Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1-L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1-L2, (b) TCP/HA constructs group/L2-L3, and (c) autogenous bone graft group/L5-L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.

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Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10 7 cells/ml) and high (3×10 7 cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10 7 cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10 7 cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

Chondrocytes obtained from osteoarthritis (OA) joints has been recognized as an abnormal cell; however, it's proven to have potential in supporting cartilage regeneration. We have isolated chondrocytes from OA joints (OAC) and expanded chondrocytes growth medium (CGM). The growth kinetic, immunophenotyping and cell multilineage differentiation were analyzed to confirm the OAC stemness. The optimal condition to developed PLGA nanofibre with ratio 50:50 were 20% concentration of PLGA, flow rate with 0.3 mL/h, 10 kv voltage and 10 cm distance from nozzle to the collector. The toxicity level, scanning electron microscopy (SEM) and q-PCR analysis was performed in the present study. OAC fulfills the minimal criteria to be known to have stem cell as the cell easily adheres to the culture plate, shows high expression (≥95%) for CD13, CD29, CD44, CD73 and CD90 and less expression (≤2%) for CD45 and HLA-DR and potentially induced to mesodermal multilineage, which is osteocytes, adipocytes and chondrocytes. Toxicity test showed no adverse effect of PLGA towards the cell. Based on the cell-PLGA nanofibre interaction, difference in fibre size will influence the proliferation of the cell. Nanofibres with 100 nm in size showed high proliferation of OAC and better gene and protein expression compared to monolayer culture. Thus, we concluded that OAC has the potential to be used in cartilage regeneration based on the presence of stem cell markers as similar to the human bone marrow. The cartilage regeneration will be more efficient if OAC cultured on 3D microenvironment as showed in the present study.