Biodegradable mesoporous delivery system for biomineralization precursors

Abstract

Scaffold supplements such as nanoparticles, components of the extracellular matrix, or growth factors have been incorporated in conventional scaffold materials to produce smart scaffolds for tissue engineering of damaged hard tissues. Due to increasing concerns on the clinical side effects of using large doses of recombinant bone-morphogenetic protein-2 in bone surgery, it is desirable to develop an alternative nanoscale scaffold supplement that is not only osteoinductive, but is also multifunctional in that it can perform other significant bone regenerative roles apart from stimulation of osteogenic differentiation. Because both amorphous calcium phosphate (ACP) and silica are osteoinductive, a biodegradable, nonfunctionalized, expanded-pore mesoporous silica nanoparticle carrier was developed for loading, storage, and sustained release of a novel, biosilicification-inspired, polyamine-stabilized liquid precursor phase of ACP for collagen biomineralization and for release of orthosilicic acid, both of which are conducive to bone growth. Positively charged poly(allylamine)-stabilized ACP (PAH-ACP) could be effectively loaded and released from nonfunctionalized expanded-pore mesoporous silica nanoparticles (pMSN). The PAH-ACP released from loaded pMSN still retained its ability to infiltrate and mineralize collagen fibrils. Complete degradation of pMSN occurred following unloading of their PAH-ACP cargo. Because PAH-ACP loaded pMSN possesses relatively low cytotoxicity to human bone marrow-derived mesenchymal stem cells, these nanoparticles may be blended with any osteoconductive scaffold with macro- and microporosities as a versatile scaffold supplement to enhance bone regeneration.

Keywords: amorphous calcium phosphate; biomineralization; collagen; mesoporous silica; osteoinductive; poly(allylamine).

Figure 1

Chemoanalytical analyses of pMSN before and after loading with PAH-ACP (N=6). Notes: Small-angle X-ray diffraction pattern of pMSN showing four well-resolved diffraction peaks at d-spacing of 5.01 (210), 2.85 (200), 2.44 (110), and 1.84 (100) nm. The three weak peaks are shown with higher magnification in the inset (A). Infrared spectra of pMSN-CTAB (prior to calcination), pMSN (after calcination), and PAH-ACP@ pMSN (B). Abbreviations: pMSN, expanded-pore mesoporous silica nanoparticles; PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; CTAB, cetyltrimethyl ammonium bromide; PAH-ACP@pMSN, PAH-ACP loaded pMSN.

Figure 2

Ultrastructure of pMSN before and after loading with PAH-ACP. Notes: Low (left image; bar: 50 nm) and high magnification (middle image; bar: 20 nm) TEM images of intact, template-free pMSN. Right image: TEM image of sectioned, epoxy resin-embedded pMSN (bar: 20 nm) (A). Low (left image; bar: 50 nm) and high magnification (middle image; bar: 20 nm) TEM images of intact PAH-ACP@pMSN. Right image: TEM image of sectioned, epoxy resin-embedded PAH-ACP@pMSN (bar: 20 nm) (B). STEM-EDS elemental mapping images of sectioned PAH-ACP@pMSN showing the distribution of Si, O, Ca, P, and N within the nanoparticles (C). AFM height and phase images and three-dimensional presentation of the surface morphology of pMSN (before loading) and PAH-ACP@pMSN (after loading) (D). Abbreviations: PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; pMSN, expanded-pore mesoporous silica nanoparticles; PAH-ACP@pMSN, PAH-ACP loaded pMSN; TEM, transmission electron microscopy; STEM-EDS, scanning transmission electron microscopy-energy dispersive X-ray analysis; AFM, atomic force microscopy.

Figure 3

Dimensional analysis, thermogravimetric analysis, and zeta potential measurements (N=6). Notes: Nitrogen adsorption–desorption isotherms (A) and pore size distribution (B) of pMSN and PAH-ACP@pMSN. Thermogravimetric analysis (C) and derivative thermogravimetric analysis (D) plots of pMSN and PAH-ACP@pMSN. Zeta potential measurements of pMSN, PAH-ACP, and PAH-ACP@pMSN (E). Abbreviations: pMSN, expanded-pore mesoporous silica nanoparticles; PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; PAH-ACP@pMSN, PAH-ACP loaded pMSN.

Figure 4

Release and dissolution of elements present in PAH-ACP@pMSN (N=6). Notes: Cumulative release profiles of Ca and P from PAH-ACP@pMSN in TBS solution for 30 days (A). Changes in Ca: P ratio of the TBS solutions containing PAH-ACP@ pMSN from 0–30 days (B). Cumulative dissolution profile of Si from PAH-ACP@pMSN in TBS solution for 30 days (C). Abbreviations: PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; pMSN, expanded-pore mesoporous silica nanoparticles; PAH-ACP@pMSN, PAH-ACP loaded pMSN; TBS, tris(hydroxymethyl)aminomethane-buffered saline.

Figure 5

Ultrastructure of PAH-ACP@pMSN-mediated intrafibrillar mineralization of reconstituted collagen. Notes: Unstained TEM images of intrafibrillar mineralization of reconstituted collagen fibrils by PAH-ACP@pMSN (A–C). Mineralization precursors released from the pMSN (open arrow) after 24 h spread across the solution (open arrowhead) and infiltrated the collagen fibrils, resulting in their partial mineralization. Pointer: unmineralized fibrils. Bar: 100 nm (A). Heavy intrafibrillar mineralization was evident after 3 days. Bar: 100 nm (B). High magnification of a heavily mineralized collagen fibril showing electron-dense mineral strands within the fibril. Bar: 20 nm. Inset: selected area electron diffraction taken from double arrowed location of the mineralized fibril showed arc-shaped 002 Debeye diffraction patterns that are characteristic of apatite deposition along the c-axis of the collagen fibril (C). Uranyl acetate-stained TEM image of a collagen fibril mineralized for 2 days by PAH-ACP released from pMSN revealed regular banding patterns within the mineralized fibril. Bar: 100 nm (D). An adjacent partially mineralized fibril showed attachment of PAH-ACP mineralization precursors (open arrowheads) to the fibril surface. Needle-shaped intrafibrillar crystallites could be seen within the partially mineralized fibril (arrow). The mesoporous characteristics of a nearby pMSN (asterisk) was obscured by staining. Abbreviations: PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; PAH-ACP@pMSN, PAH-ACP loaded pMSN; pMSN, expanded-pore mesoporous silica nanoparticles; TEM, transmission electron microscopy.

Figure 6

Unstained TEM images taken from sections of hydrophilic resin (R)-infiltrated, partially mineralized hard tissues (bovine dentin) showing collagen remineralization and silicon dissolution. Notes: Low magnification of a PAH-ACP@pMSN pretreated specimen stored in TBS for 24 h showing a 5 μm thick band of resin-infiltrated collagen matrix (RC). The PAH-ACP@pMSN nanoparticles were located along the surface of the 3-D collagen matrix (arrow). M: mineralized hard tissue. Bar: 1 μm (A). High magnification of image A showing release of mineralization precursors (pointer) from the pMSN (arrow). Bar: 20 nm (B). Low magnification of a PAH-ACP@pMSN-pretreated specimen stored in TBS for 3 months. Bar: 1 μm. The resin-sparse bottom of the collagen matrix was heavily mineralized (between open arrows). The pMSN that were originally present on top of the resin infiltrated collagen matrix (RC) were completely solubilized (open arrowhead). T: tubule-like channels characteristic of the dentin (C). High magnification images of image C, showing voids (open arrowhead) between the hydrophilic resin (R) and the resin-infiltrated collagen matrix (RC) that were originally occupied by PAH-ACP@ pMSN. Bar: 100 nm (D). Abbreviations: PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; PAH-ACP@pMSN, PAH-ACP loaded pMSN; pMSN, expanded-pore mesoporous silica nanoparticles; TBS, tris(hydroxymethyl)aminomethane-buffered saline; TEM, transmission electron microscopy.

Figure 7

Schematic summarizing the development of a biodegradable mesoporous carrier for the delivery of ACP mineralization precursors. Abbreviations: CTAB, cetyltrimethylammonium bromide; NaOH, sodium hydroxide; PAH-ACP, poly(allylamine)-stabilized amorphous calcium phosphate; PAH-ACP@ pMSN, PAH-ACP loaded pMSN; pMSN, expanded-pore mesoporous silica nanoparticles; TBS, tris(hydroxymethyl)aminomethane-buffered saline; TEOS, tetraethyl orthosilicate; TMB, 1,3,5-trime thylbenzene.