Scaffolds of Calcium Phosphate Cement Containing Chitosan and Gelatin

Calcium phosphate cements (CPCs) have potential to be used on repairing damaged bones due to their moldability, bioactivity and bioresorbability. These materials combine calcium orthophosphate powders with a liquid leading to a paste that hardens spontaneously at low temperatures. Hence, CPCs could be applied as scaffolds to support cell/tissue growth. This paper studies CPC scaffolds processing by foaming cement's liquid phase in which was added gelatin and chitosan. The former acted to increase the foam stability while the ladder acted as a foaming agent. Moreover, these polymers would enhance scaffold's biological properties by controlling material's total porosity and in vivo resorption. The method proposed led to scaffolds with 58.71% porosity with sizes ranging from 160 to 760 µm and compressive strength of 0.70MPa. After foaming, pores' size, distribution and interconnectivity changed significantly leading to a material that could be applied on bone regeneration since it would allow nutrient's transport, cell attachment and an increase in material degradation rate.


Introduction
Calcium phosphate bioceramics present several applications in orthopedics since they provide implant's structural integrity holding it in place and intact while new bone tissue is growing. Their chemical composition provides interesting properties making them biocompatible, bioactive, resorbable and osteoconductive 1 .
In this context, calcium phosphate cements (CPCs) present appropriate characteristics to be used in repairing damaged bones. CPCs were introduced in the 1990s as a commercial product and since then, different formulations and applications have been developed 2,3 . CPCs are formed by mixing calcium orthophosphate powders with a liquid phase resulting in a moldable paste that hardens spontaneously after a low temperature setting reaction [2][3][4] . One kind of CPCs is brushite cements, which are obtained after an acid-base reaction. The most common composition contains β-tricalcium phosphate (β-TCP) and an acid, for example phosphoric acid. When β-TCP reacts incompletely, final composition is a mixture of β-TCP and brushite 2 . One application of CPCs is scaffolds production, which facilitate cell infiltration and tissue growth 4,5 .
Gelatin and chitosan have been added to bone cement to optimize the foaming process during scaffolds fabrication 6,7 and to enhance materials' mechanical and biological properties. Gelatin is a biocompatible and resorbable protein obtained from bovine collagen and employed as a foaming agent [6][7][8] . Montufar et al have proposed the fabrication of CPC scaffolds by foaming gelatin in the liquid-phase of an apatite cement 4 . Foam stability was obtained by increasing wt.% of gelatin. A different path to obtain foam stability is to employ chitosan 7,9,10 , a biopolymer originated from chitin; that is non-toxic, biodegradable, and biocompatible. It is insoluble in water or in body fluid, but soluble below pH 6.5 in most of the acidic mediums. Thus, the objective of this work was to synthesize and characterize scaffolds of calcium phosphate cement by foaming the liquid phase containing gelatin and chitosan.

Calcium Phosphate Cement (CPC) and scaffolds
CPCs solid phase, β-TCP, was synthesized by solid state reaction of a stoichiometric mixture of CaCO 3 (Synth, Brazil) and CaHPO 4 (Synth, Brazil) at 1050 °C 11,12 followed by ball milling during 48 hours 12 . The resulting powder was analyzed by laser diffraction (Mastersizer 2000) and presented mean particle size of 3.6 µm and granulometric distribution between 0.770 µm and 20.843 µm.
Liquid phase consisted of an aqueous solution containing citric acid 13 (C 6 H 8 O 7 , Synth, Brazil), phosphoric acid (H 3 PO 4 , Synth, Brazil), chitosan (Sigma Aldrich, Germany) and gelatin (Synth, Brazil). Gelatin was employed as a foaming agent and chitosan was added to stabilize the foam 9,10,14 . All formulations are described in Table 1.
Powder and liquid phases were mixed for 1 min and molded into cylindrical silicone molds (20X10mm). After setting, samples were polished and demolded. For sample BC0.5Gf, the liquid phase was stirred for 1 minute using UltraTurrax Tube Disperser (IKA  -WERKE, Germany) before mixing it with the powder.
Samples chemical composition were investigated using attenuated total reflection (ATR) with iS5-Nicolet Thermo FT-IR spectrometer with a scanning range of 550 to 4000 cm -1 and resolution of 2 cm -1 .

Results and Discussions
Foaming CPC liquid phase allowed scaffold formation. As observed in Figure 1, gelatin increased material's total porosity even with no foaming. Scaffolds were obtained only with 0.5 wt.% of gelatin since higher concentrations lowered mechanical resistance and formed higher volumes of foam that prevented cement's setting. Furthermore, chitosan addition was necessary in order to maintain the foam stability by gelling CPC liquid phase and avoiding the collapse of the porous structure during cement setting 9,10 .
Samples without gelatin (B and BC) exhibited lower porosity than those containing gelatin (BC0.5G, BC1G, BC0.5Gf). As seen in Figure 1, samples B and BC0.5Gf  presented 48% and 59 % of total porosity, respectively. Besides of increasing sample total porosity, foaming also created larger and broader interconnected pores that might promote nutrients' transport and the enlargement of cell interaction surface area facilitating new bone growth 6,14,15 . This can be observed in the SEM micrographs (Figure 2) of the surface of fracture of the set cements with and without chitosan, gelatin and foaming. (BC1G) in mechanical resistance compared to sample BC. Finally, compressive strength of foamed sample was 0.70 ± 0.27MPa. Nevertheless, materials' compressive strength remained close to the range of cancellous bone (1-12 MPa) 19 . Further studies varying stirring speed and time are necessary to control material's total porosity and, consequently, to enhance material's mechanical properties. Cement setting reaction took place as expected; β-TCP has reacted with H 3 PO 4 to form brushite (CaHPO 4 .2H 2 O) as shown in the XRD patterns of Figure 3 2 and ATR-IR spectra of Figure 4 20 .
The presence of gelatin and chitosan after setting reaction could not be detect in ATR-IR analysis (Figure 4), because of their low concentration 21,22  Conclusions CPC scaffold was obtained by foaming cement liquid phase containing 0.5 wt.% of gelatin. Interconnected pores ranging from 160 to 760 µm were obtained. As expected, the compressive strength has decreased as a function of porosity increase.
Total porosity had significantly increased after foaming, directly affecting the size distribution and interconnectivity of the pores, leading to a material that could be applied to bone regeneration, since it would allow transport of nutrients, cell attachment and material's degradation rate increases. Chitosan addition was effective since it promoted foam stability and permitted scaffolds fabrication with lower contents of gelatin.  Scaffolds with pore diameters ranging from 100 to 800 µm facilitate cell attachment and proliferation 16,17 . Moreover, making scaffolds of resorbable materials enhance its degradation rate permitting new bone tissue growth and improving implant osteointegration 6,[14][15][16]18 . Fortunately, all samples containing gelatin have pore size distribution between the ideal range (100 to 800 µm). Nevertheless, foamed sample presented larger, more homogeneous and interconnected pores, ranging from 160 to 760 µm.
As expected, compressive strength decreased with porosity increase, as seen in Figure 1. Chitosan promoted a 60% decrease in compressive strength. Samples with gelatin presented a decrease of 30% (BC0.5G) and 50%