Cursos externos

Gelatin microspheres (average diameter of about 26 μm) were about hundred-fold larger than gelatin nanospheres (average diameter of 150-200 nm) (Fig. 1 and Table 1). GelA and GelB microspheres were of similar size, but GelA nanospheres were bigger than GelB nanospheres (Table 1). ζ-potential measurements confirmed the positive and negative charge of GelA and GelB nanospheres, respectively, whereas the charge of microspheres was not measurable using laser Doppler electrophoresis due to rapid sedimentation of microspheres in suspension.

The GA crosslinking process (with a fixed molar ratio of GA to [NH2]gelatin equal to 1)

consumed similar amounts of free amine groups for both GelA and GelB micro- or nanospheres, since comparable crosslinking densities were observed ranging from 40-50%. At similar crosslinking densities, nanospheres exhibited significantly higher

25 µg of BMP-2 (75 µl, 0.337 mg/ml) or ALP (25 µl, 1 mg/ml) was pipetted into a 100 μg iodogen coated 1.5 ml Eppendorf® _{vial together with 10 μl of} 125_I

(radioactivity = 1000 µCi, Perkin-Elmer, Boston, MA). The final volume was adjusted to 100 μl with 0.5 M phosphate buffer, pH 7.0. The tube was then incubated at room temperature for 10 min to allow for completion of the reaction. Subsequently, 100 μl of saturated tyrosine solution in PBS was added to react with the nonbound 125_{I. The reaction mixture was then eluted with 0.5% BSA in 1}

mM NaCl (pH 7.0) solution on a prerinsed disposable Sephadex G25M column (PD-10; Pharmacia, Uppsala, Sweden). The reaction mixture was eluted and 500 μl fractions were collected. The fraction with the highest radioactivity was used for further studies. The radiochemical purity of the 125_{I-labeled proteins was}

determined by instant thin layer chromatography (ITLC) on Gelman ITLC-SG strips (Gelman Laboratories, Ann Arbor, MI, USA) with 0.1 M citrate, pH 5.0 as the mobile phase. The radiochemical purity of BMP-2 and ALP was 97% and 93.5%, respectively. The solution containing the radiolabeled proteins were further diluted to a concentration of 12 µg/ml.

Radiolabeled BMP-2 and ALP were loaded onto gelatin spheres by diffusional loading by directly mixing protein-containing solutions with lyophilized gelatin particles. To ensure complete sorption of biomolecules by gelatin, less volume of liquid than required for complete swelling of gelatin spheres was used. The dose of both proteins loaded by gelatin was 60 ng per mg gelatin particles[28]_{. Briefly, 5 mg of}

gelatin spheres were mixed in a 1.5 ml Eppendorf® _{tube with 25 µl of the solutions}

containing 12 µg/ml 125_{I-labeled BMP-2 or ALP, obtaining colloidal gels with a solid}

content of 20 w/v%. The mixtures were stored at 4 ºC overnight to allow for complete sorption of osteogenic proteins. 1 ml PBS containing 400 ng/ml collagenase 1A and 0.001 w/v% sodium azide was added to the tube and then incubated at 37 ºC on a rotating plate (70 rpm). At each time point, 0.9 ml of supernatant was refreshed with the same volume of medium after centrifugation (2767 g, 5min) to sediment the gelatin spheres. The release of BMP-2 and ALP at each time point was monitored by measuring the residual γ-irradiation from the gelatin samples using a gamma counter after refreshing the supernatant media. Consequently, the cumulative release of BMP-2 or ALP was determined by normalizing radioactivity at each time point with starting radioactivity of each sample. The decay of I125 _{was taken into account by}

recording the remaining radioactivity of labeled proteins without loading to gelatin spheres.

2.9. Statistics

Measurements for rheological and injection tests were performed three-fold (n=3), while measurements for in vitro degradation and release study were performed fourfold (n=4). The results were depicted as average ± standard deviation. The statistical analyses were performed using GraphPad InStat software. Differences among groups were determined by one-way Analysis of Variance (ANOVA) with a Tukey (multiple comparisons) post test, and a value of p < 0.05 was considered as significantly different.

Table 1. Characteristics of lyophilized gelatin micro- and nanospheres.

Characteristics Microsphere Nanosphere

GelA GelB GelA GelB

Particle size 26±6µm 26±5µm 198±41nm 148±28 nm ζ-potential (mV) n/a n/a +9.3±0.3 -20.0±0.4 Water content (%) 80.1±1.9 75.0±1.3 89.2±0.4 87.1±0.5 Crosslinking density (%) 48.1±2.6 41.3±1.7 41.0±2.7 44.9±6.0

3. Results

3.1. Characteristics of gelatin micro- and nanospheres

Gelatin microspheres (average diameter of about 26 μm) were about hundred-fold larger than gelatin nanospheres (average diameter of 150-200 nm) (Fig. 1 and Table 1). GelA and GelB microspheres were of similar size, but GelA nanospheres were bigger than GelB nanospheres (Table 1). ζ-potential measurements confirmed the positive and negative charge of GelA and GelB nanospheres, respectively, whereas the charge of microspheres was not measurable using laser Doppler electrophoresis due to rapid sedimentation of microspheres in suspension.

The GA crosslinking process (with a fixed molar ratio of GA to [NH2]gelatin equal to 1)

consumed similar amounts of free amine groups for both GelA and GelB micro- or nanospheres, since comparable crosslinking densities were observed ranging from 40-50%. At similar crosslinking densities, nanospheres exhibited significantly higher

water contents than microspheres. This enhanced water uptake by nanospheres can be explained by their exceptionally small size and corresponding large surface area that allows for more ab- and adsorption of water inside the nanospheres as well as water entrapment in the pores formed between nanospheres.

Figure 1. SEM images of lyophilized GelA (A, C) and GelB (B, D) micro- (A, B) and nanospheres (C, D).

Figure 2. Elastic modulus G’ (A) and tan(δ) (B) of colloidal dispersions in 1 mM NaCl solution (pH 7.0) comprising similarly (GelA or GelB) or oppositely (GelA+B) charged micro- (MS) or nanospheres (NS) as a function of solid content. Microsphere dispersions of 5 w/v% solid content were excluded since these dispersions behaved as highly flowable liquid-like

materials, whereas nanosphere dispersions of 25 w/v% solid content were excluded due to the difficulty to form homogeneous gels at such high solid content.