Plasma-treated collagen functionalized with chondroitin sulfate as bioactive and nanostructured extracellular matrix mimics.

Aim: Cell microenvironment contains a plethora of information that influences cell modulation. Indeed, the extracellular matrix plays a central role in tissue development. Reproducing the cell-extracellular matrix crosstalk able to recapitulate both physical and biochemical signals is crucial to obtain functional tissue models or regenerative strategies. Materials & methods: Here, a combined method is proposed to easily functionalize collagen surface films, tailoring morphological properties. Oxygen nonthermal plasma treatment and glyco-conjugation with chondroitin sulfate are used to modify surface properties. Results: It results in higher adhesion, proliferation and morphological organization of U87 glioblastoma cells. Conclusion: Our finding suggests new promising strategies for the development of collagen-based biomaterials, which can be employed for advanced in vitro models.

Chitosan derivatives as dynamic coatings for transferrin glycoform separation in capillary electrophoresis.

Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Tyrosine glucosylation of collagen films exploiting Horseradish Peroxidase (HRP).

The development of human tissue models for regenerative medicine and animal-free drug screening requires glycosylated biomaterials such as collagen. An easy and fast biomaterial glycosylation method exploiting Horseradish Peroxidase (HRP) phenol coupling reaction is proposed. The protocol is adaptable to any polymer functionalized with phenol residues or tyrosine containing proteins. As a model the tyrosine residues on collagen films were functionalized with salidroside, a natural ß-glucoside with a phenol in the aglycone. Scanning Electron Microscope (SEM) and contact angle analysis revealed the influence of glycosylation on the sample's morphology and wettability. Preliminary biological evaluation showed the cytocompatibility of the glucosylated collagen films.

Alginate-Gelatin Self-Healing Hydrogel Produced via Static-Dynamic Crosslinking.

Alginate-gelatin hydrogels mimicking extracellular matrix (ECM) of soft tissues have been generated by static-dynamic double crosslinking, allowing fine control over the physical and chemical properties. Dynamic crosslinking provides self-healing and injectability attributes to the hydrogel and promotes cell migration and proliferation, while the static network improves stability. The static crosslinking was performed by enzymatic coupling of the tyrosine residues of gelatin with tyramine residues inserted in the alginate backbone, catalyzed by horseradish peroxidase (HRP). The dynamic crosslinking was obtained by functionalizing alginate with 3-aminophenylboronic acid which generates a reversible bond with the vicinal hydroxyl groups of the alginate chains. Varying the ratio of alginate and gelatin, hydrogels with different properties were obtained, and the most suitable for 3D soft tissue model development with a 2.5:1 alginate:gelatin molar ratio was selected. The selected hydrogel was characterized with a swelling test, rheology test, self-healing test and by cytotoxicity, and the formulation resulted in transparent, reproducible, varying biomaterial batch, with a fast gelation time and cell biocompatibility. It is able to modulate the loss of the inner structure stability for a longer time with respect to the formulation made with only covalent enzymatic crosslinking, and shows self-healing properties.

Characterization of a Bioink Combining Extracellular Matrix-like Hydrogel with Osteosarcoma Cells: Preliminary Results.

Three-dimensional (3D) bioprinting allows the production of artificial 3D cellular microenvironments thanks to the controlled spatial deposition of bioinks. Proper bioink characterization is required to achieve the essential characteristics of printability and biocompatibility for 3D bioprinting. In this work, a protocol to standardize the experimental characterization of a new bioink is proposed. A functionalized hydrogel based on gelatin and chitosan was used. The protocol was divided into three steps: pre-printing, 3D bioprinting, and post-printing. For the pre-printing step, the hydrogel formulation and its repeatability were evaluated. For the 3D-bioprinting step, the hydrogel-printability performance was assessed through qualitative and quantitative tests. Finally, for the post-printing step, the hydrogel biocompatibility was investigated using UMR-106 cells. The hydrogel was suitable for printing grids with good resolution from 4 h after the cross-linker addition. To guarantee a constant printing pressure, it was necessary to set the extruder to 37 °C. Furthermore, the hydrogel was shown to be a valid biomaterial for the UMR-106 cells' growth. However, fragmentation of the constructs appeared after 14 days, probably due to the negative osteosarcoma-cell interference. The protocol that we describe here denotes a strong approach to bioink characterization to improve standardization for future biomaterial screening and development.

3D printed tissue models: From hydrogels to biomedical applications.

The development of new advanced constructs resembling structural and functional properties of human organs and tissues requires a deep knowledge of the morphological and biochemical properties of the extracellular matrices (ECM), and the capacity to reproduce them. Manufacturing technologies like 3D printing and bioprinting represent valuable tools for this purpose. This review will describe how morphological and biochemical properties of ECM change in different tissues, organs, healthy and pathological states, and how ECM mimics with the required properties can be generated by 3D printing and bioprinting. The review describes and classifies the polymeric materials of natural and synthetic origin exploited to generate the hydrogels acting as "inks" in the 3D printing process, with particular emphasis on their functionalization allowing crosslinking and conjugation with signaling molecules to develop bio-responsive and bio-instructive ECM mimics.

3D bioprinted colorectal cancer models based on hyaluronic acid and signalling glycans.

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3'-Sialylgalactose, 6'-Sialylgalactose and 2'-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.