*Review* **Emerging Biofabrication Techniques: A Review on Natural Polymers for Biomedical Applications**

**María Puertas-Bartolomé 1,2,\* , Ana Mora-Boza 3,4,\* and Luis García-Fernández 4,5,\***

4

	- Institute of Polymer Science and Technology (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

**Abstract:** Natural polymers have been widely used for biomedical applications in recent decades. They offer the advantages of resembling the extracellular matrix of native tissues and retaining biochemical cues and properties necessary to enhance their biocompatibility, so they usually improve the cellular attachment and behavior and avoid immunological reactions. Moreover, they offer a rapid degradability through natural enzymatic or chemical processes. However, natural polymers present poor mechanical strength, which frequently makes the manipulation processes difficult. Recent advances in biofabrication, 3D printing, microfluidics, and cell-electrospinning allow the manufacturing of complex natural polymer matrixes with biophysical and structural properties similar to those of the extracellular matrix. In addition, these techniques offer the possibility of incorporating different cell lines into the fabrication process, a revolutionary strategy broadly explored in recent years to produce cell-laden scaffolds that can better mimic the properties of functional tissues. In this review, the use of 3D printing, microfluidics, and electrospinning approaches has been extensively investigated for the biofabrication of naturally derived polymer scaffolds with encapsulated cells intended for biomedical applications (e.g., cell therapies, bone and dental grafts, cardiovascular or musculoskeletal tissue regeneration, and wound healing).

**Keywords:** biofabrication; microfluidics; electrospinning; 3D printing; electrospraying; natural polymers; cell encapsulation

### **1. Introduction**

Polymeric biomaterials have been developed to provide an artificial matrix that can mimic the cell microenvironment. This artificial matrix needs to provide appropriate biophysical and structural properties (e.g., stiffness, roughness, topography, and alignment) as well as biochemical cues (e.g., signaling, growth factors, and proteins) in order to promote the native capacity of cells to adhere, migrate, proliferate, and differentiate towards the growth of new tissue [1].

Natural polymers extracted from biological systems such as plants, microorganisms, algae, or animals have been used for decades in the biomedical field. These materials retain the biochemical cues and properties necessary to improve their biocompatibility and present similar structures to the extracellular matrix (ECM) of native tissues [2–5]. Therefore, they usually present good cellular attachment, improve cellular behavior, and avoid immunological reactions, although in some cases, these properties are limited due to batch variability within production and purification processes. The most common natural polymers used in biomedical applications include polysaccharides (e.g.,

**Citation:** Puertas-Bartolomé, M.; Mora-Boza, A.; García-Fernández, L. Emerging Biofabrication Techniques: A Review on Natural Polymers for Biomedical Applications. *Polymers* **2021**, *13*, 1209. https://doi.org/ 10.3390/polym13081209

Academic Editor: Paolo Ferruti

Received: 15 March 2021 Accepted: 3 April 2021 Published: 8 April 2021

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alginate [5–7], hyaluronic acid [3,8], and chitosan [9,10]), proteins (e.g., collagen [11], silk [12,13], gelatin [14–16], and fibrin [17]), and bacterial polyesters (e.g., bacterial cellulose [18]). However, the poor mechanical strength of natural polymers frequently makes the manipulation and biofabrication process difficult. For this reason, the use of derivatives or blends with different polymers are usually required to obtain appropriate mechanical properties for their use. An example is the modification of gelatin with methacrylamide to obtain a photopolymerizable biomaterial that can be used for 3D bioprinting and microfluidics [19–22].

Actual biomedical challenges require the use of complex polymer matrixes that can mimic the native ECM and regenerate the lost or damaged tissues [23–25]. Recent advances in biofabrication techniques allow the production of a polymer matrix with biophysical and structural properties similar to the ECM, and its combination with different cell lines is capable of proliferating and differentiating into the desired tissue. Moreover, the incorporation of different growth factors or other biomolecules can improve the migration, growth, and differentiation of the cells [3,26].

Currently, numerous research lines for polymer matrix biofabrication follow two different strategies for the incorporation of the cells: (i) cell implantation on the previously formed polymer matrix and (ii) fabrication of a polymer matrix with encapsulated cells.

The first strategy was used in the last decade, and it is restricted to the method of cell implantation. Normally, these systems do not present a good integration between cells and the polymer matrix, and their efficacy for tissue regeneration depends on the physical properties of the polymer matrix such as hydrophobicity, degradation rate, or stiffness [14,27]. Among the most used techniques, we can highlight layer-by-layer [28,29], melt molding [30], photolithography [31], and self-assembling [32].

The second strategy is the most investigated in recent years, since it allows the fabrication of advanced cell-laden structures with complex cellular microenvironments. Recently, some advanced techniques (i.e., microfluidics [33,34], electrospinning [10,35], and 3D printing [36,37]) allow the integration of cells directly into the polymer matrix with the adequate physical and biological properties to imitate the ECM of the desired tissue.

This review focuses on the biofabrication techniques of microfluidics, electrospinning, and 3D printing using natural polymers. These techniques have been recently explored to create polymer matrixes with embedded cells for biomedical applications, and they are in continuous evolution, as we are going to illustrate in the present review.

### **2. Microfluidics**

Microfluidics has emerged as a powerful tool for the high throughput generation of monodisperse microgels [33,34]. Microgels are defined as 3D-crosslinked particles that provide a porous polymeric network and can recapitulate the cellular microenvironment (i.e., ECM), mimicking in vivo conditions and diffusion of nutrients and metabolic waste [38–40] (Figure 1A,B). Specifically, microgels are fabricated in microfluidic devices by the generation of polymer droplets (i.e., droplet-based microfluidics) through water/oil emulsions followed by physical or chemical crosslinking. The most frequently used geometry configurations to generate the droplets in the devices are T-junction, flow-focusing, and co-flowing (or capillary) laminar streams, which are illustrated in Figure 1C [41–44]. Microgels are especially attractive as cell carriers, because their large surface-to-volume ratio promotes efficient mass transport and enhances cell-matrix interactions, but it is important to notice that cell microencapsulation requires a polymer network that ensures cell viability during microgel preparation and adequate crosslinking chemistry to form a polymer network [45]. Microfluidics technology provides a tight control over microgel chemical properties and composition by easily tuning the flow rates and components in the microfluidic channels, being a versatile biofabrication platform, where different crosslinking strategies can be applied [41]. As mentioned, the microdroplets generated in the microfluidic devices should undergo physical (e.g., electrostatic interaction, thermal gelation, and hydrogen bond interaction) or chemical crosslinking (photopolymerization, Michael addition, and enzymatic

reaction) to form solidified microgels [38]. Physical or chemical gelation will be chosen based on different factors like the type of polymer, the strategy for tissue encapsulation, as well as the final biomedical application. In addition, different crosslinking mechanisms can be combined to fulfil the desired features of the microgel systems [38,43,46,47], and these crosslinking processes can take place inside the microfluidic device (in situ crosslinking) or after microgel collecting [38]. In this review, most recent examples of interesting processes for the microfluidics generation of cell-laden microgels prepared using natural polymers and different crosslinking strategies are exposed (Table 1).

**Figure 1.** Recapitulating the natural cellular microenvironment in biomimetic microgels using droplet-based microfluidics. (**A**) The natural cellular microenvironment is composed of different cell types, ECM, and biomolecules such as growth factors. (**B**) Droplet-based microfluidics allows for versatile and high throughput generation of cell-laden microgels that can mimic the natural cellular environment. By mixing defined amounts of selected cells, ECM, and biomolecules, the microenvironment can be designed in a bottom-up approach with defined properties [34]. (**C**) Schematic illustration of different types of droplet generators, including T-Junction, flow-focusing, and co-flow (capillary) configurations. Adapted with permission from John Wiley and Sons Copyright®.


**Table 1.** Summary of the studies exposed in this review regarding microfluidics generation of cell-laden microgels.
