A New Anorganic Equine Bone Substitute for Oral Surgery: Structural Characterization and Regenerative Potential

Abstract

Different xenogeneic inorganic bone substitutes are currently used as bone grafting materials in oral and maxillo-facial surgery. The aim of the present study was to determine the physicochemical properties and the in vivo performance of an anorganic equine bone (AEB) substitute. AEB is manufactured by applying a process involving heating at >300 °C with the aim of removing all the antigens and the organic components. AEB was structurally characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray fluorescence (XRF), and Fourier-transformed infrared (FT-IR) spectroscopy and compared to the anorganic bovine bone (ABB). In order to provide a preliminary evaluation of the in vivo performance of AEB, 18 bone defects were prepared and grafted with AEB (nine sites), or ABB (nine sites) used as a control, in nine Yucatan Minipigs. De novo bone formation, residual bone substitute, as well as local inflammatory and tissue effects were histologically evaluated at 30 and 90 days after implantation. The structural characterization showed that the surface morphology, particle size, chemical composition, and crystalline structure of AEB were similar to cancellous human bone. The histological examination of AEB showed a comparable pattern of newly formed bone and residual biomaterial to that of ABB. Overall, the structural data and pre-clinical evidence reported in the present study suggests that AEB can be effectively used as bone grafting material in oral surgery procedures.

Keywords: anorganic bone; bone formation; equine bone substitute; xenograft.

Figure 1

AEB (A) and ABB (B) particles observed with a stereo microscope.

Figure 2

SEM images (A–F) and 3D surface reconstruction (G,H) of AEB (A,C,E,G) and ABB (B,D,F,H). Scale bars: (A,B): 200 μm; (C,D): 100 μm; (E,F): 30 μm.

Figure 3

Graphical representation of the granule size distribution of AEB and ABB measured with SEM. On the x-axis the granules size in shown in millimeters, whereas on y-axis is shown the percentage of granules for each size.

Figure 4

The XRD spectra obtained for ABB (top) and AEB (bottom). Both biomaterials show the typical peaks of hydroxyapatite.

Figure 5

FTIR spectra of ABB (orange) and AEB (gray). Samples of both biomaterials exhibit main peaks around 1450–1415, 1020, 962, and 874 cm⁻¹.

Figure 6

Histological examination of grafted particles at 30 and 90 days from regenerative procedure. At 30 days AEB (**) and ABB (*) particles were surrounded by a thin layer of newly formed bone. At 90 days AEB (**) and ABB (*) particles appeared integrated in extended bony islands.

Figure 7

Box plot representation of the percentage of newly formed bone ((A) top panel) and of residual biomaterial ((B) bottom panel) in samples that were grafted with AEB or with ABB. No statistical differences are present between the two biomaterials.