SEEDING OF THE DEMINERALIZED BONE MATRIX WITH CHONDROGENIC CELLS

Aim To determine optimal approaches of demineralized bone tissue processing after preservation to ensure efficient seeding of chondrocytes. Methods Demineralized bone matrix specimens sized 1 x 1 x 1 cm³ were used in the experiment. A purification method ensuring the removal of cytotoxic substances from the matrices has been developed. It consists of a multi-stage soaking of the specimens in H₂O, 0.1H NaOH, 1N NaOH, H₂O and DPBS until a neutral pH is reached. After chemical purification (a 3-stage process), all the specimens were subjected to sonication for 1 minute at 5W to improve cell adhesion. The water was changed after each exposure. Then, the water was replaced to DPBS and the specimens were sonicated for 1 minute at 5W. After it, the sample was placed in a neutral medium (pH 7.0). The matrices undergoing sonicated procession were seeded with cells. Hyaline cartilage of minipigs was used as a source of the cells. Chondrocytes were isolated using collagenase II digestion and cultured for 20 days in the culture flasks. Passage 1 chondrocytes were seeded on the matrices. DBM were pretreated with a 1% gelatin solution to improve the efficiency of cell seeding. The microtitration viability test estimating the impact of the extract obtained during sonation cycles on cell viability was performed to determine whether these matrices may be seeded with chondrocytes. The test was performed on the lag- and log-phase cells. The effect of the extract on the cells lasted around 3 days. Results Extract-treated chondrocytes during the lag-phase showed a direct dose-dependent cytotoxic effect, compared to extract-treated chondrocytes during the log-phase. Low efficiency of DBM was associated with both, the stringent requirements for the manufacturing process of DBM and the subsequent matrices processing, including the cell growth phases. The increased cell migration depth into the matrices resulted in the disturbances of the microcirculation, leading to the insufficient cell feeding and slowed down metabolic processes. Conclusion The efficiency of DBM cell seeding depends on the matrix processing, its cytotoxic effect and architectonics. The problem of slowing down the metabolism of cells in DMB may be solved by the application of the combined purification technique, i.e. chemical and ultrasonic purification methods. The obtained results prove the necessity of using mechanical and electrical stimuli for the normal functioning of bone and cartilage tissue cells within the matrix.

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