BM-MSC-Loaded Graphene-Collagen Cryogels Ameliorate Neuroinflammation in a Rat Spinal Cord Injury Model.

A major obstacle to axonal regeneration following spinal cord injury (SCI) is neuroinflammation mediated by astrocytes and microglial cells. We previously demonstrated that graphene-based collagen hydrogels alone can decrease neuroinflammation in SCI. Their regenerative potential, however, is poorly understood and incomplete. Furthermore, stem cells have demonstrated both neuroprotective and regenerative properties in spinal cord regeneration, although there are constraints connected with the application of stem cell-based therapy. In this study, we have analyzed the regeneration capability of human bone marrow mesenchymal stem cell (BM-MSC)-loaded graphene-cross-linked collagen cryogels (Gr-Col) in a thoracic (T10-T11) hemisection model of SCI. Our study found that BM-MSC-loaded Gr-Col improves axonal regeneration, reduces neuroinflammation by decreasing astrocyte reactivity, and promotes M2 macrophage polarization. BM-MSC-loaded-Gr-Col demonstrated enhanced regenerative potential compared to Gr-Col and the injury group control. Next-generation sequencing (NGS) analysis revealed that BM-MSC-loaded-Gr-Col modulates the JAK2-STAT3 pathway, thus decreasing the reactive and scar-forming astrocyte phenotype. The decrease in neuroinflammation in the BM-MSC-loaded-Gr-Col group is attributed to the modulation of Notch/Rock and STAT5a/b and STAT6 signaling. Overall, Gene Set Enrichment Analysis suggests the promising role of BM-MSC-loaded-Gr-Col in promoting axonal regeneration after SCI by modulating molecular pathways such as the PI3/Akt pathway, focal adhesion kinase, and various inflammatory pathways.

Fabrication of a Coculture Organoid Model in the Biomimetic Matrix of Alginate to Investigate Breast Cancer Progression in a TAMs-Leading Immune Microenvironment.

The current research models of breast cancer are usually limited in their capacity to recapitulate the tumor microenvironment in vitro. The lack of an extracellular matrix (ECM) oversimplifies cell-cell or cell-ECM cross-talks. Moreover, the lack of tumor-associated macrophages (TAMs), that can comprise up to 50% of some solid neoplasms, poses a major problem for recognizing various hallmarks of cancer. To address these concerns, a type of direct breast cancer cells (BCCs)-TAMs coculture organoid model was well developed by a sequential culture method in this study. Alginate cryogels were fabricated with appropriate physical and mechanical properties to serve as an alternative ECM. Then, our previous experience was leveraged to polarize TAMs inside of the cryogels for creating an in vitro immune microenvironment. The direct coculture significantly enhanced BCCs organoid growth and cancer aggressive phenotypes, including the stemness, migration, ECM remodeling, and cytokine secretion. Furthermore, transcriptomic analysis and protein-protein interaction networks implied certain pathways (PI3K-Akt pathway, MAPK signaling pathway, etc.) and targets (TNF, PPARG, TLR2, etc.) during breast cancer progression in a TAM-leading immune microenvironment. Future studies to advance treatment strategies for BCC patients may benefit from using this facile model to reveal and target the interactions between cancer signaling and the immune microenvironment.

Dimethylsulfoniopropionate decorated cryogels as synthetic spatially structured habitats of marine bacterial communities.

In microbial consortia bacteria often settle on other organisms that provide nutrients and organic material for their growth. This is true for the plankton where microalgae perform photosynthesis and exude metabolites that feed associated bacteria. The investigation of such processes is difficult since algae provide bacteria with a spatially structured environment with a gradient of released organic material that is hard to mimic. Here we introduce the design and synthesis of a cryogel-based microstructured habitat for bacteria that provides dimethylsulfoniopropionate (DMSP) as a carbon and sulfur source for growth. DMSP, a widely distributed metabolite released by algae, is thereby made available for bacteria in a biomimetic manner. Based on a novel DMSP derived building block (DMSP-HEMA), we synthesized cryogels providing structured surfaces for settlement and delivering the organic material fueling bacterial growth. By monitoring bacterial settlement and performance we show that the cryogels represent microbial arenas mimicking the ecological situation in the plankton.

The specific biopanning of single-domain antibody against haptens based on a functionalized cryogel.

Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.

Physical confinement in alginate cryogels determines macrophage polarization to a M2 phenotype by regulating a STAT-related mRNA transcription pathway.

The immunologic response is considered to play a pivotal role in the application of biomaterial implants, and intrinsic properties of biomaterials can significantly modulate the anti-inflammatory effects. However, how physical confinement influences M2 polarization of macrophages and the relevant mechanisms have not been clearly elucidated. In this study, pore size and porosity in cryogels can be mediated by utilizing alginates with different viscosities. Cryogels of small pore size and low porosity can restrict M2 polarization of macrophages in vitro, judging from cell morphology, secretion of cytokines and expression of key M2-related genes. In comparison, cryogels of large pore size or high porosity can induce M2 polarization in vivo, resulting in the anti-inflammation effects. High-throughput RNA-seq analysis demonstrates that the mRNA surveillance pathway is key in the polarization process, and four primary transcription factors (PPAR-γ, STAT6, NF-κB, and STAT1) participate probably by competition in DNA binding to regulate M2-related gene expression. This study confirms that enough physical space inside is necessary to promote M2 polarization for the anti-inflammatory performance, which can be applied widely in the fields of tissue engineering and regenerative medicine.

Multifunctional Tissue-Adhesive Cryogel Wound Dressing for Rapid Nonpressing Surface Hemorrhage and Wound Repair.

Cryogels with tissue adhesion have great potential as wound dressings for rapid hemostasis for uncontrollable nonpressing surface hemorrhage and wound healing, but their use has not been reported previously. Herein, we designed a series of antibacterial and antioxidant tissue-adhesive cryogels based on quaternized chitosan (QCS) and polydopamine (PDA). These cryogels had good blood cell and platelet adhesion, enrichment, and activation properties for rapid nonpressing surface hemostasis and wound healing. The cryogels exhibited outstanding mechanical strength and easy removability, antioxidant activity, and NIR photothermal-enhanced antibacterial performance. The cryogels showed much better hemostasis than gauze and gelatin sponge in a standardized strip rat liver injury model, a standardized circular rabbit liver section model, and a pig skin laceration model. Furthermore, the excellent hemostatic performance of the QCS/PDA2.0 cryogel (containing 20 mg/mL QCS and 2.0 mg/mL PDA) for coagulopathic hemorrhages was confirmed in a standardized coagulation disorder rabbit circular liver section model. In addition, the QCS/PDA2.0 cryogel promoted rapid hemostasis in a deep noncompressible wound and a much better wound healing effect than a chitosan sponge and Tegaderm film in a full-thickness skin defect model. Overall, these multifunctional tissue-adhesive cryogels with excellent hemostatic performance and enhanced wound healing properties are suitable candidates for tissue-adhesive hemostat and wound healing dressings.

Preparation and characterization of cellulose nanofiber cryogels as oil absorbents and enzymatic lipolysis scaffolds.

Cellulose nanofiber (CNF) materials have received much attention as sustainable "green" materials with high mechanical properties. Their application in oil absorption and enzymatic lipolysis makes them further attractive from the perspective of environmental issues including marine pollution preservation. Herein, we prepared CNF cryogels with various surface properties, evaluated their capacities as oil absorbents and applied them as lipase-lipolysis scaffolds. Their obtained cryogels consisted of various modified CNFs and their structure and properties were investigated. Moreover, lipase-supported CNF cryogels were prepared for enzymatic lipolysis. The cryogels of protonated TEMPO-oxidized CNF showed the highest absorption capacity for olive oil, while all the CNF cryogels possessed similar absorption abilities towards water. In enzymatic lipolysis with lipase, the TEMPO-oxidized CNF (TOCN-Na+) cryogel showed the highest specific activity. The specific activities of lipase in TOCN-Na+ cryogels remained unchanged after being stored at 40 °C for 3 days.

Preparation and properties of cryogel based on poly(hydroxypropyl methacrylate).

A novel supermacroporous poly(hydroxypropyl methacrylate) (p(HPMA)) cryogel was synthesized by cryogelation method at -16 °C. In this synthesis process, HPMA was used as a monomer, and N,N'-methylenebisacrylamide (MBAAm) was used as cross-linker; the reaction was carried out in the presence of redox initiator pair N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS). The effect of monomer concentration, cross-linker content, cooling rate, and dioxane co-solvent were determined with respect to the pore structure, mechanical behavior, swelling degree, and porosity of cryogel. The ESEM images indicate that the pore wall structure of cryogels was rough; moreover, small holes were present in the pore walls of cryogels. The result of compression test indicates that cryogels can be compressed by at least 80% without any breakdown. The result of swelling kinetics indicates that cryogels attain swelling equilibrium in 10 s. Furthermore, p(HPMA)-Cu2+ cryogel was prepared by loading Cu2+ ions on functionalized poly(hydroxypropyl methacrylate)-iminodiacetic acid (p(HPMA)-IDA) cryogel. We investigated the adsorption of bovine serum albumin (BSA) on cryogels. The results indicate that compared to Freundlich isotherm, Langmuir isotherm could more suitably describe the adsorption process of BSA on cryogels. Meanwhile, the adsorption capacity of p(HPMA)-Cu2+ cryogel was significantly greater than that of p(HPMA) cryogel. The maximum adsorption capacity of BSA on p(HPMA)-Cu2+ cryogel, which was treated with 1 M Cu2+ ions, was as high as 196.87 mg/g cryogel (equivalent to 20.48 mg/mL cryogel) at 25 °C and pH = 7.8; therefore, the maximum adsorption capacity of BSA on p(HPMA)-Cu2+ cryogel was 4.35 times higher than that of p(HPMA) cryogel. Thus, the adsorption capacity of cryogels was strongly influenced by Cu2+ concentration, moreover, temperature changes clearly affected the adsorption capacity of p(HPMA)-Cu2+cryogel. The adsorption capacity at 25 °C was twice as that at 15 °C. By calculating Gibbs free energy change (∆G) of adsorption, we found that the adsorption process was spontaneous; moreover, adsorption process occurred better at higher temperature.

An in vitro evaluation of fibrinogen and gelatin containing cryogels as dermal regeneration scaffolds.

Macroporous cryogels containing mixtures of two key components of the dermal extracellular matrix, fibrinogen and collagen-derived gelatin, were evaluated for use as dermal tissue regeneration scaffolds. The infiltration of human dermal fibroblasts into these matrices was quantitatively assessed in vitro using a combination of cell culture and confocal laser scanning microscopy. The extent of cellular infiltration, as measured by the number of cells per distance travelled versus time, was found to be positively correlated with the fibrinogen concentration of the cryogel scaffolds; a known potentiator of cell migration and angiogenesis within regenerating tissue. An analysis of the proteins expressed by infiltrating fibroblasts revealed that the cells that had migrated into the interior portion of the scaffolds expressed predominantly F-actin along their cytoplasmic stress fibres, whereas those present on the periphery of the scaffolds expressed predominantly α-smooth muscle actin, indicative of a nonmotile, myofibroblast phenotype associated with wound contraction. In conclusion, the cryogels produced in this study were found to be biocompatible and, by alteration of the fibrinogen content, could be rendered more amenable to cellular infiltration.

Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.

Grafting zwitterionic polymer onto cryogel surface enhances protein retention in steric exclusion chromatography on cryogel monolith.

Cryogel monoliths with interconnected macropores (10-100µm) and hydrophilic surfaces can be employed as chromatography media for protein retention in steric exclusion chromatography (SXC). SXC is based on the principle that the exclusion of polyethylene glycol (PEG) on both a hydrophilic chromatography surface and a protein favors their association, leading to the protein retention on the chromatography surface. Elution of the retained protein can be achieved by reducing PEG concentration. In this work, the surface of polyacrylamide-based cryogel monolith was modified by grafting zwitterionic poly(carboxybetaine methacrylate) (pCBMA), leading the increase in the surface hydrophilicity. Observation by scanning electron microscopy revealed the presence of the grafted pCBMA chain clusters on the cryogel surface, but pCBMA grafting did not result in the changes of the physical properties of the monolith column, and the columns maintained good recyclability in SXC. The effect of the surface grafting on the SXC behavior of γ-globulin was investigated in a wide flow rate range (0.6-12cm/min). It was found that the dynamic retention capacity increased 1.4-1.8 times by the zwitterionic polymer grafting in the flow rate range of 1.5-12cm/min. The mechanism of enhanced protein retention on the zwitterionic polymer-grafted surface was proposed. The research proved that zwitterionic polymer modification was promising for the development of new materials for SXC applications.

Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system.

In this study, we have focused our attention on preparing supermacroporous cryogels as a potential dye-affinity adsorbent for interferon purification. For this purpose, 2-hydroxyethyl methacrylate (HEMA) and Cibacron Blue F3GA (CB) were selected as main monomer and dye-ligand. Cibacron Blue F3GA attached supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)/CB] cryogels were prepared and characterized by swelling test, scanning electron microscopy, elemental analysis, and FTIR. After that, the effecting factors such as pH, concentration, interaction time, and ionic strength on the interferon separation were evaluated. The maximum adsorption capacity of poly(HEMA)/CB cryogels was obtained as 38.2mg/g at pH 6.0. Fast protein liquid chromatography (FPLC) system was used for interferon purification from human gingival fibroblast extract. The chromatography parameters, capacity and selectivity factors, resolution and theoretical plate number were found as 7.79, 9.62, 4.23 and 554, respectively. Although some decreases in total protein content, from 320 µg to 18 µg, and interferon activity, from 2.6 × 10(3)IU to 2.2 × 10(3)IU, were determined, specific antiviral activity increased from 7.19 IU/µg to 122.2 IU/µg. The purified interferon samples have 97.6% purity determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After repeated ten adsorption-desorption cycles, no significant decrease was determined in adsorption capacity of cryogel. In result, poly(HEMA)/CB cryogels have an application potential for rapid, cheap and specific purification of interferon.

Inorganic/organic biocomposite cryogels for regeneration of bony tissues.

The present work focuses on the physical, mechanical and in vitro properties of porous inorganic/organic biocomposite scaffolds of polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC). These scaffolds are prepared by means of cryogelation technology and are intended for bone tissue engineering applications. The biocomposite cryogels have much more favorable physical and biological properties compared to the previous work of our group on the same composition in the form of pellets and foams. The optimized and heat-treated PTAC biocomposite cryogels show homogenous porosity and good mechanical properties and also exhibit the formation of a hydroxyapatite-like layer on their surface on coming in contact with simulated body fluid (SBF). Furthermore, the biocomposite cryogels showed good biocompatibility with L929 fibroblasts. Also, the influence of pre-soaking in SBF to that of non-soaked scaffolds was compared in terms of proliferation of MG-63 osteoblast-like osteosarcoma cells on these scaffolds and it was found that the pre-soaking caused a decrease in cell proliferation. Finally, the response of human osteoblasts on these scaffolds was analyzed by MTT assay, scanning electron microscopy, energy dispersive X-ray spectroscopy and micro X-ray computing tomography. The cells revealed good biocompatibility with the biocomposite cryogels and were mostly present as cell sheets on the surface with thick bundles of collagenous extracellular matrix during initial period of incubation. During later phases, the formation of calcium phosphate-like mineral deposits was observed on the surface of the cryogels suggesting a high potential of the biocomposite cryogels towards bone regeneration. Therefore, the PTAC biocomposite cryogels, due to their favorable properties and high biocompatibility with human osteoblasts can be suggested as potential scaffolds for bone tissue engineering applications.