Bone regeneration of double-canaled implant with peripheral blood derived mesenchymal stem cells: in vitro and in vivo

Abstract

Purpose: Peripheral blood (PB) is known as a source of mesenchymal stem cells (MSCs) like bone marrow (BM). It is easy to get, however, it has limited proved that the osteogenic differentiation potential of PB-derived MSCs after implant in dentistry. In this study, we characterized rabbit PB-derived MSCs compared to BM-derived MSCs in vitro and in vivo. Furthermore, we proved the osteogenic ability in the process of double-canaled implant surgery model of rabbits. Materials and methods: After collection and purification of PB and BM from New Zealand white rabbit, we isolated PB- and BM- derived MSCs. PBMSCs (peripheral blood mesenchymal stem cells) were cultured on the extracellular matrix (ECM)-coated culture plate which was made by BMMSCs and used to obtain the PBMSCs. BMMSCs (bone marrow mesenchymal stem cells) were cultured on the plastic culture plate, and used as a control. For characterization of PBMSCs, we performed morphology study, fluorescence-activated cell scanner (FACS) to analyze positive expression of MSC surface molecular CD14 and CD90, cell proliferation assay, and multiple differentiation assay in vitro comparing with BMMSCs. And then the PBMSCs were mixed with hydroxyapatite/tricalcium (HA/TCP), transplanted into immunocompro- mised mice (BALB/c), and stained with hematoxylin and eosin (H&E) to identify the osteogenic ability of the PBMSCs. For bone regeneration of the PBMSCs in double-canaled implant, HA/TCP, mix of PBMSCs and HA/TCP, and mix of BMMSCs and HA/TCP were applied to double-canaled implant, but defect double-canaled implant were not filled. During the installation into rabbit tibia, the upper and lower canal of implant were located in the cortical and marrow area respectively. After 3 weeks and 6 weeks, the sectioned implants were stained with toluidine blue and von kossa for histological examination and histomorphometric analysis. Results: The PBMSCs showed fibroblastic-like morphology, similar rate of BrdU positive cells to BMMSCs, and positively expressed CD90 (~31.8%), but negatively showed CD14 (~0.9%). For osteogenic differentiation, nodules were stained with red and black by Alizarin red S and von kossa, respectively. Lipid droplets were detected by Oil red O staining for adipogenic differentiation, and cartilage matrices were observed by Alcian blue and Safranin O staining for chondrogenic. After transplantation in vivo, the PBMSCs generated bone which were stained with H&E. In result, there was no difference bone formation area between PBMSCs (~41%) and BMMSCs (~43%). Histological examination of the double-canaled implant in PBMSCs and BMMSCs showed more mature bone, which were stained with toluidine blue and von kossa in both the upper and lower canals at 6 weeks and in the upper canal at 3 weeks. Histomorphometric analysis results proved that the PBMSCs (~16%) and BMMSCs (~17%) groups showed higher new bone (NB) than groups of HA/TCP (~12%) and defect only (~10%) in the upper canal at 3 weeks, however there was no different results of NB among all groups (defect only: ~3.7%, HA/TCP: ~4.8, BMMSCs: ~ 4.9%, PBMSCs: ~4.5%) in the lower canal. At 6 weeks, groups of the PBMSCs (upper canal: ~36%, lower canal: ~8%) and BMMSCs (upper canal: ~35%, lower canal: ~7.2%) showed higher NB than the HA/TCP (upper canal: ~21%, lower canal: ~6.0%) and defect only groups (upper canal: ~15%, lower canal: ~5.0%) in both the upper and lower canals. Conclusions: The PBMSCs have characteristics and bone regeneration ability just like BMMSCs, not only in vitro but also in vivo. ECM was effective of obtaining PBMSCs. Therefore, the PBMSCs could be the promising source for bone regeneration in dental clinical uses.