Hydrogel mechanics are a key driver of bone formation by mesenchymal stromal cell spheroids.

Mesenchymal stromal cells (MSCs) can promote tissue repair in regenerative medicine, and their therapeutic potential is further enhanced via spheroid formation. Stress relaxation of hydrogels has emerged as a potent stimulus to enhance MSC spreading and osteogenic differentiation, but the effect of hydrogel viscoelasticity on MSC spheroids has not been reported. Herein, we describe a materials-based approach to augment the osteogenic potential of entrapped MSC spheroids by leveraging the mechanical properties of alginate hydrogels. Compared to spheroids entrapped in covalently crosslinked elastic alginate, calcium deposition of MSC spheroids was consistently increased in ionically crosslinked, viscoelastic hydrogels. We previously demonstrated that intraspheroidal presentation of Bone Morphogenetic Protein-2 (BMP-2) on hydroxyapatite (HA) nanoparticles resulted in more spatially uniform MSC osteodifferentiation, providing a method to internally influence spheroid phenotype. In these studies, we observed significant increases in calcium deposition by MSC spheroids loaded with BMP-2-HA in viscoelastic gels compared to soluble BMP-2, which was greater than spheroids entrapped in all elastic alginate gels. Upon implantation in critically sized calvarial bone defects, bone formation was greater in all animals treated with viscoelastic hydrogels. Increases in bone formation were evident in viscoelastic gels, regardless of the mode of presentation of BMP-2 (i.e., soluble delivery or HA nanoparticles). These studies demonstrate that the dynamic mechanical properties of viscoelastic alginate are an effective strategy to enhance the therapeutic potential of MSC spheroids for bone formation and repair.

Tunable fibrin-alginate interpenetrating network hydrogels to support cell spreading and network formation.

Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. STATEMENT OF SIGNIFICANCE: Hydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Spatial localization of endothelial cells in heterotypic spheroids influences Notch signaling.

Cell-based therapeutic approaches are an exciting strategy to replenish compromised endothelial cell (EC) populations that contribute to impaired vasculogenesis. Co-cultures of ECs and mesenchymal stromal cells (MSCs) can enhance neovascularization over ECs alone, but the efficacy of cells is limited by rapid cell death upon implantation. Co-culture spheroids exhibit improved survival compared with monodisperse cells, yet little is known about the influence of spatial regulation of ECs within co-culture spheroids. We hypothesized that EC sprouting from co-culture spheroids is a function of EC spatial localization. We formed co-culture spheroids containing ECs and MSCs in two formats: ECs uniformly distributed throughout the spheroid (i.e., mixed) or seeded on the perimeter of the MSC core (i.e., shell). Qualitative observations suggested increased vasculogenesis for mixed co-culture spheroids compared with shell conformations as early as day 3, yet quantitative metrics did not reveal significant differences in network formation between these 3D structures. Notch3 expression demonstrated significant increases in cell-cell communication in mixed conformations compared with shell counterparts. Furthermore, knockdown of Notch3 in MSCs abrogated the vasculogenic potential of mixed spheroids, supporting its role in promoting EC-MSC contacts. This study highlights the direct impact of EC-MSC contacts on sprouting and provides insight to improve the quality of network formation. KEY MESSAGES: • Endothelial cell (EC) localization can be controlled in co-culture EC-MSC spheroids. • Mixed spheroids exhibit consistent networks compared to shell counterparts. • Differences in NOTCH3 were observed between mixed and shell spheroids. • NOTCH3 may be a necessary target for improved vasculogenic potential.

Injectable mineralized microsphere-loaded composite hydrogels for bone repair in a sheep bone defect model.

The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.

Restoring vasculogenic potential of endothelial cells from diabetic patients through spheroid formation.

INTRODUCTION: Diabetes is an emerging epidemic in the developing world and represents a major risk factor for cardiovascular disease. Among other issues, patients with diabetes suffer from diminished endothelial cell (EC) function, which contributes to impaired vasculogenesis and recovery from ischemic insult. The formation of cells into three-dimensional spheroids promotes cell survival and activates key signaling pathways through the upregulation of cell-cell contacts, providing an opportunity to overcome shortcomings associated with individual autologous cells. METHODS: We hypothesized that forming human microvascular endothelial cells (HMVECs) from diabetic patients into spheroids would restore their vasculogenic potential following upregulation of these cell-cell interactions. HMVEC spheroids were formed and suspended in fibrin gels to quantify vasculogenic potential. RESULTS: Individual HMVECs from diabetic patients exhibited similar proliferative and chemotactic potential to cells from healthy donors but reduced tubulogenesis. HMVEC spheroids formed from diabetic donors formed more sprouts than spheroids from healthy donors, and more sprouts than individual cells from either population. CONCLUSIONS: Compared to cells from healthy donors, sprout formation was more efficiently abrogated in HMVECs from diabetic patients by blocking matrix metalloproteinase activity. This study demonstrates a promising approach for restoring the diminished vasculogenic potential of endothelial cells in diabetic patients.

High-Throughput Formation of Mesenchymal Stem Cell Spheroids and Entrapment in Alginate Hydrogels.

Mesenchymal stem cells (MSCs) are a promising cell source for tissue repair and regeneration due to their multilineage capacity, potential for autologous use, and secretion of potent bioactive factors to catalyze the endogenous repair program. However, a major limitation to current cell-based tissue engineering approaches is the drastic loss of cells upon transplantation. The causation of this loss, whether due to apoptosis following a dramatic change in the microenvironment or migration away from the defect site, has yet to be determined. MSCs formed into aggregates, known as spheroids, possess a strong therapeutic advantage compared to the more commonly used dissociated cells due to their improved resistance to apoptosis and increased secretion of endogenous trophic factors. Furthermore, the use of biomaterials such as alginate hydrogels to transplant cells in situ improves cell survival, localizes payloads at the defect site, and facilitates continued instruction of cells by manipulating the biophysical properties of the biomaterial. Transplantation of MSC spheroids without a vehicle into tissue defects comprises the majority of studies to date, ceding control of spheroid function due to the cell's interaction with the native tissue extracellular matrix and abrogating the established benefits of spheroid formation. Thus, there is a significant need to consider the role of biomaterials in transplanting MSC spheroids using an appropriate carrier. In this chapter, we describe high-throughput formation of spheroids, steps for further characterization, and encapsulation in alginate hydrogels with an eye toward localizing MSC spheroids at the target site.

Evaluating 3D-printed biomaterials as scaffolds for vascularized bone tissue engineering.

There is an unmet need for a consistent set of tools for the evaluation of 3D-printed constructs. A toolbox developed to design, characterize, and evaluate 3D-printed poly(propylene fumarate) scaffolds is proposed for vascularized engineered tissues. This toolbox combines modular design and non-destructive fabricated design evaluation, evaluates biocompatibility and mechanical properties, and models angiogenesis.

Evaluation of the in vitro cytotoxicity of cross-linked biomaterials.

This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), preosteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue scaffold. The sol fraction of the PPF films was measured and then utilized to estimate cross-linking density. Cytotoxicity was evaluated using XTT assay and fluorescence imaging. Results showed that PPF supported similar cell metabolic activities of hMSC, L929, MC3T3, and cMSC compared to the noncytotoxic control, high-density polyethylene (HDPE) and were statistically different than those cultured with the cytotoxic control, a polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZCF). Results showed differing cellular responses to ZCF, the cytotoxic control. The L929 cells had the lowest cell metabolic activity levels after exposure to ZCF compared to the cell metabolic activity levels of the MC3T3, hMSC, or cMSC cells. Qualitative verification of the results using fluorescence imaging demonstrated no change in cell morphology, vacuolization, or detachment when cultured with PPF compared to HDPE or blank media cultures. Overall, the cytotoxicity response of the cells to PPF was demonstrated to be similar to the cytotoxic response of cells to known noncytotoxic materials (HDPE).