A Design of Experiments (DoE) Approach to Optimize Cryogel Manufacturing for Tissue Engineering Applications

Abstract

Marine origin polymers represent a sustainable and natural alternative to mammal counterparts regarding the biomedical application due to their similarities with proteins and polysaccharides present in extracellular matrix (ECM) in humans and can reduce the risks associated with zoonosis and overcoming social- and religious-related constraints. In particular, collagen-based biomaterials have been widely explored in tissue engineering scaffolding applications, where cryogels are of particular interest as low temperature avoids protein denaturation. However, little is known about the influence of the parameters regarding their behavior, i.e., how they can influence each other toward improving their physical and chemical properties. Factorial design of experiments (DoE) and response surface methodology (RSM) emerge as tools to overcome these difficulties, which are statistical tools to find the most influential parameter and optimize processes. In this work, we hypothesized that a design of experiments (DoE) model would be able to support the optimization of the collagen-chitosan-fucoidan cryogel manufacturing. Therefore, the parameters temperature (A), collagen concentration (B), and fucoidan concentration (C) were carefully considered to be applied to the Box–Behnken design (three factors and three levels). Data obtained on rheological oscillatory measurements, as well as on the evaluation of antioxidant concentration and adenosine triphosphate (ATP) concentration, showed that fucoidan concentration could significantly influence collagen-chitosan-fucoidan cryogel formation, creating a stable internal polymeric network promoted by ionic crosslinking bonds. Additionally, the effect of temperature significantly contributed to rheological oscillatory properties. Overall, the condition that allowed us to have better results, from an optimization point of view according to the DoE, were the gels produced at −80 °C and composed of 5% of collagen, 3% of chitosan, and 10% fucoidan. Therefore, the proposed DoE model was considered suitable for predicting the best parameter combinations needed to develop these cryogels.

Advanced Polymeric Membranes as Biomaterials Based on Marine Sources Envisaging the Regeneration of Human Tissues

“…Additionally, in research previously published by our team [ 9 , 83 , 84 ] we performed biological in vitro analysis of biomaterials formulations comprising the marine polymers used herein, namely the evaluation of cell viability, cytotoxicity, DNA content, morphology and ATP activity, using chondrocyte (ATDC5) and fibroblast (L929) cell lines. Taking this into account, the present study aims to expand the scaffolding knowledge by exploring other processing methodologies of the same marine polymers for various purposes, in this case specifically to form membranes.…”

Application of Collagen-Based Hydrogel in Skin Wound Healing

“…Most gelation has been reported to take place at physiological temperature (37 °C), but 25 °C has also been used. In addition, the gelation process has also been performed in cryo-environments (i.e., temperatures below zero) [ 38 ]. Under higher-temperature conditions, collagen molecules denature and become faster in their self-assembly behavior, leading to a decrease in the ordered structure of collagen fibers, which ultimately affects the structure of the hydrogel.…”

Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing

“…Initially, collagen and chitosan were separately solubilized in 0.5 M acetic acid [AcOH] (around pH 2.5), while fucoidan was dissolved in ultra-pure water. The polymeric solutions were prepared according to the best polymeric concentration obtained in previous work [7], i.e. 5% collagen (50 mg ml −1 ), and 10% fucoidan (100 mg ml −1 ).…”

“…In a first assessment, several 3D printing pre-tests were performed to find the most promising biomaterial ink based on the best concentration of blended three marine compounds (i.e. collagen-chitosanfucoidan, adapted from previous study [7]), and the proper processing parameters (i.e. nozzle size, printing speed, and air pressure) in order to obtain controlled printability and shape fidelity.…”

“…Compared with the traditional techniques for fabricating porous scaffolds, such as freeze-drying or electrospinning, 3D bioprinting approaches can offer reproducible structures with more precise pore size and geometry and, importantly, have the advantage that it can replicate the complex architecture and organization of native tissues, providing more personalized treatment for each clinical case [3][4][5]. However, a big challenge in the field of TE stems from involving materials from natural sources due to their variability, depending on the species, gender, age, environment, and type of polymer extraction [6,7]. For decades, the demands of TE have been filled by bioactive compounds from mammalian sources, such as collagen or hyaluronic acid mainly obtained from bovine or porcine tissues provided by the processing meat industry [8,9].…”