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Anchorage dependence, integrins, and apoptosis

Macroporous-interconnected cryostructures are prepared via cryoprocess approaches, where polymerization, crosslinking-polymerization, or even chain aggregation is primarily conducted at the sub-freezing temperature of the medium followed by thawing. Given their unique desirable properties, cryostructures have been studied as a stationary phase in chromatography to purify and separate proteins. The macroporous cryostructures with their highly interconnected large (10–100 µm) pores have various advantages, including the high surface area, sponge-like morphology, high elasticity, low-pressure drop, short diffusion path, and a temporary residence. In this review, we elaborate on the methods used in the preparation of cryostructures and the factors affecting their properties. We also highlight the underlying progress in chromatography techniques that take advantage of cryostructures as stationary phases for separating multiple biomacromolecules.

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.

Extracellular matrix (ECM) is a fundamental constituent of the cellular microenvironment, which forms a complicated three-dimensional (3D) network. ECM components and their remodeling events play a key role in regulating and advancing vasculogenesis and angiogenesis. Angiogenic factors, matrix-degrading proteases, and cell-ECM interactions can affect different stages of neovessel formation, including the growth and migration of endothelial cells (ECs) as well as tube formation. Although most of its components are proangiogenic, ECM also contains some antiangiogenic molecules that negatively affect the formation of new blood vessels. Due to the significant impact of vascularization in tissue repair and regeneration, ECM is currently being identified as an interesting structure for tissue engineering and regenerative medicine applications in which an accelerated healing process is desirable. In this regard, the use of acellular matrices has become a common therapeutic approach in the clinic to manage a wide range of diseases and disorders. This chapter introduces and discusses the role of ECM components and ECM remodeling in advancing the neovessel formation.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin β3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID₅₀ titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulated each other. Our data indicated that ITGB3, presumably as a co-receptor, played an imperative role during PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host–pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.

Survival and apoptosis are of major importance in the osteoclast life cycle. As osteoclasts have short lifespan, any alteration that prolongs their viability may cause enhanced osteoclast activity. Hence, the regulation of OC apoptosis has been recognized as a critical factor in bone remodeling. An imbalance in bone remodeling due to increased osteoclast activity leads to most adult bone diseases such as osteoporosis, rheumatoid arthritis and multiple myeloma. Therefore, manipulating osteoclast death would be a viable therapeutic approach in ameliorating bone diseases, with accelerated resorption. Over the last few decades we have witnessed the unraveling of many of the intracellular mechanisms responsible for osteoclast apoptosis. Thus, an understanding of the underlying mechanisms by which osteoclasts undergo programmed cell death and the regulators that modulate that activity will undoubtedly provide an insight into the development of pharmacological agents to treat such pathological bone diseases.

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.