Cellular remodeling of the matrix has recently emerged as a key factor in promoting neural differentiation. Most strategies to manipulate matrix remodeling focus on proteolytically cleavable cross-linkers, leading to changes in tethered biochemical signaling and matrix properties. Using peptides that are not the direct target of enzymatic degradation will likely reduce changes in the matrix and improve control of biological behavior. In this study, lamininderived peptides, IKVAV and LRE, tethered to independent sites in hyaluronic acid matrices using Michael addition and strain-promoted azide−alkyne cycloaddition are sufficient to manipulate hyaluronic acid degradation, gelatinase expression, and protease expression, while promoting neurite extension through matrix metalloprotease-dependent mechanisms in mouse embryonic stem cells encapsulated in hyaluronic acid matrices using an oxidation−reduction reaction initiated gelation. This study provides the foundation for a new strategy to stimulate matrix remodeling that is not dependent on enzymatic cleavage targets.

Polyethylene glycol (PEG) is a synthetic biocompatible polymer with many useful properties for developing therapeutics to treat spinal cord injury. Direct application of PEG as a fusogen to the injury site can repair cell membranes, mitigate oxidative stress, and promote axonal regeneration to restore motor function. PEG can be covalently or noncovalently conjugated to proteins, peptides, and nanoparticles to limit their clearance by the reticuloendothelial system, reduce their immunogenicity, and facilitate crossing the blood–brain barrier. Cross-linking PEG produces hydrogels that can act as delivery vehicles for bioactive molecules including growth factors and cells such as bone marrow stromal cells, which can modulate the inflammatory response and support neural tissue regeneration. PEG hydrogels can be cross-linked in vitro or delivered as an injectable formulation that can gel in situ at the site of injury. Chemical and mechanical properties of PEG hydrogels are tunable and must be optimized for creating the most favorable delivery environment. Peptides mimicking extracellular matrix protein such as laminin and n-cadherin can be incorporated into PEG hydrogels to promote neural differentiation and axonal extensions. Different hydrogel cross-linking densities and stiffness will also affect the differentiation process. PEG hydrogels with a gradient of peptide concentrations or Young’s modulus have been developed to systematically study these factors. This review will describe these and other recent advancements of PEG in the field of spinal cord injury in greater detail.

Hyaluronic acid functionalized with two orthogonal chemistries at different targets expedites neural maturation in vitro, while reducing inflammation in vivo.

Although preclinical models of spinal cord injury have shown that matrix inclusion in stem cell therapy leads to greater neurological improvements than that including cells alone, there has been insufficient matrix optimization for human cells. N-Cadherin influences the development and maintenance of neural tissue, but the effects of N-cadherin derived peptide His-Ala-Val-Asp-Ile (HAVDI) on the survival, neurite extension, and expression of neural differentiation markers in human induced pluripotent stem cell derived neural stems (hNSC) have not been widely examined. Using polyethylene glycol hydrogels containing a continuous gradient of HAVDI, this study identifies concentration dependent effects on hNSC survival and neural differentiation.

Treatment of MCl4(4-tBupy)2 (M = Nb, Ta) with 3 equiv of potassium 3,5-di-tert-butylpyrazolate afforded (2,2,6,6-tetramethyl-5-ketimidehept-3-en-3-imide)bis(3,5-di-tert-butylpyrazolate)chloroniobium(V) (1; 24%) and (2,2,6,6-tetramethyl-5-ketimidehept-3-en-3-imide)bis(3,5-di-tert-butylpyrazolate)chlorotantalum(V) (2; 27%) as deep red and yellow crystalline solids, respectively. Analogous treatment of NbCl4(THF)2 with 3 equiv each of 4-tert-butylpyridine and potassium 3,5-di-tert-butylpyrazolate and excess Na/Hg in diethyl ether afforded (2,2,6,6-tetramethyl-5-ketimidohept-3-en-3-imide)bis(3,5-di-tert-butylpyrazolate)niobium(V) (3, 32%) as deep red crystals. X-ray crystallography established that 1 and 3 each contain two intact η2-3,5-di-tert-butylpyrazolate ligands as well as one 3,5-di-tert-butylpyrazolate ligand that has undergone N–N bond oxidative addition to the niobium center. In 1, one of the nitrogen atoms abstracted a hydrogen atom from tetrahydrofuran solvent, whereas no hydrogen atom abstraction occurred in 3. These complexes represent rare examples of pyrazolate N–N bond cleavage. NICS calculations suggest that the niobacycles are weakly aromatic, in comparison to the highly aromatic 3,5-di-tert-butylpyrazolate ligands and pyrimidine.

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are required to understand the effects of MMP driven ECM remodeling in human systems. Although polymer [7-9] and bioactive peptide signaling [10-12] selection will alter MMP activity in the matrix, the field has traditionally focused on the use of enzymatically degradable crosslinkers to increase cellular spreading and migration by increasing pore size. [13] However, enzymatically degradable cross linkers have been found to alter cellular differentiation [14] and axonal extension in 2D culture, [15,16] which implies they have greater bioactivity than previously appreciated. To incorporate ECM remodeling as a matrix design criteria, greater consideration to the effects of all matrix components on MMP activity needs to be given. Hyaluronic acid (HA) enhances neural stem cell growth, differentiation, and axon extension. [17,18] Adding laminin to HA further increases in axon extension. [19] However, HA stimulates gelatinase (MMP 2 & 9) expression. [7,8] Gelatinase promotes axonal regeneration after CNS injury, [20] but degrades laminin. [21] Revealed cryptic sites in laminin can then participate in regulating gelatinase activity and cellular behaviors. [10,22] Use of laminin derived peptide signaling, instead of whole laminin molecules, provides more consistent bioactive signaling. Not all commonly used laminin derived peptides regulate gelatinase activity. [10,23] Laminin derived Ile-Lys-Val-Ala-Val (IKVAV) promotes neural differentiation, increases axon extension, and modulates gelatinase expression. [11,24] To further manipulate cellular responses, additional peptides are often used with IKVAV. [25] Leu-Arg-Glu (LRE), another lamininderived peptide, supports adhesion, axon guidance, and other cell behaviors not stimulated by IKVAV. [26] LRE also modulates the expression of gelatinase and inhibitors of gelatinase activity by manipulating Ca 2+ flow through Cav2.2 voltage gated Ca 2+ channels. [12] IKVAV and LRE peptide signaling in HA matrices increases protease (MMP 1 & 3) and gelatinase expression in mouse embryonic stem cells, while reducing the degradation of the HA matrix. [3] The IKVAV and LRE signaling combination is capable of affecting human induced pluripotent stem cell derived neural stem cells (hNSC) behavior in 2D culture. [27] This study examines the effect of IKVAV and LRE on hNSC,

Low neural tissue extracellular matrix (ECM) content has led to the understudy of its effects on neural cells and tissue. Hyaluronic acid (HA) and laminin are major neural ECM components, but direct comparisons of their cellular effects could not be located in the literature. The current study uses human-induced pluripotent stem-cell-derived neural stem cells to assess the effects of HA, laminin, and HA with laminin-derived peptides IKVAV and LRE on cellular morphology, attachment, neurite extension and ECM remodeling. Increased attachment was observed on HA with and without IKVAV and LRE compared to laminin. Cellular morphology and neurite extension were similar on all surfaces. Using a direct binding inhibitor of Cav2.2 voltage gated calcium channel activity, a known binding partner of LRE, reduced attachment on HA with and without IKVAV and LRE and altered cellular morphology on surfaces with laminin or IKVAV and LRE. HA with IKVAV and LRE reduced the fluorescent intensity of fibronectin staining, but did not alter the localization of ECM remodeling enzymes matrix metalloprotease 2 and 9 staining compared to HA. Overall, the data indicate HA, IKVAV and LRE have complementary effects on human-induced pluripotent stem-cell-derived neural stem cell behavior.