Kefiran cryogels as potential scaffolds for drug delivery and tissue engineering applications

https://doi.org/10.1016/j.mtcomm.2019.100554Get rights and content

Highlights

  • Kefiran biopolymer has received an increasing interest because of its safe status.

  • Diclofenac delivery by Kefiran cryogels showed a slow and sustained release.

  • hASCs seeded on Kefiran cryogels revealed that these scaffolds are biocompatible.

  • Kefiran can serve as drug delivery system and 3D platform for tissue regeneration.

Abstract

A Kefiran-based scaffold was developed using a freeze gelation technique, and its potential use for both drug delivery system and tissue engineering applications were investigated. This scaffold showed, through micro-computed tomography and scanning electron microscopy, high porosity (82.3 ± 4.4%), thick pore walls (13.4 ± 0.7 μm), aerogel form with foam-like structure, and elastic behaviour (δ = 16 ± 0.7°). Moreover, the Kefiran scaffold degradation showed a delayed profile for over 28 days. The developed scaffold allowed a slow and sustained diclofenac release over two weeks, and the human Adipose-derived Stem Cells, cultured onto the Kefiran scaffold, were metabolically active after 72 h. Therefore, our research suggests that Kefiran cryogel could be a potential candidate for drug delivery of controlled bioactive molecules and tissue engineering scaffolding.

Introduction

Tissue engineering (TE) has emerged as a new approach involving the combination of cells, biomaterial scaffolds and bioactive agents to fabricate functional new tissues replacing the damaged ones. Polymeric scaffolds are fundamental components of TE approaches, since they provide an optimal cellular microenvironment, allowing 3D (three-dimensional) cellular adhesion, growth, and differentiation [1]. In addition, these scaffolds can promote high and efficient drug-loading to specific sites, providing local delivery of the appropriate dose of drug over a given time range, reducing the drug concentration at non-target sites, and, therefore promoting tissue regeneration [2]. In order to be functional for drug delivery and TE, polymeric scaffolds need to have particular physicochemical properties, and present easy-scalable and cost-effective manufacturing methods that will determine their success in TE approaches.

Polymeric scaffolds can form 3D cryogels by a freeze gelation technique. In fact, the most common processing methods for synthetizing cryogels are freeze-drying, salt leaching, particulate leaching and lyophilisation. However, recently freeze gelation has been reported and described as a time and energy efficient technique [3]. These cryogels are sponge-like networks with interconnected macropores that are formed in moderately frozen solutions of monomeric or polymeric precursors [4]. The unique structure of cryogels, in combination with their amazing chemical and physical properties, makes them attractive for several biomedical applications, especially as scaffolds for delivery systems and tissue engineering applications [5]. In fact, these cryogels are considered potential carriers for molecules of pharmaceutical interest, growth factors and cells [4,6].

A variety of polysaccharide-based scaffolds like alginate, dextran, hyaluronic acid, gellan gum and chitosan, among others, have been recently used in TE and regenerative medicine strategies due to their excellent biocompatibility combined with their potential biodegradability [6]. Among the remarkable polysaccharides used lately in several applications, it is possible to find the water-soluble branched glucogalactan Kefiran extracted from the flora of kefir grains [7]. This natural biopolymer has received increasing interest because of its safe status and its outstanding properties for biomedical applications [[7], [8], [9]]. However, achieving translational medicine together with product commercialization requires regenerative polymers to be safe and functional, with cost-effective and mass-production manufacturing methods. Therefore, we propose the development of a new advanced functional polymer, Kefiran, manufactured with a cost-effective and easy scalable method, freeze gelation.

Our research aimed to develop and characterise a novel 3D polymeric scaffold for drug delivery and TE applications based on Kefiran cryogels. The Kefiran scaffold was characterised in terms of the microstructure (micro-CT and SEM analysis), degradation assessment, and mechanical properties (rheometer analysis). Then, Kefiran-based cryogels were evaluated also for controlled delivery of diclofenac sodium. Finally, human Adipose Stem Cells (hASCs) were cultured onto the Kefiran scaffold, and their metabolic function was measured as indicator of their viability. Kefiran scaffolds are rarely reported as drug delivery systems [10]; however, a recent study described Kefiran-alginate gel microspheres for oral delivery [11]. Nevertheless, it is the first time to our knowledge that Kefiran cryogels are evaluated for delivery of diclofenac sodium in combination with its potential use as a TE scaffold.

Section snippets

Materials

AlamarBlue® reagent, Diclofenac sodium, Kefiran, Phosphate-buffered saline, Quant-iT PicoGreen dsDNA kit, Ultrapure water.

Preparation of diclofenac sodium solution

Diclofenac sodium is the sodium salt form of diclofenac, a benzene acetic acid derivate and nonsteroidal anti-inflammatory drug (NSAID) with analgesic, antipyretic and anti-inflammatory activity. Diclofenac sodium salt was provided by Sigma-Aldrich (cat 15307-79-6, D6899). Diclofenac sodium solution was prepared at a concentration of 0.5 mg/mL in ultrapure H2O.

Production of Kefiran cryogel

Kefiran

Results and discussion

Kefiran polysaccharide with its exceptional properties could be a potential candidate for future 3D biomaterial scaffold development. This Kefiran scaffold will interact with biological environment, deliver bioactive molecules such as drugs and growth factors, and act as cell adhesion mediators to cellular functioning and differentiation towards TE. Moreover, in TE, the 3D scaffolds should have an interconnected pore structure and high porosity to guarantee cellular penetration, proliferation

Conclusion

Kefiran cryogels were evaluated as scaffolds for TE and controlled drug delivery. The developed scaffold showed stability, elastic behavior and high porosity 3D structure, capable of controlled release of diclofenac drug for two weeks. Moreover, hASCs seeded on Kefiran scaffold revealed that this platform is biocompatible, sustaining cell metabolic activity for 72 h, a fundamental feature for tissue engineering and regenerative medicine. These features make Kefiran cryogels an appealing

Declaration of Competing Interest

The authors declare no conflicts of interest

Acknowledgements

Hajer Radhouani, Cristiana Gonçalves and F. Raquel Maia were supported by the Fundação para a ciência e a Tecnologia (FCT) from Portugal, with references CEECIND/00111/2017, SFRH/BPD/94277/2013 and SFRH/BPD/117492/2016, respectively. Diana Bicho was supported through the M-ERA-NET/0001/2014 project (FCT). Joaquim. M. Oliveira would like to thank the FCT for the fund provided under the program Investigador FCT 2015 (IF/01285/2015). This work was funded by the R&D Project KOAT – Kefiran

References (26)

  • P. Kooiman

    The chemical structure of Kefiran, the water-soluble polysaccharide of the kefir grain

    Carbohydr. Res.

    (1968)
  • H. Radhouani

    Biological performance of a promising Kefiran-biopolymer with potential in regenerative medicine applications: a comparative study with hyaluronic acid

    J. Mater. Sci. Mater. Med.

    (2018)
  • H. Radhouani

    Kefiran biopolymer: evaluation of its physicochemical and biological properties

    J. Bioact. Compat. Polym.

    (2018)
  • Cited by (32)

    • Cryogels as smart polymers in biomedical applications

      2022, Advances in Biomedical Polymers and Composites: Materials and Applications
    • Medical applications of polymer/functionalized nanoparticle composite systems, renewable polymers, and polymer-metal oxide composites

      2022, Renewable Polymers and Polymer-Metal Oxide Composites: Synthesis, Properties, and Applications
    • Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment

      2021, Materials Science and Engineering C
      Citation Excerpt :

      Generally, natural biopolymers show enzyme-controlled degradability, good biocompatibility, low inflammatory potential, high chemical versatility, and similarities with the extracellular matrix [26,27]. In this context, a variety of polysaccharide-based scaffolds like alginate, dextran, hyaluronic acid, gellan gum and chitosan, among others, have been recently used in tissue engineering and regenerative medicine strategies due to their excellent biocompatibility combined with their potential biodegradability [26,28]. Among the remarkable polysaccharides used in this field, it is possible to find the water-soluble branched glucogalactan Kefiran [29,30].

    View all citing articles on Scopus
    View full text