Multiphoton imaging for quantification of mesenchymal stem cell survival and distribution in PEG granular hydrogel scaffolds post-implantation into rat cranial bone defects

Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies aimed at promoting bone regeneration due to their secretory properties and osteoblastic differentiation capacity. However, typically < 5% of delivered MSCs are retained at the healing site within days of delivery via injection. In this work, granular PEG hydrogel scaffolds were used to deliver MSCs, labeled with fluorescent Quantum Dots, into critical-sized rat calvarial bone defects. The presence, survival, and distribution of MSCs within the hydrogel scaffold were evaluated with multiphoton microscopy at 3- and 7-days post-implantation. Additionally, endogenous cell infiltration into scaffolds was quantified, and markers for M1 and M2 macrophages were identified with immunohistochemistry. This multiphoton microscopy technique provides a quantitative analysis of exogenous MSC presence and survival and allows for micron-level spatial resolution of cell distribution throughout the implanted scaffolds. When ∼750,000 MSCs were implanted in a calvarial bone defect via PEG granular hydrogel scaffolds, ∼27% and ∼8% survived 3- and 7-days post-implantation, respectively. At 3- and 7-days post-implantation, exogenous MSCs and infiltrating endogenous cells, including M1 and M2 macrophages, were well distributed throughout the scaffolds. This multiphoton microscopy technique could be used to assess biomaterial delivery systems that can improve exogenous MSC presence and survival, facilitate endogenous cell infiltration, and investigate exogenous-endogenous cell interactions for bone regeneration therapies.