Calcium Phosphate and Silicate-Based Nanoparticles: History and Emerging Trends.

Calcium phosphates (CaPs) and silicate-based bioglasses have been extensively studied since the early 1970s due to their unique capacity to bind to host bone, which led to their clinical translation and commercialization in the 1980s. Since the mid-1990s, researchers have synthesized nanoscale CaP and silicate-based particles of increased specific surface area, chemical reactivity, and solubility, which offer specific advantages compared to their bulk counterparts. This review provides a critical perspective on the history and emerging trends of these two classes of ceramic nanoparticles. Their synthesis and functional properties in terms of particle composition, size, shape, charge, dispersion, and toxicity are discussed as a function of relevant processing parameters. Specifically, emerging trends such as the influence of ion doping and mesoporosity on the biological and pharmaceutical performance of these nanoparticles are reviewed in more detail. Finally, a broad comparative overview is provided on the physicochemical properties and applicability of CaP and silicate-based nanoparticles within the fields of (i) local delivery of therapeutic agents, (ii) functionalization of biomaterial scaffolds or implant coatings, and (iii) bioimaging applications. Impact statement This review provides a critical perspective on the history and emerging trends of the two main classes of bioceramic nanoparticles, that is, calcium phosphate (CaP) and silicate-based nanoparticles. While most reviews in literature focus on either CaP or silicate-based nanoparticles, our review evaluates both classes of bioceramic nanoparticles simultaneously. This combined review offers the opportunity to analyze differences and similarities with respect to the historic development and emerging trends within both fields of bioceramics research.

Biomimetic coatings for bone tissue engineering of critical-sized defects.

The repair of critical-sized bone defects is still challenging in the fields of implantology, maxillofacial surgery and orthopaedics. Current therapies such as autografts and allografts are associated with various limitations. Cytokine-based bone tissue engineering has been attracting increasing attention. Bone-inducing agents have been locally injected to stimulate the native bone-formation activity, but without much success. The reason is that these drugs must be delivered slowly and at a low concentration to be effective. This then mimics the natural method of cytokine release. For this purpose, a suitable vehicle was developed, the so-called biomimetic coating, which can be deposited on metal implants as well as on biomaterials. Materials that are currently used to fill bony defects cannot by themselves trigger bone formation. Therefore, biological functionalization of such materials by the biomimetic method resulted in a novel biomimetic coating onto different biomaterials. Bone morphogenetic protein 2 (BMP-2)-incorporated biomimetic coating can be a solution for a large bone defect repair in the fields of dental implantology, maxillofacial surgery and orthopaedics. Here, we review the performance of the biomimetic coating both in vitro and in vivo.

Laryngotracheal reconstruction with porous titanium in rabbits: are vascular carriers and mucosal grafts really necessary?

Laryngotracheal reconstruction requires a supportive structure with a mucosal lining, which needs a vascular supply in order to regenerate properly. We investigated the necessity of a vascular carrier and mucosal graft when using porous titanium for laryngotracheal reconstruction. Surgical defects of the laryngotracheal complex in 22 rabbits were reconstructed with: (a) porous titanium implanted on a vascularized fascia combined with a buccal mucosal graft (first stage) before transposing to the neck area (second stage); (b) porous titanium implanted on a vascularized fascia (first stage) combined with a mucosal graft (second stage); (c) porous titanium on a pedicled fascia flap; and (d) porous titanium alone. The grafts were tolerated well. Re-epithelialization occurred in all groups. Normal mucosa with a submucosal layer containing vital cells was noted using the titanium implants. Blood vessels were grown in the pores of the titanium scaffold to supply the overlying mucosa. The scaffold was well integrated in the adjacent tracheal cartilage and surrounding tissues, except in the two cases that showed titanium displacement. Inflammation and granulation formation were seen in most rabbits in groups III and IV, initiated probably by the use of buccal mucosal grafts. Reconstruction of a rabbit's trachea using composites of porous titanium, mucosal grafts and a fascia flap is feasible. Titanium seems to meet the requirements needed for closing a small defect of the tracheal wall and allows for re-epithelialization. For larger defects, a vascular carrier with a mucosal graft is probably indispensable to ensure the process of re-epithelialization.

Porous beta tricalcium phosphate scaffolds used as a BMP-2 delivery system for bone tissue engineering.

Macroporous beta tricalcium phosphate (beta-TCP) scaffolds were evaluated as potential carriers and delivery systems for bone morphogenetic protein-2 (BMP-2). Chemical etching was performed to increase the available surface and thus the protein loading. X-ray diffraction and infrared spectrocopy analyses confirmed the preparation of pure beta-TCP scaffolds. Scanning electron microscopy revealed interconnected porosity (64%) and a microporous surface after chemical etching. Scaffolds loaded with 30 and 15 microg of BMP-2 were implanted respectively into the back muscles and into femoral defects (condyle and diaphysis) of rabbits for 4 weeks. Histological observations confirmed the activity of the BMP-2 released from the scaffolds. Intramuscularly, bone was formed within the BMP-2-loaded scaffold pores. In the bone defects, the effect of released BMP-2 was similarly noticeable, as evaluated by histomorphometry. The incorporation of BMP-2 resulted in an amount of newly formed bone that was 1.3 times higher than with unloaded scaffolds. The implant site, however, did not have an effect on bone formation as no statistical differences were measured between cortical (diaphysis) and trabecular (condyle) defects. These results indicate the suitability of chemically etched beta-TCP scaffolds as BMP-2 carriers, in the context of bone regeneration.

Tracheal reconstruction: mucosal survival on porous titanium.

OBJECTIVE: To investigate whether porous titanium can provide a better support for revascularization of a mucosal graft ideal for tracheal reconstruction. In patients with laryngotracheal stenosis or tumor, the mucosa with supporting structures can be damaged, resulting in a defect that has to be reconstructed. Autologous tissues like cartilage and mucosa have been used for reconstruction. The main problem has been incomplete mucosal reepithelialization. DESIGN: In the first experiment, porous titanium or ear cartilage was combined with mucosa and implanted subcutaneously in athymic mice for different periods of time. In the second experiment, using rabbits, surgically created defects were reconstructed with porous titanium and mucosa on a pedicled fascia flap using a 2-stage procedure. The implants were analyzed with emphasis on angiogenesis and mucosal survival. SUBJECTS: Male New Zealand white rabbits and nude athymic mice (BALB-c nu/nu). RESULTS: Normal mucosa having a submucosal layer with vital cells was noted on top of the titanium. Multiple blood vessels were observed extending from the muscle layer through the titanium. Cytokeratin expression was detected in the suprabasal and basal layers of the mucosal epithelium. In contrast, the mucosa on cartilage showed no vital cells and no cytokeratin expression. In the rabbit experiment, all animals survived the reconstruction. The titanium was well integrated to the adjacent tracheal cartilage and surrounding tissues, supporting a fully vital mucosa. CONCLUSIONS: Porous titanium is an inert biomaterial that provides support and allows easy revascularization of a mucosal graft. Titanium, in combination with viable autologous tissues, is a good alternative for tracheal reconstruction.

Electrolytic deposition of lithium into calcium phosphate coatings.

OBJECTIVES: Lithium ions stimulate the Wnt signaling pathway and the authors previously demonstrated that lithium enhances the proliferation of tissue cultured human mesenchymal stem cells. The aim of this study was to prepare and characterize a calcium phosphate/lithium coating by means of electrolytic deposition. It was hypothesized that the hybrid coatings would enhance the proliferation of MG63 osteoblast-like cells in vitro. METHODS: Calcium phosphate coatings were electrolytically deposited in electrolytes containing 0, 0.5 and 5g/L lithium chloride, respectively. They were characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The coating thickness, lithium content and release profile were also measured. The cell attachment and cell-doubling index of MG63 cells on these coatings were determined through a Cell Counting Kit-8. RESULTS: Lithium inhibited calcium phosphate deposition in a dose-dependent manner. Both crystallinity and thickness of the coatings were reduced with increasing lithium concentration in the electrolyte. The incorporation of lithium was 2.2 and 5.5microg/mg, respectively. The hybrid coatings demonstrated a burst lithium release within half an hour upon immersion into simulated physiological solution. Both attachment and early proliferation of MG63 cells on these hybrid coatings were enhanced. SIGNIFICANCE: These results suggest that lithium can be effectively incorporated into calcium phosphate coatings. The incorporation of lithium interferes with calcium phosphate deposition; however, it enhances the biocompatibility of the coatings.

Proliferation and differentiation of osteoblast-like MC3T3-E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings.

Biomimetic and electrolytic deposition are versatile methods to prepare calcium phosphate coatings. In this article, we compared the effects of biomimetically deposited octacalcium phosphate and carbonate apatite coatings as well as electrolytically deposited carbonate apatite coating on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. It was found that MC3T3-E1 cells cultured on the biomimetically deposited carbonate apatite coating demonstrated the greatest proliferation rate and the highest differentiation potential. Cells on the biomimetically deposited octacalcium phosphate coating had lower proliferation rate before day 7, but higher after that, than those on the electrolytically deposited carbonate apatite coating. There was no difference on the expression of early differentiation markers, that is, alkaline phosphatase activity and collagen content, between biomimetically deposited octacalcium phosphate and electrolytically deposited carbonate apatite coatings. However, higher expression of late differentiation markers, that is, osteocalcin and bone sialoprotein mRNA, was found on the biomimetically deposited octacalcium phosphate coating on day 14. These results suggest that the difference in in vitro osteoblast cell performance of calcium phosphate coatings might relate to their physicochemical properties. Biomimetic carbonate apatite coating is the most favorable surface for the proliferation and differentiation of MC3T3-E1 cells.

Osteoinductive biomaterials--properties and relevance in bone repair.

The need for bone tissue regeneration is continuously expanding due to the improvement of life quality and the consequent increase in life expectancy. Although natural bone grafts have shown excellent clinical successes, their use is associated with some important drawbacks, limited availability being one of the most important. Cell- and growth-factor based tissue engineering provides a promising alternative to natural bone grafts; however, the performance of tissue-engineered constructs often depends on the used carrier. An important challenge in the field of bone regeneration is the development of synthetic bone graft substitutes that are "intelligent" in that they are able to instruct the in vivo environment to form bone. A group of potentially "intelligent" bone graft substitutes are osteoinductive biomaterials. In this paper, background on the phenomenon of osteoinduction and an overview of synthetic biomaterials with osteoinductive potential are given. Furthermore, we elaborate on physicochemical properties of biomaterials that are of influence on their osteoinductive potential. Finally, we discuss the relevance of osteoinductivity of biomaterials in the repair of clinically relevant bone defects.

Bone ingrowth in porous titanium implants produced by 3D fiber deposition.

3D fiber deposition is a technique that allows the development of metallic scaffolds with accurately controlled pore size, porosity and interconnecting pore size, which in turn permits a more precise investigation of the effect of structural properties on the in vivo behavior of biomaterials. This study analyzed the in vivo performance of titanium alloy scaffolds fabricated using 3D fiber deposition. The titanium alloy scaffolds with different structural properties, such as pore size, porosity and interconnecting pore size were implanted on the decorticated transverse processes of the posterior lumbar spine of 10 goats. Prior to implantation, implant structure and permeability were characterized. To monitor the bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. Bone formation in the scaffolds was investigated by histology and histomorphometry of non-decalcified sections using traditional light- and epifluorescent microscopy. In vivo results showed that increase of porosity and pore size, and thus increase of permeability of titanium alloy implants positively influenced their osteoconductive properties.

Protein mapping of calcium carbonate biominerals by immunogold.

The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement, the samples are subsequently observed with scanning electron microscopy by using back-scattered electron mode. In the present case, the technique is applied to a particular example, the calcitic prisms that compose the outer shell layer of the mediterranean fan mussel Pinna nobilis. One major soluble protein, caspartin, which was identified recently, was partly de novo sequenced after enzymatic digestions. A polyclonal antibody raised against caspartin was used for its localization within and on the prisms. The immunogold localization indicated that caspartin surrounds the calcitic prisms, but is also dispersed within the biominerals. This example illustrates the deep impact of the technique on the definition of intracrystalline versus intercrystalline matrix proteins. Furthermore, it is an important tool for assigning a putative function to a matrix protein of interest.

Cross-species comparison of ectopic bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) scaffolds.

Material-induced bone formation reported in canine, bovid, suid, and primate species does not often occur in lagomorph or rodent models. In this study, we test biphasic calcium phosphate and hydroxyapatite- induced bone formation in subcutaneous pockets of mice and intramuscular pockets in rats, rabbits, and dogs. All scaffolds are of similar size, and all animals were sacrificed at 90 days post-implantation. In dogs (N = 8), all implants showed bone formation with significantly more bone formed in biphasic calcium phosphates (30 +/- 6%, N = 8) as compared to hydroxyapatite (14 +/- 5%, N = 8) (p = 0.003). Hydroxyapatite implants did not induce bone formation in mice, rats, or rabbits. Biphasic calcium phosphate induced bone in 6 of 8 scaffolds implanted in 4 rabbits and 3 of 16 scaffolds implanted in 16 mice, whereas it did not induce bone formation in any of the 8 rats. The results presented herein suggest that the incidence of material-induced bone formation varies with animal species and is related to the implant material used.

Relevance of osteoinductive biomaterials in critical-sized orthotopic defect.

Several publications have shown the phenomenon of osteoinduction by biomaterials to be real. However, whether the ability of a biomaterial to initiate bone formation in ectopic implantation sites improves the performance of such osteoinductive biomaterial in clinically relevant orthotopic sites remains unclear. No studies have been published in which osteoinductive potential of a biomaterial is directly related to its performance orthotopically. In this study, we compared osteoinductive and nonosteoinductive biphasic calcium-phosphate (BCP) ceramics ectopically and in a clinically relevant critical-sized orthotopic defect in goats. The two materials, BCP1150 and BCP1300, had similar chemical compositions, crystallinities, and macrostructures, but their microstructures differed significantly. BCP1150, sintered at a lower temperature, had a large amount of micropores, small average crystal size, and hence a high specific surface area. In contrast, BCP1300, with few micropores, had a significantly lower specific surface area as compared to BCP1150. Twelve-week intramuscular implantation in goats (n = 10) showed that bone was induced in all BCP1150 implants, while no signs of bone formation were found in any of the BCP1300 implants. After 12 weeks of implantation in a bilateral critical-sized iliac wing defect in the same goats, BCP1150 showed significantly more bone than BCP1300. In addition, the analysis of fluorochrome markers, which were administered to the animals 4, 6, and 8 weeks after implantation to follow the bone growth dynamics, showed an earlier start of bone formation in BCP1150 as compared to BCP1300. Significantly better performance of an osteoinductive ceramic in a critical-sized orthotopic defect in a large animal model in comparison to a nonosteoinductive ceramic suggests osteoinduction to be clinically relevant. Further improvement of material osteoinductive properties is thus a significant step forward in the search for alternatives for autologous bone graft.

Osteoinduction by biomaterials--physicochemical and structural influences.

Osteoinduction by biomaterials has been shown to be a real phenomenon by many investigators in the last decade. The exact mechanism of this phenomenon is, however, still largely unknown. This in vivo study in goats was performed to get insight into processes governing the phenomenon of osteoinduction by biomaterials and had four main goals: (i) to further investigate the influence of physicochemical properties and structure on biomaterial osteoinductive potential, (ii) to investigate the influence of implant size on the amount of induced bone, (iii) to investigate implantation site dependence, and (iv) to investigate changes occurring on the surface of the material after implantation. Intramuscular implantations of four different biphasic calcium phosphate ceramics, consisting of hydroxyapatite and beta-tricalcium phosphate and a carbonated apatite ceramic, indicated that, for a maximal osteoinductive potential, there is an optimal specific surface area for each material type. It was further shown that a decrease of the implant size with a half significantly decreased the relative amount of induced bone. In addition, subcutaneous implantation did not give rise to ectopic bone formation in any of the animals, while bone was induced in most animals intramuscularly. Analysis of the surfaces of the materials after subcutaneous implantation inside diffusion chambers indicated that the increased specific surface area leads to more surface reactivity, which is hypothesized to be essential for osteoinductivity by biomaterials.

Porous Ti6Al4V scaffold directly fabricating by rapid prototyping: preparation and in vitro experiment.

Three-dimensional (3D) fiber deposition (3DF), a rapid prototyping technology, was successfully directly applied to produce novel 3D porous Ti6Al4V scaffolds with fully interconnected porous networks and highly controllable porosity and pore size. A key feature of this technology is the 3D computer-controlled fiber depositing of Ti6Al4V slurry at room temperature to produce a scaffold, consisting of layers of directionally aligned Ti6Al4V fibers. In this study, the Ti6Al4V slurry was developed for the 3D fiber depositing process, and the parameters of 3D fiber depositing were optimized. The experimental results show how the parameters influence the structure of porous scaffold. The potential of this rapid prototyping 3DF system for fabricating 3D Ti6Al4V scaffolds with regular and reproducible architecture meeting the requirements of tissue engineering and orthopedic implants is demonstrated.

Electrolytic deposition of calcium phosphate/chitosan coating on titanium alloy: growth kinetics and influence of current density, acetic acid, and chitosan.

Electrolytically deposited calcium phosphate/chitosan coating demonstrated good bone marrow stromal cell attachment. The aim of this study was to understand the coating's growth kinetics as well as the effects of current density, acetic acid, and chitosan on the coating's formation. The scanning electron micrographs found that calcium phosphate crystals homogeneously distributed into chitosan aggregates as early as 30 min. X-ray diffraction patterns and Fourier transform infrared spectra demonstrated that the coating experienced a compositional conversion from octacalcium phosphate to carbonate apatite during the deposition process. Electric current influenced the deposition. Higher current density accelerated the process and induced faster and more chitosan deposition. Both acetic acid and chitosan were found to inhibit calcium phosphate deposition. Chitosan was thought to induce stronger effects than acetic acid did. Furthermore, the inhibitive effect related to their concentration in the electrolyte. When chitosan concentration increased to a certain degree, this inhibitive effect not only affected calcium phosphate deposition, but also affected its own deposition. The chitosan content within the hybrid coating was small, which could be verified through Raman spectrum. At the same time, no clear evidence of chemical reactions could be found between these two components. We considered that both components were just naturally wrapped to form as a whole.

Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics.

Calcium phosphate bioceramics are widely used in orthopedic and dental applications and porous scaffolds made of them are serious candidates in the field of bone tissue engineering. They have superior properties for the stimulation of bone formation and bone bonding, both related to the specific interactions of their surface with the extracellular fluids and cells, ie, ionic exchanges, superficial molecular rearrangement and cellular activity.

3D microenvironment as essential element for osteoinduction by biomaterials.

In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures. Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250 degrees C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (beta-TCP), was sintered at 1100, 1150 and 1200 degrees C. Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and beta-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10 microm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures. Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants. All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals. Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage.

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V.

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity.

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.