Emerging Fabrication Techniques for Engineering Extracellular Matrix Biomimetic Materials

There is need to address the challenges of organ shortage, through development of tissues and organs with alternatives to those of the allograft-kind. This illustrates the quest behind novel biofabrication strategies such as 3D bio-printing, which is necessary to create artificial multicellular tissues/organs. Several findings have been reported in this review. First, the role of ECM components in tissue regenerative medicine is presented. Different ECM components such as collagen, gelatin, elastin, fibronectin, laminins and glycosaminoglycans are concisely examined for their tissue regenerative medicine applications. Next, current state of research on extrusionbased 3D bio-printing techniques and their limitations are reviewed. For example, we show that cell viability is still a challenge with extrusion, while the use of natural polymers such as collagen in improving composites’ mechanical properties is limited. Lastly, we examine unresolved research questions necessary to advance the present state of research in the field.

unresolved research questions necessary to advance the present state of research in the field were presented.

ECM Biopolymer Components for Tissue Regeneration
Tissues and organs are not composed of solely cellular components; instead, they converge with an extracellular matrix (ECM). The structure and role of the ECM differ depending on tissue natures. The ECM provides a microenvironment that is essential for cellular functionality and regulation [21].
The ECM offers biophysical, biochemical, and biomechanical cues for cellular components.
Furthermore, it provides structural support, functions as an adhesive substrate, presents growth factors to its receptors, sequesters, stores growth factors, senses, and transduces mechanical signals. Also, the ECM is involved in regulating numerous cellular purposes for instance survival, adhesion, migration, proliferation, differentiation, and supporting cells for binding [22,23]. The ECM organizes large fibrillar 3-D nets composed of glycosaminoglycans (GAGs) and various types of proteins such as collagen, fibronectin, elastin, and laminin. Among the numerous constituents of the ECM fibrous proteins (i.e., collagen and elastin), adhesive glycoproteins (fibronectin and laminin and glycosaminoglycans (i.e., hyaluronic acid) are recognized as the main components [24] as shown in Figure 1.

2.1.Collagen for tissue regeneration:
Collagen is the most abundant structural protein in the mammalian ECM that imparts tensile strength to prevent deformation of vascular tissue [25]. Collagen provides structural properties and resilience to the injured tissue in tissue regeneration manner [26]. Collagen delivers mechanical integrity to the vasculature and other biological tissues during tissue regeneration [27]. Some ECM molecules including type I collagen and fibronectin are known to be over expressed in renal tissue regeneration, promoting fibrosis and tissue dysfunction [28]. A good balance between inflammation and ECM remodeling is essential to maintain homeostasis and to restore essential functions after tissue damage [26]. Collagens provide tensile strength, regulate cell adhesion, support chemotaxis and migration, and direct tissue development in tissue engineering process [29].
Collagen acts as scaffolds providing ECMs and tissues with their structural organization and mechanical properties in tissue regeneration. In addition to their role as tissue designers, collagens interact with cell surface receptors, and regulate numerous biological processes either as fulllength proteins or via their bioactive fragments, called matricryptins or matrikines, released by limited proteolysis.

2.2.Gelatin for tissue regeneration:
Gelatin (Gel) is one of the major components of the ECM of various tissues [30]. Gelatin is a polypeptide partially hydrolysis or denatured product of collagen biocompatible, biodegradable and lower immunogenic [31,32].
Gautam et al [33]., stated that Human osteoblasts cells exhibited effective cell adhesion and significant proliferation over the gelatin nanofibers. The in vivo analysis by Samadian et al., [34] presented that gelatin can induce the neo-bone formation, osteocyte in lacuna woven bone formation, and angiogenesis in the defect location [35].

2.3.Elastin for tissue regeneration:
Elastin is an elastic protein of ECM and usually found in skin, lungs and blood vessels. Elastin provides mechanical strength, elasticity, firmness, and suppleness to the damaged tissues in the tissue engineering procedure [36,37]. A different important ECM component that delivers elasticity to the damaged tissue is elastin, which is involved in cellular functions such as cellular attachment, proliferation, differentiation, and migration [38]. Elastin is responsible for resilience and elasticity for cell expansion, differentiation and migration in tissue regeneration [39]. It is a highly cross-linked protein that self-assembles in the ECM to form robust elastic fibers which are essential for maintaining the structural integrity of injured tissues and organs [38].Also, Elastin provides the tissues with long-range elasticity, especially with participation of elastin when it is deposited on a scaffold in the tissue regeneration.

2.4.Fibronectin for tissue regeneration:
Fibronectin is a complex ECM glycoprotein that binds to integrin and other ECM proteins such as collagen. The importance of ECM proteins in mediating bone repair in tissue engineering is evident, and fibronectin has emerged as a pivotal regulator of this process [40,41]. Fibronectin functions in several stages of tissue regeneration [42]. Fibronectin acts as a 3-D scaffold directly following trauma, guiding the assembly of additional ECM components [43]. Also, fibronectin regulates cellular behavior via integrin-binding and growth factor-binding domains, promoting downstream responses including cell recruitment, proliferation and differentiation in tissue engineering method [44]. Fibronectin mediates cellular activities by binding with receptors in the tissue regeneration procedure [45].
In various functions of Fibronectin in the tissue regeneration process includes embryonic development, fibrosis and blood clotting, providing binding sites for mammalian cells [46].
Fibronectin can bind various ECM molecules such as collagen I and III, gelatin, thrombospondin, and heparin [47]. Fibronectin typically functions through interactions with the ECM network in tissue engineering [48]. Fibronectin also plays an essential role in guiding cell behavior, guiding and polarizing the formation and the progression from rostro to caudal of these cells and tissue segments in the tissue regeneration [49]. Fibronectin interacts with many other ECM proteins as well as small molecules, glycosaminoglycans (GAGs), cell surface receptors and other FN molecules in tissue regeneration process [50]. [29] Fibronectin displays a highly specific enrichment in hair follicle stem cells (HFSC) at the onset of regeneration [51].

2.5.Laminins for tissue regeneration:
Laminins are a structurally minor component of the ECM [52], that have been shown to play important roles in facilitating cell attachment and mechano-transduction by interacting with cells through cell-membrane receptors including integrins and syndecans [53]. Laminins aid mediating processes based on receptor-and matrix-binding properties in the tissue regeneration process [31].
Laminin mediates a number of cellular functions such as cell adhesion, migration, differentiation and survival in tissue engieering [27]. Laminin usually incorporated into matrix for cell expansion, differentiation and migration [54].

2.6.Glycosaminoglycans for tissue regeneration:
GAGs are multifaceted anionic un-branched hetero-polysaccharides which represent major structural and functional ECM components of connective tissues [55]. Glycosaminoglycan chains regulate several signaling pathways in normal and pathological processes through their interactions with different classes of matrix proteins in tissue engineering process.
GAGs participate in a number of vascular events such as the regulation of vascular permeability, lipid metabolism, hemostasis, and thrombosis, but also interact with vascular cells, growth factors, and cytokines to modulate cell adhesion, migration, and proliferation [56]. Proteoglycans are composed of GAG chains that are covalently linked to the core protein and considered to have a significant role in chemical signaling among cells.

2.7.Decellularized extracellular matrix (dECM) for tissue regeneration:
Decellularization is the process of removing cells and cellular debris from a tissue or organ and isolating the extracellular matrix (ECM). Decellularized ECM is a complex configuration of natural ECM components. dECM isolated from native tissues or organs after decellularization.
The dECM contains proteins, proteoglycans, and glycosaminoglycans responsible for cell adhesion, cell remodeling, and mechano-transduction in tissue engineering [57]. Significantly, dECMs do not contain many immunogenic components that are found in the native tissue, making them attractive for cardiovascular tissue engineering and regenerative medicine applications. In addition, tissue-specific dECMs also retain the biochemical and structural properties necessary for tissue function in the regeneration process [58]. dECM provides the unique structural and functional components to support cell growth in the tissue engineering [36]. dECM broadly used for various tissue engineering and regeneration applications owing to providing ECM biological signals to cells [21].

3D ECM Bio-printing
There is need to address the challenges of organ shortage, through development of tissues and organs alternative to those of allograft-kind [59]. This illustrates the quest behind novel biofabrication strategies necessary to create artificial multi-cellular tissues/organs. To this end, research has presented 3D -printing as an alternative method of developing custom-made equipment/devices for specific and personalized treatment and diagnostics [60] in tissue engineering [61,62,63], pharmaceutical screening [63,65], and drug manufacturing [66,67].
Increasing interest on the use of 3D bio-printing technique is due to the failure of conventional techniques to fabricate constructs requiring structural, mechanical and biological complexity [Ji and Guvendiren, 2017].

Extrusion Bio-printing
Extrusion bio-printing is fundamentally the squeezing out of bioink through a nozzle by mechanical or pneumatic forces to form filaments which are systematically assembled on a surface to form 3D architectures.
It is unarguably the most widely used technique amongst other bioprinting techniques such as photo-curing-based bio-printing and droplet-based bioprinting [92]. This is because of its numerous advantages. For instance, extrusion bio-printing is used to print a wide range of biocompatible materials. It also has a wide range of viscosity in the range of 30mPa/s to 6 x 10 7 mPa/s [93], hence can be used to provide structural support due to high viscosity and for maintaining cell viability due to low viscosity. Furthermore, the setup of extrusion printing is quite simple. However, its limitation compared to droplet-based printing is that cell viability is lower.
According to Smith et al, Chang et al [94,95] pressure and diameter of nozzle affect the cell viability of extrusion bio-printed materials.

Coaxial 3D Extrusion
While current reviews are presenting in-depth discussion on coaxial wet -spinning extrusion technique for printing [68], there is need for more review studies since 3D bioprinting can be used to fabricate cell-laden constructs for applications as in-vitro models or for therapeutic applications [69]. Self-assembled multicellular heterogeneous brain tumor fibers can be produced using coaxial extrusion 3D bioprinting system with alginate/gelatin as external shell [70].
To illustrate, the coaxial bioprinting provides cell-favorable gelatin methacryloyl (GelMA) microenvironments for tissue engineering and pharmaceutical screening application. This is observed when the coaxial extrusion is used to bioprint gelatin methacryloyl/alginate core/sheath microfibers to obtain 3D microfibrous constructs. Liu et al [79] suggests that it is possible to construct cell-laden GelMA materials at low concentrations of less than 2%, to support proliferation, as well as spreading for different cell types, implies a minimization and reduction of bioprinting requirement occur. Also, Hong et al [81] suggests that coaxial bioprinting of cell-laden vascular constructs using a gelatin-tyramine bioink can be used to develop 3D tissue architecture vascularization, demonstrating a blood vessel -like structure.
Building on the work of Liu et al, Pi et al [80] confirm that tubular tissue constructs possess cell viability and undergoes proliferation and differentiation, when they examined the digitally tunable microfluidic bioprinting of multilayered cannular tissues using blend bioinks consisting GelMA,  [70] reports that it is possible to study tumor microenvironment in vitro for tumor -stromal interactions using these self-assembled fiber-based 3D models. From this study, the coaxial bioprinted tumor fibers possess high expression of the used glioma stem/progenitor cell biomarker reported. Tumor stromal cells interacts with each other, which can result to an observable fusion. Moreover, studies have shown that coaxial bioprinting provides opportunity for cell-cell interactions in 3D space [71]. Besides, the possibility of fabricating 'coreshell' cell fiber structures demonstrate their scaffolding and soft nature, which is essential for tissue engineering and drug delivery applications [72,73,74]. The importance of these structures is in their ability to aid initial cell viability, vascularization, differentiations ad tissue heterogeneity [75,76,77].
The coaxial 3D extrusion-based bioprinting is versatile [78], as the tissue constructs obtained are usually viable, and could undergo proliferation and differentiation. All of these features indicate the wide applications in biomedical engineering.

3D-Printed Electrospun Materials, and their Mechanical Properties
Electrospinning and 3D bioprinting techniques can be used to fabricate novel 3D asymmetric construct (SAC) for skin tissue regeneration [82]. Miguel et al [82] suggests that the 3D printed skin construct can be used for tissue regeneration in skin wounds healing. Producing the top layer of the skin construct is possible through electrospinning, as a result of easy cell penetration and nutrient exchange [83,84]. Then, the 3D bioprinting has capability to replace broken dermis structure through an improved sequential layer- Other studies have shown that native skin presents same mechanical properties at 5-30MPa (tensile strength), 4.6-20MPa (Young's Modulus), and elongation at break of 35-115% [85,86]. The differences between the mechanical properties of the study and those of native skin can be attributed to the use of poly (caprolactone) PCL [87]. Though this study also reports that the hydrogel display a lower mechanical performance due to the higher porosity of the top layer, it is actually difficult to relate porosity to mechanical properties [88]. While Venugopal et al [89] evaluates the influence of porosity on production of PCL/nanohdroxyapatite/collagen nanofibers and reports that an increase in the pore size (from 2-15µm to 5-50 µm) decreased the tensile strength (2.72MPa to 1.28MPa), only a negative influence is nonetheless reported.
Moving forward, Chen et al [90] also claims that the 3D electrospinning hybrid technique is innovative for development of electrospun fiber-reinforced cartilage decellularized matrix (CDM) with customizable shape and controlled inner structure scaffold for cartilage regeneration.
Collagen fibers can improve the mechanical properties of the 3D-printed scaffolds, as they display an elastic nature in wet condition. This is however outdated because current studies show that natural polymers including collagen possess low mechanical strength [91]. Adding the fibers to scaffold materials can only improve their stiffness and toughness, not necessarily the main mechanical properties. Therefore, other composite materials such as iron should be added to the composite scaffold to enhance their mechanical properties, and optimal condition for more applications. Iron ions in composite materials have been severally reported to aid mechanical stabilization of biomaterials.

Microfludics: A new -layer of control for extrusion -based 3D printing.
Serex et al. [96] was able to demonstrate multiple smart printing heads that allow the use of new materials, enhance the print resolution and printing of multi-material parts by the application of extrusion 3D bio-printing. In this study, multi-material printing was achieved by careful design of the print head composed of micro channel merging into one just before the extrusion point.
Additionally, multiplexing and switching between materials during printing was achieved by using values made from polydimethysiloxane (PDMS). The advantage of static mixer over meanderbased mixer in the formation of new materials was also demonstrated.
Further in the study, enhanced resolution was achieved by dispersing the material using sheath flow and varying the core to laminar flow. The problem of residual fluid was resolved by using highly volatile materials such as perfluoro (methydecabin) (PFD) as sheath flow.
Through a cross flow design, the authors demonstrated a print head allowing the concentration of particles in solution which can be used to increase the filler concentration of a composition just before it is printed.
Mousavi et al., [97]  Liu et al. [98] fabricated cell-loaded with gelatin methacryloyl (GelMA)/alginate core/sheath microfibers composite with 3D-micro environment. This process was adopted to reduce core bio ink printing requirements and facilitates the fabrication of the call at low concentration.
Importantly, the biopolymer composite was printed using a co-axial nozzle -assisted 3D bioprinting. A favourable fabrication of was made by co-axial extrusion bioprinting of GelMA/alginate core/ sheath microfibers. Interestingly, the GelMA hydrogel was used as a core, while the alginate and sheath serves a template support. The novel technique provides extremely low concentrations of the cell microenvironment against the conventional bioprinting strategies.
More so, the method afforded a high degree of the encapsulated cell.

Perspectives and Future Research
1. How can neurite extension test beds be further adapted to understand the role of chemical molecules for stimulating cellular interaction and regeneration in nerve tissue engineering? [101].

2.
How can dorsal root ganglia (DRG) test beds be modified to ensure they utilize controlled release of growth factors?
3. What are the dose-dependent and gradient effects associated with the use of glial cell linederived neurotrophic factor (GDNF) platform? [102] 4. What is the potential mechanism of genes in regulating signaling pathways?

5.
How do external stimuli affect the interaction of specific genes in a tightly controlled microenvironment? [103] 6. How is endothelial cell viability related to jet velocity and cell penetration in research involving cell biology? [104] 7. How can modular hyaluronic acid and gelatin-based hydrogel platform be utilized for numerous applications in biomedicine? [105] 8. What is the effect of PCL scaffold pore sizes on the mechanical strength and cell retaining capacity of chitosan thermo-gel incorporated with 3D-printed poly (Ɛ-caprolactone) scaffold?

9.
What is the effect of chitosan thermogel on in-vivo application of chitosan-based 3D printed PCL scaffolds in load bearing bone defects?

Conclusion
Current research on biomedical application of several ECM components in tissue regenerative medicine, and emerging extrusion-based fabrication techniques have been reviewed in this work.
New insights on improving the mechanical stability involving the use of iron ions have also been discussed. Having reviewed current advances, we propose that the coaxial-based extrusion method is versatile and of more potential significance in tissue regenerative medicine. The field is expected to evolve more with

Conflict of Interest
The authors have no known conflict to declare