Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization.

Hydrogel matrices with angiogenic properties are much desirable for therapeutic vascularization strategies, namely to provide vascular supply to ischemic areas, transplanted cells, or bioengineered tissues. Here we report the pro-angiogenic effect of fibrin (Fb) functionalization with the T1 sequence from the angiogenic inducer CCN1, forseeing its use in the injured brain and spinal cord. Fibrin functionalization with 40 µM of T1 peptide effectively improved cellular sprouting of human brain microvascular endothelial cells (hCMEC/D3) in the absence of vascular endothelial growth factor (VEGF), without impacting the viscoelastic properties of Fb, cell viability, or proliferation. The pro-angiogenic effect of immobilized T1 was potentiated in the presence of VEGF and partially mediated through α6ß1 integrin. The tethering of T1 also enhanced sprouting of human cord blood-derived outgrowth endothelial cells (OEC). Still, to elicit such effect, a higher input T1 concentration was required (60 µM), in line with the lower protein levels of α6 and ß1 integrin subunits found in OEC comparing to hCMEC/D3, prior to embedment in Fb gel. Finally, the ability of T1-functionalized Fb in inducing cappilary invasion in vivo was assessed using the CAM assay, which evidenced a significant increase in the number of newly formed vessels at sites of implantation of T1-functionalized Fb, in the absence of soluble angiogenic factors. Overall these results demonstrate the potential of T1 peptide-presenting gels for use in therapeutic vascularization approaches. Considering T1 neurite-extension promoting capability and pro-angiogenic properties, T1-functionalized Fb hydrogels are particularly promising for application in the injured central nervous system.

An affinity-based approach to engineer laminin-presenting cell instructive microenvironments.

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices.

Synthetic matrix enhances transplanted satellite cell engraftment in dystrophic and aged skeletal muscle with comorbid trauma.

Muscle satellite cells (MuSCs) play a central role in muscle regeneration, but their quantity and function decline with comorbidity of trauma, aging, and muscle diseases. Although transplantation of MuSCs in traumatically injured muscle in the comorbid context of aging or pathology is a strategy to boost muscle regeneration, an effective cell delivery strategy in these contexts has not been developed. We engineered a synthetic hydrogel-based matrix with optimal mechanical, cell-adhesive, and protease-degradable properties that promotes MuSC survival, proliferation, and differentiation. Furthermore, we establish a biomaterial-mediated cell delivery strategy for treating muscle trauma, where intramuscular injections may not be applicable. Delivery of MuSCs in the engineered matrix significantly improved in vivo cell survival, proliferation, and engraftment in nonirradiated and immunocompetent muscles of aged and dystrophic mice compared to collagen gels and cell-only controls. This platform may be suitable for treating craniofacial and limb muscle trauma, as well as postoperative wounds of elderly and dystrophic patients.

Biomimetic synthetic self-assembled hydrogels for cell transplantation.

The development of three-dimensional matrices capable of recapitulating the main features of native extracellular matrix and contribute for the establishment of a favorable microenvironment for cell behavior and fate is expected to circumvent some of the main limitations of cell-based therapies. In this context, self-assembly has emerged as a promising strategy to engineer cell-compatible hydrogels. A wide number of synthetically-derived biopolymers, such as proteins, peptides and DNA/RNA, with intrinsic ability to self-assemble into well-defined nanofibrous structures, are being explored. The resulting hydrogels, in addition to closely resembling the architecture of native cellular microenvironments, present a versatile and dynamic behavior that allows them to be designed to undergo sol-to-gel transition in response to exogenous stimulus. This review presents an overview on the state-of-the-art of the different strategies being explored for the development of injectable synthetic self-assembled hydrogels for cell transplantation and/or recruitment of endogenous cells, with an emphasis on their biological performance, both in vitro and in vivo. Systems based on peptides are the most widely explored and have already generated promising results in pre-clinical in vivo studies involving different repair/regenerative scenarios, including cartilage, bone, nerve and heart. On the other hand, systems based on DNA and hybrid hydrogels are now emerging for application in the biomedical field with high potential. Finally, the main challenges hampering the translation of these systems to the clinic and the issues that need to be addressed for these to progress from bench-to-bedside are discussed.