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, 10, 201
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Osteogenic Enhancement Between Icariin and Bone Morphogenetic Protein 2: A Potential Osteogenic Compound for Bone Tissue Engineering

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Osteogenic Enhancement Between Icariin and Bone Morphogenetic Protein 2: A Potential Osteogenic Compound for Bone Tissue Engineering

Xin Zhang et al. Front Pharmacol.

Abstract

Icariin, a typical flavonol glycoside, is the main active component of Herba Epimedii, which was used to cure bone-related diseases in China for centuries. It has been reported that Icariin can be delivered locally by biomaterials and it has an osteogenic potential for bone tissue engineering. Biomimetic calcium phosphate (BioCaP) bone substitute is a novel drug delivery carrier system. Our study aimed to evaluate the osteogenic potential when Icariin was internally incorporated into the BioCaP granules. The BioCaP combined with Icariin and bone morphogenetic protein 2 (BMP-2) was investigated in vitro using an MC3T3-E1 cell line. We also investigated its efficacy to repair 8 mm diameter critical size bone defects in the skull of SD male rats. BioCaP was fabricated according to a well-established biomimetic mineralization process. In vitro, the effects of BioCaP alone or BioCaP with Icariin and/or BMP-2 on cell proliferation and osteogenic differentiation of MC3T3-E1 cells were systematically evaluated. In vivo, BioCaP alone or BioCaP with Icariin and/or BMP-2 were used to study the bone formation in a critical-sized bone defect created in a rat skull. Samples were retrieved for Micro-CT and histological analysis 12 weeks after surgery. The results indicated that BioCaP with or without the incorporation of Icariin had a positive effect on the osteogenic differentiation of MC3T3-E1. BioCaP with Icariin had better osteogenic efficiency, but had no influence on cell proliferation. BioCap + Icariin + BMP-2 showed better osteogenic potential compared with BioCaP with BMP-2 alone. The protein and mRNA expression of alkaline phosphatase and osteocalcin and mineralization were higher as well. In vivo, BioCaP incorporate internally with both Icariin and BMP-2 induced significantly more newly formed bone than the control group and BioCaP with either Icariin or BMP-2 did. Micro-CT analysis revealed that no significant differences were found between the bone mineral density induced by BioCaP with icariin and that induced by BioCaP with BMP-2. Therefore, co-administration of Icariin and BMP-2 was helpful for bone tissue engineering.

Keywords: Icariin; MC3T3-E1; biomimetic calcium phosphate; bone morphogenetic protein 2; critical-sized bone defect.

Figures

FIGURE 1
FIGURE 1
Surgical procedure. 8 mm critical sized defect was created was made on the scalp of the rats (A–D), the granules were randomly implanted into the defects and covered by Bio-gide membranes (E–G). The incision was closed in layers (H).
FIGURE 2
FIGURE 2
Two-dimensional (2D) images of BioCaP and bone by Micro-CT (A). Bone (red) and BioCaP (white) were separated by the analysis software (B).
FIGURE 3
FIGURE 3
The proliferation of MC3T3-E1 cells stimulated by BioCaP alone or BioCaP with Icariin or/and BMP-2. The absorbance was measured on days 1, 4, and 7. Mean values (n = 3 samples per group) were represented together with the standard deviation. Error bars denote the standard deviation. P < 0.05, ∗∗P < 0.01, ∗∗without lines means versus control (C). C, Control; BioCaP, biomimetic calcium phosphate; I, Icariin; B, BMP-2.
FIGURE 4
FIGURE 4
Osteogenic differentiation results of MC3T3-E1 cells after treatment with BioCaP alone or BioCaP with Icariin or/and BMP-2. (A) The ALP activities were determined by colorimetric assay on days 1, 4, and 7. (B) The OCN expressions on days 4, 7, and 10. (C) Macroscopic images of alzarin red stained 3, 4, and 5 weeks after five groups treatment. (D) The mineralization results on 3, 4, and 5 weeks. Mean values (n = 3 samples per group) were represented together with the standard deviation normalized by total cellular protein. Error bars denote the standard deviation. P < 0.05, ∗∗P < 0.01, ∗∗without lines means versus control (C). C, Control; BioCaP, biomimetic calcium phosphate; I, Icariin; B, BMP-2.
FIGURE 5
FIGURE 5
The time course changes in mRNA expression of (A) ALP, (B) BMP-2 (C) Col1, (D) OCN, (E) Runx2 in MC3T3-E1 on days 1, 4, and 7. Mean values (n = 3 samples per group) were represented together with the standard deviation. Error bars denote the standard deviation. P < 0.05, ∗∗P < 0.01, ∗∗without lines means versus control (C). C, Control; BioCaP, biomimetic calcium phosphate; I, Icariin; B, BMP-2.
FIGURE 6
FIGURE 6
(A) The Micro-CT images of the bone defects in scalp of the rats 12 weeks after treatment with BioCaP, Icariin-int. BioCaP, BMP-2-int. BioCaP, and Icariin + BMP-2-int. BioCaP. (B) The volume of newly formed bone within the bone defect after 12 weeks postoperation for each group which is analyzed by Micro-CT. Mean value (n = 6 specimens per group ) are represented together with the standard deviation. ∗∗P < 0.01. (C) The bone mineral density (mg HA/cm3) of the newly formed bone within the bone defect after 12 weeks postoperation for each group which is analyzed by Micro-CT. Mean value (n = 6 specimens per group) are represented together with the standard deviation.
FIGURE 7
FIGURE 7
Representative histological micrographs of bone defect of each group at 12 weeks after implantation. The BioCaP granules materials (M) were surround by newly formed bone (B) in Icariin-int. BioCaP, BMP-2-int. BioCaP, and Icariin + BMP-2-int. BioCaP Groups. The slices were surface-stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine Blue. (A) The panoramic images of the defects of four experiment groups. (B) Scale bar = 200 μm. (C) Scale bar = 100 μm. The vascular (black arrows) was observed in all the groups. (D) The volume of newly formed bone within the bone defect after 12 weeks postoperation for each group which is analyzed by histology. Mean value (n = 6 specimens per group) are represented together with the standard deviation. ∗∗P < 0.01

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