ALLOGENIC MESENCHYMAL STEM CELLS FROM PERIPHERAL BLOOD REGENERATE CRITICAL-SIZED BONE DEFECT IN RABBIT ULNAE

Abstract

Introduction: Peripheral blood derived mesenchymal stem cells (PBMSCs) are multipotent cells capable of forming bone, cartilage, fat, and other connective tissues. Bone marrow derived mesenchymal stem cells (BMMSCs) have promoted repair a critical-sized bone defect in several animal models including mouse, rat, rabbit, and dog. The aim of this study was to investigate whether or not the use of allogenic BMMSCs and PBMSCs could regenerate a critical-sized bone defect in rabbit ulnae.

Methods: Rabbit peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation method and cultured at a density of 100,000/ cm2 in flasks with DMEM 15% FCS. Colony forming efficiency (CFE) was calculated and their multipotential differentiations into bone, cartilage, and fat were examined under different induction conditions. Specific differentiation markers were examined using cytochemistry and immunocytochemistry methods in the PBMSCs. Critical-sized ulna bone defects, 20 mm in length, were created in the mid-diaphysis of both ulnae in twelve 6 month old NZW rabbits. The ulnar defects were treated as the following 5 groups: empty control (n=4), PBMSCs/Skelite (multi-phase porous calcium phosphate resorbable substitute, EBI Company, USA) (n=5), BMMSCs/Skelite (n=4), PBMNCs/Skelite (n=5), and Skelite alone (n=5). All animals were sacrificed 12 weeks after treatment. The bone regeneration was evaluated by regular radiography, and all samples were subject to peripheral quantitative computed tomography (pQCT) and histological examination at the end point.

Results: The CFE of PBMSCs ranged from 1.2 to 13 per million mononuclear cells. Spindle and polygonal shaped cells were found in the primary PBMSCs colony, showing similar differentiation potential with BMMSCs. Mineralized bone nodules formed under osteogenic media were positive for Alizarin Red S staining in the PBMSCs. Chondrogenic differentiation was identified in serum free media containing TGF-¦Â1 (10 ng/ml), with type II collagen expression and Alcian blue positive nodule formation. Adipocytic differentiation was tested with or without adipogenic media, with positive Oil Red O staining for lipid accumulation and CEBP¦Á expression in the PBMSCs. After twelve weeks implantation, the ulnar defects were not healed in the empty control group; the total bone density in PBMSCs/Skelite and BMMSCs/Skelite treated defects were greater than that of PBMNCs/Skelite and Skelite alone treated groups (p< 0.05), with higher score of X-ray evaluation (p< 0.05). Histologically, there were a greater amount of new bone present in both the PBMSCs/Skelite and BMMSCs/Skelite treated groups compared to the PBMNCs/Skelite and Skelite alone treated groups.

Conclusions: This study demonstrated that PBMSCs were multipotent cells; allogenic PBMSCs loaded onto porous calcium phosphate resorbable substitute had enhanced bone regeneration of a critical-sized segmental defect in the rabbit ulna. PBMSCs may be a new source of osteogenic stem cells for bone regeneration and tissue engineering, and further investigations are undergoing to clarify their functions.