Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2.

In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic protein-2 (rhBMP-2). Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and 4.0 mg/ml. HY accelerated cellular proliferation when compared with cultures in the absence of HY at both Days 2 and 7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higher than under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml. When BMSc were cultivated under HY at concentrations of 0, 1.0 and 4.0 mg/ml and its 12 combinations with rhBMP-2 at concentrations of 0 and 10 ng/ml and Dex (+, -) at both Days 2 and 7, cellular responses were examined by 3H-thymidine incorporation into DNA, cellular alkaline phosphatase (ALP) activity, and pro-collagen type I C-terminal propeptide production. HY accelerated cellular proliferation irrespective of the presence of Dex and rhBMP-2. HY increased expression of ALP activity at Day 7, whereas had inhibitory effect at Day 2. HY and Dex showed an interaction on expression of ALP acitivity irrespective of the HY dose by Day 7. Collagen synthesis was inhibited by HY irrespective of the presence of other factors at both Days 2 and 7. When BMSc were cultivated with HY of 4.0 mg/ml alone, its combinations with Dex (+) and 10 ng/ml rhBMP-2, and with DMEM/FBS alone, expression of bone-related marker genes was evaluated by real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) analysis. Osteocalcin was up-regulated under both rhBMP-2 and HY-Dex-rhBMP-2 at Day 2, as also under 4 mg/ml HY, Dex, HY-Dex, Dex-rhBMP-2, and HY-Dex-rhBMP-2 by Day 7. Type 1alpha1 collagen was induced by rhBMP-2 on Day 2, and by Dex-rhBMP-2 on Day 7. Osteonectin and type X collagen was only marginally induced by HY at Day 2. Type 1alpha1 collagen and type X collagen were down-regulated in the presence of 4 mg/ml HY by Day 7. These results suggest that HY stimulates BMSc proliferation, osteocalcin gene expression, and a secretion of enzymes such as that of ALP activity in vitro. More importantly, HY can interact with Dex and rhBMP-2 to generate direct and specific cellular effects, which could be of major importance in bone tissue engineering.