Abstract Adult stem cells reside in unique niches that provide vital cues for their survival, self-renewal and differentiation. In order to better understand the contribution of substrate stiffness ...to neural stem/progenitor cell (NSPC) differentiation and proliferation, a photopolymerizable methacrylamide chitosan (MAC) biomaterial was developed. Photopolymerizable MAC is particularly compelling for the study of the central nervous system stem cell niche because Young's elastic modulus ( EY ) can be tuned from less than 1 kPa to greater than 30 kPa. Additionally, the numerous free amine functional groups enable inclusion of biochemical signaling molecules that, together with the mechanical environment, influence cell behavior. Herein, NSPCs proliferated on MAC substrates with Young's elastic moduli below 10 kPa and exhibited maximal proliferation on 3.5 kPa surfaces. Neuronal differentiation was favored on the softest surfaces with EY < 1 kPa as confirmed by both immunohistochemistry and qRT-PCR. Oligodendrocyte differentiation was favored on stiffer scaffolds (>7 kPa); however, myelin oligodendrocyte glycoprotein (MOG) gene expression suggested that oligodendrocyte maturation and myelination was best on <1 kPa scaffolds where more mature neurons were present. Astrocyte differentiation was only observed on <1 and 3.5 kPa surfaces and represented less than 2% of the total cell population. This work demonstrates the importance of substrate stiffness to the proliferation and differentiation of adult NSPCs and highlights the importance of mechanical properties to the success of scaffolds designed to engineer central nervous system tissue.
Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve ...prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable.
Display omitted
•Colloidal aggregates form above a critical aggregation concentration, yet can be re-solubilized to monomers with detergents.•The formation of colloidal drug aggregates impact ...biological outcomes.•Colloidal aggregates can lead to false positives in drug screens.•Current efforts to stabilize and exploit colloidal drug aggregates are highlighted.
It is well known that small molecule colloidal aggregation is a leading cause of false positives in early drug discovery. Colloid-formers are diverse and well represented among corporate and academic screening decks, and even among approved drugs. Less appreciated is how colloid formation by drug-like compounds fits into the wider understanding of colloid physical chemistry. Here we introduce the impact that colloidal aggregation has had on early drug discovery, and then turn to the physical and thermodynamic driving forces for small molecule colloidal aggregation, including the particulate nature of the colloids, their critical aggregation concentration-governed formation, their mechanism of protein adsorption and subsequent inhibition, and their sensitivity to detergent. We describe methods that have been used extensively to both identify aggregate-formers and to study and control their physical chemistry. While colloidal aggregation is widely recognized as a problem in early drug discovery, we highlight the opportunities for exploiting this phenomenon in biological milieus and for drug formulation.
The central nervous system (CNS) has a limited capacity to spontaneously regenerate following traumatic injury or disease, requiring innovative strategies to promote tissue and functional repair. ...Tissue regeneration strategies, such as cell and/or drug delivery, have demonstrated promising results in experimental animal models, but have been difficult to translate clinically. The efficacy of cell therapy, which involves stem cell transplantation into the CNS to replace damaged tissue, has been limited due to low cell survival and integration upon transplantation, while delivery of therapeutic molecules to the CNS using conventional methods, such as oral and intravenous administration, have been limited by diffusion across the blood-brain/spinal cord-barrier. The use of biomaterials to promote graft survival and integration as well as localized and sustained delivery of biologics to CNS injury sites is actively being pursued. This review will highlight recent advances using biomaterials as cell- and drug-delivery vehicles for CNS repair.
The role of microglia in spinal cord injury (SCI) remains poorly understood and is often confused with the response of macrophages. Here, we use specific transgenic mouse lines and depleting agents ...to understand the response of microglia after SCI. We find that microglia are highly dynamic and proliferate extensively during the first two weeks, accumulating around the lesion. There, activated microglia position themselves at the interface between infiltrating leukocytes and astrocytes, which proliferate and form a scar in response to microglia-derived factors, such as IGF-1. Depletion of microglia after SCI causes disruption of glial scar formation, enhances parenchymal immune infiltrates, reduces neuronal and oligodendrocyte survival, and impairs locomotor recovery. Conversely, increased microglial proliferation, induced by local M-CSF delivery, reduces lesion size and enhances functional recovery. Altogether, our results identify microglia as a key cellular component of the scar that develops after SCI to protect neural tissue.
Abstract Traumatic injury to the spinal cord causes cell death, demyelination, axonal degeneration, and cavitation resulting in functional motor and sensory loss. Stem cell therapy is a promising ...approach for spinal cord injury (SCI); however, this strategy is currently limited by the poor survival and uncontrolled differentiation of transplanted stem cells. In an attempt to achieve greater survival and integration with the host tissue, we examined the survival and efficacy of adult brain-derived neural stem/progenitor cells (NSPCs) injected within a hydrogel blend of hyaluronan and methyl cellulose (HAMC) into a subacute, clinically relevant model of rat SCI. Prior to use, HAMC was covalently modified with recombinant rat platelet-derived growth factor-A (rPDGF-A) to promote oligodendrocytic differentiation. SCI rats transplanted with NSPCs in HAMC-rPDGF-A showed improved behavioral recovery compared to rats transplanted with NSPCs in media. Rats with NSPC/HAMC-rPDGF-A transplants had a significant reduction in cavitation, improved graft survival, increased oligodendrocytic differentiation, and sparing of perilesional host oligodendrocytes and neurons. These data suggest that HAMC-rPDGF-A is a promising vehicle for cell delivery to the injured spinal cord.
Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that ...aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the “enhanced permeability and retention (EPR) effect” in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.
Design of three-dimensional biomimetic scaffolds Owen, Shawn C.; Shoichet, Molly S.
Journal of biomedical materials research. Part A,
15 September 2010, Letnik:
94A, Številka:
4
Journal Article
Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved ...difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase-barstar and streptavidin-biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar-SHH and biotin-CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.
A key challenge in engineering functional tissues in vitro is the limited transport capacity of oxygen and nutrients into the tissue. Inducing vascularization within engineered tissues is a key ...strategy to improving their survival in vitro and in vivo. The presence of vascular endothelial growth factor (VEGF) in a three-dimensional porous collagen scaffold may provide a useful strategy to promote vascularization of the engineered tissue in a controlled manner. To this end, we investigated whether immobilized VEGF could promote the invasion and assembly of endothelial cells (ECs) into the collagen scaffolds. We conjugated VEGF onto collagen scaffolds using
N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride chemistry, and measured the concentrations of immobilized VEGF in collagen scaffolds by direct VEGF enzyme-linked immunosorbent assay. We demonstrated that immobilized VEGF (relative to soluble VEGF) promoted the penetration and proliferation of ECs in the collagen scaffold, based on results of cell density analysis in histological sections, immunohistochemistry, XTT proliferation assay, glucose consumption and lactate production. Furthermore, we observed increased viability of ECs cultured in scaffolds with immobilized VEGF relative to soluble VEGF. This research demonstrates that immobilization of VEGF is a useful strategy to promote the invasion and proliferation of ECs into a scaffold, which may in turn lead to a vascularized scaffold.
PDF
