New macroporous calcium phosphate glass ceramic for guided bone regeneration

Abstract

This work describes a method to obtain macroporous resorbable glass and glass ceramic scaffolds with controlled biodegradability for tissue engineering applications. The constructs consisted of glass and glass ceramics in the system P₂O₅–CaO–Na₂O–TiO₂ and they were prepared by foaming a slurry of glass particles by addition of a H₂O₂ solution, and subsequent sintering of the porous structures obtained. Different thermal treatments were applied to control the degree of devitrification of the glass. The resultant materials showed a porosity percentage between 40% and 55% with a wide variety of pores ranging from 20 to 500 μm in diameter as determined by SEM and Image Analysis. The resulting constructs were predominantly formed by a vitreous phase, although small amounts of calcium metaphosphate and pyrophosphates were detected by X-ray diffraction and Raman spectroscopy after the sintering process. The biological response was also evaluated by means of the MTT test, the material showed a non-cytotoxic effect.

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.