Preparation of cell aggregates incorporating gelatin hydrogel microspheres containing bone morphogenic protein-2 with different degradabilities.

The objective of this study is to evaluate the survival and functions of cells in cell aggregates incorporating gelatin hydrogel microspheres (GM) containing bone morphogenic protein-2 (BMP2). Gelatin was dehydrothermally crosslinked in a water-in-oil emulsion state at 140 °C for various time periods to prepare GM with different degradabilities. BMP2 was dropped onto the GM freeze dried, followed by leaving at 25 °C to obtain GM containing BMP2 (GM-BMP2). MC3T3-E1 cells were cultured with GM-BMP2 and GM in round U-bottom wells of 96-multiwell microplates which had been coated with poly (vinyl alcohol) (PVA), to allow to form cell aggregates containing GM-BMP2 and GM, respectively. Higher MC3T3-E1 cell proliferation and the L-lactic acid/glucose ratio were observed for MC3T3-E1 cell aggregates cultured with the GM of slower degradation. The runt-related transcription factor 2 (RUNX2) messenger ribonucleic acid (mRNA) expression, alkaline phosphatase (ALP) activity, and calcium content of MC3T3-E1 cells in cell aggregates were assayed to evaluate their osteogenic differentiation. When cultured for 7 days with GM-BMP2 or free BMP2, the RUNX2 mRNA expression and ALP activity were higher for MC3T3-E1 cell aggregates cultured with the GM-BMP2 of faster degradation than those of free BMP2 added into the medium. After 21 days culture, the ALP activity and calcium content were higher for the GM-BMP2 of medium degradation compared with other experimental groups. It is concluded that BMP2 of GM-BMP2 incorporated in the cell aggregates enhanced the osteogenic differentiation of cells compared with free BMP2 added externally. The degradability of GM-BMP2 affected the extent of osteogenic differentiation.