Fabrication of poly(vinyl alcohol)-Carrageenan scaffolds for cryopreservation: Effect of composition on cell viability

Highlights

• PVA-Carrageenan scaffolds were prepared via freeze-gelation technique.

• 3 dimensional highly porous, interconnected lamellar structure observed in SEM.

• All the scaffold constructs were found to be hemocompatible.

• The scaffolds exhibited excellent cell viability after 15 days of cryopreservation.

Abstract

The present investigation reports the fabrication of three dimensional (3D), interconnected, highly porous, biodegradable scaffolds using freeze-gelation technique. The hydrogels prepared with different ratios (5:5, 6:4, 7:3, 8:2 and 9:1) of poly(vinyl alcohol) (PVA) and Carrageenan (Car) was lyophilized to obtain their respective scaffolds. The PVA-Car scaffolds were further characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The prepared scaffolds were found to be biodegradable and highly compatible with hemoglobin. Further, normal keratinocyte (HaCaT) and osteosarcoma (Saos-2) cells seeded on PVA-Car scaffolds were cryopreserved for 15 days and their viability was checked at regular interval of 3 days (0, 3, 6, 9, 12, 15 days) through MTT assay and fluorescence microscopy. Overall, the collective results indicate the scaffold constructs with 7:3 and 8:2 PVA-Car ratios possess ideal characteristics for tissue engineering applications and for long term cryopreservation of cells.

Introduction

In tissue engineering an ideal scaffold should possess the properties of hydrophobicity, visco-elasticity and biocompatibility to support cellular proliferation and other metabolic activities. Ultimately, the scaffold should be able to mimic the extra cellular matrix (ECM) of the body and upon implantation they should degrade slowly to promote the specific tissue regeneration (Yoon & Ji, 2005). Scaffolds can be prepared from different approaches such as physical (freeze gelation) and chemical (photo-irradiation, atom transfer radical polymerization) cross linking methods. The physical cross linking method possess some disadvantages such as time consuming preparation step with temporary cross linking whereas in chemical cross linking route the polymerization reaction is very rapid and sometimes permanent junctions are formed due to covalent bonds, but they posses some disadvantages such as use of toxic cross linking agents which might be detrimental to the cells inside the body environment (Hollister, 2005, Takezawa, 2003). The primary motive in tissue engineering is to improve the overall health of the concerned organ/tissue in the ailing patients by implanting a cell seeded scaffold with additional growth factors at the concerned site which could assist spatial and progressive organization of the tissues in parity with scaffold degradation (Cheung, Lau, Lu, & David, 2007). The scaffolds upon implantation allow cells to secrete their own ECM and adjacent tissue gets dissolved steadily. Thus, this approach of tissue engineering requires cells seeded scaffolds in large quantities which in turn may take several weeks to substitute the tissue expansion. So, to overcome this problem cryopreservation of the cells seeded scaffolds could be an alternative approach. Cryopreservation is a long term biopreservation method using cryoprotective agents such as dimethylsulfoxide (DMSO) that maintains in vivo conditions and is one of the most important research aspects of modern tissue engineering and regenerative medicine (Bissoyi, Pramanik, Panda, & Sarangi, 2014). Generally, cryopreservation of cells are carried out by two methods, i.e., vitrification and slow freezing process. Vitrification is an ultra rapid method of cryopreservation where high concentration of cryoprotectant is used with rapid cooling rate up to −196 °C within 1 s from room temperature. Therefore due to this rapid rate of cooling and warming along with higher cryoprotectant concentrations this could be potentially harmful to the cells seeded scaffolds. In slow freezing process instead of directly transferring the scaffolds to very low temperature they are eventually incubated at different temperature conditions from 4 °C to −20 °C and eventually to lower temperatures. However this method also poses some serious drawbacks such as formation of intracellular and extracellular ice crystals which could damage the cellular structure and machinery of the cells (Danasouri & Selman, 2005). Polysaccharides should be utilized for scaffolding materials, as they can mimic the chemical composites of native ECM, which contains many negatively charged functional groups such as COOH, NH₂, and SO₃H (Li et al., 2015). PVA [CH₂CH(OH)]_n is a water soluble polymer having biodegradable and biocompatible properties. Due to its excellent mechanical and weight bearing properties it is widely used for porous scaffold fabrication (Colosi et al., 2013; Pal, Banthia, & Majumdar, 2006; Puppi et al., 2010). The native ECM contains sulfated polysaccharides such as chondroitin sulfate that binds and enriches growth factors which is very essential for cell differentiation, but as chondroitin sulfate is very costly and has poor gelling capacity therefore there are very limited reports on their applicability in scaffolding materials. Thus in this scenario Carrageenan (Car) can be used as an alternative to substitute the chondroitin sulfate. It is a naturally found hydrophilic polysaccharide that are present in the ECM of some species of red algae (Nijenhuis, 1997). Car is primarily composed of a group of linear anionic-sulfated polysaccharides, linked by glycosidic bonds between galactose and anhydrogalactose. The sulphonyl groups associated with Car provides its anticoagulant property. Car has also shown its importance in the production of macroporous composites for bone tissue engineering and wound dressing (Sharma, Bhat, Vishnoi, Nayak, & Kumar, 2013).

In the present investigation Car was selected as a candidate to form composite scaffolds with PVA because they are highly flexible molecules that have the ability to shape into different formats at room temperature due to its thermosensitive characteristics as well as to provide strength to the PVA polymer as PVA gels after lyophilization produces porous scaffolding materials which have very limited mechanical strength. Thus the incorporation of Car to the PVA polymer not only provides mechanical strength but also makes it more biocompatible as the sulphated disaccharides present in Car resembles naturally occurring glycosaminoglycans which are very prominent components of connective tissues (Kudo, Ishida, Syuu, Sekine, & Ikeda-Fukazawa, 2014; Qian and Zhang, 2011, Zhang et al., 2015). To the best knowledge of the authors till now there are no reports on fabrication of PVA-Car scaffolding materials for cryopreservation of cells up to 15 days of incubation which brings the novelty of the present work. In the current study porous biodegradable scaffolds were prepared without using any external cross linking agents thereby only keeping them in −20 °C for 24 h and 12 h of thawing temperature. Further the effect of cell viability upon cryopreservation of the constructed scaffolds seeded with Saos-2 cells and HaCat cells with different incubation period was studied.