Tissue engineering of long bones with a vascular matrix in a bioreactor

Background. The purpose of this study was to assess the feasibility of tissue engineering a vascular, osteogenic construct. Methods. The capillary system of a vascular biologic matrix (BioVaM) was seeded. with human bone marrow stromal Cells (hBMSC) that were differentiated in an endothelial cell-specific media. Within two layers of the vascular matrix, hBMSC were either cultivated with demineralized bone matrix (DBM) or liquefied small intestine submucosa (SIS). After 3 and 6 weeks in a perfusion bioreactor, endothelial cell phenotype and cell adherence were investigated with immunohistology. The contents of alkaline phosphatase (ALP) and osteocalcin (OC) were determined and a load to failure test was executed. Histologic differences were analyzed using H&E and von Kossa staining. Results. The capillary system of the matrix stained positive for endothelial markers. ALP activity increased in DBM after 3 and 6 weeks (2.2 +/- 1.0 and 2.4 +/- 0.6 U/l/mug protein, p<0.05). Osteocalcin levels were highest for DBM (23.5 +/- 21.1 and 11.1 +/- 4.5 ng/ml, p<0.05). Tissue strength was enhanced in SIS and DBM after 6 weeks (15.1 +/- 3.7 N/mm(2) and 17.0 +/- 5.0 N/mm(2), p<0.05). Tissue morphology resembled osteons in DBM and SIS cultures; however, areas that stained positive for phosphate were more often found in the DBM group. Conclusion. The capillary system of the matrix could be seeded with BMSC in vitro. Seeding of hBMSC mixed with DBM or SIS within a vascular matrix led to enhanced tissue strength and to osteon-like tissue structures. Osteogenic differentiation was highest when DBM was used.