Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Abstract

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC-chitosan and CPC-chitosan-fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC-chitosan-fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC-chitosan-fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.

Fig. 1

hBMSCs encapsulated in alginate on day 1: (A) live cells in alginate alone; (B) live cells in CPC control; (C) live cells in CPC–chitosan; (D) live cells in CPC–chitosan–fiber. hBMSCs encapsulated in alginate on day 21: (E) live cells in alginate alone; (F) live cells in CPC control; (G) live cells in CPC–chitosan; (H) live cells in CPC–chitosan–fiber.

Fig. 2

Live/dead results of encapsulated hBMSCs inside alginate hydrogel beads, CPC, CPC–chitosan and CPC–chitosan–fiber constructs (means ± SD, n = 6). (A) Percentage of live cells on days 1, 7, 14 and 21. (B) Live cell density inside the four different constructs on days 1, 7, 14 and 21. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test, family confidence coefficient 0.95).

Fig. 3

Wst-1 assay results for encapsulated hBMSCs inside alginate hydrogel beads, CPC, CPC–chitosan and CPC–chitosan–fiber constructs (means ± SD, n = 6). The viability of hBMSCs was quantified by recording the absorbance at 450 nm as an indication of dehydrogenase activity. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test, family confidence coefficient 0.95).

Fig. 4

ALP activity of encapsulated hBMSCs inside alginate hydrogel beads, and inside CPC, CPC–chitosan and CPC–chitosan–fiber constructs (means ± SD, n = 6). The ALP value was normalized to the DNA concentration, with units of mM pNpp min⁻¹/µg DNA. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test, family confidence coefficient 0.95).

Fig. 5

Mineralization by hBMSCs in hydrogel beads in the CPC–chitosan–fiber construct on (A) day 7 and (B) day 14, cultured in osteogenic medium. The live cell culture was stained with xylenol orange, which stains mineral a red color (means ± SD, n = 6). (C) hBMSC mineralization area fraction, which is the area of stained mineralization divided by the total area of the field of view of the image. This was done for the encapsulated hBMSCs in alginate hydrogel beads, in CPC, in CPC–chitosan and in CPC–chitosan–fiber constructs. Dissimilar letters in (C) indicate values that are significantly different (Tukey’s multiple comparison test, family confidence coefficient 0.95).

Fig. 6

SEM showing significant mineral formation via hBMSCs. (A) Mineral synthesized by hBMSCs encapsulated in alginate hydrogel beads on day 21 and (B) mineral synthesized by hBMSCs encapsulated in beads in the CPC–chitosan–fiber construct on day 21.

Fig. 7

Powder XRD pattern of minerals. XRD patterns of (A) the hBMSC-synthesized minerals collected on day 21 from beads in the CPC–chitosan–fiber constructs and (B) a known hydroxyapatite, formed by calcium phosphate cement conversion and provided by Dr. Shozo Takagi of the National Institute of Standards and Technology.