Engineering peptide-modified alginate-based bioinks with cell-adhesive properties for biofabrication.

Alginate (ALG) and its oxidised form alginate-dialdehyde (ADA) are highly attractive materials for hydrogels used in 3D bioprinting as well as drop-on-demand (DoD) approaches. However, both polymers need to be modified using cell-adhesive peptide sequences, to obtain bioinks exhibiting promising cell-material interactions. Our study explores the modification of ALG- and ADA-based bioinks with the adhesive peptides YIGSR (derived from laminin), RRETEWA (derived from fibronectin) and IKVAV (derived from laminin) for 3D bioprinting. Two coupling methods, carbodiimide and Schiff base reactions, were employed to modify the polymers with peptides. Analytical techniques, including FTIR and NMR were used to assess the chemical composition, revealing challenges in confirming the presence of peptides. The modified bioinks exhibited decreased stability, viscosity, and stiffness, particularly-ADA-based bioinks in contrast to ALG. Sterile hydrogel capsules or droplets were produced using a manual manufacturing process and DoD printing. NIH/3T3 cell spreading analysis showed enhanced cell spreading in carbodiimide-modified ADA, Schiff base-modified ADA, and PEG-Mal-modified ADA. The carbodiimide coupling of peptides worked for ADA, however the same was not observed for ALG. Finally, a novel mixture of all used peptides was evaluated regarding synergistic effects on cell spreading which was found to be effective, showing higher aspect ratios compared to the single peptide coupled hydrogels in all cases. The study suggests potential applications of these modified bioinks in 3D bioprinting approaches and highlights the importance of peptide selection as well as their combination for improved cell-material interactions.

Architecture of ß-lactoglobulin coating modulates bioinspired alginate dialdehyde-gelatine/polydopamine scaffolds for subchondral bone regeneration.

In this study, we developed polydopamine (PDA)-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds for subchondral bone regeneration. These polymeric scaffolds were then coated with ß-Lactoglobulin (ß-LG) at concentrations of 1 mg/ml and 2 mg/ml. Morphological analysis indicated a homogeneous coating of the ß-LG layer on the surface of network-like scaffolds. The ß-LG-coated scaffolds exhibited improved swelling capacity as a function of the ß-LG concentration. Compared to ADA-GEL/PDA scaffolds, the ß-LG-coated scaffolds demonstrated delayed degradation and enhanced biomineralization. Here, a lower concentration of ß-LG showed long-lasting stability and superior biomimetic hydroxyapatite mineralization. According to the theoretical findings, the single-state, representing the low concentration of ß-LG, exhibited a homogeneous distribution on the surface of the PDA, while the dimer-state (high concentration) displayed a high likelihood of uncontrolled interactions. ß-LG-coated ADA-GEL/PDA scaffolds with a lower concentration of ß-LG provided a biocompatible substrate that supported adhesion, proliferation, and alkaline phosphatase (ALP) secretion of sheep bone marrow mesenchymal stem cells, as well as increased expression of osteopontin (SPP1) and collagen type 1 (COL1A1) in human osteoblasts. These findings indicate the potential of protein-coated scaffolds for subchondral bone tissue regeneration. STATEMENT OF SIGNIFICANCE: This study addresses a crucial aspect of osteochondral defect repair, emphasizing the pivotal role of subchondral bone regeneration. The development of polydopamine-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds, coated with ß-Lactoglobulin (ß-LG), represents a novel approach to potentially enhance subchondral bone repair. ß-LG, a milk protein rich in essential amino acids and bioactive peptides, is investigated for its potential to promote subchondral bone regeneration. This research explores computationally and experimentally the influence of protein concentration on the ordered or irregular deposition, unravelling the interplay between coating structure, scaffold properties, and in-vitro performance. This work contributes to advancing ordered protein coating strategies for subchondral bone regeneration, providing a biocompatible solution with potential implications for supporting subsequent cartilage repair.

Bovine serum albumin-modified 3D printed alginate dialdehyde-gelatin scaffolds incorporating polydopamine/SiO2-CaO nanoparticles for bone regeneration.

Three-dimensional (3D) printing allows precise manufacturing of bone scaffolds for patient-specific applications and is one of the most recently developed and implemented technologies. In this study, bilayer and multimaterial alginate dialdehyde-gelatin (ADA-GEL) scaffolds incorporating polydopamine (PDA)/SiO2-CaO nanoparticle complexes were 3D printed using a pneumatic extrusion-based 3D printing technology and further modified on the surface with bovine serum albumin (BSA) for application in bone regeneration. The morphology, chemistry, and in vitro bioactivity of PDA/SiO2-CaO nanoparticle complexes were characterized (n = 3) and compared with those of mesoporous SiO2-CaO nanoparticles. Successful deposition of the PDA layer on the surface of the SiO2-CaO nanoparticles allowed better dispersion in a liquid medium and showed enhanced bioactivity. Rheological studies (n = 3) of ADA-GEL inks consisting of PDA/SiO2-CaO nanoparticle complexes showed results that may indicate better injectability and printability behavior compared to ADA-GEL inks incorporating unmodified nanoparticles. Microscopic observations of 3D printed scaffolds revealed that PDA/SiO2-CaO nanoparticle complexes introduced additional topography onto the surface of 3D printed scaffolds. Additionally, the modified scaffolds were mechanically stable and elastic, closely mimicking the properties of natural bone. Furthermore, protein-coated bilayer scaffolds displayed controllable absorption and biodegradation, enhanced bioactivity, MC3T3-E1 cell adhesion, proliferation, and higher alkaline phosphatase (ALP) activity (n = 3) compared to unmodified scaffolds. Consequently, the present results confirm that ADA-GEL scaffolds incorporating PDA/SiO2-CaO nanoparticle complexes modified with BSA offer a promising approach for bone regeneration applications.

A review of glycosaminoglycan-modified electrically conductive polymers for biomedical applications.

The application areas of electrically conductive polymers have been steadily growing since their discovery in the late 1970s. Recently, electrically conductive polymers have found their way into biomedicine, allowing the realization of many relevant applications ranging from bioelectronics to scaffolds for tissue engineering. Extracellular matrix components, such as glycosaminoglycans, build an important class of biomaterials that are heavily researched for biomedical applications due to their favorable properties. Due to their highly anionic character and the presence of sulfate groups in glycosaminoglycans, these biomolecules can be employed to functionalize conductive polymers, which enables the tailorability and improvement of cell-material interactions of conductive polymers. This review paper gives an overview of recent research on glycosaminoglycan-modified conductive polymers intended for biomedical applications and discusses the effect of different biological dopants on material characteristics, such as surface roughness, stiffness, and electrochemical properties. Moreover, the key findings of the biological characterization in vitro and in vivo are summarized, and remaining challenges in the field, particularly related to the modification of electrically conductive polymers with glycosaminoglycans to achieve improved functional and biological outcomes, are discussed. STATEMENT OF SIGNIFICANCE: The development of functional biomaterials based on electrically conductive polymers (CPs) for various biomedical applications, such as neural regeneration, drug delivery, or bioelectronics, has been increasingly investigated over the last decades. Recent literature has shown that changes in the synthesis procedure or the chosen dopant could adjust the resulting material characteristics. Hence, an interesting approach lies in using natural biomolecules as dopants for CPs to tailor the biological outcome. This review comprehensively summarizes the state of the art in the field of glycosaminoglycan-modified electrically conductive polymers for the first time, particularly highlighting the effect of the chosen dopant on material characteristics, such as surface morphology or stiffness, electrochemical properties, and consequently, cell-material interactions.

How to Determine a Suitable Alginate for Biofabrication Approaches using an Extensive Alginate Library?

Alginate-based hydrogels are a promising class of biomaterials due to their usability, biocompatibility, and high water-binding capacity which is the reason for their broad use in biofabrication. One challenge of these biomaterials is, however, the lack of cell adhesion motifs. This drawback can be overcome by oxidizing alginate to alginate dialdehyde (ADA) and by subsequent cross-linking with gelatin (GEL) to fabricate ADA-GEL hydrogels, which offer improved cell-material interactions. The present work investigates four pharmaceutical grade alginates of different algae sources and their respective oxidized forms regarding their molecular weight and M/G ratio using 1H NMR spectroscopy and gel permeation chromatography. In addition, three different methods for determining the degree of oxidation (% DO) of ADA, including iodometric, spectroscopic, and titration methods, are applied and compared. Furthermore, the aforementioned properties are correlated with the resulting viscosity, degradation behavior, and cell-material interactions to predict the material behavior in vitro and thus choose a suitable alginate for an intended application in biofabrication. In the framework of the present work, easy and practicable detection methods for the investigations of alginate-based bioinks were summarized and shown. In this regard, the success of oxidation of alginate was confirmed by the three aforementioned methods and was further proven by solid-state 13C NMR, for the first time in the literature, that only guluronic acid (G) was attacked during the oxidation, leading to the formation of hemiacetals. Furthermore, it was shown that ADA-GEL hydrogels of alginates with longer G-blocks are more suitable for long-term experiments due to their stability over an incubation period of 21 days, while ADA-GEL hydrogels of alginates with longer mannuronic acid (M)-blocks are more suitable for short-term applications such as sacrificial inks due to their extensive swelling and subsequent loss of shape. Finally, it was proven that the M/G ratio did not show any influence on the biocompatibility or printability of the investigated alginate-based hydrogels. The physicochemical findings provide an alginate library for tailored application in biofabrication.

Targeted Printing of Cells: Evaluation of ADA-PEG Bioinks for Drop on Demand Approaches.

A novel approach, in the context of bioprinting, is the targeted printing of a defined number of cells at desired positions in predefined locations, which thereby opens up new perspectives for life science engineering. One major challenge in this application is to realize the targeted printing of cells onto a gel substrate with high cell survival rates in advanced bioinks. For this purpose, different alginate-dialdehyde-polyethylene glycol (ADA-PEG) inks with different PEG modifications and chain lengths (1-8 kDa) were characterized to evaluate their application as bioinks for drop on demand (DoD) printing. The biochemical properties of the inks, printing process, NIH/3T3 fibroblast cell distribution within a droplet and shear forces during printing were analyzed. Finally, different hydrogels were evaluated as a printing substrate. By analysing different PEG chain lengths with covalently crosslinked and non-crosslinked ADA-PEG inks, it was shown that the influence of Schiff's bases on the viscosity of the corresponding materials is very low. Furthermore, it was shown that longer polymer chains resulted in less stable hydrogels, leading to fast degradation rates. Several bioinks highly exhibit biocompatibility, while the calculated nozzle shear stress increased from approx. 1.3 and 2.3 kPa. Moreover, we determined the number of cells for printed droplets depending on the initial cell concentration, which is crucially needed for targeted cell printing approaches.