Megakaryocytes (MKs) can induce proliferation of calvarial osteoblasts [Ciovacco et al., 2009], but this same phenomenon has not been reported for bone marrow stromal populations from long bones. Bone marrow contains several types of progenitor cells which can be induced to differentiate into multiple cell types. Herein, we examined mesenchymal stromal cell proliferation and osteoblastic differentiation when rabbit or mouse MK were cultured with i) rabbit bone marrow stromal cells, ii) rabbit dental pulp stromal cells, or iii) mouse bone marrow stromal cells. Our results demonstrated that rabbit and mouse stromal cells co-cultured with rabbit MK or mouse MK, have significant increases in proliferation on day 7 by 52%, 46%, and 24%, respectively, compared to cultures without MK. Conversely, alkaline phosphatase (ALP) activity was lower at various time points in these cells when cultures contain MK. Similarly, calcium deposition observed at day 14 rabbit bone marrow and dental pulp stromal cells and day 21 mouse bone marrow stromal cells was 63%, 69%, and 30% lower respectively, when co-cultured with MK. Gene expression studies reveal transcriptional changes broadly consistent with increased proliferation and decreased differentiation. Transcript levels of c-fos (associated with cell proliferation) trended higher after 3, 7, and 14 days in culture. Also, expression of alkaline phosphatase, osteonectin, osterix, and osteopontin, which are markers for osteoblast differentiation, showed MK-induced decreases in a cell type and time dependent manner. Taken together, these data suggest that MK play a role in stromal cell proliferation and differentiation, from multiple sites/locations in multiple species. This article is protected by copyright. All rights reserved.

Successful regeneration of the cranium in patients suffering from cranial bone defects is an integral step to restore craniofacial function. However, restoration of craniofacial structure has been challenging due to its complex geometry, limited donor site availability, and poor graft integration. To address these problems, we investigated the use of a thiol-acrylate hydrogel as a cell carrier to facilitate cranial regeneration. Thiol-acrylate hydrogels were formulated with 5-15 wt% poly(ethylene glycol)-diacrylate (PEGDA) and 1-9 mm dithiothreitol (DTT). The degradation rate, swelling ratio, and shear modulus of the resulting hydrogel were first characterized. Then, pre-osteoblast-like cells (MC3T3-E1) were encapsulated in the hydrogel and cultured for up to 21 d. Our results demonstrate that compared to samples formulated from 15 wt% PEGDA, 5 wt% PEGDA samples showed lower storage modulus at day 10 (0.7 kPa versus 8.3 kPa), 62.7% higher in weight change after soaking for 10 d. While the 5 wt% PEGDA group showed an 85% weight loss between day 10 and 21, the 15 wt% PEGDA group showed a 5% weight gain in the same time period. Cell viability with 15 wt% PEGDA and 5 mm DTT hydrogel decreased by 41.3% compared to 5 wt% PEGDA and 5mM DTT gel at day 7. However, histological analysis of cells after 21 d in culture revealed that they had pericellular mineral deposition indicating that the cells were differentiating into osteoblasts lineage in all experimental groups. This study shows that thiol-acrylate hydrogels can be tailored to achieve different degradation rates, in order to enhance cell viability and differentiation. Thus, the findings of this study provide a fundamental understanding for the application of thiol-acrylate hydrogels in cranial bone regeneration.