining more weight following exposure to SBF. A similar trend was also found (as in the case of SBF), with the amount of deposition and length of deposition time depending on the hydrophilicity/hydrophobicity of the polymer. These results suggest that the nucleation process is due to calcium and phosphate, and the absence of crystal growth inhibitors allows for the rapid nucleation of HAp. In both cases, the mineralization process was favored on hydrophilic surfaces (static water contact angle of 56–65°) versus hydrophobic surfaces (71–86°).

The alignment of MC3T3-E1 osteoblasts on steps of slip traces introduced by dislocation motion

Bone tissue shows a highly anisotropic microstructure comprising biological apatite and collagen fibrils produced by the mutual activities of bone cells, which dominates its mechanical function. Accordingly, directional control of osteoblasts is crucial for forming anisotropic bone tissue. A new approach was proposed for controlling cell directionality by using crystallographic slip traces caused by dislocation glide. Dislocations were introduced into α-titanium single crystals by plastic deformation of (011¯0)[21¯1¯0] slip system, inducing a step-like structure with acute angles between the surface normal and the slip plane. Topographical properties of step patterning, including step interval and step height, could be controlled by varying the compressive plastic strain. The step geometry introduced by plastic deformation strongly influenced osteoblast elongation, and it aligned preferentially along slip traces. Ti substrates under 10% plastic strain with step height of approximately 300 nm and step interval of 10 μm induced osteoblast alignment most successfully. Actin stress fibers elongated parallel to slip traces, with polarized vinculin accumulation between steps.

Biological apatite (BAp) crystallographic orientation and texture as a new index for assessing the microstructure and function of bone regenerated by tissue engineering

Recently, there have been remarkable advances in medical techniques for regenerating bone defects. To determine the degree of bone regeneration, it is essential to develop a new method that can analyze microstructure and related mechanical function. Here, quantitative analysis of the orientation distribution of biological apatite (BAp) crystallites by a microbeam X-ray diffractometer system is proposed as a new index of bone quality for the evaluation of regenerated bone microstructure. Preferential alignment of the BAp c-axis in the rabbit ulna and skull bone, regenerated by controlled release of basic fibroblast growth factor (bFGF) was investigated. The BAp c-axis orientation was evaluated by the relative intensity between the (002) and (310) diffraction peaks, or the three-dimensional texture for the (002) peak. It was found that new bone in the defects was initially produced without preferential alignment of the BAp c-axis, and subsequently reproduced to recover towards the original alignment. In other words, the BAp density recovered prior to the BAp orientation. Perfect recovery of BAp alignment was not achieved in the ulna and skull defects after 4weeks and 12weeks, respectively. Apparent recovery of the macroscopic shape and bio-mineralization of BAp was almost complete in the ulna defect after 4weeks. However, an additional 2weeks was required for complete repair of BAp orientation. It is finally concluded that orientation distribution of BAp crystallites offers an effective means of evaluating the degree of microstructural regeneration, and also the related mechanical function, in regenerated hard tissues.

A Comparative Study of Carbonate Determination in Human Teeth Using Raman Spectroscopy

Carbonate determination in dental apatites such as dentine and enamel is important for studying the dynamics of dental caries and developmental defects of these tissues. Traditionally, these determinations have been performed by acidic digestion with the subsequent measurement of released carbon dioxide gas. As an alternative, Raman spectroscopy has been used for the determination of carbonate in synthetic carbonated apatites with at least four analytical methods published thus far. However, these methods have not been applied to biological apatites. The aim of this comparative study was to test the suitability of these four methods for the determination of B-type carbonate in human enamel and dentine. A method for determining the A-type carbonate content of enamel using the Raman technique is also presented. Raman spectra were obtained from 10 human enamel and dentine samples and analysed with each of the four methods using either a single or multiple ν₁(PO₄^3–) band spectral fitting model. Each of the methods resulted in a different determination for the carbonate content when using the same measurement data. The method that used the full-width-at-half-maximum of the ν₁(PO₄^3–) band to determine the B-type carbonate concentration was found to be in best agreement with (i) the results (using the acid digestion method) of teeth collected from the same sample population and (ii) previously reported values for both enamel and dentine. The use of a multiple-band spectral fitting model produced the highest determination precision (particularly in the case of dentine).

Evidence of polyphosphates and their distribution in active biological apatite mineralization sites of stingray jaws

Evidence of polyphosphates and their distribution in active biological apatite mineralization sites of stingray jaws

Testing of Brushite (CaHPO4·2H2O) in Synthetic Biomineralization Solutions and In Situ Crystallization of Brushite Micro‐Granules

Conventional flat plate‐shaped brushite, dicalcium phosphate dihydrate, CaHPO4·2H2O), produced by reacting Ca‐chloride and alkali phosphate salt solutions, were found to undergo a maturation process (changing their Ca/P molar ratio from 0.8 to the theoretical value of 1) similar to those seen in biological apatites. Water lily (WL)‐shaped brushite crystals were produced in nonstirred aqueous solutions at room temperature in 24 h, by using precipitated calcite and NH4H2PO4 as the starting chemicals. The hydrothermal transformation of WL‐type brushite into octacalcium phosphate (OCP) or Ca‐deficient hydroxyapatite (CDHA) was tested at 37°C by using four different biomineralization solutions, including Tris‐buffered SBF (synthetic body fluid) and sodium lactate‐buffered SBF solutions. All four solutions used in this study consumed the starting brushite in 1 week and caused transformation into a biphasic mixture of nanocrystalline OCP and CDHA of high surface area. WL‐type brushite crystals when synthesized in the presence of small amounts of Zn2+ ions resulted in the formation of, for the first time, spherical micro‐granules of brushite. Synthesis of brushite in spherical form was difficult prior to this study.

In-situ generation of chitosan/hydroxyapatite composite microspheres for biomedical application

Chitosan/hydroxyapatite (HA) composite microspheres were firstly fabricated in a water-in-oil (W/O) emulsion by in situ generation of the HA nanoparticles in the chitosan matrix. The inorganic component in the composite microspheres was identified as poorly crystalline nano-HA containing carbonate ions, similar to biological apatite. The SEM and TEM studies indicated that the nano-HA particles were uniformly distributed in the chitosan matrix, and the resulting composite microspheres displayed a good dispersity and an average particle size of about 10μm. The as-obtained chitosan/HA composite microspheres with uniform microstructures may have more potential for both bone tissue regeneration and drug delivery applications, when compared to those prepared by the traditional mixing method.

Efficient method to produce biomineralizated nanohydroxyapatite/vertically aligned multiwalled carbon nanotube scaffolds

In this paper, we introduce a new biomimetic mineralization method employing nanohydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube (HA/VAMWCNT) nanocomposites as highly stable template materials. The biomineralization was obtained after HA/VAMWCNT nanocomposites were soaked in simulated body fluid (SBF) solution. Structural analysis revealed that the polycrystalline biological apatites were formed due to the high crystallinity of the produced nanohydroxyapatite (HA). These new nanocomposites are a very promising nanobiomaterial due to their excellent calcification in vitro process.

In situ amino acid functionalization and microstructure formation of hydroxyapatite nanoparticles synthesized at different pH by precipitation route

The effects of in situ amino acids functionalization on hydroxyapatite (HAp) and subsequent variation in pH during the synthesis in the precipitation method was undertaken. The acidic pH during HAp synthesis supported the growth of ordered micro-structures whereas basic pH enhanced the surface porosity. The characteristic nature of amino acids had significantly changed the HAp nanoparticle size and smaller HAp nanoparticles were synthesized in the presence of polar-charged amino acid as compared to uncharged-polar amino-acids. The non-polar nature of amino acid supported the growth of order microstructures of HAp whereas polar characteristic was favorable to develop highly porous structures. Significant reduction in the relative intensity of attenuated total reflectance of Fourier transform infrared (ATR-FTIR) spectral absorption peak associated with CO32− functional groups was observed in HAp synthesized at basic pH conditions. Thus, retaining lower pH value of solution during HAp synthesis may have chemical functionalities similar to the biological apatite such as carbonate functional group. The strong ATR-FTIR bands associated with NH2⋯H+ symmetric/asymmetric stretching vibrations from amino acids functional moieties indicates the possibility of the preferentially attachment of carboxylate group of the amino acids with the surfaces of HAp nanoparticles.

Biomineralization of Superhydrophilic Vertically Aligned Carbon Nanotubes

Vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. In this paper, we introduce a new biomimetic mineralization routine employing superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in simulated body fluid solution. Detailed structural analysis reveals that the polycrystalline biological apatites formed due to the -COOH terminations attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that COOH termination sites can play a significant role in biomimetic mineralization. These new nanocomposites are very promising as nanobiomaterials due to the excellent human osteoblast adhesion.

Biomineralization and Size Control of Stable Calcium Phosphate Core–Protein Shell Nanoparticles: Potential for Vaccine Applications

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.

Evaluation of Bone Quality in Mandible of Young M-CSF Deficient-Induced Osteopetrotic Mouse

The preferred crystallographic orientation of the biological apatite (BAp) c-axis has been shown to be one of the important bone quality indices that sensitively reflect in vivo stress distribution and dominate bone mechanical functions. The BAp orientation is expected to be regulated by bone modeling or remodeling by osteoblasts and osteoclasts whose primary functions are bone formation and absorption, respectively. Mouse with macrophage colony-stimulating factor (M-CSF) deficiency-induced osteopetrosis (op/op mouse) is a suitable animal model to elucidate the role of osteoclasts in the development of BAp orientation. In this study, the mandibles of 5-week-old mice were used because their mandible is subjected to complicated stresses including a biting stress locally applied just around the roots of the teeth and a bending stress applied along the mesiodistal axis of the mandibular body, and the response to the stress distribution is important to the formation of BAp orientation. The normal mouse mandible (control) has a one-dimension preferred BAp orientation in the mesiodistal direction, but just near the tooth root, the direction of BAp orientation changes locally to that of the tooth root responding to a biting stress. In the op/op mouse, the preferred BAp orientation only along the mesiodistal direction is found, but the degree is quite lower than that in normal mice. Moreover, the effect of biting was not observed in op/op mice because these mice are devoid of teeth eruption and are unable to bite. This suggests that M-CSF plays a critical role in forming the optimal BAp orientation, and therefore, the op/op mouse without osteoclasts cannot fully develop the appropriate bone microstructure in response to in vivo stress distribution, although BAp orientation is very sensitive to local in vivo stresses in normal animals with normal osteoclast function.

Regeneration of Bone Mass and Bone Quality around Implant with Grooves for Aligning Bone Cells in Rabbit Hindlimb Bones

It was reported that one-dimensionally elongated pores in implants promote the production of new bone tissue possessing both high bone density and the preferential alignment of biological apatite (BAp) c-axis/collagen as a bone quality parameter. This finding indicates that the anisotropic orientation and/or migration of osteoblasts guided by the grooved-pore surface affected the establishment of the anisotropic microstructure of bone tissue. In this study, a grooved polytetrafluoroethylene (Teflon) implant, which may have a role in regulating osteoblast arrangement, was prepared to investigate the relationship between cell behavior and bone microstructure. A cylindrical Teflon implant with 8 grooves on its side was prepared. The width and depth of the groove cross-section were 0.5 and 0.75 mm, respectively. Each implant was inserted in a drill-hole defect created on a rabbit femur such that the groove direction was parallel or perpendicular to the long bone axis in which the BAp c-axis aligns one-dimensionally. The Young’s modulus of Teflon is approximately 0.5 GPa, much lower than that of bone; therefore, the effects of applied stress can be eliminated in this model. The oriented new bone was preferentially produced along the grooved surface. The alignment direction of the BAp c-axis was almost parallel to the grooved surface even near the surface vertically aligned to the long bone axis. The geometry of the implant surface can control the organization of BAp alignment through the arrangement of osteoblasts to orient and subsequently to migrate along the surface direction; hence, implant geometry, particularly the groove, is considered an important factor controlling the BAp orientation of regenerated bone tissues.

Alignment of Biological Apatite Crystallites at First Molar in Human Mandible Cortical Bone

The aim of this study was to quantitatively clarify the c-axis alignment of biological apatite (BAp) crystallites (hereafter referred to as BAp alignment) in the cortical bone of the human mandible first molar. Six mandible specimens were collected from the cadavers of six dentulous Japanese adults (mean age, 63.0±12.1 years) held at the Department of Anatomy, Tokyo Dental College. A microbeam x-ray diffraction system was used to determine BAp alignment in the mesiodistal direction. Bone mineral density (BMD) was also measured using 3-dimensional trabecular structure measurement software. The results showed that the degree of BAp alignment in the mesiodistal direction was low in the alveolar area and high at the base of the mandible, suggesting that BAp alignment in the alveolar area is affected by occlusal force. Moreover, it was observed that the correlation between BAp alignment and BMD was small, indicating that BAp alignment and BMD could be independent factors. Therefore, determining BAp alignment was important in the evaluation of bone quality, including bone strength.

A novel glass ionomer cement containing MgCO3 apatite induced the increased proliferation and differentiation of human pulp cells in vitro

This study aimed to investigate the in vitro biological response of human dental pulp cells to glass ionomer cement (GIC, Fuji IX GP(®)) containing 2.5% magnesium carbonate apatite (MgCO(3)Ap). MgCO(3)Ap was synthesized by wet method and characterized using FT-IR, XPS, and SEM. Fuji IX GP(®) served as a control. Test and control cements were prepared by encapsulated mixing the powder with Fuji IX-liquid (P/L=3.6:1). Eluates from cements extracted by 1 mL culture medium were collected at day 1, 7 and 14, and used for WST-1 proliferation assay. For ALPase activity, cells were maintained with cements in transwells, harvested and enzyme activity was measured at day 1, 4, 7, 14, and 21. We found a higher cell proliferation and increased ALPase activity by pulp cells in the test group compared to the control. This suggests the potential of GIC containing this novel biological apatite as a restorative material for pulp-dentin regeneration.