Human perivascular stem cell-based bone graft substitute induces rat spinal fusion

Abstract

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair.

Keywords: Adventitial cell; Mesenchymal stem cell; Osteogenesis; Pericyte; Perivascular stem cell; Tissue engineering.

Figure 1.

Manual palpation. Acellular control was compared with three concentrations of hPSC-based bone graft substitutes. Spines were harvested 4 weeks postoperative and analyzed by manually applying flexion and extension forces against the L4/L5 vertebrae. (A): An average score of ≥4 was considered fused. (B): Fusion was apparent in 20% (1 of 5) of control-treated samples and 100% (6 of 6), 80% (4 of 5), and 100% (6 of 6) in 0.15 × 10⁶, 0.50 × 10⁶, and 1.50 × 10⁶ hPSC-treated animals, respectively. ∗∗, p ≤ .01 compared with control. No significant difference in fusion scores or rates was observed between hPSC-treated groups. Abbreviation: hPSC, human perivascular stem cell.

Figure 2.

Micro-computed tomography (micro-CT) analysis. (A): Reconstructions of high-resolution micro-CT scans, shown in frontal and axial planes. (B–F): Micro-CT quantifications were next performed for bone mineral density (B), fractional bone volume (C), trabecular thickness (D), trabecular number (E), and trabecular spacing (F). ∗∗, p ≤ .01 compared with control. No significant difference in micro-CT parameters was observed between hPSC-treated groups. Abbreviations: BMD, bone mineral density; BV/TV, fractional bone volume; hPSC, human perivascular stem cell; Tb. N, trabecular number; TB. Sp, trabecular spacing; Tb. Th, trabecular thickness.

Figure 3.

Biomechanical/finite element analysis. (A): A uniform compressive stress force of 0.5 MPa was applied on the superior surface of the L4/L5 spinal segment. (Gy scale bar indicates all values exceeding 25 MPa). (B): Cuboidal specimens from newly formed bone were next assessed for von Mises stress. (C): Quantification of cuboidal segments of newly formed bone for mean von Mises stress. Abbreviation: hPSC, human perivascular stem cell.

Figure 4.

Histological analyses. (A, B): Coronal sections of spinal fusion stained with hematoxylin and eosin (A) and Masson’s trichrome (B). (C): Quantification for fractional bone area. (D, E): Representative osteocalcin (D) and bone sialoprotein, immunohistochemical staining, and quantification (E). Yellow arrows = hypertrophic chondrocytes; green arrow = osteoid; black scale bar = 0.5 cm; blue scale bar = 100 μm; green scale bar = 50 μm. ∗, p ≤ .05; ∗∗, p ≤ .01 compared with control. A slight dose-dependent effect was observed in histomorphometric analysis between hPSC-treated groups. Abbreviations: BSP, bone sialoprotein; H&E, hematoxylin and eosin; hPSC, human perivascular stem cell; OCN, osteocalcin.

Figure 5.

Species-specific MHC analyses. (A, B): Immunohistochemical staining of human- (A) or rat-specific (B) MHC. (C, D): Quantifications for osteoblasts (C) and osteocytes per higher power field (D). Blue scale bar = 100 μm. ∗, p ≤ .05 compared with rat-specific control values; ∗∗, p ≤ .01 compared with rat-specific control values; ##, p ≤ .01 compared with human-specific control values. A significant dose-dependent effect was observed between hPSC-treated groups. Abbreviations: hMHC, human-specific major histocompatibility complex; HPF, higher power field; hPSC, human perivascular stem cells; MHC, major histocompatibility complex; Ob, osteoblasts; Ot, osteocytes; rMHC, rat-specific major histocompatibility complex.

Figure 6.

Bone labeling analyses. (A): Representative fluorescent microscopy pictures of calcein/calcein-labeled mineralization fronts of each treatment group. (B): Quantification of relative fluorescence of calcein/calcein mineralization fronts. (C): Representative fluorescent microscopy pictures of calcein/demeclocycline-labeled mineralization fronts. Green, calcein; orange, demeclocycline. A significant dose-dependent effect was observed in calcein staining between hPSC-treated groups. Abbreviation: hPSC, human perivascular stem cell.