We demonstrate the ability to spatially pattern biochemical signals into nanofibrous materials using thiol-ene reactions of thiolated molecules to presented norbornene groups. This approach is used to pattern three molecules independently within one scaffold, pattern signals with depth through a scaffold, and to spatially control cell adhesion and morphology. Keywords nanofibers; patterning; electrospinning; tissue engineering; alignment Biomaterials are being developed to investigate and control cellular interactions with their surroundings; however, the majority of engineered systems present signals (e.g. mechanics, topography, adhesion) in a spatially uniform manner, despite the role that spatially controlled signals -both biophysical and biochemical -play in a number of different processes in vivo. [1][2][3] For example, spatial organization of soluble signals occurs as early as gastrulation when morphogen gradients activate various signaling pathways (e.g. sonic hedgehog, WnT, activin) [4][5][6][7] to guide tissue development. This organization continues in adult tissues where spatial regulation of growth factors directly influences processes such as chondrogenesis [8] , angiogenesis [9] , and immune responses. [10] Beyond soluble factors, spatially controlled signaling of insoluble extracellular matrix (ECM) proteins is also important, as localized fibronectin deposition directs neural crest formation [11] and tumor HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript angiogenesis, [12] vitronectin expression in the ventral neural tube promotes motor neuron differentiation, [13] and spatial alignment of collagen fibers contributes to soft tissue functions of the cornea, [14] articular cartilage, [15] and arterial wall. [16] Notably, these ECM proteins all contain amino acid sequences known to induce integrin-mediated cell adhesion, [17] suggesting a broader role for cell adhesion in spatially dictating cell behavior.A variety of patterning techniques have been previously developed to engineer materials with precisely defined features, including the patterning of ECM proteins. [18] Studies using microcontact printing, a technique where proteins are "stamped" onto a substrate using a preformed master mold, have indicated that spatial patterning of cell adhesive proteins influences cellular processes including spreading, differentiation, proliferation, and death. [19,20] Other patterning techniques such as soft lithography, [21] 3-D printing, [22] and microfluidic devices [23] have also been successful in forming patterns and gradients of ECM proteins to control cell-material interactions. In particular, photopatterning has emerged as a promising technique in which a desired reaction (e.g. crosslinking, [24] bond scission, [25] covalent attachment [26] ) is spatially controlled to specific regions exposed to light, [27] without associated changes in surface topography that are typical of mechanical techniques like microcontact printing, 3-D printing, and soft lithog...
Electrospun nanofibers are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases (MMPs). Here, we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.
Despite recent progress in the treatment of rheumatoid arthritis (RA), many patients still fail to achieve remission or low disease activity. An imbalance between auto-reactive effector T cells (Teff) and regulatory T cells (Treg) may contribute to joint inflammation and damage in RA. Therefore, restoring this balance is a promising approach for the treatment of inflammatory arthritis. Accordingly, our group has previously shown that the combination of TGF-βreleasing microparticles (MP), rapamycin-releasing MP, and IL-2-releasing MP (TRI MP) can effectively increase the ratio of Tregs to Teff in vivo and provide disease protection in several preclinical models. In this study TRI MP was evaluated in the collagen-induced arthritis (CIA) model. Although this formulation has been tested previously in models of destructive inflammation and transplantation, this is the first model of autoimmunity for which this therapy has been applied. In this context, TRI MP effectively reduced arthritis incidence, the severity of arthritis scores, and bone erosion. The proposed mechanism of action includes not only reducing CD4 + T cell proliferation, but also expanding a regulatory population in the periphery soon after TRI MP administration. These changes were reflected in the CD4 + T cell population that infiltrated the paws at the onset of arthritis and were associated with a reduction of immune infiltrate and inflammatory myeloid cells in the paws. TRI MP administration also reduced the titer of collagen antibodies, however the contribution of this reduced titer to disease protection remains uncertain since there was no correlation between collagen antibody titer and arthritis score.
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