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Materials (Basel)
2019 Jun 11;1211:. doi: 10.3390/ma12111881.
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In Vitro and In Vivo Evaluation of Starfish Bone-Derived β-Tricalcium Phosphate as a Bone Substitute Material.
Ishida H
,
Haniu H
,
Takeuchi A
,
Ueda K
,
Sano M
,
Tanaka M
,
Takizawa T
,
Sobajima A
,
Kamanaka T
,
Saito N
.
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We evaluated starfish-derived β-tricalcium phosphate (Sf-TCP) obtained by phosphatization of starfish-bone-derived porous calcium carbonate as a potential bone substitute material. The Sf-TCP had a communicating pore structure with a pore size of approximately 10 μm. Although the porosity of Sf-TCP was similar to that of Cerasorb M (CM)-a commercially available β-TCP bone filler-the specific surface area was roughly three times larger than that of CM. Observation by scanning electron microscopy showed that pores communicated to the inside of the Sf-TCP. Cell growth tests showed that Sf-TCP improved cell proliferation compared with CM. Cells grown on Sf-TCP showed stretched filopodia and adhered; cells migrated both to the surface and into pores. In vivo, vigorous tissue invasion into pores was observed in Sf-TCP, and more fibrous tissue was observed for Sf-TCP than CM. Moreover, capillary formation into pores was observed for Sf-TCP. Thus, Sf-TCP showed excellent biocompatibility in vitro and more vigorous bone formation in vivo, indicating the possible applications of this material as a bone substitute. In addition, our findings suggested that mimicking the microstructure derived from whole organisms may facilitate the development of superior artificial bone.
Figure 1. Photographs of the analyzed materials. (a) Cerasorb M (CM). (b) starfish-derived β-tricalcium phosphate (Sf-TCP) after sieving.
Figure 2. Surface images obtained by scanning electron microscopy. Magnification: ×1000. (a) CM. Arrows indicate pores. (b) Sf-TCP. Scale bar: 10 µm.
Figure 3. Adsorption-desorption isotherms. The blue line indicates adsorption, and the red line indicates desorption. (a) CM; (b) Sf-TCP.
Figure 5. Surface SEM image of Sf-TCP cultured with MC3T3-E1 cells. Magnification: ×3000. The arrows show the scaly structure on the surface, and the triangles show cell filopodia.
Figure 6. Hematoxylin and eosin (HE) staining. Hematoxylin stains cell nuclei blue, while eosin stains the cytoplasm, connective tissue, and other extracellular substances pink or red. Brain tissues are shown in the lower portion of the images, and the outer side of the skull is shown in the upper portion of the images. Boxed parts are prosthetic materials. Scale bar: 200 μm.
Figure 7. Blood vessel structures (red boxed area) were identified by hematoxylin and eosin (HE) staining.
Figure 8. Masson’s trichrome (MT)-stained images taken with Vectra3. Aniline blue stains fibrous tissue blue, Masson liquid stains cells red, and Orange G stains blood cells orange. (a) CM implant at 4 weeks. (b) Sf-TCP implant at 4 weeks. The blue boxes indicate the area quantified in Figure 10.
Figure 9. Images of Masson’s trichrome (MT)-stained sections and analysis images for quantification. Representative images showing cells and fibrous tissue existing mainly around CM or Sf-TCP pores. Cells appear more uniformly scattered at 8 weeks than at 4 weeks. (a,b) CM images with MT staining. (c,d) CM images for analysis. (e,f) Sf-TCP images with MT staining. (g,h) Sf-TCP images for analysis. Aniline blue staining (blue) shows fibrous tissue, and Masson liquid and Orange G staining (red) show cells. Scale bar: 50 μm.
Figure 11. Image of erythrocytes observed inside Sf-TCP. (a) 4 weeks. (b) 8 weeks. Red circles indicate erythrocytes observed in Sf-TCP. Scale bar: 20 μm.
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