Bone colonization of beta-TCP granules incorporated in brushite cements

Injectable calcium phosphate hydraulic cements are known to have a high clinical potential in bone reconstruction for mini-invasive orthopaedic surgery, interventional radiology, and rheumatology. Previous in vivo experiments in rabbit have shown that the presence of beta-TCP granules in injectable bone cement help maintain the transient biomechanical function of the implanted bone and promote the formation of good-quality new bone. Histomorphometric analysis of two brushite hydraulic cement (BHC) mixtures selected from previous results (referred to in this work as BHC-A and BHC-B) was performed at three postoperative delays (0, 12, and 24 weeks): histomorphometric analysis of bone colonization within beta-TCP shows that, just before implantation, the beta-TCP granule area is significantly higher in BHC-B; the residual granule area decreases steadily over time in BHC-A, whereas it goes through a maximum of 30% at 12 weeks in BHC-B; the residual granule porosity increases steadily up to 35% in BHC-A, whereas it goes through a maximum of 35% at 12 weeks and decreases somewhat until 24 weeks in BHC-B. New bone formation within granules appears higher in BHC-A (58% Area) compared to BHC-B (38% area) at 12 weeks. At 24 weeks bone colonization levels off in both cements at about 50% area. Irrespective of the cement matrix composition, beta-TCP granules contribute actively to the conduction of new bone formation.     

In vitro biodegradation of three brushite calcium phosphate cements by a macrophage cell-line

Depending upon local conditions, brushite (CaHPO4 x 2 H2O) cements may be largely resorbed or (following hydrolysis to hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the osteoclast-like cell cultures, ultrastructural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement degradation in vitro.     

The use of RANKL-coated brushite cement to stimulate bone remodelling

Calcium phosphate cements were first proposed as synthetic bone substitutes over two decades ago, however, they are characterised by slow chemical or cellular resorption and a slow osteointegration. In contrast, bone autograft has been shown to stimulate osteoclastogenesis and angiogenesis resulting in active bone remodelling and rapid graft incorporation. Therefore, we aimed to develop a biomaterial able to release a key stimulator of the bone remodelling process, cytokine RANKL. Cylinders of brushite cement, hydroxyapatite cement and sodium alginate were loaded with RANKL either by incorporation into the cement or by coating the material with soluble RANKL. To test the biological activity of these formulations, we assessed their effectiveness in inducing osteoclast formation from RAW 264.7 monocytic cell line. Only brushite and hydroxyapatite cements coated with RANKL allowed for retaining sufficient biological activity to induce osteoclast formation. Most efficient was coating 40 mg cylinder of brushite cement with 800 ng RANKL. We have found that RANKL-coated brushite cement exhibits osteoclastogenic activity for at least 1 month at 37 degrees C. Thus, we developed a formulation of brushite cement with RANKL - a synthetic bone graft that is similar to autografts in its ability to actively induce osteoclastogenesis.     

Beta-tricalcium phosphate release from brushite cement surface

Different in vivo studies demonstrated that brushite cements are biocompatible, bioresorbable, and osteoconductive. However, the decay of brushite cements has been scarcely studied even though it may be of great concern for clinical applications in highly blood-perfused regions. This work was elaborated to elucidate factors that determine brushite cement surface disintegration. For that, brushite cements were modified using in their preparation different aqueous solutions of phosphoric, glycolic, tartaric, and citric acids in concentrations that were reported to improve the cement properties. Two-viscosity enhancing polysaccharides, chondroitin-4 sulfate and hyaluronic acid, were also assayed. Thereafter, pre- and set cement samples were immersed in distilled water for 24 h. The cement-solid weight loss, microstructure, liquid phase viscosity, mean size of the released particles, and zeta potential were analyzed using X-ray diffraction, FTIR spectroscopy, light scattering, scanning electron microscopy and optical microscopy. It was found that the particles released from the cement surface were beta-TCP, and their amount depends on the carboxylic acid used in the preparation of the cement. The addition of hyaluronic acid and chondroitin-4 sulfate decreased the amount of released particles from the surface of the set brushite cement made with citric acid. Furthermore, the hyaluronic acid increased significantly the viscosity of the citric acid solution and the cement paste prepared with this liquid phase showed a pronounced step down in particle release. In this study, we showed that the water solubility of calcium carboxylate and the viscosity of mixing liquid may dictate the superficial disintegration of brushite cements.     

Biologically mediated resorption of brushite cement in vitro

A new calcium phosphate cement is reported, which sets to form a matrix consisting of brushite, dicalcium pyrophosphate dihydrate and an amorphous phase following the mixture of beta-tricalcium phosphate with an aqueous pyrophosphoric acid solution. This reactant combination set within a clinically relevant time-frame (approximately 10 min) and exhibited a higher compressive strength (25 MPa) than previously reported brushite cements. The in vitro degradation of the beta-tricalcium phosphate-pyrophosphoric acid cement was tested in both phosphate buffered saline and bovine serum. The pyrophosphate ion containing cement reported here was found not to be hydrolysed to form hydroxyapatite in vitro like beta-tricalcium phosphate-orthophosphoric acid solution cements. This finding is significant since the formation of hydroxyapatite by hydrolysis is thought to retard in vivo degradation of brushite cements. When aged in bovine serum, the cement lost considerably more mass than when aged in phosphate buffered saline, indicating that proteins, most likely phosphatase enzymes played an important role in the degradation. As pyrophosphate ions are thought to be the source of orthophosphate ions during bone mineralisation, this new class of bone cement offers a route to new degradable synthetic bone grafting materials.     

Novel bioactive composite bone cements based on the beta-tricalcium phosphate-monocalcium phosphate monohydrate composite cement system

Bioactive composite bone cements were obtained by incorporation of tricalcium silicate (Ca3SiO5, C3S) into a brushite bone cement composed of beta-tricalcium phosphate [beta-Ca3(PO4)2, beta-TCP] and monocalcium phosphate monohydrate [Ca(H2PO4)2.H2O, MCPM], and the properties of the new cements were studied and compared with pure brushite cement. The results indicated that the injectability, setting time and short- and long-term mechanical strength of the material are higher than those of pure brushite cement, and the compressive strength of the TCP/MCPM/C3S composite paste increased with increasing aging time. Moreover, the TCP/MCPM/C3S specimens showed significantly improved in vitro bioactivity in simulated body fluid and similar degradability in phosphate-buffered saline as compared with brushite cement. Additionally, the reacted TCP/MCPM/C3S paste possesses the ability to stimulate osteoblast proliferation and promote osteoblastic differentiation of the bone marrow stromal cells. The results indicated that the TCP/MCPM/C3S cements may be used as a bioactive material for bone regeneration, and might have significant clinical advantage over the traditional beta-TCP/MCPM brushite cement.     

Effect of cold-setting calcium- and magnesium phosphate matrices on protein expression in osteoblastic cells

Bone loss due to accidents or tissue diseases requires replacement of the structure by either autografts, allografts, or artificial materials. Reactive cements, which are based on calcium phosphate chemistry, are commonly used in nonload bearing areas such as the craniofacial region. Some of these materials are resorbed by the host under physiological conditions and replaced by bone. The aim of this study was to test different calcium and magnesium cement composites in vitro for their use as bone substitution material. Phase composition of calcium deficient hydroxyapatite (Ca(9) (PO(4) )(5) HPO(4) OH), brushite (CaHPO(4) ·2H(2) O), and struvite (MgNH(4) PO(4) ·6H(2) O) specimens has been determined by means of X-ray diffraction, and compressive strength was measured. Cell growth and activity of osteoblastic cells (MG 63) on the different surfaces was determined, and the expression of bone marker proteins was analyzed by western blotting. Cell activity normalized to cell number revealed higher activity of the osteoblasts on brushite and struvite when compared to hydroxyapatite and also the expression of osteoblastic marker proteins was highest on brushite scaffolds. While brushite sets under acidic conditions, formation of struvite occurs under physiological pH, similar to hydroxyapatite cements, providing the possibility of additional modifications with proteins or other active components.     

Doxycycline sustained release from brushite cements for the treatment of periodontal diseases

Doxycycline (DOXY) is a wide spectrum antibiotic used in the treatment of dental, periodontal, and bone infections. Brushite cements are calcium phosphate biomaterials especially interesting for bone regeneration processes. In this work, we describe the preparation of a brushite cement containing DOXY and the drug release from the cement. DOXY solutions were mixed with the cement powder and after a 50% burst release in the first 12 h, a slow and controlled release was achieved over 3.5 days. The release of DOXY hyclate was controlled by both, diffusion and Ca(2+) interaction. Formation of DOXY-Ca(2+) chelates was detected in the cement structure using solid state fluorescence. The brushite cement loaded with DOXY hyclate had antibacterial activity against periodontal pathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides frosthytus. This new biomaterial may be helpful for the treatment of periodontal diseases.     

Vertical bone augmentation with granulated brushite cement set in glycolic acid

Brushite cements are a biocompatible materials that are resorbed in vivo. A new cement composed of a mixture of monocalcium phosphate (MCP) and beta-tricalcium phosphate (beta-TCP) that sets using glycolic acid (GA) was synthesized and characterized. After setting, the cement composition, derived from X-ray diffraction, was 83 wt % brushite and 17 wt % beta-TCP with an average brushite crystal size of about 2.6 +/- 1.4 microm. The cement has a diametral tensile strength of 2.9 +/- 0.7 MPa. Granules prepared from the set-cement were used as grafting material in bone defects on rabbit calvaria for evaluating in vivo its bone regeneration capacity. Considerable cement resorption, improvement in the bone mineral density, and bone neoformation was observed after 4 weeks of the granules' implantation.     

Biocompatibility and resorption of a brushite calcium phosphate cement

A hydraulic calcium phosphate cement with beta-tricalcium phosphate (TCP) granules embedded in a matrix of dicalcium phosphate dihydrate (DCPD) was implanted in experimentally created defects in sheep. One type of defect consisted of a drill hole in the medial femoral condyle. The other, partial metaphyseal defect was located in the proximal aspect of the tibia plateau and was stabilized using a 3.5 mm T-plate. The bone samples of 2 animals each per group were harvested after 2, 4, 6 and 8 weeks. Samples were evaluated for cement resorption and signs of immediate reaction, such as inflammation, caused by the cement setting in situ. Differences regarding these aspects were assessed for both types of defects using macroscopical, radiological, histological and histomorphometrical evaluations. In both defects the brushite matrix was resorbed faster than the beta-TCP granules. The resorption front was followed directly by a front of new bone formation, in which residual beta-TCP granules were embedded. Cement resorption occurred through (i) extracellular liquid dissolution with cement disintegration and particle formation, and (ii) phagocytosis of the cement particles through macrophages. Signs of inflammation or immunologic response leading to delayed new bone formation were not noticed at any time. Cement degradation and new bone formation occurred slightly faster in the femur defects.     

The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: brushite vs. monetite

Dicalcium phosphate dihydrate (brushite) is an osteoconductive biomaterial with great potential as a bioresorbable cement for bone regeneration. Preset brushite cement can be dehydrated into dicalcium phosphate anhydrous (monetite) bioceramics by autoclaving. This heat treatment results in changes in the physical characteristics of the material, improving in vivo bioresorption. This property is a great advantage in bone regeneration; however, it is not known how autoclaving brushite preset cement might improve its capacity to regenerate bone. This study was designed to compare brushite bioceramics with monetite bioceramics in terms of physical characteristics in vitro, and in vivo performance upon bone implantation. In this study we observed that monetite bioceramics prepared by autoclaving preset brushite cements had higher porosity, interconnected porosity and specific surface area than their brushite precursors. In vitro cell culture experiments revealed that bone marrow cells expressed higher levels of osteogenic genes Runx2, Opn, and Alp when the cells were cultured on monetite ceramics rather than on brushite ones. In vivo experiments revealed that monetite bioceramics resorbed faster than brushite ones and were more infiltrated with newly formed bone. In summary, autoclaving preset brushite cements results in a material with improved properties for bone regeneration procedures.     

The effect of hyaluronic acid on brushite cement cohesion

The improvement of calcium phosphate cement (CPC) cohesion is essential for its application in highly blood perfused regions. This study reports the effectiveness of hyaluronic acids of different molecular weights in the enhancement of brushite cement cohesion. The cement was prepared using a powder phase composed of a mixture of β-tricalcium phosphate and monocalcium phosphate monohydrate, whereas the liquid phase was formed by 0.5 M citric acid solution modified by the addition of hyaluronic acid of different molecular weights. It was found that medium and high molecular weight hyaluronic acid enhances the cement cohesion and scarcely affects the cement mechanical properties. However, concentrations >0.5% (w/v) were less efficient to prevent the cement disintegration. It is concluded that hyaluronic acid could be applied efficiently to reduce brushite cement disintegration.     

Calcium carbonate-calcium phosphate mixed cement compositions for bone reconstruction

The feasibility of making calcium carbonate-calcium phosphate (CaCO(3)-CaP) mixed cements, comprising at least 40% (w/w) CaCO(3) in the dry powder ingredients, has been demonstrated. Several original cement compositions were obtained by mixing metastable crystalline CaCO(3) phases with metastable amorphous or crystalline CaP powders in aqueous medium. The cements set within at most 1 h at 37 degrees C in atmosphere saturated with water. The hardened cement is microporous and exhibits weak compressive strength. The setting reaction appeared to be essentially related to the formation of a highly carbonated nanocrystalline apatite phase by reaction of the metastable CaP phase with part or almost all of the metastable CaCO(3) phase. The recrystallization of metastable CaP varieties led to a final cement consisting of a highly carbonated poorly crystalline apatite analogous to bone mineral associated with various amounts of vaterite and/or aragonite. The presence of controlled amounts of CaCO(3) with a higher solubility than that of the apatite formed in the well-developed CaP cements might be of interest to increase resorption rates in biomedical cement and favors its replacement by bone tissue. Cytotoxicity testing revealed excellent cytocompatibility of CaCO(3)-CaP mixed cement compositions.     

Control of in vivo mineral bone cement degradation

The current study aimed to prevent the formation of hydroxyapatite reprecipitates in brushite-forming biocements by minimizing the availability of free Ca²⁺ ions in the cement matrix. This was achieved by both maximizing the degree of cement setting to avoid unreacted, calcium-rich cement raw materials which can deliver Ca²⁺ directly to the cement matrix after dissolution, and by a reduction in porosity to reduce Ca²⁺ diffusion into the set cement matrix. In addition, a biocement based on the formation of the magnesium phosphate mineral struvite (MgNH₄PO₄·6H₂O) was tested, which should prevent the formation of low-solubility hydroxyapatite reprecipitates due to the high magnesium content. Different porosity levels were fabricated by altering the powder-to-liquid ratio at which the cements were mixed and the materials were implanted into mechanically unloaded femoral defects in sheep for up to 10 months. While the higher-porosity brushite cement quantitatively transformed into crystalline octacalcium phosphate after 10 months, slowing down cement resorption, a lower-porosity brushite cement modification was found to be chemically stable with the absence of reprecipitate formation and minor cement resorption from the implant surface. In contrast, struvite-forming cements were much more degradable due to the absence of mineral reprecipitates and a nearly quantitative cement degradation was found after 10 months of implantation.     

Of the in vivo behavior of calcium phosphate cements and glasses as bone substitutes

The use of injectable self-setting calcium phosphate cements or soluble glass granules represent two different strategies for bone regeneration, each with distinct advantages and potential applications. This study compares the in vivo behavior of two calcium phosphate cements and two phosphate glasses with different composition, microstructure and solubility, using autologous bone as a control, in a rabbit model. The implanted materials were alpha-tricalcium phosphate cement (cement H), calcium sodium potassium phosphate cement (cement R), and two phosphate glasses in the P(2)O(5)-CaO-Na(2)O and P(2)O(5)-CaO-Na(2)O-TiO(2) systems. The four materials were osteoconductive, biocompatible and biodegradable. Radiological and histological studies demonstrated correct osteointegration and substitution of the implants by new bone. The reactivity of the different materials, which depends on their solubility, porosity and specific surface area, affected the resorption rate and bone formation mainly during the early stages of implantation, although this effect was weak. Thus, at 4 weeks the degradation was slightly higher in cements than in glasses, especially for cement R. However, after 12 weeks of implantation all materials showed a similar degradation degree and promoted bone neoformation equivalent to that of the control group.     

Resorption patterns of calcium-phosphate cements in bone

Two calcium phosphate cements, one monophasic and the other biphasic, have been used as bone void filler in a sheep model. The cements were injected into a slot defect in the proximal tibia and into a cylindrical defect in the distal femur. In this study, we focused on the resorption pattern of the two cement formulations and the subsequent biologic reaction. Bone remodeling occurred synchronously with the resorption of the implant material in a creeping substitution process. Cracks and pores in the monophasic cement were filled with osseous tissues. The biphasic cement showed faster resorption of the matrix. The more slowly resorbing granules were surrounded by newly grown bone, thus providing an inverse scaffold for cancellous bone regeneration. In highly loaded areas, the long-term support function of the fixation appears to be critical. Because cortical bridging of the defects was seen in only one case, it can be concluded that calcium-phosphate cements are preferentially suitable as cancellous bone substitute materials.     

Osteogenic biphasic calcium sulphate dihydrate/iron-modified α-tricalcium phosphate bone cement for spinal applications: In vivo study

In this study, the biocompatibility and the osteogenic features of a new iron-modified α-tricalcium phosphate (IM/α-TCP) and calcium sulphate dihydrate (CSD) biphasic cement (IM/α-TCP/CSD-BC) have been investigated in terms of the in vivo cement resorption, bone tissue formation and host tissue response on sheep animal model. Histological evaluation performed on undecalcified cement–bone specimens assessed the in vivo behaviour. It has been shown that the new IM/α-TCP/CSD-BC has the ability to produce firm bone binding in vivo (i.e. bioactivity). Qualitative histology proved cement biocompatibility, osteoconduction and favourable resorption, mainly through a macrophage-mediated mechanism. The results showed that the new cements have biocompatible and osteogenic features of interest as possible cancellous bone replacement biomaterial for minimally invasive spinal surgery applications.     

Setting properties and in vitro bioactivity of strontium-enriched gelatin-calcium phosphate bone cements

Strontium is known to reduce bone resorption and stimulate bone formation. We have investigated the effect of strontium on the setting properties and in vitro bioactivity of a biomimetic gelatin-calcium phosphate bone cement. Gelatin-alpha-TCP powders, with a gelatin content of 15 wt %, were prepared by grinding and sieving the solid compounds obtained by casting gelatin aqueous solutions containing alpha-TCP. 5 wt % of CaHPO(4).2H(2)O were added to the cement powders before mixing with the liquid phase, with a L/P ratio of 0.3 mL/g. Strontium was added as SrCl(2).6H(2)O in different amounts up to 5 atom %. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The presence of strontium affects both the initial and the final setting times of the cements, which increase with the ion content. The microstructural modifications observed in the SEM micrographs of the fractured surfaces are in agreement with the increase of the total porosity, and with the slight reduction of the compressive strength of the aged cements, on increasing strontium content. The rate of transformation of alpha-TCP into calcium deficient hydroxyapatite increases on increasing strontium content. SEM reveals that MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, strontium stimulates Alkaline Phosphatase activity, Collagen type I, osteocalcin, and osteoprotegerin expression.     

Preparation, physical-chemical characterisation and cytocompatibility of calcium carbonate cements

The feasibility of calcium carbonate cements involving the recrystallisation of metastable calcium carbonate varieties has been demonstrated. Calcium carbonate cement compositions presented in this paper can be prepared straightforwardly by simply mixing water (liquid phase) with two calcium carbonate phases (solid phase) which can be easily obtained by precipitation. An original cement composition was obtained by mixing amorphous calcium carbonate and vaterite with an aqueous medium. The cement set and hardened within 2h at 37 degrees C in an atmosphere saturated with water and the final composition of the cement consisted mostly of aragonite. The hardened cement was microporous and showed poor mechanical properties. Cytotoxicity tests revealed excellent cytocompatibility of calcium carbonate cement compositions. Calcium carbonates with a higher solubility than the apatite formed for most of the marketed calcium phosphate cements might be of interest to increase biomedical cement resorption rates and to favour its replacement by bone tissue.     

Strontium modified biocements with zero order release kinetics

Strontium-substituted beta-TCP with the general formula Ca((3-x))Sr(x)(PO(4))(2) (0相似文献