Stem Cell-Encapsulated Self-Setting Calcium Phosphate Scaffolds for Bone Regeneration

Due to its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. However, the CPC setting reaction may be cytotoxic to human mesenchymal stem cells (hMSCs). Objectives: (1) Encapsulate hMSCs into alginate hydrogel beads and combine with high strength CPC-chitosan and CPC-chitosan-fiber scaffolds; (2) Culture encapsulated hMSCs to differentiate down the osteogenic lineage and measure viability, differentiation and mineral formation. Methods: Alginate hydrogel was used to encapsulate and protect cells in CPC. hMSCs were mixed into three pastes: conventional CPC-control, CPC-chitosan, and CPC-chitosan reinforced with degradable poly(lactide-co-glycolide) fibers. Cell viability was assayed via live/dead staining and Wst-1 assay. Differentiation was measured by ALP activity, mineral staining, SEM and XRD. Results: Cell viability (mean±sd; n=5) after 21 d showed no significant difference between cells encapsulated in alginate alone (80.41±9.15)% or CPC control (74.49±4.87)%, CPC-chitosan (71.53±8.89)% and CPC-chitosan-fibers (69.26±5.43)% (p>0.1). ALP activity was highest at 14 d for all materials with activity for cells in alginate alone at (14.71 ± 4.05) significantly greater than CPC-control (8.97±1.67), CPC-chitosan (7.84±1.71), and CPC-chitosan-fibers (6.37±1.63) (p<0.05). When stained with xylenol orange, deposited mineral increased for all materials from 1 d to 21 d. At 21 d, mineralization increased significantly for CPC control (3.64±0.79), CPC-chitosan (4.08±0.95), and CPC-chitosan-fibers (4.56±0.94) compared with 14 d (p<0.05), while there was very little change for alginate alone (4.99±0.74) (p>0.05). SEM microscopy and powder x-ray diffraction confirmed the presence of poorly-crystalline hydroxyapatite-like mineral deposited by the cells. Conclusion: Encapsulation of hMSCs in alginate hydrogel protected the cells from the CPC setting reaction and allowed them to undergo osteogenic differentiation. The high strength, self-hardening CPC-chitosan scaffold is promising to be a delivery vehicle for stem cells to promote dental and craniofacial bone regeneration. Supported by NIH R01 DE14190 and Maryland Stem Cell Research Fund.