Development of a bioreactor-based connective tissue production line (TISSUE REACTOR)

- Improved emulsions starting from a given nutrient solution, delivered by partner 5 - Emulsions containing either 40% w/v Perfluorodecalin or 80% w/v Perfluorotributylamine as core oxygen carrier promising a sufficient oxygen delivery capacity; the first emulsion is based on FKT-I solution, the second on pure water to be mixed with a double concentrated FKT-I solution by partner 5 - Delivery of two litres of latter emulsion to partner 5 for testing on physical and physiological parameters in cell culture assays and in novel fixed bed reactors concerning their effectiveness in supporting bone-cell growth under oxygen limited conditions - Average particle size of the emulsions between 150 and 250nm. - Long-term storability at ambient temperature for more than one year. - Development of a novel sterile production chamber for the production of PFC emulsions under sterile conditions. - Readyness of delivery of the emulsions in a litre scale to partner 7 for degradation studies of polymeric scaffolds. - Rheology studies of the emulsions for the use together with polymeric scaffolds. - Surface charge studies of PFC emulsions. - Ultrapurification of perfluorocarbons and purity analysis.

In this study a sterilization validation was performed on Porous PLGA Scaffolds (UT026-UT045). The study was performed according to the requirements of ISO/TR 13409. This ISO technical report describes a method of substantiating the suitability of 25kGy as a sterilization dose for radiation sterilization of products with an average bioburden of less than 1000 colony forming units (CFU) that are manufactures in small quantities (less than 1000 product units). The method of ISO/TR 13409 is based on method 1 as described in ISO 11137. ISO 11137 requires a relatively large number of product items, drawn from separate production batches, be used to establish the sterilization dose. Since this is not feasible in case of products, which are under development and/or new, ISO/TR 13409 provides guidance on how to allow substantiation of 25kGy as an appropriate sterilization dose.

Procedures have been optimised for the isolation and culture of bovine and human endothelial cells, smooth muscle cells and pericytes. These cells have been plated onto three-dimensional PLGA scaffolds, CaP ceramics and PLGA-CaP composite scaffolds and maintained in static culture, in dynamic cultures and in bioreactors for up to 6 weeks. Using cell biology assays, biochemical assays, SEM analysis and immunohistochemical staining, we have shown that these cells adhere to the scaffolds, proliferate and deposit an extensive extracellular matrix containing type IV collagen, fibronectin and thrombospondin. However, marked contraction of the PLGA scaffolds was observed when either pericytes alone or co-cultures of pericytes or smooth muscle cells and endothelial cells were plated on the scaffolds, indicating that this scaffold is not suitable for use in bioreactors or in vivo. This contraction was not observed when cells were plated on CaP ceramics or on PLGA-CaP composite scaffolds, demonstrating that the PLGA scaffolds had been strengthened by modification with CaP. Human endothelial cells did not attach, proliferate or form a contact-inhibited monolayer on these scaffolds to the same extent as bovine cells, suggesting that conditions still need to be optimised for the growth of these cells on these scaffolds.

Preparation of apatite coatings on several substrates, mostly polymeric ones, using a well known coating method, the biomimetic method. Substrates provided by other partners of the consortium (3, 4 and 8) were coated by a biomimetic method. The obtained coatings were characterized by SEM / EDS and by XRD. Production of apatite nanoparticles and their use on the preparation of porous apatite particles. Apatite nanosized particles were produced by a wet chemical method, involving a close control of the hydrotermal conditions. The morphology of the particles was characterized by SEM and TEM. Their specific surface area was evaluated by BET. Their phase composition was analyzed by XRD and the Ca/P ratio was estimated by EDS. Porous apatite particles were prepared by spray drying a suspension of the previously prepared apatite nanoparticles. The spray dried particles were characterized by SEM and their specific area was evaluated by BET.

CAM Implants initially provided a macroporous calcium phosphate (CaP) ceramic scaffold with interconnective pore structure and high porosity to support bone culture growth and allow bioreactor development as well as dynamic bone culture development. During the project CAM Implants improved their existing scaffold by optimising: - Porosity, pore size and pore-wall-thickness; - CaP phase distribution, trace metals content and degree of carbonation, as well as - Cell adhesion, osteoclastic resorbability and surface topography in order to obtain a biodegradable second generation scaffold. Those improved scaffolds were made available to the project partners.

Gamma irradiation was found to irreversibly damage the polymer components of tissue engineering scaffolds resulting in increased degradation rates and increased rate of loss of mechanical strength when degraded. Ethylene oxide sterilised scaffolds showed less damage and a slower rate of loss of mechanical strength when degraded similar to that of virgin scaffolds. In vitro tests with and without serum and/or cells showed significant differences in degradation and mechanical behaviour. Data has been obtained that will enable degradation and mechanical behaviour to be calculated for the bioreactor and in vivo studies. With this data candidate the PLGA/CaP scaffolds degradation and mechanical state can be controlled by pre-treatment with an in vitro post sterilisation technique, optimising it for implantation. A purpose built plasma sterilisation test rig was constructed. Plasma characteristics for plasma sterilisation were found to proportional to the gas flow rates and the power intensity used. Increased amounts of hard UV were observed when lower percentages of oxygen were used in a nitrogen plasma. Plasma sterilisation in the glow was found to kill bacterial spores but at the same time result in thermal degradation of polymer scaffolds. Using the afterglow of the plasma was found to result in the destruction in bacterial spores without thermal damage to PLGA based scaffolds. Therefore two important outcomes have come from this work that will be of interest to the scientific and industrial community: - Data on suitable characterisation profiles of a degradable polymer scaffolds for use through bioreactors to an implant allowing prediction of a polymer (in this case PLGA) state of degradation and mechanical performance at key time points in its delivery. - The potential to sterilize a 3-D porous polymer construct or composite using plasma sterilisation rather than EtO sterilisation which has a temperature c.a. 50?C which is too close to the Tg of some of the key degradable polymers. Plasma sterilisation effectiveness is strongly dependent on the interconnectivity and dimensions of the construct.

Provisional Results of the development of a continously perfused fixed-bed bio-reactor system, tailored to the specific requirements of bone cells in order to obtain a bone tissue construct in vitro were obtained: In close connection to WP1 and 2 the shape of the reactor was defined. Based on the modularity and miniaturisation of the system, it is also necessary to develop some peripheric compounds like DO and pH-Probes with small size, fittings and measurement for small flowrates and peristaltic pumps for small volumes. Due to these reasons meredos has first developed a special reactor concept and the necessary electronics, which can be used for the following applications with the change of only few elements and low expenditures: The vessel-variety is the prerequisite for successful test series with various scaffolds and culture media and for realisation of an organic unity of all elements such as vessel, sampling system, scaffold holder for different shapes, miniaturized sensors, variable flow rates within the microliter range with new miniaturized peristaltic pumps. Furthermore this concept is new on the market and meredos has applied for patent.

Within the project different partner produce or modify three different scaffold types based on polymers, CaP or both. mnemoScience will obtain quantitative data about the chemical composition, the thermal and mechanical properties, the structure and the in vitro degradation behaviour of the scaffolds. The applied methods have to be optimised and standardised. The aim is to carry out a complete and reproducible characterisation with a preferably low number of samples as fast as possible and as economical as possible. The establishment of a standard quality control system will be completed with the design of a quality report sheet for giving an objective overview of the materials properties and the quality of the scaffolds. The standardised quality control system can be applied not only on bone substitutes, but also on such for other tissue substitutes and generally for porous cell carriers. The end-users of the results are the manufacturer of the scaffolds and the user of the scaffolds. Manufacturer are settled in the chemical or medical-technical industry, user are settled in the biotechnological and biomedical sector. The results are relevant for ensuring a certain scaffold quality, to increase the safety for the application of the material or to decrease the risk of a material defect.

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.

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