INTRODUCTION: Cartilage is a tissue with a limited regeneration success. Damage cartilage usually repairs as a fibrocartilage. In articular surfaces the presence of fibrocartilage regions results in impaired functions and pain to the patients. The autologous chondrocyte transplantation is a technique for articular cartilage repair, viewing the formation of a tissue with the same biomechanical characteristics of the preexisting tissue. This technique has been applied to repair damage cartilage with success. It briefly consists in: I. take a biopsy of non-damaged cartilage form the patient through arthroscopy; II. digest this tissue to isolate the chondrocytes; III. culture the chondrocytes to obtain enough cells to fill the defect; IV. return the cells to the patient after a surgical preparation of the damaged tissue. The tissue formed from the transplant has the morphology and biochemical composition of the hyaline cartilage and it behaves just as the normal articular cartilage. However, the technique has its limitations, e.g. the demanding surgical technique described in the literature, the age of the patients, less than 50 years, or diseases like degenerative lesions, tumors and infections. The technique requires a cell culture laboratory working closely together with the orthopedic surgeons, since the transportation of the cartilage samples to the laboratory is an integral step the technique. However, the hospital is not always located in the same country as the cell culture laboratory, resulting in several hours of the transport. The purpose of the present work was to evaluate the effects of the transport time on the ultrastructure of the cartilage samples. We examined cartilage tissue at different times after the biopsy and with two different fixatives, one preserving the cytoplasmic organells of the cells and the other for optimal demonstration of the components of the extracellular matrix.
Experimental Method: Articular cartilage was obtained during chondroplastic arthroscopic surgery from a patient with severe knee accident. After surgery, cartilage tissue which had been resected for treatment of the lesions on the femoral condyles and the patella were collected from the intercondylar fossa. These cartilage samples were transported between two Brazilian cities (100 km of distance) in the same conditions used to transport biopsies for autologous chondrocyte transplantation (transportation medium containing HAMF12:DME and 1% penicillin/streptomycin, at 4ºC to 9ºC). The samples were fixed at 2 different times: 6 and 24 hours after the arthroscopy.

The samples were fixed in glutaraldehyde (2,5% in cacodylate buffer), post fixed in osmium tetroxide (1% in cacodylate buffer), dehydrated in an ethanol series followed by propylene oxide and inclusion in resin (Epon 812). As a variation of this protocol we used 1% tannic acid in the first fixation solution. Semithin sections were stained with toluidine blue and observed by standard light microscopy at initial magnifications of up to x 100. Ultrathin sections were contrasted by uranyl acetate and lead citrate examined on a JEOL 906 transmission microscope.

RESULTS AND DISCUSSION: Cartilage samples fixed 6 hours after arthroscopy presented nuclei with normal appearance of their condensation figures, cristae of mitochondria, and microfilaments in the cytoplasm. The endoplasmatic reticulum, however, showed distended areas. The extracellular matrix contained collagen fibers and a reticulated meshwork characteristic for cartilage tissue. Samples fixed after 24 hours were similar in appearance, however, some phagocytotic bodies were present in the cytoplasm and numerous vesicles were observed along the cytoplasmatic membrane, pinocytic or secretion vesicles. The presence of an increased number of vesicle at the cytoplasmatic membrane could suggest that the cells are remodeling the extracellular matrix.

CONCLUSION: The semithin sections indicate that the tissues examined may be derived form different levels of the cartilage tissue. Chondromes contained one to several cells, which suggests some reaction to the cartilage lesion set by the trauma. From these results we conclude that cartilage tissue from arthroscopic chondroplasty is not ideal to study the changes in the tissue after excision without additional information on the origin of the tissue (anatomical location, level within the cartilage layer, and histological changes caused by the trauma). In contrast, the ultrastructure evaluation appears to be a powerful method to follow changes in cartilage after the excision of biopsies for the autologous cell therapy.

REFERENCES: Brittberg M, Lindhal A, Nilsson A, Ohlsson C, Isaksson O, Peterson L,1994. New Engl J Med, 331: 889-895.Mandelbaum BR, Browne JE, Fu F, Micheli L, Mosely JB, ErggeletvC, Minas T, Peterson L.1998. Am J Sports Med, 26:853-861.

ACKNOWLEDGMENTS AND SOURCES OF FOUNDING STATED: We thank the Electronic Microscopy Laboratory, UNICAMP, for technical support. GMReis Biológica supported this study.