Suck eci x 2006.cdr

Institut für Technische Chemie
Partner in der Forschung
der Universität Hannover
Institut für Technische Chemie, Callinstr. 3, 30167 Hannover
Application of a new rotating bed reactor system for cultivating 3D bone constructs
Kirstin Suck1, Larissa Behr1, Martijn van Griensven2, Hans Hoffmeister3, Thomas Scheper1,
1Institut für Technische Chemie der Universität Hannover, Callinstr. 3, 30167 Hannover, Germany Boltzmann Institut für experimentelle und klinische Traumatologie, Donaueschingenstr. 13, 1200 Wien, Austria erk GmbH, Ziegeleistr. 7, 16727 Eichstädt, Germany Abstract
Aim of the project is the development and testing of a rotating-bed bioreactor for the generationof bone tissue. Large bone defects caused by tumors, infectious diseases or trauma result in a medical need for bone regeneration. The principle of bone tissue engineering is to seedosteoblasts, precursor or stem cells onto an appropriate 3D matrix and to cultivate the cell exhaust air seeded scaffold in vitro in a suitabel biorector system. The generated tissue can be implantated into the defect of the patient.
During the differentiation of bone tissue different osteoblastic markers (e.g alkalinephosphatase AP) are expressed. Finally, the cells are embedded in the extracellular matrix and sparging begin to mineralise by depositing mineral along and within collagen fibrils. The suitability of twodifferent 3D macroporous scaffolds (Sponceram® and Sponceram bone tissue was investigated under static and dynamic conditions.
Sponceram® scaffolds
Material and Methods
Scaffolds: Macroporous ceramic Sponceram®
Cell seeding: Scaffolds were incubated for 24 h in medium at 37°C, 5 % Co . Either primary
osteoblasts or MC3T3-E1 cells were seeded on each scaffold in 96-well plates for 30 min at Standard medium: DMEM, 10 % FCS, antibiotics
Differentiation medium: Standard medium + 1 µM dexamethasone, 10 mM b-glycerol-
Differentiation medium + BMP-2 (10 ng/ml)
Cell viability was assayed using MTT-test.
Alkaline phosphatase (AP) was determined by an assay based on the hydrolysis of p-
nitrophenyl phosphate to p-nitrophenol.
Pre-screening
Bioreactor cultivation
(static condition)
(dynamic conditions)
Samples of Sponceram® of approximately 3 mm x The BIOSTAT® B plus RBS 500 was designed as a 3 mm x 4 mm were preconditioned for 24 h in cell multi-purpose high density cell culturing device for culture medium at 37°C, 5 % CO . Subsequently, anchorage-dependent cell lines and primary cells.
1.5 x 10 MC3T3-E1 cells in 80 µl medium were In this study the bioreactor was equipped with 4 seeded on each scaffold in 96-well dishes for 30 min Sponceram® carrier discs for each cultivation. Cell at gentle stirring at 37°C, 5 % CO . Non attached inoculation was carried out with a total volume of 2 ml cells were removed and the wells were filled up with cell suspension/disc with a cell density of 1 1 200 µl medium. The cultivations were performed for disc. To allow adhesion onto the Sponceram® surface the reactor was filled with 300 ml of differentiation The cells were cultivated in three different media: medium 30 min after cell inoculation. The bioreactor standard medium, differentiation medium and features a unique technology for improved oxygen and differentiation medium containing BMP-2. The nutrient supply through the alternating exposure to effect of the media composition on cell viability and medium and gas phase. The cultivation was performed the differentiation process was investigated and at 37°C, 2 rpm and a pH of 7.3 for 25 days.
MC3T3-E1
Primary Osteoblasts
Results for the cell viability (MTT-test) of MC3T3-E1 cells During the bioreactor cultivation the cell growth of the cultured on Sponceram in standard medium, differentiation osteobalsts was monitored by the determination of glucose medium and BMP-2 medium show a high cell viability during consumption using the YSI 2700 (Yellow Springs the first ten days. The cell viabilty attained a plateau phase Instruments, USA). Cells were cultivated on Sponceram followed by a decrease after 10 days due to high confluence and Sponceram /HA using differentiation medium. Total glucose consumption after 25 days: 14.05 g.
Glucose consumption during the bioreactor cultivation The differentiation process of the preosteoblastic The mineralisation of the extracellular matrix determination of the early osteogenic marker alkaline phosphatase (AP). The highest enzyme activity was Kossa. In each picture scaffolds are shown achieved at day 5 in BMP-2 medium due to the after bioreactor cultivation for 25 days (right) differentiation induction of the growth factor.
in comparison to the control matrix (left). The Scanning electron micrographs (SEM) show the cell morphology After bioreactor cultivation of the primary of MC3T3-E1 cells cultured on Sponceram .
osteoblasts a dense layer of cells and parallel under static conditions in 96-well dishes display that the cells grew structures of ECM fibrils can be observed in well inside the macroporous structure of Sponceram® showing a the SEM pictures. The ceramic material is no cuboid morphology of osteoblasts-like cells. Cells were cultured longer visible. Moreover, the formation of for 10 days. They grew as a network having intercellular contacts Conclusion
In summary, this study demonstrated that the newly developed ceramic material Sponceram® is an appropiate scaffold for the cultivation of MC3T3-E1 cells. The macroporous structure ofthe scaffold contributed to a fast cell attachment and proliferation. The ultimate shape of the 3D structure supports the differentiation process of preosteoblastic cells even in the absenceof BMP-2. The rotating-bed bioreactor BIOSTAT® Bplus RBS features a unique technology for improved oxygen and nutrient supply for the cells through the alternating exposure tomedium and gas phase. Therefore, the BIOSTAT® Bplus RBS equipped with Sponceram® discs provides an optimal environment for bone tissue generation. Primary osteoblasts weresuccessfully cultivated on Sponceram® and Sponceram /HA for 25 days in the bioractor system. The analyses revealed a bone tissue-like generation of a fibril structured mineralised Acknowledgement
The authors like to thank Dr. A. Feldhoff and F. Steinbach (University of Hannover) for the SEM pictures und Prof. W. Sebald for kindly donating BMP-2.

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