Laser Sintering of Nano 1393 Glass Scaffolds : Microstructure , Mechanical Properties and Bioactivity

As the only bioactive material that can bond with both hard tissues and soft tissues, bioactive glass has become much important in the field of tissue engineering. 13-93 bioactive glass scaffolds were fabricated via selective laser sintering (SLS). It was focused on the effects of laser sintering on microstructure and mechanical properties of the scaffolds. The experimental results showed that the sintered layer gradually became dense with the laser power increasing and then some defects occurred, such as macroscopic caves. The optimum compressive strength and fracture toughness were 21.43±0.87 MPa and 1.14±0.09 MPa.m, respectively. In vitro bioactivity showed that there was the bone-like apatite layer on the surface of the scaffolds after soaking in simulated body fluid (SBF), which was further evaluated by Fourier transform infrared spectroscopy (FTIR). Moreover, cell culture study showed MG-63 cells adhered and spread well on the scaffolds, and proliferated with increasing time in cell culture. These indicated excellent bioactivity and biocompatibility of nano 13-93 glass scaffolds.


Introduction
Metals, polymers and ceramics have long been widely used for implant materials [1].However, these materials suffer from several limitations, such as immunological response, poor bonding properties and stress concentrations damage due to the use of bone screws [2][3][4].Bioactive glass was a promising bone scaffold biomaterial because of the ability to form a secure physical bonding with bone and soft tissue [5].Additionally, the degradation products of bioactive glass could excite the gene expression of osteoblasts, promote the production of growth factors and stimulate cell proliferation [6][7][8].Nano 13-93 glass has bioactive and resorbent properties as a third-generation bioactive material.It can be used to assist or enhance the body's own natural reparative capacity.Furthermore, it contains high SiO 2 content which can slow down action rates after implantation [9,10].
Porous scaffolds were usually manufactured by conventional methods such as foam replication, melt molding and freeze drying.These techniques could generate porous architecture which was required for bone scaffolds.However, the pores lack of precision and reproducibility.Rapid prototyping (RP) techniques [11][12][13], which cannot only overcome these shortcomings, but manufacture highly complex shapes and completely interconnection pore networks.SLS is a powder based RP technique [14][15][16].In the SLS process, the 3D CAD models are sectioned into 2D layers and the laser beam selectively scans the thin layers in powder bed, layer-by-layer, to form solid 3D objects.
In this study, porous nano 13-93 glass scaffolds were fabricated via SLS.The influence of SLS process parameters on microstructure and mechanical properties was investigated with scanning electron microscopy (SEM), a universal testing system, a Vickers microindenter, etc.Moreover, bioactivity and biocompatibility of the scaffolds were evaluated in vitro.

Material
Nano 13-93 glass with a nominal chemical composition of 53% SiO 2 , 4% P 2 O 5 , 20% CaO, 5% MgO, 6% Na 2 O and 12% K 2 O in wt.% was prepared by mixing distilled water and high-purity chemical reagents dissolved in ethanol and stirred until the solution became clear.The solution was transferred to large Teflon containers, which were placed in an oven for aging at 60 ο for 72 h.Then, the aged gel was transferred into another Teflon vessel and dried at 110 o for 48 h.Finally, the obtained dried nano 13-93 glass powders were calcined at 600 o for 2 h by a heating and cooling rate of 2 o / min and milled to reduce aggregation.

Fabrication method
The 13-93 glass scaffolds were prepared by using a home-made SLS system which had been reported in our previous study [17,18].During sintering process, the variation of laser power was investigated in this paper and main process parameters were scan speed of 100 mm•min -1 , layer thickness of 0.1 mm and laser spot diameter of 1.0 mm.

Mechanical properties
The compressive strength of the scaffolds was measured using a universal mechanical tester (Shanghai Zhuoji Instruments CO.,Ltd,China).The crosshead speed was set at 0.5 mm•min -1 .Five samples for each laser power were tested, and the compressive strength was determined as a mean±SD.The fracture toughness of the scaffolds was tested by using a Vickers microindenter (HXD-1000TM/LCD, Digital Micro Hardness Tester, Shanghai Taiming Optical Instrument Co., Ltd.) with a load of 4.9 N, applying on the polished samples embedded in epoxy.The average values and standard deviations were calculated from five tests for each laser power.The fracture toughness K ic was determined using the equation (1): K ic =0.0824Pc -3/2 (1) Where P is the indentation load and c is the length of the induced crack.

In-vitro bioactivity evaluation
In vitro bioactivity of the scaffolds was investigated by immersing them in SBF solution which has inorganic ion concentrations similar to those of human blood plasma.The SBF solution was prepared by dissolving reagent chemicals of NaCl, KCl, NaHCO 3 , and Na 2 SO 4 into distilled water.The SBF was adjusted to physiological pH (pH=7.25)by HCl solution and buffered bytris(hydroxymethyl) aminomethane at 37 o .The immersing experiment was carried out in a shaking incubator for 7 days.After the preselected immersing time, the scaffolds were removed from SBF, rinsed with deionized water followed by drying in vacuum desiccators.SEM was carried out to observe the surface of the scaffolds for mineralization.

Cell culture
The in vitro biocompatibility of the scaffolds was tested using MG63 cells (human osteogenic sarcoma) (ATCC, Rochville, MD).Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum (FBS), 100 Uml-1 penicilillin and 100 μg/mL strptomycin at 37 0 C. Before confluence, cells were harvested from the culture plate with trypsin/EDTA.The scaffolds were sterilized with 75% ethanol and maintainedin normal medium for 1 h preconditioning soak.
MG63 cells were seeded onto the top of pre-wetted scaffolds at a concentration of 5×10 3 cells/cm 2 in a 12-well plate (well area 3.8 cm 2 ).After that, the cells/scaffold constructs were kept in a humidified incubator at 37 0 C/5% CO 2 for 3 and 6 days, respectively.Cells were fixed with 2.5% glutaraldehyde, followed by 1% osmium tetroxide.The scaffolds with attached cells were then washed with phosphate-buffered saline (PBS) to remove loosely bound cells and dehydrated through a graded ethanol series.The morphological results of cell culturing were observed with SEM.

3.1Microstructure
The surface morphologies of the scaffolds fabricated at various laser power were shown in Fig. 1.The initial 13-93 glass (Fig. 1a) was spherical with average particle size of 100 nm.The surface of scaffolds sintered at 3 W was shown in Fig. 1b.There were only a few 13-93 glass particles melted and bonded together, and the individual particles could still be obviously identified.At a laser power of 5 W (Fig. 1c), there was mass bonding between the particles.Meanwhile, few individual particles could be identified.As the laser power increased to 7 W (Fig. 1d), particles obtained sufficient laser energy and tightly bonded together, a dense surface was obtained.While the laser power was further increased to 9W (Fig. 1e), a number of macroscopic caves inside the scaffold wall were formed due to the fact that high laser power leaded to the sharp change of temperature, which stopped the gas coming out during fusion.Therefore, the laser power range was 5 to 9W.

Mechanical properties
Scaffolds were fabricated at the laser power of 5, 6, 7, 8 and 9 W. The compressive strength and fracture toughness of the scaffolds sintered at various laser power were shown in Fig. 2. The results clearly revealed that the compressive strength and fracture toughness increased with the increasing of laser power from 5 W to 7 W.The optimum compressive strength and fracture toughness were 21.43±0.87MPa and 1.14±0.09MPa.m 1/2 , respectively.While the laser power further increased, the compressive strength and fracture toughness gradually decreased.Therefore, the optimal laser power was 7 W.

XRD analysis
The XRD pattern of 13-93 glass scaffolds sintered at 7 W was shown in Fig. 3.The result showed that no crystallization occurred, which indicated that the 13-93 glass maintained amorphous state after laser sintering.

Scaffold fabrication
A cube 13-93 glass scaffold (12 mm×12 mm×12 mm) was fabricated under the laser power of 7 W using established process parameters (Fig. 4).After removal of the unsintered powder, the interconnected porous structure could be easily recognized.

In-vitro bioactivity evaluation
SEM micrographs of 13-93 glass scaffolds after soaking in SBF for 7 days were shown in Fig. 5a.Compared to the scaffold before SBF (Fig. 1d), there was a fine particulate surface layer after SBF.At higher magnification, the precipitates showed a network of nanocrystallines, not unlike the rod-like hydroxyapatite crystals in human bone.
FTIR spectra of the scaffolds surfaces before and after soaking in SBF were shown in Figs. 5 (b,c).IR spectra were recorded in the range 4000~400 cm -1 .Before soaking in SBF (Fig. 5b), the absorption band at 471 cm -1 corresponded to Si-O-Si bending mode [19,20], 1038 cm -1 3corresponded to unsymmetry flex vibration (ν3) of PO [21].The broadband centered at 3400 cm -1 corresponded to O-H band [22].After soaking in SBF (Fig. 5c), the intensity of the silicate absorption band decreased and a couple of well-defined additional peaks appeared at 606 and 573 cm −1 , corresponding to P-O bending vibrations [23].In addition, some carbonate was observed around 1500 cm -1 [24], which indicated that the rodlike apatite crystals on the surface of the scaffolds were synthetic carbonate hydroxyapatite [25].

Cell culture
The morphologies of the MG-63 cells cultured on the scaffolds for 3 and 6 days were shown in Fig. 6, respectively.After being cultured for 3 days (Fig. 6a), a few MG-63 cells were observed on the surface of the scaffolds, most of them were separated and spherical, which indicated that the cells were at initial adhesion stage.After being cultured for 6 days (Fig. 6b), MG-63 cells showed a sharp increase in number, and in physical contact with the neighboring cells.The results indicated the good biocompatibility of the 13-93 glass scaffolds.

Conclusions
Porous nano 13-93 glass scaffolds were successfully fabricated using SLS technology.The mechanical properties measurements showed that the compressive strength and fracture toughness increased with the laser power increasing to 7 W.The optimum compressive strength and fracture toughness were 21.43±0.87MPa and 1.14±0.09MPa.m 1/2 , respectively.However, the mechanical strength decreased owing to the occurrence of macroscopic caves with the laser power further increasing.In-vitro bioactivity showed bonelike apatite layer formed on the surface after immersion in SBF.Moreover, MG-63 cells were able to adhere and grow well on the glass scaffolds.The results indicated that the nano 13-93 glass scaffold is a promising candidate for bone repair and regeneration.

Fig. 2 .
Fig. 2. The effect of laser power on compressive strength and fracture toughness of 13-93 glass samples.

Fig. 5 .
Fig. 5. (a) Surface morphology of 13-93 glass scaffolds after immersion in SBF for 7 days: lower magnification image and higher magnified image.FT-IR spectra of 13-93 glass scaffolds (b) before and (c) after soaking in SBF.