Megakaryocytes enhances proliferation but delayed differentation in stem cells

摘要

Statement of Purpose: For several years, cranial bone defect owing to congenital malformations, accidents, severe trauma, and tumor surgery, has been a major challenge to reconstruct and to restore the functions of broken bone tissues. Traditional treatment approaches using natural bone grafting methods, e.g. autografis and allografls come with many limitations, such as donor site morbidity and inconsistent clinical results. Current advances in Tissue Engineering (TE) and Regenerative Medicine have promising by using innovative techniques to address this critical clinical challenge. In the past, TE approach utilizing bone morphogenetic protein-2 (BMP-2) in conjunction with scaffold and mesenchymal stem cells to facilitate cranial bone regeneration has shown the ability to reconstruct a critical bony defect. However, recent studies revealing serious side effects such as higher risk of cancer, ectopic bone formation, and osteolysis of BMP-2 demand the TE community to seek a safe and effective alternative growth factor to BMP-2. Recently, we found that human-recombinant thrombopoietien (hrTPO), the main growth factor of megakaryocytes (MKs), can indirectly stimulate the proliferation of osteoblasts through MKs1. Our in vivo results showed that TPO allow for a more physiologically-contoured callus formation in mouse, rat, and minipigs, but at a later time point compared to BMP-2. In this study, we aim to further investigate this observation by studying the effects of MKs on the proliferation and differentiation of stem cells harvested from rabbit bone marrow (BMSC) and dental pulp tissues (DPSC). Materials and Methods: Under a protocol approved by the Institutional Animal Care and Use Committee at the Indiana University School of Medicine, a 2 kg New Zealand rabbits were sacrificed to serve as cell donors. To isolate BMSCs, femora and tibia were collected and then bone marrow was flashed in DMEM medium supplemented with 2% FBS and 1% penicillin -streptomycin (PenStrp). For DPSCs, the rabbit's incisors were extracted and the tissue was digested in 10% collagenase type Ⅱ and cells were isolated from the dental pulp. BMSCs and DPSCs were cultured in 10% FBS, 1 % PenStrp incubated at 37C°, 5% CO2 and proliferated. Another 4 kg New Zealand rabbit were sacrificed and the bone marrows were used as the MKs source. After processing, rhTPO were added at 100 ng/mL and cells were cultured in 10% FBS, 1% PenStrp and incubated for 7 days to stimulate MKs. BMSCs and DPSCs were trypsinized and co-cultured in 24 well plate with MKs at 1:1 ratio with 15,000 cells per well in control (CTR) DMEM medium and ostegenic (OST) medium (DMEM supplemented with 50 μg/mL ascorbic acid, 10 mM beta-glycerol phosphate, and 10 nM dexamethasone). BMSCs and DPSCs without MKs were also cultured in OST and CTR medium. AlamarBlue, ALP, Alazrin Red, and RT-PCR assays were preformed to evaluate cells viability, and differentiation at different time-points. Results and Discussion: AlarmarBlue cells proliferation assay showed that BMSCs and DPSCs co-cultured with MKs demonstrated significantly higher cell proliferation comparing to groups cultured without MKs in both OST and CTR medium, although BMSCs and DPSCs cultured in OST medium show slightly lower in proliferation. Interestingly, BMSCs and DPSCs co-cultured with MKs illustrated lower ALP activities in compare with other groups cultured without MKs, specifically the cell group cultured in OST medium . Although MKs seem to delay stem cell differentiation; the cells cultured without MKs in OST medium showed increasing in calcium at day 14 and decreasing at Day 21 while cells cultured with MKs shows increasing in calcium at day 21. Eventually, the cells in OST medium with or without MKs were able to differentiate into osteoblasts Conclusion: In this study, we investigated the effects of MKs have on BMSCs and DPSCs. Our results show that MKs significantly increased the early proliferation, but delayed the differentiation of BMSCs and DPSCs, indicating a potential mechanism responsible for the delayed healing observed in our in vivo study.