JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2008; 2: 400–407. Published online 29 August 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.110 Cultivation of human bone marrow stromal cells on three-dimensional scaffolds of mineralized collagen: influence of seeding density on colonization, proliferation and osteogenic differentiation Anja Lode # *, Anne Bernhardt # and Michael Gelinsky Max Bergmann Center for Biomaterials, Technische Universit¨ at Dresden, Institute of Materials Science, Budapester Strasse 27, D-01069 Dresden, Germany Abstract In this study human bone marrow stromal cells (hBMSCs) were cultured on three-dimensional porous scaffolds of biomimetically mineralized collagen type I developed for bone engineering. Three different cell numbers were used for seeding of the nanocomposites, and the impact of the seeding density on proliferation and osteogenic differentiation of hBMSCs was investigated. In addition, the effect of the seeding cell number on seeding efficiency and distribution of the cells within the scaffolds was studied. Our data revealed that the open and interconnecting porosity of the mineralized collagen scaffolds allows a very efficient seeding for all seeding densities tested. Although penetration of the cells into the interior of the scaffolds was demonstrated for all seeding densities, the application of higher cell numbers resulted in a better colonization also of the deeper scaffold regions. A substantial influence of the seeding density was observed on proliferation and osteogenic differentiation of hBMSCs. Thus, the highest proliferation rate and specific alkaline phosphatase activity was found for the cell matrix constructs seeded with the lowest density. RT–PCR analyses revealed a higher expression of alkaline phosphatase and bone sialoprotein II at lower seeding densities; however, expression of osteopontin was unaffected by the seeding cell number. Our results demonstrated that the seeding density might be an important factor for the development of optimal cell matrix constructs for bone tissue engineering. Copyright  2008 John Wiley & Sons, Ltd. Received 4 June 2008; Accepted 5 June 2008 Keywords mineralized collagen; mesenchymal stem cells; bone marrow stromal cells; proliferation; osteogenic differentiation; alkaline phosphatase; seeding density; reverse transcriptase PCR 1. Introduction Bone substitutes for the treatment of osseous defects are in high demand. The use of autologous bone graft, the current gold standard as osteogenic bone replacement *Correspondence to: Anja Lode, Max Bergmann Centre for Biomaterials, Technische Universit¨ at Dresden, Institute of Materials Science, Budapester Strasse 27, D-01069 Dresden, Germany. E-mail: anja.lode@nano.tu-dresden.de # These authors contributed equally to this paper. material, is restricted by the limited amount of bone which can be harvested and considerable donor site morbidity. Therefore, a variety of synthetic bone graft materials have been developed. However, for the repair of large defects, an osteogenic potential of the bone graft sub- stitute is required which will be provided only by viable cells. The combination of artificial biodegradable matrices with autologous cells according to the tissue engineering (TE) concept is a promising strategy to overcome the limitations of autologous bone graft. For this approach, Copyright  2008 John Wiley & Sons, Ltd.