Comparison Of The Efficacy Of Bone Marrow Mononuclear Cells And Bone Mesenchymal Stem Cells In The Treatment Of Osteoarthritis In A Sheep Model

ObjectiveTo evaluate the therapeutic efficacy of uncultured bone marrow mononuclear cells (BMMCs) and bone mesenchymal stem cell (BMSCs) in an osteoarthritis (OA) model of sheep.Methods1. Fresh bone marrow was extracted from the superior iliac crest of under general anesthesia, and BMSCs were obtained by the whole bone marrow culture method. The shape and adherent growth of BMSCs of passage2was observed under inverted phase contrast microscope. The cells were induced to adipogenic, osteogenic and chondrogenic lineages. Intracellular lipid vesicles from the adipogenic monolayer cultures were stained with oil red O. Cytoplasmic alkaline phosphatase from differentiated osteoblasts was stained with ALP solution. Safranin-O was used to detect chondrogenesis of BMSCs.2. Eighteen sheep were randomly divided into three treatment groups:PBS (N=6), PBS+BMSCs (N=6), and PBS+BMMCs (N=6). Two contralateral joints of each group were selected at random as the control group (N=6). OA was induced in the right knee joint of all donor animals via complete anterior cruciate ligament (ACL) transection and medial meniscectomy. After12weeks, concentrated BMMCs obtained from autologous bone marrow harvested from anterior iliac crest or a single dose of autologous bone mesenchymal stem cells (BMSCs) suspended in phosphate-buffered saline (PBS) was delivered to the injured knee via direct intra-articular injection. Animals of the PBS group received vehicle alone.Knees of the four groups were compared macroscopically with international cartilage repair society (ICRS) scores and histologically with H&E and safranin-O staining8weeks after injection. Glycosaminoglycan (GAG) contents normalized to cartilage wet weight were measured at lesions of cartilage from medial condyle of the femur head. Gene expression levels of type II collagen (Col2Al), Aggrecan and matrix metalloproteinase-13(MMP-13) in cartilage were measured based on RT-PCR and prostaglandin E2(PGE2), Tumor Necrosis Factor-α (TNF-α) and Transforming Growth Factor beta (TGF-β) concentrations in synovial fluid were determined with enzyme linked immunosorbent assays (ELISA) at8weeks after injection.Results1. BMSCs were purified and expanded to form confluent cultures of adherent cells with a fibroblastic morphology. Osteogenesis of BMSCs was confirmed by the presence of ALP staining in the cytoplasm. The BMSCs exposed to adipogenic medium were strongly positive with oil red O staining. Chondrogenesis of BMSCs were confirmed by safranin-O staining.2. At8weeks post cell transplantation, partial cartilage repair was observed in the cell therapy, but not the PBS group (P<0.05). The BMSCs group showed higher regeneration of cartilage and lower proteoglycan loss than the BMMCs group (P<0.05). Concentrated BMMCs injection led to a weaker treatment effect, but also inhibited PGE2, TNF-a and TGF-β levels in synovial fluid and promoted higher levels of Aggrecan and Col2A1and downregulation of MMP-13in sheep chondrocytes compared with BMSCs, but still showed a significant treatment effect when compared with the PBS group(P<0.05).ConclusionsBMSCs showed therapeutic efficacy in a sheep model of OA. Despite a weaker therapeutic potential, the easier and faster process of collection and isolation of BMMCs supports their utility as an effective alternative for OA treatment in the clinic.