Fabrication Techniques for Scaffolds Applied in Regenerative Medicine

Abstract

Tissue engineering strategies in regenerative medicine combine cells, scaffolds, and growth factors to regenerate and reconstruct pathologically damaged tissues such as periodontium, bone, nerves, cartilage skin, heart valves, and various other organs. Scaffolds have a major role as they provide a three-dimensional environment for tissue regeneration. They act as an extracellular matrix that favors the ingrowth of new cells thereby assisting the regeneration of target tissues. Various properties of scaffolds like scaffold architecture, surface topography, biodegradability, mechanical properties, and manufacturing process are important to achieve optimal results in tissue engineering. Scaffold fabrication can be achieved by conventional as well as non-conventional current manufacturing techniques. Solvent casting, phase separation, particulate-leaching, gas foaming, freeze-drying, and electrospinning are conventional methods for fabricating scaffolds. The architecture of these scaffolds greatly depends on processing techniques. Fused deposition modeling, hydrogel processing, selective laser sintering, decellularization techniques, three dimensional printing, and bioprinting, are current techniques for scaffold fabrication. The chapter will give an overview of each fabrication technique and will aid biomedical engineers to select the ideal fabrication technique for specific applications in the field of regenerative medicine.

1. Introduction

Regenerative medicine is a broad field that focuses on self-healing and tissue engineering is an integral part of it. Langer and Vacanti have described tissue engineering as “an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve function or a whole organ” [1]. Generally, three strategies are adopted for the creation of new tissues: cells or cell substitutes, tissue-inducing substances, and cells placed on or within matrices known as scaffolds [2]. Scaffolds are engineered to cause desirable cellular interactions thereby contributing to the formation of new functional tissues. They mimic the extra-cellular matrix and form a microenvironment that serves purposes like cell attachment, migration, diffusion of vital nutrients, etc. The clinical success of tissue engineering largely depends upon scaffold architecture, material composition, and fabrication techniques.

The specific requirements that a scaffold must-have is as follows. They should have high porosity and appropriate pore size to enable active cell seeding and nutrient diffusion. They should be biodegradable with a degradation rate similar to the tissue formation rate. Adequate structural integrity is needed to maintain a three-dimensional environment. Both synthetic and natural materials are used for scaffold fabrication. The synthetic ones include polylactic acid (PLA) [3], polyglycolic acid (PGA), polycaprolactone (PCL), and poly-lactic-co-glycolic acid [4] each with its tailored degradation rates. The tunability and biocompatibility are advantageous for scaffold fabrication [4]. Natural materials can be proteinaceous such as collagen and fibrin or polysaccharides like chitosan and glycosaminoglycans (GAG). Decellularised tissues may also function as scaffolds. They are formed by chemically extracting cells from tissues leaving behind an extracellular matrix but can possess serious immunologic complications (Table 1).

Premade porous scaffolds	Decellularised extracellular matrix	Cell sheets	Cell encapsulated in hydrogel
Raw materials fabricated into porous scaffolds	Decellularised native tissues	ECM secretion on confluent cells forms cell sheets	Cell mixing on monomer solution
↓	↓	↓	↓
Cells seeded to form cell seeded scaffolds	Porous scaffolds	Laminated to multiple cell sheets	Cell encapsulated in hydrogel
↓	↓	↓	↓
Implantation	Cell seeded scaffolds	Implantation	Injection
	↓
	Implantation

Table 1.

Different scaffolding approaches in tissue engineering.

2. Scaffold fabrication

The design and fabrication of scaffolds depend upon the mechanical, biological, and physicochemical requirements of the scaffold. The pore size, pore interconnectivity, degradation statistics, stability, etc. are taken into consideration during the fabrication process [5]. The 2D scaffold fabrication is more accurate and easy with good control over the physicochemical properties. Fabrication of 3D scaffolds is challenging and requires advanced bio-printing and bio assembly which is automated. Computer assisted designing and machining technologies help in such 3D designs as sponges, meshes, and foams. 3D technologies have further revolutionized into 4D printing techniques which are quite expensive.

Several scaffold fabrication techniques have been described so far based on the biomaterial used, the tissue intended to regenerate as well as the purpose of the scaffold. The techniques can be broadly classified into conventional and advanced types.

3. Conventional techniques

Conventional fabrication technologies include solvent casting/ particle leaching, thermally induced phase separation, gas forming, freeze drying, melt molding, sol-gel method and electrospinning [6].

3.1 Solvent casting/particle leaching

Here, a polymer is dissolved in a highly volatile solvent into which a porogen is uniformly distributed. The porogen can be an organic compound like gelatin, collagen, or glucose microspheres or inorganic water soluble salts like KCl or NaCl. The polymer-solvent-porogen mixture is cast into a mold. When the solvent is evaporated, a composite network of polymer with entrapped porogen is obtained. Then using a suitable solvent, the porogen is dissolved which creates a porous scaffold. The porosity can be altered by altering the composition of the polymer mixture or the volume fraction of porogen.

The technique produces scaffolds with a degree of porosity between 50–90% and pore sizes ranging from 5 to 600 μm. The pore sizes can also be tuned by altering the porogen content or its size and shape [7]. The process is relatively easy and of low cost. The long processing time, variations in pore interconnectivity, and poor mechanical properties are the disadvantages [8]. The thickness of the scaffold is also limited. Hazards from residual solvent may also be anticipated.

3.2 Thermally induced phase separation/TIPS

The polymer to be used like PLA is dissolved in an easily sublimable solvent with a low melting point like dioxane. The mixture is then quenched to induce phase separation and polymer rich and polymer poor phases are formed. When the mixture is cooled below the melting point of the solvent and then subjected to vacuum drying, sublimation takes place leaving behind a porous scaffold. The scaffolds obtained have high porosity of more than 90% with uniform pore size and good pore interconnectivity. But uniform pore distribution cannot be attained and the reproducibility of scaffolds is also unattainable [6]. Since the technique uses lower temperatures and there is the complete removal of the organic solvent, TIPS is particularly employed for bioactive heat labile drugs. Thermoplastics are mainly processed by the TIPS technique. The process is relatively inexpensive also.

3.3 Gas foaming

This technique uses pressurized and non-pressurized gases instead of organic solvents for porosity formation. In the polymer solution, gases are introduced by in situ generation of gas bubbles known as nucleation. Nucleation can be achieved by a chemical blowing agent or by a physical blowing agent. Agents like sodium bicarbonate act as chemical blowing agents whereas gases like nitrogen and carbon dioxide are used as physical blowing agents [9]. The evolution of gases results in void formation and formation of a porous matrix.

By this method, hazards related to the organic solvents are eliminated. Highly porous scaffolds are obtained with the preservation of the bioactive species. Porosity is approximately 85% and pore size is approximately 100μm. This method applies to both hydrophobic and hydrophilic polymers [10]. The disadvantages include lack of precise control over pore size and poor pore interconnectivity. The processing time is also longer [11].

3.4 Freeze drying/Lyophilisation

In freeze drying, a polymer solution is prepared by dissolving the polymer in an organic solvent or organic solvent-water emulsion. The polymer solution is mold cast and frozen below its triple point by liquid nitrogen or by refrigeration. The frozen mixture is then subjected to two step drying process. The primary and secondary drying by sublimation removes the formed ice crystals and pores are formed in their places [12].

It’s a commonly used method where almost dry and highly porous scaffolds with high interconnectivity are made. The porosity obtained is more than 90% and pore sizes range from 20 to 200 μm. Irregular pore sizes are obtained but pore sizes can be controlled by altering the temperature, drying time or polymer concentration. The procedure is expensive and time consuming [13].

3.5 Melt molding

This is the technique used for thermoplastic polymers where the polymers are melted and cast into a suitable 3D mold. The structure of the mold conforms to the defect. Porosity will be introduced by methods like gas foaming or particulate leaching. Later, the scaffolds are freeze dried. Most commonly used polymer for melt molding is PLGA due to its low glass transition temperature. PLGA is mixed with gelatin microspheres and cast in a Teflon mold. The mold is then heated above the glass transition temperature of PLGA which allows the incorporation of biomaterial through the gelatin forming a composite. The composite is then placed in water so that the water soluble microspheres get dissolved leaving behind a porous structure [14].

The method is simple with precise control over pore size and pore interconnectivity. There is no use of organic solvents that is detrimental to cell growth and differentiation. The disadvantage is that thermolabile drugs cannot be incorporated because of the high temperatures during melt molding [15].

The most commonly used strategy in melt molding is the injection molding technique. It’s a highly economical, productive and flexible technique.

3.6 Sol-gel method

The principle behind the sol-gel process is hydrolysis and polycondensation. The precursors are organic or inorganic metal compounds like metal alkoxides or metal chlorides. These are dissolved in water or organic solvent where they undergo hydrolysis and polycondensation to form a colloid solution. The resultant solution is then cast into 3D molds. The low viscosity of sol makes it easier to cast into any particular shape. Then in the 3D mold, the gelation process starts with interactions between the contents forming 3D networks. The resultant product is dried in the mold itself and later subjected to gentle heating for solidification of the matrix. Dehydration or chemical stabilization is done later to create ultra-stable porous material [16].

This technique uses low temperatures but longer processing time and the high cost of the raw materials precludes its wide application. The sol-gel method is particularly employed for the fabrication of bioactive glasses and bioceramics.

3.7 Electrospinning

The most commonly employed technique to produce nanofibers is electrospinning. In this spinning, technique electrostatic forces are used to produce fibrous scaffolds from biocompatible polymers. It’s a rapid and simple technique where high voltage electric current is applied to a molten polymer solution that is extruded out of a fine needle. The ultra-fine polymer fibers thus generated are deposited on a grounded collector. The method uses two electrodes, one placed into the spinning polymer solution and the other attached to the collector. When an electric charge is applied to the molten polymer contained in the capillary tube, a charged jet of fluid is ejected. The discharged jet solidifies when traveling in the air and forms a polymer fiber that is deposited onto a grounded metallic collector [17].

The formed scaffold mimics the extracellular matrix and has a high surface to volume ratio. Electrospinning can process a wide range of polymers and can fabricate micro and nano scaffolds with high porosities.

4. Advanced techniques/rapid prototyping (RP)

Rapid prototyping comprises those advanced manufacturing processes that enable the creation of tailor-made patient-specific scaffolds useful in tissue engineering. This unique process helps in the direct manufacturing of scaffolds from data obtained by computer aided designing (CAD) models, CT or MRI. RP is also known as solid free-form fabrication or additive manufacturing. Since there is a layer-by-layer deposition of the material, precise spatial control is possible over the polymer structure and desirable mechanical properties can be achieved [18].

The first 3D printing technology was reported by Hull et al, in 1986. They described a 3D scaffold fabrication technique that used UV light exposure for layer-by-layer fabrication of constructs [19]. Because it’s a layer-by-layer fabrication technique, the precision of constructs was high, unlike the conventional procedure. Over the years, other additive manufacturing techniques were developed.

The basic technique is as follows. A computer aided three-dimensional design of the scaffold is produced preferably from the data obtained through CT or MRI. The design is then transferred to a standard tessellation language (STL) [14] format where it can be sliced into thin horizontal cross-sections. The resultant data obtained is then transferred to the RP machine which fabricates the scaffold by layer-by-layer deposition of the polymer material.

4.1 Stereolithography

Stereolithography is an additive manufacturing technique that solidifies resin into 3D scaffolds by photopolymerization. The principle of stereolithography is the selective curing of a photosensitive resin/polymer by using an ultraviolet [20] laser. The main components of the system are a tank containing photosensitive liquid polymer, a built platform for depositing the cured resin, a UV laser for radiation and a dynamic mirror system. With the help of UV laser, a layer of photosensitive resin is deposited on the platform. Once the initial layer is deposited and solidified, the platform is lowered vertically and a second layer is placed over the first layer. In this manner, a 3D scaffold is created. After cleaning off the uncured resin, post curing is done for the scaffold using UV light. The process uses data from an STL file, where a 3D model is converted into 2D slices, based on which layer-by-layer deposition occurs.

The curing reaction of resin is an exothermic reaction that is initiated by UV light. During the curing process, two transition stages are noticed – gelation and vitrification. In gelation, the liquid resin changes to rubbery consistency with increased viscosity. In vitrification, the rubber resin changes to glassy solid resin [21].

Four generations of stereolithography approaches have been described so far. The first generation technique described by Hull uses a laser beam to cure the liquid resin [19] the second generation technique is known as projection stereolithography where each layer is simultaneously cured by photo mask technology. Here, once a thin layer of resin is deposited, the resin is illuminated through a photo mask for curing. The uncured portions are removed, refilled with resin and again cured. The process is repeated until the desired pattern is obtained. The need for several masks and the precision required during mask alignment makes this a time consuming process and therefore less desirable [22].

In 2015, Tumbleton et al, described the third generation approach which is the continuous lithography technique. It is based on continuous liquid interface production (CLIP) with which the print speed of scaffolds can be reduced from hours to minutes [23]. A recent innovation in this context is the volumetric stereolithography that fabricates 3D geometries and is useful in processing high viscous resins.

Uniformity in pore structure and pore interconnectivity can be obtained. The viability of cells and the presence of growth factors can be maintained. The technique is quite expensive.

4.2 Selective laser sintering (SLS)

An additive manufacturing technique in which laser is used as the heat source to fuse the powder particles is known as SLS. This is also a layer-by-layer formation technique based on computer assisted pre-designed architectures. A computer controlled laser beam like a CO₂ laser is used on powdered materials. The heat from the laser sinters/ fuses the powder and this continues in a layer-by-layer manner to build 3D constructs. Cells or biomaterials cannot be incorporated into scaffolds due to the higher temperature involved [9].

High precision and control over scaffold fabrication are possible with a high degree of porosity and mechanical strength. Only thermally stable materials can be processed by this method. Removal of residual material is also tedious as the compound materials are in powdered form [24].

4.3 Fused deposition modeling (FDM)

The most popular and widely used additive manufacturing technique is fused deposition modeling. They are used for metals and thermoplastic materials. Here, a temperature controlled extruder and nozzle help in depositing the thermoplastic material onto a platform to produce 3D scaffolds. The material in the form of filament is driven through the extruder using rollers and gets converted into a molten form. Through the nozzle, a thin layer is deposited precisely and sequentially. The extrusion and deposition are entirely under the control of a computer aided tool. The deposition takes place on the surface of a base that is movable vertically which allows it to be lowered after the deposition of each layer, thereby adding more layers on top. Layer-by-layer deposition of the thin filament takes place which cools on exposure to air resulting in the fusion of these filaments to form scaffolds of desired 3D architecture [9].

The scaffolds obtained have high mechanical strength and a high degree of porosity. The size and structure of the pores can be adjusted [25]. As the technique uses high temperatures, non-thermoplastic materials cannot be processed by FDM.

4.4 Three dimensional bioprinting

A recently developing technology that uses bioinks and living cells to print 3D structures is bioprinting. In this additive manufacturing technique also, layer-by-layer development of tissues and organs is accomplished in a bottom-up manner. Every attempt is made to mimic the normal tissues in both form and function. Advantages include the creation of patient-specific tailor-made constructs which mimic the concerned tissues or organs. Since the process is automated, precision can be achieved in the organization of cells and extracellular matrix. Layer-by-layer construction also ensures good pore interconnectivity. But the technique is also expensive and complex [26]. The bio ink used for bioprinting is a composite made of cells and other biomaterials. They can also be made from natural and synthetic polymers such as alginate, collagen, hydroxyapatite, polyethylene glycol,l etc.

The different strategies involved in 3D bioprinting are ink-jet printing, extrusion printing, and laser assisted bioprinting [27]. The basic steps in all strategies are the same with a pre-processing, processing and post-processing phases. In the pre-processing stage, the bio images of the target tissue are obtained via CT or MRI, followed by the construction of accurate 3D models using CAD. The data thus obtained will be converted to 2D stacks based on which the bio ink is selected and fed to the bioprinter. In the processing phase, actual bioprinting is done in a layer-by-layer manner using any of the four strategies mentioned earlier. Post-processing phase is a maturation phase in a bioreactor, followed by structural and functional characterization of the constructs [28].

Bioprinting can be done as a scaffold based and scaffold free approach. In the scaffold based approach, the cellular deposition takes place on a biomaterial matrix in the form of a 3D construct. The construct should closely mimic the extracellular matrix so that cells will grow and populate it. In scaffold free approach, cells or tissues will be directly laid down to form patterns like spheroids or honeycombs [29].

4.4.1 Ink-jet based bioprinting

In this type, the bioink is deposited over a biopaper substrate. The technique is digitally controlled and has a non-contact printing pattern. Two ways of ink-jet printing are continuous ink-jet printing and the drop-on-demand (DOD) technique [30].

In continuous ink-jet (CIJ) printing, pressure is applied on the bioink to force it out of a nozzle as a continuous jet. The ejected jet is subjected to an electric field to deflect it onto a substrate. The excess droplets are deflected to a gutter for collection and re-use. In DOD inkjet printing, the droplets are produced on demand. Instead of the continuous pressure CIJ method, here a pulsed pressure is used. DOD technique is more favored because there is no reuse of the bioink as in the CIJ method, thereby avoiding the risk of contamination [31].

The DOD technique is further categorized into two types, thermal method and piezoelectric method. In thermal method, a pulsed electric current is applied to a heating element that vaporizes the ink creating pressure by the vapor. This pressure forces the bioink through the nozzle onto the substrate [32]. But in the piezoelectric method, a pulsed voltage is applied onto the piezoelectric transducer that creates pressure. Exposure to high temperatures is occurring only for a few microseconds and so the viability of cells is not compromised [33]. The printing characteristics depend on the viscosity of the ink and the size of the droplets, which in turn depends on factors like nozzle size, nozzle to substrate distance, temperature gradient, frequency of current etc.

4.4.2 Extrusion based bioprinting

This is a direct ink writing method where the material is continuously extruded out of the nozzle for layer-by-layer deposition and the creation of 3D architecture. Here, either mechanical force from a piston or pneumatic forces are used for extrusion of bioink through the nozzle onto the substrate. The whole process is computer controlled and produces small streams of bioink in contrast to the droplets of inkjet method [34]. This technique is favorable for highly viscous bioink types and also for high cell densities. The drawback is that the high forces for extrusion may affect cell viability and distorts the cellular structures [20].

4.4.3 Laser assisted 3D bioprinting

This is a non-contact direct writing method where the deposition of bioink onto the substrate is facilitated by a pulsed laser beam. The three main elements of laser assisted technique are a pulsed laser source, a ribbon coated with heat sensitive bioink and a substrate for printing [30]. Laser source can be UV or near UV wavelength lasers. Volatilization of the bioink on the ribbon takes place with the application of laser which propels a high speed jet of cell laden bioink onto the substrate. The substrate here is a plate made of quartz or materials that allows laser transmission [35].

Sometimes, a sacrificial interlayer is placed between the bioink and the ribbon, which has laser absorbing properties, thereby assisting in maintaining cell viability. Coating the substrate surface with a natural biopolymer can also help, by facilitating cell attachment and growth. The two approaches described are the LIFT and MAPLE DW. LIFT uses high energy pulsed laser whereas the latter uses a low powered pulsed laser. In LIFT, the sacrificial layer is made of metal or metal oxides. High energy pulsed laser can cause rapid thermal expansion of this sacrificial layer that causes high speed propulsion of bioink onto the substrate. Biological laser processing or BioLP is a variant technique where the sacrificial layer is a hydrogel.

The living cells can either be embedded inside the polymeric matrix or imprinted onto the matrix. The viability of cells is affected by the energy of the laser, the thickness of sacrificial layer and the viscosity of bioink. Thicker sacrificial layers and viscous bioink favored cell viability whereas higher energy of laser compromised it.

The factors to be considered during 3D bioprinting are the following. The choice of bioink should be accurate to favor the formation of a desirable 3D structure. Low viscosity bioinks should be preferred as high viscosity bioinks can compromise the cell viability during the extrusion process [36]. Most of the systems can only print one type of bioink during one deposition process. Attempts including the use of multi nozzle bioprinters were tried to extrude different types of bioink during a single deposition process. Moreover, the structure once printed cannot transform itself in response to biological stimuli. This is in contrast to the nature of native tissues that are highly responsive and dynamic [37]. Taking all these into consideration happened the emergence of four dimensional printing techniques.

5. Four dimensional printing

Four dimensional printing is the most advanced technology for the fabrication of multi material scaffolds. They are tailor-made and patient-specific with the potential to change shape over time [38]. They are mostly made of those materials that respond to stimuli and undergo dynamic configuration in response. Thus, they are of high use in large bony defects. Their development is still in infancy, because of challenges like the need to cope with the dynamic transformation of native tissues [38].

6. Conclusion

Since tissue engineering is an interdisciplinary field, the developments happening in scaffold fabrication techniques have an important role in determining success in this field. The fabrication techniques have evolved from conventional ones to the current 3D bioprinting methods where tissues and organs are engineered. Each technique has its pros and cons. 3D printing technology has and will play an important role in the field of scaffold development. But the development of a scaffold that can match the dynamic responses of the native tissues is still unattained. Studies related to the nanoarchitecture of the scaffolds as well as the incorporation of bioactive molecules and their sustained release are still in their infancy and are areas of potential research.

Conflict of interest

The author declares no conflict of interest.