Synergistic interplay between the two major bone minerals, hydroxyapatite and whitlockite nanoparticles, for osteogenic differentiation of mesenchymal stem cells

Abstract

The inorganic part of human bone is mainly composed of hydroxyapatite (HAP: Ca₁₀(PO₄)₆(OH)₂) and whitlockite (WH: Ca₁₈Mg₂(HPO₄)₂(PO₄)₁₂) minerals, where the WH phase occupies up to 20-35% of total weight. These two bone minerals have different crystal structures and physicochemical properties, implying their distinguished role in bone physiology. However, until now, the biological significance of the presence of a certain ratio between HAP and WH in bone is unclear. To address this fundamental question, bone mimetic scaffolds are designed to encapsulate human mesenchymal stem cells (MSCs) for assessing their osteogenic activity depending on different ratios of HAP and WH. Interestingly, cellular growth and osteogenic differentiation are significantly promoted when MSCs are grown with a 3-1 ratio of HAP and WH nanoparticles, which is similar to bone. One of the reasons for this synergism between HAP and WH in hydrogel scaffolds is that, while WH nanoparticles can enhance osteogenic differentiation of MSCs compared to HAP, WH counterintuitively decreases the mechanical stiffness of nanocomposite hydrogels and hinders the osteogenic activity of cells. Taken together, these findings identify the optimal ratio between two major minerals in bone mimetic scaffolds to maximize the osteogenic differentiation of MSCs.

Statement of significance: Human bone minerals are composed of HAP and WH inorganic nanoparticles which have different material properties. However, the reason for the coexistence of HAP and WH in human bone is not fully identified, and HAP and WH composite biomaterial has not been utilized in the clinic. In this study, we have developed bone mimetic HAP and WH nanocomposite hydrogel scaffolds with various ratios. Importantly, we found out that HAP can promote the mechanical stiffness of the composite hydrogel scaffolds while WH can enhance the osteogenic activity of stem cells, which together induced synergism to maximize osteogenic differentiation of stem cells when mixed into 3-1 ratio that is similar to human bone.

Keywords: Bone materials; Hydroxyapatite; Mesenchymal stem cells; Osteogenic differentiation; Whitlockite.

Fig. 1

The two major inorganic components of bone: hydroxyapatite (HAP: Ca₁₀(PO₄)₆(OH)₂) and whitlockite (WH: Ca₁₈Mg₂(HPO₄)₂(PO₄)₁₂) nanocrystallites. a) Schematic of bone structure ranging from the nanometer to micrometer scales, showing that bone tissue consists of cylindrical osteon units at the microscale, which are composed of collagen nanofibers with HAP and WH bone mineral particles at the nanoscale. b) Amount (weight percent) of WH in the inorganic part of hard tissues, calculated based on the magnesium amount. Data were obtained from previous publications, with different colors representing different references (Orange circle: Driessens et al. [16], Magenta triangle: Gabriels et al. [10], red triangle: Carlstrom et al. [16], purple triangle: Breibart et al. [11], blue triangle: Wooddard et al. [12], yellow triangle: Duckworth et al. [13], green triangle: Schraer et al. [14], blue rectangle: Long et al. [15] c) Schematic of experiments to identify optimal conditions in the stem cell niche for osteogenic differentiation of human mesenchymal stem cells (MSCs) with regard to the different ratios of HAP and WH.

Fig. 2

Engineered bone-mimetic nanocomposite hydrogel scaffolds. FESEM observation of a) synthesized WH nanoparticles with rhombohedral shape, b) HAP nanoparticles with needle shape, and c) microporous structured GelMA hydrogel scaffold after freeze drying. d-f) The nanoscale features of composite hydrogel scaffolds, showing that rhombohedral shaped WH nanoparticles and needle-shaped HAP nanoparticles are homogeneously dispersed in the GelMA hydrogel. Insets in each panels show the EDS area scan analysis, where WH incorporated GelMA hydrogel had a Mg peak, and HAP incorporated GelMA exhibited a higher ratio of the Ca and P peak intensity. g) XRD analysis of HAP-incorporated GelMA hydrogel and WH-incorporated GelMA hydrogel, confirming that their crystal phase is maintained after fabrication. h) FT-IR analysis result of HAP-incorporated GelMA hydrogel and WH-incorporated GelMA hydrogel, confirming that the chemical groups of HAP, WH and GelMA hydrogel remain intact after fabrication. i) The Young’s modulus of the composite hydrogel scaffolds depending on different concentrations and ratios of HAP and WH in GelMA hydrogel were calculated from compression tests. The green dotted line indicates the stiffness of pure GelMA hydrogel (~23 kPa). (*p<0.05, **p<0.01)

Fig. 3

Cellular viability, proliferation, and spreading in composite hydrogel scaffolds with different ratios of HAP and WH. a–d) Viability (a–b) and proliferation (c–d) of cells grown in 3D composite hydrogel scaffolds with different ratios of HAP and WH, in two media conditions (DMEM and osteoinductive media) were compared. e–k) Spreading of cells in 3D composite hydrogel scaffolds depending on different ratios of HAP and WH at day 7 was observed under confocal microscopy and quantified based on their spreading area. Cellular nuclei and actin were stained with DAPI (blue) and phalloidin (green). (*p<0.05, **p<0.01)

Fig. 4

Effect of ratios between HAP and WH on osteogenic activity of MSCs that are encapsulated in bone mimetic 3D HAP/WH nanocomposite hydrogel scaffolds. a) Fluorescence images of immunostained MSCs encapsulated in 3D composite hydrogel scaffolds with different ratio of HAP and WH. Osteogenic marker expression of MSCs (ALP, OCN, OPN, RUNX2) were compared after grown in DMEM media conditions for 2 weeks. b) Osteogenic protein expression levels of MSCs in panel a were quantified by using ImageJ software and dividing the area of stained images of experimental groups by control groups. The result indicated that cells grown in scaffolds with HAP and WH nanoparticles which were mixed in a 3 to 1 ratio had the highest level of osteogenic gene expression. c) Relative osteogenic gene expressions of MSCs were evaluated by real-time quantitative PCR analysis and calculated by 2^−ΔΔCt method, after 2 weeks of culturing in 3D composite hydrogel scaffolds. d) Fluorescence images of immunostained MSCs that were grown in 3D composite hydrogel scaffolds with different ratio of HAP and WH for 2 weeks under osteoinductive media conditions. e) Quantified osteogenic protein expression levels of MSCs from panel d, based on the same calculation method as mentioned above. f) Relative osteogenic gene expression of MSCs that were cultured in 3D HAP/WH composite hydrogel scaffold for 2 weeks under osteogenic media conditions. (*p<0.05, **p<0.01)

Fig. 5

Effect of stiffness of composite hydrogel scaffold and the optimal ratio between HAP and WH for directing osteogenic differentiation of MSCs. a) Stiffness of GelMA hydrogel scaffold was tuned by controlling UV exposure time, to assess the effect of the scaffold stiffness on the osteogenic activity of cells grown in it. b) Relative osteogenic gene expression of MSCs cultured in 3D GelMA hydrogel scaffolds with different stiffness level showed that cellular osteogenic activity was generally enhanced as the stiffness of hydrogel scaffold increased, in a range of 13-26 kPa. c) Optimal ratio between HAP and WH for inducing osteogenic differentiation of MSCs was obtained in a narrowed range of 65%HAP+35%WH to 85%HAP+15%WH, by using quantitative real-time PCR. The result showed that the osteogenic activities of cells were more upregulated when the ratio between HAP and WH is in a range of 75%HAP+25%WH to 85%HAP+15%WH. (*p<0.05, **p<0.01)