Piedad N. De Aza

Comparison of three hydroxyapatite/β‐tricalcium phosphate/collagen ceramic scaffolds: An in vivo study

Calcium-phosphate ceramics, which have a composition similar to bone mineral, represent a potentially interesting synthetic bone graft substitute. In the present study, three porous hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP)/collagen ceramic scaffolds were developed, characterized, and tested for their bone repairing capacity and osteoinductive potential in a New Zealand Rabbit model. The ratio of the ceramic components HA/-TCP/collagen varied from 40/30/30 to 50/20/30 and 60/20/20 (in wt %), respectively. None of the ceramic scaffolds succeeded in completely bridging the 6 mm calvarian defect with new bone after 60 days implantation. 60/20/20 ceramic scaffolds showed significantly more bone formation in the pores and in the periphery of the graft than the other two materials. Histomorphometric analysis revealed that the 40/30/30 scaffold produced best bone-to-implant contact (67.23 ± 0.34% with higher quality, closer contact) in comparison with 50/20/30 (54.87 ± 0.32%), and 60/20/20 (48.53 ± 0.31%). Both physicochemical and structural properties of the ceramic composites affected their in vivo behavior, either dependently or independently, emphasizing the importance of assessing bone repair parameters individually. The scaffolds may offer clinical applications in reconstructive surgery for treating bone pathologies.

Physical properties, mechanical behavior, and electron microscopy study of a new α-TCP block graft with silicon in an animal model.

This study reports the characterization process and in vivo application of a new block bone graft of α-TCP with silicate in three different percentages in the aim of determining the influence of the silicate. Three groups of cylindrical implants (6 ± 0.01 mm diameter, 8 ± 0.01 mm length) with varying Si composition were studied: A: 3 wt % C(2) S; B: 1.5 wt % C(2) S; C: 100 wt % TCP-0 wt % C(2) S. These were implanted randomly in critical size defects in New Zealand rabbits. X-ray diffraction analysis was performed to determine the crystalline phases of the different compositions. Histomorphometric analysis produced one measurement of bone-to-implant contact. Comparing the α-TCPss ceramics, the trial found improved mechanical properties due to the silicon content in solid solution as well as densification. Previous studies have shown that the mechanical strengths of sintered ceramics correlate to densification as well as grain size and mechanical properties. Because of its mechanical and biological behavior, the study has shown α-TCP with C(2) S to be an alternative to other bone graft substitutes for use in bone reconstructive surgery in the fields of veterinary, medicine, and oral and maxillofacial surgery.

Phenotypic differences during the osteogenic differentiation of single cell-derived clones isolated from human lipoaspirates

Osteogenic precursors can be obtained from mesenchymal stem cells, which can be isolated from different sources, including adipose tissue. Optimal osteogenic differentiation in in vitro conditions and the selection of the potential precursors that could be further used in bone regeneration still have two main questions left to solve, viz. the heterogeneity of the mesenchymal cell population and the presence of a basal transcription level of several characteristic genes of the osteogenic lineage, which makes rapid and effective comparisons during cell differentiation difficult. Single‐cell clones were isolated and expanded from human lipoaspirate cells. Osteogenic differentiation was induced and studied in defined media, using four representative isolated cell clones showing differences in the basal expression of a set of characteristic osteogenic genes. The clones showing a low constitutive expression of these genes were able to display comparatively higher levels of mineralization. In addition, the cells from these clones displayed a characteristic pattern of bundle fibres of collagen during osteogenic induction and showed a higher potency to differentiate towards the adipogenic lineage. These results demonstrate that specific multipotent precursors can be isolated from human lipoaspirate cells with a higher differentiation potential, allowing the maturation of specific lineages in a shorter time. These results additionally demonstrate that, since the basal expressions of the several genes were used as osteogenic markers, a phenotypic biochemical analysis should always be utilized to study optimal osteogenesis conditions. Copyright

αTCP ceramic doped with dicalcium silicate for bone regeneration applications prepared by powder metallurgy method: In vitro and in vivo studies

This study reports on the in vitro and in vivo behavior of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 or 3.0 wt % of dicalcium silicate (C2 S). The ceramics were successfully prepared by powder metallurgy method combined with homogenization and heat treatment procedures. All materials were composed of a single-phase, αTCP in the case of a pure material, or solid solution of C2 S in αTCP for the doped αTCP, which were stable at room temperature. The ceramics were tested for bioactivity in simulated body fluid, cell culture medium containing adult mesenchymal stem cells of human origin, and in animals. Analytical scanning electron microscopy combined with chemical elemental analysis was used and Fourier transform infrared and conventional histology methods. The in vivo behavior of the ceramics matched the in vitro results, independently of the C2 S content in αTCP. Carbonated hydroxyapatite (CHA) layer was formed on the surface and within the inner parts of the specimens in all cases. A fully mineralized new bone growing in direct contact with the implants was found under the in vivo conditions. The bioactivity and biocompatibility of the implants increased with the C2 S content in αTCP. The C2 S doped ceramics also favoured a phase transformation of αTCP into CHA, important for full implant integration during the natural bone healing processes. αTCP ceramic doped with 3.0 wt % C2 S showed the best bioactive in vitro and in vivo properties of all the compositions and hence could be of interest in specific applications for bone restorative purposes.

The effects of Ca2SiO4-Ca3(PO4)2 ceramics on adult human mesenchymal stem cell viability, adhesion, proliferation, differentiation and function.

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 wt.% or 3.0 wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73(+), CD90(+), CD105(+), CD34(-) and CD45(-) onto the 3 substrates for 30 days. Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5 wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.

Influence of thermal treatment on the "in vitro" bioactivity of wollastonite materials.

The aim of this work was to study the influence of the composition and thermal treatment of the in vitro bioactivity of wollastonite materials obtained by sol–gel method. For this purpose, gels in the system SiO2–CaO were obtained applying calcium nitrate and tetraethoxysilicate as precursors. The gels were heated to 700°C and then sintered up to 1400°C. The bioactivity of the gel-derived materials in simulated body fluid (SBF) was investigated and characterized. Additional changes in ionic concentration, using inductively couple plasma atomic emission spectroscopy (ICP-AES), were determined. The results showed that all materials obtained were bioactive and indicate that the absence of phosphorous in the material composition is not an essential requirement for the development of a Hydroxyapatite layer. The bioactivity was influenced by the thermal treatment, the different phases (glass-phase, wollastonite and pseudowollastonite) as well as the porous size. On the gel-derived materials the bioactivity decreased with the sintering temperature.

Morphological and Structural Study of a Novel Porous Nurse’s A Ceramic with Osteoconductive Properties for Tissue Engineering

The characterization process of a new porous Nurse’s A ceramic and the physico chemical nature of the remodeled interface between the implant and the surrounding bone were studied after in vivo implantation. Scaffolds were prepared by a solid-state reaction and implanted in New Zealand rabbits. Animals were sacrificed on days 15, 30, and 60. The porous biomaterial displayed biocompatible, bioresorbable, and osteoconductive capacity. The degradation processes of implants also encouraged osseous tissue ingrowths into the material’s pores, and drastically changed the macro- and microstructure of the implants. After 60 healing days, the resorption rates were 52.62% ± 1.12% for the ceramic and 47.38% ± 1.24% for the residual biomaterial. The elemental analysis showed a gradual diffusion of the Ca and Si ions from the materials into the newly forming bone during the biomaterial’s resorption process. The energy dispersive spectroscopy (EDS) analysis of the residual ceramic revealed some particle categories with different mean Ca/P ratios according to size, and indicated various resorption process stages. Since osteoconductive capacity was indicated for this material and bone ingrowth was possible, it could be applied to progressively substitute an implant.

Biodegradation process of α-tricalcium phosphate and α-tricalcium phosphate solid solution bioceramics in vivo: a comparative study.

This article reports the structure and morphology of the in vivo interface between implants composed of either a tricalcium phosphate (αTCP) or αTCP doped with 3.0 wt% dicalcium silicate (αTCP(ss)) ceramic, and natural bone of rabbit tibias. Both interfaces developed a new bone layer in direct contact with the implants after 4 and 8 weeks of implantation. The specimens were examined using analytical scanning and transmission electron microscopy, up to the lattice plane resolution level. Degradation processes of the implants developed at the interfaces encouraged osseous tissue ingrowth into the periphery of the material, changing the microstructure of the implants. The ionic exchange initiated at the implant interface with the environment was essential in the integration process of the implant, through a dissolution–precipitation–transformation mechanism. The interfaces developed normal biological and chemical activities and remained reactive over the 8-week period. Organized collagen fibrils were found at the αTCP(ss)/bone interface after 4 weeks, whereas a collagen-free layer was present around the Si-free αTCP implants. These findings suggest that the incorporation of silicate ions into αTCP ceramic promotes processes of the bone remodeling at the bone/αTCP(ss) interface, hence the solubility rate of the aTCP(ss) material decreased.

Novel Resorbable and Osteoconductive Calcium Silicophosphate Scaffold Induced Bone Formation

This aim of this research was to develop a novel ceramic scaffold to evaluate the response of bone after ceramic implantation in New Zealand (NZ) rabbits. Ceramics were prepared by the polymer replication method and inserted into NZ rabbits. Macroporous scaffolds with interconnected round-shaped pores (0.5-1.5 mm = were prepared). The scaffold acted as a physical support where cells with osteoblastic capability were found to migrate, develop processes, and newly immature and mature bone tissue colonized on the surface (initially) and in the materials interior. The new ceramic induced about 62.18% ± 2.28% of new bone and almost complete degradation after six healing months. An elemental analysis showed that the gradual diffusion of Ca and Si ions from scaffolds into newly formed bone formed part of the biomaterials resorption process. Histological and radiological studies demonstrated that this porous ceramic scaffold showed biocompatibility and excellent osteointegration and osteoinductive capacity, with no interposition of fibrous tissue between the implanted material and the hematopoietic bone marrow interphase, nor any immune response after six months of implantation. No histological changes were observed in the various organs studied (para-aortic lymph nodes, liver, kidney and lung) as a result of degradation products being released.

Bioeutectic® Ceramics for Biomedical Application Obtained by Laser Floating Zone Method. In vivo Evaluation

In this study, the Bioeutectic® blocks were inserted into the critical size defects of eight rabbits, using both tibiae, and the physical and chemical nature of the remodeled interface between the Bioeutectic® implants and the surrounding bone were performed at four and 15 months. The results showed a new fully mineralized bone growing in direct contact with the implants. The ionic exchange, taking place at the implant interface with the body fluids was essential in the process of the implant integration through a dissolution-precipitation-transformation mechanism. The study found the interface biologically and chemically active over the 15 months implantation period. The osteoblastic cells migrated towards the interface and colonized the surface at the contact areas with the bone. The new developed apatite structure of porous morphology mimics natural bone.