Human Perivascular Stem Cell-based Bone Graft Substitute Induces Rat Spinal Fusion

Background: Adipose tissue is an attractive source of mesenchymal stem cells (MSC) due to its abundance and accessibility. Previous studies have defined a population of native MSC termed perivascular stem cells (PSC), purified from diverse human tissues, including adipose tissue. Human (h)PSC are a homologous population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting (FACS), with properties identical to those of culture identified MSC. The evidence supporting the use of human perivascular stem cells (hPSC) for bone tissue engineering is based on our prior studies: firstly, pancreas- (and other organ-) derived human pericytes exhibit robust in vitro osteogenic differentiation and intramuscular bone formation and angiogenesis[31]; next, adipose-derived hPSC form significantly increased intramuscular bone compared to patient-matched unpurified cells and demonstrate in vivo trophic and angiogenic effects[32,33], and lastly adipose-derived hPSC exhibited improved calvarial bone defect healing as compared to unsorted SVF[34]. Methods: Athymic rats(n=5-6 per treatment group) were randomly distributed into four treatment groups of escalating human PSC(hPSC) dose, delivered on a demineralized bone matrix scaffold. After four weeks, animals were sacrificed and spinal fusion was assessed by manual palpation, high-resolution micro-Computed Tomography(microCT) and histology. Computerized biomechanical assays were performed using finite element analysis(FEA). Finally, immunohistochemistry, with anti-human/anti-rat Major Histocompatibility Complex Class (MHC) Class I, was performed to assay the origin of newly formed bone. Results: Treatment with hPSC successfully induced 80-100% fusion by four weeks compared to acellular-treated controls (20% fusion), confirmed by microCT and histological analyses. Biomechanically, it was observed that hPSC treatment induced more sound bone, capable of withstanding significantly higher force compared to control. Lastly, using immunostaining, it was observed that rat-specific osteoblasts outnumbered human-specific osteoblasts by a ratio of 3.9-10:1. Likewise, rat-specific osteocytes outnumbered human-specific osteocytes by a ratio of 7.8-30.8:1, indicating that hPSC operate to a large degree via paracrine signaling. Immunohistochemistry for species-specific antigens verified that hPSC play a direct role in bone formation, but also that hPSC play a role in paracrine support of host osteoprogenitor cell recruitment and/or differentiation.Conclusion: hPSC are a readily available MSC population that effectively forms bone without requirements for culture, pre-differentiation or exogenous cytokine stimulation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair