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Cartilage has limited capacity for self-regeneration and none of the current treatments or therapies that are used to rectify damaged cartilage are ideal or permanent. In recent years, tissue engineering technique has emerged as an alternative and promising tool for the replacement or repair of the damaged &/or diseased cartilage tissue. Approaches used in cartilage tissue engineering involve the mechano-induction of mesenchymal stem cells to emulate the forces experienced by cells in vivo towards clonal differentiation to chondrocytes which are capable of mimicking the compositional and mechanical properties of the native cartilage, and the achievement of the functional cartilage ââ‚¬" scaffold implant for which a spinner flask bioreactor vessel is used.
In the proposed spinner flask bioreactor system a stirring element will be located at the bottom of the container to ensure the mixing of the liquid culture medium along with perfused flow of media for mechano-induction of Mesenchymal stem cell to promote cell adherence, cell migration and differentiation to cartilage tissue. The design uses a minimum number of parts that are sterilisable and the system has been designed to minimize the introduction of contaminants by enclosing the complete setup in UV protected glass chamber. In addition, Magnetic Activated Cell Seeding technique is incorporated into the system for achieving high efficiency cell seeding.
Keywords: Spinner flask, MSC, Chondrocytes, Perfusion bioreactor, Magnetic Activated Cell Seeding, cartilage regeneration
Joint pain is a major cause of disability in middle-aged and older people. Pain usually results from degeneration of the joint's cartilage due to primary osteoarthritis or from trauma causing loss of cartilage . Since cartilage shows very little tendency for self-repair, the injuries to cartilage are maintained for years and can eventually lead to further degeneration (secondary osteoarthritis) . Hyaline cartilage, the type of cartilage found in joints, provides stable movement with less friction than any prosthetic replacement, and can alter its properties in response to divergences in loading. Although appearing to be a simple, vascular matrix, hyaline cartilage possesses properties such as resistance to compression and the ability to distribute loads that cannot be fully replaced by any other tissue or device designed to date [3, 4].A variety of bioreactor technologies have been documented for the culture of engineered tissues for in vivo implantation. All culture methods involve isolation of MSCs and then cell seeding on three-dimensional scaffolds. The bioreactors used to facilitate the development of new tissue in vitro by providing appropriate stimuli to the cultured cells. One of the simplest bioreactor designs is the spinner flask [5-10]. Scaffolds seeded with cells are attached to needles hanging from the cover of the flask. Sufficient medium is added to cover the scaffolds. Mixing of the medium is maintained with a magnetic stir bar at the bottom of the flask. The fluid dynamic environment within the stirred medium has been well characterized [5,11]. The perfusion bioreactor offers enhanced transport of nutrients because it allows medium to be transported through the interconnected pores of the scaffolds. It has been reported that Murine K8 osteosarcoma cells seeded on three-dimensional collagen sponges and cultured in a flow perfusion bioreactor has enhanced viability, alkaline phosphatase activity, and mineralization . Rat marrow stromal cells seeded on three-dimensional porous biodegradable polymer foams and cultured in a perfusion bioreactor resulted in better cell uniformity when compared with a spinner flask and a rotating wall vessel bioreactor .The spinner flask bioreactor is one of the earliest dynamic environments for the development of articular cartilage tissue having limitations such as not able to provide the mechano-induction environment, separate system for media exchange leading to more complexity and chance of contamination. The flow perfusion bioreactor although effective in 2D dimensional mass transfer is not efficient in mass transfer to both the internal and external dimensions of the scaffold, proper hydrodynamic pressure is not maintained in the capillaries as they are not uniform throughout the scaffold leading to pressure changes in scaffold internal structure . Also one of the major limitations of the perfusion bioreactor is blockage of internal capillary due to cell mass growth which needs to be regulated or replace thereby greatly increasing the chance of contamination. An innovative Magnetic activated cell seeding techniques involving specified antibody labelled magnetic beads is incorporated into the proposed spinner flask bioreactor system to further strengthen the cell seeding technique for overall high efficiency of the proposed system.
2. Spinner Flask Bioreactor system
The results of the earlier study indicate that currently available bioreactors are inadequate for cell growth and mineralized matrix production throughout the seeded scaffolds. The study also indicated that enhanced medium delivery and mechanical forces may potentially increase cell differentiation . However, while bioreactors such as the spinner flask mitigated external diffusional limitations and internal diffusional limitations, that is, the inability of medium and other nutrients to penetrate within the porous network of the scaffold, remained which is taken care in this prototype with the help of a peristaltic single channel pump for developing perfusion phenomenon. This resulted cell growth and matrix production confined to the external aspects of each scaffold . For this reason, it was desirable to investigate flow perfusion culture as an alternative method that could potentially mitigate these limitations, especially for metabolically active cells such as osteoblasts. In a flow perfusion bioreactor, medium is pumped through each scaffold continuously. In this manner, medium is delivered throughout each cultured scaffold (Figure 1).
FIG. 1. Diagram of spinner flask Bioreactor system . The media reservoir supplies the media by gravity to the peristaltic pump which is the distributed over the two perfusion chambers attached to the wall of the bioreactor.
A flow perfusion bioreactor offers several advantages for culturing scaffolds for tissue engineering. It provides enhanced delivery of nutrients throughout the entire scaffold by mitigating both external and internal diffusional limitations as fresh medium is not only delivered to each scaffold, but also throughout the internal structure of each scaffold. In addition, it offers a convenient way of providing mechanical stimulation to the cells by way of fluid shear stress (bone cells are known to be stimulated by mechanical signals) [13,14]. The amount of shear stress experienced by cells cultured in a flow perfusion system can be varied simply by varying the flow rates through the system. Of course, depending on the porous structure, the local shear stresses experienced by individual cells will be variable and depend on the scaffold microarchitecture. A flow perfusion bioreactor thus extends the benefits offered by the spinner flask bioreactor.
2.2 Design Philosophy
To investigate the use of flow perfusion culture for cartilage tissue engineering, a flow perfusion culture system is proposed. However, there are several aspects were considered for a successful design of a flow perfusion system. First, it must deliver the flow through the scaffolds, minimizing the non-perfusing flow that goes around each cultured scaffold, otherwise, it offers little advantage over the mixing provided by a reactor such as a spinner flask. Secondly, it must have a repeatable, controllable, and consistent rate of flow delivered to the constructs to be cultured. For comparison both within and between experiments, consistency must be maintained to correctly draw conclusions about the results obtained. It must also be able to be sterilized and to maintain sterile condition throughout the culture period. Again, contamination can alter the results, causing incorrect conclusions to be made. Finally, the system should be easy to operate.
The flow perfusion bioreactor is proposed to be made of Plexiglas as it is sterilisable by autoclave and offers the advantage of being relatively transparent, allowing the contents of each flow chamber to be visualized through the block. In addition, it is readily machinable to allow for the fabrication of the unit. The flow system body consists of a spinner flask machined into one block. The scaffold is held in a cassette attached to wall of spinner flask. Medium is drawn from the media reservoir, pumped through flow chamber, with the help of peristaltic pump. This flow system circuit is illustrated in Figure 2.
FIG.2. A. Perfusion chambers with scaffold tightly held in cassette to experience maximum hydrodynamic force; B. Perfusion being lost due to a non-perfusing flow.
The tubing used to connect each component in the circuit is made up of platinum-cured silicone tubing (Master flex tubing; Cole Parmer). The pump driving the flow is a single -channel peristaltic pump (Cole Parmer). This pump gives accurate and consistent flow rates from 0.1 to 10 mL/min with the tubing size used in our system (Cole Parmer L/S 16). Due to the peristaltic nature of the pump and the relatively lower mechanical durability of silicone tubing, neoprene tubing (Pharmed tubing; Cole Parmer), a more rigid but non-gas-permeable tubing, is used for a short segment of the circuit within the pump. The entire system is readily sterilisable. The flow system body with the cassettes, all made of Plexiglas, must be sterilized by ethylene oxide. The tubing, reservoir flasks, and all other parts are sterilisable by autoclaving. Because of this, the majority of the flow system can be assembled and sterilized as one piece before placing the seeded scaffolds into the system. This aspect of the design enhances usability and also limits handling of components during start-up, thereby lessening opportunities for the introduction of contaminants.
2.4 Magnetic Activated Cell Seeding
Apart from the use of the hydrodynamic force for the seeding of differentiated chondrocytes into the scaffold, we here by propose a different approach of magnetic activated cell seeding for more efficient outcome.
In this design, the strong magnet from BD Biosciences will be used underneath the scaffold holding cassette and the chondrocytes will be bound to biotin labelled CD antibodies which is specific to chondrocytes cell surface receptors CD151. Subsequently the biotin labelled antibody will be conjugated to streptavidin magnetic beads.
The magnet will then exert a force to pull the magnetic beads with cells into the scaffold as shown in Figure 3.
MAGNETIC ACTIVATED CELL SEEDING IN SCAFFOLD
Streptavidin CD Marker
Biotin labeled cells
High power Magnet
Magnetic Bead (Streptavidin Labeled)
FIG.3.Magnetic Force Pull The Streptavidin Labeled Magnetic Beads With Biotin Labeled Cells Into The Scaffold Thus We Achieve A High Level Of Efficient Seeding
A flow perfusion system design has been proposed with the intention to provide uniform perfusion flow through each scaffold by the use of its cassette system for holding the individual scaffolds and a singlechannel pump with each flow chamber isolated on its own pumping circuit it will be possible to repeatedly deliver accurate, controlled flow rates. All components of the flow system are sterilisable and set-up has been designed with UV enclosement chamber to minimize the introduction of contaminants. In addition, the system has been simplified to include a minimum number of parts, allowing for ease of handling, cost effectiveness and less modes of contamination. The magnetic assisted cell seeding is also integrated into the proposed design for higher efficiency of the cell seeding and subsequent implant development.