The frequency of CTPs in bone marrow is quite low and this rarity highlights the need for clinically relevant selection strategies to optimize the impact of these cells in a graft site. This project represents the first exploration of the potential utility of using the extracellular matrix niche around a cell as a potential marker for positive or negative selection.
We hypothesized that hyaluronan represents a distinguishing feature of the in vivo niche of one or more subsets of the heterogeneous population of CTPs in bone marrow, and would therefore provide a means for enrichment of CTPs from a fresh bone marrow aspirate. HA-based magnetic separation resulted in the isolation of a cell fraction enriched in highly proliferative CTPs.
We also hypothesized that positive selection of CTPs based on hyaluronan (HA) will increase the efficacy of a bone graft in vivo due to increased concentration and prevalence of CTPs in a graft site. In order to test this hypothesis in vivo in a biologically relevant large animal model, we designed a new magnetic separation system to process larger volumes of marrow and defined a preferred method (drip soaking) for loading of HA-positive cells onto scaffolds.
The canine femoral defect model provides a method for comparison of bone grafting materials using four cylindrical bone defects in the canine femur. Defects were grafted using either HA-positive cells or heparinized bone marrow aspirate (hBMA). Both defects grafted with MS-processed HA-positive cells and hBMA showed robust bone formation at 4 weeks, but the amount of total bone mineral in the defect was not different between the two groups as assessed by microCT. Using histomorphometric analysis, both the area of new bone formation and vascular sinusoids was significantly greater when MS processing was used.
In aggregate, these data support the initial hypotheses of these projects. They provide the first proof-of-concept that HA can be used as a marker for selection of CTPs and that rapid intraoperative processing of bone marrow using HA can improve the outcome of local bone regeneration in vivo.