Influence of the sintering atmosphere on the physico-chemical properties and the osteoclastic resorption of ß-tricalcium phosphate cylinders.

One of the most widely used materials for bone graft substitution is ß-Tricalcium phosphate (ß-TCP; ß-Ca3(PO4)2). ß-TCP is typically produced by sintering in air or vacuum. During this process, evaporation of phosphorus (P) species occurs, leading to the formation of a calcium-rich alkaline layer. It was recently shown that the evaporation of P species could be prevented by co-sintering ß-TCP with dicalcium phosphate (DCPA; CaHPO4; mineral name: monetite). The aim of this study was to see how a change of sintering atmosphere could affect the physico-chemical and biological properties of ß-TCP. For this purpose, three experimental groups were considered: ß-TCP cylinders sintered in air and subsequently polished to remove the surface layer (control group); the same polished cylinders after subsequent annealing at 500 °C in air to generate a calcium-rich alkaline layer (annealed group); and finally, ß-TCP cylinders sintered in a monetite-rich atmosphere and subsequently polished (monetite group). XPS analysis confirmed that cylinders from the annealed group had a significantly higher Ca/P molar ratio at their surface than that of the control group while this ratio was significantly lower for the cylinders from the monetite group. Sintering ß-TCP in the monetite-rich atmosphere significantly reduced the grain size and increased the density. Changes of surface composition affected the activity of osteoclasts seeded onto the surfaces, since annealed ß-TCP cylinders were significantly less resorbed than ß-TCP cylinders sintered in the monetite-rich atmosphere. This suggests that an increase of the surface Ca/P molar ratio leads to a decrease of osteoclastic resorption. STATEMENT OF SIGNIFICANCE: Minimal changes of surface and bulk (< 1%) composition have major effects on the ability of osteoclasts to resorb ß-tricalcium phosphate (ß-TCP), one of the most widely used ceramics for bone substitution. The results presented in this study are thus important for the calcium phosphate community because (i) ß-TCP may have up to 5% impurities according to ISO and ASTM standards and still be considered to be "pure ß-TCP", (ii) ß-TCP surface properties are generally not considered during biocompatibility assessment and (iii) a rationale can be proposed to explain the various inconsistencies reported in the literature on the biological properties of ß-TCP.

Cell-free, quantitative mineralization measurements as a proxy to identify osteoinductive bone graft substitutes.

Some synthetic bone graft substitutes (BGS) can trigger ectopic bone formation, which is the hallmark of osteoinduction and the most important prerequisite for the repair of large bone defects. Unfortunately, measuring or predicting BGS osteoinductive potential based on in vitro experiments is currently impossible. A recent study claimed that synthetic BGS can induce bone formation ectopically if they create a local homeostatic imbalance during their in vivo mineralization. This raised the hope that a simple cell free in vitro mineralization experiment would correlate with osteoinduction. The aim of the present study was therefore to assess the ability of a quantitative in vitro mineralization test to predict and rank the osteoinductive potential of BGS. Eight calcium phosphate BGS already tested ectopically in 9 different in vivo studies were used for that purpose. The experiment was able to identify materials that are reliably osteoinductive from those that are not, but was inaccurate in ranking the osteoinductive materials between each other. Chemical contaminants (Ca2+, Mg2+, H+, OH-, PO43-) present in some of the BGS affected the in vitro mineralization experiment results, but not in a direction that could explain the different rankings. In conclusion, this study suggests that an in vitro experiment can be used as a fast and reliable screening tool to identify osteoinductive BGS and underline the need to study ionic contaminants on calcium phosphate BGS.

ß-tricalcium phosphate for bone substitution: Synthesis and properties.

ß-tricalcium phosphate (ß-TCP) is one the most used and potent synthetic bone graft substitute. It is not only osteoconductive, but also osteoinductive. These properties, combined with its cell-mediated resorption, allow full bone defects regeneration. Its clinical outcome is sometimes considered to be "unpredictable", possibly due to a poor understanding of ß-TCP physico-chemical properties: ß-TCP crystallographic structure is not fully uncovered; recent results suggest that sintered ß-TCP is coated with a Ca-rich alkaline phase; ß-TCP apatite-forming ability and osteoinductivity may be enhanced by a hydrothermal treatment; ß-TCP grain size and porosity are strongly modified by the presence of minute amounts of ß-calcium pyrophosphate or hydroxyapatite impurities. The aim of the present article is to provide a critical, but still rather comprehensive review of the current state of knowledge on ß-TCP, with a strong focus on its synthesis and physico-chemical properties, and their link to the in vivo response. STATEMENT OF SIGNIFICANCE: The present review documents the richness, breadth, and interest of the research devoted to ß-tricalcium phosphate (ß-TCP). ß-TCP is synthetic, osteoconductive, osteoinductive, and its resorption is cell-mediated, thus making it one of the most potent bone graft substitutes. This comprehensive review reveals that there are a number of aspects, such as surface chemistry, crystallography, or stoichiometry deviations, that are still poorly understood. As such, ß-TCP is still an exciting scientific playground despite a 50 year long history and > 200 yearly publications.

In vitro response of mesenchymal stem cells to biomimetic hydroxyapatite substrates: A new strategy to assess the effect of ion exchange.

Biomaterials can interact with cells directly, that is, by direct contact of the cells with the material surface, or indirectly, through soluble species that can be released to or uptaken from the surrounding fluids. However, it is difficult to characterise the relevance of this fluid-mediated interaction separately from the topography and composition of the substrate, because they are coupled variables. These fluid-mediated interactions are amplified in the case of highly reactive calcium phosphates (CaPs) such as biomimetic calcium deficient hydroxyapatite (CDHA), particularly in static in vitro cultures. The present work proposes a strategy to decouple the effect of ion exchange from topographical features by adjusting the volume ratio between the cell culture medium and biomaterial (VCM/VB). Increasing this ratio allowed mitigating the drastic ionic exchanges associated to the compositional changes experienced by the material exposed to the cell culture medium. This strategy was validated using rat mesenchymal stem cells (rMSCs) cultured on CDHA and beta-tricalcium phosphate (ß-TCP) discs using different VCM/VB ratios. Whereas in the case of ß-TCP the cell response was not affected by this ratio, a significant effect on cell adhesion and proliferation was found for the more reactive CDHA. The ionic exchange, produced by CDHA at low VCM/VB, altered cell adhesion due to the reduced number of focal adhesions, caused cell shrinkage and further rMCSs apoptosis. This was mitigated when using a high VCM/VB, which attenuated the changes of calcium and phosphate concentrations in the cell culture medium, resulting in rMSCs spreading and a viability over time. Moreover, rMSCs showed an earlier expression of osteogenic genes on CDHA compared to sintered ß-TCP when extracellular calcium fluctuations were reduced. STATEMENT OF SIGNIFICANCE: Fluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of biomimetic hydroxyapatite, which makes the in vitro characterisation of cell-material interactions especially challenging. The present work proposes a novel and simple strategy to explore the mechanisms of interaction of biomimetic and sintered calcium phosphates with mesenchymal stem cells. The effects of topography and ion exchange are analysed separately by modifying the volume ratio between cell culture medium and biomaterial. High ionic fluctuations interfered in the maturation of focal adhesions, hampering cell adhesion and leading to increased apoptosis and reduced proliferation rate.

Hydrogen-substituted ß-tricalcium phosphate synthesized in organic media.

ß-Tricalcium phosphate (ß-TCP) platelets synthesized in ethylene glycol offer interesting geometries for nano-structured composite bone substitutes but were never crystallographically analyzed. In this study, powder X-ray diffraction and Rietveld refinement revealed a discrepancy between the platelet structure and the known ß-TCP crystal model. In contrast, a model featuring partial H for Ca substitution and the inversion of P1O4 tetrahedra, adopted from the whitlockite structure, allowed for a refinement with minimal misfits and was corroborated by HPO42- absorptions in Fourier-transform IR spectra. The Ca/P ratio converged to 1.443 ±â€…0.003 (n = 36), independently of synthesis conditions. As a quantitative verification, the platelets were thermally decomposed into hydrogen-free ß-TCP and ß-calcium pyrophosphate which resulted in a global Ca/P ratio in close agreement with the initial ß-TCP Ca/P ratio (ΔCa/P = 0.003) and with the chemical composition measured by inductively coupled plasma (ΔCa/P = 0.003). These findings thus describe for the first time a hydrogen-substituted ß-TCP structure, i.e. a Mg-free whitlockite, represented by the formula Ca21 - x(HPO4)2x(PO4)14 - 2x, where x = 0.80 ±â€…0.04, and may have implications for resorption properties of bone regenerative materials.

The Effects of Crystal Phase and Particle Morphology of Calcium Phosphates on Proliferation and Differentiation of Human Mesenchymal Stromal Cells.

Calcium phosphate (CaP) ceramics are extensively used for bone regeneration; however, their clinical performance is still considered inferior to that of patient's own bone. To improve the performance of CaP bone graft substitutes, it is important to understand the effects of their individual properties on a biological response. The aim of this study is to investigate the effects of the crystal phase and particle morphology on the behavior of human mesenchymal stromal cells (hMSCs). To study the effect of the crystal phase, brushite, monetite, and octacalcium phosphate (OCP) are produced by controlling the precipitation conditions. Brushite and monetite are produced as plate-shaped and as needle-shaped particles, to further investigate the effect of particle morphology. Proliferation of hMSCs is inhibited on OCP as compared to brushite and monetite in either morphology. Brushite needles consistently show the lowest expression of most osteogenic markers, whereas the expression on OCP is in general high. There is a trend toward a higher expression of the osteogenic markers on plate-shaped than on needle-shaped particles for both brushite and monetite. Within the limits of CaP precipitation, these data indicate the effect of both crystal phase and particle morphology of CaPs on the behavior of hMSCs.

Design of an inorganic dual-paste apatite cement using cation exchange.

The use of hydraulic calcium phosphate cements (CPCs) as bone substitute is impaired by their relatively poor handling due to the need to mix a powder and a liquid during surgery. The aim of the present study was to assess the possibility to design CPCs as inorganic dual-paste cements, where both pastes would be stable for years, but would react as soon as they are mixed together. Results showed that aqueous pastes of α-tricalcium phosphate (α-TCP) powder could be stabilized for up to a year at room temperature by the use of 0.1 M Mg chloride solution. Adding a calcium chloride solution in a 1:4 volume ratio activated α-TCP pastes provided the Ca/Mg ratio was larger than one. Mechanistic investigations suggest that Ca ions can displace Mg cations adsorbed at the surface of α-TCP particles to initiate α-TCP transformation to calcium-deficient hydroxyapatite and concomitant paste hardening. The compressive strength (29 MPa) was similar to that of commercial formulations (5-80 MPa). Other divalent cations (Ba, Ni, Sr) had a similar effect although with a different degree of efficacy.

Synthesis of spherical calcium phosphate particles for dental and orthopedic applications.

Calcium phosphate materials have been used increasingly in the past 40 years as bone graft substitutes in the dental and orthopedic fields. Accordingly, numerous fabrication methods have been proposed and used. However, the controlled production of spherical calcium phosphate particles remains a challenge. Since such particles are essential for the synthesis of pastes and cements delivered into the host bone by minimally-invasive approaches, the aim of the present document is to review their synthesis and applications. For that purpose, production methods were classified according to the used reagents (solutions, slurries, pastes, powders), dispersion media (gas, liquid, solid), dispersion tools (nozzle, propeller, sieve, mold), particle diameters of the end product (from 10 nm to 10 mm), and calcium phosphate phases. Low-temperature calcium phosphates such as monetite, brushite or octacalcium phosphate, as well as high-temperature calcium phosphates, such as hydroxyapatite, ß-tricalcium phosphate or tetracalcium phosphate, were considered. More than a dozen production methods and over hundred scientific publications were discussed.

Synthetic calcium phosphate ceramics for treatment of bone fractures.

Bone is a complex natural material with outstanding mechanical properties and remarkable self-healing capabilities. The mechanical strength is achieved by a complex structure of a mineral part comprising apatitic calcium phosphate crystals embedded in an organic matrix. Bone also contains several types of cells constantly replacing mature bone with new bone. These cells are able to seal fractures and fill gaps with new bone in case of structural damage. However, if a defect exceeds a critical size, surgery is necessary to fill the void with a spacer in order to prevent soft tissue from growing into the defect and delaying the healing process. The spacers, also known as bone grafts, can either be made of fresh bone from the patient, of processed bone from donor organisms, or of synthetic materials chemically similar to the mineral part of bone. Synthetic bone void fillers are also known as bone graft substitutes. This review aims at explaining the biological and chemical background that lead to the development of synthetic bone graft substitutes and gives an overview of the current state of development. It also highlights the multidisciplinary nature of biomaterials research, which combines cell biology and medicine with chemistry, mineralogy, crystallography, and mechanical engineering.