Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers

Woo Seob Kim, Joseph P. Vacanti, Linda Cima, David Mooney, Joseph Upton, Wolfgang C. Puelacher, Charles A. Vacan. Plastic and Reconstructive Surgery, Vol. 94, No. 2, pp. 233-237. August 1994.

Introduction During plastic and reconstructive surgery, inorganic materials are often implanted to provide support to produce the desired aesthetic or reconstructive effect. However, these inorganic materials are often recognized as foreign bodies by the body's immune system, leading to infection and ultimate rejection. If cartilage is to be safely and successfully implanted in patients, it must be immune-capatible. In this study, a team of researchers and medical doctors designed biodegradable polymers in a variety of shapes (e.g. triangle, rectangle, cross, cylinder), seeded them with chondrocytes, and observed the specimens during a 12-week time course. The chondrocytes were observed to take on the shape of their scaffolds with little evidence of resorption by the body or overgrowth beyond the bounds of the scaffold. These results suggest that autogenous cartilage, developed from a patient's own cells and grown on a pre-designed scaffold holds promise for use in the fields of plastic and reconstructive surgery.

Materials and Methods Researchers began with an entanglement of polyglycolic acid fabric (PGA), whose fibers are randomly arranged. This was then submersed in a 2% (w/v) solution of poly-L-lactic acid (PLLA), which chemically bonds the fibers of PGA by forming cross-linkages. After removal from solution, the polymer constructs were cut into the desired geometric shapes: a triangle,a rectangle, and a cross.

Cartilage was harvested from a bovine source and washed in povidone-iodine 10% solution. Chondrocytes were subsequently harvested and concentrated to 5x107 cells per mL. 100uL of this solution was seeded into the scaffolds and supplemented with 10% fetal calf serum and an assortment of amino acids and antibiotics. Cell nutrients were labeled with BrdU to allow researchers to identify implanted cells. The cell-polymer constructs were incubated in 5% carbon dioxide at 37 degrees Celsius, with the culture medium replaced every 3 days. After a week, the cell-polymer constructs were subcutaneously implanted into the dorsum of mice. For the cylindrical cartilage scaffolds, sheets of the polymer construct were cut into rectangles and seeded with chondrocytes that were incubated for a week. These polymer-cell constructs were then wrapped around 2.5 cm long silicone rods of 3mm diameter before being subcutaneously implanted into the dorsum of mice. After 3, 6, 9, and 12 weeks, mice were sacrificed and specimens evaluated with histological stains. Results and Discussion Gross examination of the non-cylindrical scaffolds (12 triangles, 12 rectangles, 12 crosses) showed that all scaffolds were replaced with chondrocytes that retained approximately the same shape as the scaffold. Two of the 12 cylindrical constructs became infected and were extruded through the skin -- the other 10 remained subcutaneous and showed replacement be chondrocytes, with retention of scaffold shape.

Histological staining showed that cells found on the periphery of the implants were more disordered and less mature than those located centrally, which exhibited deeper basophilic staining that suggests increased mucopolysaccharide concentration characteristic of mature cartilage. Immunohistochemical staining found BrdU labeling in the periphery of the cartilage, suggesting that cells located in this region were produced after in-vitro culturing. By week 3, the polymer fibers of the polymer-cell implants had begun degrading, with degradation continuing throughout the 12-week time course of the study.

Critique While this study represents a promising advance in creation and manipulation of autologous cartilage constructs, there are still several problems that must be addressed before this technique becomes feasible for use in human patients. To begin with, the time course of this study is relative short (12 weeks), whereas most cartilaginous implants are expected to last the duration of a patient's life. It is unclear if the shape of the cartilage may change past the 12 week mark; if the cartilage was to begin to lose its shape or lose its structural integrity, these implants would no longer be appropriate for use in plastic and reconstructive surgery. Researchers must also address the mechanical properties of cartilage formed on these scaffolds -- do they have the structural integrity of naturally-occuring cartilage? Can the mechanical properties be varied as a function of experimental parameters (e.g. density of injected cells, growth medium conditions)? This is an important aspect to address because cartilage used in different parts of the body may require different mechanical properties. A third problem the researchers may consider addressing is the immunoreactivity of their implants. It is interesting to note that in this study, chondrocytes harvested from calves were injected into scaffolds that were then implanted into mice. No mention of immune reactions is noted besides the two cylindrical scaffolds that were extruded. It is unclear if this immune reaction is due to the calf chondrocytes or perhaps the scaffold. Though 12 weeks is conceivably sufficiently long for an immune response to be elicited and noticed, long-term effects are unlikely to be noticed during the time course of this study. In conclusion, this paper presents a novel, perhaps revolutionary way for cartilaginous implants to be used in plastic and reconstructive surgery. However, before human implantation becomes even remotely feasible, researchers and medical doctors must take into account a host of factors that may influence the feasibility of such a design.