This study introduces an implantable scaffold-free (SF) cartilage tissue construct that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were isolated from the articular cartilages from knees of domestic pigs (2-week old) and monolayer-cultured for 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 microg/mL of ascorbic acid. Briefly treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), an intact chondrocytes/ECM membrane, as a cell sheet was released from the plate bottom and subsequently centrifuged into a pellet-type construct. Each was grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 14, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 4) were manually created on the femoro-patellar groove of the left porcine knee and 1-week-cultured SF construct was implanted as an allograft for a month. One defect (#1) was an empty control and the remaining three received different recipes; construct only (#2) or 0.25% trypsin/EDTA-treated first and then construct and collagen gel (#3) or construct and collagen gel (#4). While the total cell numbers significantly increased by 2 weeks and then remained stable, cell viability stayed in the mid-70% range through the entire culture period. Biochemical assay found continuous glycosaminoglycan (GAG) accumulation. Histology exhibited that cell distribution was even in the construct and GAG intensity became stronger and uniform with time. Real-time reverse transcription polymerase chain reaction (RT-PCR) results showed that phenotypic stability peaked at 2 weeks, which was arable to that of freshly isolated chondrocytes. Upon analysis of the retrieved implants, some promising results were witnessed in the defects (#3) retaining not only their intact mass but also chondrocytic morphology with lacuna formation.

This study introduces an implantable scaffold‐free cartilage tissue construct (SF) that is composed of chondrocytes and their self‐produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full‐thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16‐week old) and 3 week‐cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold‐free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold‐free engineered cartilage. When implanting tissue‐engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.

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Two passaged (P2) immature porcine articular chondrocytes were used to fabricate an engineered cartilage tissue in an in vitro scaffold-free system with or without insulin like growth factor 1 (IGF-1). This study shows the possibility of the fabrication of structurally regular neocartilage tissue using passaged chondrocytes in the scaffold-free system with insulin like growth factor-1(IGF-1).

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