New macroporous calcium phosphate glass ceramic for guided bone regeneration
Introduction
Bone replacements are frequently required to substitute damaged tissue due to any trauma, disease or surgery. Some of the therapies employed in order to solve these problems are the use of autografts, allografts or xenografts.
Even though the use of these grafts have had satisfactory results under certain conditions, they present some limitations like the material availability, donor site morbidity, anatomic problems and the risk of inducing transmissible diseases among others.
Due to the numerous restrictions biologic grafts present, the development of synthetic materials and their employment in tissue engineering is an option to trabecular bone grafting. Such synthetic substitutes should be especially created to stimulate bone regeneration and support the newly formed tissue. There are two different general strategies in Tissue Engineering: the first one consists in the elaboration of 3-D scaffolds where the cells are seeded in vitro in order to promote the secretion of extra-cellular matrix and the formation of tissue in vitro, before implantation. The second approach consists in the development of porous interconnected materials with the capacity to host the osseous cells and guide the bone regeneration in vivo. In this framework, several materials, most of them polymers and some ceramics [1], [2], [3], [4], [5], [6], [7] have been proposed for the development of these scaffolds. Biological glasses based in calcium phosphate represent an interesting option as a biodegradable material for the elaboration of tissue engineering constructs.
One of the features a 3-D scaffold must fulfill is a controlled resorbability. The scaffold degradation should be concomitant with the tissue regeneration. In general, crystalline hydroxyapatite results to have a reabsorbability rate too low for this aim given that it is nearly insoluble at normal physiological pH [8]. Phosphate glasses and particularly glasses in the system P2O5–CaO–Na2O–TiO2 [9], have demonstrated to present a controlled solubility [10]. Moreover, their chemical composition is close to that of the bone mineral phase which confers them an advantage over polymeric scaffolds since this fact can affect positively the material–cell interaction. In addition, previous studies have revealed the biocompatibility of these glasses. In vitro cultures have shown that these formulations elicit no cytotoxicity [11].
Porosity is another relevant feature these scaffolds must fulfill. The ideal structures must be formed by an interconnected porous network with a wide variety of pores sizes, large pores that allow tissue ingrowth and vascularization of the new formed tissue, and pores in the microporous range to promote protein adhesion and consequently cell adhesion and proliferation.
Different methods for the elaboration of these 3-D vitreous or ceramic constructs have been proposed such as the introduction of diverse kinds of porosifiers, foaming agents and emulsifiers, the solid free form fabrication technique and the impregnation of a skeleton of porous polyurethane foam with ceramic emulsions, among others [2], [4], [12], [13], [14], [15], [16], [17].
This work describes a method to obtain porous glass and glass ceramic constructs in the system P2O5–CaO–Na2O–TiO2. The method consists in the foaming of a slurry of glass particles by addition of H2O2, and the subsequent sintering of the porous structures obtained. This method has been applied both to ceramics [18] and to bioactive glasses [17]. However, none of these materials are resorbable. The application of it to phosphate glasses could allow for the fabrication of macroporous resorbable constructs, with controlled biodegradability.
Different parameters are studied, which affect both the architecture/microstructure of the porous constructs and the relative amounts of amorphous/crystalline phases present, and which therefore will condition the resorbability of the scaffold. Besides, cell cultures are carried out to evaluate the cellular response of one of the constructs developed. Given that the phosphate glass used in this study is a soluble glass, the cytotoxicity test was performed not only in direct contact with the material but also with material extracts according to ISO-10993-5.
Section snippets
Preparation of the scaffolds
A 23 factorial design was carried out to evaluate the influence of three different factors: percentage of H2O2 solution into the slurry, the sintering temperature and the sintering time. The experiment design is summarized in Table 1.
For the elaboration of the porous scaffolds, calcium phosphate glass particles with the following molar composition, 44.5%P2O5–44.5%CaO–6%Na2O–5%TiO2 were used. For the preparation of the glasses CaCO3, NH4H2PO4, Na2CO3 and TiO2 were used as the basic reagents. The
Material characterization
Fig. 1 shows the fracture surface of specimens with the two different percentages of H2O2 sintered at 540°C for 3 h. The samples with 40% w/w of H2O2 show an structure mainly formed by elongated pores as a result of the interconnection of smaller pores. In the case of the structure of the 60% H2O2 scaffolds (Figs. 1c and d), pores with rounded shape and larger size were the predominant. Besides, a higher degree of interconnectivity than in the materials with 40% w/w of H2O2 could be observed.
Discussion
In this work, new-glass ceramic porous scaffolds based in calcium phosphate glasses have been developed. The incorporation of H2O2 as the foaming agent was chosen for the elaboration of these tissue engineering constructs. Basically, in this technique, the porosity is produced as a consequence of the decomposition process undergone by the H2O2 when it is heated at 60°C. The released oxygen tends to form bubbles in the interior of the glass+H2O2 slurry, originating in this way the porous
Conclusions
Foaming with H2O2 is an interesting method to obtain porous glasses and vitro-ceramics. By varying the amount of H2O2 incorporated and the thermal treatment, the percentages of porosity, pore size and percentages of crystallinity can be modulated.
Besides, the material was shown to be non-cytotoxic; thus, further studies should be performed in order to gain a better understanding of the material–cell interaction in 3-D conditions.
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