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.

Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro.

Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x10⁶ cells/ml, in Myelocult^TM medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x10⁵ cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations.

Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations.

Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications.