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Increased Osteoblast Adhesion on Nanoparticulate Crystalline Hydroxyapatite Functionalized With KRSR

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Increased Osteoblast Adhesion on Nanoparticulate Crystalline Hydroxyapatite Functionalized With KRSR

Michael Nelson et al. Int J Nanomedicine.

Abstract

The present in vitro study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate for novel orthopedic applications. Specifically, nano-crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by hydrothermal treatment for 2 hours at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of control conventional HA particles (or those currently used in orthopedics with micron diameters), the aforementioned calcium phosphate particles were pressed into compacts and sintered at 1100 degrees C for 2 hours. All calcium phosphate-based particles were fully characterized. Results showed that although there was an initial weight gain for all the compacts studied in this experiment, higher eventual degradation rates up to 3 weeks were observed for nano-amorphous calcium phosphate compared with nano-crystalline HA which was higher than conventional HA. Peptide functionalization (with the cell adhesive peptide lysine-arginine-serine-arginine [KRSR] and the non-cell-adhesive peptide lysine-serine-arginine-arginine [KSRR]) was accomplished by means of a three-step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The peptide functionalization was fully characterized. Results demonstrated increased osteoblast (bone-forming cell) adhesion on non-functionalized and functionalized nano-crystalline HA compacts compared with nano amorphous calcium phosphate compacts; both increased osteoblast adhesion compared with conventional HA. To further exemplify the novel properties of nano crystalline HA, results also showed similar osteoblast adhesion between non-functionalized nano crystalline HA and KRSR functionalized conventional HA. Thus, results provided evidence that nanocrystalline HA should be further studied for orthopedic applications.

Figures

Figure 1
Figure 1
Reaction scheme for functionalizing peptides on calcium phosphate-based materials. In the present study, peptides were KRSR (experimental) and KSRR (negative control). Abbreviations: APTES, amino-functional organosilane; DMF, dimethylformamide; SMP, N-succinimidyl-3-maleimido propionate.
Figure 2
Figure 2
Scanning electron microscopy images of calcium phosphate-based compacts. Increased compact surface roughness was observed on (a) nano-amorphous calcium phosphate compared with (b) nano-crystalline HA and (c) conventional HA. Bars = 10 μm.
Figure 3
Figure 3
Degradation profile of calcium phosphate-based materials in DMEM. Data = mean ± SEM; n = 3; * p <0.01 (compared with respective substrate at 0 days); ** p <0.01 (compared with conventional HA weight loss over the entire time period); and *** p <0.01 (compared with nano-crystalline HA weight loss over the entire time period). Abbreviations: HA, hydroxyapatite.
Figure 4
Figure 4
X-ray photoelectron spectroscopy analysis of Ca 2p, P 2p for KRSR peptide-functionalized compared with non-functionalized materials. Abbreviations: CPS, counts per second; HA, hydroxyapatite.
Figure 5
Figure 5
X-ray photoelectron spectroscopy analysis of Si 2p, N 1s, S 2p for KRSR peptide functionalized compared with non-functionalized materials. Abbreviations: CPS, counts per second; HA, hydroxyapatite.
Figure 6
Figure 6
Increased osteoblast adhesion on nano-crystalline HA compacts functionalized with KRSR. Data = mean ± SEM; n = 3; * p <0.01 (compared with respective plain substrate); **p <0.01 (compared with respective conventional HA functionalization); and *** p <0.01 (compared with respective nano-crystalline HA functionalization). Abbreviations: HA, hydroxyapatite; SMP, N-succinimidyl-3-maleimido propionate.
Figure 7
Figure 7
Scanning electron microscopy images of osteoblasts attached to calcium phosphate-based compacts. Increased cell spreading was observed on nano-crystalline HA compared with nano-amorphous calcium phosphate and conventional HA. Bars = 10 μm. Abbreviations: HA, hydroxyapatite.

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