Repair of Cartilage Defects by Using In Vitro Differentiated Mesenchymal Stem Cells In Vivo: Experimental Study in Rabbits.

The repair of cartilaginous defect remains a significant clinical challenge. Damaged cartilage has a limited potential for repair and large defects do not heal spontaneously. Bone marrow-derived mesenchymal stem cells (MSC) possess excellent regenerative capacity, the ability to proliferate rapidly in culture, and the ability to differentiate into a wide variety of chondrogenic, osteogenic, and myogenic cell types. In this study, the effect of MSC on cartilage formation at ear cartilage defect was investigated in the rabbit ear model in vivo. For this purpose, male New Zealand rabbits were used standard laboratory conditions and animals were anesthetized with ketamine HCl and sevoflurane. Five milliliters of the bone marrow was aspirated from the posterior iliac crest, separated with Ficoll-Hypaque and mononuclear cells were cultured in DMEM including 10%(v/v) autologous serum, antibiotics at 37°C in a humidified atmosphere of 5% CO₂, changing the media every 3 days. After reaching 80% to 90% confluence approximately 2 weeks after seeding, the adherent cells were released from dishes with 0.25% trypsin, and seeded onto fresh plates for twice. After the twice-passaged the cells became nearly confluent, MSC differentiation was checked with morphologically and vimentin positivity. Lateral skin incisions were done at the back of the rabbit ear and dorsal perichondrium was incised to expose the ear cartilage. Cartilages, sized 1x1 cm, were removed while protecting the anterior perichondrium to create 4 ear pockets per rabbit. After hemostasis, 1x1 cm bi-perichondrial pockets that are devoid of cartilage were filled with oxidized cellulose (Group I) or oxidized cellulose plus 1 x 10⁶ of MSC (Group II) or 1 x 10⁶ of MSC (Group III) or sodium chloride (Group IV as control). Oxidized cellulose was used as a carrier material. The quality of the new cartilage formation on each side was determined with serial biopsy specimens on the 30^th, 60^th and 90^th days from each rabbit. Then the specimens were stained with hematoxylin-eosin and saphranin-O. Macroscopic evaluation of the defect area was also performed for each time period. On day 30 in the control group, there were few chondroblast formations with fibrosis. In the experimental group (Group II and III), there was a dense chondroblastic formation filling the pocket between the perichondrium and spreading beyond the normal cartilage. On day 60 in the control group, the result of most specimens was similar to that of those obtained on day 30 with intense fibrosis. In the experimental group, neocartilage was twice as thick as the normal cartilage and in most parts it consisted of mature chondrocytes with vacuoles in their cytoplasm. In oxidized cellulose group, chondrogenesis was limited and intermingled with fibrosis in all time periods. At day 90, defect site was translucent and soft in the control group; however, in the experimental group the defect site was hard with an irregular surface and without translucency. These data shows that allogeneic culture expanded bone marrow-derived MSC have a chondrogenic potential and may be used successfully to repair cartilaginous defects without any significant immunological reaction and superior to the perichondrium guided chondrogenesis.