Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template-mediated by heparin

Biomimetic synthesis of carbonated apatites with good biocompatibility is a promising strategy for the broadening application of apatites for bone tissue engineering. Most researchers were interested in collagen or gelatin-based templates for synthesis of apatite minerals. Inspired by recent findings about the important role of polysaccharides in bone biomineralization, here we reported that heparin, a mucopolysaccharide, was used to synthesize carbonated apatites in vitro. The results indicated that the Ca/P ratio, carbon content, crystallinity and morphology of the apatites varied depending on the heparin concentration and the initial pH value. The morphology of apatite changed from flake-shaped to needle-shaped, and the degree of crystallinity decreased with the increasing of heparin concentration. Biocompatibility of the apatites was tested by proliferation and alkaline phosphatase activity of MC3T3-E1 cells. The results suggested that carbonated apatites synthesized in the presence of heparin were more favorable to the proliferation and differentiation of MC3T3-E1 cells compared with traditional method. In summary, the heparin concentration and the initial pH value play a key role in the chemical constitution and morphology, as well as biological properties of apatites. These biocompatible nano-apatite crystals hold great potential to be applied as bioactive materials for bone tissue engineering.     

Physico-chemical properties of nanocrystalline apatites: Implications for biominerals and biomaterials

Nanocrystalline apatites play an important role in biomineralisation and they are used as bioactive biominerals for orthopaedic applications. One of the most interesting characteristics of the nanocrystals, evidenced by spectroscopic methods, is the existence of a structured surface hydrated layer, well developed in freshly formed precipitates, which becomes progressively transformed into the more stable apatitic lattice upon ageing in aqueous media. The hydrated layer is very fragile and irreversibly altered upon drying. Several routes leading to different apatite compositions are found in biological systems. The loosely bound ions of the hydrated layer can be easily and reversibly substituted by other ions in fast aqueous ion exchange reactions. These ions can either be included in the growing stable apatite lattice during the ageing process or remain in the hydrated layer. The adsorption properties of nanocrystals appear to be strongly dependent on the composition of the hydrated layer and on ageing. The surface reactivity of the apatite nanocrystals can play a part in different biomaterials and could explain the setting reactions of biomimetic calcium phosphate cements and the possibility of obtaining adherent nanocrystalline coatings on different substrates.     

Analysis of bovine-derived demineralized bone extracts

Demineralized bone factors are capable of stimulating bone regeneration through an osteoinductive mechanism and thus it has been recognized as a good bone graft. In this study, a kind of demineralized bone extracts (DBX) derived from bovine tibia by a chemical route. The extracts thus obtained were analyzed for their bio-chemical and physical properties using various techniques and results provided quite interesting insights into the demineralization process. There is no significant evidence of mineral phase associated with the connective tissue detected during chemical as well as physical testing, indicating the formation of DBX. This kind of bone extracts may used as a bone graft material and as a substrate for the growth of biomimetic apatites.     

Cell Surface Receptor Targeted Biomimetic Apatite Nanocrystals for Cancer Therapy

Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO‐24 monoclonal antibody (mAb) directed against the Met/Hepatocyte Growth Factor receptor (Met/HGFR), which is over‐expressed on different types of carcinomas and thus represents a useful tumor target. The chemical‐physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL‐16 gastric carcinoma line) or not expressing (NIH‐3T3 fibroblasts) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL‐16) but not in the ones that do not express it (NIH‐3T3). Moreover they discharge DOXO in the targeted GTL‐16 cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic applications in nanomedicine.     

Formation of bone-like apatite layer on chitosan fiber mesh scaffolds by a biomimetic spraying process

Bone-like apatite coating of polymeric substrates by means of biomimetic process is a possible way to enhance the bone bonding ability of the materials. The created apatite layer is believed to have an ability to provide a favorable environment for osteoblasts or osteoprogenitor cells. The purpose of this study is to obtain bone-like apatite layer onto chitosan fiber mesh tissue engineering scaffolds, by means of using a simple biomimetic coating process and to determine the influence of this coating on osteoblastic cell responses. Chitosan fiber mesh scaffolds produced by a previously described wet spinning methodology were initially wet with a Bioglass((R))-water suspension by means of a spraying methodology and then immersed in a simulated body fluid (SBF) mimicking physiological conditions for one week. The formation of apatite layer was observed morphologically by scanning electron microscopy (SEM). As a result of the use of the novel spraying methodology, a fine coating could also be observed penetrating into the pores, that is clearly within the bulk of the scaffolds. Fourier Transform Infrared spectroscopy (FTIR-ATR), Electron Dispersive Spectroscopy (EDS) and X-ray diffraction (XRD) analysis also confirmed the presence of apatite-like layer. A human osteoblast-like cell line (SaOs-2) was used for the direct cell contact assays. After 2 weeks of culture, samples were observed under the SEM. When compared to the control samples (unmodified chitosan fiber mesh scaffolds) the cell population was found to be higher in the Ca-P biomimetic coated scaffolds, which indicates that the levels of cell proliferation on this kind of scaffolds could be enhanced. Furthermore, it was also observed that the cells seeded in the Ca-P coated scaffolds have a more spread and flat morphology, which reveals an improvement on the cell adhesion patterns, phenomena that are always important in processes such as osteoconduction.     

Ion exchanges in apatites for biomedical application

The modification of the composition of apatite materials can be made by several processes corresponding to ion exchange reactions which can conveniently be adapted to current coatings and ceramics and are an alternative to setting up of new synthesis methods. In addition to high temperature thermal treatments, which can partly or almost totally replace the monovalent OH^– anion of stoichiometric hydroxyapatite by any halogen ion or carbonate, aqueous processes corresponding to dissolution-reprecipitation reactions have also been proposed and used. However, the most interesting possibilities are provided by aqueous ion exchange reactions involving nanocrystalline apatites. These apatites are characterised by the existence on the crystal surface of a hydrated layer of loosely bound mineral ions which can be easily exchanged in solution. This layer offers a possibility to trap mineral ions and possibly active molecules which can modify the apatite properties. Such processes are involved in mineralised tissues and could be used in biomaterials for the release of active mineral species.     

Biomimetic mineral coatings in dental and orthopaedic implantology

Biomimetic techniques are used to deposit coatings of calcium phosphate upon medical devices. The procedure is conducted under near-physiological, or “biomimetic”, conditions of temperature and pH primarily to improve their biocompatibility and biodegradability of the materials. The inorganic layers generated by biomimetic methods resemble bone mineral, and can be degraded within a biological milieu. The biomimetic coating technique involves the nucleation and growth of bone-like crystals upon a pretreated substrate by immersing this in a supersaturated solution of calcium phosphate under physiological conditions of temperature (37°C) and pH (7.4). The method, originally developed by Kokubo in 1990, has since undergone improvement and refinement by several groups of investigators. Biomimetic coatings are valuable in that they can serve as a vehicle for the slow and sustained release of osteogenic agents at the site of implantation. This attribute is rendered possible by the near-physiological conditions under which these coatings are prepared, which permits an incorporation of bioactive agents into the inorganic crystal latticework rather than their mere superficial adsorption onto preformed layers. In addition, the biomimetic coating technique can be applied to implants of an organic as well as of an inorganic nature and to those with irregular surface geometries, which is not possible using conventional methodologies.     

Development of Biomimetic Needle-like Apatite Nanocrystals by a Simple New Method

A new method of calcium nitrate and sodium phosphate as reactants was employed to prepare biomimetic apatite nanocrystals by a simple heating treatment in water. The structure and properties of the apatite crystals were investigated by TEM, XRD, IR, ICP and TG. It is found that the apatite nanocrystals contain OH-, CO32-, Na+ and HPO42- ions in their crystal structure. The crystal water is removed during heating from 200℃ to 400℃. CO32-and HPO42- are decomposed at 600℃ to 800℃, also there is lattice vvater lost at this temperature stage. The morphology of the apatite nanocrystals is needle-like with a length less than 80 nm. The size and crystallinity of the apatite nanocrystals increase with vvater treatment temperature and time. Compared to the apatite crystals sintered at 800℃, water treated apatite nanocrystals are poorly crystallized apatite. The results indicate that the apatite nanocrystals have similarity in composition, structure, morphology and crystallinity to that of bone apatite crystal     

Osteoblast cell response to surface-modified carbon nanotubes

In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.     

Carbonate and fluoride incorporation in synthetic apatites: Comparative effect on physico-chemical properties and in vitro bioactivity in fetal bovine serum

CO₃- and/or F-substituted apatites have been considered as potential bone substitution material for dental and orthopedic applications. The objective of this study was to compare physico-chemical properties and in vitro bioactivity in fetal bovine serum (FBS) of apatites containing CO₃ and/or F. The results showed that CO₃ and F in apatites have opposite effects on crystallinity and solubility. Calcium deficient hydroxyapatite (HA) and F-substituted apatite (FA) partially transformed to beta-tricalcium phosphate (β-TCP) at temperature 950 °C. After immersion in FBS for 10 days, calcium deficient HA, FA, and CO₃-substituted apatite (CHA) and CO₃- and F-substituted apatite (CFA) pellet surfaces all showed formation of apatite.     

19F and 31P NMR spectroscopy of calcium apatites

Glass ceramics that include apatite crystals are used as implant materials. Because most of these glass ceramics are comprised of fluoride-containing glass compositions, the included apatites could be hydroxyapatite, fluorapatite or fluoride-substituted hydroxyapatite. However, these apatites differ in regard to their solubility and thermal stability. The purpose of the current study was to determine the possibilities of distinguishing between these apatities. High resolution solid-state ¹⁹F and ³¹P magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of two fluorapatites, a hydroxyapatite and a fluoridated hydroxyapatite have been obtained. Using ³¹P NMR investigations it is possible to distinguish between calcium apatites and other calcium phosphates, but the distinction between fluoride-containing apatite and hydroxyapatite is not possible. However, ¹⁹F high-resolution solid-state NMR investigations permit the distinction between these various apatites. The results of the NMR investigations could be used for the characterization of glass ceramics. The application of those results was demonstrated using a newly developed apatite-containing glass ceramic.     

Adsorption on apatitic calcium phosphates for drug delivery: interaction with bisphosphonate molecules

Bisphosphonates (BPs) are well established as an important class of drugs for the treatment and prevention of several bone disorders including osteoporosis. This work investigated the interaction of two bisphosphonates, risedronate and tiludronate, with several apatitic supports, a well-crystallised hydroxyapatite (HA) and nanocrystalline apatites with varying maturation times, chemical composition and surface characteristics. The purpose was to fully understand the adsorption mechanism and desorption process, by the evaluation of the effect of several physicochemical parameters (temperature, pH and concentration of calcium and phosphate ions). Whatever the nature of the BP and the structure and composition of the apatite, the adsorption of such anti-resorptive agents can be well described as an ion exchange-reaction between phosphates species on the apatitic surface and BP molecules in solution. However, the parameters of adsorption can vary depending on the physicochemical conditions of the adsorption reaction. In addition, the structure and composition of the apatitic surface also influence the adsorption properties. Finally, BPs molecules are slowly released from apatitic supports, because most of the adsorbed molecules are irreversibly bound and not spontaneously released by dilution or simple washing. Moreover, similar to their adsorption, the release of bisphosphonates is strongly affected not only by the chemical properties of the molecule, but also by the chemical and structural characteristics of the apatitic substrates. The understanding of the adsorption and release processes provides fundamental tools for the development of drug delivery systems using apatite materials.     

Biomimetic mineralization of electrospun poly(lactic-co-glycolic acid)/multi-walled carbon nanotubes composite scaffolds in vitro

Biomimetic mineralization is an effective method to improve the biocompatibility and bone inductivity of certain materials. In this study, composite scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and multi-walled carbon nanotubes (MWNTs) were prepared by electrospinning. Subsequently, the scaffolds were immersed in a simulated body fluid (1.5 × SBF) at 37 °C for 7, 14 and 21 days for biomimetic mineralization. Scanning electron microscopy, Raman spectroscopy, and X-ray diffraction were used for characterization. It was found that the electrospun scaffolds had extremely resemblant structural morphology to the natural extracellular matrix. After mineralization, apatite crystals were deposited on the PLGA/MWNTs composite scaffolds. The mineralized PLGA/MWNTs composites may be potentially useful in tissue engineering applications, particularly as scaffolds for bone tissue regeneration.     

Investigations on the in vitro bioactivity of swift heavy oxygen ion irradiated hydroxyapatite

Poly(propylene fumarate) (PPF) is an ultraviolet-curable and biodegradable polymer with potential applications for bone regeneration. In this study, we designed and fabricated three-dimensional (3D) porous scaffolds based on a PPF polymer network using micro-stereolithography (MSTL). The 3D scaffold was well fabricated with a highly interconnected porous structure and porosity of 65%. These results provide a new scaffold fabrication method for tissue engineering. Surface modification is a commonly used and effective method for improving the surface characteristics of biomaterials without altering their bulk properties that avoids the expense and long time associated with the development of new biomaterials. Therefore, we examined surface modification of 3D scaffolds by applying accelerated biomimetic apatite and arginine-glycine-aspartic acid (RGD) peptide coating to promote cell behavior. The apatite coating uniformly covered the scaffold surface after immersion for 24 h in 5-fold simulated body fluid (5SBF) and then the RGD peptide was applied. Finally, the coated 3D scaffolds were seeded with MC3T3-E1 pre-osteoblasts and their biologic properties were evaluated using an MTS assay and histologic staining. We found that 3D PPF/diethyl fumarate (DEF) scaffolds fabricated with MSTL and biomimetic apatite coating can be potentially used in bone tissue engineering.     

Nanostructural analysis of trabecular bone

The mechanical properties of bone are dictated by the size, shape and organization of the mineral and matrix phases at multiple levels of hierarchy. While much is known about structure–function relations at the macroscopic level, less is known at the nanoscale, especially for trabecular bone. In this study, high resolution transmission electron microscopy (HRTEM) was carried out to analyze shape and orientation of apatite crystals in murine femoral trabecular bone. The distribution and orientation of mineral apatites in trabecular bone were different from lamellar bone and the c-axis of the tablet-like mineral apatite crystals in trabecular bone was arranged with no preferred orientation. The difference in the orientation distribution of apatite crystals of trabecular bone in the present study compared with that of lamellar bone found in the literature can be attributed to the more complex local stress state in trabecular bone. Apatite crystals were also found to be multi-crystalline, not single crystalline, from dark field image analysis. From the observations of this study, it is suggested that Wolff’s law can be applicable to the nanostructural orientation and distribution of apatite crystals in trabecular bone. It was also found that small round crystalline particles observed adjacent to collagen fibrils were similar in size and shape to the apatite crystals in biomimetically nucleated synthetic amorphous calcium phosphate, which suggests that they are bone mineral apatite nuclei.     

Human osteoblasts adhesion and proliferation on magnesium-substituted tricalcium phosphate dense tablets

Tricalcium phosphate (TCP) is recognized as a promising bone replacement material due to its high bioactivity and resorbable properties. To mimic biological apatites, incorporation of magnesium (Mg) in TCP was proposed. Mg-substituted TCP (β-TCMP) and β-TCP dense tablets were obtained by pressing and sintering at 1,000°C Mg-substituted calcium deficient apatite (Mg-CDA) and commercial TCP, respectively. The materials were characterized using X-ray diffraction, infrared spectroscopy and electron microscopy. Human osteoblast cells (SaOs2) were seeded onto the sintered tablets for 4 h, 24 h and 7 days. Results showed that Mg-CDA was completely transformed into β-TCMP. Moreover, β-TCMP stimulated adhesion and proliferation of human osteoblast cells. Consequently, the magnesium incorporation on calcium deficient apatites followed by sintering at 1,000°C seems to be a useful path to obtain biocompatible and non cytotoxic dense tablets with TCP structure with potential application on bone engineering.     

仿生矿化法制备可降解羟基磷灰石/氧化细菌纤维素复合材料

细菌纤维素是具有天然纳米网状结构的支架材料,对其进行氧化改性后可获得可调控的降解性能。通过仿生矿化氧化改性的细菌纤维素支架,制备了可降解羟基磷灰石/氧化细菌纤维素复合骨组织工程支架材料。观察并分析了仿生矿化过程氧化细菌纤维素的降解和羟基磷灰石的形成,并通过SEM、EDS、XRD对羟基磷灰石在可降解氧化细菌纤维素支架上沉积进行了表征,矿化7天的羟基磷灰石/氧化细菌纤维素复合材料表面和内部均有磷灰石形成,测得磷灰石的钙磷比为1.75,主要为羟基磷灰石,伴有少量碳羟磷灰石。结果表明,使用仿生矿化法成功获得了一种新型可降解羟基磷灰石/氧化纤维素复合材料支架。     

Preparation of core–shell poly(l-lactic) acid-nanocrystalline apatite hollow microspheres for bone repairing applications

In this paper, hybrid inorganic–organic core–shell hollow microspheres, made of poly(l-lactic acid) (PLLA) and biomimetic nano apatites (HA), were prepared from biodegradable and biocompatible substances, suitable for bone tissue applications. Preparation is started from Pickering emulsification, i.e., solid particle-stabilized emulsions in the absence of any molecular surfactant, where solid particles adsorbed to an oil–water interface. Stable oil-in-water emulsions were produced using biomimetic 20?nm sized HA nanocrystals as particulate emulsifier and a dichloromethane (CH₂Cl₂) solution of PLLA as oil phase. Hybrid hollow PLLA microspheres at three different HA nanocrystals surface coverage, ranging from 10 to 50?μm, were produced. The resulting materials were completely characterized with spectroscopic, calorimetric and microscopic techniques and the cytocompatibility was established by indirect contact tests with both fibroblasts and osteoblasts and direct contact with these latter. They displayed a high level of cytocompatibility and thus represent promising materials for drug delivery systems, cell carriers and scaffolds for regeneration of bone useful in the treatment of orthopaedic, maxillofacial and dental fields.     

Nanocrystalline apatites in biological systems: characterisation,structure and properties

Nanocrystalline apatitic calcium phosphates play a crucial role in calcified tissues and biomaterials. One of the most interesting characteristics of biomimetic apatite nanocrystals is the existence of a surface hydrated layer essentially related to their formation process in solution. This hydrated layer shows specific spectroscopic characteristics. It seems to exist in its nascent state only in wet samples and is altered on drying. This surface‐hydrated layer progressively disappears as the stable apatite domains develop. The surface ions can be rapidly and reversibly exchanged in solution, mainly with selected bivalent species. The exchange reactions clearly reveal the existence of two domains: the relatively inert apatite core and the very reactive surface‐hydrated domains. The structure of the hydrated layer has been shown to be reversibly affected by the constituting ions. Such a surface layer in bone apatite nanocrystals could participate actively in homeostasis and probably other regulation processes. The specificity of biomimetic apatite nanocrystals also opens interesting possibilities in materials science. The mobility of the mineral ions on the crystal surface, for example, allows strong bonding and interactions either with other crystals or with different substrates. Inter‐crystalline interactions have been described as a “crystal fusion” process in vivo and they could be involved in the setting reaction of biomimetic calcium phosphate cements. Ceramic‐like materials using the surface interaction capabilities of the nanocrystals can be produced at very low temperature (below 200 °C). The surface‐hydrated layer could also be involved in interactions with macromolecules and polymeric materials or in the coating of implants. The ion exchange and adsorption capabilities of the nanocrystals could probably be used for drug release, offering a range of possible behaviours.