The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site.

Small intestinal submucosa (SIS) membranes as a decellularized tissue are known to be a natural nanofibrous biomaterial mainly made of type I collagen fibers and containing some growth factors (fibroblast growth factor 2 and transforming growth factor β) desired in tissue engineering. Here we show that the SIS membranes can promote the formation of bone mineral hydroxylapatite (HAP) crystals along the collagen fibers constituting the membranes from a HAP-supersaturated solution. The resultant biomineralized HAP-SIS scaffolds were found to promote the attachment, growth and osteogenic differentiation of mesenchymal stem cells (MSCs) in both basal and osteogenic media by the evaluation of osteogenic marker formation. More importantly, the HAP-SIS scaffolds could induce the osteogenic differentiation in the basal media without osteogenic supplements due to the presence of HAP crystals in the scaffolds. Histological characterization of the MSC-seeded scaffolds showed that HAP-SIS scaffolds are biocompatible and promote the formation of new tissue in vitro. The biomineralized SIS membranes mimic some aspects of natural bone in terms of the composition and nanostructures and can find potential use in bone tissue engineering.