Silicon substituted hydroxyapatite/VEGF scaffolds stimulate bone regeneration in osteoporotic sheep

Abstract

Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and fostered osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis. STATEMENT OF SIGNIFICANCE: For the first time, the in vivo behavior of scaffolds made of silicon substituted hydroxyapatites (SiHA) has been evaluated under osteoporosis conditions. In order to optimize the bone regeneration properties of these bioceramics, 3D macroporous scaffolds have been manufactured by robocasting and implanted in osteoporotic sheep. Our experimental design shed light on the important issue of the biological response of nano-sized bioceramics vs highly crystalline bioceramics, as well as on the importance of coupling vascularization and bone growth processes by decorating SiHA scaffolds with vascular endothelial growth factor.

Keywords: In vivo test; Macroporous scaffold; Osteoporosis; Silicon substituted hydroxyapatite; VEGF.

Figure 1

SEM micrographs of NanoSiHA (a,c,e) and SiHA (b,d,f) at different magnifications.

Figure 2

Proliferation of EC₂ endothelial cells on NanoSiHA and SiHA scaffolds without (white) and with (black) adsorbed VEGF after 7 days of culture. Cell proliferation was analyzed by measuring absorbance at 450 nm by CCK-8 protocol. *For each material, comparisons between conditions without and with VEGF. Statistical significance: *p < 0.05; **p < 0.01.

Figure 3

Morphology evaluation by SEM of EC₂ endothelial cells on NanoSiHA and SiHA scaffolds without and with adsorbed VEGF after 7 days of culture.

Figure 4

Proliferation and differentiation of MC3T3 pre-osteoblasts on NanoSiHA and SiHA scaffolds without (white) and with (black) adsorbed VEGF after 7 days of culture. Cell proliferation was analyzed by measuring absorbance at 450 nm by CCK-8 protocol. Cell differentiation was evaluated by measuring ALP activity which was normalized to the cell protein content. *For each material, comparisons between conditions without and with VEGF. ^ф Comparison between NanoSiHA and SiHA. Statistical significance: *p < 0.05, ^фффp < 0.005.

Figure 5

Morphology evaluation by SEM of MC3T3 pre-osteoblasts on NanoSiHA and SiHA scaffolds without and with adsorbed VEGF after 7 days of culture.

Figure 6

CT scan images obtained after 12 weeks of implantation.

Figure 7

Histological images after 12 weeks of implantation of SiHA (a and b), SiHA/VEGF (b and c), NanoSiHA (e and f) and NanoSiHA/VEGF (g and h). The highlighted regions in low magnification images correspond to the ones with high magnification. The inset in Figure 7f shows inflammatory components found in this sample.

Figure 8

Histomorphometrical studies for the different scaffolds implanted in osteoporotic sheep. (a) Ossification volume, (b) trabeculae thickness, (c) presence of osteoblasts, (d) presence of osteoclasts, (e) amount of inflammatory component and (f) vascularization degree. Statistical significance: *p < 0.05, **p < 0.01 and ***p < 0.005.