gms | German Medical Science

Objectives: Large bone defects denote a major socioeconomic issue. In this context, the concept of bone tissue engineering represents an innovative, promising cell-based strategy for effective bone regeneration. Within this project a fully tissue-engineered vascularized bone implant consisting of a hard tissue and an artificial vascular component was developed and evaluated in vitro.

Methods: Cylindrical, porous scaffolds made of beta-tricalciumphosphate (b-TCP, hard tissue component) were seeded with mesenchymal stromal cells and cultured under static conditions for 7 days. At the same time artificial vessels were produced by fibrin precipitation from plasma by means of an automated system. Tubular fibrin segments were generated in a high velocity rotating custom-made mold. During the generation process fibrin segments were seeded with endothelial and smooth muscle cells differentiated from circulating vascular progenitor cells that were isolated previously from blood. Thereafter, segments were placed in a pulsatile bioreactor and cultured for 7 days. Subsequently, b-TCP scaffold and artificial vessel were combined and placed in a special-designed bioreactor enabling perfusion of the vascular component.

The experiment was repeated 3 times and run with 3 groups: (1) scaffold without vessel, static cultivation in tube; (2) scaffold with vessel, static cultivation within bioreactor and (3) scaffold with vessel, perfusion of the vessel within bioreactor. After 14 days of cultivation oxygen concentration was measured at different positions within the b-TCP scaffolds. Cell viability and distribution was assessed by fluorescent live/dead staining whereas cell differentiation and cell outgrowth was analyzed by CD31 and von Willebrand factor (vWF) antibody staining. Additionally, histological analyses of the artificial vessels were performed.

Results and Conclusion: Perfusion of artificial vessels increased oxygen concentration in b-TCP scaffolds leading to homogeneous cell distribution and enhanced cell viability as verified by live/dead staining. Cells expressing CD31 were only found on vessel-containing scaffolds of experiment 1 that were cultured dynamically in the bioreactor suggesting that cells were grown out from the artificial vessel into the pores of the b-TCP scaffold. Von Willebrand factor was present in all scaffolds from all experiments. Histological analyses revealed enhanced expression of vWF, endothelial nitric oxide synthase and collagen type 4 in perfused vessels as compared to not perfused ones.

Due to several general conditions results of the 3 experiments were not consistent. Standardization of construct production processes will help to overcome this problem. Nevertheless, our investigations suggest that it is possible to generate a fully tissue-engineered vascularized bone implant in vitro suitable for connection to the host vasculature system during implantation.