Highly Stable Amorphous (Pyro)phosphate Aggregates: Pyrophosphate as a Carrier for Bioactive Ions and Drugs in Bone Repair Applications

Abstract

Pyrophosphate is widely used as an iron supplement because of its excellent complexation and hydrolysis ability; however, there are few reports on the use of pyrophosphate in active ionophores for bone repair. In this research, we proposed a simple and efficient ultrasonic method to prepare magnesium-calcium (pyro)phosphate aggregates (AMCPs). Due to strong hydration, AMCPs maintain a stable amorphous form even at high temperatures (400 °C). By changing the molar ratio of calcium and magnesium ions, the content of calcium and magnesium ions can be customized. AMCPs had surface negativity and complexing ability that realized the controlled release of ions (Ca²⁺, Mg²⁺, and P) and drugs (such as doxorubicin) over a long period. Pyrophosphate gave it an excellent bacteriostatic effect. Increasingly released Mg²⁺ exhibited improved bioactivity though the content of Ca²⁺ decreased. While Mg²⁺ content was regulated to 15 wt %, it performed significantly enhanced stimulation on the proliferation, attachment, and differentiation (ALP activity, calcium nodules, and the related gene expression of osteogenesis) of mouse embryo osteoblast precursor cells (MC3T3-E1). Furthermore, the high content of Mg²⁺ also effectively promoted the proliferation, attachment, and migration of human umbilical vein endothelial cells (HUVECs) and the expression of angiogenic genes. In conclusion, pyrophosphate was an excellent carrier for bioactive ions, and the AMCPs we prepared had a variety of active functions for multiscenario bone repair applications.

Figure 1

Chemical formula of pyrophosphate and the possible formula of AMCPs and the preparation procedure.

Figure 2

Qualitative analysis of AMCPs. (a) XRD curves and (c) Raman spectrum of AMCPs and (d) Raman spectrum of A3M1CP at three random locations; (e) ³¹P NMR of AMCPs dissolved in deuterium oxide.

Figure 3

Microstructure and morphology of AMCPs. (a) SEM of AMCPs and (b) EDS of A3M1CP; (c) TEM of AMCPs and (d) EDS of an A3M1CP single agglomerated particle.

Figure 4

Thermal stability of AMCPs. (a) TG-DSC curves; (b) XRD and (c) FTIR results of products sintered at 400 °C.

Figure 5

DOX loading and release of AMCPs. (a) Zeta potential of AMCPs; (b) DOX-load percent within 20 h and (c) TG-DSC results of DOX-loaded AMCPs; (d) DOX release ability of AMCPs (converted to adsorption percent) at pH 7.4 and (e) pH 4.5.

Figure 6

Ion release of AMCPs. (a) Release of Ca²⁺, (b) Mg²⁺, and (c) P at pH 7.4; (d) pH variety at different points when immersed in PBS at pH 7.4.

Figure 7

Induced mineralization precipitation of AMCPs in vitro. (a) SEM of deposits for AMCPs and elemental map of A3M1CP in (b); (c) XRD and (d) FTIR of mineral products; (e) weight changes of AMCPs in SBF (pH 7.4) at 7, 14, and 21 d.

Figure 8

Antimicrobial activity of AMCPs. (a) Photographs of the bacterial colony; (b) statistical results of the colony number (**: P < 0.01, ****: P < 0.0001).

Figure 9

Evaluation of osteogenic bioactivity of AMCPs. (a) Cell proliferation at 1, 3, and 5 d cultured by material extraction medium; (b) confocal images of cell fluorescence staining treated for 24 h by material extraction medium (63× oil, the scale bar is 10 μm).

Figure 10

Evaluation of osteogenic differentiation promoting of AMCPs. (a) ALP staining. (b) ARS results and mineralized nodules appeared orange or purplish red. All the images were obtained at the same multiple sites. (c) Expression of genes associated with osteogenesis at 1 and 7 d (ns: no significance, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Figure 11

Evaluation of angiogenesis capacity. (a) Confocal images of cell fluorescence staining treated for 24 h by material extraction medium (63× oil, the scale bar is 10 μm). (b) Cell proliferation at days 1, 3, and 5 cultured by material extraction medium. (c) Cell repair and migration within 2 days (10× , the scale bar is 100 μm); all the images were obtained at the same multiple sites. (d) Expression of genes associated with osteogenesis at 1 and 7 days (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).