Marco A. Lopez-Heredia

Influence of the pore generator on the evolution of the mechanical properties and the porosity and interconnectivity of a calcium phosphate cement.

Porosity and interconnectivity are important properties of calcium phosphate cements (CPCs) and bone-replacement materials. Porosity of CPCs can be achieved by adding polymeric biodegradable pore-generating particles (porogens), which can add porosity to the CPC and can also be used as a drug-delivery system. Porosity affects the mechanical properties of CPCs, and hence is of relevance for clinical application of these cements. The current study focused on the effect of combinations of polymeric mesoporous porogens on the properties of a CPC, such as specific surface area, porosity and interconnectivity and the development of mechanical properties. CPC powder was mixed with different amounts of PLGA porogens of various molecular weights and porogen sizes. The major factors affecting the properties of the CPC were related to the amount of porogen loaded and the porogen size; the molecular weight did not show a significant effect per se. A minimal porogen size of 40 μm in 30 wt.% seems to produce a CPC with mechanical properties, porosity and interconnectivity suitable for clinical applications. The properties studied here, and induced by the porogen and CPC, can be used as a guide to evoke a specific host-response to maintain CPC integrity and to generate an explicit bone ingrowth.

Bulk properties and bioactivity assessment of porous polymethylmethacrylate cement loaded with calcium phosphates under simulated physiological conditions

Polymethylmethacrylate (PMMA) cements are widely used in spinal surgery. Nevertheless, these types of cements present some documented drawbacks. Therefore, efforts have been made to improve the properties and biological performance of solid PMMA. A porous structure would seem to be advantageous for anchoring purposes. This work studied the bulk physicochemical, mechanical and interconnectivity properties of porous PMMA cements loaded with various amounts of calcium phosphate (CaP). As a measure of bioactivity, changes of PMMA cements under simulated physiological conditions were studied in a calcium phosphate solution for 0, 3, 7, 14, 21 and 28 days. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), micro-computed tomography (μ-CT) and mechanical compression tests were performed to characterize the morphology, crystallographic and chemical composition, interconnectivity and mechanical properties, respectively. SEM allowed observing the result of loading CaP into the porous PMMA, which was corroborated by XRD, FTIR and μ-CT. No interference of the CaP with the PMMA was detected. μ-CT described similar interconnectivity and pore distribution for all CaP percentages. Mechanical properties were not significantly altered by the CaP percentages or the immersion time. Hence, porous PMMA was effectively loaded with CaP, which provided the material with properties for potential osteoconductivity.

Processing and in vivo evaluation of multiphasic calcium phosphate cements with dual tricalcium phosphate phases.

Calcium phosphate cements (CPCs) use the simultaneous presence of several calcium phosphates phases. This is done to generate specific bulk and in vivo properties. This work has processed and evaluated novel multiphasic CPCs containing dual tricalcium phosphate (TCPs) phases. Dual TCPs containing α- and β-TCP phases were obtained by thermal treatment. Standard CPC (S-CPC) was composed of α-TCP, anhydrous dicalcium phosphate and precipitated hydroxyapatite, while modified CPC (DT-CPC) included both α- and β-TCP. Physicochemical characterization of these CPCs was based on scanning electron microscopy, X-ray diffraction, specific surface area (SSA) and particle size (PS) analysis and mechanical properties. This characterization allowed the selection of one DT-CPC for setting time, cohesion and biological assessment compared with S-CPC. Biological assessment was carried out using a tibial intramedullary cavity model and subcutaneous pouches in guinea pigs. Differences in the surface morphology and crystalline phases of the treated TCPs were detected, although PS analysis of the milled CPC powders produced similar results. SSA analysis was significantly higher for DT-CPC with α-TCP treated at 1100°C for 5h. Poorer mechanical properties were found for DT-CPC with α-TCP treated at 1000°C. Setting time and cohesion, as well as the in vivo performance, were similar in the selected DT-CPC and the S-CPC. Both CPCs created the desired host reactions in vivo.

The effect of ball milling grinding pathways on the bulk and reactivity properties of calcium phosphate cements

Calcium phosphate cements (CPCs) are significant alternatives to autologous bone grafting. CPCs can be composed of biphasic or multiphase calcium phosphate (CaP) compounds. A common way to process CPCs is by ball milling. Ball milling can be used for grinding or mechanosynthesis. The aim of this study was to determine the effect of well-defined ball milling grinding parameters, applied via different milling pathways, on the properties of CPCs. Starting CaP compounds used included α-tricalcium phosphate, dicalcium phosphate anhydrous and precipitated hydroxyapatite. Scanning electron microscopy showed changes in the powder morphology, which were related to the behavior of the starting CaP materials. Specific surface area (SSA) and particle size (PS) measurements exposed the effect of ball milling on the CaP compounds and CPC powders. X-ray diffraction revealed no effect of ball milling pathways or milling time on the composition of CPCs or the starting materials, but affected their crystallographic properties. No contamination of the milling media or transformation into an amorphous calcium phosphate compound was found. The milling pathways affected setting and cohesion. Fourier transform infrared spectroscopy (FTIR) revealed differences on the CPC v₄-PO₄³⁻ bands according to the interaction, created between the CaP compounds by the milling pathways. FTIR confirmed that the milling pathways changed the crystallographic properties. This study demonstrates that the pathways used for milling grinding modify the PS, SSA, and crystallographic properties of the powders, without affecting their composition. These modifications affected the bulk and reactivity properties of the CPCs by creating different setting and cohesion behaviors.

Bulk physicochemical, interconnectivity, and mechanical properties of calcium phosphate cements-fibrin glue composites for bone substitute applications

Calcium phosphate cements (CPCs) and fibrin glue (FG) are used for surgical applications. Their combination is promising to create bone substitutes able to promote cell attachment and bone remodeling. This study proposes a novel approach to create CPC-FG composites by simultaneous CPC setting and FG fibrinogenesis. CPC-FG composites were obtained by mixing CPC powders, i.e. α-tricalcium phosphate, dicalcium phosphate anhydrous and precipitated hydroxyapatite, with FG powder components, i.e. fibrinogen and thrombin, and a 2% Na(2) HPO(4) solution. To study the effect of FG quantity and fibrinogenesis kinetics, long and fast setting FGs were evaluated in amounts of 0.125, 0.250, and 0.500 mL on CPC-FG composites. Physicochemical, interconnectivity, and mechanical properties were measured. Scanning electron microscopy, Micro-computed tomography (μ-CT), X-ray diffraction, and Fourier transform Infrared spectroscopy (FTIR) analyzed morphology, structure, crystallographic, and chemical composition, respectively. FG fibrinogenesis was performed within the CPC. FTIR confirmed this and its interfacial bonding with CPC. μ-CT confirmed a good FG distribution. FG addition affected the CPC when compared with pristine CPC. Adding FG to CPC changed their morphology, density, porosity, setting, cohesion, injectability, interconnectivity, crystallographic and chemical composition and mechanical properties. Moreover, 0.500 mL of long setting FG modified the observed fracture behavior of the CPC-FG.

Composites of gellan gum hydrogel enzymatically mineralized with calcium-zinc phosphate for bone regeneration with antibacterial activity.

Gellan gum hydrogels functionalized with alkaline phosphatase were enzymatically mineralized with phosphates in mineralization medium containing calcium (Ca) and zinc (Zn) to improve their suitability as biomaterials for bone regeneration. The aims of the study were to endow mineralized hydrogels with antibacterial activity by incorporation of Zn in the inorganic phase, and to investigate the effect of Zn incorporation on the amount and type of mineral formed, the compressive modulus of the mineralized hydrogels and on their ability to support adhesion and growth of MC3T3‐E1 osteoblast‐like cells. Mineralization medium contained glycerophosphate (0.05 m) and three different molar Ca:Zn ratios, 0.05:0, 0.04:0.01 and 0.025:0.025 (all mol/dm3), hereafter referred to as A, B and C, respectively. FTIR, SAED and TEM analysis revealed that incubation for 14 days caused the formation of predominantly amorphous mineral phases in sample groups A, B and C. The presence of Zn in sample groups B and C was associated with a drop in the amount of mineral formed and a smaller mineral deposit morphology, as observed by SEM. ICP–OES revealed that Zn was preferentially incorporated into mineral compared to Ca. Mechanical testing revealed a decrease in compressive modulus in sample group C. Sample groups B and C, but not A, showed antibacterial activity against biofilm‐forming, methicillin‐resistant Staphylococcus aureus. All sample groups supported cell growth. Zn incorporation increased the viable cell number. The highest values were seen on sample group C. In conclusion, the sample group containing the most Zn, i.e. group C, appears to be the most promising. Copyright

In Vivo Osteogenesis Assessment of a Tricalcium Phosphate Paste and a Tricalcium Phosphate Foam Bone Grafting Materials

Calcium phosphates (CaPs) are synthetic bone grafting materials. CaPs are an alternative to overcome the drawbacks present with autologous bone grafting and/or xenograft materials. Among the CaPs, tricalcium phosphate (TCP) stands out as a good candidate due to its physicochemical properties. The clinical performance of β-TCP has already been proven and established. Nevertheless, the format in which TCP is delivered is also important in terms of clinical handling. This work assessed the in vivo performance of TCP-based bone grafting materials with different formats. Materials studied were a TCP paste (TCP-P), a TCP foam (TCP-F) and TCP granules (TCP-G). A sheep scapula model was used to evaluate the osteogenic performance of these bone grafting materials. All materials performed well in terms of bone regenerative capacity and material resorption. However, TCP-P and TCP-F displayed a more pronounced initial material resorption and also exhibited better handling properties compared to TCP-G. TCP-based materials with improved handling properties, such as TCP-P and TCP-F, which at the same time possess the advantageous properties of β-TCP are suitable bone substitute materials for grafting and reconstruction of bone defects in numerous clinical applications.

Effect of Stressed and Unstressed Cell Culture Environments on the Viability of MC3T3 Cells with Calcium Phosphates

The effect of Fetal Bovine Serum (FBS) deprivation on survival and apoptosis of osteoblasts cultured on various calcium phosphates was studied. Test materials were two calcium alkali orthophosphates (materials denominated: GB9 and GB14), which were compared to β-tricalcium phosphate (TCP). Tissue culture polystyrene (PS) served as control. Test materials were characterized by X-Ray Diffraction and scanning electron microscopy. An apoptotic challenge assay entailing serum withdrawal was applied: MC3T3-E1 osteoblasts were cultured for 72h on the test materials in serum containing medium, followed by incubation in serum free medium for another 24h. Serum withdrawal is an apoptotic challenge, which creates a stressed environment. Cells cultured on the test specimens in serum containing medium served as control. The TUNEL Assay was employed to quantify the percentage of apoptotic cells. GB9 and GB14 displayed a significantly lower percentage of apoptotic cells than TCP. TCP had significantly fewer apoptotic cells than PS. The percentage of apoptotic cells on GB9 and GB14 was less than 10%, while the number of apoptotic cells found on the untreated control specimens ranged between 5 and 7%. These findings indicate that GB9 and GB14 endow osteoblasts cultured on them with a decreased sensitivity to apoptosis, which corresponds well with the results of previous in vitro and in vivo studies.