Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing

Abstract

Mesenchymal stem cells (MSCs) have great therapeutic potential for the repair of nonhealing bone defects, because of their proliferative capacity, multilineage potential, trophic factor secretion and lack of immunogenicity. However, a major challenge to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesize that forming MSCs into more physiologic three-dimensional spheroids, rather than employing dissociated cells from two-dimensional monolayer culture, will enhance their survival when exposed to a harsh microenvironment but maintain their osteogenic potential. MSC spheroids were formed by using the hanging drop method with increasing cell numbers. Compared with larger spheroids, the smallest spheroids, which contained 15,000 cells, exhibited increased metabolic activity, reduced apoptosis and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free medium and 1 % oxygen. Compared with identical numbers of dissociated MSCs in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more vascular endothelial growth factor. Moreover, fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. Thus, MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential while maintaining similar osteogenic potential to dissociated MSCs entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair.

Fig. 1

MSC spheroid diameter, metabolic activity, viability, and proliferation can be controlled by cell number. (a) Spheroid diameter increases linearly with increasing cell number (n=12). (b) The smaller 15,000-cell spheroids exhibited increased metabolic activity, as measured by Alamar Blue, than the larger 30,000-cell and 60,000-cell spheroids (*p<0.05 vs. 30,000-cell spheroid, ****p<0.0001 vs. 60,000-cell spheroid; n=6). (c) The smaller 15,000-cell spheroids exhibited reduced apoptosis, as measured by caspase 3/7 activity, than the larger 30,000-cell and 60,000-cell spheroids (***p<0.001 vs. 30,000-cell and 60,000-cell spheroids; n=6). (d) EdU (green) and Hoechst (blue) staining revealed the cells in the 15,000-cell spheroid (left) are more proliferative than those in the larger 30,000-cell (middle) and 60,000-cell spheroids (right). Scale bar represents 500 μm. (e) Toluidine Blue staining indicated the 15,000-cell spheroids are more compact than the larger 30,000-cell and 60,000-cell spheroids. Scale bar represents 500 μm.

Fig. 2

MSC spheroids entrapped in a fibrin gel and cultured in SD/H conditions exhibit enhanced survival and VEGF secretion compared to dissociated MSC. (a) Caspase 3/7 activity in MSC spheroids compared to dissociated cells (**p<0.01 vs. spheroids at day 0, ****p<0.0001 vs. spheroids, n=4). (b) VEGF secretion in spheroids compared to dissociated cells (**p<0.01 vs. spheroids, ****p<0.0001 vs. spheroids, n=4). (c–h) H&E staining of cells entrapped in fibrin gels over time. Arrows denote cells; scale bar represents 500 μm.

Fig. 3

Osteogenic potential of MSC spheroids and adherent monolayers is influenced by timing of osteogenic induction. Osteogenic potential was assessed 7 days after spheroid formation. (a) When cells were cultured in αMEM prior to spheroid formation in OM (- OM during 10 day induction), ALP activity was significantly lower in spheroidal culture compared to adherent monolayers. However, ALP activity was not statistically different between the two groups if both spheroid and adherent monolayers received osteogenic media throughout the experiment (****p<0.0001 vs. spheroids, n=4). (b) Calcium content significantly increased in both MSC spheroids and adherent monolayers as the duration of culture in OM increased (**p<0.01 vs. spheroids, ****p<0.0001 vs. spheroids, n=4).

Fig. 4

MSC spheroids entrapped in fibrin gels exhibit comparable osteogenic potential to dissociated MSC. (a) ALP activity was statistically indistinguishable between spheroids and dissociated cells entrapped in fibrin gels at all time points (n=3). (b) Total calcium content was statistically indistinguishable between MSC spheroids and dissociated cells entrapped in fibrin gels at all time points (n=3). (c–j) H&E staining of cells entrapped in fibrin gels over time. Scale bar represents 200 μm.