Modulation of Stiffness-Dependent Macrophage Inflammatory Responses by Collagen Deposition.

Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

Low-sugar universal mRNA vaccine against coronavirus variants with deletion of glycosites in the S2 or stem of SARS-CoV-2 spike messenger RNA (mRNA).

Since the outbreak of Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) in 2019, more than 15 million spike protein sequences have been identified, raising a new challenge for the development of a broadly protective vaccine against the various emerging variants. We found that the virus, like most other human viruses, depends on host-made glycans to shield the conserved epitopes on spike protein from immune response and demonstrated that deletion of the glycan shields exposed highly conserved epitopes and elicited broadly protective immune responses. In this study, we identified 17 conserved epitopes from 14 million spike protein sequences and 11 of the conserved epitopes are in the S2 domain, including the six most conserved epitopes in the stem region. We also demonstrated that deletion of the glycosites in the spike messenger RNA (mRNA) S2 domain or the stem region exposed the highly conserved epitopes and elicited broadly protective immune responses, particularly CD-8+ T cell response against various SARS-CoV-2 variants, and other human coronaviruses including MERS, SARS viruses, and those causing common cold.

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine.

Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

Evaluating Anti-tumor Immune Responses of Protein Nanoparticle-Based Cancer Vaccines.

Cancer vaccines displaying tumor-associated antigens (TAAs) train the immune system for enhanced tumor recognition and elimination. Nanoparticle-based cancer vaccines are ingested and processed by dendritic cells, which subsequently activate antigen-specific cytotoxic T cells, allowing them to identify and eliminate tumor cells displaying these TAAs. Here, we describe the procedures to conjugate TAA and adjuvant to a model protein nanoparticle platform (E2), followed by assessment of vaccine performance. Utilizing a syngeneic tumor model, the efficacy of in vivo immunization was determined by cytotoxic T lymphocyte assays and IFN-γ ELISpot ex vivo assays to measure tumor cell lysis and TAA-specific activation, respectively. In vivo tumor challenge directly allows evaluation of anti-tumor response and survival over time.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1-E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo- and heterosubtypic cross-reactivity is also observed for conjugated H1-E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein-protein conjugation strategy may have utility for future vaccine development against other human pathogens.

Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display.

Cancer vaccine immunotherapy facilitates the immune system's recognition of tumor-associated antigens, and the biomolecular design of these vaccines using nanoparticles is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed towards vaccine efficacy, participation by highly cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We examined vaccines based on the E2 protein nanoparticle platform that delivered encapsulated TLR9 agonist bacterial-like DNA (CpG1826 or CpG1018) or TLR7 agonist viral ssRNA to determine their efficacy over free agonists in activating both mDCs and pDCs for antigen presentation. Although mDCs were only activated by nanoparticle-encapsulated TLR9 agonists, pDCs were activated by all the individually tested constructs, and CpG1826 was shown to induce pDC cytokine production. Transfer of secreted factors from pDCs that were stimulated with a vaccine formulation comprising peptide antigen and CpG1826 enhanced mDC display of the antigen, particularly when delivered in nanoparticles. Only when treated with nanoparticle-conjugated vaccine could pDCs secrete factors to induce antigen display on naïve mDCs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing effective cancer vaccines, and demonstrate the advantage of using nanoparticle-based vaccine delivery.

Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging.

Bioluminescence imaging has advantages over fluorescence imaging, such as minimal photobleaching and autofluorescence, and greater signal-to-noise ratios in many complex environments. Although significant achievements have been made in luciferase engineering for generating bright and stable reporters, the full capability of luciferases for nanoparticle tracking has not been comprehensively examined. In biocatalysis, enhanced enzyme performance after immobilization on nanoparticles has been reported. Thus, we hypothesized that by assembling luciferases onto a nanoparticle, the resulting complex could lead to substantially improved imaging properties. Using a modular bioconjugation strategy, we attached NanoLuc (NLuc) or Akaluc bioluminescent proteins to a protein nanoparticle platform (E2), yielding nanoparticles NLuc-E2 and Akaluc-E2, both with diameters of ∼45 â€‹nm. Although no significant differences were observed between different conditions involving Akaluc and Akaluc-E2, free NLuc at pH 5.0 showed significantly lower emission values than free NLuc at pH 7.4. Interestingly, NLuc immobilization on E2 nanoparticles (NLuc-E2) emitted increased luminescence at pH 7.4, and at pH 5.0 showed over two orders of magnitude (>200-fold) higher luminescence (than free NLuc), expanding the potential for imaging detection using the nanoparticle even upon endocytic uptake. After uptake by macrophages, the resulting luminescence with NLuc-E2 nanoparticles was up to 7-fold higher than with free NLuc at 48 â€‹h. Cells incubated with NLuc-E2 could also be imaged using live bioluminescence microscopy. Finally, biodistribution of nanoparticles into lymph nodes was detected through imaging using NLuc-E2, but not with conventionally-labeled fluorescent E2. Our data demonstrate that NLuc-bound nanoparticles have advantageous properties that can be utilized in applications ranging from single-cell imaging to in vivo biodistribution.

Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Synthesis of Azido-Globo H Analogs for Immunogenicity Evaluation.

Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.

Nanopillar Templating Augments the Stiffness and Strength in Biopolymer Films.

Natural load-bearing mammalian tissues, such as cartilage and ligaments, contain ∼70% water yet can be mechanically stiff and strong due to the highly templated structures within. Here, we present a bioinspired approach to significantly stiffen and strengthen biopolymer hydrogels and films through the combination of nanoscale architecture and templated microstructure. Imprinted submicrometer pillar arrays absorb energy and deflect cracks. The produced chitosan hydrogels show nanofiber chains aligned by nanopillar topography, subsequently templating the microstructure throughout the film. These templated nanopillar chitosan hydrogels mechanically outperform unstructured flat hydrogels, with increases in the moduli of ∼160%, up to ∼20 MPa, and work at break of ∼450%, up to 8.5 MJ m-3. Furthermore, the strength at break increases by ∼350%, up to ∼37 MPa, and it is one of the strongest hydrogels yet reported. The nanopillar templating strategy is generalizable to other biopolymers capable of forming oriented domains and strong interactions. Overall, this process yields hydrogel films that demonstrate mechanical performance comparable to that of other stiff, strong hydrogels and natural tissues.

Advancements in protein nanoparticle vaccine platforms to combat infectious disease.

Infectious diseases are a major threat to global human health, yet prophylactic treatment options can be limited, as safe and efficacious vaccines exist only for a fraction of all diseases. Notably, devastating diseases such as acquired immunodeficiency syndrome (AIDS) and coronavirus disease of 2019 (COVID-19) currently do not have vaccine therapies. Conventional vaccine platforms, such as live attenuated vaccines and whole inactivated vaccines, can be difficult to manufacture, may cause severe side effects, and can potentially induce severe infection. Subunit vaccines carry far fewer safety concerns due to their inability to cause vaccine-based infections. The applicability of protein nanoparticles (NPs) as vaccine scaffolds is promising to prevent infectious diseases, and they have been explored for a number of viral, bacterial, fungal, and parasitic diseases. Many types of protein NPs exist, including self-assembling NPs, bacteriophage-derived NPs, plant virus-derived NPs, and human virus-based vectors, and these particular categories will be covered in this review. These vaccines can elicit strong humoral and cellular immune responses against specific pathogens, as well as provide protection against infection in a number of animal models. Furthermore, published clinical trials demonstrate the promise of applying these NP vaccine platforms, which include bacteriophage-derived NPs, in addition to multiple viral vectors that are currently used in the clinic. The continued investigations of protein NP vaccine platforms are critical to generate safer alternatives to current vaccines, advance vaccines for diseases that currently lack effective prophylactic therapies, and prepare for the rapid development of new vaccines against emerging infectious diseases. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Screening for the Proteins That Can Interact with Grouper Nervous Necrosis Virus Capsid Protein.

Nervous necrosis virus (NNV) can infect many species of fish and has an 80-100% mortality rate. NNV capsid protein (NNVCP) is the only structural protein of NNV, but there are few studies on the protein-protein interaction between NNVCP and the host cell. To investigate NNV morphogenesis, native NNV capsid protein (NNVCP) was used to screen for protein-protein interactions in this study. The results identified that 49 grouper optic nerve proteins can interact with NNVCP and may function as putative receptor or co-receptor, cytoskeleton, glucose metabolism and ATP generation, immunity, mitochondrial ion regulation, and ribosomal proteins. Creatine kinase B-type (CKB) is one of those 49 optic nerve proteins. CKB, a kind of enzyme of ATP generation, was confirmed to interact with NNVCP by far-Western blot and showed to colocalize with NNVCP in GF-1 cells. Compared to the control, the expression of CKB was significantly induced in the brain and eyes infected with NNV. Moreover, the amount of replication of NNV is relatively high in cells expressing CKB. In addition to providing the database of proteins that can interact with NNVCP for subsequent analysis, the results of this research also verified that CKB plays an important role in the morphogenesis of NNV.

Effects of Surface-Bound Collagen-Mimetic Peptides on Macrophage Uptake and Immunomodulation.

The interaction between collagen/collagen-like peptides and the commonly expressed immune cell receptor LAIR-1 (leukocyte-associated immunoglobulin-like receptor-1) regulates and directs immune responses throughout the body. Understanding and designing these interactions within the context of biomaterials could advance the development of materials used in medical applications. In this study, we investigate the immunomodulatory effects of biomaterials engineered to display a human collagen III-derived ligand peptide (LAIR1-LP) that targets LAIR-1. Specifically, we examine the effects of LAIR1-LP functionalized surfaces on uptake of polymeric particles and cell debris by macrophages polarized toward inflammatory or healing phenotypes. We observed that culture of macrophages on LAIR1-LP functionalized surfaces increased their uptake of PLGA micro- and nano-particles, as well as apoptotic fibroblasts, while reducing their secretion of TNFα in response to LPS/IFNγ pro-inflammatory stimulation, when compared to cells seeded on control surfaces. To investigate the role of LAIR-1 in the observed LAIR1-LP-induced effects, we used siRNA to knock down LAIR-1 expression and found that cells lacking LAIR-1 exhibited enhanced particle uptake on LAIR1-LP and control surfaces. Furthermore, analysis of gene expression showed that LAIR-1 knockdown led to increase expression of other receptors involved in cell uptake, including CD-36, SRA-1, and beta-2 integrin. Together, our study suggests that LAIR1-LP enhances macrophage uptake potentially through interactions with collagen-domain binding surface receptors, and inhibits inflammation through interaction with LAIR-1.

An Antigen-Delivery Protein Nanoparticle Combined with Anti-PD-1 Checkpoint Inhibitor Has Curative Efficacy in an Aggressive Melanoma Model.

Immune checkpoint inhibition is a promising alternative treatment to standard chemotherapies; however, it fails to achieve long-term remission in a significant portion of patients. A previously developed protein nanoparticle-based platform (E2 nanoparticle) delivers cancer antigens to increase antigen-specific tumor responses. While prior work has focussed on prophylactic conditions, the objectives in this study are therapeutic. It is hypothesized that immune checkpoint inhibition, when augmented by antigen delivery using E2 nanoparticles containing CpG oligonucleotide 1826 (CpG) and a glycoprotein 100 (gp100) melanoma antigen epitope (CpG-gp-E2), would synergistically elicit antitumor responses. To identify a regimen primed for obtaining effective treatment results, immune benchmarks in the spleen and tumor are examined. Conditions that lead to significant immune activation, including increases in gp100-specific interferon gamma (IFN-𝜸), CD8 T cells in the spleen, tumor-infiltrating CD8 T cells, and survival time are identified. Based on the findings, the resulting combination of CpG-gp-E2 and anti-programmed cell death protein 1 (anti-PD-1) treatment in tumor-challenged mice yield significantly increased long-term survival; more than 50% of the mice treated with combination therapy were tumor-free, compared with 0% and ≈5% for CpG-gp-E2 and anti-PD-1 alone, respectively. Evidence of a durable antitumor response is also observed upon tumor rechallenge, pointing to long-lasting immune memory.

Metabolite Responsive Nanoparticle-Protein Complex.

Stimuli-responsive polymers are an efficient means of targeted therapy. Compared to conventional agents, they increase bioavailability and efficacy. In particular, polymer hydrogel nanoparticles (NPs) can be designed to respond when exposed to a specific environmental stimulus such as pH or temperature. However, targeting a specific metabolite as the trigger for stimuli response could further elevate selectivity and create a new class of bioresponsive materials. In this work we describe an N-isopropylacrylamide (NIPAm) NP that responds to a specific metabolite, characteristic of a hypoxic environment found in cancerous tumors. NIPAm NPs were synthesized by copolymerization with an oxamate derivative, a known inhibitor of lactate dehydrogenase (LDH). The oxamate-functionalized NPs (OxNP) efficiently sequestered LDH to produce an OxNP-protein complex. When exposed to elevated concentrations of lactic acid, a substrate of LDH and a metabolite characteristic of hypoxic tumor microenvironments, OxNP-LDH complexes swelled (65%). The OxNP-LDH complexes were not responsive to structurally related small molecules. This work demonstrates a proof of concept for tuning NP responsiveness by conjugation with a key protein to target a specific metabolite of disease.

Extracellular Matrix-Based Strategies for Immunomodulatory Biomaterials Engineering.

The extracellular matrix (ECM) is a complex and dynamic structural scaffold for cells within tissues and plays an important role in regulating cell function. Recently it has become appreciated that the ECM contains bioactive motifs that can directly modulate immune responses. This review describes strategies for engineering immunomodulatory biomaterials that utilize natural ECM-derived molecules and have the potential to harness the immune system for applications ranging from tissue regeneration to drug delivery. A top-down approach utilizes full-length ECM proteins, including collagen, fibrin, or hyaluronic acid-based materials, as well as matrices derived from decellularized tissue. These materials have the benefit of maintaining natural conformation and structure but are often heterogeneous and encumber precise control. By contrast, a bottom-up approach leverages immunomodulatory domains, such as Arg-Gly-Asp (RGD), matrix metalloproteinase (MMP)-sensitive peptides, or leukocyte-associated immunoglobulin-like receptor-1(LAIR-1) ligands, by incorporating them into synthetic materials. These materials have tunable control over immune cell functions and allow for combinatorial approaches. However, the synthetic approach lacks the full natural context of the original ECM protein. These two approaches provide a broad range of engineering techniques for immunomodulation through material interactions and hold the potential for the development of future therapeutic applications.

Protein-based nanoparticles in cancer vaccine development.

Peptide and protein-based cancer vaccines usually fail to elicit efficient immune responses against tumors. However, delivery of these peptides and proteins as components within caged protein nanoparticles has shown promising improvements in vaccine efficacy. Advantages of protein nanoparticles over other vaccine platforms include their highly organized structures and symmetry, biodegradability, ability to be specifically functionalized at three different interfaces (inside and outside the protein cage, and between subunits in macromolecular assembly), and ideal size for vaccine delivery. In this review, we discuss different classes of virus-like particles and caged protein nanoparticles that have been used as vehicles to transport and increase the interaction of cancer vaccine components with the immune system. We review the effectiveness of these protein nanoparticles towards inducing and elevating specific immune responses, which are needed to overcome the low immunogenicity of the tumor microenvironment.

Tailoring Collagen to Engineer the Cellular Microenvironment.

Collagen is the most abundant protein in the extracellular matrix (ECM), and it can direct the behavior of the neighboring cells. By customizing properties of collagen, it is possible to control the cells that interact with it. Utilizing a bottom-up strategy, modular gene fragments are assembled and recombinantly processed to create collagen-mimetic variants that modulate proteolytic degradation, cell adhesion, and mechanical characteristics. The removal of the native MMP cleavage site results in MMP-1 resistant collagen. By introducing additional MMP-susceptible sequences, the degradation characteristics of collagen molecules are modified. Additional non-native functionality is also introduced into the collagen, including the IKVAV sequence, which has been implicated in neurite outgrowth. This mutation, which disrupts the Gly-X-Y tripeptide repeat of collagen, does not prevent the formation of triple-helical collagen. Non-native cysteines and the integrin binding sequence GFOGER are combined in the collagen, and encapsulation of normal human lung fibroblasts within collagen hydrogels are tested. Cells remain spherical, when encapsulated within hydrogels of collagen variants in which the native integrin binding sites are removed, but cell adhesion is restored with the introduction of non-native GFOGER binding sequences. This modular collagen system allows for the combination of multiple functionalities, and it enables the production of biomimetic scaffolds with customizable characteristics to modulate cellular microenvironments.

Recombinant collagen scaffolds as substrates for human neural stem/progenitor cells.

Adhesion to the microenvironment profoundly affects stem cell functions, including proliferation and differentiation, and understanding the interaction of stem cells with the microenvironment is important for controlling their behavior. In this study, we investigated the effects of the integrin binding epitopes GFOGER and IKVAV (natively present in collagen I and laminin, respectively) on human neural stem/progenitor cells (hNSPCs). To test the specificity of these epitopes, GFOGER or IKVAV were placed within the context of recombinant triple-helical collagen III engineered to be devoid of native integrin binding sites. HNSPCs adhered to collagen that presented GFOGER as the sole integrin-binding site, but not to IKVAV-containing collagen. For the GFOGER-containing collagens, antibodies against the ß1 integrin subunit prevented cellular adhesion, antibodies against the α1 subunit reduced cell adhesion, and antibodies against α2 or α3 subunits had no significant effect. These results indicate that hNSPCs primarily interact with GFOGER through the α1ß1 integrin heterodimer. These GFOGER-presenting collagen variants also supported differentiation of hNSPCs into neurons and astrocytes. Our findings show, for the first time, that hNSPCs can bind to the GFOGER sequence, and they provide motivation to develop hydrogels formed from recombinant collagen variants as a cell delivery scaffold. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1363-1372, 2018.