gms | German Medical Science

Objective: Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) and osteoblasts (OB) for clinical use in bone engineering. However on a cellular and molecular level, the processes surrounding new bone formation remain poorly characterized. It was hypothesized that bone cell origin, ossification type, and degree of vascularisation and bone neoformation is dependent on the nature and commitment of transplanted cells as well as supplemented growth factors such as rhBMP-7.

Methods: MPC were isolated from ovine bone marrow, OB from tibial compact bone. Cells were seeded onto mPCL-TCP cylindrical scaffolds at a density of 250.000 and cultured for 4 weeks under osteogenic conditions prior to implantation in to NOD/SCID mice. Subcutaneously transplanted specimens included scaffolds with OB, Scaffold with MSC with and without 5 μg of rhBMP-7 (Stryker) administrated in 54 μl of tisseel (Baxter) to the inner duct of the scaffold intraoperatively. Cells were either non-labeled or marked with BrdU. Controls included scaffolds only, scaffold-tisseel, scaffold-tisseel-BMP. A number of 6 specimens was transplanted per group. After sacrifice, samples were compression tested (Instron microtester), scanned in a μCT scanner (Scanco), and processed for either paraffin (n=3) or PMMA embedding (n=3). H&E, von Kossa, TRAP and alcian blue staining were performed as well as immunohistochemistry for osteocalcin, collagen II, and vWF. Histology sections were quantified using Image J software to quantify the amount of mineralization in a given area of section. Labeled cells were visualized using a BrdU detection kit (Zymed). Statistical analysis was carried out using the student’s t test and p values <0.05 were considered significant.

Results and conclusions: Quantitative μCT analysis showed no bone formation in the scaffold only or scaffold-tisseel group and only scattered bone formation in scaffolds seeded with MSC. More deposited bone was observed when scaffolds or MSC seeded scaffolds were combined with rhBMP-7. However, significantly higher amounts of bone were observed in OB seeded scaffolds and even more when additionally stimulated with rhBMP-7. These results were also reflected in the compressive stiffness values and quantified v. Kossa staining. The formation of different tissue types within the transplanted constructs included mineralized bone positive for osteocalcin, adipose tissue and fibrous connective tissue with clear blood vessel formation. Alcian blue and collagen II staining suggested endochondral bone formation for groups including MSC while rather intramembraneous ossification was found in OB groups. A higher osteoclastic activity was found in all groups including rhBMP-7.

In conclusion, the study demonstrates that the origin, type and degree of ossification as well as vascularisation is strongly dependent on the commitment of transplanted cells and additionally supplied growth factors such as rhBMP-7.