Decellularization of ram cardiac tissue via supercritical CO2

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Abstract

The decellularization method efficacy varies depending on the tissue type. There are commonly used physical, chemical and enzymatic methods. Most of the methods affect the extracellular matrix properties that will in turn alter the cell fate when the biological scaffold is recellularised. We aimed to optimize the decellularization of ram cardiac via scCO2 extraction to preserve the moisture and the components of the ECM during the decellularization procedure. In literature decellularization studies focus on heart vessels. In this study, we aimed to decellularize and recellularize ram cardiac tissue for the first time. Besides we obtained a decellularized tissue without cells on it with a mechanical strength similar to native ECM. Decellularized ram cardiac tissue is expected to be animal-based biomaterial for candidate biomedical applications in vitro and in vivo further studies.

Introduction

Among the people around the age of 70, 10% of them suffer from heart failure while 1–2% of the adult population has heart-related health problems. Currently, heart transplantation and left ventricular assist device (VAD) implantation are the common treatment modalities for end-stage heart failure [1]. However, the long-standing organ waiting list limits the heart transplantation for many people. In general, pharmacological treatments are insufficient for more than 50% of patients with heart disease. Therefore, heart disease treatments should be personalized by designing patient-specific cardiac tissues [2]. Most of the tissue engineering studies include various techniques for reconstruction of heart tissues such as scaffolds with pro-angiogenic factors and/or micro-fabricated scaffolds for cellular guidance. However, the mentioned techniques have not been successful yet for reorganizing the natural vasculature of the native tissue. Besides, functional microvasculature cannot be created by existing scaffold production techniques such as 3-D printing [3]. Therefore, decellularization technologies have been popular in recent years to obtain a natural 3D tissue/organ scaffold. Decellularization is a novel technique to remove the cells, genetic materials such as DNA/RNA from native ECM while protecting biochemical, biomechanical and structural properties of ECM. The main advantage of the decellularization of heart is to obtain the organ with its complex 3D architecture for reconstruction [4].

Generally, the decellularized matrices are obtained by the lysis of the cell membrane and removing the cellular component from ECM. The decellularization efficiency is evaluated by taking into consideration the total protein amount and the amount of double-stranded DNA. The amount of double-stranded DNA should be less than 50 ng per mg ECM dry weight and the length of the DNA fragment should be less than 200 bp [5]. The broadly used chemical approaches for decellularization involve sodium deoxycholate, Triton X-100 and 3- [(3-chola-midopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) [6]. The sodium deoxycholate (SDS) is an ionic detergent that is effective for the solubilization of both nuclear and cytoplasmic membranes, however, it tends to disrupt the protein-protein interactions. Thus, SDS may cause damage on glycosaminoglycans (GAGs) which is the main structural element of ECM providing the mechanical strength to the tissues [7]. On the other hand, Triton X-100 is a non-ionic detergent and has a relatively mild effect on the ECM structure compared to the other detergents. It does not have a role on protein-protein interactions but affects the lipid-lipid and lipid-protein interactions. Thus, proteins of the ECM remain in the functional construction compared to the other detergents when treated with Triton X-100 [8]. The zwitterionic detergents present the properties of both non-ionic and ionic detergents. The most famous member is the CHAPS. It is generally used for the decellularization of blood vessels. CHAPS treatment ends up with a significant decrease of the resistance to vascular pressure [9].

Another type of decellularization process is the enzymatic approach carried out with nucleases, calcium chelating agents and protease digestions. The enzymatic method is particularly useful for the selective cleaning of cell debris and undesired ECM building blocks. Trypsin is a well-known and highly preferred enzyme that cleaves the peptide bonds between the amino acids. Generally, enzymatic procedure is not effective on its own. Thus, it needs to be combined with chemical methods such as ethylenediaminetetraacetic acid (EDTA) for an efficient removal of the cellular components [10].

The physical methods consisting freezing, mechanical pressure, electroporation and agitation are also used for decellularization. The matrix adhesive proteins of cells are broken with physical methods [4]. The freeze-thaw method is a widely used technique. It is mainly effective for the decellularization of tendon, ligamentous and nerve tissues [10]. The tissues are frozen at −86 °C and then the temperature is rapidly raised to 37 °C. The ECM structure is minimally affected compared to other methods. Depending on the tissue or organ of interest, specific protocols may be performed by varying the temperature or varying the number of freeze-thaw cycles. However, it is not an efficient method in itself and must be combined with other decellularization methods to achieve an efficient decellularization. However, a multistep process is time-consuming, endangers the mechanical structure, and biochemical components of ECM [11].

In this study, a novel method for the decellularization of tissues is performed which is not a multi-step procedure and time-consuming. A supercritical fluid extraction (SFE) is the process of separating the components of a material from its matrix which the cell represents the material and the ECM represents the matrix in this study. We used the supercritical carbon dioxide (scCO2) for the extraction process. At standard temperature and pressure CO2 is in the gas state. However, when the temperature and pressure are increased above the critical point for scCO2 is obtained. The CO2 gas becomes a supercritical fluid which gains unique behaviors between gas and liquid. The critical point is 7.2 MPa/ 37 °C for scCO2 and the critical coordinates of CO2 are Tc = 31.06 °C and P = 7.38 MPa [12]. Supercritical fluids have densities as liquids, viscosities as gases and diffuses intermediate to that of a liquid and a gas. The scCO2 above 31.1 °C (304 K) and 73.4 bar (7.3 MPa) can penetrate to the tissues, dissolve the cells and provide the removal of cells from the tissues easily [13]. Moreover, the conditions can be changed, and calibrated by altering temperature and pressure [14]. Besides, it does not affect the cross-linked proteins and does not remain cytotoxic residual chemicals behind [13]. Additionally; supercritical CO2 has also been used in various biomedical applications such as extraction of biologic molecules, sterilization of natural/synthetic biomaterials and materials [15], [16], [17], [18]. The decellularization of a material with scCO2 treatment would be completed remarkable faster compared to the chemical methods which ends in hours rather than days due to having the intermediate features of both gases and liquids [18]. Therefore transport coefficient is the most significant property for scCO2 that indicates a high transfer and permeability rate due to the viscosity of the supercritical fluid that is almost equal to a gas. Additionally, the content of collagen and elastin is not affected, and ultimately the mechanical strength is not changed during the decellularization by utilizing supercritical CO2 [4]. Supercritical CO2 is a non-flammable, non-toxic, relatively inert material and has desirable solvent characteristics. On the other hand, scCO2 is nonpolar and needs ethanol to entrain the charged molecules such as phospholipids [19]. The usage of ethanol with scCO2 provides efficient decellularization especially, on cornea, aorta, adipose tissues and esophagus [20], [21]. In literature, other decellularization protocols that benefit from ethanol as the decellularization solvent have reported tissue dehydration [22]. Hydration of a tissue is essential for the viability of the scaffold. Thus, for the decellularization process, hydration must be prevented. Porta et al., have discussed about the conventional and innovative supercritical technologies for monolithic scaffold production, besides the supercritical systems used in biomedical technologies for production of nanoparticles and micro/nano devices. They have especially mentioned the critical biological and mechanical signals delivered from the scaffold to the cells which significantly affects the cell fate on the scaffold [23]. In this study, we reduced or even eliminate dehydration of the tissues caused by scCO2 treatment by presaturating scCO2 with ethanol prior to treatment in the extraction unit.

Section snippets

Methods

Supercritical fluid extraction system (Applied Separations-Speed SFE), Ram cardiac tissue sample is supplied from a local slaughterhouse, DNA purification kit (RTA lab, Genomic DNA purification kit), Nanodrop (Thermo Scientific), Coomassie Brilliant Blue G-250 for Bradford Assay, pure ethanol, Elabscience Collagen ELISA Kit, ELISA Reader (Infinite 200 PRO), Scanning Electron Microscopy(SEM) (Zeiss Evo LS10), H&E staining (Vector Laboratories) and Perkin Elmer DMA 8000 Dynamic Mechanical

In vitro analysis

Cell viability and recellularization: L929 (Mouse Fibroblast) cell line (American Type Culture Collection (ATCC) utilized for cell viability and adhesion tests. Cell line was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) with 10% Fetal bovine serum (FBS, Gibco) in a 5% CO2 humidified air incubator, maintained at 37 °C. When the 80% cell confluency is determined, cells washed with PBS and trypsinized with 0.25%Trypsin-EDTA for passaging and seeding each time. MTT assay was then used

Decellularization of ram cardiac tissue via supercritical CO2 and the hybrid method

The ram cardiac tissue was decellularized with scCO2(1 H) for 1 h, scCO2(4 H) for 4 h treated with scCO2 for 1 h/TritonX-100 for 2 h (2HT-1 H) and treated with scCO2 for 1 h/incubated overnight with TritonX-100(OT-1 H).

Scaffold characterization

The surface morphology of the obtained scaffolds was characterized by scanning electron microscopy. The total DNA and protein concentrations were determined. The collagen type 1 and type 3 content for each sample was examined. The Hematoxylin&Eosin (H&E) staining were performed

Discussion

In the study, the aim was decellularization of ram cardiac tissue via by optimizing scCO2 treatment conditions that were not reported previously. During this study, removing major cellular components, in the meantime, maintaining the mechanical strength of ECM will be achieved by optimization of decellularization conditions. For this purpose, the efficacy of SFE via the supercritical CO2 method and a hybrid method (scCO2 and Triton X) were optimized. In the study, we compared the

Conclusion

Decellularization is nowadays a popular method to obtain tissue or organs instead of human donors for transplantation. Animal-based biomaterials' biomechanical behavior can be controlled easily by modification of the decellularization process. In this study, ram cardiac tissue was chosen as a model due to its structural morphology and material characteristics in literature first time. The herein described method revealed that the scCO2 and hybrid treated cardiac tissue mimics 3-dimensional

Ethics Approval and Consent to Participate

Not applicable.

Funding

The author(s) received no specific funding for this work.

Consent for Publication

All quotations were mentioned in this manuscript with proper citation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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