Effect of Chemistry on Osteogenesis and Angiogenesis Towards Bone Tissue Engineering Using 3D Printed Scaffolds

Abstract

The functionality or survival of tissue engineering constructs depends on the adequate vascularization through oxygen transport and metabolic waste removal at the core. This study reports the presence of magnesium and silicon in direct three dimensional printed (3DP) tricalcium phosphate (TCP) scaffolds promotes in vivo osteogenesis and angiogenesis when tested in rat distal femoral defect model. Scaffolds with three different interconnected macro pore sizes were fabricated using direct three dimensional printing. In vitro ion release in phosphate buffer for 30 days showed sustained Mg²⁺ and Si⁴⁺ release from these scaffolds. Histolomorphology and histomorphometric analysis from the histology tissue sections revealed a significantly higher bone formation, between 14 and 20% for 4-16 weeks, and blood vessel formation, between 3 and 6% for 4-12 weeks, due to the presence of magnesium and silicon in TCP scaffolds compared to bare TCP scaffolds. The presence of magnesium in these 3DP TCP scaffolds also caused delayed TRAP activity. These results show that magnesium and silicon incorporated 3DP TCP scaffolds with multiscale porosity have huge potential for bone tissue repair and regeneration.

Keywords: 3D printing; Angiogenesis; Bone histomorphometry; Calcium phosphates; Injury/fracture healing; Matrix mineralization; Osteogenesis.

Figure 1

(a) Schematic representation of the 3D printing (3DP); (b) Photograph of the MgO and SiO₂ doped TCP scaffolds. Smaller scaffolds with 350 μm designed pore size were used for rat model in vivo; (c) XRD patterns of 3DP pure TCP and Mg-Si doped TCP scaffolds.

Figure 2

Surface morphology of 3DP pure TCP and Mg-Si doped TCP sintered at 1250 °C: Pure TCP with low (a) and high (b) magnification, Mg-Si doped TCP with low (c) and high (d) magnification.

Figure 3

(a) Photomicrograph of 3DP pure (i & ii) and Mg-Si doped TCP (iii & iv) scaffolds showing the development of new bone formation inside the interconnected macro pores of the 3DP scaffolds after 16 (i & iii) and 20 (ii & iv) weeks in rat distal femur model. Hematoxylin and Eosin (H&E) staining of transverse section. BM = Bone marrow; Arrows indicate the interface between scaffold and host bone; Star (*) indicates acellular regions derived from the scaffold. Color description: Black = Bone marrow; Pink/Reddish = New/old bone; Yellowish = acellular regions derive from scaffold; (b) Histomorphometric analysis of bone area fraction (total newly formed bone area/total area, %) from 800 μm width and 800 μm height H&E stained tissue sections (**p < 0.05, *p > 0.05, n=8).

Figure 4

Histomorphometric analysis of TRAP activity (TRAP positive area/total area, %) from 800 μm width and 800 μm height TRAP stained tissue sections (**p < 0.05, *p > 0.05, n=8).

Figure 5

Photomicrograph of vWF stained tissue sections showing blood vessel formation after 4, 8 and 12 weeks in 3DP pure TCP scaffolds and Mg-Si doped TCP scaffolds. Arrows indicate newly formed blood vessels inside scaffolds. vWF positive signals are brown with hematoxylin counterstaining.

Figure 6

(a) Histomorphometric analysis showing new blood vessel area comparisons between pure TCP and Mg-Si doped TCP (vWF positive area/total area, %) from 200 μm width and 200 μm height vWF stained tissue sections (**p < 0.05, *p > 0.05, n=8); Cumulative Mg²⁺ (b) and Si⁴⁺ (c) release in the phosphate buffer (pH 7.4) from Mg-Si doped 3DP TCP scaffolds.

Figure 7

Schematic representation showing accelerated osteogenesis and angiogenesis process triggered by Mg²⁺ and Si⁴⁺ ions from Mg-Si doped multiscale porous 3DP tricalcium phosphate tissue engineered scaffold when implanted on a defect site.