Uniform deposition of protein incorporated mineral layer on three-dimensional porous polymer scaffolds

Abstract

Inorganic-organic hybrid materials designed to facilitate bone tissue regeneration use a calcium phosphate mineral layer to encourage cell adhesion, proliferation, and osteogenic differentiation. Mineral formed on porous materials is often discontinuous through the thickness of the scaffold. This study aimed to uniformly coat the pores of three-dimensional (3D) porous, polymer scaffolds with a bone-like mineral layer in addition to uniformly incorporating a model protein within this mineral layer. A filtration system designed to induce simulated body fluid flow through the interstices of 3D polylactic-co-glycolic acid scaffolds (10-mm diameter x 2-mm thickness) illustrated that a uniform, continuous mineral layer can be precipitated on the pore surfaces of a 3D porous structure within 5 days. MicroCT analysis showed increased mineral volume percent (MV%) (7.86 +/- 3.25 MV%, p = 0.029) and continuous mineralization of filtered scaffolds compared with two static control groups (floating, 0.16 +/- 0.26 MV% and submerged, 0.20 +/- 0.01 MV%). Furthermore, the system was effective in coprecipitating a model protein, bone sialoprotein (BSA), within the mineral layer. A 10-fold increase in BSA incorporation was seen when coprecipitated filtered scaffolds (1308 +/- 464 microg) were compared to a submerged static control group (139 +/- 45 microg), p < 0.001. Confocal microscopy visually confirmed uniform coprecipitation of BSA throughout the thickness of the filtration scaffolds. The designed system enables 3D mineralization through the thickness of porous materials, and provides the option of including coprecipitated biomolecular cues within the mineral layer. This approach of providing a 3D conductive and osteoinductive environment could be conducive to bone tissue regeneration.

Figure 1

Schematic showing the filtration device. The device consists of a Delrin^® mold (A), aluminum base (B), thermocouple (C), temperature controller (D), and heating element (E). The Delrin^® mold is connected to the actuator of an Instron 8521 servohydraulic system (F) and cyclically loaded via an aluminum rod (G). The SBF level and maximum and minimum positions that the Delrin^® mold is displaced to are labeled on the figure. Holes in the Delrin^® mold that did not have a PLGA scaffold were plugged with rubber stoppers. 1 L of SBF solution is housed in the aluminum base during mineralization. The Delrin^® mold can simultaneously cast sixteen 10–mm diameter scaffolds (or twenty-five 5-mm diameter scaffolds, not shown).

Figure 2

Representative MicroCT images of the filtration group (a–c) and the submerged control group (d–f). These images represent top (a,d), side (b,e), and center cross-sections (c,f). A greater amount of mineral formed on the filtration scaffolds in comparison to the submerged control group. Renderings of the floating control group are not shown because mineral present in the images was minimal (<0.2 MV%). All images were created in MicroView^® using the Isosurface tool (voxel size = 16 μm, threshold = 1000, surface quality factor = 0.55).

Figure 3

MV% for the filtration, floating control, and submerged control groups demonstrated the filtration group mineralized the greatest amount. (*p = 0.029 vs. both controls). MV% was calculated from MicroCT data using MicroView^® at a threshold of 1000.

Figure 4

Volumetric mineral analysis of the MicroCT images (threshold = 1000) showing the filtration scaffolds have a uniform mineral profile throughout all six regions of the scaffolds analyzed. The MV% for 6 concentric shell volumes show the control mineralized scaffolds have at minimum 60% of the mineral present in the 2 outer shells (shells 1 and 2). The floating control group shell volumes were statistically different from one another (n = 4, p = 0.002), whereas the submerged control and filtration groups did not show significance (n = 3, p = 0.051 and n = 4, p = 0.104, respectively). *Statistical significance found, p < 0.050.

Figure 5

FTIR spectra for filtration, floating control, and submerged control groups showing the mineral deposited on the scaffolds was characteristic of hydroxyapatite with carbonate peaks (ν_{3c P–O} 1032 cm⁻¹, ν_{1 P–O} 962 cm ⁻¹ ν_{4a O–P–O} 602 cm⁻¹, and ν_{4c O–P–O} 561 cm⁻¹ [filtration group only]) and carbonated apatite ( ${\nu }_{1{\text{CO}}_3^{2-}}$ 1465 cm⁻¹ [filtration and floating control groups]). Stronger peaks for the filtration group indicate the presence of more mineral.

Figure 6

XRD spectra for all three groups (filtration, floating control, submerged control) show characteristic apatite peaks between 25.9° and 26.8°, between 31.8° and 32.7°, at 40.1°, and between 45° and 55° when compared to the hydroxyapatite standard. A similar type of mineral was precipitated over the 5 days of filtration (1, 3, and 5 days). To assess if the mineral formed within the center of the filtration scaffolds was similar to the mineral formed on the surface of the filtration scaffolds, 5-day scaffolds were sectioned transversely and analyzed (filtration center 5 days). The mineral formed on the interior surface of 5-day filtration scaffolds showed a similar spectrum to the mineral formed on the outer surface of the same scaffolds at 5 days, verifying that a similar mineral formed throughout the depth of the scaffold.

Figure 7

Quantitative coprecipitated BSA values (μg) showing greater protein incorporation in the filtered scaffolds. The amount of BSA incorporated was quantified using UV Spectrophotometry at 494nm wavelength to detect the FITC–BSA. (*Statistically significant compared to submerged control group, p < 0.001).

Figure 8

Representative confocal microscopy images of transverse sections of filtration (a) and submerged control (b) groups coprecipitated with BSA for 3 days. The mosaic images were compiled from multiple images of the center cross-section for each scaffold in each group (n = 4 per group). The incorporation of BSA is shown by the fluorescence of FITC conjugated to the BSA. The filtration scaffolds showed uniform BSA coprecipitation throughout. A typical mineral shell, evidenced by more FITC seen on the left and right edges, is seen for the submerged control group. The presence of a mineral shell also supports less BSA incorporation. Autofluorescence of the mineral was not detected, shown by a representative image of a center cross-section of a scaffold mineralized via filtration for 5 days using the 4× SBF/2× SBF regimen precipitated without protein (c). Original images were taken at 10×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 9

MV% for the filtration and submerged control coprecipitation groups demonstrated the filtration group mineralized the greatest amount (*p < 0.001). MV% was calculated from MicroCT data using MicroView^® at a threshold of 1000.