This report is a study about bioreactor design for bone tissue generation. Bioreactors are used for in vitro expansion of cells on a scaffold. This scaffold is later implanted into the body for clinical treatments. The aim of this report is to design a bioreactor that will be able to replicate in vivo loading conditions on a bone tissue. Several bioreactor systems have been studied such as perfusion flow, rotating wall and spinner flask. Advantages and disadvantages of these bioreactor systems were assessed and perfusion flow system is chosen to continue with for the design.
Tissue engineering holds a potential to reduce risks and complications that could be caused by autograft or allograft surgeries. Autograft causes complications in 30% of the surgeries. Whereas, allograft can result in immune response by the body to the bone of the cadaver and spread disease. By using person’s own stem cells, refusal of the bone structure by the body is eliminated. A scaffold is used as the base structure of the bone tissue where a cell source is seeded and grown. Scaffold with cell source are cultured to increase cell proliferation on the scaffold and initiate differentiation of stem cells into osteoblasts. This scaffold is later implanted into the body at the injury site with cells.
Bioreactors are used at the process of proliferation and differentiation due to their ability to allow a continuous supply of nutrients to the cells. Another important characteristic of bone tissue is that it is under constant pressure. Therefore, the scaffold to be used must be biodegradable in vivo and at the same time able to bear load during the degradation of scaffold material and regeneration of bone tissue. A bioreactor should enable automation of the process therefore reduce labour intensity, minimise risk of contamination and reduce costs related to in vitro cell culture.
There is a tendency of formation of a nutrient gradient in the scaffolds of static cultures. Oxygen, glucose and other nutrients are able to reach the cells at the outer surface. However, consumption of these nutrients is faster than the diffusion rate. Therefore, cells closer to the centre of the scaffold, lack some of these nutrients necessary for cell growth, as a result cell death occurs. Bioreactors are employed to offer a controlled environment and dynamic culture for cell population expansion. Also, mechanical stimulation via fluid shear stress causes further bone differentiation and mineralization. Consequently, use of bioreactors improve in vitro cell culture.
As mentioned before, the main task of this report is to design a bioreactor capable to replicating in vivo loading conditions experienced by bone cells in the body. This bioreactor is to produce a cancellous/spongy bone with the dimensions of 1cm x 1cm x 1cm. One of the most important challenge is the fact that tissue engineered bone cannot bear load. However, bone is an adaptive material and bone cells change over time with regards to applied loading. Therefore, bioreactor to be designed is required to apply mechanical stimuli to be able to obtain bone tissues that can withstand load.
Spinner flask is a simple bioreactor system containing suspended scaffolds in the culture with stir bar located at the bottom to circulate media around. This type of bioreactors gives better results in some cases when compared to static culture and rotating wall bioreactors. That is believed to be due to convective transport of nutrients and increased concentration of oxygen throughout the scaffold. Even though spinner flask systems give a better diffusion of nutrients, there still is a nutrient gradient that becomes very important as the scaffold gets bigger. Therefore, spinner flask systems are not desirable for big scaffold structures. High stirring rates increases the shear due to stirring and can affect the osteoblastic differentiation of cells.
Rotating wall bioreactors are essentially comprised of two concentric cylinders in which the inner one is immobile and used for gas exchange and the outer one rotates. In between these cylinders, there is the culture media and scaffolds moving freely within. Studies found out that although certain aspects of this system could give a better yield, in some respect, compared to static culture, other bioreactor systems achieved better results.
Perfusion bioreactor systems use a pump to perfuse media, directly or indirectly, through the scaffold. They are able to perfuse media through the scaffold effectively without creating any nutrient gradient. Generally, perfusion bioreactor design include a pump, tubing circuit, media reservoir and a perfusion cartridge. The shear force applied on the scaffold due to media flow is much greater than other bioreactor systems ability to create such shear forces. Therefore, media flow is important not only to deliver nutrients to cells all around the scaffold, but also to stimulate cells with shear stress for growth and production of certain products.
4.1) Proposed Designed
Based on literature review carried out, bioreactor design is chosen to be a perfusion flow system. Figure 1 shows the perfusion cartridge. Perfusion cartridge is custom made to tightly fit the scaffold in. The full open side is where a cyclic load will be applied. Opposite side is kept close for counter balancing the load. Top and bottom sides of the perfusion cartridge are open with an indentation to hold the scaffold firmly during media flow. The flow will be coming from the top and exiting through the bottom. Other two sides of the perfusion cartridge are kept closed.
Perfusion flow bioreactors are more versatile with their designs. Improvements to their designs can further improve their effect on cell proliferation and differentiation. Shear stresses due to fluid flow and mechanical loads can be added and manipulated for mechanical stimulation. This is a very important point as a load bearing bone tissue is yet to be achieved using a bioreactor.
Perfusion cartridge can easily slide into the bioreactor for experiments (see Figure 2). This design is implemented to enable usage of different sizes of scaffolds with the same bioreactor. Instead of building a new bioreactor from scratch, a perfusion cartridge can be built to hold the scaffold. Top plate of the bioreactor has three holes into the flow chamber. The one in the middle is the inlet for the media fluid. Other two are reserved for sensors. Some of these sensors can be pH sensor, thermometer and dissolved oxygen (DO) meter which measures the proportion of oxygen present in the fluid.
These sensors could be connected to a controller and then to the same computer that commands the mechanical stimulation. By this way, sensor operations could be automatized resulting in decreased labour intensity (see Figure 5). The bottom plate has two exits. One is for the fluid to be removed and the other one is the outlet to remove any bubbles that might be present in the fluid using a bubble trap. The overall design of the bioreactor is designed to be thick to preserve the temperature and prevent any possible leakage of the fluid. Drawings of the design can be seen in Figure 1-4.
4.2) Design Parameters
Perfusion cartridge is fitted into the flow chamber in order to hold the scaffold firmly in place. It is found out that scaffold can lose some material over time due to media flow. Therefore, perfusion cartridge was found necessary to be implemented to prevent any problems that may arise due to movement of the scaffold. The minimum flow chamber size would be the same as the dimensions of the scaffold to be used which is 1cm x 1cm x 1cm.
Flow rates of the nutrients depends on the shear stress to be applied on the cells in the perfusion chamber. Shear stress range on 0.01 to 1 dyn/cm2 is chosen. This is the range in which shear stress is either too small to be effective or is big enough to damage the cells. Shear stress is calculated by the equation Τw = (8µVpore)/δ where Vpore is the mean pore velocity, δ is the mean pore diameter and µ is the viscosity. Vpore is given by (Q/Aε) where Q is the volumetric flow rate, A is the area of the flow chamber and ε is the scaffold porosity. For calculating Vpore from maximum and minimum shear stresses, µ is taken to be 0.01 g/cm s assuming a laminar flow of the medium and δ is 0.05 cm when ε is 0.7. Vpore maximum and minimum are obtained as 0.625 cm/s and 0.00625 cm/s, respectively. Then volumetric flow rate is calculated to be 0.2625 mL/min and 26.25 mL/min for minimum and maximum flow rates assuming a scaffold porosity (ε) of 0.7 when area (A) is 1cm2.
Mechanical stimulation of the scaffold is carried out by mechanical compression and shear stress created due to fluid flow. Shear stress created due to fluid flow is calculated above. Mechanical load is applied through a plate onto the surface of the scaffold as can be seen on Figure 2. A load cell and a motor is attached to the loading plate. Load cell is used to measure the applied load to the scaffold so that the power from the motor can be adjusted. Connecting the motor and the load cell to a computer could help automatize the process. Load magnitude and intervals in which the load would applied can be input into the system and reduce labour intensity. The load that will be applied should not be exaggerated as excessive mechanical stimuli can damage the cells. At the same time it should not be too low that it won’t have an effect on it. Therefore, the force to be applied should be studied and calculated properly o enhance the effect it will have on the process.
4.3) Material Selection
Stainless steel is one of the most preferred material for the construction of the bioreactor. It is resistant to corrosion, can withstand high temperatures and pressures and is biocompatible. It is also an expensive material. This design is relatively small therefore it does not have too much of an effect. Stainless steel bioreactors can also be cleaned and sterilised easily using an autoclave. This material suit the versatility of the design as any size of scaffold smaller than 1cm x 1cm x 1cm can be used.
There are two types of materials that are used for scaffolds. They are biodegradable and non-biodegradable materials. Their only difference is whether they degrade in the body once they are implanted into the body. Titanium is one of the most preferred non-biodegradable material for scaffolds. Whereas, there is a big variety of choice for biodegradable materials for scaffolds. Scaffold porosity has an effect on the cell proliferation and cell differentiation as well as production of certain proteins and minerals. Another important thing to note is the cell culture to be used.
Different cells also react differently to varying scaffold materials. For this case scaffold with a porosity of 70% was chosen. It is found that as scaffold porosity increases cell proliferation and cell differentiation increases too. Given all these factors that affect the growth of the tissue and uncertainty of how exactly, it is hard to choose a material for the scaffold. However, based on previous experiments, Polyglycolic acid and collagen mixture can be chosen as it is found to give higher ALP activity and cell uniformity compare to other bioreactor systems. Also same level of osteocalcin expression and cell number was recorded.
In conclusion, there needs to be further research and experiments to be able to replicate in vivo loading conditions in bioreactors. In general, recent studies with different cells and different materials for scaffold have shown that using bioreactors enhance cell survival and differentiation. Also, it creates an environment for this process where many parameters are controllable. Perfusion flow bioreactors hold a potential for tissue maturation and bone formation in vitro and in vivo. In the future, culture systems could be obtained through this method for bone substitutes and applied clinical use.
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