Sandra Sánchez-Salcedo

In vitro structural changes in porous HA/β-TCP scaffolds in simulated body fluid

Porous scaffolds of biphasic calcium phosphate (hydroxyapatite/beta-tricalcium phosphate (beta-TCP)) have been fabricated and changes induced both in phase composition and porous architecture by immersion in simulated body fluid (SBF) under static and orbital stirring conditions have been studied. The starting porous scaffolds exhibit a low and randomized micro- and mesoporosity, an interconnected macroporosity centered at 100 and 0.6microm, a fractal connectivity of D=2.981 and total percent porosity of ca. 80%. After immersion for up to 60days the micro- and mesoporosity increase slightly, which could be attributed to dissolution of the beta-TCP phase confirmed by transmission electron microscopy. The effects of the change in the porous framework with SBF immersion time favor the bioactive behavior of the tested materials, inducing a nucleation and growth of a nanocrystalline apatite phase as the interconnected macroporosity centered at 0.6microm is reduced. The macroporosity centered at 100microm is still stable after 60days in SBF. Therefore, these biphasic calcium phosphate porous scaffolds combine bioactive behavior with the stability of interconnected macroporosity over large periods of soaking time in SBF under static and orbital stirring conditions.

Hierarchical pore structure of calcium phosphate scaffolds by a combination of gel-casting and multiple tape-casting methods.

The objective of this work was to design hierarchical pore structure scaffolds with potential applications in bone tissue regeneration. For that purpose, a bioceramic material such as biphasic calcium phosphate, which consists of a mixture of hydroxyapatite and beta-tricalcium phosphate, was selected. Multilayer pieces (MLP) with hierarchical pore structure were developed employing a new technique that combines gel casting and adding porogens, using multiple tape-casting methods. Pieces with functionally graded porosity were fabricated using porogens with different sizes. The porogens used were Porlat K85 and Porlat K86 with diameters <150 microm and 150-300 microm, respectively. Two types of sintered tapes, with different porosity, no cracking and enough interconnection size were selected. MLP with hierarchical pore structure were designed by the multiple tape-casting method. Interconnected pores whose sizes increase from interior tapes (1.6-3.6 microm) towards exterior tapes (20-51.5 microm) and interpenetration between tapes were achieved. Delamination or cracking were not observed after heat treatment. The flexural strength of pieces was investigated by the three-point bending test. This new combination of methods offers the possibility of manufacturing scaffolds with interconnected pore sizes ranging from 1.6 to 51.5 microm.

Biocompatibility markers for the study of interactions between osteoblasts and composite biomaterials

Biphasic calcium phosphate (BCP), a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has attracted attention as an excellent bone graft substitute. Mixtures of ceramics with agarose, as natural biodegradable binder, have been recently performed in order to increase the flexibility of the ceramic component and to facilitate the biomaterial preparation. In previous studies we have evaluated the response of both L929 fibroblasts and Saos-2 osteoblasts to hydroxyapatite-betaTCP/agarose disks observing a higher sensitivity of osteoblasts to this biomaterial. In the present study, the use of specific fluorescent probes and antibodies has allowed to evaluate different cell function parameters as biocompatibility markers for the cell/biomaterial interaction of Saos-2 osteoblasts cultured for 7 days on hydroxyapatite-betaTCP/agarose disks. The cell cycle subG(1) fraction, the exposition of phosphatidylserine on the outside surface of the plasma membrane and the analysis of plasma membrane integrity versus cell size, indicate that the interaction with the biomaterial induces a light increase of apoptosis in osteoblasts without producing cell necrosis. The high percentage of viable cells on the biomaterial and the preservation of endothelial nitric oxide synthase (eNOS) expression, eNOS activity and mitochondrial membrane potential (Deltapsi(m)), demonstrate the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.

Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells

Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and beta-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell-particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca2+ uptake, allowing the development of a functional osteoblast phenotype.

Influence of surface porosity and pH on bacterial adherence to hydroxyapatite and biphasic calcium phosphate bioceramics.

Hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramic materials are widely employed as bone substitutes due to their porous and osteoconductive structure. Their porosity and the lowering of surrounding pH as a result of surgical trauma may, however, predispose these materials to bacterial infections. For this reason, the influence of porosity and pH on the adherence of common Gram-positive bacteria to the surfaces of these materials requires investigation. Mercury intrusion porosimetry measurements revealed that the pore size distribution of both bioceramics had, on a logarithmic scale, a sinusoidal frequency distribution ranging from 50 to 300 nm, with a mean pore diameter of 200 nm. Moreover, total porosity was 20 % for HA and 50 % for BCP. Adherence of Staphylococcus aureus and Staphylococcus epidermidis was studied at a physiological pH of 7.4 and at a pH simulating bone infection of 6.8. Moreover, the effect of pH on the zeta potential of HA, BCP and of both staphylococci was evaluated. Results showed that when pH decreased from 7.4 to 6.8, the adherence of both staphylococci to HA and BCP surfaces decreased significantly, although at the same time the negative zeta-potential values of the ceramic surfaces and both bacteria diminished. At both pH values, the number of S. aureus adhered to the HA surface appeared to be lower than that for BCP. A decrease in pH to 6.8 reduced the adherence of both bacterial species (mean 57 %). This study provides evidence that HA and BCP ceramics do not have pores sufficiently large to allow the internalization of staphylococci. Their anti-adherent properties seemed to improve when pH value decreased, suggesting that HA and BCP bioceramics are not compromised upon orthopaedic use.

In vitro behaviour of adult mesenchymal stem cells seeded on a bioactive glass ceramic in the SiO2–CaO–P2O5 system

This work describes the evaluation of a glass ceramic (55S41C4P-1300) as a potential substrate for bone tissue engineering. For that purpose, the capacity of mesenchymal stem cells (MSCs), isolated from rabbit bone marrow, to adhere, proliferate and differentiate into osteoblast (OBs) with or without 55S41C4P-1300 was investigated. Two types of culture medium, i.e. growth medium (GM) and osteogenic medium (OM), were evaluated. The bioactive 55S41C4P-1300, containing pseudowollastonite, wollastonite, tricalcium phosphate and crystoballite as crystalline phases, was obtained by heat treatment of a sol-gel glass (55SiO(2), 41CaO, 4P(2)O(5) (mol.%)) at 1300 degrees C. The results showed that the MSCs adhered, spread, proliferated and produced mineralized extracellular matrix on 55S41C4P-1300 regardless of the culture medium used. As the same time, they showed an osteoblastic phenotype, and this phenomenon was accompanied by the gradual diminution of the marker CD90 expression. The 55S41C4P-1300 was able to induce the differentiation of MSCs into OBs in the same way as OM without glass ceramic. This effect increased with the combination of 55S41C4P-1300 with OM. The glass ceramic evaluated in this work is bioactive, cytocompatible and capable of promoting the differentiation of MSCs into OBs. For that reason, it could be regarded as a suitable matrix in tissue engineering for bone tissue regeneration.

Inhibition of bacterial adhesion on biocompatible zwitterionic SBA-15 mesoporous materials

In this manuscript in vitro bacterial adhesion assays using Escherichia coli on different SBA-15 nanostructured ceramics have been performed. For this purpose pure silica, NH(2) or COOH monofunctionalized, and NH(2)/COOH bifunctionalized SBA-15 mesoporous materials have been used. Material characterization reveals that both NH(2)/COOH and NH(2) functionalized SBA-15 materials exhibit a zwitterionic character due to the presence of -NH(3)(+)/COO(-) or -NH(3)(+)/SiO(-) moieties, respectively. In vitro adhesion assays have been carried out at the pH at which the zwitterionic nature of both of these samples is preserved, i.e. pH 5.5. The results show that the presence of both positive and negative moieties with an overall neutral charge leads to reduced E. coli adhesiveness. In vitro tests with cultured human Saos-2 osteoblasts have been carried out to evaluate the biocompatibility of the different materials at the physiological pH of 7.4. The results demonstrate that all materials exhibit good biocompatibility, with Saos-2 osteoblasts adhering, proliferating and maintaining their morphological and functional characteristics. This novel family of zwitterionic mesoporous materials opens up promising expectations in diverse biomedical applications, such as preventing some side-effects associated with bone implant infections.

Upgrading Calcium Phosphate Scaffolds for Tissue Engineering Applications

The research on ceramic scaffolds for bone tissue engineering is, nowadays, one of the newest and most attractive topics in the field of materials for biomedical applications. These scaffolds are aimed to provide supporting or even enhance the reparative capacity of body. Biphasic calcium phosphates (BCPs) and silicon doped BCP are very interesting candidates to be used as materials for scaffolds fabrication in bone tissue engineering. BCPs and silicon doped BCP consist of a mixture of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) or HA and α-tricalcium phosphate (α-TCP), respectively. For the regenerative purposes BCPs show better performance than HA because of the higher solubility of β-TCP compound, which facilitate the subsequent bone ingrowth in the implant. On the other, silicon doped BCP involve silicon that substituted into the apaptite crystal lattice for phosphorous with the subsequent charge imbalance. HA/α-TCP based bioceramics exhibits an important improvement of the bioactive behaviour with respect to non-substituted apatites. This work reviews the procedures to synthesise and fabricate scaffolds based on HA/β-TCP and silicon stabilised HA/α-TCP. Special attraction has been paid in the different synthesis methods and to the shaping of final scaffolds. By knowing the scaffold features at the crystallinity and macrostuctural level, the biocompatibility and clinical performance can be better understood, which will be also considered in this review.

In vitro antibacterial capacity and cytocompatibility of SiO2–CaO–P2O5 meso-macroporous glass scaffolds enriched with ZnO

Zn2+ ions exhibit osteogenic, angiogenic and antimicrobial properties. For this reason, they are often added in small amounts to bioceramics being investigated for bone tissue engineering. In this paper, the cytocompatibility and antibacterial properties of 80% SiO2-15% CaO-5% P2O5 (mol%) mesoporous bioactive glass (MBG) scaffolds substituted with 4.0% and 7.0% of ZnO were studied and compared with the Zn-free scaffold. Cell proliferation, morphology, differentiation and cytotoxic effects of Zn2+ ions released from the samples were examined by culturing human osteoblast-like cells (HOS) osteoblasts both in the presence of sample extracts and on the scaffold surface. The bacterial inhibition capacity of the scaffolds was explored by using Gram-positive Stapylococcus aureus bacteria, responsible for numerous infections in orthopedic surgery, to simulate a severe infection. Our results show that the Zn-MBG scaffolds possess a hierarchical meso-macropore structure suitable for osteoblast growth. Furthermore, the amount of Zn2+ released from the scaffold with 4.0% ZnO was found to be more favorable for HOS cell development than that released from the scaffold including 7.0% ZnO. Zn2+ released to the medium from both scaffolds exhibited antibacterial properties against S. aureus. Thus, the cytocompatibility and the antibacterial ability exhibited by the MBG scaffold containing 4.0% ZnO make it a suitable candidate for bone regeneration applications.

L929 fibroblast and Saos‐2 osteoblast response to hydroxyapatite‐βTCP/agarose biomaterial

Biphasic calcium phosphate, a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has been successfully used as an excellent bone graft substitute because of the HA capacity for direct interaction with bone and the beta-TCP resorption properties. Agarose has been recently mixtured with ceramics as natural biodegradable binder to increase the biomaterial flexibility facilitating its placement into the bone defect. In this study, the behavior of L929 fibroblasts and Saos-2 osteoblasts cultured on hydroxyapatite-betaTCP/agarose disks has been evaluated. Both cell types adhere and proliferate on the biomaterial surface maintaining their characteristic morphology. Transitory changes on cell cycle, size, and complexity are observed. The biomaterial induces apoptosis in Saos-2 osteoblasts but not in fibroblasts. A transitory stimulation of fibroblast mitochondrial activity is observed. This effect remains in osteoblasts after 9 days of culture showing a higher sensitivity of this cell type. However, the intracellular reactive oxygen species content and the lactate dehydrogenase release of Saos-2 osteoblasts indicate that hydroxyapatite-betaTCP/agarose does not induce oxidative stress in this cell type and confirm the integrity of the osteoblast plasma membrane. These results underline the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.