Bioactive composite bone cement based on α-tricalcium phosphate/tricalcium silicate

Silicon compounds are known as bioactive materials that are able to bond to the living bone tissue by inducing an osteogenic response through the stimulation and activation of osteoblasts. To improve the bioactive and mechanical properties of an α-Ca(3)PO(4)-based cement, the effects of the addition of Ca(3 SiO(5) (C(3)S) on physical, chemical, mechanical, and biological properties after soaking in simulated body fluid (SBF) were studied. The morphological and structural changes of the material during immersion were analyzed by X-ray diffraction and scanning electron microscopy. The results showed that it is possible to increase the compressive strength of the cement by adding 5% of C(3)S. Higher C(3)S contents enhance bioactivity and biocompatibility by the formation of a dense and homogeneous hydroxyapatite layer within 7 days; however, compressive strength decreases drastically as a consequence of delayed hydrolysis of α-Ca(3)(PO(4) (2). An increment in setting times and degradation rate of composites containing C(3)S was also observed.     

Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway

Immune reactions play important roles in determining the in vivo fate of bone substitute materials, either in new bone formation or inflammatory fibrous tissue encapsulation. The paradigm for the development of bone substitute materials has been shifted from inert to immunomodulatory materials, emphasizing the importance of immune cells in the material evaluation. Macrophages, the major effector cells in the immune reaction to implants, are indispensable for osteogenesis and their heterogeneity and plasticity render macrophages a primer target for immune system modulation. However, there are very few reports about the effects of macrophages on biomaterial-regulated osteogenesis. In this study, we used β-tricalcium phosphate (β-TCP) as a model biomaterial to investigate the role of macrophages on the material stimulated osteogenesis. The macrophage phenotype switched to M2 extreme in response to β-TCP extracts, which was related to the activation of calcium-sensing receptor (CaSR) pathway. Bone morphogenetic protein 2 (BMP2) was also significantly upregulated by the β-TCP stimulation, indicating that macrophage may participate in the β-TCP stimulated osteogenesis. Interestingly, when macrophage-conditioned β-TCP extracts were applied to bone marrow mesenchymal stem cells (BMSCs), the osteogenic differentiation of BMSCs was significantly enhanced, indicating the important role of macrophages in biomaterial-induced osteogenesis. These findings provided valuable insights into the mechanism of material-stimulated osteogenesis, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of bone substitute materials.     

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.     

Evaluation of host inflammatory responses of β-tricalcium phosphate bioceramics caused by calcium pyrophosphate impurity using a subcutaneous model

Implantation of synthetic materials into body elicits inflammatory host responses that limit medical device integration and biological performance. Since the effective use of biomaterials in vivo requires good biocompatibility and bio-functionality, it is vital that we assess the inflammatory reactions provoked by various implanted biomaterials. In chemical precipitation of β-tricalcium phosphate [β-Ca?(PO?)?, β-TCP], the impurity of calcium pyrophosphate (Ca?P?O?, CPP) will easily appear if the preparation conditions are not well controlled. To test the influences of CCP-impurity on the biocompatibility of the material, four groups of β-TCP ceramic samples doped with 0.5-10 wt % of CCP impurity, and pure β-TCP and CCP samples were fabricated and implanted in rat subcutaneous site for one, two, and four weeks. The host tissue responses to the ceramics were evaluated by histomorphometric analysis, and the results were compared with pure β-TCPbioceramics. The results show that the CPP impurity can elicit and stimulate the inflammatory responses at the tissue/implant interface. Moreover, with the increase of CPP doping amount, the inflammation increases apparently. However, the pure β-TCP bioceramics only present slight post-implantation inflammatory responses. The influence of the CPP doping on the inflammatory responses is mainly related to a microparticles release because of an insufficient sintering of β-TCP by CPP doping. The microparticle release could be at the origin of local inflammation and cell/tissue damages. Therefore, to obtain perfect biocompatibility and high quality β-TCP bioceramics, it is important to avoid and control the CPP impurity in the preparation of β-TCP powders and bioceramics.     

Bioactivity and mechanical properties of collagen composite membranes reinforced by chitosan and β-tricalcium phosphate

In this study, we analyzed the effects of varying concentrations of chitosan (CS) and β-tricalcium phosphate [β-TCP, Ca(3) (PO(4) )(2) ] on the mechanical and cell-adhesion properties of a collagen (CG) matrix for use in guided bone regeneration (GBR). Three different CS concentrations (0.5, 1, and 2%) and five different contents of β-TCP (0, 17, 29, 38, and 44%) were investigated. The composite membranes were analyzed by scanning electron microscopy and cell-adhesion, flexural-strength, and tear-strength assays. The results show that the cell-adhesion and mechanical properties of the composite membranes improved with increasing β-TCP and CS contents, yielding suitable levels of the adhesion of cells and adequate mechanical stability to ensure successful GBR. The CS adhered to the microsized β-TCP, which was distributed uniformly in the composite membranes. The β-TCP and CS have no negative effect on the cell morphology, viability, and proliferation and possess good biocompatibility. This study demonstrates that β-TCP/CS/CG composite membranes are good candidates for GBR membranes in future applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Controllable dual-release of dexamethasone and bovine serum albumin from PLGA/β-tricalcium phosphate composite scaffolds

Localized dual-drug delivery from biodegradable scaffolds is an important strategy in tissue engineering. In this study, porous poly(L-lactide-co-glycolide) (PLGA)/β-tricalcium phosphate scaffolds containing both dexamethasone (Dex) and bovine serum albumin (BSA) were prepared by incorporating Dex-loaded and BSA-loaded microspheres into the scaffolds. PLGA microspheres containing Dex or BSA were prepared by spray-drying and double emulsion/solvent evaporation, respectively. In vitro release studies indicated that microspheres prepared from PLGA in 3:1 molar ratio of L-lactide/glycolide and 89.5 kDa relative molecular mass showed prolonged release profiles compared with those prepared from PLGA in 1:1 L-lactide/glycolide molar ratio and 30.5 kDa relative molecular mass. Additionally, introduction of poly(ethylene glycol) in the PLGA chain could improve the encapsulation efficiency and reduce the release rate. Based on the above results, controllable dual-release of Dex and BSA with relatively higher or lower release rate was achieved by incorporating Dex-loaded and BSA-loaded microspheres with different release profiles into the PLGA/β-tricalcium phosphate scaffolds.     

Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate – Chitosan composite scaffold

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, has excellent osteoconductivity, and can be resorbed and replaced by new bone. However, its low strength limits CPC to non-stress-bearing repairs. Chitosan could be used to reinforce CPC, but mesenchymal stem cell (MSC) interactions with CPC-chitosan scaffold have not been examined. The objective of this study was to investigate MSC proliferation and osteogenic differentiation on high-strength CPC-chitosan scaffold. MSCs were harvested from rat bone marrow. At CPC powder/liquid (P/L) mass ratio of 2, flexural strength (mean ± sd; n = 5) was (10.0 ± 1.1) MPa for CPC-chitosan, higher than (3.7 ± 0.6) MPa for CPC (p < 0.05). At P/L of 3, strength was (15.7 ± 1.7) MPa for CPC-chitosan, higher than (10.2 ± 1.8) MPa for CPC (p < 0.05). Percentage of live MSCs attaching to scaffolds increased from 85% at 1 day to 99% at 14 days. There were (180 ± 37) cells/mm² on scaffold at 1 day; cells proliferated to (1808 ± 317) cells/mm² at 14 days. SEM showed MSCs with healthy spreading and anchored on nano-apatite crystals via cytoplasmic processes. Alkaline phosphatase activity (ALP) was (557 ± 171) (pNPP mM/min)/(μg DNA) for MSCs on CPC-chitosan, higher than (159 ± 47) on CPC (p < 0.05). Both were higher than (35 ± 32) of baseline ALP for undifferentiated MSCs on tissue-culture plastic (p < 0.05). In summary, CPC-chitosan scaffold had higher strength than CPC. MSC proliferation on CPC-chitosan matched that of the FDA-approved CPC control. MSCs on the scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. Hence, the stronger CPC-chitosan scaffold may be useful for stem cell-based bone regeneration in moderate load-bearing maxillofacial and orthopedic applications.     

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.     

Human urine-derived stem cells can be induced into osteogenic lineage by silicate bioceramics via activation of the Wnt/β-catenin signaling pathway

Human urine-derived stem cells (USCs) have great application potential for cytotherapy as they can be obtained by non-invasive and simple methods. Silicate bioceramics, including calcium silicate (CS), can stimulate osteogenic differentiation of stem cells. However, the effects of silicate bioceramics on osteogenic differentiation of USCs have not been reported. In this study, at first, we investigated the effects of CS ion extracts on proliferation and osteogenic differentiation of USCs, as well as the related mechanism. CS particles were incorporated into poly (lactic-co-glycolic acid) (PLGA) to obtain PLGA/CS composite scaffolds. USCs were then seeded onto these scaffolds, which were subsequently transplanted into nude mice to analyze the osteogenic differentiation of USCs and mineralization of extracellular matrix formed by USCs in vivo. The results showed that CS ion extracts significantly enhanced cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and expression of certain osteoblast-related genes and proteins. In addition, cardamonin, a Wnt/β-catenin signaling inhibitor, reduced the stimulatory effects of CS ion extracts on osteogenic differentiation of USCs, indicating that the observed osteogenic differentiation of USCs induced by CS ion extracts involves Wnt/β-catenin signaling pathway. Furthermore, histological analysis showed that PLGA/CS composite scaffolds significantly enhanced the osteogenic differentiation of USCs in vivo. Taken together, these results suggest the therapeutic potential of combining USCs and PLGA/CS scaffolds in bone tissue regeneration.     

Silicon-stabilized α-tricalcium phosphate and its use in a calcium phosphate cement: characterization and cell response

α-Tricalcium phosphate (α-TCP) is widely used as a reactant in calcium phosphate cements. This work aims at doping α-TCP with silicon with a twofold objective. On the one hand, to study the effect of Si addition on the stability and reactivity of this polymorph. On the other, to develop Si-doped cements and to evaluate the effect of Si on their in vitro cell response. For this purpose a calcium-deficient hydroxyapatite was sintered at 1250°C with different amounts of silicon oxide. The high temperature polymorph α-TCP was stabilized by the presence of silicon, which inhibited reversion of the β→α transformation, whereas in the Si-free sample α-TCP completely reverted to the β-polymorph. However, the β-α transformation temperature was not affected by the presence of Si. Si-α-TCP and its Si-free counterpart were used as reactants for a calcium phosphate cement. While Si-α-TCP showed faster hydrolysis to calcium-deficient hydroxyapatite, upon complete reaction the crystalline phases, morphology and mechanical properties of both cements were similar. An in vitro cell culture study, in which osteoblast-like cells were exposed to the ions released by both materials, showed a delay in cell proliferation in both cases and stimulation of cell differentiation, more marked for the Si-containing cement. These results can be attributed to strong modification of the ionic concentrations in the culture medium by both materials. Ca-depletion from the medium was observed for both cements, whereas continuous Si release was detected for the Si-containing cement.     

Vertical alveolar ridge augmentation with β-tricalcium phosphate and autologous osteoblasts in canine mandible

A tissue-engineered bone has become a viable alternative to autologous bone for bone augmentation in atrophy alveolar ridge. The aim of the present study was to evaluate porous β-tricalcium phosphate (β-TCP) combined with autologous osteoblasts to augment edentulous alveolar ridge in a canine model. Autologous osteoblasts were expanded and combined with β-TCP scaffold to fabricate a tissue-engineered bone. 12 bilateral alveolar ridge augmentation surgeries were carried out in 6 beagle dogs with the following 3 groups: β-TCP/osteoblasts, β-TCP alone and autogenous iliac bone control (n = 4 per group). Sequential fluorescent labeling and radiographs were used to compare new bone formation and mineralization in each group. 24 weeks later, animals were sacrificed and non-decalcified and decalcified sections were evaluated histologically and histomorphometrically. Results indicated that the tissue-engineered bone dramatically enhanced new bone formation and mineralization, increase the new bone area, and maintain the height and thickness of the augmented alveolar ridge when compared with β-TCP alone group. More importantly, the tissue-engineered bone achieved an elevated bone height and thickness comparable to that of autogenous iliac bone graft. This study demonstrated the potential of porous β-TCP as a substrate for autogenous osteoblasts in bone tissue engineering for alveolar ridge augmentation.     

β-TCP/MCPM-based premixed calcium phosphate cements

Novel premixed calcium phosphate cements (CPCs) were prepared by combining cement liquids comprised of glycerol or polyethylene glycol with CPC powders that consisted of β-tricalcium phosphate (β-TCP) and monocalcium phosphate monohydrate (MCPM). Changing the powder to liquid mass ratio enabled the setting time to be regulated, and improved the compressive strength of the CPCs. Although some ratios of the new premixed CPCs had long setting times, these ranged from 12.4 to 27.8 min which is much shorter than the hour or more reported previously for a premixed CPC. The premixed CPCs had tolerable washout resistance before final setting, and the cements had strengths matching that of cancellous bone (5–10 MPa); their maximum compressive strength was up to 12 MPa. The inflammatory response around the premixed CPCs implanted in the subcutaneous tissue in rabbits was more prominent than that of apatite cement. These differences might be due to the much faster resorption rate of the premixed CPCs.     

Synthesis, mechanical and biological characterization of ionic doped carbonated hydroxyapatite/β-tricalcium phosphate mixtures

The influence of ionic substituents in calcium phosphates intended for bone and tooth replacement biomedical applications is an important research topic, owing to the essential roles played by trace elements in biological processes. The present study investigates the mechanical and biological evaluation of ionic doped hydroxyapatite/β-tricalcium phosphate mixtures which have been prepared by a simple aqueous precipitation method. Heat treating the resultant calcium phosphates in a carbonated atmosphere led to the formation of ionic doped carbonated hydroxyapatite/β-tricalcium phosphate mixtures containing the essential ions of biological apatite. The structural analysis determined by Rietveld refinement confirmed the presence of hydroxyapatite as the main phase, together with a considerable amount of β-tricalcium phosphate. Such phase assemblage is essentially due to the influence of substituted ions during synthesis. The results from mechanical tests proved that carbonate substitutions are detrimental for the mechanical properties of apatite-based ceramics. In vitro proliferation assays of osteoblastic-like cells (MC3T3-E1 cell line) to powders revealed that carbonate incorporation can either delay or accelerate MC3T3 proliferation, although reaching the same proliferation levels as control cells after 2 weeks in culture. Further, the powders enable pre-osteoblastic differentiation in a similar manner to control cells, as indirectly measured by ALP activity and Type-I collagen medium secretion.     

Degradable properties of lam inated composite scaffolds of β-tricalcium phosphate/poly[L-lactic acid]

The crux of tissue engineering is to construct highly porous three-dimensionalscaffold with desired cells on it.Because the scaffold serves as both a physical sup-port and an adhesive substrate for isolated cells during in vitro culture and subse-quent implantation,the materials for scaffold should be non-toxic and biocompati-ble,highly porous and processable into devices of variousshapes.Atpresent,PLA,Ca/P bioglasses and ceramicshave been extensively used in tissue engineering.PLAhas a rel…     

Proliferation and differentiation of periosteum cells induced by icariin北大核心

BACKGROUND: Icariin has a broad prospect for promoting cell proliferation. Differentiation direction of periosteal cells is uncertain, but the cells are easy to be induced by ultrasound, oxygen or bone morphogenetic protein 7 (BMP7). Periosteal cells have been applied in bone tissue engineering; however, icariin effects on the proliferation and differentiation of periosteal cells is little reported. OBJECTIVE: To investigate the effect of icariin on the proliferation and differentiation of human periosteal cells, thus providing theoretical basis for icariin applied in bone tissue engineering. METHODS: The human periosteum was obtained and the primary cells were isolated in vitro. After culture and expansion, periosteal cells were cultured in 24-well plates, and induced by 0.001, 0.01 and 0.1 mg/L icariin and 50 µg/L BMP7, respectively. The corresponding avsorbance values of different groups were detected. The levels of alkaline phosphatase and calcium nodules in periosteal cells were measured at 1, 3, 5 and 7 days, and the mRNA levels of osteocalcin, osteopontin and Runx2 were detected at 3, 5 and 7 days. RESULTS AND CONCLUSION: The periosteal cells proliferated well after induction with icariin, and could proliferate well in different concentrations of icariin and the positive control group (P < 0.05). Compared with the control group, the periosteal cells induced by icariin were able to produce more alkaline phosphatase and calcium nodules (P < 0.05). The mRNA expression of osteocalcin, osteopontin and Runx2 in periosteal cells could be up-regulated by icariin (P < 0.05). These findings imply that icariin can promote proliferation and differentiate of periosteal cells into osteoblasts, and it can be used as an inducer for the preparation of seed cells in bone tissue engineering. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Synthesis and structural characterization of strontium- and magnesium-co-substituted β-tricalcium phosphate

The synthesis of five different Sr²⁺- and Mg²⁺-co-substituted β-tricalcium phosphate (β-TCP) has been obtained by heating the calcium-deficient apatites above 800 °C. With the investigated concentrations of Sr²⁺ and Mg²⁺ from the present study, no additional phases other than β-TCP have been detected. The synthesized powders have been characterized by X-ray diffraction, Fourier transform infrared spectrometry, elemental analysis and Rietveld refinement studies. The co-substitution of Sr²⁺ and Mg²⁺ in the β-TCP has resulted in the formation of crystalline β-TCP at hexagonal setting (space group R3c). The reduction of lattice a- and c-axis parameters with the combined substitution of Sr²⁺ and Mg²⁺ in the β-TCP has been found evident from the present results. Sr²⁺ has been found occupying the Ca(1,2,3,4) sites and Mg²⁺ was found at the sixfold coordinated Ca(5) site of β-TCP structure.     

Structural and spectroscopic characterization of a series of potassium- and/or sodium-substituted β-tricalcium phosphate

In this paper, we report X-ray diffraction investigations as well as Raman and solid-state (31)P and (23)Na magic angle spinning nuclear magnetic resonance (NMR) characterization of three series of calcium orthophosphates. The general formulae of the studied compounds are Ca(10.5-x/2)M(x)(PO(4))(7), where M=K or Na and x=0, 0.25, 0.50, 0.75, 1.0; and Ca(10)K(x)Na(1-x)(PO(4))(7), where x=0, 0.25, 0.5, 0.75, 1.0. These calcium orthophosphates are found to be isostructural with β-tricalcium phosphate (β-TCP, Ca(3)(PO(4))(2)) with the substitution of some calcium sites by potassium and/or sodium cations. The unit cell parameters vary continuously with the level of substitution, a characteristic of these solid solutions. The Raman spectra show the different vibrational bands of the phosphate groups PO(4), while the NMR chemical shifts are sensitive to the non-equivalent phosphorus and sodium ions present in these substituted samples. As both Raman and NMR spectroscopies are local probes, they offer tools to distinguish between these different phosphorus and phosphate groups, according to their structural site and local environment, especially the type of cation substituent. A convenient decomposition of the Raman and NMR spectra into Gaussian-Lorentzian components leads us to propose an assignment of the main observed bands of these substituted β-TCPs.     

Fabrication and characterization of novel nano hydroxyapatite/β-tricalcium phosphate scaffolds in three different composition ratios

The biphasic calcium phosphate (BCP) concept was introduced to overcome disadvantages of single phase biomaterials. Different composition ratios of BCP bioceramics have been studied, yet controversies regarding the effects of ratio on biomaterial behavior still exist. In this study, BCP scaffolds were prepared from nano hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) that were synthesized via a solid state reaction. Three different composition ratios of pure BCP and collagen-based BCP scaffolds (%HA/%β-TCP; 30/70, 40/60, and 50/50) were produced using a polymeric sponge method. Physical and mechanical properties of all materials and scaffolds were investigated. SEM showed overall distribution of both macropores (80-200 μm) and micropores (0.5-2 μm) with high interconnected porosities. Total porosity of pure BCP (90% ± 3%) was found to be higher than collagen-based BCP (85% ± 2%). It was observed that following sintering process, dimensional shrinkage of large scaffolds (39% ± 4%) was lower than small ones (42% ± 5%) and scaffolds with high HA ratios (50%) experienced higher dimensional changes than those with higher β-TCP (70%) ratios (45% ± 3% and 36% ± 1%, respectively). Compressive strength of both groups was less than 0.1 MPa and collagen coating had almost no influence on mechanical behavior. Further studies may improve the physical properties of these scaffolds and investigate their exact biological behaviors.     

Effect of strontium and gelatin on the reactivity of α-tricalcium phosphate

The hydrolysis reaction of α-tricalcium phosphate (α-TCP) is of great interest because of its widespread use in the preparation of biomaterials for hard tissue repair. The aim of this study was to investigate how this reaction is influenced by the presence of a bioactive ion, Sr²⁺, and of a biopolymer, gelatin, which were previously reported to affect the setting reaction of α-TCP-based cements. Hydrolysis experiments were carried out at different Sr²⁺ concentrations (0, 5, 10, 20 at.%) in solutions at different gelatin concentrations (0, 0.1, 0.5, 1.0 wt.%). The results indicate that Sr²⁺ delays the conversion of α-TCP into calcium-deficient hydroxyapatite (CDHA). The structural and morphological modifications of CDHA obtained from solutions at increasing Sr²⁺ concentrations indicate that during hydrolysis strontium enters the structure of CDHA, where it partially substitutes for calcium. On the contrary, α-TCP hydrolysis rate increases on increasing gelatin concentration. Gelatin promotes conversion of α-TCP into octacalcium phosphate, and strongly interacts with the nucleating and growing crystals.