Scaffolds in tissue engineering

2.1 Characteristics of a biologically active scaffold

A 3-D, well defined porous structure to make the surface-to-volume ratio high for seeding of cells as many as possible.

2.2 Roles of a scaffold

Framework: It can work as a framework to support cellular migration into the defect from surrounding tissues. This is important when a fibrin clot is important.

2.3 Categories/classification of scaffolds

Naturally derived scaffolds: Acellular dermis and amniotic membrane

2.4 Design and production of scaffolds

There have been various methods to produce porous materials which are to be used as scaffolds in the field of tissue engineering and regenerative medicine.

Manipulation of fibres into nonwoven and woven structures [12].

2.5 Development of scaffolds is based on selection of biomaterial or on the production of the scaffold

Biomaterials-based approaches examples include:

Biomaterials that have been frequently used for other implications (eg. Polylactic acid-polyglycolic acid) [16]

2.6 (A) Conventional techniques

A significant number of scaffolds have been developed conventionally for drug delivery, but they have subsequently been used in 3D cell culture in the context of tissue engineering [18]. The traditional techniques of scaffold fabrication like solvent casting/particulate leaching are intended to define the scaffold shape and pore size but are mostly limited to the prior the scaffold internal design or connectivity of the void space [19].

2.7 (B) Solvent Casting & Particulate Leaching (SCPL)

In this technique, a solvent combined with uniformly distributed salt particles of a certain size is used to dissolve the polymer solution. The solvent evaporates leaving a matrix containing salt particles. The matrix is then submerged in water, and the salt leaches away to form a structure with high porosity. The solvent casting with particle leaching only fits thin membranes of thin wall three-dimensional specimens; otherwise, the soluble particles cannot be separated from within the polymer matrix [20]. Scaffolds developed by this method have a porosity between 50% and 90% [21].

The advantage is that the produced scaffold is of high porosity and with the capability of tuning the pore size, which makes it appropriate for the development and growth of the 3D cell [22]. Also this technique is relatively easy and low cost. One of the drawbacks of this fabrication technique is its time consumption since it only uses thin membranes. Layers of porous sheets allow only a defined number of pore networks between them and may, therefore, limit its suitability to use because of the limited porous size [23]. This technique applies various toxic solvents which take a lot of time to evaporate (days or weeks).

2.8 (C) Freeze-drying

The process of freeze-drying is also known as lyophilization. It involves the use of a synthetic polymer that is first dissolved in an appropriate solvent. After dissolution, the polymer solution is cooled under the freezing point, resulting in a solid solvent that is evaporated by sublimation to leave a solid scaffold with numerous interconnected pores [24]. In this technique, when the solution is cooled to freezing point, the solutes can be separated in the ice phase resulting in a small porous structure characterized by a “fence” of matter surrounding the ice. The scaffolds are achieved after consequent drying; by simple dissolving and freeze-drying, the macro porosity corresponds to the empty area initially occupied by ice crystals. The advantage is that the capability of obviating high temperatures that could decrease the activity of integrated biological factors [25].

Min and Lee [28], applied this technique for a three-dimensional scaffold fabrication using chitosan nanoparticles [26]. Freeze-drying technique is a more suitable method in biomedical application because of the use of water and ice crystals instead of an organic solvent during scaffold fabrication; however, this methodology is challenged in the fabrication of hierarchical structured scaffolds such as vascular systems in biomedicine [27]. As this method also uses cytotoxic solvents for mixing the polymer; hence, the fabricated scaffold needs to be washed repeatedly to remove the solvent and to minimize cell death.

2.9 (D) Thermal-Induced Phase Separation TIPS

TIPS is a low-temperature method designed to force phase separation via the temperature alternate related to setting the homogeneous polymer solution with a high temperature in a decrease temperature environment to induce phase separation so that a polymer-rich phase, as well as a poor polymer phase, is achieved [28]. A porous scaffold structure can be achieved when the solvent is eliminated with the aid of freeze-drying leaving a relatively porous, nanoscale fibrous network. This method can be utilized for the construction of the thermoplastic crystalline polymer scaffold. Low temperature can be utilized for the integration of bioactive molecules within the fibrous scaffold material.

Blaker et al. [34] proposed a new fabrication approach via the use of TIPS to obtain microspheres for TE and drug delivery; this technique made it practical to adjust the pore sizes to allow inclusion of fillers and drugs [29]. The main drawback of this technique is that limited materials can be used in fabrication and inadequate resolution.

2.10 (E) Electrospinning

Electrospinning is known as a technique for making fibres from a solution by using electricity. This technique is vital for developing nanofibrous scaffolds in tissue engineering. Electrospinning is a very complicated technique in which charging liquid under high voltage leads to the interaction between the surface tension and electrostatic repulsion that causes droplets on spinneret to erupt and stretch. A standard electrospinning system consists of four main components: a spinner with a syringe pump, a metallic needle, a high-voltage power supply, and a grounded collector [28, 30]. Even though electrospinning is a simple and quick method in fabrication of nanofibrous scaffolds, there still exists a challenge in fabrication of scaffolds with complex structures such as homogeneous distribution of pores, hence limited applications in biomedicine [31, 32].

2.11 (F) Rapid-prototyping

Rapid prototyping (RP) technologies, also known as solid free-from fabrication (SFF), are a set of manufacturing processes that can generate direct forms directly from computer-aided design (CAD) models of an object without needing specific tooling or knowledge. The RP systems combine powder, liquid, and sheet materials to make parts compared to machining methods (e.g., milling and drilling). Layer by layer, rapid prototyping machine can produce wood, ceramic, plastic and metal objects using thin horizontal cross sections from a computer-generated model [33]. RP scaffold fabrication technique enables manufacturing of designs with precise spatial control over polymer structure to deal with some of the challenges in traditional production methods [34]. The main benefit of these techniques is that they enable the production of customized and patient-specific scaffolds suitable for tissues and organs in question [30]. The basic RP techniques include 3D printing (3DP), fused deposition modelling (FDM), selective laser sintering (SLS), and stereolithography [28, 29].

2.12 (G) Stereolithography

Stereolithography method is basically used to creating solid, three-dimensional objects by consecutively printing a thin layer of ultraviolet (UV) curable material layer-by-layer. A stereolithography system has four main components, namely, a tank with a photosensitive liquid resin, a transferable built platform, a UV laser for radiating resin, and a dynamic mirror system. The process begins with a UV laser by depositing a layer of photosensitive liquid resin on the platform. After the solidification of the initial layer, the platform is lowered vertically. A second layer is then placed on the first layer; the process is repeated until a 3D scaffold is created. Finally, the uncured resin is cleaned off, and the scaffold is post cured under UV light.

Therefore, this method overcomes the challenges related to wastage in subtractive fabrication methods. Limitations in the process of photopolymerization, requiring massive amounts of monomers and post polymerization treatment to improve monomer conversion [35].

OPEN IN VIEWER

Fig. 1

Stereolithography

Gas foaming technique is a technique that has been evolved to cope with using high temperature and organic cytotoxic solvents. This technique uses relatively inert gas foaming agents such as carbon dioxide or nitrogen to pressurize modelled biologically degradable polymer with water or fluoroform until they are saturated or full of gas bubbles. This technique usually produces structures like a sponge with a pore size of 30 to 700μm and a porosity up to 85% [29]. The scaffold is made from multiple layers of adjacent microfilaments. It is utilized to process thermoplastic biopolymers. Generally, form several studies, FDM is useful in the scaffold design under different aspects of scaffold fabrication. The main drawback in this technique is the need for preformed consistent-sized fibres to feed through rollers and nozzle; it also has limitations in its application to biodegradable polymers.

2.14 (I) Selective Laser Sintering (SLS)

This technique was developed in 1986 by Texas University of Austin [36]. This technique uses laser as the power source to sinter powdered material defined by a 3D model in thin layers. Due to the use of a laser, this technique has been utilized to make various materials, such as polymers, metals, or ceramics [37]. The efficiency of this technique has been shown in the fabrication of scaffold using ultrahigh-molecular-weight polyethylene and in fabricating of bio nanocomposite microspheres composing of that PLLA that can efficiently produce microspheres carbonated hydroxyapatite (CHAP) nanospheres within a poly (L-lactide) (PLLA) matrix, in order to build tissue engineered scaffold [38].

The advantage is that it provides excellent user control over the microstructures of the produced scaffold by adapting various SLS process parameters such as percentage compositions of physically mixed polymer/composite powder blends to be utilized to obtain the preferred scaffold properties. The disadvantage is that additional procedure is required to remove injected powder at high operating temperature after processing the spin of the phase [39].

2.15 (J) Three-dimensional printing (3DP)

3DP is a process of creating tools and functional prototype features directly from the computer models. It is performed by applying the powdered material in layers and the selective fusion of the powder by “inkjet,” where the adhesive is printed. After continuous deposition of the layers, the unbound powder is taken out, yielding a complex 3D object. This process can be utilized to make ceramic, metal, and metal/ceramic composite part. The 3DP process can directly or indirectly function in printing the actual part or a mould [40]. 3DP is a new fabrication method for tissue engineering that can be utilized for precise control of scaffold structure at the micron level.

The success involves the ability to strictly follow the structure of the natural tissue and the mechanical characteristics of the scaffold, the scaffolds produced by 3DP technique have limited emulating of the nanoscale extracellular matrix properties of the tissue they aim to replace [41].

2.16 Bioprinting

Bioprinting is a 3DP technique, defined as “using material transfer processes for developing a biological pattern and assembly of relevant materials, cells, molecules, tissues, and biodegradable biomaterials with a prescribed structure to achieve some biological functions” [41]. The introduction of solvent-free, aqueous-based systems allows the direct printing of biomaterials on three-dimensional scaffolds for transplantation with/without seeded cells. It enables personalized medicine by using the technical form of cell growth. The technologies of 3D bioprinting can be classified into two types, namely, acellular and cellular constructs. In comparison with cellular bioprinting, acellular bioprinting can deliver a higher accuracy and greater shape complexity because it has less restrictive fabrication conditions than methods requiring the cell viability maintenance. Cellular bioprinting integrates cells and other bioagents with the material during the production process to fabricate living tissue constructs.

Therefore, the conditions and optimization of parameters in the construction of these constructs vary depending on existence or inexistence of living cells as well as biological substances. There are numerous different ways of 3D bioprinting, among which autonomous self-assembly, biomimicry, and minitissue building block are based on [42].

2.17 A. Inkjet bioprinting

Inkjet bioprinting is known as a noncontact technique that uses picolitre bioink droplets to construct 2D or 3D structures layered onto a substrate. Thermal ink jetting, acoustic wave jetting, and electro-hydrodynamic jetting are typical examples of material jetting techniques. These techniques have several advantages, such as low costs because of its similarity to the structures of a commercial printer, high speed of printing with the capability of supporting parallel work mode, and high cell viability. However, the major challenges are that the method includes the narrow material selectivity, the frequent print head clogging and keeping the biological material in liquid form for droplet formation [42].

2.18 B. Laser assisted bioprinting

Laser-assisted bioprinting is a technique based on laser-induced forward transfer. A typical system includes a pulsed laser beam coupled with a focusing system; a “ribbon” with donor transport support covered with a layer of gold or titanium able to absorb laser energy and a cell- and- hydrogel-containing layer of biological material; and a receiving substrate facing the ribbon. The laser-assisted bioprinter directs laser pulses on the laser-absorbing gold layer of the ribbon leading high-pressure bubble, which in turn drives the cell-containing materials to the collector substrate. One of the benefits of this method is that it has nothing to do with the problem of nozzle clogging with cells or material because it is nozzle free [41].