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Development of cell culture protocols for bone tissue culture in rabbit and human

The cell culture protocols were sucessfully developed based on original protocols in the rat system-using alpha MEM supplemented with ascorbic acid, beta glycerophosphate and dexamethasone in a 15% FCS supplementation. The optimized media for rabbit were based on the ZKT I formulation, where higher growth rates could be obtained. Supplementation with ascorbic acid (long acting), beta glycerophosphate and dexamethasone could be kept as in previous protocols. The supplementation with specific growth factors (BMPs) allowed the reduction of serum concentrations. In the human system, ZKT I proved most beneficial for the separation of the expansion and differentiation of cells, the fundamental underlying strategy in this project, and Ca-ion concentration could be identified as an additional factor to modulate differentiation and mineralization. The differentiation of human cells along the osteogenic pathway was investigated using a microarray, which was specifically developed for this project, and which allowed to identify the gene expression pattern as the cells differentiated along the osteogenic pathway within the various media employed.

A continuously perfused fixed-bed bioreactor system could be developed successfully which allowed for online monitoring and control of temperature, perfusion rate, pH, pO2, as well as circulation rate. Furthermore, a routine probing of the medium was possible to measure glucose and lactate levels, important to assess the metabolic status of the cells. The bioreactor system, which could be operated with a medium volume as low as 30 ml allowed for the fitting of cylindrical ceramic or composite carriers of 1 cm diameter and supported bone growth in vitro with a superior performance as in static or even dynamic bone cultures as described in the literature as reference models. The use of PFC emulsions affected cell attachment in the rabbit model and was therefore put on hold until non-attachment-affecting PFC emulsions are available.

The production of in vitro rabbit bone tissue constructs was successfully achieved in the allogeneic model, as originally proposed. Production in the autologous model could not be performed, as viable cells after explantation could not be provided for two main reasons:
-- Altered age of the donor animals (animals needed to be older) and
-- The punctuation of bone marrow in the rabbit femur did not allow for sufficient yield of cells as compared to the employed explantation protocol of the allogeneic system. Human bone tissue constructs could be successfully generated using human bone marrow cells as well as human trabecular bone-derived cells, the latter of which are currently in co-culturing experiments with human endothelial cells in the bioreactor system.

Therefore four important outcomes have come from this work that will be of interest to the scientific and industrial community:
-- Cell culture protocols for rabbit as well as human bone tissue engineering approaches that aim for the seraration of expansion and differentiation as well as low serum supplementation.
-- Analytical microarray-based technology to monitor osteogenic differentiation in bone cultures
-- A continuously perfused fixed-bed bioreactor system with online control of perfusion, pO2, pH, circulation and temperature for optimized culturing of tissue-engineered bone.
-- The production of bone tissue constructs in both rabbit and human.

Reported by

Abteilung Zellkuturtechnik (ZKT)
Mascheroder Weg 1
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