**1. Introduction**

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally; according to World Health Organization (WHO) more than 17.9 million people die from such causes every year—an estimated 31% of all deaths worldwide [1]. Simultaneously, the estimated healthcare cost for cardiovascular disease in Europe reaches to 169 billion € [2]. It is well established that more than 80% of CVD deaths occur in low-and middle-income countries compared to high-income countries [3,4]. Therefore, the need to reduce the economic burden is not debatable. CVD includes a wide group of complex disorders, namely peripheral arterial disease (PAD), coronary heart disease (CHD), cerebrovascular disease and rheumatic heart disease [2].

Current high-cost therapies include conventional tissue engineering, cell therapy and medical approaches [5,6]. In valve repairment, autografts are widely used by harvesting from autologous cell sources, namely parts of a patient's body with the advent of low risk of thromboembolism and prosthetic valve infection [7]. The end stage heart failure is treated by allografting a heart from a donor, while some valve replacement surgeries employ bovine or porcine heart valves. Xenografts, though, impute other undesirable properties, including cytotoxicity and calcification [8]. Conclusively, the aforementioned grafts have their set of drawbacks, including shortage of donor organs, mechanical mismatches, anticoagulation therapy and immune rejection [9]. Synthetic valves and vascular grafts can also be implanted to treat CVD; however, the high structural durability and low rate of reoperation is outweighed by the increased risk of anticoagulation complications in patients with long life expectancy [10]. Small-diameter vascular grafts (SDVGs) constructed from synthetic polymers and decellularized matrices are promising in the field of reconstructive

**Citation:** Kozaniti, F.K.; Metsiou, D.N.; Manara, A.E.; Athanassiou, G.; Deligianni, D.D. Recent Advancements in 3D Printing and Bioprinting Methods for Cardiovascular Tissue Engineering. *Bioengineering* **2021**, *8*, 133. https://doi.org/10.3390/ bioengineering8100133

Academic Editors: Efstathios Michalopoulos and Panagiotis Mallis

Received: 21 August 2021 Accepted: 24 September 2021 Published: 27 September 2021

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surgery; however, further evaluation and in vivo implementations are mandatory in order to be applied to therapeutic approaches in CVD [2].

Therefore, one showcasing solution will be the overarching focus on identifying alternative tissue treatments that preserve natural tissue without deleterious side effects.

The increased demand on recovery of damaged cardiovascular tissues in combination with the demand for low-cost but effective constructions, heralds new methods in tissue engineering [11]. Three-dimensional (3D) printing and bioprinting are the recent promising methods that successfully regenerate various organs, scaffolds and blood vessels, which can be used for replacing partly or thoroughly natural organs in the human body [12,13]. With the advent of additive manufacturing, 3D bioprinting technology employs a layer-bylayer approach which enables precise control over multiple compositions (biomaterials) and spatial distributions (cells) resulting in architectural construction accuracy [14]. Biomaterials that have been employed in 3D printing for cardiovascular tissue engineering are major natural or synthetic hydrogels, or decellularized matrices in order to mimic the dense vascular network that supports the cardiac tissue, by providing an interconnected porous network that enables cells to migrate, proliferate and receive vital nutrients and adequate oxygen supply [2,11,15,16]. This takes into consideration the survival distance limitation for cells which is no further than 100 ÷ 200 μm away from blood vessels [17]. Alginate and collagen are the most commonly used hydrogels in bioprinting following gelatin methacrylate, fibrinogen and gelatin [18]. Cell viability, proliferation and morphology after printing are crucially affected by characteristics of the selected bioink [19]. The challenge of bioink design is the improvement of printability without detracting cell viability [20]. Biomaterial-based hydrogels are capable of cell encapsulation. The major cell types that integrate the cardiac tissue are cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts. Furthermore, computational simulation with a patient's anatomical data and features is compulsory for an integrated patient-specific bioprinted construct.

The current review article, briefly outlines the basic principles and growing applications of 3D bioprinting, highlighting key developments and in vivo implementation in the field of cardiovascular tissue engineering. More specifically, 3D-printing definitions are presented, and the 3D construct stages of manufacturing are investigated followed by the computational and experimental designing approach. In addition, different techniques of bioprinting and constitution of the inks are presented thoroughly. In closing, this review outlines recent innovative in vivo studies on 3D bioprinted applicable therapeutic approaches in CVD.

#### **2. From 3D Printing to the New Era of 3D Bioprinting**

#### *2.1. Three-Dimensional Printing—Additive Manufacturing*

The terms "3D printing" and "additive manufacturing" are usually confused. According to the American Society for Testing and Materials (ASTM), additive manufacturing is the process of joining materials using 3D model data layer by layer in contrast to subtractive manufacturing methodologies, such as traditional machining [21,22], whereas "3D printing" is defined as object fabrication through the deposition of a material with the help of a print head, nozzle or another printer technology [21]. The two terms are often used synonymously, especially considering they are low end in price and/or overall capability [21].

Moroni et al. tried to define "3D printing" based on the appearance of the printing process with cell-laden inks [23]. According to them, in this additive manufacturing technology a jet of binder is directed at a powder bed to define a pattern. A slice of solid material is formed after the binding of the solvent to the powder. A new layer of powder is set and by repeating this process the scaffold is build layer by layer. This definition was based on the first patent for 3D printing of Sachs et al., in which a binder solution was deposited in a powder bed according to a Computer Aided Design (CAD) model [24]. However, in the recent research of Marti et al., 3D printing is referred as the process of

additive production of 3D objects, which starts from a 3D digital model [25]. Thus, the term 3D printing is still used instead of additive manufacturing for the sake of simplicity.

#### Computational Stage—Preparation of 3D Printing

Computational methods are widely used to study tissue engineered constructs. However, the entrance of computer designing is essential in the field of tissue bioengineering due to personalized medicine. The main idea is to produce a specialized human part for each specific patient, thus contributing to a more efficient and low-cost tissue engineering [26]. The main purpose of this strategy is to create a tissue engineered scaffold with similar mechanical and biological properties concerning the defective tissue [20]. This procedure includes the following steps.

The tissue defect is digitally visualized using imaging machines, particularly CT scan (Computer Tomography scan), MRI (Magnetic Resonance Imaging) and ultrasound scan. The next step is to create a scaffold that readily supports the formation of the new tissue. The architecture of the scaffold can be meticulously designed using Computer Aided Design (CAD), a feasible way to manipulate the design parameters of tissue porosity, dimensions and biological-related properties. The scaffold can now be integrated into the 3D model of the defective tissue. Subsequently, bioink will be fabricated by assessing the proper materials, the cell types and bioactive molecules, and the location and requirements of the injured area of the patient. Eventually, using bioprinting technology the cell seeded construct can be manufactured and then placed in a cell culture or implanted directly into the patient [27].

Three-dimensional printing enables the precise fabrication of computationally designed scaffolds with increased accuracy, flexibility and reproducibility. These methods allow scientists to conduct low-cost parametric studies in order to create the most functional construct for the addressed medical issue. The structural design and the mechanical behavior under different conditions of the small diameter composite vascular grafts can easily be optimized by using computational methods.

Computational methods integrate the 3D printing methods and provide the following advantages:


A crucial issue in 3D printed tissue engineering is that the new formatted tissue may not develop adequate vascularisation for long-term survival [12]. The 3D printed construct must provide nutrients and growth factors to cells in order to proliferate [15]. However, key parameters that affect the vascularisation and need to be overcome are the shear stress from blood flow and the wall shear stress [13]. A feasible way to anticipate this challenge is through computational fluid dynamics simulations. Nowadays, personalized medicine is widely applicable and therefore three-dimensional hemodynamic simulations could contribute to the diagnosis of CVDs in order to demonstrate the needs of each patient [30]. Conclusively, flow simulations support the design and additive manufacturing techniques to produce patient-specific 3D printed biodegradable scaffolds, thus being tailored to the individual patient based on their predicted post-surgical response [31].

## *2.2. Bioprinting*

Three-dimensional bioprinting has emerged as an advanced and novel process in the field of tissue engineering and regenerative medicine. Notably, researchers' interest in bioprinting is also evident in their efforts to define the term. According to Groll et al., Moroni et al. and Lee et al., bioprinting could be defined as the production of bio-engineered structures through computer-aided transfer processes in order to pattern and assemble living and non-living materials with a prescribed 2D or 3D organization [32–34].

Currently, the use of biomaterials in regenerative medicine and cardiovascular engineering faces challenges, including host inflammatory responses, immunogenicity, biomaterial degradation and toxicity of degradation products, that may affect the long-term function of the engineered tissue construct [35–37]. Therefore, the innovative biomaterialfree method of bioprinting is gaining attention in the scientific society.

Three-dimensional bioprinting has been expected to be a promising method in tissue engineering because of the ability to control precisely the geometry and the amount of biomaterials during construct fabrication [37]. More specifically, this technique can fully incorporate cells into hydrogels that satisfactorily mimic the microenvironment of the extracellular matrix (ECM) and directly print onto the targeted host location [38]. In cardiovascular engineering there are multilateral problems that need to be overcome in order to achieve an integrated 3D bioprinted model [39,40].

Cell viability and vascularization of printed tissues are key factors which determine the effectiveness of bioprinted tissues. Another impediment that needs to be overcome is the promotion of mass transfer of nutrients and oxygen into bioprinted scaffolds, including adhesion molecules and factors that induce angiogenesis [41]. The vascular tissues need substitutes with specific physical characteristics. For example, high stiffness is not favourable like in the cases of bone and cartilage tissues. On the contrary, vascular substitutes must be malleable enough to be shaped correctly in order to regenerate vessels [18]. A schematic representation of the bioprinting process and most recruited bioprinters is illustrated in Figure 1.

**Figure 1.** Schematic representation of bioprinters. ( **A**) Development of bioengineered vascular grafts with computer assisted software. Different types of bioprinters (**B**), microextrusion bioprinter ( **C**) and laser-assisted bioprinter ( **D**). (**E**) Production and transplantation of bioengineered vascular grafts. Piezoel: Piezoelectric.

#### 2.2.1. Bioprinting Methods

Several studies and reviews conclude that there are three main types of bioprinting modalities: Droplet-based, extrusion-based and laser-based techniques. [14,42]. Herein, the widely applicable techniques with their set of benefits and drawbacks are briefly introduced in Table 1.
