**1. Introduction**

Cardiovascular diseases are the most common cause of mortality in high-income countries [1] and are estimated to be responsible for one in three deaths worldwide [2]. Amongst them, aortic stenosis (AS) is the most common heart valve lesion encountered in clinical practice and affects 2% to 5% of adults over the age of 65 years [3]. Furthermore, narrowing at various level of the left ventricle may also affect the pediatric population, which shows a prevalence of 0.22 per 1000 live births [4]. The currently available surgical intervention is the replacement of the diseased valve either with biological prostheses or mechanical devices. The major limitations associated with the former are short post-surgical

**Citation:** Faggioli, M.; Moro, A.; Butt, S.; Todesco, M.; Sandrin, D.; Borile, G.; Bagno, A.; Fabozzo, A.; Romanato, F.; Marchesan, M.; et al. A New Decellularization Protocol of Porcine Aortic Valves Using Tergitol to Characterize the Scaffold with the Biocompatibility Profile Using Human Bone Marrow Mesenchymal Stem Cells. *Polymers* **2022**, *14*, 1226. https://doi.org/10.3390/polym14061226

Academic Editors: Antonia Ressler and Inga Urlic

Received: 21 February 2022 Accepted: 14 March 2022 Published: 17 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

lifespan (10 years) and low aptness in young patients [5,6]; the latter have longer durability but require lifelong anticoagulation therapy, which results in chronic complications.

Xenogeneic substitutes, i.e., chemically treated acellular valves, can be an alternative. Two main advantages are associated with these valves: first, their large availability; second, after implantation, they can stimulate mechanisms of tissue regeneration and healing, which favor specific cell types to grow and differentiate [7,8]. This is due to the presence of tissue-specific biochemical factors that are still preserved, even after decellularization. Indeed, the immunogenic responses triggered by biological tissues are still a hurdle that can be limited by improving decellularization protocols.

Decellularization involves the use of chemical/biological agents, physical stimulation, and/or a combination of both. Generally, decellularization protocols are applied through perfusion or under mechanical agitation to allow chemicals (mainly detergents, e.g., SDS, Triton, and sodium deoxycholate) or enzymes (e.g., trypsin) penetrating the tissue or the whole organ. Numerous studies demonstrated that decellularization is efficient for removing the DNA content, with an acceptable level of damage to the ECM composition, architecture, bioactivity, and mechanical properties [9,10]. The removal of nuclear materials is of primary importance since high levels of residual DNA can trigger severe immunological responses. Therefore, it is crucial to minimize the DNA content while avoiding potential structural damage to tissue/organ during decellularization [11].

In the present study, we propose a new efficient, low cytotoxic decellularization protocol (based on current data), which is based on a new detergent, Tergitol, which is eco friendly and highly degradable. The previously frequently applied Triton-100, is included in the candidate list by the European Chemical Agency (ECHA) because of its endocrine toxicity [12]; the alternative detergents were proposed by the same authorities, and we selected Tergitol due to its surfactant properties and rapid degradability. To our best of knowledge, the systemic toxicity of Tergitol has not been reported previously; however, it was reported to be toxic in the *C. elegans* model elsewhere [13]. The new decellularization protocol includes a cocktail of protease inhibitors, freezing and thawing cycles, and the application of Tergitol and sodium cholate in various steps along the whole process. Decellularization was applied to porcine aortic valves: the acellular matrices were characterized regarding their histological, immunological, biochemical, and biomechanical properties and shown to have significant perseverance.

ECMs are prone to cell attachment: this indicates scaffold biocompatibility and the possibility for tissue integration and remodeling in vivo. A well-preserved structure can provide a favorable environment for cell repopulation: the crosstalk between ECMs and cells can also promote the structural organization, directing cell functions. Understanding the process underlying the cross talk between cells and ECMs will lead to the design of the remodeling process of the engineered heart valves [14].

### **2. Materials and Methods**

### *2.1. Tissue Procurement and Preservation*

Fresh porcine hearts (20 in total) were harvested from 6–8 month-old adult pigs from the local abattoir (F.lli Guerriero S.r.l, Villafranca Padovana, Italy) within 2 h from death. The aortic roots were dissected from the heart and, after removing fat and blood remnants, washed with phosphate buffered saline (PBS, Sigma, Saint Louis, MO, USA), dried on blotting paper, and frozen in organ plastic bags at −80 ◦C until use.

### *2.2. Decellularization Procedure*

The Tergitol-based decellularization protocol was evolved from the TRICOL protocol previously reported by our group [15]. Briefly, tissues were thawed at RT for 3 h, before initiating the decellularization procedure. Then, they were submerged in 500 mL sterile jars in agitation at 4 ◦C with proteases inhibitors cocktail for 8 h, afterwards with 1% Tergitol (Sigma) at RT for the next 12 h; hypertonic/hypotonic shocks (8 h each cycle at RT) followed, and finally they were treated with 0.4% sodium cholate (Sigma) for 16 h in the dark at

RT. After decellularization, 2 cycles of washing with PBS (1X) were performed. Ethanol (4%, Carlo Erba, Cornaredo, Milano, Italy) and peracetic acid (0.1%, Sigma) solution was used for bioburden reduction. Finally, an endonuclease (Benzonase 25k U, Sigma, E1014) was applied to remove nucleic acids for 48 h at 37 ◦C.

### *2.3. DNA Extraction and Quantification*

Native (*n* = 4) and decellularized tissues (*n* = 4) were lyophilized with the Speed vac SPD130DLX (Thermo Scientific). Dry tissue samples (5–10 mg each) were used for DNA extraction that was performed according to the procedures described in the DNeasy Blood & Tissue Kit (Qiagen®, 69506, Valencia, CA, USA). Briefly, tissues were incubated in proteinase K solution and the lysis buffer ATL was added overnight in thermomixer at 56 ◦C. DNA concentrations were measured by using Nanodrop One (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit (QubitTM 1X dsDNA HS Assay Kit, Q33231, Waltham, MA, USA) fluorescent-based assays.
