Bioreactor Systems - Case Study Example

REPLICATION OF THE IN VIVO LOADED BONE CELLS IN THE BIOREACTOR Bioreactors are generally defined as the devices which are used for the production of the biological compounds under tightly controlled and closely monitored environment. The operating conditions such as pH, Temperature, pressure, nutrient supply and waste removal are highly regulated. Bioreactor systems play an important role in the tissue engineering. Tissue engineering refers to the generation of the artificial three-dimensional tissues, which are used as a powerful tool in the regenerative medicine and for drug screening. The proliferation of cells is the first step in the tissue culture. It is followed by the dedifferentiation of the cells. This process produces only a minimal concentration of the cells. Hence the use of micro carrier cultures is done for the well-mixed bioreactor systems. The defects in the skeleton system are a big impediment to the normal functioning of the human system. The autogenous bone transplantation is practiced for the bone replacement so far. (Schieker, 2006).This method has increased risk of surgery and post operative morbidality to the patients. This method also has the disadvantage of limited quantity and the secondary operational procedure costs. The calcium phosphate ceramics is widely used for the production of the bone supplement. The hydroxyapatites of the calcium phosphate ceramics have considerable clinical use because of their chemical and relvant crystallographic structures to bone. The cell based therapy aims at developing the 3D biohybrid structure of a scaffold and cultured cells. The bioreactors that are established for the microbes and mammalian cultures are not suitable for the 3D structure constructs. Hence the need for a tissue specific bioreactor design arose. To restore back the skeleton function, the bone tissue engineering is used. The production of tissue engineered piece of spongy bone is one of the challenging fields in bone tissue engineering. The three key elements of bone tissue engineering are osteogenic progenitor cells, osteoinductive growth factors and osteoconductive matrices. Osteoblasts are the differentiated cells that arise from the osteoprogenitor cells. These cells are very mobile and they change the shape and size according to the matrix of the bone. These cells appear with in the vascular tissue. These cells are responsible for the synthesis and the secretion of the organic matrix of the bone. Bone remodeling and the bone reformation are the two types of the tissue engineering processes that can are possible with the osteoblast cells. (Dusting, 2003). Adult progenitor cells are harvested from the bone marrow and they are then isolated. These cells are then allowed to grow on the medium under appropriate conditions of Temperature, pH, CO2 and O2 levels. The cells will then expand and grow. Cell actions and their responses to various environmental effects such as electrical, mechanical, structural and chemical are mediated by the low protein molecules and they are commonly referred as growth factors. Scaffolds are the temporary matrices for the bone growth and they provide a specific environment and architecture for the tissue development. The scaffolds must also favor cellular attachment, growth and differentiation of the cells. These scaffolds must also have a fully porous environment with a good inter connecting network for the development and differentiation of the osteoblast cells. The pore size should not increase above 10μm. (Schieker, 2006).When a scaffold is given to the cells, they will adhere and proliferate in vitro. The scaffolds vary in their material chemistry, geometry, structure, mechanical properties and the degradation. The structure of the scaffold determines the transport of the nutrients, degradation and mechanical properties. The scaffold material should be an US food and Drug Administration approved one. To achieve a perfect tissue replacement system, the rate of biodegradation should match the rate of extracellular matrix deposition and it should not produce any toxic substances as products during degradation. The medium should be supplied by the dynamic method for maximum concentration of the live cells. These cells must fulfill the two criteria. The integration of the cells to the host cartridge and the integration with the subchondral bone. Hence the use of a biphasic scaffold can solve the problem. The development of a functional graft requires excellent support of the nutrients to the cells, with the support of the mechanical forces that direct the cellular activity and phenotype (Chang, 2005). The dynamic loading of the chondrocytes was found to have high seeding efficiencies. For higher efficiency and uniformity in seeder either spinner flask bioreactor or perfusion chamber is preferred. Among the two, the perfusion chamber bioreactor has more advantages for increasing the cell content and the matrix content. (Chang, 2005). Similarly the use of the rotating wall vessel bioreactor has found to be very efficient and they produce the cartridge that is very similar to the native.The different types of the flows that are needed for the long term supply are transfusion, perfusion, circulation and convention. Of these optimal perfusion ensures the optimum concentration of the nutrients and the metabolites. The main advantage of the perfusion chamber is that it they perfuse the nutrient medium directly into the pores of the 3D-scaffold. The direct supply of the medium reduces the mass transfer limitations in the periphery and the pores. The use of pump for the continuous perfusion of medium to the seed cultures is achieved well in the perfusion bioreactor. Thus perfusion chamber bioreactor is more advantageous than the other reactor. The reduction of mass transfer limitations and the application of the mechanical stress are notable characteristics of this reactor. In this bioreactor, the medium is pumped through each scaffold continuously, Therefore the nutrient requirements and the mechanical simulation for the cells are provided continuously enabling them to proliferate in the matrix. The advantage of this system is that it delivers the medium through the scaffolds and thus reducing the non perfusing flow, it also has a repeatable, controllable and consistent rate of flow of the medium to the cells. It is also sterilizable easily and hence can be used for the continuous operation and flow of the medium. Sterilization is the most important concept behind the bioreactor and bioprocess. The contamination can alter the cells and will result in the incorrect conclusions. This perfusion bioreactor is easy to operate as there are only a very few extraneous factors to monitor and control. The cassettes must be sterilized with the ethylene di-oxide and the other parts such as tubing, reservoirs and the other parts of the bioreactor must be autoclaved under steam at high pressure. The bioreactor should be transparent to enable us to view all the blocks inside the reactor. The flow chambers must also be machined for one block only. The cassette should be kept in between the o-rings thus enabling us a good control of the medium flow. The cassettes must be designed such that the opening design of the cassette must have a tight tolerance to the dimensions of the scaffolds being cultured. (Gregory et. al, 2003). The medium should be allowed to flow into the reactor obeying the gravitational force.Ths scaffold must be designed as a press-fit, as the cassettes can be varied to study the different scaffolds. The cassettes should be mechanically fixed and specified for the different diameter scaffolds. The cassette and the scaffold must be fitted very tight. This is done with the help of the O-rings. The neoprene O-ring must be kept above and below the cassette and tightly packed.The use of the low protein binding tubes and the non corrosive curing are also an important factor to be considered for designing. Hence the silicon tubes and platinum curing will be the best option. The next is the use of the stable polymer that can withstand the mechanical stress, biocompatible and has excellent osteoconductive properties. These scaffolds must be designed to allow diffusion of the nutrients to the cells that are transplanted and should also guide the cell organization, attachment and migration. The biopolymers such as starch, fibrin, collagen, chitosan can be used. These biopolymers have one disadvantage. They are not mechanically stable during the process. Hence the search for a good biopolymer is done. Synthetic bioresorbable polymers such as poly- ξ-caprolactone, poly hydroxyl acids, poly lactic acid, poly glycolic acid, poly 3-hydroxy butyric acid-co-3-Hydroxyvaleric acid, hyaluronic acid properties and their capability were studied and found to have a greater mechanical stability and very slow degrading capacity. These polymer based scaffolds can be used extensively. The ceramic based scaffolds are also widely used. (Matthias, 2006).The scaffold diameter must be larger than the flow chamber. This will be beneficial for the perfusion chamber designs. As the in vitro development of the bone matrix has been achieved, the in vivo development is targeted here. Moreover the in vivo bone formation and vascular cell differentiation are independent processes. Another important factor that determines the design of the bioreactor is the oxygen supply to the system. The oxygen is supplied to the cells only by diffusion process. This oxygen diffusion cannot be directly measured. So the oxygen diffusion phenomena are connected to the mass transfer limitations and a mathematical model is designed accordingly. As the density of the cells increases, the oxygen supply decreases as the molecule cannot penetrate deep into the cells. The studies have shown that the flow rate has no significant effect on the oxygen profile within the vessel wall as the mass transfer resistance is within the vessel wall and not on the boundary layer. In order to look for how much amount of oxygen is present and required by the outer region of the medium which is not flowing, the one dimensional diffusion equation is used. From these studies, it was estimated that the flow rate of the liquid medium should be 6 ml/min, the diffusion coefficient of the oxygen in the membrane should be 8.9 mm2 /hour, and the diffusion coefficient of oxygen in the medium should be 11.16 mm2 /hour. The oxygen concentration for the adherent cells should vary from 0.12mM to 0.06mM for a depth of 0-1mm. The seeding cell concentration should be around 10 6 cells/ mm3. The vessel thickness, inner radius and length also has minor importance in the design of the bioreactor. (Portner and Giese, 2007). The rate of compression is another important factor that needs to be designed for the effective osteoblast adhesion to the scaffold. The mechanical compression at a range of 200-250 kPa must be provided. The deep zones can receive the compression pressure of about 700-900 kPa. Similarly the amount of shear stress can be varied by altering the flow rate of the system. The dynamic compression of the cartilage cells results in the deformation of the cells and their extracelluar matrix. It also alters the hydrostatic pressurization of the tissue fluid, pressure gradients, the accompanying flow of fluid inside the tissue, streaming potentials and the currents. The change in the flow rate of the medium and the gas exchange will play a significant role in the osteoblast formation. (Gregory et. al, 2003). Several non-mechanical stimuli are applied to the 3D structure to induce the formation of the cartilage like properties. but it was found that oxygen tension and exogenous supplementation have found to have very less effect on the proliferation of the cells. the medium formulation is the next challenge in the tissue engineering. A sufficient supply of the nutrients together with the toxic removal must be the target for the long term culture preservation and maintenance. The change in the pH was observed during the aging of the medium. Hence the pH should be checked periodically for the osteoblast growth. The L-ascorbic acid, Beta-Glycerophosphate, dexamethasone can be used for the osteoblast growth (Lewandrowski, 2002). The use of l-ascorbic acid acts as an enzyme cofactor and anti oxidant. The L-ascorbic acid also acts as a stimulant for the transcription, translation and posttranslational process. Beta-Glycerophosphate and ascorbate acts as inducers for the osteogenic differentiation. The Beta-Glycerophosphate induces the bone matrix deposition and this deposition increases with the increase in time. The nodal formation which is the connecting layer is formed by the dexamethasone. The in vivo modeling forces and flows are identified and are quantified by the in vitro method. References: Chang, CH 2005, Novel bioreactors for osteochondral tissue Engineering, Biomedical Engineering Applications basis and Communications, vol.17, pp.38-43. Dusting J et. al 2006, A Fluid Dynamics Approach to Bioreactor Design for Cell and Tissue Culture, Wiley Publications. Gregory BN et. al 2003, Design of a Flow Perfusion Bioreactor System for Bone Tissue- Engineering Applications, Tissue Engineering, vol. 9, no. 3. Lewandrowski, KU 2002, Tissue Engineering and Biodegradable Equivalents: Scientific and Clinical Applications, CRC Press. Portner, R and Giese, C 2007, An Overview on Bioreactor Design, prototyping and Process Control for Reproducible Three-Dimensional Tissue Culture, Wiley publications. Read More