Cryopreservation of network bioactivity and multi-lineage stromal cell differentiation potential within three-dimensional synthetic hydrogels.

Despite progress in many aspects of controlling cell behavior within synthetic three-dimensional hydrogels, approaches to cryopreserve these systems - encompassing the protection of both encapsulated cell viability and network bioactive functions - are lacking. Here, we demonstrate the retention of encapsulated human mesenchymal stromal cell (hMSC) viability following in situ cryopreservation regardless of cell line, material system, or storage duration. Further, the preservation extends to network bioactive functions, with hMSCs cryopreserved within degradable and adhesive hyaluronic-acid (HA) based hydrogels exhibiting degradation-mediated spreading within the gels equivalent to their non-frozen counterparts. Finally, the platform cryopreservation protocol preserves multi-lineage cellular differentiation capacity, with encapsulated hMSCs in non-degradable and adhesive/degradable HA-based hydrogels undergoing rates of adipogenesis and osteogenesis, respectively, equivalent to those in non-frozen gels on a per-cell basis. Collectively, these findings indicate a versatile platform technology that contributes to an increased understanding of three-dimensional cell-matrix interactions, and which may enable the indefinite cryopreservation of tissue engineering constructs for clinical applications.

Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels.

While significant progress has been made in directing the behavior of cells encapsulated within three-dimensional (3D) covalently crosslinked hydrogels, the capacity of these materials to support in situ cryopreservation of cells directly within the gels has not been assessed. Here, we demonstrate the retention of human mesenchymal stem cell (hMSC) viability within hyaluronic acid (HA) and polyethylene glycol based hydrogels via a facile gradual cooling and freezing protocol. Encapsulated cell viability was retained at similar rates in both materials systems regardless of initial duration in culture or adhesive ligand incorporation, indicating the versatility of the approach. Additionally, the cryopreservation protocol maintains stem cell differentiation potential; incubation in adipogenic differentiation media induced equal rates of hMSC adipogenesis in freeze-thawed and non-frozen HA based hydrogels on a per-cell basis. Collectively, these findings highlight the cryopreservation protocol as a platform technology that, in addition to contributing to an increased understanding of three-dimensional cell-matrix interactions, could enable the long-term preservation of tissue engineering constructs for clinical applications.

Esterified Trehalose Analogues Protect Mammalian Cells from Heat Shock.

Trehalose is a disaccharide produced by many organisms to better enable them to survive environmental stresses, including heat, cold, desiccation, and reactive oxygen species. Mammalian cells do not naturally biosynthesize trehalose; however, when introduced into mammalian cells, trehalose provides protection from damage associated with freezing and drying. One of the major difficulties in using trehalose as a cellular protectant for mammalian cells is the delivery of this disaccharide into the intracellular environment; mammalian cell membranes are impermeable to the hydrophilic sugar trehalose. A panel of cell-permeable trehalose analogues, in which the hydrophilic hydroxyl groups of trehalose are masked as esters, have been synthesized and the ability of these analogues to load trehalose into mammalian cells has been evaluated. Two of these analogues deliver millimolar concentrations of free trehalose into a variety of mammalian cells. Critically, Jurkat cells incubated with these analogues show improved survival after heat shock, relative to untreated Jurkat cells. The method reported herein thus paves the way for the use of esterified analogues of trehalose as a facile means to deliver high concentrations of trehalose into mammalian cells for use as a cellular protectant.

Quantification of the Disaccharide Trehalose from Biological Samples: A Comparison of Analytical Methods.

Trehalose is a disaccharide that is biosynthesized by many different organisms subjected to extreme conditions, such as dehydration, heat, oxidative stress, and freezing. This disaccharide allows organisms to better survive these environmental stresses; however, the mechanisms by which trehalose exerts its protective effects are not well understood. Methods to accurately measure trehalose from different organisms will help us gain better understanding of these protective mechanisms. In this study, three experimental approaches for the quantification of trehalose from biological samples were compared: an enzymatic trehalose assay (Trehalose Assay Kit; Megazyme International), a high-performance liquid chromatography coupled with refractive index detection-based assay, and a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay. Limits of detection and quantification for each assay were compared, as were the dynamic ranges for all three assays. The percent recoveries for known amounts of trehalose spiked into bacterial and mammalian cellular lysates were also determined for each of the assays. Finally, endogenous trehalose produced by Escherichia coli cells was detected and quantified using these assays. Results from this study indicate that an LC-MS/MS-based assay is the most direct and sensitive method for the quantification of low concentrations of trehalose from biological samples; however, the enzymatic assay is suitable for the rapid quantification of higher concentrations of trehalose when an LC-MS/MS is unavailable.

Fibrous hyaluronic acid hydrogels that direct MSC chondrogenesis through mechanical and adhesive cues.

Electrospinning has recently gained much interest due to its ability to form scaffolds that mimic the nanofibrous nature of the extracellular matrix, such as the size and depth-dependent alignment of collagen fibers within hyaline cartilage. While much progress has been made in developing bulk, isotropic hydrogels for tissue engineering and understanding how the microenvironment of such scaffolds affects cell response, these effects have not been extensively studied in a nanofibrous system. Here, we show that the mechanics (through intrafiber crosslink density) and adhesivity (through RGD density) of electrospun hyaluronic acid (HA) fibers significantly affect human mesenchymal stem cell (hMSC) interactions and gene expression. Specifically, hMSC spreading, proliferation, and focal adhesion formation were dependent on RGD density, but not on the range of fiber mechanics investigated. Moreover, traction-mediated fiber displacements generally increased with more adhesive fibers. The expression of chondrogenic markers, unlike trends in cell spreading and cytoskeletal organization, was influenced by both fiber mechanics and adhesivity, in which softer fibers and lower RGD densities generally enhanced chondrogenesis. This work not only reveals concurrent effects of mechanics and adhesivity in a fibrous context, but also highlights fibrous HA hydrogels as a promising scaffold for future cartilage repair strategies.

Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels.

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Integration and regression of implanted engineered human vascular networks during deep wound healing.

The ability of vascularized constructs to integrate with tissues may depend on the kinetics and stability of vascular structure development. This study assessed the functionality and durability of engineered human vasculatures from endothelial progenitors when implanted in a mouse deep burn-wound model. Human vascular networks, derived from endothelial colony-forming cells in hyaluronic acid hydrogels, were transplanted into third-degree burns. On day 3 following transplantation, macrophages rapidly degraded the hydrogel during a period of inflammation; through the transitions from inflammation to proliferation (days 5-7), the host's vasculatures infiltrated the construct, connecting with the human vessels within the wound area. The growth of mouse vessels near the wound area supported further integration with the implanted human vasculatures. During this period, the majority of the vessels (∼60%) in the treated wound area were human. Although no increase in the density of human vessels was detected during the proliferative phase, they temporarily increased in size. This growth peaked at day 7, the middle of the proliferation stage, and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the vasculatures including the transplanted human vessels generally regressed, and few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 µm² (including the human ones), remained in the healed wound. Overall, this study offers useful insights for the development of vascularization strategies for wound healing and ischemic conditions, for tissue-engineered constructs, and for tissue regeneration.

Spatial control of cell-mediated degradation to regulate vasculogenesis and angiogenesis in hyaluronan hydrogels.

Matrix remodeling is crucial for neovascularization, however its utilization to control this process in synthetic biomaterials has been limited. Here, we utilized hyaluronic acid (HA) hydrogels to spatially control cellular remodeling during vascular network formation. Specifically, we exploited a secondary radical polymerization to alter the ability of cells to degrade the hydrogel and utilized it to create spatial patterning using light initiation. We first demonstrated the ability of the hydrogel to either support or inhibit in vitro vasculogenesis of endothelial colony-forming cells (ECFCs) or angiogenesis from ex ovo chorioallantoic membranes. We showed that vascular tube branching and sprouting, which required matrix metalloproteinases (MMPs)-dependent remodeling, could be achieved in hydrogels formed by primary addition-crosslinking only. Although ECFCs expressed higher levels of MMPs in the hydrogels with the secondary radical crosslinking, the generated kinetic chains disabled cell-mediated remodeling and therefore vascular formation was arrested at the vacuole and lumen stage. We then patterned hydrogels to have regions that either permitted or inhibited cell-mediated degradation during in vitro vasculogenesis or angiogenesis. Our ability to control degradation cues that regulate vascular tube formation is important for the study of vascular biology and the application of synthetic biomaterials in tissue regeneration.

Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix.

Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells (ECFCs) to form efficient vascular networks. Entrapped ECFCs underwent tubulogenesis dependent on the cellular interactions with the HA hydrogel during each stage of vascular morphogenesis. Vacuole and lumen formed through integrins α(5)ß(1) and α(V)ß(3), while branching and sprouting were enabled by HA hydrogel degradation. Vascular networks formed within HA hydrogels containing ECFCs anastomosed with the host's circulation and supported blood flow in the hydrogel after transplantation. Collectively, we show that the signaling pathways of vascular morphogenesis of ECFCs can be precisely regulated in a synthetic matrix, resulting in a functional microvasculature useful for the study of 3-dimensional vascular biology and toward a range of vascular disorders and approaches in tissue regeneration.

Patterning network structure to spatially control cellular remodeling and stem cell fate within 3-dimensional hydrogels.

The spatially directed 3-dimensional (3D) remodeling of synthetic materials may be useful to regionally control cell behavior. In this work, we developed a process to synthesize hyaluronic acid hydrogels using multiple modes of crosslinking applied sequentially; a primary addition reaction to introduce protease degradable peptide crosslinks, then a UV light-induced secondary radical reaction (enabling spatial control) to introduce non-degradable kinetic chains. These differential network structures either permitted (primary crosslinking only, "-UV") or inhibited (sequential crosslinking, "+UV") cellular remodeling. This behavior was validated by controlling the outgrowth from chick aortic arches or the spreading of encapsulated mesenchymal stem cells (MSCs), where only -UV regions permitted arch outgrowth and MSC spreading. Additionally, network structures dictated adipogenic/osteogenic MSC fate decisions, with spatial control, by controlling encapsulated MSC spreading. This manipulation of microenvironmental cues may be valuable for advanced tissue engineering applications requiring the spatial control of cells in 3D.

Tuning hydrogel properties for applications in tissue engineering.

Biomaterial design is an important component towards tissue engineering applications. There are many parameters that may be adjusted including physical properties (i.e., degradation and mechanics) and chemical properties (e.g., adhesion and cellular interactions). These design components may dictate the success or failure of a tissue engineering approach. Our group is particularly interested in the use of swollen hydrogels as cell carriers. One material that is used to fabricate hydrogels is hyaluronic acid (HA), which is found in many tissues in the body. Here, we show the control over hydrogel degradation, both in the bulk and locally to cells to control both the distribution of extracellular matrix by cells and whether or not a cell spreads in the hydrogels. These signals are important in the final structure and mechanical properties of engineered tissues, and potentially the differentiation of encapsulated stem cells.

Cellular encapsulation in 3D hydrogels for tissue engineering.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.). The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures.

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials.

Synthesis, in vitro degradation, and mechanical properties of two-component poly(ester urethane)urea scaffolds: effects of water and polyol composition.

The development of minimally invasive therapeutics for orthopedic clinical conditions has substantial benefits, especially for osteoporotic fragility fractures and vertebral compression fractures. Poly(ester urethane)urea (PEUUR) foams are potentially useful for addressing these conditions because they cure in situ upon injection to form porous scaffolds. In this study, the effects of water concentration and polyester triol composition on the physicochemical, mechanical, and biological properties of PEUUR foams were investigated. A liquid resin (lysine diisocyanate) and hardener (poly(epsilon-caprolactone-co-glycolide-co-DL-lactide) triol, tertiary amine catalyst, anionic stabilizer, and fatty acid-derived pore opener) were mixed, and the resulting reactive liquid mixture was injected into a mold to harden. By varying the water content over the range of 0.5 to 2.75 parts per hundred parts polyol, materials with porosities ranging from 89.1 to 95.8 vol-% were prepared. Cells permeated the PEUUR foams after 21 days post-seeding, implying that the pores are open and interconnected. In vitro, the materials yielded non-cytotoxic decomposition products, and differences in the half-life of the polyester triol component translated to differences in the PEUUR foam degradation rates. We anticipate that PEUUR foams will present compelling opportunities for the design of new tissue-engineered scaffolds and delivery systems because of their favorable biological and physical properties.