In this study, we derivatized type I collagen without altering its triple helical conformation to allow for facile hydrogel formation via Micheal addition of thiols to methacrylates without the addition of other crosslinking agents. This method provides the flexibility needed for fabrication of injectable hydrogels or pre-fabricated implantable scaffolds, using the same components by tuning the modulus from Pa to kPa. Enzymatic degradability of the hydrogels can be also easily fine tuned by variation of the ratio and type of cross-linking component. The structural morphology reveals lamellar structure mimicking native collagen fibrils. The versatility of this material is demonstrated by its use as a pre-fabricated substrate for culturing human corneal epithelial cells, and as an injectable hydrogel for 3-D encapsulation of cardiac progenitor cells.Keywords: bio-orthogonal chemistry, tissue engineering, pre-fabricated scaffolds, injectable scaffolds, cell compatible. IntroductionThe extracellular matrix (ECM) provides mechanical support as well as instructive signals for cell development, migration, proliferation, survival and function. Natural biopolymers derived from the ECM are therefore by nature, very biocompatible and bio-interactive. [1][2][3] The most abundant is collagen that has extensively been used to prepare scaffolds for tissue repair and engineering. This structural ECM component has, however limited number of functional groups that can be used for direct crosslinking. 4, 5 The main functional groups are amine and carboxylic acids, which allows collagen to crosslinked (e.g. via UV, thermal heating, carbodiimides, epoxy or aldehyde crosslinkers). Conversely, synthetic polymers such as poly (ethylene) glycols, poly (lactic acids), poly (methacryl/acryl amides) etc.), are easily chemically modified for facile processing than collagen.6 However, synthetic polymers have issues with biocompatibility, degradability and do not completely integrate within the host.7-9 Hence, synthetic routes to introduce biocompatible crosslinkable modifications on the protein structure, (reactive moieties), are highly desirable for the development of the next generation of regenerative materials for tissue engineering. Particularly, introducing reactive moieties in collagen would expand its functionality allowing for development of a wider range of scaffolds that can serve as regeneration templates. 10, 11 Several routes to render collagen more processable while retaining its in vitro/ in vivo or clinical bio interactive capacity for promoting tissue regeneration have been explored. [12][13][14] The simplest method is blending collagen with natural or synthetic polymers (such as hyaluronic acid, chitosan, poly(ethylene oxide), polylactic acid, and polyglycolic acid) to fabricate scaffolds. 5, 15 Crosslinked collagen-chitosan hydrogels, which were mechanically stronger than collagen alone, promoted angiogenesis and has been used for islet transplantations in murine models. 16 Li et al.co-polymerized collagen with lam...

BACKGROUND: Respiratory viral infections can increase the risk of chronic lung allograft dysfunction after lung transplantation, but the mechanisms are unknown. In this study, we determined whether symptomatic respiratory viral infections after lung transplantation induce circulating exosomes that contain lung-associated self-antigens and assessed whether these exosomes activate immune responses to self-antigens. METHODS: Serum samples were collected from lung transplant recipients with symptomatic lower-and upper-tract respiratory viral infections and from non-symptomatic stable recipients. Exosomes were isolated via ultracentrifugation; purity was determined using sucrose cushion; and presence of lung self-antigens, 20S proteasome, and viral antigens for rhinovirus, coronavirus, and respiratory syncytial virus were determined using immunoblot. Mice were immunized with circulating exosomes from each group and resulting differential immune responses and lung histology were analyzed. RESULTS: Exosomes containing self-antigens, 20S proteasome, and viral antigens were detected at significantly higher levels (p < 0.05) in serum of recipients with symptomatic respiratory viral infections (n = 35) as compared with stable controls (n = 32). Mice immunized with exosomes from recipients with respiratory viral infections developed immune responses to self-antigens, fibrosis, small airway occlusion, and significant cellular infiltration; mice immunized with exosomes from controls did not (p < 0.05). CONCLUSIONS: Circulating exosomes isolated from lung transplant recipients diagnosed with respiratory viral infections contained lung self-antigens, viral antigens, and 20S proteasome and elicited immune responses to lung self-antigens that resulted in development of chronic lung allograft dysfunction in immunized mice.

Extracellular matrix proteins like collagen promote regeneration as implants in clinical studies. However, collagens are large and unwieldy proteins, making small functional peptide analogs potentially ideal substitutes. Self-assembling collagen-like-peptides conjugated with PEG-maleimide were assembled into hydrogels. When tested pre-clinically as corneal implants in mini-pigs, they promoted cell and nerve regeneration, forming neo-corneas structurally and functionally similar to natural corneas. IntroductionResident stem cells capable of regeneration are present in almost every organ in the body but frequently cannot achieve the repair needed after damage by injury or disease. The limiting factor is often the extracellular matrix (ECM) surrounding the cells. During damage, the ECM is frequently replaced by scar tissue, which does not provide the required structural integrity and inhibits regeneration of functional tissue. 1 The replacement of scar tissue with ECM or biomaterials that mimic its structure and function could therefore promote regeneration. 1,2 Regenerating damaged tissues and organs, including corneas, could potentially mitigate the need for organ transplantation, which currently faces acute worldwide shortage and immune rejection issues. We have previously shown in human corneal transplantation clinical studies that cell-free corneal ECM mimics made from recombinant human collagen (RHC) stimulated regeneration of the human cornea, an organ that normally does not regenerate on its own. 3,4 However, RHC replicates human collagen, which, like many other ECM biopolymers, is comprised of large proteins and is difficult to manipulate. Smaller units of complex proteins, particularly those capable of self-assembly have been examined as controllable ECM mimics, as they can form a wide range of structures including nanofibres. 5 Collagen-like peptides (CLPs), also known as collagen-mimetic peptides (CMPs), have recently been investigated as potential alternatives to collagen, as they can self-assemble and form triple helical nanofibers like collagen. [6][7][8][9][10][11][12][13][14] In order to stabilize the triple helices of CLPs, polymer templates that can link the three peptide chains together with sufficient flexibility to allow for proper packing of the chains with correct amino acid register have been tested. 7 More recent designs have used collagen peptides as the physical crosslinks for the polymer system through triple helix formation. 6,7 CLPs and CLP-polymer systems have now been tested in vitro as engineered 3D scaffolds on their own, 8 conjugated to polyethylene glycol (PEG) backbones, 9-11 and complexed with bioactive factors 12,13 or localization agents such as gold nanoparticles. 14 However, the majority of studies have been confined to in vitro testing with cells.The trend in regenerative medicine is now towards the use of complex, naturally derived ECM macromolecules, as intact decellularised scaffolds or as processed and purified concentrated liquids or finely ground powder that are the...