Tissue Enginering and its Application in Arthritis

Dr. James Patrick Abulencia '97

Department of Chemical Engineering

Tissue engineering, also known as regenerative medicine, is a multi disciplinary field that is rapidly growing. The fundamental premise of this technology is to replace damaged or diseased tissue within a patient's body with a functional equivalent. Three important considerations must be made in order to successfully accomplish this. First, the new issue formed must be biologically relevant. In other words, it must be able to accomplish the same tasks of the healthy predecessor. For example, if skin is to be replaced on a burn victim, the replacement tissue must provide the functions of normal, healthy skin (i.e. a barrier for infection, prevent the patient from becoming dehydrated, and help in regulating their temperature). Second, the new tissue must be able to withstand the same biomechanical forces as the healthy predecessor. If a vein or artery is being designed, the final product must be able to withstand the fluid shear stresses applied by the flowing blood. The third and final consideration is that the new tissue must be sustainable. Once placed on site, it must be able to become self sufficient (i.e. obtain nutrients, and have the ability to regenerate and adapt) to avoid any invasive procedures for the patient post implantation.

The design of functional tissue often begins with a decision on the cell source and substrate that will be used. Traditional tissue culture technique proliferate cells in flat 2-D monolayers. However, this is very limited for tissue engineering purposes because a 3-D environment to match the geometry of the damaged or diseased tissue is required. To address this, a structure called a scaffold is used as a substrate to provide a relevant 3-D space for cells to attach. Scaffolds used for tissue engineering are generally made of biodegradable polymers. As the attached cells proliferate, they form their own independent functional structure which no longer requires the original substrate. Scaffold geometry varies from a mesh-like pattern, to one that looks like a porous sponge. Regardless of geometry, a major challenge of tissue engineering is obtaining a uniform spatial distribution of cells within the scaffold. More specifically, it is difficult to direct cells towards the inner interstices of these scaffolds.

The cell source chosen for a particular tissue engineering application requires careful thought. One attractive candidate that has wide applicability are mesenchymal stem cells (MSCs). These cells offer three advantages. First, these cells can be harvested from several sources such as bone marrow, muscle, and adipose. Thus, there is essentially an unlimited supply. Second, stem cells are plastic. In other words, they can differentiate into many different cell types. Finally, MSCs are derived from an adult rather than a fetus. As a consequence, any political and religious controversy associated with embryonic stem cell work can be circumvented.

After a cell source and substrate are chosen, the process of engineering functional tissue beings with the “seeding” phase. The goal here is to provide an opportunity for cells to attach to the scaffolds. One device used to accomplish this task is called a spinner flask. In this case, polymer scaffolds are immobilized on metal rods within a cell suspension agitated with a magnetic stir bar. The problem with this particular method is the high rate of shear stress imposed on the cells, which may potentially cause death. A second device that is used is called the rotating wall bioreactor designed by NASA's biotechnology group. Here, cell suspension is rotated about a horizontal axis in such a way that the polymer scaffolds inside remain suspended. The net effect is that a condition of zero gravity is created, thus simulating the behavior of cells in outer space. More importantly, however, is that fluid shear and turbulence is minimized, thus creating a gentler environment for cells to attach and proliferate onto the scaffold. The process of cell seeding typically occurs over two to three days.

The second stage of the tissue engineering process is called the “culture” phase. After cell attachment is achieved from the seeding phase, the goal becomes proliferation within the scaffold into functional tissue. Methods similar to those used in the seeding phase may be used here (i.e. spinner flask and rotating wall bioreactor). However, the difference is complete cell media (i.e. culture media without the cells) is used, and is performed over a longer period of time (~30 days).

Arthritis is a disease in which tissue engineering appears to be a promising application. Healthy joints are lined with a layer of cartilage that surrounds a very narrow gap of liquid called synovial fluid. This configuration provides a nearly frictionless surface for movement. Beneath the cartilage layer is bone, and is the primary source of structure and support for t he patient. Arthritis progresses by initially attacking the cartilage surface, and ultimately penetrating into the underlying bone itself. As a consequence, a small defect or hole is created into the bone, thus disrupting its ability to act as the primary load bearing structure for the patient. Because of this weakness, stresses normally assumed by the bone transfers to ancillary supporting tissues such as nearby muscles and ligaments. This, in turn, puts an unnecessary burden to those tissues, thus increasing the probability for their own damage. Most important, however, is the pain that a patient experiences when weight is placed on the defect in the joint.

A tissue engineering approach for treating arthritis aims to fill the defect created by the disease. The aforementioned considerations must be addressed when designing a process for this undertaking. First is biological relevance. The defect encompasses two distinct cell types, namely cartilage and bone. The cartilage cells on the top layer of the defect must create a frictionless surface, while the bone beneath must be strong and rigid. Second is the ability to withstand biomechanical forces. Cartilage experiences pressure upon weight bearing, as well as fluid shear during ambulation, while bone primarily experiences compressive forces. The final issue is sustainability. This is a challenge because there is no vascularity within the joint space, so all sustainability and waste removal for the surface cartilage must occur through diffusion within the synovial fluid.

In this work, MSCs were seeded on chitosan and poly-glycolic acid (PGA) scaffolds using a spinner flask and rotating wall bioreactor. MSCs were chosen because both cartilage and bone can be derived from this starting material. Thus, the need to work with two separate cell populations was eliminated. Chitosan is a derivative of chitin, a byproduct of the shells of mollusks and exoskeletons of insects. Its main advantages are its biocompatibility (because of its natural source) and biodegradability. When processed as a cell scaffold, it has the geometry of a porous sponge. In contrast, PGA scaffolds are the result of a benchtop polymerization reaction, and has the geometry of an intertwined mesh. Despite its artificial character, PGA scaffolds dissolve within 3-4 weeks in culture, thus creating the possibility of a scaffold free implant. The cells were seeded over a period of two days, and analyzed 1) for the number of cells attached by measuring the amount of DNA present, and 2) the cell distribution by examining the superficial surface and interior of the scaffolds using scanning electron microscopy (SEM). The DNA analysis revealed that more cells attached onto the PGA and chitosan scaffolds for the spinner flask technique versus the rotating wall bioreactor technique. Despite the greater number of cells that attached on the PGA scaffolds, the cells appear to form flat sheets in the spinner flask technique, while the cells better retained their shape using the rotating wall bioreactor technique. This may be a consequence of the higher magnitude of fluid shear forces seen in the spinner flask methodology.

Because of the versatility of MSCs to differentiate into several cell types, future work examining their use in other tissue engineering applications merits further study. The ways to direct these cells into a particular cell type (e.g. cartilage cells, bone cells, skin cells) and assemble into functional tissue still needs to be explored.