Implantation of Autologous Endothelial Progenitor Cells (EPCs) as a Conduit for Enhanced Vascularized Bone Regeneration
Statement of the problem: Effective bone graft regeneration requires rapid neovascularization of implanted grafts to ensure the survival of cells in the early post-implantation phase. Here, we investigated the incorporation of autologous endothelial progenitor cells (EPCs) for enhanced vascularization and bone regeneration in vivo.
Materials and methods: Cell
Culture. EPCs were isolated from
New Zealand rabbit peripheral blood,1 and
cultured in endothelial growth media (EGM2). Human umbilical vein endothelial cells (HUVECs, ATTC) were cultured in EGM2. Protein
Expression. Western blot was performed to
evaluate protein expression of key endothelial cell markers, CD31 and
von Willabrand Factor (vWF). Matrigel
2D assay. Angiogenic
potential of endothelial cells was determined via Matrigel
2D assay. Samples were incubated with calcien-AM,
and imaged using confocal microscopy. In
Vitro Culture of Cell-Seeded Constructs. 2.5x105
EPCs were seeded on scaffolds and cultured for 2 days.2
Immunostaining of CD31 and vWF was performed, and imaged via confocal-microscopy. In Vivo Studies. Pre-vascularized
grafts (scaffolds seeded/cultured with EPCs), and control/acellular
grafts (scaffolds not seeded with cells) were implanted in
a rabbit ulnar bone defect model (5x105
cells/graft). MicroCT was utilized to evaluate new
bone formation, and histology (PMMA embedded; Goldner's
Trichrome staining) was used to quantify neovascularization at defect
site.
Methods of data analysis: For quantification of bone mass in rabbit bone defect study (n=6/group), we utilized MicroCT, which was calibrated to a stepped hydroxyapatite phantom (mg HA/cm3). For quantification of vascularization, we counted vascular structures in ten random images/group (20X-magnification). For vascular and bone quantification analysis, a two-way analysis of variance (ANOVA) was preformed. Error is reported as the standard deviation and significance was determined using a probability value of p<0.05.
Results: To confirm endothelial expression of the isolated peripheral blood-derived EPCs, we used HUVECs as a control for typical endothelial cell expression. As seen in Fig. 1A, EPCs have similar high angiogenic abilities as HUVECs, and uniformly participated in elaborate network formations when seeded on Matrigel. There was no significant difference between formed branch points and total tube length in comparison to HUVECs. In addition, EPCs express endothelial markers CD31 and vWF in culture (Fig. 1B), as well as when seeded on biodegradable 3D porous scaffolds (Fig. 1C).
Two day cell-seeded constructs were implanted in a rabbit ulnar defect to evaluate their angiogenic and osteogenic potential in vivo. After 12 weeks, we observed enhanced bone formation in EPC-seeded-grafts via quantitative MicroCT (~27%) (Fig. 2A,B), and histological Trichrome staining (Fig. 2C,D). Significant enhancement of vascularization was observed in EPC group as compared to acellular (~47%) (Fig. 2D).
Conclusions: Insufficient vascularization of bone grafts hinders bone regeneration. Here, we successfully isolated EPCs from peripheral blood, a clinically-relevant cell source. EPCs display stable endothelial phenotype, express key endothelial markers, and demonstrate excellent vasculogenic performance. Further, implantation of EPCs resulted in significantly enhanced vascularization and bone regeneration in a rabbit critical-sized bone defect model. The fundamental implications of this study are evident in addressing a grand challenge in vascularized bone regeneration for oral/maxillofacial surgery applications.
References:
1. Amini et al. J Orthop Res. 30(9):1507-15.
2. Amini et al. Tissue Eng A. 18(13-14):1376-88.