Synergistic enhancement of ectopic bone formation by supplementation of freshly isolated marrow cells with purified MSC in collagen–chitosan hydrogel microbeads

ABSTRACT

Purpose: Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration. Methods: Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen–chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum. Results: MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples. Conclusions: These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.

Keywords:

• Bone formation
• bone marrow mononuclear cells (BMMC)
• chitosan
• collagen
• ectopic
• hydrogel
• mesenchymal stem cells

Acknowledgments

The Microscope and Image Analysis Laboratories (MIL) facility at the University of Michigan provided assistance with laser scanning confocal microscopy. In the Orthopaedic Research Laboratories at the University Michigan, Bonnie Nolan provided assistance with rat bone marrow harvesting, Kathy Sweet provided assistance with animal handling and in vivo implants, John Baker provided assistance with histology, and Basma Khoury, Dana Begun, and Ethan Daley provided assistance with microCT scanning and image/data analysis.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Research reported in this publication was supported in part by the “Large Bone Defect Healing (LBDH)” Consortium of the AO Foundation and by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award numbers R21AR062709 and R01AR062636. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.