Conventional reconstructive surgery has employed autologous, biological, and artificial materials in an effort to reestablish normal human anatomy and function, however, often trigger immunological or organ-specific complications. Tissue engineering seeks to overcome the inherent problems of introduced materials by generating tissue that conforms to site-specific anatomy and function. To facilitate tissue regeneration, several studies have been performed using various biomaterials in association with different cell types (i.e. osteoblasts, osteoclasts and endothelial cells) under 2D and 3D conditions for bone repair, however, there have been few reports investigating the osteogenic differentiation of hMSC under MG conditions.
The Rotary Wall Vessel (RWV) is a device designed to simulate microgravity conditions, however, we and others have utilized this system for the RWV culture of cells [17, 18]. This system encompasses the features of low-shear stress, high mass transfer environment, comparable to the weightless state that is characteristic of blood, lymphatic fluid, or even in the bone marrow [19, 20]. Additionally, this system has been extremely useful in determining cellular interactions between cancer and host bone marrow stromal cells , and in studying therapeutic options for many pathological situations, which include radiation resistance, phenotypic differentiation, and response to targeted drug delivery. In this study we utilized the RWV system, which models microgravity (MG) conditions, with a gelatin sponge scaffold, to explore the possibility that hMSC, with an appropriate scaffold, will facilitate cell growth and organization, as well as directed differentiation, and that it can be utilized for regeneration of bone cells.
Previous studies have shown that gelatin scaffold supports the differentiation of hMSC or other stem cells into multiple lineages including osteogenic, chondrogenic, and adipogenic on 2D surfaces [11, 22, 23]. However, differentiation of hMSC cultured under modeled microgravity has not been determined. Our initial results indicate that hMSC cultured in the RWV system with gelatin scaffold support randomized adherence to the gelatin scaffold with near complete cell uptake within the gelatin-based spheroid as measured by DNA content after 3 days of culture. These findings are supported by previous reports that numerous scaffolds including microcarrier beads or collagen-coated sponges support cell attachment in vitro. Although collagen is a major component of the bone matrix, we did not observe significant cell attachment with collagen-coated scaffolds (data not shown). Thus, we utilized uncoated gelatin sponges for the remainder of our studies.
Upon histological examination, 3 day control spheroids showed increased cell organization with a homogeneous multi-cellular tissue-like structure that completely coated the 1x1x1 mm scaffold. A high proliferative index, determined by Ki-67 staining, was seen as well. Interestingly, we did not observe any necrosis, which is typical of RWV engineered tissue-like structures greater than 200 μm in size in bioreactor systems [25, 26]. Thus, it is possible that the gelatin sponge facilitates diffusion of nutrients and oxygen, not typically observed with more rigid scaffold designs. Surprisingly, these findings are in contrast to a study  that reported a decrease in the viability and proliferation of cells cultured in the RWV bioreactor under modeled MG conditions. Although we did find a decrease in attachment of free floating cells, viability and proliferation remained through all experimental time points.
In order to determine the effects of MG on hMSC differentiation, we further characterized the spheroids over a 10 day period. Early differentiation (3 days) was not observed since undifferentiated stem cell markers CD44, CD133, and CD166 were robustly expressed and typical markers associated with lineage-specific differentiation (von Kossa, oil red O, collagen II, aggrecan) were not detected. Indeed, we did detect isolated lipid deposits, (oil red O staining) within the spheroids, suggesting the spheroids were pre-adipogenic. However, given the relatively low oil red O staining and presence of stem cells markers, it appears that after 3 days of MG, hMSCs largely remain undifferentiated.
The pluripotent potential of spheroids was further demonstrated after 10 day culture under defined differentiation conditions (shown to promote adipogenic, chondrogenic, and osteogenic differentiation, respectively). As expected, we observed early lipid deposits (3 days) and strong lipid deposits in 10 day control and adipogenic cultured cells. These findings are similar to Zayzafoon’s study  that concluded that hMSC, cultured on microcarrier beads in MG, undergo adipogenic differentiation opposed to other lineages including cells cultured in divergent differentiation-inducing conditions. The authors further concluded that the adipogenic lineage represents the default pathway of hMSC cultured under MG conditions. However, our findings do not completely reflect this hypothesis. hMSC cultured under osteogenic conditions, after 10 days exhibited robust von Kossa staining, suggesting calcification and mineralization, and VDR staining which is responsible for mineral stability [28–30]. Additionally, we observed ALP expression, which is produced by osteoblastic cells and represents a definitive marker for early osteogenesis [31, 32]. Loss of stem cell markers CD133 and CD166 expression in all differentiation conditions further support the pluripotency of 3 day spheroids and the commitment to cellular differentiation after 10 days.
Osteogenic differentiation of mouse bone marrow stromal cells in RWV system has been previously reported . Conditioned media from mature osteoblasts were able to induce increased osteoclastogenesis and bone resorption in mouse bone marrow cultures via indirect stimulation of osteoclast formation and activity by regulating osteoblast secretion of regulatory factors such as RANKL and OPG. VDR regulates the expression of bone matrix proteins and promotes osteoclast differentiation by inducing the expression of RANKL. We observed an increase in VDR expression and constant expression of ERp60, a membrane-associated receptor implicated in the rapid actions of 1,25(OH)2D , in 3 day and 10 day spheroids (control or osteogenic condition), along with increased ALP expression and mineralization. These findings suggest that exogenous or endogenous soluble ligands (i.e. RANKL/OPG) are required to override the adipogenic default lineage and promote osteoblast formation of hMSC in MG conditions. Our observation of a low level of ALP expression after 10 days, even without osteogenic conditions, further supports a directive role of gelatin scaffold. Indeed, since the biocompatibility of gelatin likely facilitates cell attachment, which is required to promote differentiation and proliferation, it likely plays a role in promoting osteogenic properties of hMSC under MG conditions
In conclusion, this study provides greater insight into the effects of MG on hMSC osteogenic differentiation. Furthermore, this model establishes a rapid culture method that overcomes the bone diminishing effects of MG on hMSC osteogenesis and provides a system to determine the cellular and molecular mechanisms that are associated with this decline. Although the pluripotency of MG-cultured hMSC within the gelatin scaffold has yet to be verified in vivo in regard to bone regeneration, our system provides undifferentiated, cell-coated, and implantable constructs for tissue engineering and ex vivo modeled organogenesis.