Cartilage Tissue Engineering
Our tissue engineering studies are conducted in collaboration with the Cellular Engineering Laboratory of Professor Clark Hung.
There are many challenges to engineering successful cartilage replacements. These include identifying suitable cell sources, media supplements, bioreactor design features, implantation procedures, postsurgical treatment, etc. Each of these challenges is critical to a successful tissue engineering strategy and progress toward this goal is advancing along these parallel tracks. The specific challenges addressed in our studies are the importance of the mechanical properties and geometry of engineered cartilage constructs and strategies for successful implantation.
Because the load support and frictional mechanisms of cartilage are so intimately dependent on its mechanical properties and because cartilage has a limited ability for repair in situ, our strategy for engineering cartilage constructs has focused on reproducing the normal properties of native tissue under in vitro conditions before implantation. One hypothesis is that dynamic loading of chondrocyte-seeded constructs can promote mechanical properties that approach those of native tissue. In our earliest cartilage tissue engineering study, cylindrical agarose gels seeded with primary bovine chondrocytes were subjected to dynamic loading in unconfined compression at 1 Hz, for 3 hours per day over a period of 4 weeks. A control group of cells was cultured under similar conditions, without loading. The equilibrium modulus of the constructs increased from 5 kPa at day 0 to 100 kPa for loaded constructs and 15 kPa for free-swelling controls at day 28. In comparison, the native tissue from which the cells were obtained has an equilibrium modulus of ~280 kPa. We showed for the first time that dynamic loading can be effective for promoting better mechanical properties in engineered cartilage. One potential advantage of dynamic loading of cylindrical disks in unconfined compression is that this loading configuration promotes compressive strains in the axial direction and tensile strains in the radial and circumferential directions, similar to the native environment of articular cartilage.
In a subsequent study, we showed that tissue growth and its response to dynamic loading also was dependent on cell seeding density and nutrient supply. Mechanical properties improved considerably when increasing the cell seeding density from 10 million to 60 million cells per mL, and the nutrient supply from 10 to 20% fetal bovine serum. After 56 days in culture, the equilibrium modulus of constructs in the high cell seeding density and high nutrient supply group was 190 kPa in the dynamically loaded constructs and 85 kPa in the free-swelling controls. Therefore, the dynamically loaded constructs had a modulus equal to 2/3 of the native tissue. Similarly, the dynamic modulus at 1 Hz was 1600 kPa in the dynamically loaded constructs and 870 kPa in the free-swelling controls. In comparison, in similar testing conditions, the native tissue has a dynamic modulus of 7000 kPa. Interestingly, unlike the mechanical properties of the tissue constructs, glycosaminoglycan (GAG) content and collagen content were found to be the same in free-swelling and dynamically loaded samples. These results suggest that ultrastructural matrix organization, and matrix products other than GAG and collagen may have a substantial influence on the tissue's mechanical properties. These studies formed a significant component of the dissertation of Dr. Robert Mauck.
The results of these studies are encouraging because they show the mechanical properties of tissue engineered constructs approaching those of native tissue. While simultaneously exploring alternative strategies to further improve mechanical properties of engineered constructs, such as growth factor supplementation, we have also considered the challenge of implanting tissue constructs into osteoarthritic joints. Because loss of cartilage in degenerative joint disease usually spans a large percentage of the articular surface, our long-term strategy is to consider replacing the entire articular layer with an anatomically shaped tissue-engineered construct. We have shown the feasibility of generating constructs in the shape of the human patella, using three-dimensional geometric data derived from stereophotogrammetric measurements. The basic approach is to create anatomically shaped molds using computer-aided design techniques to form chondrocyte-seeded gels into the desired shape. These constructs can maintain their original shape for several weeks in free-swelling culture even as the matrix elaborates.
To anchor anatomically shaped cartilage layers into the native subchondral bone of a joint, it may be necessary to engineer osteochondral constructs in which the cartilage layer already is anchored into a bony substrate. In our studies, bovine trabecular bone was machined into the shape of the subchondral bone surface of a human patella using a computer-controlled milling machine. Chondrocyte-seeded agarose gel was cast into the trabecular space to form an anatomically shaped osteochondral construct. These constructs were cultured for up to 35 days under free-swelling conditions, showing progressive elaboration matrix products from the periphery to the center. These studies were a component of Dr. Eric Lima's dissertation.
With such large constructs, it becomes apparent that nutrient diffusion limitations pose a considerable challenge for uniform matrix development as evidenced by the GAG distribution on histological sections. Increasing the nutrient supply to large constructs can be performed by adding channels that eventually fill up with tissue, and that demonstrate considerably improvement in material properties and matrix production at the center of the constructs. These studies represent a significant component of Dr. Liming Bian's dissertation.