Biofunctionalized Calcium-Phosphate Cement Seeded with Stem Cells for Bone Engineering

Objective: Stem cell-encapsulating microbeads could be mixed into a paste such as calcium phosphate cement (CPC), where the microbeads could protect the cells from the mixing/injection forces.  After CPC is placed, the microbeads could quickly degrade to release the cells throughout the scaffold, while creating macropores.  The objectives of this study were to (1) construct alginate-fibrin microbeads encapsulating human umbilical-cord mesenchymal stem cells (hUCMSCs) embedded in the surface of novel biofunctionalized CPC; and (2) investigate microbead degradation, cell release, and osteodifferentiation on CPC.

Method: Hydrogel microbeads with sizes of 100-500 µm were fabricated with 1×10⁶ hUCMSCs/mL.  CPC was biofunctionalized with fibronectin (Fn) and Arg-Gly-Asp (RGD) for the first time.  Four scaffolds were tested: CPC mixed with Fn, CPC mixed with RGD, CPC grafted with RGD, and CPC without Fn or RGD (control).

Result: The degradable microbeads released hUCMSCs at 7-day, and the released cells successfully attached to CPC.  Biofunctionalizing CPC with Fn or RGD greatly improved cell attachment.  CPC grafted with RGD had live cell density at 21-day of (1070±40) cells/mm², higher than (115±20) cells/mm² for CPC control (p<0.05).  The released hUCMSCs underwent osteodifferentiation.  Alkaline phosphatase gene expression increased from 1 at 1-day to 14.3 at 7-day (p<0.05).  Osteocalcin increased from 1 at 1-day to 12.7 at 21-day (p<0.05).  The released cells attached to CPC and synthesized bone minerals, with mineralization (nM) increasing from (0.2±0.1) at 7-day to (7.8±1.0) at 21-day (p<0.05).

Conclusion: Alginate-fibrin microbeads embedded in CPC surface were able to quickly release the hUCMSCs which attached to biofunctionalized CPC.  Incorporating Fn and RGD into CPC greatly improved cell function, and CPC grafted with RGD had the fastest cell proliferation.  The released cells on CPC differentiated into the osteogenic lineage and synthesized bone minerals.  The novel biofunctionalized CPC with hUCMSC-encapsulating microbeads is promising to enhance bone regeneration in dental, craniofacial and orthopedic applications.